RfpA, RfpB, and RfpC are the Master Control Elements of Far-Red Light Photoacclimation (FaRLiP)

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

Zhao, Chi; Gan, Fei; Shen, Gaozhong

Terrestrial cyanobacteria often occur in niches that are strongly enriched in far-red light (FRL; λ > 700 nm). Some cyanobacteria exhibit a complex and extensive photoacclimation response, known as FRL photoacclimation (FaRLiP). During the FaRLiP response, specialized paralogous proteins replace 17 core subunits of the three major photosynthetic complexes: Photosystem (PS) I, PS II, and the phycobilisome. Additionally, the cells synthesize both chlorophyll (Chl) f and Chl d. Using biparental mating from Escherichia coli, we constructed null mutants of three genes, rfpA, rfpB, and rfpC, in the cyanobacteria Chlorogloeopsis fritschii PCC 9212 and Chroococcidiopsis thermalis PCC 7203. The resulting mutantsmore » were no longer able to modify their photosynthetic apparatus to absorb FRL, were no longer able to synthesize Chl f, inappropriately synthesized Chl d in white light, and were unable to transcribe genes of the FaRLiP gene cluster. We conclude that RfpA, RfpB, and RfpC constitute a FRL-activated signal transduction cascade that is the master control switch for the FaRLiP response. FRL is proposed to activate (or inactivate) the histidine kinase activity of RfpA, which leads to formation of the active state of RfpB, the key response regulator and transcription activator. RfpC may act as a phosphate shuttle between RfpA and RfpB. Our results show that reverse genetics via conjugation will be a powerful approach in detailed studies of the FaRLiP response.« less

The Consulting Challenge: A Case Competition

ERIC Educational Resources Information Center

Sachau, Daniel A.; Naas, Patricia A.

2010-01-01

The Consulting Challenge is a yearly case competition in which teams of graduate students respond to a request for proposals (RFP) for consulting services. The case and RFP are based on a problem that a host organization has experienced. Over 3 days, students meet with representatives of the host organization, analyze data, prepare a proposal for…

Improving Evaluation in Nonprofit Organizations: A Study of How Evaluation Requests for Proposals Are Developed, Responded to, and Fulfilled

ERIC Educational Resources Information Center

Matthews, Nakia S.

2015-01-01

It is often necessary for nonprofit organizations (NPOs) to formulate a Request for Proposal (RFP) to procure essential contracted services. This is most common when seeking the services of an external evaluator. Since most NPOs do not have internal evaluation staff, developing an appropriate RFP can be quite challenging. With limited literature…

State Assessment Program Item Banks: Model Language for Request for Proposals (RFP) and Contracts

ERIC Educational Resources Information Center

Swanson, Leonard C.

2010-01-01

This document provides recommendations for request for proposal (RFP) and contract language that state education agencies can use to specify their requirements for access to test item banks. An item bank is a repository for test items and data about those items. Item banks are used by state agency staff to view items and associated data; to…

Service Delivery to the Handicapped: The Role of the Federal Procurement Process. Part II. Selecting Contractors by Competitive Award.

ERIC Educational Resources Information Center

Donaldosn, William S.; Stephens, Thomas M.

1979-01-01

Sections address the RFP/IFB (Request for Proposals/Invitation for Bids) process and procedures for selecting the "best" contract. In reviewing the federal procurement code, and the recent decision of the Government Accounting Office, particularly regarding the NIMIS (National Instructional Materials Information System contract), inconsistencies…

Theoretical study on the laser-driven ion-beam trace probe in toroidal devices with large poloidal magnetic field

NASA Astrophysics Data System (ADS)

Yang, X.; Xiao, C.; Chen, Y.; Xu, T.; Yu, Y.; Xu, M.; Wang, L.; Wang, X.; Lin, C.

2018-03-01

Recently, a new diagnostic method, Laser-driven Ion-beam Trace Probe (LITP), has been proposed to reconstruct 2D profiles of the poloidal magnetic field (Bp) and radial electric field (Er) in the tokamak devices. A linear assumption and test particle model were used in those reconstructions. In some toroidal devices such as the spherical tokamak and the Reversal Field Pinch (RFP), Bp is not small enough to meet the linear assumption. In those cases, the error of reconstruction increases quickly when Bp is larger than 10% of the toroidal magnetic field (Bt), and the previous test particle model may cause large error in the tomography process. Here a nonlinear reconstruction method is proposed for those cases. Preliminary numerical results show that LITP could be applied not only in tokamak devices, but also in other toroidal devices, such as the spherical tokamak, RFP, etc.

Networking and knowledge exchange to promote the formation of transdisciplinary coalitions and levels of agreement among transdisciplinary peer reviewers.

PubMed

Lobb, Rebecca; Petermann, Lisa; Manafo, Elizabeth; Keen, Deb; Kerner, Jon

2013-01-01

Funding for transdisciplinary chronic disease prevention research has increased over the past decade. However, few studies have evaluated whether networking and knowledge exchange activities promote the creation of transdisciplinary teams to successfully respond to requests for proposals (RFPs). Such evaluations are critical to understanding how to accelerate the integration of research with practice and policy to improve population health. To examine (1) the extent of participation in pre-RFP activities among funded and nonfunded transdisciplinary coalitions that responded to a RFP for cancer and chronic disease prevention initiatives and (2) levels of agreement in proposal ratings among research, practice, and policy peer reviewers. Descriptive report of a Canadian funding initiative to increase the integration of evidence with action. Four hundred forty-nine representatives in 41 research, practice, and policy coalitions who responded to a RFP and whose proposals were peer reviewed by a transdisciplinary adjudication panel. The funder hosted 6 national meetings and issued a letter of intent (LOI) to foster research, practice, and policy collaborations before issuing a RFP. All provinces and territories in Canada were represented by the coalitions. Funded coalitions were 2.5 times more likely than nonfunded coalitions to submit a LOI. A greater proportion of funded coalitions were exposed to the pre-RFP activities (100%) compared with coalitions that were not funded (68%). Overall research, practice, and policy peer reviewer agreement was low (intraclass correlation 0.12). There is widespread interest in transdisciplinary collaborations to improve cancer and chronic disease prevention. Engagement in networking and knowledge exchange activities, and feedback from LOIs prior to submission of a final application, may contribute to stronger proposals and subsequent funding success. Future evaluations should examine best practices for transdisciplinary peer review to facilitate funding of proposals that on balance have both scientific rigor and are relevant to the real world.

Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins

NASA Astrophysics Data System (ADS)

Krumholz, Arie; Shcherbakova, Daria M.; Xia, Jun; Wang, Lihong V.; Verkhusha, Vladislav V.

2014-02-01

Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8 mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo.

Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

PubMed Central

Sun, Chongyun; Li, Chao; Wang, Xiaochen; Liu, Haican; Zhang, Pingping; Zhao, Xiuqin; Wang, Xinrui; Jiang, Yi; Yang, Ruifu; Wan, Kanglin; Zhou, Lei

2015-01-01

Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a “probe dropout” manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories. PMID:26599667

A Holistic Compliance Model for Capture Teams: A Grounded Theory Approach

ERIC Educational Resources Information Center

Anderson, Duane C.

2013-01-01

By the time a Department of Defense (DOD) Request for Proposal (RFP) is awarded, its initial targets have moved, in many cases so much as to render any replies by the defense industry to an RFP useless. Requirements tend to evolve during the development of systems specifications. This may be caused in part by new information acquired during the…

[An experimental study on a slow-release complex with rifampicin-polylactic-co-glycolic acid-calcium 
phosphate cement].

PubMed

Wu, Jianhuang; Ding, Zhou; Lei, Qing; Li, Miao; Liang, Yan; Lu, Tao

2016-09-28

To prepare the slow-release complex with rifampicin (RFP)-polylactic-co-glycolic acid (PLGA)-calcium phosphate cement (CPC) (RFP-PLGA-CPC complex), and to study its physical and chemical properties and drug release properties in vitro.
 The emulsification-solvent evaporation method was adopted to prepare rifampicin polylactic acid-glycolic acid (RFP-PLGA) slow-release microspheres, which were divided into 3 groups: a calcium phosphate bone cement group (CPC group), a CPC embedded with RFP group (RFP-CPC group), and a PLGA slow-release microspheres carrying RFP and the self-curing CPC group (RFP- PLGA-CPC complex group). The solidification time and porosity of materials were determined. The drug release experiments in vitro were carried out to observe the compressive strength, the change of section morphology before and after drug release. 
 The CPC group showed the shortest solidification time, while the RFP-PLGA-CPC complex group had the longest one. There was statistical difference in the porosity between the CPC group and the RFP-CPC group (P<0.05); Compared to the RFP-PLGA-CPC complex group, the porosity in the CPC group and the RFP-CPC group were significantly changed (both P<0.01). There was significant difference in the compressive strength between the RFP- PLGA-CPC complex group and the CPC group (P<0.01), while there was significant difference in the compressive strength between the RFP-CPC group and the CPC group (3 days: P<0.05; 30 and 60 days: P<0.01). The change of the compressive strength in the CPC was not significant in the whole process of degradation. The sizes of PLGA microspheres were uniform, with the particle size between 100-150 μm. The microspheres were spheres or spheroids, and their surface was smooth without the attached impurities. There was no significant change in the section gap in the CPC group after soaking for 3 to 60 days. The microstructure change in the RFP-CPC group was small, and the cross section was formed by small particles. The pores of section in the RFP-PLGA-CPC complex group increased obviously, and PLGA microspheres gradually disappeared until the 60th day when there were only empty cavities left. The RFP-PLGA-CPC complex group had no obvious drugs sudden release, and the cumulative drug release rate was nearly 95% in the 60 days. The linear fitting was conducted for the drug release behavior of the complex, which was in accordance with zero order kinetics equation F=0.168×t.
 The porosity of RFP-PLGA-CPC complex is significantly higher than that of CPC, and it can keep slow release of the effective anti-tuberculosis drugs and maintain a certain mechanical strength for a long time.

The two Dictyostelium discoideum autophagy 8 proteins have distinct autophagic functions.

PubMed

Meßling, Susanne; Matthias, Jan; Xiong, Qiuhong; Fischer, Sarah; Eichinger, Ludwig

2017-06-01

Autophagy is a highly conserved cellular degradation pathway which is crucial for various cellular processes. The autophagic process is subdivided in the initiation, autophagosome maturation and lysosomal degradation phases and involves more than forty core and accessory autophagy-related (ATG) proteins. Autophagy 8 (ATG8, in mammals LC3) is a well-established marker of autophagy and is linked to the autophagic membrane from initiation until fusion with the lysosome. We generated single and double knock-out mutants of the two Dictyostelium paralogues, ATG8a and ATG8b, as well as strains that expressed RFP-ATG8a and/or GFP-ATG8b, RFP-ATG8b, RFP-GFP-ATG8a or RFP-GFP-ATG8b in different knock-out mutants. The ATG8b¯ mutant displayed only subtle phenotypic changes in comparison to AX2 wild-type cells. In contrast, deletion of ATG8a resulted in a complex phenotype with delayed development, reduced growth, phagocytosis and cell viability, an increase in ubiquitinylated proteins and a concomitant decrease in proteasomal activity. The phenotype of the ATG8a¯/b¯ strain was, except for cell viability, in all aforementioned aspects more severe, showing that both proteins function in parallel during most analysed cellular processes. Immunofluorescence analysis of knock-out strains expressing either RFP-GFP-ATG8a or RFP-GFP-ATG8b suggests a crucial function for ATG8b in autophagosome-lysosome fusion. Quantitative analysis of strains expressing RFP-ATG8a, RFP-ATG8b, or RFP-ATG8a and GFP-ATG8b revealed that ATG8b generally localised to small and large vesicles, whereas ATG8a preferentially co-localised with ATG8b on large vesicles, indicating that ATG8b associated with nascent autophagosomes before ATG8a, which is supported by previous results (Matthias et al., 2016). Deconvoluted confocal fluorescence images showed that ATG8b localised around ATG8a and was presumably mainly present on the outer membrane of the autophagosome while ATG8a appears to be mainly associated with the inner membrane. In summary, our data show that ATG8a and ATG8b have distinct functions and are involved in canonical as well as non-canonical autophagy. The data further suggest that ATG8b predominantly acts as adapter for the autophagy machinery at the outer and ATG8a as cargo receptor at the inner membrane of the autophagosome. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

48 CFR 415.207 - Handling proposals and information.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 48 Federal Acquisition Regulations System 4 2013-10-01 2013-10-01 false Handling proposals and... Proposals RFP Offeror 1. To the best of my knowledge and belief, no conflict of interest exists that may.... 3, 2005] ...

48 CFR 415.207 - Handling proposals and information.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 48 Federal Acquisition Regulations System 4 2011-10-01 2011-10-01 false Handling proposals and... Proposals RFP Offeror 1. To the best of my knowledge and belief, no conflict of interest exists that may.... 3, 2005] ...

48 CFR 415.207 - Handling proposals and information.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Handling proposals and... Proposals RFP Offeror 1. To the best of my knowledge and belief, no conflict of interest exists that may.... 3, 2005] ...

48 CFR 415.207 - Handling proposals and information.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 48 Federal Acquisition Regulations System 4 2012-10-01 2012-10-01 false Handling proposals and... Proposals RFP Offeror 1. To the best of my knowledge and belief, no conflict of interest exists that may.... 3, 2005] ...

48 CFR 415.207 - Handling proposals and information.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 48 Federal Acquisition Regulations System 4 2014-10-01 2014-10-01 false Handling proposals and... Proposals RFP Offeror 1. To the best of my knowledge and belief, no conflict of interest exists that may.... 3, 2005] ...

Software Product Lines: Report of the 2010 US Army Software Product Line Workshop

DTIC Science & Technology

2010-06-01

requirements and statement of work ( SOW ) tasks can be in- cluded in the request for proposal (RFP) and the contract. 2.2.1 Basic Product Line Acquisition... SOW tasks in Figure 1. Two additional tasks (at the third tier level) ac- count for sustaining the production capability over the life cycle and...Acquisition Strategy RFP and SOW Initial Product Line Scope Product Line Business Case Capability Description Document Teaming Product Line

Progress in Design and Construction of the Optical Communications Laser Laboratory

NASA Technical Reports Server (NTRS)

Wilson, K. E.; Britcliffe, M.; Golshan, N.

1999-01-01

The deployment of advanced hyperspectral imaging and other Earth sensing instruments on board Earth observing satellites is driving the demand for high-data-rate communications. Optical communications meet the required data rates with small, low mass, and low-power communications packages. JPL, as NASA's lead center in optical communications, plans to construct a 1-m Optical Communications Telescope Laboratory (OCTL) at its Table Mountain Facility (TMF) complex in the San Gabriel Mountains of Southern California. The design of the building has been completed, and the construction contractor has been selected. Ground breaking is expected to start at the beginning of the 1999 TMF construction season. A request for proposal (RFP) has been issued for the procurement of the telescope system. Prior to letting the RFP we conducted a request for information with industry for the telescope system. Several vendors responded favorably and provided information on key elements of the proposed design. These inputs were considered in developing the final requirements in the RFP. Keywords: Free space optical communications, lasercom, telescopes, ground stations, adaptive optics, astrometry, Table Mountain Facility

Enhancing the biophysical properties of mRFP1 through incorporation of fluoroproline

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

Deepankumar, Kanagavel; Nadarajan, Saravanan Prabhu; Ayyadurai, Niraikulam

2013-11-01

Graphical abstract: Enhancing the biophysical properties of mRFP1 through incorporation of (2S, 4R)-4-fluoroproline at proline residues after mutating non-permissive site Pro63 into Ala. -- Highlights: •We incorporate (4S)-FP into mRFP1 led to insoluble protein. •Whereas, incorporation of (4R)-FP resulted in soluble but lost its fluorescence. •mRFP1-P63A mutant accommodate (4R)-FP and gave soluble protein with fluorescence. •Moreover mRFP1-P63A[(4R)-FP] showed enhanced biophysical properties of protein. -- Abstract: Here we enhanced the stability and biophysical properties of mRFP1 through a combination of canonical and non-canonical amino acid mutagenesis. The global replacement of proline residue with (2S, 4R)-4-fluoroproline [(4R)-FP] into mRFP1 led to solublemore » protein but lost its fluorescence, whereas (2S, 4S)-4-fluoroproline [(4S)-FP] incorporation resulted in insoluble protein. The bioinformatics analysis revealed that (4R)-FP incorporation at Pro63 caused fluorescence loss due to the steric hindrance of fluorine atom of (4R)-FP with the chromophore. Therefore, Pro63 residue was mutated with the smallest amino acid Ala to maintain non coplanar conformation of the chromophore and helps to retain its fluorescence with (4R)-FP incorporation. The incorporation of (4R)-FP into mRFP1-P63A showed about 2–3-fold enhancement in thermal and chemical stability. The rate of maturation is also greatly accelerated over the presence of (4R)-FP into mRFP1-P63A. Our study showed that a successful enhancement in the biophysical property of mRFP1-P63A[(4R)-FP] using non-canonical amino acid mutagenesis after mutating non-permissive site Pro63 into Ala.« less

New transmission planning methodology for requesting proposals for wind generation

NASA Astrophysics Data System (ADS)

Isaacs, Andrew L.

The increasing interest in renewable energy technologies during the last decade has caused conventional transmission and generation expansion planning methodologies to be strained and in some cases abandoned. This is due both to the quantity of generator interconnection requests and the constraints imposed by deregulated energy industry structures. One technique used to control the influx of renewable generation while maintaining competitive principles is a Request for Proposals (RFP). However, lack of transmission planning due to a disconnection between generation and transmission owners, difficulty in identifying viable projects, and high risk for proponents stand as obstacles to the goals of an RFP. This research proposes a procedure which minimizes the effect of these obstacles; meeting the purchaser requirements for low price and combining conventional planning concepts with feedback from competitive structures. The general features of the method include definition of generation limits and study area, expansion plan design, transmission cost evaluation, optimal selection of requested generation levels, and final selection of successful proponents. The method is shown to be effective in creating an RFP where proponents are well-informed and provided with cost certainty to reduce bid price, buyers are able to determine end costs of their energy, and good expansion planning principles are maintained. A case study using a real system in New Mexico demonstrates these concepts.

Solar Requests for Proposals | State, Local, and Tribal Governments | NREL

Science.gov Websites

who have a lot of experience in this area may handle the process on their own while others may decide greatest number of respondents and give the agency the greatest number of options. Solar RFP Elements As to the selection criteria. Sources Solar Powering Your Community: Key Elements of Solar Requests for

Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.

PubMed

Subach, Fedor V; Patterson, George H; Renz, Malte; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V

2010-05-12

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

75 FR 72830 - Medicare Program; Quality Improvement Organization (QIO) Contracts: Solicitation of Proposals...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-26

... information relating to the RFP. ADDRESSES: Proposals for the contracts must be submitted to the Centers for...] Medicare Program; Quality Improvement Organization (QIO) Contracts: Solicitation of Proposals From In-State... QIOs' contracts. It also specifies the period of time in which in-State QIOs may submit a proposal for...

Confirmation of germ-line transmission in the red fluorescence protein (RFP) transgenic cloned male cat.

PubMed

Cho, Su-Jin; Lee, Young S; Lee, Jae-Ik; Bang, Jae-Il; Yang, Jing; Klassen, Henry; Kong, Il-Keun

2010-12-01

The production of transgenic animals is highly desirable for biotechnology and basic research. We investigated the reproductive ability of a red fluorescence protein (RFP) transgenic cloned male cat (RFP TG cat) by natural mating with a domestic female cat. The RFP expression levels were examined in early embryogenesis, tissues from 45-day-old two fetuses, and four RFP TG cat offspring. The RFP gene was detected in tissue samples from one dead kitten, including several organs and the skin. Also, under a fluorescent light source, we were able to directly detect the RFP expression of in in vitro-produced blastocysts derived with sperm from the RFP TG cat. These results indicate that the RFP TG cat exhibits normal reproductive fertility, stable germ-line transmission of the RFP transgene, and characteristic RFP expression in its offspring. We isolated feline neural progenitor cells from a 45-day-old fetus derived from the natural mating of the RFP TG cat with a domestic female cat. Isolated brain and retinal progenitor cells were successfully passaged at least four times post isolation (day 23), and showed a high RFP expression level. This method of producing genetically modified cloned cats will be important for generating biomedical models of human diseases.

76 FR 66169 - Office of Advocacy and Outreach Federal Financial Assistance Programs

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-26

...) Information about how to obtain proposal forms and the instructions for completing such forms. (14...) Regulatory information. (8) Definitions. (9) Minimum and maximum budget requests and whether proposals... Content of a proposal. The RFP provides instructions on how to access a funding opportunity. The funding...

FY 2018 Grant Announcement: FY2018 Support for Geospatial Analysis Support

EPA Pesticide Factsheets

The U.S. Environmental Protection Agency’s (EPA) Chesapeake Bay Program Office (CBPO) is announcing a Request for Proposals (RFP) for applicants to provide the Chesapeake Bay Program (CBP) partners with a proposal for providing geospatial analysis support

The new millennium program: Fast-track procurements

NASA Astrophysics Data System (ADS)

Metzger, Robert M.

1996-11-01

The National Aeronautics and Space Administration's (NASA's) New Millennium Program (NMP) has embarked on a technology flight-validation demonstration program to enable the kinds of missions that NASA envisions for the 21st century. Embedded in this program is the concept of rapid mission development supported by a fast-track procurement process. This process begins with the decision to initiate a procurement very early in the program along with the formation of a technical acquisition team. A close working relationship among the team members is essential to avoiding delays and developing a clear acquisition plan. The request for proposal (RFP) that is subsequently issued seeks a company with proven capabilities, so that the time allotted for responses from proposers and the length of proposals they submit can be shortened. The fast-track procurement process has been demonstrated during selection of NMP's industrial partners and has been proven to work.

Bifurcation in the MHD behaviour of a self-organizing system: the reversed field pinch (RFP)

NASA Astrophysics Data System (ADS)

Cappello, S.

2004-12-01

Within the framework of MHD modelling the RFP is shown to develop turbulent or laminar regimes switching from the former to the latter in a continuous way depending on the strength of dissipative forces (the higher they are the more laminar is the corresponding regime). In either of these cases interesting features can be observed such as the occurrence of quasi-periodic relaxation events involving reconnection processes, or the formation of stationary helical symmetric configurations. The first case corresponds to the conventional turbulent dynamo in the RFP where perturbations with multiple helical harmonic content are present. The second case corresponds to a global single helical deformation of the current channel. This simpler configuration is associated with a laminar electrostatic dynamo field and may also be found as a solution of a helical Ohmic equilibrium problem where a finite beta is necessary. The continuity of the transition between the two regimes suggests that the simple helical symmetric solution can provide a fruitful intuitive description of the RFP dynamo in general. Many of the MHD predictions are in good agreement with experimental findings and suggest possible improvements for the confinement properties of the RFP configuration.

Nonlinear 3D MHD verification study: SpeCyl and PIXIE3D codes for RFP and Tokamak plasmas

NASA Astrophysics Data System (ADS)

Bonfiglio, D.; Cappello, S.; Chacon, L.

2010-11-01

A strong emphasis is presently placed in the fusion community on reaching predictive capability of computational models. An essential requirement of such endeavor is the process of assessing the mathematical correctness of computational tools, termed verification [1]. We present here a successful nonlinear cross-benchmark verification study between the 3D nonlinear MHD codes SpeCyl [2] and PIXIE3D [3]. Excellent quantitative agreement is obtained in both 2D and 3D nonlinear visco-resistive dynamics for reversed-field pinch (RFP) and tokamak configurations [4]. RFP dynamics, in particular, lends itself as an ideal non trivial test-bed for 3D nonlinear verification. Perspectives for future application of the fully-implicit parallel code PIXIE3D to RFP physics, in particular to address open issues on RFP helical self-organization, will be provided. [4pt] [1] M. Greenwald, Phys. Plasmas 17, 058101 (2010) [0pt] [2] S. Cappello and D. Biskamp, Nucl. Fusion 36, 571 (1996) [0pt] [3] L. Chac'on, Phys. Plasmas 15, 056103 (2008) [0pt] [4] D. Bonfiglio, L. Chac'on and S. Cappello, Phys. Plasmas 17 (2010)

Design study to simulate the development of a commercial freight transportation system

NASA Technical Reports Server (NTRS)

Batill, Stephen M.; Costello, Kevin; Pinkelman, Jim

1992-01-01

The Notre Dame Aerospace Engineering senior class was divided into six design teams. A request for proposals (RFP) asking for the design of a remotely piloted vehicle (RPV) was given to the class, and each design team was responsible for designing, developing, producing, and presenting an RPV concept. The RFP called for the design of commercial freight transport RPV. The RFP provided a description of a fictitious world called 'Aeroworld'. Aeroworld's characteristics were scaled to provide the same types of challenges for RPV design that the real world market provides for the design of commercial aircraft. Fuel efficiency, range and payload capabilities, production and maintenance costs, and profitability are a few of the challenges that were addressed in this course. Each design team completed their project over the course of a semester by designing and flight testing a prototype, freight-carrying remotely piloted vehicle.

Near-infrared fluorescent proteins for multicolor in vivo imaging

PubMed Central

Shcherbakova, Daria M.; Verkhusha, Vladislav V.

2013-01-01

Near-infrared fluorescent proteins are in high demand for in vivo imaging. We developed four spectrally distinct fluorescent proteins, iRFP670, iRFP682, iRFP702, and iRFP720, from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging in living cells and mice using standard approaches. Five iRFPs including previously engineered iRFP713 allow multicolor imaging in living mice with spectral unmixing. PMID:23770755

A core functional region of the RFP1 promoter from Chinese wild grapevine is activated by powdery mildew pathogen and heat stress.

PubMed

Yu, Yihe; Xu, Weirong; Wang, Jie; Wang, Lei; Yao, Wenkong; Xu, Yan; Ding, Jiahua; Wang, Yuejin

2013-01-01

RING-finger proteins (RFP) function as ubiquitin ligases and play key roles in plant responses to biotic and abiotic stresses. However, little information is available on the regulation of RFP expression. Here, we isolate and characterize the RFP promoter sequence from the disease-resistant Chinese wild grape Vitis pseudoreticulata accession Baihe-35-1. Promoter-GUS fusion assays revealed that defense signaling molecules, powdery mildew infection, and heat stress induce VpRFP1 promoter activity. By contrast, the RFP1 promoter isolated from Vitis vinifera was only slightly induced by pathogen infection and heat treatment. By promoter deletion analysis, we found that the -148 bp region of the VpRFP1 promoter was the core functional promoter region. We also found that, in Arabidopsis, VpRFP1 expressed under its own promoter activated defense-related gene expression and improved disease resistance, but the same construct using the VvRFP1 promoter slightly improve disease resistance. Our results demonstrated that the -148 bp region of the VpRFP1 promoter plays a key role in response to pathogen and heat stress, and suggested that expression differences between VpRFP1 and VvRFP1 may be key for the differing disease resistance phenotypes of the two Vitis genotypes.

Sample RFP for Architectural Services, 2000.

ERIC Educational Resources Information Center

Arizona State School Facilities Board, Phoenix.

This document presents a sample request for proposal that Arizona school districts can use when requesting architectural services, from the general request requirements to response information and signature sheet. General proposal requirements cover such areas as information on special terms and conditions, the scope of architectural services…

7 CFR 2500.018 - Electronic submission.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 15 2014-01-01 2014-01-01 false Electronic submission. 2500.018 Section 2500.018... Pre-Award: Solicitation and Proposals § 2500.018 Electronic submission. Applicants and awardees are encouraged, but not required, to submit proposals and reports in electronic form as prescribed in the RFP...

7 CFR 2500.018 - Electronic submission.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 15 2012-01-01 2012-01-01 false Electronic submission. 2500.018 Section 2500.018... Pre-Award: Solicitation and Proposals § 2500.018 Electronic submission. Applicants and awardees are encouraged, but not required, to submit proposals and reports in electronic form as prescribed in the RFP...

7 CFR 2500.018 - Electronic submission.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 15 2013-01-01 2013-01-01 false Electronic submission. 2500.018 Section 2500.018... Pre-Award: Solicitation and Proposals § 2500.018 Electronic submission. Applicants and awardees are encouraged, but not required, to submit proposals and reports in electronic form as prescribed in the RFP...

Tulane/Xavier Universities hazardous materials in aquatic environments of the Mississippi River Basin. Quarterly progress report, October 1, 1995--December 31, 1995

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

NONE

1995-12-31

To provide the necessary administrative support to assure that the scientific and educational goals of the project are obtained and to assure that all Department of Energy reporting requirements and requests are fulfilled.The grant reporting is divided into three aspects: Collaborative Cluster projects, Initiation projects and Education projects. A cluster project is one or more closely related collaborative, multidisciplinary research projects in which a group of investigators employs a synergistic approach to the solution of problems in the same general area of research. An initiation project typically involves a single investigator. The purpose of the project is to undertaken pilotmore » work, lasting no more than one year, which will lead to the successful submission of an externally-funded proposal or the development of a collaborative cluster project. The education projects are designed to develop courses with emphasis on environmental studies and/or to train students in areas of environmental research. Accomplishments this quarter are: (1) Issued a request for proposals (RFP) to complete work during the next year of the project. Forty-two proposals were received in response to the RFP. (2) Selected and convened a panel of experts to review the proposals and make funding recommendations. (3) Assigned each proposal to two or more reviewers and sent copies of all proposals to each review panelist. (4) Interfaced with Oak Ridge National Laboratory regarding QA/QC issues related to sampling and analysis for the project. (5) Held numerous phone conversations with DOE representatives regarding funding allocations procedure changes as a result of the delays in the Federal budget and appropriations process this year. This quarterly report briefly describes research projects ongoing in this program.« less

Enhanced rifampicin delivery to alveolar macrophages by solid lipid nanoparticles

NASA Astrophysics Data System (ADS)

Chuan, Junlan; Li, Yanzhen; Yang, Likai; Sun, Xun; Zhang, Qiang; Gong, Tao; Zhang, Zhirong

2013-05-01

The present study aimed at developing a drug delivery system targeting the densest site of tuberculosis infection, the alveolar macrophages (AMs). Rifampicin (RFP)-loaded solid lipid nanoparticles (RFP-SLNs) with an average size of 829.6 ± 16.1 nm were prepared by a modified lipid film hydration method. The cytotoxicity of RFP-SLNs to AMs and alveolar epithelial type II cells (AECs) was examined using MTT assays. The viability of AMs and AECs was above 80 % after treatment with RFP-SLNs, which showed low toxicity to both AMs and AECs. Confocal Laser Scanning Microscopy was employed to observe the interaction between RFP-SLNs and both AMs and AECs. After incubating the cells with RFP-SLNs for 2 h, the fluorescent intensity in AMs was more and remained longer (from 0.5 to 12 h) when compared with that in AECs (from 0.5 to 8 h). In vitro uptake characteristics of RFP-SLNs in AMs and AECs were also investigated by detection of intracellular RFP by High performance liquid chromatography. Results showed that RFP-SLNs delivered markedly higher RFP into AMs (691.7 ng/mg in cultured AMs, 662.6 ng/mg in primary AMs) than that into AECs (319.2 ng/mg in cultured AECs, 287.2 ng/mg in primary AECs). Subsequently, in vivo delivery efficiency and the selectivity of RFP-SLNs were further verified in Sprague-Dawley rats. Under pulmonary administration of RFP-SLNs, the amount of RFP in AMs was significantly higher than that in AECs at each time point. Our results demonstrated that solid lipid nanoparticles are a promising strategy for the delivery of rifampicin to alveolar macrophages selectively.

Overview of MST Research

NASA Astrophysics Data System (ADS)

Chapman, B. E.

2017-10-01

MST progress in advancing the RFP for (1) fusion plasma confinement with ohmic heating and minimal external magnetization, (2) predictive capability in toroidal confinement physics, and (3) basic plasma physics is summarized. Validation of key plasma models is a program priority, which is enhanced by programmable power supplies (PPS) to maximize inductive capability. The existing PPS enables access to very low plasma current, down to Ip =0.02 MA. This greatly expands the Lundquist number range S =104 -108 and allows nonlinear, 3D MHD computation using NIMROD and DEBS with dimensionless parameters that overlap those of MST plasmas. A new, second PPS will allow simultaneous PPS control of the Bp and Bt circuits. The PPS also enables MST tokamak operation, thus far focused on disruptions and RMP suppression of runaway electrons. Gyrokinetic modeling with GENE predicts unstable TEM in improved-confinement RFP plasmas. Measured fluctuations have TEM properties including a density-gradient threshold larger than for tokamak plasmas. Turbulent energization of an electron tail occurs during sawtooth reconnection. Probe measurements hint that drift waves are also excited via the turbulent cascade in standard RFP plasmas. Exploration of basic plasma science frontiers in MST RFP and tokamak plasmas is proposed as part of WiPPL, a basic science user facility. Work supported by USDoE.

Factors Affecting Aerosol Radiative Forcing from Both Production-based and Consumption-based View

NASA Astrophysics Data System (ADS)

Wang, J.; Lin, J.; Ni, R.

2017-12-01

Aerosol radiative forcing (RF) is determined by emissions and various chemical-transport-radiative processes in the atmosphere, a multi-factor problem whose individual contributors have not been well quantified. This problem becomes more complicated when taking into account the role of international trade, which means reallocated aerosol RF due to separation of regions producing goods and emissions and regions consuming those goods. Here we analyze major factors affecting RF of secondary inorganic aerosols (SIOAs, including sulfate, nitrate and ammonium), primary organic aerosol (POA) and black carbon (BC), extending the work of Lin et al. (2016, Nature Geoscience). We contrast five factors determining production-based (RFp, due to a region's production of goods) and consumption-based (RFc, due to a region's consumption) forcing by 11 major regions, including population size, per capita output, emission intensity (emission per output), chemical efficiency (mass per unit emission) and radiative efficiency (RF per unit mass). Comparing across the 11 regions, East Asia produces the strongest RFp and RFc of SIOA and BC and the second largest RFp and RFc of POA primarily due to its high emission intensity. Although Middle East and North Africa has low emissions, its RFp is strengthened by its largest chemical efficiency for POA and BC and second largest chemical efficiency for SIOA. However, RFp of South-East Asia and Pacific is greatly weakened by its lowest chemical efficiency. Economic trade means that net importers (Western Europe, North America and Pacific OECD) have higher RFc than RFp by 50-100%. And such forcing difference is mainly due to the high emission intensity of the exporters supplying these regions. For North America, SIOA's RFc is 50% stronger than RFp, for that emission intensity of SIOA is 5.2 times in East Asia and 2.5 times in Latin America and Caribbean compared with that in North America, and the chemical efficiency in the top four exporters are 1.4-2.1 times of North America. For East Asia, the RFc of SIOA is 20% lower than RFp due to the relatively low emission intensity and chemical efficiency of the top two exporters (Pacific OECD and Western Europe). Overall, economic, emission and atmospheric factors all play important roles in differentiating regions' RFp and RFc.

36 CFR 18.8 - How are lease proposals solicited and selected if the Director issues a Request for Proposals?

Code of Federal Regulations, 2010 CFR

2010-07-01

... Request for Qualifications (RFQ). The purpose of an RFQ is to select a “short list” of potential offerors... recycling; and (v) Any other criteria the RFP may specify. (2) If the property to be leased is an historic...

36 CFR 18.8 - How are lease proposals solicited and selected if the Director issues a Request for Proposals?

Code of Federal Regulations, 2013 CFR

2013-07-01

... Request for Qualifications (RFQ). The purpose of an RFQ is to select a “short list” of potential offerors... recycling; and (v) Any other criteria the RFP may specify. (2) If the property to be leased is an historic...

36 CFR 18.8 - How are lease proposals solicited and selected if the Director issues a Request for Proposals?

Code of Federal Regulations, 2012 CFR

2012-07-01

... Request for Qualifications (RFQ). The purpose of an RFQ is to select a “short list” of potential offerors... recycling; and (v) Any other criteria the RFP may specify. (2) If the property to be leased is an historic...

36 CFR 18.8 - How are lease proposals solicited and selected if the Director issues a Request for Proposals?

Code of Federal Regulations, 2014 CFR

2014-07-01

... Request for Qualifications (RFQ). The purpose of an RFQ is to select a “short list” of potential offerors... recycling; and (v) Any other criteria the RFP may specify. (2) If the property to be leased is an historic...

36 CFR 18.8 - How are lease proposals solicited and selected if the Director issues a Request for Proposals?

Code of Federal Regulations, 2011 CFR

2011-07-01

... Request for Qualifications (RFQ). The purpose of an RFQ is to select a “short list” of potential offerors... recycling; and (v) Any other criteria the RFP may specify. (2) If the property to be leased is an historic...

Technical Leadership Development Program - Year 2

DTIC Science & Technology

2012-02-01

Why Projects Fail Pennotti Wed 2:45-3:00 Break Wed 3:00-4:30 Project: AR2D2: RFP Robinson UNCLASSIFIED Contract Number: H98230-08-D-0171 DO 02...Project: AR2D2 RFP 12. Customer Expectation-1: Lecture: Why Systems Fail 13. Customer Expectation-2: Case Study: Process Automation 14...01 February 2012 UNCLASSIFIED 65 Syllabus Segment 12: Why Systems Fail (Lecture) Time: 1.5 hours Responsible: Mike Pennotti Support

Community for Data Integration 2014 annual report

USGS Publications Warehouse

Langseth, Madison L.; Chang, Michelle Y.; Carlino, Jennifer; Birch, Daniella D.; Bradley, Joshua; Bristol, R. Sky; Conzelmann, Craig; Diehl, Robert H.; Earle, Paul S.; Ellison, Laura E.; Everette, Anthony L.; Fuller, Pamela L.; Gordon, Janice M.; Govoni, David L.; Guy, Michelle R.; Henkel, Heather S.; Hutchison, Vivian B.; Kern, Tim; Lightsom, Frances L.; Long, Joseph W.; Longhenry, Ryan; Preston, Todd M.; Smith, Stan W.; Viger, Roland J.; Wesenberg, Katherine; Wood, Eric C.

2015-10-02

To achieve these goals, the CDI operates within four applied areas: monthly forums, annual workshop/webinar series, working groups, and projects. The monthly forums, also known as the Opportunity/Challenge of the Month, provide an open dialogue to share and learn about data integration efforts or to present problems that invite the community to offer solutions, advice, and support. Since 2010, the CDI has also sponsored annual workshops/webinar series to encourage the exchange of ideas, sharing of activities, presentations of current projects, and networking among members. Stemming from common interests, the working groups are focused on efforts to address data management and technical challenges including the development of standards and tools, improving interoperability and information infrastructure, and data preservation within USGS and its partners. The growing support for the activities of the working groups led to the CDI’s first formal request for proposals (RFP) process in 2013 to fund projects that produced tangible products. As of 2014, the CDI continues to hold an annual RFP that creates data management tools and practices, collaboration tools, and training in support of data integration and delivery.

7 CFR 4284.913 - Evaluation criteria and weights.

Code of Federal Regulations, 2011 CFR

2011-01-01

... established in the applicable RFP. (b) Working Capital Grants—(1) Business viability. Proposals will be evaluated on the basis of the technical and economic feasibility and sustainability of the venture and the...

7 CFR 4284.913 - Evaluation criteria and weights.

Code of Federal Regulations, 2010 CFR

2010-01-01

... established in the applicable RFP. (b) Working Capital Grants—(1) Business viability. Proposals will be evaluated on the basis of the technical and economic feasibility and sustainability of the venture and the...

Intrepid: A Mission to Pluto

NASA Technical Reports Server (NTRS)

Behling, Michael; Buchman, Donald; Marcus, Andres; Procopis, Stephanie; Wassgren, Carl; Ziemer, Sarah

1990-01-01

A proposal for an exploratory spacecraft mission to Pluto/Charon system was written in response to the request for proposal for an unmannned probe to pluto (RFP). The design requirements of the RFP are presented and under the guidance of these requirements, the spacecraft Intrepid was designed. The RPF requirement that was of primary importance is the minimization of cost. Also, the reduction of flight time was of extreme importance because the atmosphere of Pluto is expected to collapse close to the Year 2020. If intrepid should arrive after the collapse, the mission would be a failure; for Pluto would be only a solid rock of ice. The topics presented include: (1) scientific instrumentation; (2) mission management, planning, and costing; (3) power and propulsion subsystem; (4) structural subsystem; (5) command, control, and communications; and (6) attitude and articulation control.

Dynamics of red fluorescent dental plaque during experimental gingivitis--A cohort study.

PubMed

van der Veen, Monique H; Volgenant, Catherine M C; Keijser, Bart; Ten Cate, Jacob Bob M; Crielaard, Wim

2016-05-01

The dynamics of red fluorescent plaque (RFP) in comparison to clinical plaque and bleeding scores were studied during an experimental gingivitis protocol in a cohort of healthy participants. Forty-one participants were monitored for RFP before (24h plaque), during 14 days plaque accumulation (days 2, 5, 9, 14) and after 7 days recovery (24h plaque). RFP was assessed on fluorescence photographs of the vestibular aspect of the anterior teeth (cuspid to cuspid) in the upper and lower jaw. Clinical plaque and bleeding were assessed at days -14, 0, 14 and 21. RFP of 24h plaque was reproducible (days -14, 0), then increased during 14 days plaque accumulation and returned to baseline after 7 days recovery. Groups of low, moderate and high RFP formers were statistically significantly different at all times even already at baseline. The individual RFP response during 14 days plaque accumulation correlated well with RFP of 24h plaque (days -14, 0). RFP correlated moderate to well with clinical plaque at days -14, 0, 14 and 21. From day 2 of the gingivitis challenge RFP correlated with bleeding at day 14. RFP provided an objective measure of oral hygiene status. Given the correlation with clinical parameters found, the amount of RFP after 24h plaque accumulation was indicatory for the inflammatory response during a prolonged period of no oral hygiene. This trial was registered at the public trial register ​of the Central Committee on Research Involving Human Subjects (CCMO) under number NL51111.029.14 CLINICAL SIGNIFICANCE: This paper shows the association between RFP after 24h plaque accumulation and inflammatory response after a prolonged period of no oral hygiene. Red plaque fluorescence can be used to identify subjects at risk for developing gingival inflammation. Copyright © 2016 Elsevier Ltd. All rights reserved.

NMCI: History, Implementation, and Change

DTIC Science & Technology

2006-09-01

RAS Remote Access Service RFP Request for Proposal SAME Similar to Automated Maintenance Environment SDG Site Deployment Guide...instead of participants individually with different sections of the organization maintaining their own information supply , and when they need something

78 FR 66320 - Approval and Promulgation of Air Quality Implementation Plans; Ohio: Bellefontaine; Determination...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-05

... available control measures, a reasonable further progress (RFP) plan, and contingency measures for failure... proposal because the Agency views this as a noncontroversial submittal and anticipates no adverse comments...

[Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3].

PubMed

Wang, Wan; Zhang, Qing; Zhao, Runpeng; Xu, Xuewei; Xing, Yingru; Zhang, Rongbo; Wu, Jing; Hu, Dong

2015-09-01

To establish murine macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3). A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264.7 cells. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment. The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.

24 CFR 943.115 - What programs are covered under this subpart?

Code of Federal Regulations, 2011 CFR

2011-04-01

... Annual Contributions Contract (ACC) with HUD; and (2) PHA administration of grants to the PHA in... to an ACC entered under the Request for Proposals (RFP) published May 19, 1999 (64 FR 27358); (2...

24 CFR 943.115 - What programs are covered under this subpart?

Code of Federal Regulations, 2010 CFR

2010-04-01

... Annual Contributions Contract (ACC) with HUD; and (2) PHA administration of grants to the PHA in... to an ACC entered under the Request for Proposals (RFP) published May 19, 1999 (64 FR 27358); (2...

RANT Building Upgrade Pre-Proposal Meeting

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

Thompson, Melinda Ann; Meadows, Darren W.; Rodriguez, Roger R.

This presentation is designed to provide an overview of the project scope and RFP; discuss interfaces for the construction project; highlight key expectations and deliverables from the subcontractor; and address questions and provide clarification of the project scope.

Analysis of Stroma Labeling During Multiple Passage of a Sarcoma Imageable Patient-Derived Orthotopic Xenograft (iPDOX) in Red Fluorescent Protein Transgenic Nude Mice.

PubMed

Kiyuna, Tasuku; Murakami, Takashi; Tome, Yasunori; Kawaguchi, Kei; Igarashi, Kentaro; Miyake, Kentaro; Kanaya, Fuminori; Singh, Arun; Eilber, Fritz C; Hoffman, Robert M

2017-10-01

A patient-derived orthotopic xenograft (PDOX) model of undifferentiated pleomorphic sarcoma (UPS) was previously established that acquired red fluorescent protein (RFP)-expressing stroma by growth in an RFP transgenic nude mouse. In the present study, an imageable PDOX model (iPDOX) of UPS was established by orthotopic implantation in the biceps femoris of transgenic RFP nude mice. After the tumors grew to a diameter of 10 mm, they were harvested and the brightest portion of the tumors were subsequently orthotopically transplanted to both RFP and non-colored nude mice. The UPS PDOX tumor was again transplanted to RFP transgenic and non-colored nude mice, and finally a 3rd passage was made in the same manner. Five UPS tumors from each passage in both RFP and non-colored mouse models were harvested. The FV1,000 confocal microscope was used to visualize and quantitate the RFP area of the resected tumors. The average percent fluorescent area in the first passage of RFP mice was 34 ± 22%; in the second passage, 34 ± 20%; and 36 ± 11% in the third passage of RFP transgenic nude mice. The average tumor RFP area in the first passage from RFP mice to non-colored mice was 20 ± 7%; in the second passage, 28 ± 11%; in the third passage was 27 ± 13%. The present results demonstrate the extensive and stable acquisition of stroma by the UPS-tumor growing orthotopically in transgenic RFP nude mice (iPDOX). This model can be used for screening for effective drugs for individual patients and drug discovery. J. Cell. Biochem. 118: 3367-3371, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Air Force KC-X Tanker Aircraft Program: Background and Issues for Congress

DTIC Science & Technology

2009-12-07

24, 2009, the Department of Defense (DOD) announced its proposed strategy for conducting a new competition between Boeing and a team consisting of...acquire a new tanker over the last several years have ultimately failed. DOD’s proposed new KC-X acquisition competition strategy poses several...5 DOD’s Proposed New KC-X Competition Strategy ...............................................................5 Response to the Draft RFP

77 FR 54877 - Request for Proposals (RFP): Farm Labor Housing Technical Assistance Grants

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-06

... corporate felony convictions and corporate federal tax delinquencies. To comply with these provisions, all..., statistical records, and all other records pertinent to the grant for a period of at least 3 years after the...

Structural characterization of acylimine-containing blue and red chromophores in mTagBFP and TagRFP fluorescent proteins.

PubMed

Subach, Oksana M; Malashkevich, Vladimir N; Zencheck, Wendy D; Morozova, Kateryna S; Piatkevich, Kiryl D; Almo, Steven C; Verkhusha, Vladislav V

2010-04-23

We determined the 2.2 A crystal structures of the red fluorescent protein TagRFP and its derivative, the blue fluorescent protein mTagBFP. The crystallographic analysis is consistent with a model in which TagRFP has the trans coplanar anionic chromophore with the conjugated pi-electron system, similar to that of DsRed-like chromophores. Refined conformation of mTagBFP suggests the presence of an N-acylimine functionality in its chromophore and single C(alpha)-C(beta) bond in the Tyr64 side chain. Mass spectrum of mTagBFP chromophore-bearing peptide indicates a loss of 20 Da upon maturation, whereas tandem mass spectrometry reveals that the C(alpha)-N bond in Leu63 is oxidized. These data indicate that mTagBFP has a new type of the chromophore, N-[(5-hydroxy-1H-imidazole-2-yl)methylidene]acetamide. We propose a chemical mechanism in which the DsRed-like chromophore is formed via the mTagBFP-like blue intermediate. (c) 2010 Elsevier Ltd. All rights reserved.

Trade Space Analysis: Rotational Analyst Research Project

DTIC Science & Technology

2015-09-01

POM Program Objective Memoranda PM Program Manager RFP Request for Proposal ROM Rough Order Magnitude RSM Response Surface Method RSE ...response surface method (RSM) / response surface equations ( RSEs ) as surrogate models. It uses the RSEs with Monte Carlo simulation to quantitatively

48 CFR 15.203 - Requests for proposals.

Code of Federal Regulations, 2010 CFR

2010-10-01

... information regarding cost comparisons between Government and contractor performance. (c) Electronic commerce... RFP shall specify the electronic commerce method(s) that offerors may use (see subpart 4.5). (d... and suitability of electronic commerce methods; and (iv) Adequacy of administrative procedures and...

GPP Webinar: Solar Utilization in Higher Education Networking & Information Sharing Group: RFP, Contract, and Administrative Issues Discussion

EPA Pesticide Factsheets

This presentation from a Solar Utilization in Higher Education Networking and Information webinar covers contracts, Request for Proposals (RFPs), and administrative issues related to solar project development in the higher education sector.

VpRFP1, a novel C4C4-type RING finger protein gene from Chinese wild Vitis pseudoreticulata, functions as a transcriptional activator in defence response of grapevine

PubMed Central

Yu, Yihe; Xu, Weirong; Wang, Shengyi; Xu, Yan; Li, Hui'e; Wang, Yuejin; Li, Shuxiu

2011-01-01

RING finger proteins comprise a large family and play important roles in regulation of growth and development, hormone signalling, and responses to biotic and abiotic stresses in plants. In this study, the identification and functional characterization of a C4C4-type RING finger protein gene from the Chinese wild grapevine Vitis pseudoreticulata (designated VpRFP1) are reported. VpRFP1 was initially identified as an expressed sequence tag (EST) from a cDNA library constructed from leaves of V. pseudoreticulata inoculated with the grapevine powdery mildew Uncinula necator. Sequence analysis of the deduced VpRFP1 protein based on the full-length cDNA revealed an N-terminal nuclear localization signal (NLS) and a C-terminal C4C4-type RING finger motif with the consensus sequence Cys-X2-Cys-X13-Cys-X1-Cys-X4-Cys-X2-Cys-X10-Cys-X2-Cys. Upon inoculation with U. necator, expression of VpRFP1 was rapidly induced to higher levels in mildew-resistant V. pseudoreticulata plants. In contrast, expression of VpRFP1 was down-regulated in mildew-susceptible V. vinifera plants. Western blotting using an antibody raised against VpRFP1 showed that VpRFP1 was also induced to higher levels in V. pseudoreticulata plants at 12–48 hours post-inoculation (hpi). However, there was only slight increase in VpRFP in V. vinifera plants in the same time frame, even though a more significant increase was observed at 96–144 hpi in these plants. Results from transactivation assays in yeast showed that the RING finger motif of VpRFP1 exhibited some activity of transcriptional activation; however, no activity was seen with the full-length VpRFP1. Overexpression of VpRFP1 in Arabidopsis plants was found to enhance resistance to Arabidopsis powdery mildew Golovinomyces cichoracearum, which seemed to be correlated with increased transcript levels of AtPR1 and AtPR2 in the pathogen-infected tissues. In addition, the Arabidopsis transgenic lines showed enhanced resistance to a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Taken together, the results suggested that VpRFP1 may be a transcriptional activator of defence-related genes in grapevines. PMID:21862480

Preliminary design of nine high speed civil transports

NASA Technical Reports Server (NTRS)

Sandlin, Doral; Vantriet, Robert; Soban, Dani; Hoang, TY

1992-01-01

Sixty senior design students at Cal Poly, SLO have completed a year-long project to design the next generation of High Speed Civil Transports (HSCT). The design process was divided up into three distinct phases. The first third of the project was devoted entirely to research into the special problems associated with an HSCT. These included economic viability, airport compatibility, high speed aerodynamics, sonic boom minimization, environmental impact, and structures and materials. The result of this research was the development of nine separate Requests for Proposal (RFP) that outlined reasonable yet challenging design criteria for the aircraft. All were designed to be technically feasible in the year 2015. The next phase of the project divided the sixty students into nine design groups. Each group, with its own RFP, completed a Class 1 preliminary design of an HSCT. The nine configurations varied from conventional double deltas to variable geometry wings to a pivoting oblique wing design. The final phase of the project included a more detailed Class 2 sizing as well as performance and stability and control analysis. Cal Poly, San Luis Obispo presents nine unique solutions to the same problem: that of designing an economically viable, environmentally acceptable, safe and comfortable supersonic transport.

Community for Data Integration 2013 Annual Report

USGS Publications Warehouse

Chang, Michelle Y.; Carlino, Jennifer; Barnes, Christopher; Blodgett, David L.; Bock, Andrew R.; Everette, Anthony L.; Fernette, Gregory L.; Flint, Lorraine E.; Gordon, Janice M.; Govoni, David L.; Hay, Lauren E.; Henkel, Heather S.; Hines, Megan K.; Holl, Sally L.; Homer, Collin G.; Hutchison, Vivian B.; Ignizio, Drew A.; Kern, Tim J.; Lightsom, Frances L.; Markstrom, Steven L.; O'Donnell, Michael S.; Schei, Jacquelyn L.; Schmid, Lorna A.; Schoephoester, Kathryn M.; Schweitzer, Peter N.; Skagen, Susan K.; Sullivan, Daniel J.; Talbert, Colin; Warren, Meredith Pavlick

2015-01-01

grow overall USGS capabilities with data and information by increasing visibility of the work of many people throughout the USGS and the CDI community. To achieve these goals, the CDI operates within four applied areas: monthly forums, annual workshop/webinar series, working groups, and projects. The monthly forums, also known as the Opportunity/Challenge of the Month, provide an open dialogue to share and learn about data integration efforts or to present problems that invite the Community to offer solutions, advice, and support. Since 2010, the CDI has also sponsored annual workshops/webinar series to encourage the exchange of ideas, sharing of activities, presentations of current projects, and networking among members. Stemming from common interests, the working groups are focused on efforts to address data management and technical 2 challenges, including the development of standards and tools, improving interoperability and information infrastructure, and data preservation within USGS and its partners. The growing support for the activities of the working groups led to the CDI’s first formal request for proposals (RFP) process in 2013 to fund projects that produced tangible products. Today the CDI continues to hold an annual RFP that create data management tools and practices, collaboration tools, and training in support of data integration and delivery.

The philosophy of benchmark testing a standards-based picture archiving and communications system.

PubMed

Richardson, N E; Thomas, J A; Lyche, D K; Romlein, J; Norton, G S; Dolecek, Q E

1999-05-01

The Department of Defense issued its requirements for a Digital Imaging Network-Picture Archiving and Communications System (DIN-PACS) in a Request for Proposals (RFP) to industry in January 1997, with subsequent contracts being awarded in November 1997 to the Agfa Division of Bayer and IBM Global Government Industry. The Government's technical evaluation process consisted of evaluating a written technical proposal as well as conducting a benchmark test of each proposed system at the vendor's test facility. The purpose of benchmark testing was to evaluate the performance of the fully integrated system in a simulated operational environment. The benchmark test procedures and test equipment were developed through a joint effort between the Government, academic institutions, and private consultants. Herein the authors discuss the resources required and the methods used to benchmark test a standards-based PACS.

A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells.

PubMed

Scabone, Camila María; Frigerio, Lorenzo; Petruccelli, Silvana

2011-10-01

To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.

Near-infrared fluorescent protein iRFP713 as a reporter protein for optogenetic vectors, a transgenic Cre-reporter rat, and other neuronal studies

PubMed Central

Richie, Christopher T.; Whitaker, Leslie R.; Whitaker, Keith W.; Necarsulmer, Julie; Baldwin, Heather A.; Zhang, Yajun; Fortuno, Lowella; Hinkle, Josh J.; Koivula, Pyry; Henderson, Mark J.; Sun, Wenzhi; Wang, Kai; Smith, Jeffrey C.; Pickel, Jim; Ji, Na; Hope, Bruce T.; Harvey, Brandon K.

2017-01-01

Background The use of genetically-encoded fluorescent reporters is essential for the identification and observation of cells that express transgenic modulatory proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted. New Method Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function. We have generated a set of adeno-associated vectors in which iRFP713 has been fused to optogenetic channels, and can be expressed constitutively or Cre-dependently. Results iRFP713 is detectable when expressed in neurons both in vitro and in vivo without exogenously supplied chromophore biliverdin. Neuronally-expressed iRFP713 has similar properties to GFP-like fluorescent proteins, including the ability to be translationally fused to channelrhodopsin or halorhodopsin, however, it shows superior photostability compared to EYFP. Furthermore, electrophysiological recordings from iRFP713-labeled cells compared to cells labeled with mCherry suggest that iRFP713 cells are healthier and therefore more stable and reliable in an ex vivo preparation. Lastly, we have generated a transgenic rat that expresses iRFP713 in a Cre-dependent manner. Conclusions Overall, we have demonstrated that iRFP713 can be used as a reporter in neurons without the use of exogenous biliverdin, with minimal impact on viability and function thereby making it feasible to extend the capabilities for imaging genetically-tagged neurons in slices and in vivo. PMID:28380331

Red fluorescence of dental plaque in children -A cross-sectional study.

PubMed

Volgenant, Catherine M C; Zaura, Egija; Brandt, Bernd W; Buijs, Mark J; Tellez, Marisol; Malik, Gayatri; Ismail, Amid I; Ten Cate, Jacob M; van der Veen, Monique H

2017-03-01

The relation between the presence of red fluorescent plaque and the caries status in children was studied. In addition, the microbial composition of dental plaque from sites with red fluorescent plaque (RFP) and from sites with no red fluorescent plaque (NFP) was assessed. Fluorescence photographs were taken from fifty children (6-14 years old) with overnight plaque. Full-mouth caries scores (ICDAS II) were obtained. The composition of a saliva sample and two plaque samples (RFP and NFP) was assessed using 16S rDNA sequencing. At the site level, no clinically relevant correlations were found between the presence of RFP and the caries status. At the subject level, a weak correlation was found between RFP and the caries status when non-cavitated lesions were included (r s =0.37, p=0.007). The microbial composition of RFP differed significantly from NFP. RFP had more anaerobes and more Gram-negative bacterial taxa. The most discriminative operational taxonomic units (OTUs) for RFP were Corynebacterium, Leptotrichia, Porphyromonas and Selenomonas, while the most discriminative OTUs for NFP were Neisseria, Actinomyces, Streptococcus and Rothia. There were no clinical relevant correlations in this cross-sectional study between the presence of RFP and (early) caries lesions. There were differences in the composition of these phenotypically different plaque samples: RFP contained more Gram-negative, anaerobic taxa and was more diverse than NFP. The study outcomes provide more insight in the possibilities to use plaque fluorescence in oral health risk assessments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pharmaceutical study of suppository formulations for improved in vivo kinetics of rifampicin.

PubMed

Taki, Hisashi; Ogawa, Kenji; Nikai, Toshiaki

2008-06-01

To study rifampicin (RFP) suppository formulations that were prepared by adding an absorption promoter and a holding agent to conventional rifampicin suppositories in order to achieve higher blood drug levels. The subjects were 3 healthy volunteers who gave consent to participate in this study. Suppository formulations were prepared by adding sodium caprinate (the absorption promoter) to 600 mg, 750 mg, or 900 mg of RFP at about 3% of the suppository weight and sodium alginate (the holding agent) at 25% of the RFP content. Serum samples collected at 2, 6, and 10 hours after insertion of a suppository were subjected to RFP concentration analysis. In subject no. 1, the maximum concentration was 0.807 Lg/ml, 1.093 microg/ml, and 1.291 microg/ml after administration of a suppository containing 600 mg, 750 mg, or 900 mg of RFP, respectively. Since an injectable RFP formulation has not been approved in Japan, formulation studies of RFP suppositories are important to achieve a better clinical response and to prevent the development of resistance. The present formulation that delivered a blood concentration of RFP considerably higher than 1 microg/ml was considered to have therapeutic potential. Its clinical utility will be examined in further formulation studies.

Microgrid-Ready Solar PV - Planning for Resiliency

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

Booth, Samuel S

2017-10-17

This fact sheet provides background information on microgrids with suggested language for several up-front considerations that can be added to a solar project procurement or request for proposal (RFP) that will help ensure that PV systems are built for future microgrid connection.

Understanding District-Charter Collaboration Grants. Final Report

ERIC Educational Resources Information Center

Tuttle, Christina; McCullough, Moira; Richman, Scott; Booker, Kevin; Burnett, Alyson; Keating, Betsy; Cavanaugh, Michael

2016-01-01

In November 2012, the Bill & Melinda Gates Foundation invested in seven innovative district-charter partnerships with "the potential capacity and commitment to accelerate student college ready rates through deep collaboration and sharing of best practices" (District-Charter Collaboration Grant Request for Proposal [RFP]). These…

Status of the Landsat Data Continuity Mission

NASA Technical Reports Server (NTRS)

Irons, James R.; Ochs, William R.

2004-01-01

Efforts to begin implementing a successor mission to Landsat 7, called the Landsat Data Continuity Mission (LDCM), suffered a set back in 2003. NASA and the Department of Interior (DOI)/U.S. Geological Survey (USGS) currently manage the Landsat Program as an interagency partnership. The two agencies had planned to purchase data meeting LDCM specifications from a privately owned and commercially operated satellite system beginning in March, 2007. This approach represented a departure from the traditional procurement of a government owned and operated satellite system. NASA, however, cancelled a Request-for-Proposals (RFP) for providing the required data after an evaluation of proposals received from private industry. NASA concluded that the proposals failed to meet a key objective and expectation of the RFP, namely, to form a fair and equitable partnership between the Government and private industry. Alternative strategies for implementing an LDCM are now under consideration. The Executive Office of the President formed an interagency working group on the LDCM following the RFP cancellation. The working group is considering other options for implementing a successor system to Landsat 7 consistent with the Land Remote Sensing Policy Act of 1992 (Public Law 102-555). This Act lists four management options for consideration: 1) private sector funding and management; 2) an international consortium; 3) funding and management by the U.S. Government; and 4) a cooperative effort between the US. Government and the private sector. The working group is currently attempting to minimize the risk of a Landsat data gap through development of a strategy that leads to a Landsat 7 successor mission. The selected strategy and the status of the mission will be presented at the Symposium.

Concentration of clarithromycin and 14-R-hydroxy-clarithromycin in plasma of patients with Mycobacterium avium complex infection, before and after the addition of rifampicin.

PubMed

Yamamoto, F; Harada, S; Mitsuyama, T; Harada, Y; Kitahara, Y; Yoshida, M; Nakanishi, Y

2004-02-01

Clarithromycin (CAM) and rifampicin (RFP) have both been recognized to be effective antibiotic agents against Mycobacterium avium complex (MAC) infection. Rifamycin derivatives including RFP and rifabutin modulate the CAM metabolism by inducing the hepatic cytochrome p-450 3A4. To clarify the effect of RFP on the CAM metabolism, we measured the plasma concentration of CAM and 14-R-hydroxyclarithromycin (M-5), the major metabolite of CAM, in 9 patients suffering from MAC infection before and after the addition of RFP. After the addition of RFP, the mean plasma concentration of CAM significantly decreased, while that of M-5 did not. In addition, the amount of CAM + M-5 concentration also significantly decreased. As M-5 is less effective against MAC infection than CAM, more attention should thus be paid to the plasma CAM concentration in patients administered CAM and RFP concomitantly.

The inhibition of hepatic bile acids transporters Ntcp and Bsep is involved in the pathogenesis of isoniazid/rifampicin-induced hepatotoxicity.

PubMed

Guo, Yao Xue; Xu, Xue Fei; Zhang, Qi Zhi; Li, Chun; Deng, Ye; Jiang, Pei; He, Lei Yan; Peng, Wen Xing

2015-01-01

Co-treatment of isoniazid (INH) and rifampicin (RFP) is well known for clinically apparent liver injury. However, the mechanism of INH/RFP-induced liver injury is controversial. Emerging evidence shows links between inhibition of bile acids transporters and drug-induced liver injury (DILI). The present study investigates whether sodium taurocholate cotransporting polypeptide (NTCP/Ntcp; SLC10A1) and bile salt export pump (BSEP/Bsep; ABCB11) are involved in the anti-tuberculosis medicines induced liver injury. ICR female mice were intragastrically treated with INH (50 or 100 mg/kg), RFP (100 or 200 mg/kg), or the combination of INH/RFP (50 + 100 mg/kg or 100 + 200 mg/kg) for 14 consecutive days. Liver histopathological examination, serum biochemical and liver malondialdehyde tests were evaluated. Apparent histopathological alterations and hepatic oxidative stress showed in INH (100 mg/kg), RFP (200 mg/kg) and their combination group. The hepatoxic effect was also indicated by increased serum biomarkers, such as aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), direct bilirubin (DBil), total bilirubin (TBil) and total bile acids (TBA). Both doses of INH/RFP administration significantly down-regulated the expression of Ntcp and Bsep in liver. Furthermore, the combination of INH and RFP displayed stronger effect on the expression of Ntcp compared with the corresponding dose of INH or RFP alone. In conclusion, down-regulated expression of hepatic Ntcp and Bsep might play an important role in the development of INH and RFP induced liver injury.

Conceptual design proposal: HUGO global range/mobility transport aircraft

NASA Technical Reports Server (NTRS)

Johnston, Tom; Perretta, Dave; Mcbane, Doug; Morin, Greg; Thomas, Greg; Woodward, Joe; Gulakowski, Steve

1993-01-01

With the collapse of the former Soviet Union and the emergence of the United Nations actively pursuing a peace keeping role in world affairs, the United States has been forced into a position as the world's leading peace enforcer. It is still a very dangerous world with seemingly never ending ideological, territorial, and economic disputes requiring the U.S. to maintain a credible deterrent posture in this uncertain environment. This has created an urgent need to rapidly transport large numbers of troops and equipment from the continental United States (CONUS) to any potential world trouble spot by means of a global range/mobility transport aircraft. The most recent examples being Operation Desert Shield/Storm and Operation Restore Hope. To meet this challenge head-on, a request for proposal (RFP) was developed and incorporated into the 1992/1993 AIAA/McDonnell Douglas Corporation Graduate Team Aircraft Design Competition. The RFP calls for the conceptual design and justification of a large aircraft capable of power projecting a significant military force without surface transportation reliance.

Formation of the spinal network in zebrafish determined by domain-specific Pax genes

PubMed Central

Ikenaga, Takanori; Urban, Jason M.; Gebhart, Nichole; Hatta, Kohei; Kawakami, Koichi; Ono, Fumihito

2012-01-01

In the formation of the spinal network, various transcription factors interact to develop specific cell types. Using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) was inserted in the pax8 gene. RFP insertion marked putative pax8-lineage cells with fluorescence and inhibited pax8 expression in homozygous embryos. Pax8 homozygous embryos displayed defects in the otic vesicle, as previously reported in studies using morpholinos. The pax8 homozygous embryos survived to adulthood in contrast to mammalian counterparts that die prematurely. RFP is expressed in the dorsal spinal cord. Examination of the axon morphology revealed that RFP (+) neurons include Commissural Bifurcating Longitudinal (CoBL) interneurons, but other inhibitory neurons such as Commissural Local (CoLo) interneurons and Circumferential Ascending (CiA) interneurons do not express RFP. We examined the effect of inhibiting pax2a/pax8 expression on interneuron development. In pax8 homozygous fish, the RFP (+) cells undergo differentiation similar to that of pax8 heterozygous fish, and the swimming behavior remained intact. In contrast, the RFP (+) cells of pax2a/pax8 double mutants displayed altered cell fates. CoBLs were not observed. Instead, RFP (+) cells exhibited axons descending ipsilaterally: a morphology resembling that of V2a/V2b interneurons. PMID:21452218

Formation of the spinal network in zebrafish determined by domain-specific pax genes.

PubMed

Ikenaga, Takanori; Urban, Jason M; Gebhart, Nichole; Hatta, Kohei; Kawakami, Koichi; Ono, Fumihito

2011-06-01

In the formation of the spinal network, various transcription factors interact to develop specific cell types. By using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) was inserted into the pax8 gene. RFP insertion marked putative pax8-lineage cells with fluorescence and inhibited pax8 expression in homozygous embryos. Pax8 homozygous embryos displayed defects in the otic vesicle, as previously reported in studies with morpholinos. The pax8 homozygous embryos survived to adulthood, in contrast to mammalian counterparts that die prematurely. RFP is expressed in the dorsal spinal cord. Examination of the axon morphology revealed that RFP(+) neurons include commissural bifurcating longitudinal (CoBL) interneurons, but other inhibitory neurons such as commissural local (CoLo) interneurons and circumferential ascending (CiA) interneurons do not express RFP. We examined the effect of inhibiting pax2a/pax8 expression on interneuron development. In pax8 homozygous fish, the RFP(+) cells underwent differentiation similar to that of pax8 heterozygous fish, and the swimming behavior remained intact. In contrast, the RFP(+) cells of pax2a/pax8 double mutants displayed altered cell fates. CoBLs were not observed. Instead, RFP(+) cells exhibited axons descending ipsilaterally, a morphology resembling that of V2a/V2b interneurons. Copyright © 2010 Wiley-Liss, Inc.

75 FR 74517 - Approval and Promulgation of Implementation Plans; State of California; 2008 San Joaquin Valley...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-30

...EPA is proposing to approve in part and disapprove in part state implementation plan (SIP) revisions submitted by California to provide for attainment of the 1997 annual and 24-hour fine particulate matter (PM2.5) national ambient air quality standards (NAAQS) in the San Joaquin Valley (SJV) nonattainment area. The SIP revisions are the SJV 2008 PM2.5 Plan (revised 2010) and portions of the 2007 State Strategy (revised 2009). Specifically, EPA is proposing to approve the emissions inventories as meeting the requirements of the Clean Air Act and EPA's fine particle implementing rule and to approve commitments to implement specific measures and meet specific aggregate emissions reductions by the San Joaquin Valley Air Pollution Control District and the California Air Resource Board. In addition, we are proposing to find that volatile organic compounds are a PM2.5 attainment plan precursor in the SJV for which controls should be evaluated. EPA is proposing to disapprove the attainment demonstration. EPA is also proposing to disapprove the reasonably available control measures/reasonably available control technology demonstration, the air quality modeling, the reasonable further progress (RFP) demonstration, the contingency measures, and the attainment and RFP conformity motor vehicle emissions budgets. EPA is also proposing to not grant California's request to extend to April 5, 2015 the deadline for the SJV nonattainment area to attain the 1997 PM2.5 NAAQS.

Runaway of energetic test ions in a toroidal plasma

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

Eilerman, S., E-mail: eilerman@wisc.edu; Anderson, J. K.; Sarff, J. S.

2015-02-15

Ion runaway in the presence of a large-scale, reconnection-driven electric field has been conclusively measured in the Madison Symmetric Torus reversed-field pinch (RFP). Measurements of the acceleration of a beam of fast ions agree well with test particle and Fokker-Planck modeling of the runaway process. However, the runaway mechanism does not explain all measured ion heating in the RFP, particularly previous measurements of strong perpendicular heating. It is likely that multiple energization mechanisms occur simultaneously and with differing significance for magnetically coupled thermal ions and magnetically decoupled tail and beam ions.

7 CFR 1783.14 - What are the eligibility criteria for RFP loan recipients?

Code of Federal Regulations, 2010 CFR

2010-01-01

...) RURAL UTILITIES SERVICE, DEPARTMENT OF AGRICULTURE (CONTINUED) REVOLVING FUNDS FOR FINANCING WATER AND WASTEWATER PROJECTS (REVOLVING FUND PROGRAM) Revolving Fund Program Loans § 1783.14 What are the eligibility.... (b) The loan recipient must be unable to finance the proposed project from their own resources or...

50 CFR 648.56 - Scallop research.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Scallop research. 648.56 Section 648.56... Scallop Fishery § 648.56 Scallop research. (a) Annually, the Council and NMFS shall prepare and issue a Request for Proposals (RFP) that identifies research priorities for projects to be conducted by vessels...

Potential impact radius formulae for flammable gases other than natural gas subject to 49 CFR 192 : final report.

DOT National Transportation Integrated Search

2005-06-01

This report was prepared in accordance with the Statement of Work and proposal submitted in : response to RFP for Technical Task Order Number 13 (TTO 13) entitled Potential Impact Radius : Formulae for Flammable Gases Other Than Natural Gas. : ...

FY 2018 Grant Announcement: FY2018 support for Small Water Grant (SWG) and Innovative and Nutrient Sediment Reduction (INSR) Grant

EPA Pesticide Factsheets

The U.S. Environmental Protection Agency’s Chesapeake Bay Program Office is announcing a request for proposals (RFP) for supporting both the:1.Small Watershed Grants (SWG) program; and 2.Innovative Nutrient and Sediment Reduction (INSR) grants program.

Responding to an RFP: A Vendor's Viewpoint.

ERIC Educational Resources Information Center

Kington, Robert A.

1987-01-01

Outlines factors used by online vendors to decide whether to bid on RFPs (requests for proposals) for library automation systems, including specifications for software, hardware or performance requirements not met by the vendor; specifications based on competitors' systems; the size and complexity of the request itself; and vendors' time…

MULTIPLE PROJECTIONS SYSTEM (MPS): USER'S MANUAL VERSION 2.0

EPA Science Inventory

The document is a user's manual for Multiple Projections System (MPS) Version 2.0, based on the 3% reasonable further progress (RFP) tracking system that was developed in FY92/FY93. The 3% RFP tracking system is a Windows application, and enhancements to convert the 3% RFP track...

75 FR 40857 - Webinar About Advanced Defense Technologies RFP

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-14

... Defense Technologies RFP. Please visit http://www.sba.gov/clusters/index.html for more information. The RFP may be found on http://www.fedbizopps.gov . LOGISTICAL INFORMATION: The webinar will be held on Monday, July 19, 2010. For details, please visit http://www.sba.gov/clusters/index.html . SUPPLEMENTARY...

40 CFR 51.1009 - Reasonable further progress (RFP) requirements.

Code of Federal Regulations, 2010 CFR

2010-07-01

... between the base year and the attainment year. (e) For a multi-State nonattainment area, the RFP plans for each State represented in the nonattainment area must demonstrate RFP on the basis of common multi... State in a multi-State nonattainment area must ensure that the sources within its boundaries comply with...

Expression of membrane-associated proteins within single emulsion cell facsimiles.

PubMed

Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

2012-07-07

MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

Multi-color fluorescence imaging of sub-cellular dynamics of cancer cells in live mice

NASA Astrophysics Data System (ADS)

Hoffman, Robert M.

2006-02-01

We have genetically engineered dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in the cytoplasm in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed of the cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery or heart injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse where extreme elongation of the cell body as well as the nucleus occurred. The migration velocities of the dual-color cancer cells in the capillaries were measured by capturing individual images of the dual-color fluorescent cells over time. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT)-GFP-RFP cells were injected in the portal vein of nude mice. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, MMT-GFP-RFP cells injected into the portal vein mostly survived and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells. With the ability to continuously image cancer cells at the subcellular level in the live animal, our understanding of the complex steps of metastasis will significantly increase. In addition, new drugs can be developed to target these newly visible steps of metastasis.

Change of Paradigm for the Reversed Field Pinch

NASA Astrophysics Data System (ADS)

Escande, D. F.

2010-11-01

The reversed field pinch (RFP) is a magnetic configuration germane to the tokamak, but it produces most of its magnetic field by the currents flowing inside the plasma; external coils provide only a small edge toroidal field whose sign is reversed with respect to the central one, whence the name of the configuration. Because of the presence of magnetic turbulence and chaos, the RFP had been considered for a long period as a terrible confinement configuration. However, recently a change of paradigm occurred for this device. Indeed, when the toroidal current is increased in the RFX-mod RFP in Padua (Italy), a self-organized helical state with an internal transport barrier (ITB) develops, and a broad zone of the plasma becomes hot (above 1 keV for a magnetic field above 0.8 T). The present theoretical picture of the RFP mainly comes from three-dimensional nonlinear visco-resistive MHD simulations whose dynamics has strong similarities with the experimental one, and triggered the experimental search for RFP states with improved confinement. The RFP ohmic state involves a helical electrostatic potential generating, as an electric drift, the so-called dynamo velocity field. The magnetic topology can bifurcate from a magnetic island to kink-like magnetic surfaces with higher resilience to magnetic chaos. This theoretical scenario was found to be relevant when ITB's enclosing a broad hot domain were discovered. The ITBs occur in the vicinity of the maximum of the safety factor. The new paradigm for the RFP supports its reappraisal as a low-external field, non-disruptive, ohmically heated approach to magnetic fusion, exploiting both self-organization and technological simplicity. Furthermore the RFP has the same Greenwald density limit as the tokamak, and it is an excellent test bed for the efficient control of multiple resistive wall modes. Its helical magnetic structure makes it germane to the stellarator too. As a result the RFP is also useful to bring support to the present two main lines of magnetic confinement.

Rifampicin Resistance and Multidrug-Resistant Tuberculosis Detection Using Xpert MTB/RIF in Wuhan, China: A Retrospective Study.

PubMed

Huang, Hai; Zhang, Yanlin; Li, Sheng; Wang, Jun; Chen, Jun; Pan, Zhiyun; Gan, Hui

2018-06-01

The Xpert MTB/RIF test (Cepheid, Sunnyvale, CA) can simultaneously detect the Mycobacterium tuberculosis (MTB) complex DNA and rifampicin (RFP) resistance and can rapidly determine RFP resistance and predict multidrug-resistant tuberculosis (MDR-TB). In this study, we analyzed clinical examination results of a hospital specializing in TB treatment in Wuhan, Hubei, China, and examined the use of traditional culture and drug-sensitive test (DST) results as a gold standard to assess the diagnosis value of the Xpert MTB/RIF test in RFP resistance and MDR-TB. A total of 2,910 specimens were received in the Mycobacteriology Laboratory, Wuhan Pulmonary Hospital, for Xpert MTB/RIF testing between December 2013 and December 2014. After the results were reviewed by exclusion criteria, 1,066 Xpert test results were eligible for our study. We then compared the Xpert test results with sputum acid-fast bacilli staining, cultures, and DST results. In total, Xpert correctly identified 96.71% (147/152) RFP-resistant TB and 98.25% (898/914) RFP-sensitive TB specimens. Of the 147 RFP-resistant TB specimens detected by Xpert, 122 MDR-TB (82.99%) were identified by traditional culture and DST techniques. Xpert can simultaneously detect MTB and RFP resistance with high sensitivity and specificity. Thus, Xpert testing aids in saving a considerable amount of time in the diagnosis and treatment of MDR-TB.

Delivering radiology supplies just-in-time.

PubMed

Clinton, M

1999-01-01

The radiology department at Dartmouth Hitchcock Medical Center (DHMC) adopted a just-in-time (JIT) inventory management system in 1992, reducing the volume of its in-house inventory of radiology supplies from a value of $400,000 to $16,000, just enough for four to five days of activity. An asset manager, the only person authorized to order supplies, was given responsibility for maintaining the department's supply of fixed and consumable assets. The first step in implementing the new system was to identify the supplies needed, standardize them and determine how often deliveries would be made. The JIT implementation team developed a request for proposal (RFP) that incorporated the standardized list of supplies. Three radiology supply vendors were invited to respond to the RFP. The team later determined that only one vendor was capable of implementing the JIT program. A three-year contract was awarded to that vendor. As that three-year contract reached completion, DHMC offered the JIT program to its eight affiliate hospitals and four outpatient clinics. The team decided to re-bid the contract for the entire network, which collectively performed 700,000 radiology exams annually. The new RFP encompassed 90 percent of the network's consumable supplies and offered customized delivery for each facility. The team identified eight criteria necessary for the evaluation of each vendor response to the RFP, rather than use price as the only consideration. The company that won the three-year contract furnished 90 percent of the radiology supplies for the DHMC network, allowing even further savings by the network, particularly for the smaller facilities and clinics. The program is continually monitored, adjusted and enhanced in order to incorporate changing departmental needs.

75 FR 40856 - Federal Register Meeting Notice; Webinar About Regional Innovation Clusters RFP

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-14

... potential Offerors about the Regional Innovation Clusters RFP. For more information please go to http://www.sba.gov/clusters/index.html . The RFP may be found on http://www.fedbizopps.gov . Logistical Information: The webinar will be held on Thursday, July 15, 2010. For details, please visit http://www.sba.gov...

Students Head over Heels over NASA Challenge

ERIC Educational Resources Information Center

Evans, Eric; Artusio-Glimpse, Aly; Zaczesk, Bryan; Dawson, Ross

2010-01-01

In November 2008, a mass email sent by the department chairman arrived in the inboxes of the imaging and photographic technology students at the Rochester Institute of Technology. A great opportunity was in store for four lucky students. The department had received a request for proposals (RFP) sent by NASA to colleges around the United States…

But I'm an engineer—not a contracts lawyer!

NASA Astrophysics Data System (ADS)

Warner, Mark; Bass, Harvey

2012-09-01

Industrial partners, commercial vendors, and subsystem contractors play a large role in the design and construction of modern telescopes. Because many telescope projects carry relatively small staffs, engineers are often required to perform the additional functions of technical writing, cost estimating, and contract bidding and negotiating. The skills required to carry out these tasks are not normally taught in traditional engineering programs. As a result, engineers often learn to write Request for Proposals (RFPs), select vendors, and negotiate contracts by trial-and-error and/or by adapting previous project documents to match their own requirements. Typically, this means that at the end of a contract the engineer has a large list of do's, don'ts, and lessons learned for the next RFP he or she must generate. This paper will present one such engineer's experience writing and bidding proposal packages for large telescope components and subsystems. Included are: thoughts on structuring SOWs, Specs, ICDs, and other RFP documents; modern methods for bidding the work; and systematic means for selecting and negotiating with a contractor to arrive at the best value for the project.

Commercial Decommissioning at DOE's Rocky Flats

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

Freiboth, C.; Sandlin, N.; Schubert, A.

2002-02-25

Due in large part to the number of nuclear facilities that make up the DOE complex, DOE-EM work has historically been paperwork intensive and driven by extensive regulations. Requirements for non-nuclear facilities are often grouped with those of nuclear facilities, driving up costs. Kaiser-Hill was interested in applying a commercial model to demolition of these facilities and wanted to apply necessary and sufficient standards to the work activities, but avoid applying unnecessary requirements. Faced with demolishing hundreds of uncontaminated or non-radiologically contaminated facilities, Kaiser-Hill has developed a subcontracting strategy to drastically reduce the cost of demolishing these facilities at Rockymore » Flats. Aiming to tailor the demolition approach of such facilities to more closely follow commercial practices, Kaiser-Hill recently released a Request for Proposals (RFP) for the demolition of the site's former central administration facility. The RFP significantly reduced requirements for compliance with specific DOE directives. Instead, the RFP required subcontractors to comply with health and safety requirements commonly found in the demolition of similar facilities in a commercial setting. This resulted in a number of bids from companies who have normally not bid on DOE work previously and at a reduced cost over previous approaches. This paper will discuss the details of this subcontracting strategy.« less

A novel method to visually determine the intracellular pH of xenografted tumor in vivo by utilizing fluorescent protein as an indicator.

PubMed

Tanaka, Shotaro; Harada, Hiroshi; Hiraoka, Masahiro

2015-09-04

The alkalization of intracellular pH (pHin) advances together with enhancement of aerobic glycolysis within tumor cells (the Warburg effect), and that is responsible for the progression of tumor malignancy together with hypoxia and angiogenesis. But how they correlate each other during tumor growth is poorly understood, partly due to the lack of suitable imaging methods. In present study, we propose a novel method to visually determine the pHin of tumor xenograft model from fluorescent image ratios. We utilized tandemly-linked two fluorescent proteins as a pH indicator; yellow fluorescent protein (YFP, pH sensitive) as an indicator, and red fluorescent protein (RFP, pH insensitive) as a reference. This method can eliminate the influence of optical factors from tissue as well as of the diverse expression level of pH indicator in the grafted cells. In addition, that can be operated by filter-based fluorescent imagers that are generally used in small animal study. The efficacy of the pH indicator, RFP-YFP, was confirmed by studies using recombinant protein in vitro and HeLa cells expressing RFP-YFP in vivo. Furthermore, we prepared nude mice subcutaneously xenografted HeLa cells expressing RFP-YFP cells as tumor model. The image ratios (YFP/RFP) of the tumor at the day 5 after surgery clearly showed the heterogeneous distribution of diverse pHin cells in the tumor tissue. Concomitantly acquired angiography using near-infrared fluorescence (680 nm for emission) also indicated that the relative alkaline pHin cells located in the region far from tumor vessels in which tumor aerobic glycolysis would be facilitated by progression of hypoxia and nutrient starvation. Applying the present method for a multi-wavelength imaging concerning pO2 and/or nutrient starvation states in addition to pHin and angiogenesis would provide valuable information about complicated alteration of tumoral cell states during tumorigenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Requirements for Space Settlement Design

NASA Astrophysics Data System (ADS)

Gale, Anita E.; Edwards, Richard P.

2004-02-01

When large space settlements are finally built, inevitably the customers who pay for them will start the process by specifying requirements with a Request for Proposal (RFP). Although we are decades away from seeing the first of these documents, some of their contents can be anticipated now, and provide insight into the variety of elements that must be researched and developed before space settlements can happen. Space Settlement Design Competitions for High School students present design challenges in the form of RFPs, which predict basic requirements for space settlement attributes in the future, including structural features, infrastructure, living conveniences, computers, business areas, and safety. These requirements are generically summarized, and unique requirements are noted for specific space settlement locations and applications.

Oocytes express an endogenous red fluorescent protein in a stony coral, Euphyllia ancora: a potential involvement in coral oogenesis

PubMed Central

Shikina, Shinya; Chiu, Yi-Ling; Chung, Yi-Jou; Chen, Chieh-Jhen; Lee, Yan-Horn; Chang, Ching-Fong

2016-01-01

To date,the molecular and cellular mechanisms underlying coral sexual reproduction remain largely unknown. We then performed a differential screen to identify genes related to oogenesis in the stony coral Euphyllia ancora. We identified a clone encoding a novel red fluorescent protein cDNA of E. ancora (named EaRFP). Microscopic observation and quantitative RT-PCR revealed that EaRFP is almost exclusively expressed in the ovary of the adult coral. The combination of the ovarian-cell separation method and the RT-PCR analysis revealed that the oocytes, but not the ovarian somatic cells, are the cells expressing EaRFP. Immunohistochemical analysis revealed that the expression of EaRFP starts in the early stage of the oocyte and continues until the maturation period. Furthermore, recombinant EaRFP was shown to possess an H2O2 degradation activity. These results raise the possibility that EaRFP plays a role in protecting the oocytes from oxidative stress from the early to late stages of oogenesis. The present study provides not only the first evidence for the potential involvement of FPs in coral oogenesis but also an insight into a cellular strategy underlying coral sexual reproduction. PMID:27167722

Oocytes express an endogenous red fluorescent protein in a stony coral, Euphyllia ancora: a potential involvement in coral oogenesis.

PubMed

Shikina, Shinya; Chiu, Yi-Ling; Chung, Yi-Jou; Chen, Chieh-Jhen; Lee, Yan-Horn; Chang, Ching-Fong

2016-05-11

To date,the molecular and cellular mechanisms underlying coral sexual reproduction remain largely unknown. We then performed a differential screen to identify genes related to oogenesis in the stony coral Euphyllia ancora. We identified a clone encoding a novel red fluorescent protein cDNA of E. ancora (named EaRFP). Microscopic observation and quantitative RT-PCR revealed that EaRFP is almost exclusively expressed in the ovary of the adult coral. The combination of the ovarian-cell separation method and the RT-PCR analysis revealed that the oocytes, but not the ovarian somatic cells, are the cells expressing EaRFP. Immunohistochemical analysis revealed that the expression of EaRFP starts in the early stage of the oocyte and continues until the maturation period. Furthermore, recombinant EaRFP was shown to possess an H2O2 degradation activity. These results raise the possibility that EaRFP plays a role in protecting the oocytes from oxidative stress from the early to late stages of oogenesis. The present study provides not only the first evidence for the potential involvement of FPs in coral oogenesis but also an insight into a cellular strategy underlying coral sexual reproduction.

DLEU2 encodes an antisense RNA for the putative bicistronic RFP2/LEU5 gene in humans and mouse.

PubMed

Corcoran, Martin M; Hammarsund, Marianne; Zhu, Chaoyong; Lerner, Mikael; Kapanadze, Bagrat; Wilson, Bill; Larsson, Catharina; Forsberg, Lars; Ibbotson, Rachel E; Einhorn, Stefan; Oscier, David G; Grandér, Dan; Sangfelt, Olle

2004-08-01

Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, suggesting that a nonclassical tumor-suppressor mechanism may be involved. Here, we present data showing that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in the mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5. Copyright 2004 Wiley-Liss, Inc.

GPR41/FFAR3 and GPR43/FFAR2 as cosensors for short-chain fatty acids in enteroendocrine cells vs FFAR3 in enteric neurons and FFAR2 in enteric leukocytes.

PubMed

Nøhr, Mark K; Pedersen, Maria H; Gille, Andreas; Egerod, Kristoffer L; Engelstoft, Maja S; Husted, Anna Sofie; Sichlau, Rasmus M; Grunddal, Kaare V; Poulsen, Steen Seier; Han, Sangdon; Jones, Robert M; Offermanns, Stefan; Schwartz, Thue W

2013-10-01

The expression of short-chain fatty acid receptors GPR41/FFAR3 and GPR43/ free fatty acid receptor 2 (FFAR2) was studied in the gastrointestinal tract of transgenic monomeric red fluorescent protein (mRFP) reporter mice. In the stomach free fatty acid receptor 3 (FFAR3)-mRFP was expressed in a subpopulation of ghrelin and gastrin cells. In contrast, strong expression of FFAR3-mRFP was observed in all cholecystokinin, glucose-dependent insulinotropic peptide (GIP), and secretin cells of the proximal small intestine and in all glucagon-like peptide-1 (GLP-1), peptide YY, and neurotensin cells of the distal small intestine. Throughout the colon and rectum, FFAR3-mRFP was strongly expressed in the large population of peptide YY and GLP-1 cells and in the neurotensin cells of the proximal colon. A gradient of expression of FFAR3-mRFP was observed in the somatostatin cells from less than 5% in the stomach to more than 95% in the rectum. Substance P-containing enterochromaffin cells displayed a similar gradient of FFAR3-mRFP expression throughout the small intestine. Surprisingly, FFAR3-mRFP was also expressed in the neuronal cells of the submucosal and myenteric ganglia. Quantitative PCR analysis of fluorescence-activated cell sorting (FACS) purified FFAR3-mRFP positive cells confirmed the coexpression with the various peptide hormones as well as key neuronal marker proteins. The FFAR2-mRFP reporter was strongly expressed in a large population of leukocytes in the lamina propria of in particular the small intestine but surprisingly only weakly in a subpopulation of enteroendocrine cells. Nevertheless, synthetic ligands specific for either FFAR3 or FFAR2 each released GLP-1 from colonic crypt cultures and the FFAR2 agonist mobilized intracellular Ca²⁺ in FFAR2 positive enteroendocrine cells. It is concluded that FFAR3-mRFP serves as a useful marker for the majority of enteroendocrine cells of the small and large intestine and that FFAR3 and FFAR2 both act as sensors for short-chain fatty acids in enteroendocrine cells, whereas FFAR3 apparently has this role alone in enteric neurons and FFAR2 in enteric leukocytes.

Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET.

PubMed

Shcherbakova, Daria M; Cox Cammer, Natasha; Huisman, Tsipora M; Verkhusha, Vladislav V; Hodgson, Louis

2018-06-01

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Förster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.

Ohmic ignition with high engineering beta based on the RFP

NASA Astrophysics Data System (ADS)

Sarff, J. S.; Anderson, J. K.; Chapman, B. E.; McCollam, K. J.

2017-10-01

The RFP configuration allows the possibility of ohmic ignition for fusion energy, eliminating the need for auxiliary heating by rf or neutral beam injection. Complex plasma-facing antennas and NBI sources are therefore not required, simplifying the difficult fusion materials challenge. While all toroidal configurations require a volume-average 〈 B 〉 >= 5 T, the field strength at the magnet in the RFP is only Bcoil 3T since plasma current generates almost all of the field. Engineering beta is therefore maximized. We summarize access to ohmic ignition by examining a Lawson-like power balance for an RFP fusion plasma comparable to the ARIES-AT advanced tokamak, which generates neutron wall loading Pn / A 5 MW/m2. The required energy confinement for ohmic ignition in an RFP is similar to that for a tokamak. Confinement in MST is comparable to a same-size, same-field tokamak plasma, but 〈 B 〉 in MST is only 1/20th that required for fusion. While transport could ultimately be dominated by micro turbulence, extrapolation of stochastic transport using Lundquist number scaling for MHD tearing indicates standard RFP confinement (not enhanced by current profile control) could be sufficient to access ohmic ignition. This bolsters the possibility for steady-state inductive sustainment using oscillating field current drive. The high beta and classical energetic ion confinement measured in MST also bolster the RFP's fusion potential. Work supported by U.S. DoE.

[Survival of bone marrow mesenchymal stem cells and periodontal ligament stem cells in cell sheets].

PubMed

An, Kangkang; Liu, Hongwei

2014-11-01

To evaluate the survival of bone marrow mesenchymal stem cells (BMMSC) and periodontal ligament stem cells (PDLSC) in BMMSC/PDLSC cell sheets which transplanted ectopically into subcutaneous dorsum of nude mice. The canine BMMSC and PDLSC from primary culture were tranfected with lentiviral vectors carrying green fluorescent protein (GFP) gene (Lentivirus-GFP) or red fluorescent protein (RFP) gene (Lentivirus-RFP) respectively. The immunophenotypes of GFP-labeled BMMSC and RFP-labeled PDLSC were identified by flow cytometry. Adipogenic and osteogenic differentiation of them were detected by alizarin red or oil red O respectively. Then, both GFP-labeled BMMSC cell sheets and RFP-labeled PDLSC cell sheets were fabricated respectively using normal culture dish (6 cm) after stimulation of extracellular matrix formation. Each was enveloped by collagen membrane (Bio-Gide) and then transplanted into the subcutaneous dorsum of nude mice. In vivo non-invasive biofluorescence imaging(BFI) was performed at 1, 2, 4 and 8 w post-tranplantation to trace and quantify the survival and growth of RFP-labeled PDLSC and GFP-labeled BMMSC via the BFI system of the NightOWL. The fluorescence intensity change of GFP/RFP signal was monitored and compared. The mice were sacrificed 8 weeks after cell sheets transplantation and the survival of stem cells was verified by fluorescence immunohistochemistry. The flow cytometry showed that GFP-labeled BMMSC positively expressed CD29, CD44, CD34, STRO-1 were 93.07%, 92.84%, 3.23%, 67.67%, and RFP-labeled PDLSCs were 89.91%, 88.47%, 6.04%, 74.11%, respectively. Both of them had the potency of differentiating into osteoblasts and adipocytes. The stemness of both of them was almost same. After being transplanted into nude mice, the signal strength of GFP(BMMSC) was weaker and weaker in 1, 2, 4 and 8 w [(83.1±3.1)×10(6), (65.1±2.3)×10(6), (51.5 ± 2.3)×10(6), (33.8 ± 2.0)×10(6) ph/s, respectively.]. The signal strength of RFP(PDLSC) was weakenedin 1, 2 and 4 w [(53.8±3.0)×10(6), (42.2±2.6)×10(6), (34.5±2.1)×10(6) ph/s], then recovered in 8 w ([ 45.1±2.9)×10(6) ph/s]. The signal strength of RFP(PDLSC) was signifcantly stronger in 8 w than in 4 w(P < 0.01). The survival of RFP-labeled PDLSC was significant higher than that of GFP-labeled BMMSC. After 8 weeks, lots of RFP-labeled PDLSC were observed by microscope, but less GFP-labeled BMMSC were observed. Histometric analysis revealed that the survival of stem cells in the RFP-labeled PDLSC cell sheets was significantly higher than that of in the GFP-labeled BMMSCs cell sheets.

One-Step and Two-Step Facility Acquisition for Military Construction: Project Selection and Implementation Procedures

DTIC Science & Technology

1990-08-01

the guidance in this report. 1-4. Scope This guidance covers selection of projects suitable for a One-Step or Two-Step approach, development of design...conducted, focus on resolving proposal deficiencies; prices are not "negotiated" in the common use of the term. A Request for Proposal (RFP) states project ...carefully examines experience and past performance in the design of similar projects and building types. Quality of

Improving Teacher Quality 2007 Grant Awards. Commission Report 07-23

ERIC Educational Resources Information Center

California Postsecondary Education Commission, 2007

2007-01-01

The Improving Teacher Quality Program recently concluded its 2007 competition to select grantees who will provide high-quality teacher professional development over the next several years. Teachers in grades K-2 were the focus of this year's Request for Proposals (RFP). As required in the last two rounds of competition, the selected projects must…

Design of a turbofan powered regional transport aircraft

NASA Technical Reports Server (NTRS)

1991-01-01

The majority of the market for small commercial transport aircraft is dominated by high-efficiency, propeller-driven aircraft of non-U.S. manufacture. During the past year senior student design teams at Purdue developed and then responded to a Request For Proposal (RFP) for a regional transport aircraft. The RFP development identified promising world markets and their needs. The students responded by designing aircraft with ranges of up to 1500 n.m. and passenger loads of 50 to 90. During the design project, special emphasis was placed upon keeping acquisition cost and direct operating costs at a low level while providing passengers with quality comfort levels. Twelve student teams worked for one semester developing their designs. Several of the more successful designs and those that placed a high premium on innovation are described. The depth of detail and analysis in these student efforts are also illustrated.

The rostral parvicellular reticular formation neurons mediate lingual nerve input to the rostral ventrolateral medulla.

PubMed

Ishizuka, Ken'Ichi; Satoh, Yoshihide

2012-08-16

In rats that had been anesthetized by urethane-chloralose, we investigated whether neurons in the rostral part of the parvicellular reticular formation (rRFp) mediate lingual nerve input to the rostral ventrolateral medulla (RVLM), which is involved in somato-visceral sensory integration and in controlling the cardiovascular system. We determined the effect of the lingual nerve stimulation on activity of the rRFp neurons that were activated antidromically by stimulation of the RVLM. Stimulation of the lingual trigeminal afferent gave rise to excitatory effects (10/26, 39%), inhibitory effects (6/26, 22%) and no effect (10/26, 39%) on the RVLM-projecting rRFp neurons. About two-thirds of RVLM-projecting rRFp neurons exhibited spontaneous activity; the remaining one-third did not. A half (13/26) of RVLM-projecting rRFp neurons exhibited a pulse-related activity, suggesting that they receive a variety of peripheral and CNS inputs involved in cardiovascular function. We conclude that the lingual trigeminal input exerts excitatory and/or inhibitory effects on a majority (61%) of the RVLM-projecting rRFp neurons, and their neuronal activity may be involved in the cardiovascular responses accompanied by the defense reaction. Copyright © 2012 Elsevier B.V. All rights reserved.

A Color-coded Imageable Syngeneic Mouse Model of Stromal-cell Recruitment by Metastatic Lymphoma.

PubMed

Matsumoto, Takuro; Suetsugu, Atsushi; Shibata, Yuhei; Nakamura, Nobuhiko; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

2015-09-01

A syngeneic color-coded imageable lymphoma model has been developed to visualize recruitment of host stromal cells by malignant lymphoma during metastasis. The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were initially established. EL4-RFP cells were subsequently injected into the tail vein of C57/BL6-GFP transgenic mice. EL4-RFP metastasis was observed in the lymph nodes of the upper mediastinum and in the liver 28 days after cell injection. Large EL4-RFP liver metastases in C57/BL6-GFP mice contained GFP-expressing stromal cells derived from the host. In addition, EL4-RFP lymphoma metastasis was formed in peri-gastric lymph nodes, which were also enriched in host GFP-expressing cells. Furthermore, EL4-RFP lymphoma cells were also observed in the peripheral blood and bone marrow of C57/BL6-GFP transgenic mice, where they were associated with GFP-expressing host cells. Lymph node, liver and bone marrow metastases were found approximately 4 weeks after transplantation and all RFP-expressing metastases were highly enriched in GFP-expressing host stromal cells. This model of malignant lymphoma can be used to study early tumor development, metastasis, and the role of the stroma, as well as for discovery and evaluation of novel therapeutics for this treatment-resistant disease. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Procurement Tips When Buying from Web Firms.

ERIC Educational Resources Information Center

Ullman, Nona

2002-01-01

Provides four suggestions to follow when purchasing from web-based companies: Put in place a strong RFP process; conduct due diligence of the finalists; use contractual terms to mitigate risk; and clearly set project expectations and timeliness. (PKP)

LASER BIOLOGY AND MEDICINE: Application of laser fluorimetry for determining the influence of a single amino-acid substitution on the individual photophysical parameters of a fluorescent form of a fluorescent protein mRFP1

NASA Astrophysics Data System (ADS)

Banishev, A. A.; Vrzheshch, E. P.; Shirshin, E. A.

2009-03-01

Individual photophysical parameters of the chromophore of a fluorescent protein mRFP1 and its two mutants (amino-acid substitution at position 66 - mRFP1/ Q66C and mRFP1/Q66S proteins) are determined. For this purpose, apart from conventional methods of fluorimetry and spectrophotometry, nonlinear laser fluorimetry is used. It is shown that the individual extinction coefficients of the chromophore of proteins correlate (correlation coefficient above 0.9) with the volume of the substituted amino-acid residue at position 66 (similar to the positions of the absorption, fluorescence excitation and emission maxima).

Computational Omics Funding Opportunity | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the NVIDIA Foundation are pleased to announce funding opportunities in the fight against cancer. Each organization has launched a request for proposals (RFP) that will collectively fund up to $2 million to help to develop a new generation of data-intensive scientific tools to find new ways to treat cancer.

Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells.

PubMed

Zhou, Cuihong; Zhong, Wu; Zhou, Jun; Sheng, Fugeng; Fang, Ziyuan; Wei, Yue; Chen, Yingyu; Deng, Xiaoyan; Xia, Bin; Lin, Jian

2012-08-01

Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP(+)RFP(+) puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.

Transgenic nude mice ubiquitously expressing fluorescent proteins for color-coded imaging of the tumor microenvironment.

PubMed

Hoffman, Robert M

2014-01-01

We have developed a transgenic green fluorescent protein (GFP) nude mouse with ubiquitous GFP expression. The GFP nude mouse was obtained by crossing nontransgenic nude mice with the transgenic C57/B6 mouse in which the β-actin promoter drives GFP expression in essentially all tissues. In the adult mice, many organs brightly expressed GFP, including the spleen, heart, lungs, spleen, pancreas, esophagus, stomach, and duodenum as well as the circulatory system. The liver expressed GFP at a lesser level. The red fluorescent protein (RFP) transgenic nude mouse was obtained by crossing non-transgenic nude mice with the transgenic C57/B6 mouse in which the beta-actin promoter drives RFP (DsRed2) expression in essentially all tissues. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, liver, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. The cyan fluorescent protein (CFP) nude mouse was developed by crossing nontransgenic nude mice with the transgenic CK/ECFP mouse in which the β-actin promoter drives expression of CFP in almost all tissues. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescence signals of all internal organs, which vary in intensity. The GFP, RFP, and CFP nude mice when transplanted with cancer cells of another color are powerful models for color-coded imaging of the tumor microenvironment (TME) at the cellular level.

Minimum inhibitory concentration and killing properties of rifampicin against canine Staphylococcus pseudintermedius isolates from dogs in the southeast USA.

PubMed

Ho, Karen K; Conley, Austin C; Kennis, Robert A; Hathcock, Terri L; Boothe, Dawn M; White, Amelia G

2018-05-29

Meticillin-resistant (MR) staphylococcal pyoderma in dogs has led to increased use of alternate antibiotics such as rifampicin (RFP). However, little information exists regarding its pharmacodynamics in MR Staphylococcus pseudintermedius. To determine the minimum inhibitory concentration (MIC) and killing properties of RFP for canine Staphylococcus pseudintermedius isolates. The MIC of RFP was determined using the ETEST ® for 50 meticillin-susceptible (MS) and 50 MR S. pseudintermedius isolates collected from dogs. From these isolates, two MS isolates (RFP MIC of 0.003 and 0.008 μg/mL, respectively) and two MR isolates (RFP MIC of 0.003 and 0.012 μg/mL, respectively) were subjected to time-kill studies. Mueller-Hinton broth was supplemented with RFP at 0, 0.5, 1, 2, 4, 8, 16 and 32 times the MIC for 0, 2, 4, 10, 16 and 24 h. The number of viable colony forming units in each sample was determined using a commercial luciferase assay kit. The MIC 50 and MIC 90 were the same for MS and MR isolates, at 0.004 μg/mL and 0.008 μg/mL, respectively. Rifampicin kill curves were not indicative of concentration-dependency, suggesting time-dependent activity. Two isolates (MS 0.003 and 0.008 μg/mL) exhibited bacteriostatic activity, whereas two others (MR 0.003 and 0.012 μg/mL) exhibited bactericidal activity. This study demonstrated that MS and MR S. pseudintermedius isolates were equally susceptible to rifampicin and that dosing intervals should be designed for time-dependent efficacy. These data can support pharmacokinetic studies of RFP in dogs with susceptible infections caused by S. pseudintermedius. © 2018 ESVD and ACVD.

Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

PubMed Central

Telford, William G.; Shcherbakova, Daria M.; Buschke, David; Hawley, Teresa S.; Verkhusha, Vladislav V.

2015-01-01

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo. PMID:25811854

Multiparametric flow cytometry using near-infrared fluorescent proteins engineered from bacterial phytochromes.

PubMed

Telford, William G; Shcherbakova, Daria M; Buschke, David; Hawley, Teresa S; Verkhusha, Vladislav V

2015-01-01

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo.

Coorientation Theory and Assessment of the RFP Solution to Client/Service Learner Matchmaking

ERIC Educational Resources Information Center

Rogers, Cathy; Andrews, Valerie

2013-01-01

Tensions that result from varying expectations of service learners and clients/community partners are as common as the pedagogical practice of service learning in public relations courses. The matchmaking process between instructors and clients can influence expectations; however, the literature includes little guidance about the process of client…

An Autophagic Flux Probe that Releases an Internal Control.

PubMed

Kaizuka, Takeshi; Morishita, Hideaki; Hama, Yutaro; Tsukamoto, Satoshi; Matsui, Takahide; Toyota, Yuichiro; Kodama, Akihiko; Ishihara, Tomoaki; Mizushima, Tohru; Mizushima, Noboru

2016-11-17

Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3ΔG, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is degraded by autophagy, while RFP-LC3ΔG remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3ΔG probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms. Copyright © 2016 Elsevier Inc. All rights reserved.

[Establishment of an iRFP and luciferase dual-color fluorescence-traced hepatocellular carcinoma transplantation model in nude mice].

PubMed

Li, Hongjun; Yang, Tianhua; Huang, Yanping; Liu, Mingzhu; Qin, Zhongqiang; Chu, Fei; Li, Zhenghong; Li, Yonghai

2017-11-01

Objective To establish a hepatocellular carcinoma xenograft model in nude mice which could stably express gene and be monitored dynamically. Methods We first constructed the lentiviral particles containing luciferase (Luc) and near-infrared fluorescent protein (iRFP) and puromycin resistance gene, and then transduced them into the HepG2 hepatoma cells. The cell line stably expressing Luc and iRFP genes were screened and inoculated into nude mice to establish xenograft tumor model. Tumor growth was monitored using in vivo imaging system. HE staining and immunohistochemistry were used to evaluate the pathological features and tumorigenic ability. Results HepG2 cells stably expressing iRFP and Luc were obtained; with the engineered cell line, xenograft model was successfully established with the features of proper tumor developing time and high rate of tumor formation as well as typical pathological features as showed by HE staining and immunohistochemistry. Conclusion Hepatocellular carcinoma model in nude mice with the features of stable gene expression and dynamical monitoring has been established successfully with the HepG2-iRFP-Luc cell line.

Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675.

PubMed

Konold, Patrick E; Yoon, Eunjin; Lee, Junghwa; Allen, Samantha L; Chapagain, Prem P; Gerstman, Bernard S; Regmi, Chola K; Piatkevich, Kiryl D; Verkhusha, Vladislav V; Joo, Taiha; Jimenez, Ralph

2016-08-04

Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S1 lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift.

Extended Stokes shift in fluorescent proteins: chromophore-protein interactions in a near-infrared TagRFP675 variant.

PubMed

Piatkevich, Kiryl D; Malashkevich, Vladimir N; Morozova, Kateryna S; Nemkovich, Nicolai A; Almo, Steven C; Verkhusha, Vladislav V

2013-01-01

Most GFP-like fluorescent proteins exhibit small Stokes shifts (10-45 nm) due to rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-infrared derivative of the red fluorescent protein mKate, named TagRFP675, exhibits the Stokes shift, which is 30 nm extended comparing to that of the parental protein. In physiological conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addition, its emission maximum strongly depends on the excitation wavelength. Structures of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacts with the protein matrix via an extensive network of hydrogen bonds capable of large flexibility. Based on the spectroscopic, biochemical, and structural analysis we suggest that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties.

Model for the loop voltage of reversed field pinches

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

Jarboe, T.R.; Alper, B.

1987-04-01

A simple model is presented that uses the concept of helicity balance to predict the toroidal loop voltage of reversed field pinches (RFP's). Data from the RFP's at Culham (Plasma Phys. Controlled Fusion 27, 1307 (1985)) are used to calibrate and verify the model. The model indicates that most of the helicity dissipation occurs in edge regions that are outside the limiters or in regions where field lines contact the walls. The value of this new interpretation to future RFP and spheromak experiments is discussed.

Research, practice, and policy partnerships in pan-Canadian coalitions for cancer and chronic disease prevention.

PubMed

Manafò, Elizabeth; Petermann, Lisa; Lobb, Rebecca; Keen, Deb; Kerner, Jon

2011-01-01

To describe the development stages of the Coalitions Linking Action and Science for Prevention (CLASP) initiative of the Canadian Partnership Against Cancer to support research, practice, and policy coalitions focused on cancer and chronic disease prevention in Canada. Coalitions Linking Action and Science for Prevention was implemented in 3 stages. This article describes Stage 1 that consisted of an online concept-mapping consultation process, 3 topic specific networking and consultation workshops, and 3 context-specific networking, coalition development, and planning meetings. These were all completed using a participatory engagement approach to encourage knowledge exchange across jurisdictions and sectors in Canada. Toronto, Ontario; Calgary, Alberta; Montreal, Québec; and Ottawa, Ontario. More than 500 researchers, practitioners, and policy specialists were invited to take part in the first stage activities. (1) Participant-identified high-priority opportunities for strategic collaboration; (2) Cross-jurisdictional and cross-sector representation; and (3) Participant feedback on the CLASP processes and activities. Participants from Stage 1 activities were distributed across all provinces/territories; 3 jurisdictional levels; and research, practice, and policy sectors. Ninety priority opportunities for strategic collaboration were identified across all 3 workshops. Participants provided detailed feedback about transparency of the RFP (Request for Proposals) application process, support needed to level the playing field for potential applicants, and valuable suggestions for the adjudication process. Coalitions Linking Action and Science for Prevention engaged hundreds of research, practice, and policy experts across Canada focusing social-behavioral, clinical, and environmental and occupational opportunities for cancer and chronic disease prevention. Given the extent of expert and jurisdictional engagement, the substantial Partnership investment in a participatory engagement approach to RFP development and potential applicant response suggests that efforts to link cancer and chronic disease prevention efforts across jurisdictions and through research, practice, and policy collaboration may require this type of a priori investment in networking, communication, coordination, and collaboration.

Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method.

PubMed

Beilharz, Katrin; van Raaphorst, Renske; Kjos, Morten; Veening, Jan-Willem

2015-10-01

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

Konyashov, Vadim V.; Krasnov, Alexander M.

Results are provided of the experimental investigation of radioactive fission product (RFP) release, i.e., krypton, xenon, and iodine radionuclides from fuel elements with initial defects during long-term (3 to 5 yr) irradiation under low linear power (5 to 12 kW/m) and during special experiments in the VK-50 vessel-type boiling water reactor.The calculation model for the RFP release from the fuel-to-cladding gap of the defective fuel element into coolant was developed. It takes into account the convective transport in the fuel-to-cladding gap and RFP sorption on the internal cladding surface and is in good agreement with the available experimental data. Anmore » approximate analytical solution of the transport equation is given. The calculation dependencies of the RFP release coefficients on the main parameters such as defect size, fuel-to-cladding gap, temperature of the internal cladding surface, and radioactive decay constant were analyzed.It is shown that the change of the RFP release from the fuel elements with the initial defects during long-term irradiation is, mainly, caused by fuel swelling followed by reduction of the fuel-to-cladding gap and the fuel temperature. The calculation model for the RFP release from defective fuel elements applicable to light water reactors (LWRs) was developed. It takes into account the change of the defective fuel element parameters during long-term irradiation. The calculation error according to the program does not exceed 30% over all the linear power change range of the LWR fuel elements (from 5 to 26 kW/m)« less

A Manpower Model for U.S. Navy Operational Contracting

DTIC Science & Technology

2012-06-01

Accomplishment Time RFP Request For Proposal SAF/FM Air Force Financial Management SAP Simplified Acquisition Procedures SAT Simplified...conformance and seller’s release of claim (Garrett, 2007). 2. Contract Size and its Effect on Workload Simplified acquisition procedures ( SAP ) were...the SAP dollar threshold. 14 The drastic reduction in KO workload through the use of SAP is unmatched by any federal authorization that came

Project Management of Army Aircraft Survivability Equipment

DTIC Science & Technology

1976-11-01

develop- ment trade-offs; and request for proposal (RFP) reviews involving advanced development (AD), engineering development (ED), and pro- ducibility...Safety: Ensur~e that adequate consideration is given safetyf throughout the life cycle of aviation surviva- bility equipment. -13. Project Manager Training...rate management effort to assure an orderly progression through its life cyIcle. Many of the items carry an urgent priority for worldwide applicatic

Land remote sensing commercialization: A status report

NASA Technical Reports Server (NTRS)

Bishop, W. P.; Heacock, E. L.

1984-01-01

The current offer by the United States Department of Commerce to transfer the U.S. land remote sensing program to the private sector is described. A Request for Proposals (RFP) was issued, soliciting offers from U.S. firms to provide a commercial land remote sensing satellite system. Proposals must address a complete system including satellite, communications, and ground data processing systems. Offerors are encouraged to propose to take over the Government LANDSAT system which consists of LANDSAT 4 and LANDSAT D'. Also required in proposals are the market development procedures and plans to ensure that commercialization is feasible and the business will become self-supporting at the earliest possible time. As a matter of Federal Policy, the solicitation is designed to protect both national security and foreign policy considerations. In keeping with these concerns, an offeror must be a U.S. Firm. Requirements for data quality, quantity, distribution and delivery are met by current operational procedures. It is the Government's desire that the Offeror be prepared to develop and operate follow-on systems without Government subsidies. However, to facilitate rapid commercialization, an offeror may elect to include in his proposal mechanisms for short term government financial assistance.

Prolonged QRS duration on surface electrocardiogram is associated with left ventricular restrictive filling pattern.

PubMed

Erdoğan, Turan; Durakoğlugil, Murtaza Emre; Çiçek, Yüksel; Çetin, Mustafa; Duman, Hakan; Şatiroğlu, Ömer; Çelik, Şükrü

2017-03-01

Prolonged QRS duration is associated with decreased left ventricular (LV) systolic function. However, the relation between LV restrictive filling pattern (RFP) and QRS duration has not been investigated yet. The purpose of our study was to assess this relationship. We analyzed standard 12-lead surface electrocardiogram (ECG) of 155 consecutive patients. Mitral inflow and septal tissue velocities were obtained using the apical 4-chamber view with pulsed Doppler echocardiography. Patients were divided into 2 groups according to measured deceleration time (DT): restrictive (with DT ≤130 ms) or non-restrictive (with DT >130 ms). QRS duration was significantly longer in the restrictive group than in the non-restrictive group (0.101 vs. 0.090 s, p  < 0.0001). QRS duration of >0.10 s was highly specific (82.6%), but modestly sensitive (64.7%), for the prediction of LV RFP. Multivariate analyses demonstrated that E/A ratio, peak E, peak A, septal e', and a' velocities were significantly associated with RFP. Prolonged QRS duration (>0.10 s) obtained from a standard resting 12-lead ECG is associated with LV RFP. However, the relationship of QRS duration with RFP was not independent of echocardiographic parameters.

Women and family poultry production in rural Africa.

PubMed

Gueye, E H

2000-02-01

Poultry production has existed for many generations in Africa, and almost every village household keeps chickens. The rural family poultry (RFP) are generally raised in free-range and/or backyard systems, which are traditional extensive husbandry systems. The development of an intensive poultry production has been the goal of the African government over the years. Despite efforts aiming for such goal, RFP is still very important in African countries that are both poor and net importers of food. It is a valuable asset because it can contribute significantly in alleviating poverty, securing food supply, and promoting gender equality. In view of this, interventions to improve RFP production systems should take into account the sociocultural issues, specifically gender-based aspects. It is noted that such interventions might, in addition to food security and poverty alleviation, also serve to promote gender equality. RFP development programs should be more women-friendly in order to facilitate women's participation, as RFP production in the region is generally a woman's business. Moreover, efforts to empower village women has to be envisaged cautiously as there is a serious risk of men taking over once the poultry sector becomes more profitable.

A preliminary design proposal for a maritime patrol strike aircraft: MPS-2000 Condor

NASA Technical Reports Server (NTRS)

1994-01-01

The four member graduate design team assembled to submit a proposal for the 1993/1994 RFP at the University of Kansas has designed a four seat, variable swept wing, twin turbofan aircraft with STOL capabilities. The aircraft is named the MPS-2000 Condor and is capable of carrying air-to-surface or air-to-air weapon systems along with attack and surveillance radar and IRF systems. The aircraft has a cruise range of 800 nautical miles, a loiter of 4 hours, and a dash speed of 500 kts.

Particle Energization via Tearing Instability with Global Self-Organization Constraints

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

Sarff, John; Guo, Fan

The presentation reviews how tearing magnetic reconnection leads to powerful ion energization in reversed field pinch (RFP) plasmas. A mature MHD model for tearing instability has been developed that captures key nonlinear dynamics from the global to intermediate spatial scales. A turbulent cascade is also present that extends to at least the ion gyroradius scale, within which important particle energization mechanisms are anticipated. In summary, Ion heating and acceleration associated with magnetic reconnection from tearing instability is a powerful process in the RFP laboratory plasma (gyro-resonant and stochastic processes are likely candidates to support the observed rapid heating and othermore » features, reconnection-driven electron heating appears weaker or even absent, energetic tail formation for ions and electrons). Global self-organization strongly impacts particle energization (tearing interactions that span to core to edge, global magnetic flux change produces a larger electric field and runaway, correlations in electric and magnetic field fluctuations needed for dynamo feedback, impact of transport processes (which can be quite different for ions and electrons), inhomogeneity on the system scale, e.g., strong edge gradients).« less

Color-Coded Imaging of Syngeneic Orthotopic Malignant Lymphoma Interacting with Host Stromal Cells During Metastasis.

PubMed

Matsumoto, Takuro; Suetsugu, Atsushi; Hasegawa, Kosuke; Nakamura, Miki; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

2016-04-01

The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. In a previous study, EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were established and injected into the tail vein of C57/BL6 green fluorescent protein (GFP) transgenic mice. Metastasis was observed at multiple sites which were also enriched with host GFP-expressing stromal cells. In the present study, our aim was to establish an orthotopic model of EL4-RFP. In the present study, EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice as an orthotopic model of lymphoma. Resultant primary tumor and metastases were imaged with the Olympus FV1000 scanning laser confocal microscope. EL4-RFP metastasis was observed 21 days later. EL4-RFP tumors in the spleen (primary injection site), liver, supra-mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized by color-coded imaging. EL4-RFP metastases in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Small tumors were observed in the spleen, which were rich in host stromal cells. In the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. Color-coded imaging of the lymphoma microenvironment suggests an important role of stromal cells in lymphoma progression and metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Plasma metalloproteinase-9 and restrictive filling pattern as major predictors of outcome in patients with ischemic cardiomyopathy.

PubMed

Bajraktari, Gani; Miccoli, Mario; Buralli, Simona; Fontanive, Paolo; Elezi, Shpend; Metelli, Maria Rita; Baggiani, Angelo; Dini, Frank Lloyd

2012-10-01

Assessment of plasma matrix metalloproteinase-9 (MMP-9) and Doppler markers of increased left ventricular (LV) filling pressure may be added to risk stratify patients with ischemic cardiomyopathy (IC). Therefore, we aimed at investigating the value of plasma MMP-9 and restrictive filling pattern (RFP) in IC patients. Eighty-eight consecutive patients hospitalized for heart failure (LV ejection fraction ≤ 40%) due to IC were enrolled. A complete M-mode and two-dimensional echo-Doppler examination were performed. Patients were defined as having RFP if they had a mitral E wave deceleration time<150 ms. Plasma MMP-9 and N-terminal protype-B natriuretic peptide levels were assessed at the time of the index echocardiogram. The end point was all-cause mortality or hospitalization for worsening HF. Follow-up period was 25 ± 17 months. Median value of MMP-9 was 714 ng/ml. On univariate analysis, a number of measurements predicted the composite end point: NYHA class>2, RFP, MMP-9>60.5 ng/ml, LV ejection fraction<27%, anemia, pulmonary pressure ≥ 35 mm Hg, N-terminal protype-B natriuretic peptide>1742 pg/ml, and glomerular filtration rate<60 ml/min/1.73 m(2). Independent variables of outcome were anemia (HR=1.9, p=0.031), and the combination of plasma MMP-9 and RFP (HR=3.2, p=0.004). On Kaplan-Meier survival curves, patients with elevated MMP-9 levels and RFP had the lowest event-free survival rate (log-rank: 29.0, p<0.0001). The net reclassification improvement showed a significant increase in the prediction model when elevated MMP-9 and RFP were added to the base model that included clinical, biochemical and echocardiographic parameters (p<0.0001). MMP-9 levels and RFP have an incremental predictive value to risk classify IC patients. Copyright © 2012. Published by Elsevier B.V.

Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor.

PubMed

Fan, Yichong; Makar, Merna; Wang, Michael X; Ai, Hui-Wang

2017-09-01

Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor-TrxRFP1-to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

Development of redox-sensitive red fluorescent proteins for imaging redox dynamics in cellular compartments.

PubMed

Fan, Yichong; Ai, Hui-wang

2016-04-01

We recently reported a redox-sensitive red fluorescent protein, rxRFP1, which is one of the first genetically encoded red-fluorescent probes for general redox states in living cells. As individual cellular compartments have different basal redox potentials, we hereby describe a group of rxRFP1 mutants, showing different midpoint redox potentials for detection of redox dynamics in various subcellular domains, such as mitochondria, the cell nucleus, and endoplasmic reticulum (ER). When these redox probes were expressed and subcellularly localized in human embryonic kidney (HEK) 293 T cells, they responded to membrane-permeable oxidants and reductants. In addition, a mitochondrially localized rxRFP1 mutant, Mito-rxRFP1.1, was used to detect mitochondrial oxidative stress induced by doxorubicin-a widely used cancer chemotherapy drug. Our work has expanded the fluorescent protein toolkit with new research tools for studying compartmentalized redox dynamics and oxidative stress under various pathophysiological conditions.

Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET.

PubMed

Subach, Fedor V; Zhang, Lijuan; Gadella, Theodorus W J; Gurskaya, Nadya G; Lukyanov, Konstantin A; Verkhusha, Vladislav V

2010-07-30

We have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or nonfluorescent state (OFF state), respectively. The ON and OFF states exhibit absorbance maxima at 567 and 440 nm, respectively. Due to the photoswitchable absorbance, rsTagRFP can be used as an acceptor for a photochromic Förster resonance energy transfer (pcFRET). The photochromic acceptor facilitates determination of a protein-protein interaction by providing an internal control for FRET. Using pcFRET with EYFP as a donor, we observed an interaction between epidermal growth factor receptor and growth factor receptor-binding protein 2 in live cells by detecting the modulation of both the fluorescence intensity and lifetime of the EYFP donor upon the ON-OFF photoswitching of the rsTagRFP acceptor. 2010 Elsevier Ltd. All rights reserved.

Multimodal Fluorescence and Bioluminescence Imaging Reveals Transfection Potential of Intratracheally Administered Polyplexes for Breast Cancer Lung Metastases.

PubMed

Geyer, Antonia; Taschauer, Alexander; Alioglu, Fatih; Anton, Martina; Maier, Julia; Drothler, Elisabeth; Simlinger, Manuela; Yavuz, Sümeyye; Sami, Haider; Ogris, Manfred

2017-12-01

Local delivery of anticancer agents or gene therapeutics to lung tumors can circumvent side effects or accumulation in non-target organs, but accessibility via the alveolar side of the blood-air barrier remains challenging. Polyplexes based on plasmid and linear polyethylenimine (LPEI) transfect healthy lung tissue when applied intravenously (i.v.) in the mouse, but direct delivery into the lungs results in low transfection of lung tissue. Nevertheless, LPEI could offer the potential to transfect lung tumors selectively, if accessible from the alveolar side. This study combined near infrared fluorescent protein 720 (iRFP720) and firefly luciferase as reporter genes for detection of tumor lesions and transfection efficiency of LPEI polyplexes, after intratracheal microspraying in mice bearing 4T1 triple negative breast cancer lung metastases. Simultaneous flow cytometric analysis of iRFP720 and enhanced green fluorescent protein expression in vitro demonstrated the potential to combine these reporter genes within transfection studies. Polyplex biophysics was characterized by single nanoparticle tracking analysis (NTA) to monitor physical integrity after microspraying in vitro. 4T1 cells were transduced with iRFP720-encoding lentivirus and evaluated by flow cytometry for stable iRFP720 expression. Growth of 4T1-iRFP720 cells was monitored in Balb/c mice by tomographic near infrared imaging, tissue and tumor morphology by computed tomography and magnetic resonance imaging. In 4T1-iRFP720 tumor-bearing mice, intratracheal administration of luciferase-encoding plasmid DNA by LPEI polyplexes resulted in successful tumor transfection, as revealed by bioluminescence imaging.

Electron Bernstein Wave Studies in MST

NASA Astrophysics Data System (ADS)

Seltzman, Andrew; Anderson, Jay; Forest, Cary; Nonn, Paul; Thomas, Mark; Almagri, Abdulgader; Chapman, Brett; Dubois, Ami; Goetz, John; McCollam, Karsten

2015-11-01

The RFP plasma is inaccessible to ECRH, requiring the electron Bernstein wave (EBW) for edge localized heating and current drive. MST is capable of generating RFPs or overdense tokamaks with Bt(0) ~ 0.08-0.14T in which a 5.55 GHz RF source (450kW, 2ms pulse) can heat at fundamental and harmonic EC resonances. The design of a suitable antenna is challenging in the RFP due to a magnetic field geometry that requires a low-field-side launch. The small vacuum gap between the close-fitting conducting shell and plasma leads to substantial antenna-plasma interaction. A minimized port hole size is required to limit error fields. Even so the port hole induced magnetic field perturbation in the antenna near-field that affects the mode conversion process and introduces EC resonances. A 5cm diameter cylindrical antenna centered in 5cm and 11cm diameter portholes is used. A multi-chord time-resolved x-ray detector and GENRAY ray tracing verifies EBW heating at higher harmonics in an MST tokamak with 10-40keV detected x-ray energies. Evidence of RF-induced emission from absorption at higher harmonics (4th / 5th) in low current RFP discharges has been observed. Simultaneous reflected power changes correspond to termination of x-ray emission indicating power limits. Work supported by USDOE.

The SIMPSONS project: An integrated Mars transportation system

NASA Astrophysics Data System (ADS)

Kaplan, Matthew; Carlson, Eric; Bradfute, Sherie; Allen, Kent; Duvergne, Francois; Hernandez, Bert; Le, David; Nguyen, Quan; Thornhill, Brett

In response to the Request for Proposal (RFP) for an integrated transportation system network for an advanced Martian base, Frontier Transportation Systems (FTS) presents the results of the SIMPSONS project (Systems Integration for Mars Planetary Surface Operations Networks). The following topics are included: the project background, vehicle design, future work, conclusions, management status, and cost breakdown. The project focuses solely on the surface-to-surface transportation at an advanced Martian base.

AFTOMS Technology Issues and Alternatives Report

DTIC Science & Technology

1989-12-01

color , resolu- power requirements, physi- tion; memory , processor speed; cal and weather rugged- IAN interfaces, etc,) f,: these ness. display...Telephone and Telegraph 3 CD-I Compact Disk - Interactive CD-ROM Compact Disk-Read Only Memory CGM Computer Graphics Metafile CNWDI Critical Nuclear...Database Management System RFP Request For Proposal 3 RFS Remote File System ROM Read Only Memory 3 S SA-ALC San Antonio Air Logistics Center 3 SAC

Solar Photovoltaic and Liquid Natural Gas Opportunities for Command Naval Region Hawaii

DTIC Science & Technology

2014-12-01

Utilities Commission xii PV Photovoltaic Pwr Power RE Renewable Energy Re-gas Regasification RFP Request For Proposal RMI Rocky... forecasted LS diesel price and the forecasted LNG delivered-to-the- power -plant cost. The forecast for LS diesel by FGE from year 2020–2030 is seen...annual/html/epa_08_01.html Electric Power Research Institute. (July, 2010). Addressing solar photovoltaic operations and maintenance challenges: A

The SIMPSONS project: An integrated Mars transportation system

NASA Technical Reports Server (NTRS)

Kaplan, Matthew; Carlson, Eric; Bradfute, Sherie; Allen, Kent; Duvergne, Francois; Hernandez, Bert; Le, David; Nguyen, Quan; Thornhill, Brett

1992-01-01

In response to the Request for Proposal (RFP) for an integrated transportation system network for an advanced Martian base, Frontier Transportation Systems (FTS) presents the results of the SIMPSONS project (Systems Integration for Mars Planetary Surface Operations Networks). The following topics are included: the project background, vehicle design, future work, conclusions, management status, and cost breakdown. The project focuses solely on the surface-to-surface transportation at an advanced Martian base.

Non-axisymmetric Flows and Transport in the Edge of MST

NASA Astrophysics Data System (ADS)

Miller, Matthew Charles

Magnetic reconnection occurs in plasmas all throughout the universe and is responsible for spectacular and perplexing phenomena. In the Madison Symmetric Torus (MST) reversed field pinch (RFP), reconnection occurs as quasi-periodic bursts of tearing instabilities (saw-teeth), which give rise to a number of processes that affect the RFP's global behavior and confinement. This work examines the structure of turbulent plasma flow in the edge region and its role in affecting momentum and particle transport through the use of several insertable probes and novel ensemble techniques. Very few measurements exist of tearing mode flow structures. The flow structure has now been measured for m = 0 modes and is in good agreement with theoretical expectations for nonlinear resistive MHD calculated for the RFP using DEBS and NIMROD. The flows are predicted and measured to be different than the classical Sweet-Parker picture with symmetric inward flows. The flow fluctuations have a profound effect on momentum transport, which is trans- ported rapidly at the crash. This work advances the understanding of this process by measuring the Reynolds stress associated with turbulent flow. Combined with measurements of the Maxwell stress, a new picture for magnetic self-organization in the RFP via two-fluid physics has emerged. The Reynolds and Maxwell stresses are measured to be an order of magnitude larger than the rate of change in inertia but oppositely directed such that they almost cancel. Two-fluid effects are significant because of the relationship be- tween the Maxwell stress and the Hall dynamo, a term only existing in two-fluid theories. This relationship inextricably couples the momentum dynamics with the current dynamics. Indeed, the parallel momentum profile exhibits a relaxation at the crash akin to the relaxation seen in the parallel current density profile. Tearing modes also drive particle transport. Fluctuation-induced particle flux is resolved through a crash by measuring it directly as < neur>. The flux increases dramatically during a crash and is non-axisymmetric. Between crashes, the transport from tearing is small, which agrees with previous measurements that identified electrostatic transport as dominant at that time.

Tolerance of a knotted near infrared fluorescent protein to random circular permutation

PubMed Central

Pandey, Naresh; Kuypers, Brianna E.; Nassif, Barbara; Thomas, Emily E.; Alnahhas, Razan N.; Segatori, Laura; Silberg, Jonathan J.

2016-01-01

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFP to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified twenty seven circularly permuted iRFP that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants were discovered that initiated translation within the PAS and GAF domains. Circularly permuted iRFP retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a similar quantum yield as iRFP, but exhibited increased resistance to chemical denaturation, suggesting that the observed signal increase arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step towards the creation of near-infrared biosensors with expanded chemical-sensing functions for in vivo imaging. PMID:27304983

Prolonged QRS duration on surface electrocardiogram is associated with left ventricular restrictive filling pattern

PubMed Central

Erdoğan, Turan; Durakoğlugil, Murtaza Emre; Çiçek, Yüksel; Çetin, Mustafa; Duman, Hakan; Şatiroğlu, Ömer; Çelik, Şükrü

2017-01-01

Background Prolonged QRS duration is associated with decreased left ventricular (LV) systolic function. However, the relation between LV restrictive filling pattern (RFP) and QRS duration has not been investigated yet. The purpose of our study was to assess this relationship. Methods We analyzed standard 12-lead surface electrocardiogram (ECG) of 155 consecutive patients. Mitral inflow and septal tissue velocities were obtained using the apical 4-chamber view with pulsed Doppler echocardiography. Patients were divided into 2 groups according to measured deceleration time (DT): restrictive (with DT ≤130 ms) or non-restrictive (with DT >130 ms). Results QRS duration was significantly longer in the restrictive group than in the non-restrictive group (0.101 vs. 0.090 s, p < 0.0001). QRS duration of >0.10 s was highly specific (82.6%), but modestly sensitive (64.7%), for the prediction of LV RFP. Multivariate analyses demonstrated that E/A ratio, peak E, peak A, septal e’, and a’ velocities were significantly associated with RFP. Conclusions Prolonged QRS duration (>0.10 s) obtained from a standard resting 12-lead ECG is associated with LV RFP. However, the relationship of QRS duration with RFP was not independent of echocardiographic parameters. PMID:28932490

Initial results from the rebuilt EXTRAP T2R RFP device

NASA Astrophysics Data System (ADS)

Brunsell, P. R.; Bergsåker, H.; Cecconello, M.; Drake, J. R.; Gravestijn, R. M.; Hedqvist\\ad{2 }, A.; Malmberg, J.-A.

2001-11-01

The EXTRAP T2R thin shell reversed-field pinch (RFP) device has recently resumed operation after a major rebuild including the replacement of the graphite armour with molybdenum limiters, a fourfold increase of the shell time constant, and the replacement of the helical coil used for the toroidal field with a conventional solenoid-type coil. Wall-conditioning using hydrogen glow discharge cleaning was instrumental for successful RFP operation. Carbon was permanently removed from the walls during the first week of operation. The initial results from RFP operation with relatively low plasma currents in the range Ip = 70-100 kA are reported. RFP discharges are sustained for more than three shell times. Significant improvements in plasma parameters are observed, compared to operation before the rebuild. There is a substantial reduction in the carbon impurity level. The electron density behaviour is more shot-to-shot reproducible. The typical density is ne = 0.5-1×1019 m-3. Monitors of Hα line radiation indicate that the plasma wall interaction is more toroidally symmetric and that there is less transient gas release from the wall. The minimum loop voltage is in the range Vt = 28-35 V, corresponding to a reduction by a factor of two to three compared to the value before the rebuild.

Validation of MHD Models using MST RFP Plasmas

NASA Astrophysics Data System (ADS)

Jacobson, C. M.; Chapman, B. E.; den Hartog, D. J.; McCollam, K. J.; Sarff, J. S.; Sovinec, C. R.

2017-10-01

Rigorous validation of computational models used in fusion energy sciences over a large parameter space and across multiple magnetic configurations can increase confidence in their ability to predict the performance of future devices. MST is a well diagnosed reversed-field pinch (RFP) capable of operation with plasma current ranging from 60 kA to 500 kA. The resulting Lundquist number S, a key parameter in resistive magnetohydrodynamics (MHD), ranges from 4 ×104 to 8 ×106 for standard RFP plasmas and provides substantial overlap with MHD RFP simulations. MST RFP plasmas are simulated using both DEBS, a nonlinear single-fluid visco-resistive MHD code, and NIMROD, a nonlinear extended MHD code, with S ranging from 104 to 105 for single-fluid runs, and the magnetic Prandtl number Pm = 1 . Validation metric comparisons are presented, focusing on how normalized magnetic fluctuations at the edge b scale with S. Preliminary results for the dominant n = 6 mode are b S - 0 . 20 +/- 0 . 02 for single-fluid NIMROD, b S - 0 . 25 +/- 0 . 05 for DEBS, and b S - 0 . 20 +/- 0 . 02 for experimental measurements, however there is a significant discrepancy in mode amplitudes. Preliminary two-fluid NIMROD results are also presented. Work supported by US DOE.

Posterior lateral hypothalamic axon terminals are in contact with trigeminal premotor neurons in the parvicellular reticular formation of the rat medulla oblongata.

PubMed

Notsu, Kazuki; Tsumori, Toshiko; Yokota, Shigefumi; Sekine, Joji; Yasui, Yukihiko

2008-12-09

This study was performed to understand the anatomical substrates of hypothalamic modulation of jaw movements. After cholera toxin B subunit (CTb) injection into the parvicellular reticular formation (RFp) of the rat medulla oblongata, where many trigeminal premotor neurons have been known to exist, numerous CTb-labeled neurons were found in the posterior lateral hypothalamus (PLH) bilaterally with a clear-cut ipsilateral dominance. After ipsilateral injections of biotinylated dextran amine (BDA) into the PLH and CTb into the motor trigeminal nucleus (Vm), the prominent distribution of BDA-labeled axon terminals around CTb-labeled neurons was found in the RFp region just ventral to the nucleus of the solitary tract and medial to the spinal trigeminal nucleus ipsilateral to the injection sites. Within the neuropil of the RFp, BDA-labeled axon terminals made an asymmetrical synaptic contact predominantly with dendrites and additionally with somata of the RFp neurons, some of which were labeled with CTb. It was further revealed that these BDA-labeled axon terminals were immunoreactive for vesicular glutamate transporter 2. The present data suggest that the PLH plays an important role in the control of jaw movements by exerting its glutamatergic excitatory action upon RFp neurons presynaptic to trigeminal motoneurons.

Anomalous transport theory for the reversed field pinch

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

Terry, P.W.; Hegna, C.C; Sovinec, C.R.

1996-09-01

Physically motivated transport models with predictive capabilities and significance beyond the reversed field pinch (RFP) are presented. It is shown that the ambipolar constrained electron heat loss observed in MST can be quantitatively modeled by taking account of the clumping in parallel streaming electrons and the resultant self-consistent interaction with collective modes; that the discrete dynamo process is a relaxation oscillation whose dependence on the tearing instability and profile relaxation physics leads to amplitude and period scaling predictions consistent with experiment; that the Lundquist number scaling in relaxed plasmas driven by magnetic turbulence has a weak S{sup {minus}1/4} scaling; andmore » that radial E{times}B shear flow can lead to large reductions in the edge particle flux with little change in the heat flux, as observed in the RFP and tokamak. 24 refs.« less

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

PubMed Central

Shen, Yi; Lai, Tiffany; Campbell, Robert E.

2015-01-01

Abstract. The inherent advantages of red-shifted fluorescent proteins and fluorescent protein-based biosensors for the study of signaling processes in neurons and other tissues have motivated the development of a plethora of new tools. Relative to green fluorescent proteins (GFPs) and other blue-shifted alternatives, red fluorescent proteins (RFPs) provide the inherent advantages of lower phototoxicity, lower autofluorescence, and deeper tissue penetration associated with longer wavelength excitation light. All other factors being the same, the multiple benefits of using RFPs make these tools seemingly ideal candidates for use in neurons and, ultimately, the brain. However, for many applications, the practical utility of RFPs still falls short of the preferred GFPs. We present an overview of RFPs and RFP-based biosensors, with an emphasis on their reported applications in neuroscience. PMID:26158012

CXCR2 inverse agonism detected by arrestin redistribution.

PubMed

Kredel, Simone; Wolff, Michael; Wiedenmann, Jörg; Moepps, Barbara; Nienhaus, G Ulrich; Gierschik, Peter; Kistler, Barbara; Heilker, Ralf

2009-10-01

To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria-derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a "tandem" (td) construct for pseudo-monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td-RFP611 (Arr-td-RFP611) or enhanced green fluorescent protein (EGFP; Arr-EGFP), were found to colocalize with internalized fluorescently labeled Gro-alpha a few minutes after Gro-alpha addition. Intriguingly, however, Arr-td-RFP611 and Arr-EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2-activating ligand. Under these conditions, Arr-td-RFP611 showed a largely homogeneous cytosolic distribution, whereas Arr-EGFP segregated, to a large degree, into granular spots. These observations indicate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr-EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC(50) value similar to that observed for Arr-EGFP redistribution. Thus, the redistribution assay, when based on Arr-EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr-td-RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism.

Allelism analysis of BrRfp locus in different restorer lines and map-based cloning of a fertility restorer gene, BrRfp1, for pol CMS in Chinese cabbage (Brassica rapa L.).

PubMed

Zhang, Huamin; Wu, Junqing; Dai, Zihui; Qin, Meiling; Hao, Lingyu; Ren, Yanjing; Li, Qingfei; Zhang, Lugang

2017-03-01

In Chinese cabbage, there are two Rf loci for pol CMS and one of them was mapped to a 12.6-kb region containing a potential candidate gene encoding PPR protein. In Chinese cabbage (Brassica rapa), polima cytoplasmic male sterility (pol CMS) is an important CMS type and is widely used for hybrid breeding. By extensive test crossing in Chinese cabbage, four restorer lines (92s105, 01s325, 00s109, and 88s148) for pol CMS were screened. By analyzing the allelism of the four restorer lines, it was found that 92s105, 01s325, and 00s109 had the same "restorers of fertility" (Rf) locus (designated as BrRfp1), but 88s148 had a different Rf locus (designated as BrRfp2). For fine mapping the BrRfp1 locus of 92s105, a BC 1 F 1 population with 487 individuals and a BC 1 F 2 population with 2485 individuals were successively constructed. Using simple sequence repeat (SSR) markers developed from Brassica rapa reference genome and InDel markers derived from whole-genome resequencing data of 94c9 and 92s105, BrRfp1 was mapped to a 12.6-kb region containing a potential candidate gene encoding pentatricopeptide repeat-containing protein. Based on the nucleotide polymorphisms of the candidate gene sequence between the restoring and nonrestoring alleles, a co-segregating marker SC718 was developed, which would be helpful for hybrid breeding by marker-assisted screening and for detecting new restorer lines.

R/L, a double reporter mouse line that expresses luciferase gene upon Cre-mediated excision, followed by inactivation of mRFP expression.

PubMed

Jia, Junshuang; Lin, Xiaolin; Lin, Xia; Lin, Taoyan; Chen, Bangzhu; Hao, Weichao; Cheng, Yushuang; Liu, Yu; Dian, Meijuan; Yao, Kaitai; Xiao, Dong; Gu, Weiwang

2016-10-01

The Cre/loxP system has become an important tool for the conditional gene knockout and conditional gene expression in genetically engineered mice. The applications of this system depend on transgenic reporter mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. To develop a sensitive assay for monitoring Cre-mediated DNA excisions in mice, we generated Cre-mediated excision reporter mice, designated R/L mice (R/L: mRFP(monomeric red fluorescent protein)/luciferase), express mRFP throughout embryonic development and adult stages, while Cre-mediated excision deletes a loxP-flanked mRFP reporter gene and STOP sequence, thereby activating the expression of the second reporter gene luciferase, as assayed by in vivo and ex vivo bioluminescence imaging. After germ line deletion of the floxed mRFP and STOP sequence in R/L mice by EIIa-Cre mice, the resulting luciferase transgenic mice in which the loxP-mRFP-STOP-loxP cassette is excised from all cells express luciferase in all tissues and organs examined. The expression of luciferase transgene was activated in liver of RL/Alb-Cre double transgenic mice and in brain of RL/Nestin-Cre double transgenic mice when R/L reporter mice were mated with Alb-Cre mice and Nestin-Cre mice, respectively. Our findings reveal that the double reporter R/L mouse line is able to indicate the occurrence of Cre-mediated excision from early embryonic to adult lineages. Taken together, these findings demonstrate that the R/L mice serve as a sensitive reporter for Cre-mediated DNA excision both in living animals and in organs, tissues, and cells following necropsy.

75 FR 31709 - Approval and Promulgation of Air Quality Implementation Plans; Maryland; Reasonable Further...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-04

... Emission Inventory, Contingency Measures, Reasonably Available Control Measures, and Transportation... Implementation Plan (SIP) to meet the 2002 base year emissions inventory, the reasonable further progress (RFP) plan, RFP contingency measure, and reasonably available control measure (RACM) requirements of the...

Space Station reference configuration description

NASA Technical Reports Server (NTRS)

1984-01-01

The data generated by the Space Station Program Skunk Works over a period of 4 months which supports the definition of a Space Station reference configuration is documented. The data were generated to meet these objectives: (1) provide a focal point for the definition and assessment of program requirements; (2) establish a basis for estimating program cost; and (3) define a reference configuration in sufficient detail to allow its inclusion in the definition phase Request for Proposal (RFP).

The Leishmania donovani histidine acid ecto-phosphatase LdMAcP: insight into its structure and function.

PubMed

Papadaki, Amalia; Politou, Anastasia S; Smirlis, Despina; Kotini, Maria P; Kourou, Konstadina; Papamarcaki, Thomais; Boleti, Haralabia

2015-05-01

Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP-mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP-His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP-mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence.

The development of the conditionally replication-competent adenovirus: replacement of E4 orf1-4 region by exogenous gene.

PubMed

Nam, Jae-Kook; Lee, Mi-Hyang; Seo, Hae-Hyun; Kim, Seok-Ki; Lee, Kang-Huyn; Kim, In-Hoo; Lee, Sang-Jin

2010-05-01

Tumor or tissue specific replicative adenovirus armed with a therapeutic gene has shown a promising anti-cancer therapeutic modality. However, because the genomic packaging capacity is constrained, only a few places inside it are available for transgene insertion. In the present study, we introduce a novel strategy utilizing the early E4 region for the insertion of therapeutic gene(s). We constructed the conditionally replication-competent adenovirus (CRAd), Ad5E4(mRFP) by: (i) replacing the E4/E1a promoter by the prostate-specific enhancer element; (ii) inserting mRFP inside the E4orf1-4 deletion region; and (iii) sub-cloning enhanced green fluorescent protein controlled by cytomegalovirus promoter in the left end of the viral genome. Subsequently, we evaluated its replication abilities and killing activities in vitro, as well as its in vivo anti-tumor efficacy in CWR22rv xenografts. When infected with Ad5E4(mRFP), the number and intensity of the mRFP gene products increased in a prostate cancer cell-specific manner as designed, suggesting that the mRFP gene and E4orfs other than E4orf1-4 were well synthesized from one transcript via alternative splicing as the recombinant adenovirus replicated. As expected from the confirmed virus replication capability, Ad5E4(mRFP) induced cell lysis as potent as the wild-type adenovirus and effectively suppressed tumor growth when tested in the CWR22rv xenografts in nude mice. Furthermore, Ad5E4(endo/angio) harboring an endostatin-angiostatin gene in E4orf1-4 was able to enhance CRAd by replacing mRFP with a therapeutic gene. The approach employed in the present study for the insertion of a therapeutic transgene in CRAd should facilitate the construction of CRAd containing multiple therapeutic genes in the viral genome that may have the potential to serve as highly potent cancer therapeutic reagents. Copyright (c) 2010 John Wiley & Sons, Ltd.

77 FR 5032 - National Cancer Institute; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-01

... Initiatives; RFA and RFP Concept Reviews; and Scientific Presentations. Place: National Institutes of Health... Group(s); and Budget Presentations; Reports of Special Initiatives; RFA and RFP Concept Reviews; and Scientific Presentations. Place: National Institutes of Health, Building 31, 31 Center Drive, 6th Floor, Conf...

Survival, growth performance and immune capacity of the juvenile lined seahorse Hippocampus erectus fed with rifampicin-treated copepods.

PubMed

Lin, Tingting; Wang, Changbo; Liu, Xin; Gao, Fen; Xiao, Dongxue; Zhang, Dong; Zhu, Xialian

2017-06-19

Gastrointestinal disease is one of the most serious diseases in cultured seahorse juveniles. Treatment with antimicrobials of live food (i.e. copepods and Artemia) that is used to feed the juveniles may be a promising measure to alleviate the occurrence of gastrointestinal disease. However, relevant investigations are rare. In the present study, we first investigated the antimicrobial efficacies on bacteria within copepods that were treated with 4 antimicrobials, including 3 antibiotics (i.e. enrofloxacin hydrochloride, oxytetracycline and rifampicin [RFP]) that are approved for use in aquaculture and 1 disinfectant (i.e. povidone iodine). We then assessed the effects of copepods treated with the antimicrobial that had the best antimicrobial efficacy on survival, growth performance and immune capacity of juvenile lined seahorses Hippocampus erectus. The results showed that RFP had the best antimicrobial efficacy on both Pseudoalteromonas spp. and Vibrio spp., 2 dominant bacteria with potential pathogenicity within the copepods; the proper concentration of RFP was 6 mg l-1. Moreover, H. erectus juveniles fed with RFP-treated copepods demonstrated an improved survivorship and immune capacity and had a lower abundance of pathogenic bacteria within their gastrointestinal tracts compared to juveniles fed with untreated copepods. These results suggest that treating live food with RFP is a potential measure for reducing the incidence of gastrointestinal disease in seahorse juveniles.

48 CFR 873.110 - Solicitation provisions.

Code of Federal Regulations, 2010 CFR

2010-10-01

... DEPARTMENT SUPPLEMENTARY REGULATIONS SIMPLIFIED ACQUISITION PROCEDURES FOR HEALTH-CARE RESOURCES 873.110... information, as described in 873.112, is to be used to select a contractor under an RFQ or RFP for health-care... officer may insert the provision at 852.273-73, Evaluation—health-care resources, in the RFQ or RFP in...

48 CFR 873.110 - Solicitation provisions.

Code of Federal Regulations, 2011 CFR

2011-10-01

... DEPARTMENT SUPPLEMENTARY REGULATIONS SIMPLIFIED ACQUISITION PROCEDURES FOR HEALTH-CARE RESOURCES 873.110... information, as described in 873.112, is to be used to select a contractor under an RFQ or RFP for health-care... officer may insert the provision at 852.273-73, Evaluation—health-care resources, in the RFQ or RFP in...

7 CFR 1783.15 - What are the terms of RFP loans?

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 12 2011-01-01 2011-01-01 false What are the terms of RFP loans? 1783.15 Section 1783.15 Agriculture Regulations of the Department of Agriculture (Continued) RURAL UTILITIES SERVICE, DEPARTMENT OF AGRICULTURE (CONTINUED) REVOLVING FUNDS FOR FINANCING WATER AND WASTEWATER PROJECTS (REVOLVING...

7 CFR 1783.15 - What are the terms of RFP loans?

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 12 2014-01-01 2013-01-01 true What are the terms of RFP loans? 1783.15 Section 1783.15 Agriculture Regulations of the Department of Agriculture (Continued) RURAL UTILITIES SERVICE, DEPARTMENT OF AGRICULTURE (CONTINUED) REVOLVING FUNDS FOR FINANCING WATER AND WASTEWATER PROJECTS (REVOLVING...

7 CFR 1783.15 - What are the terms of RFP loans?

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 12 2012-01-01 2012-01-01 false What are the terms of RFP loans? 1783.15 Section 1783.15 Agriculture Regulations of the Department of Agriculture (Continued) RURAL UTILITIES SERVICE, DEPARTMENT OF AGRICULTURE (CONTINUED) REVOLVING FUNDS FOR FINANCING WATER AND WASTEWATER PROJECTS (REVOLVING...

7 CFR 1783.15 - What are the terms of RFP loans?

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 12 2013-01-01 2013-01-01 false What are the terms of RFP loans? 1783.15 Section 1783.15 Agriculture Regulations of the Department of Agriculture (Continued) RURAL UTILITIES SERVICE, DEPARTMENT OF AGRICULTURE (CONTINUED) REVOLVING FUNDS FOR FINANCING WATER AND WASTEWATER PROJECTS (REVOLVING...

78 FR 41901 - Determination of Attainment for the West Central Pinal Nonattainment Area for the 2006 Fine...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-12

... reasonably available control measures (RACM), a reasonable further progress (RFP) plan, and contingency... for implementation of reasonably available control measures (RACM), reasonable further progress (RFP..., Arizona Department of Environmental Quality, requested that EPA determine whether the West Central Pinal...

Preparing for Science at Sea - a Chief Scientists Training Cruise on Board the RV Sikuliaq

NASA Astrophysics Data System (ADS)

Coakley, B.; Pockalny, R. A.

2017-12-01

As part of their education, marine geology and geophysics students spend time at sea, collecting, processing and interpreting data to earn their degrees. While this is a critical component of their preparation, it is an incomplete introduction to the process of doing science at sea. Most students are unfamiliar with the proposal process. Many students spend their time at sea performing assigned tasks without responsibility or participation in cruise planning and execution. In December 2016, we conducted a two-week-long, NSF-funded "Chief Scientist Training Cruise" aboard the R/V Sikuliaq designed to complete their introduction to seagoing science by giving the students the opportunity to plan and execute surveys based hypotheses they formulated. The educational process began with applicants responding to a request for proposals (RFP), which provided a framework for the scientific potential of the cruise. This process continued training through two days of workshops and presentations at the Hawai'i Institute of Geophysics. The students used existing data to define hypotheses, plan surveys, and collect/analyze data to test their hypothesis. The survey design was subject to the time constraints imposed by the ship schedule and the physical constraints imposed by the ship's equipment. The training and sea time made it possible to address all of steps of the scientific process, including proposal writing. Once underway, the combination of conducting the planned surveys and attending daily presentations helped familiarize the students with at-sea operations, the equipment on board the RV Sikuliaq, and the process of writing proposals to NSF for sea-going science. Questionnaires conducted prior to the cruise and in the final days before arriving in port document the success of this training program for developing the abilities and confidence in identifying significant scientific problems, preparing proposals to secure funding, and planning and directing ship surveys.

Regional flood probabilities

USGS Publications Warehouse

Troutman, Brent M.; Karlinger, Michael R.

2003-01-01

The T‐year annual maximum flood at a site is defined to be that streamflow, that has probability 1/T of being exceeded in any given year, and for a group of sites the corresponding regional flood probability (RFP) is the probability that at least one site will experience a T‐year flood in any given year. The RFP depends on the number of sites of interest and on the spatial correlation of flows among the sites. We present a Monte Carlo method for obtaining the RFP and demonstrate that spatial correlation estimates used in this method may be obtained with rank transformed data and therefore that knowledge of the at‐site peak flow distribution is not necessary. We examine the extent to which the estimates depend on specification of a parametric form for the spatial correlation function, which is known to be nonstationary for peak flows. It is shown in a simulation study that use of a stationary correlation function to compute RFPs yields satisfactory estimates for certain nonstationary processes. Application of asymptotic extreme value theory is examined, and a methodology for separating channel network and rainfall effects on RFPs is suggested. A case study is presented using peak flow data from the state of Washington. For 193 sites in the Puget Sound region it is estimated that a 100‐year flood will occur on the average every 4.5 years.

Equipment evaluation for low density polyethylene encapsulated nitrate salt waste at the Rocky Flats Plant

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

Yamada, W.I.; Faucette, A.M.; Jantzen, R.C.

1993-08-30

Mixed wastes at the Rocky Flats Plant (RFP) are subject to regulation by the Resource Conservation and Recovery Act (RCRA). Polymer solidification is being developed as a final treatment technology for several of these mixed wastes, including nitrate salts. Encapsulation nitrate salts with low density polyethylene (LDPE) has been the preliminary focus of the RFP polymer solidification effort. Literature reviews, industry surveys, and lab-scale and pilot-scale tests have been conducted to evaluate several options for encapsulating nitrate salts with LDPE. Most of the effort has focused on identifying compatible drying and extrusion technologies. Other processing options, specifically meltration and non-heatedmore » compounding machines, were also investigated. The best approach appears to be pretreatment of the nitrate salt waste brine in either a vertical or horizontal thin film evaporator followed by compounding of the dried waste with LDPE in an intermeshing, co-rotating, twin-screw extruder. Additional pilot-scale tests planned for the fall of 1993 should further support this recommendation. Preliminary evaluation work indicates that meltration is not possible at atmospheric pressure with the LDPE (Chevron PE-1409) provided by RFP. However, meltration should be possible at atmospheric pressure using another LDPE formulation with altered physical and rheological properties: Lower molecular weight and lower viscosity (Epoline C-15). Contract modifications are now in process to allow a follow-on pilot scale demonstration. Questions regarding changed safety and physical properties of the resultant LDPE waste form due to use of the Epoline C-15 will be addressed. No additional work with non-heated mixer compounder machines is planned at this time.« less

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines

PubMed Central

Vuong, Helen E.; de Sevilla Müller, Luis Pérez; Hardi, Claudia N.; McMahon, Douglas G.; Brecha, Nicholas C.

2015-01-01

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) line with three catecholamine-related Cre recombinase lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ~6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium somal diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines were generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing. PMID:26335381

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines.

PubMed

Vuong, H E; Pérez de Sevilla Müller, L; Hardi, C N; McMahon, D G; Brecha, N C

2015-10-29

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing. Published by Elsevier Ltd.

76 FR 70078 - Approval and Promulgation of Implementation Plans and Designation of Areas for Air Quality...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-10

... reasonable further progress (RFP), RACM, and contingency measures under section 172(c)(9). The General... EPA's view of applicable requirements for purposes of evaluating redesignation requests when an area... subject to an RFP requirement, the requirement to submit the section 172(c)(9) contingency measures is not...

Numerical studies of the reversed-field pinch at high aspect ratio

NASA Astrophysics Data System (ADS)

Sätherblom, H.-E.; Drake, J. R.

1998-10-01

The reversed field pinch (RFP) configuration at an aspect ratio of 8.8 is studied numerically by means of the three-dimensional magnetohydrodynamic code DEBS [D. D. Schnack et al., J. Comput. Phys. 70, 330 (1987)]. This aspect ratio is equal to that of the Extrap T1 experiment [S. Mazur et al., Nucl. Fusion 34, 427 (1994)]. A numerical study of a RFP with this level of aspect ratio requires extensive computer achievements and has hitherto not been performed. The results are compared with previous studies [Y. L. Ho et al., Phys. Plasmas 2, 3407 (1995)] of lower aspect ratio RFP configurations. In particular, an evaluation of the extrapolation to the aspect ratio of 8.8 made in this previous study shows that the extrapolation of the spectral spread, as well as most of the other findings, are confirmed. An important exception, however, is the magnetic diffusion coefficient, which is found to decrease with aspect ratio. Furthermore, an aspect ratio dependence of the magnetic energy and of the helicity of the RFP is found.

Modeling of Resistive Wall Modes in Tokamak and Reversed Field Pinch Configurations of KTX

NASA Astrophysics Data System (ADS)

Han, Rui; Zhu, Ping; Bai, Wei; Lan, Tao; Liu, Wandong

2016-10-01

Resistive wall mode is believed to be one of the leading causes for macroscopic degradation of plasma confinement in tokamaks and reversed field pinches (RFP). In this study, we evaluate the linear RWM instability of Keda Torus eXperiment (KTX) in both tokamak and RFP configurations. For the tokamak configuration, the extended MHD code NIMROD is employed for calculating the dependence of the RWM growth rate on the position and conductivity of the vacuum wall for a model tokamak equilibrium of KTX in the large aspect-ratio approximation. For the RFP configuration, the standard formulation of dispersion relation for RWM based on the MHD energy principle has been evaluated for a cylindrical α- Θ model of KTX plasma equilibrium, in an effort to investigate the effects of thin wall on the RWM in KTX. Full MHD calculations of RWM in the RFP configuration of KTX using the NIMROD code are also being developed. Supported by National Magnetic Confinement Fusion Science Program of China Grant Nos. 2014GB124002, 2015GB101004, 2011GB106000, and 2011GB106003.

Breeding patterns of the JE vector Culex gelidus and its insect predators in rice cultivation areas of northern peninsular Malaysia.

PubMed

Abu Hassan, A; Hamady, D; Tomomitsu, S; Michael, B; Jameel S L, A S

2010-12-01

Japanese encephalitis (JE) virus activity is an important cause of viral encephalitis in Southeast Asia. In Malaysia, JEV activity has been first detected in Culex gelidus in 1976. Since then, no study has fully addressed the seasonal dynamics of this mosquito. As irrigated rice production expands, the incidence of JEV vectors, particularly Cx. gelidus is expected to increase. We surveyed Penang Island to determine the breeding patterns of Cx. gelidus and their potential insect predators, in relation to habitat/niche and rice growing period. Six rice fields proper (RFP) and related drainage canals (DC) were visited through three cultivation cycles (CCs) over 17 months. Weekly visits were performed to each of the 36 sites and mosquito larvae and aquatic insects were sampled from RFP and DCs using dippers. Culex gelidus was abundant in RFP and almost absent in DCs. Its densities usually were high during the first and 3rd CC and when the RFs were in Fp, Pp and Gp. In DCs, the mosquito was abundant during Mp, e.g., 2nd CC. Predators, especially those belonging to the families Corixidae, Coenagrionidae and Dytiscidae, were more present in RFP. Predator numbers usually were high during the first CC; in some cases predator abundance peaked during other CCs, e.g., corixids and dysticids. In RFP, neither corixids nor coenagrionids showed any positive correlation with densities of Cx. gelidus. However, dytiscids' population peaked when the mosquito densities were on the rise. These observations suggest that Cx. gelidus is active during the period of rice cultivation. Operational vector control through bio-control or with insecticides near the end of the rice cultivation season in RFP may prove beneficial in reducing the density of Cx. gelidus, but also the amount of bio-agent or insecticide applied on riceland.

Design study to simulate the development of a commercial transportation system

NASA Technical Reports Server (NTRS)

1991-01-01

Seven teams of senior-level Aerospace Engineering undergraduates were given a Request for Proposals (RFP) for a design concept of a remotely piloted vehicle (RPV). The RPV designs were intended to simulate commercial transport aircraft within the model of 'Aeroworld.' The Aeroworld model was developed so that the RPV designs would be subject to many of the engineering problems and tradeoffs that dominate real-world commercial air transport designs, such as profitability, fuel efficiency, range vs. payload capabilities, and ease of production and maintenance. As part of the proposal, each team was required to construct a prototype and validate its design with a flight demonstration.

Work Breakdown Structure (WBS) and WBS dictionary. [advanced x ray astrophysics facility (AXAF)

NASA Technical Reports Server (NTRS)

Cocuzzo, F.

1994-01-01

This document is intended to provide the framework for defining all work to be accomplished under the proposed contract for the mission support program as defined by Section J-1 Statement of Work (SOW) of the RFP No.8-1-3-TA-50030 dated 11/2/93. The time period of performance of this contract will be from 3/1/94 through to the end of fiscal 1999. In addition to providing the framework for the integration of the cost, schedule, and manpower planning necessary to accomplish this work it establishes the association among the WBS elements, the SOW, and the Technical Proposal.

SOFIA Science Working Group

NASA Technical Reports Server (NTRS)

Zmuldzinas, J.

1997-01-01

The SOFIA Science Working Group was established to help develop the plans and specifications for the next-generation airborne observatory ("SOFIA"), which is now under development. The P.I. has developed several astronomical instruments for the Kuiper Airborne Observatory, NASA's previous airborne astronomy platform (which was decommisioned in 1995 in preparation for SOFIA). SOFIA, which will be a 747 SP aircraft carrying a 2.7 meter diameter telescope, is a joint project sponsored by NASA and DLR (the German space agency), and is now under development by a consortium including Universities Space Research Association (USRA), Raytheon, Sterling Software, and United Airlines. Rather than develop the SOFIA observatory in-house, NASA decided to privatize the project by issuing a Request for Proposals (RFP). The respondents to this RFP were consortia of private organizations which together had the required facilities and expertise to be able to carry out the project; the winner was the group led by USRA. One of the main roles of the SSWG was to help develop the technical specifications for the SOFIA observatory. In particular, the SSWG provided advice to NASA on the specifications that were written into the RFP, particularly those which had an important impact on the scientific productivity of the observatory. These specifications were discussed at the meetings of the SSWG, which were held primarily at NASA/Ames (in California) and at NASA Headquarters (in Washington DC). Apart from these meetings, members of the SSWG were expected to perform more detailed analyses of the impact of certain parameters and specifications on the performance of astronomical instruments. The SSWG ended its activities with the selection of the USRA team in January 1997.

Strengthening Refugee Families: Designing Programs for Refugee and Other Families in Need.

ERIC Educational Resources Information Center

Scheinfeld, Daniel; Wallach, Lorraine B.; Langendorf, Trudi

This manual details the principles and practices of the Refugee Families Program (RFP) in Chicago and the issues that have arisen in connecting these families to the educational, medical, welfare, and other institutions that can provide the family-strengthening support they need. The approach of RFP is to work with families through their…

Effects of the resistivity profile on the formation of a reversed configuration and single helicity states in compressible simulations of the reversed-field pinch

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

Onofri, M.; Malara, F.

2013-10-15

Compressible magnetohydrodynamics simulations of the reversed-field pinch (RFP) are presented. Previous simulations of the RFP, including density and pressure evolution, showed that a stationary state with a reversed toroidal magnetic field could not be obtained, contrary to the results produced with numerical codes neglecting density and pressure dynamics. The simulations described in the present paper show that including density and pressure evolution, a stationary RFP configuration can be obtained if the resistivity has a radial profile steeply increasing close to the wall. Such resistivity profile is more realistic than a uniform resistivity, since the temperature at the wall is lowermore » than in the plasma core.« less

Sabot high speed interceptor AE 4273 aircraft design

NASA Technical Reports Server (NTRS)

Dober, Dave; Al-Hashel, Waleed; Baldocchi, Bob; Berg, Tim; Lindsay, Curt; Mcatee, Aaron; Sergent, Dan; Dunbrack, Harry

1992-01-01

Today's carrier based deck launched intercept (DLI) mission is a vital one that is aimed at protecting the carrier battle group and detering potential adversaries. The assets deployed on our carrier decks are able to complete this mission but with very limited range. The waverider concept has great potential to increase the range of this carrier based mission. As a result, a request for proposals (RFP) was developed which contains design requirements for an aircraft that can complete this mission through the utilization of waverider technology.

Corporate Memory and Spacecraft Course Development

NASA Technical Reports Server (NTRS)

1999-01-01

The Florida Space Grant Consortium (FSGC) entered into a cooperative agreement with NASA's Kennedy Space Center (KSC) to manage two programs, (1) Corporate Memory and (2) Spacecraft Course Development. The FSGC, with its central office at the University of Florida, administered these programs. In conjunction with KSC, the FGSC was responsible for the release of the Request for Proposals (RFP's) and its distribution to all its affiliates. FSGC received the applications, forwarded them to KSC for evaluations, and awarded grants to the institutions chosen by KSC.

Development of LHX (Light Helicopter Family) MANPRINT (Manpower and Personnel Integration) Issues

DTIC Science & Technology

1988-09-01

for Proposal (RFP) plus references to pertinent studies and analyses where available. In addition, cmments on the S MP and 13 reccmneded new critical...A - 2a *.-0La0La -cm La -a-C-ama aa cam-c 2-3-0 ’O3- 33 3J4m ~J- -, a a -J - a La a - a 30 am aCaLac mccc-a- aLacta r2LaLa ractaB "ooo au- ~c0--J

Non-invasive imaging of prostate cancer progression in nude mice using iRFP gene reporter

NASA Astrophysics Data System (ADS)

Zhu, Banghe; Wu, Grace; Robinson, Holly; Wilganowski, Nathaniel; Sevick-Muraca, Eva M.

2013-03-01

Prostate cancer (PCa) is the second most common cancer in US men. Metastasis is the final step of tumor progression and remains the primary cause of PCa death. Hence preclinical, orthotopic models of PCa metastasis are necessary to develop new therapeutics against metastatic disease. Yet unlike irrelevant subcutaneous tumor models, the deployment of orthotopic models of cancer metastasis in drug research and development is limited by the inability to longitudinally monitor cancer progression/regression in response to administration of experimental pharmaceuticals. Recently, a nearinfrared fluorescent protein (iRFP) was created for deeper imaging [1]. Imaging prostate tumor growth and lymph node metastasis in nude mice therefore becomes possible using this new fluorescent gene reporter. In this study, we first developed an intensified CCD (ICCD)-based iRFP fluorescence imaging device. Then human PCa PC3 cell lines expressing iRFP gene reporter were orthotopically implanted in male Nu/Nu mice at 8-10 weeks old. After 6-10 weeks, in vivo, in situ and ex vivo fluorescence imaging was performed. In vivo iRFP fluorescence imaging showed that the detected fluorescence concentrated at the prostate and became stronger over time, indicating the growth of implanted PCa. Fluorescence was non-invasively detected at locations of prostate-draining lymph nodes as early as 5 weeks post implantation, indicating the metastasis to lymph nodes. In situ and ex vivo fluorescence imaging demonstrated that the detected signals from PCa and lymph nodes were correlated with cancer positive status of tissues as assessed through standard pathology.

Imaging Nuclear-Cytoplasmic Dynamics in Primary and Metastatic Colon Cancer in Nude Mice.

PubMed

Hasegawa, Kosuke; Suetsugu, Atsushi; Nakamura, Miki; Matsumoto, Takuro; Aoki, Hitomi; Kunisada, Takahiro; Bouvet, Michael; Shimizu, Masahito; Hoffman, Robert M

2016-05-01

Colon cancer frequently results in metastasis to the liver, where it becomes the main cause of death. However, the cell cycle in primary tumors and metastases is poorly understood. We developed a mouse model of liver metastasis using the human colon cancer cell line HCT-116, which expresses green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP). HCT-116 GFP-RFP cells were injected into the spleen of nu/nu nude mice. HCT-116-GFP-RFP cells subsequently formed primary tumors in the spleen, as well as metastatic colonies in the liver and retroperitoneum by 28 days after cell transplantation. Using an Olympus FV1000 confocal microscope, it was possible to clearly image mitosis of the dual-colored colon cancer cells in the primary tumor as well as liver and other metastases. Multi-nucleate cancer cells, in addition to mono-nucleate cancer cells and their mitosis, were observed in the primary tumor and metastasis. Multi-nucleate HCT-116-GFP-RFP cells were also observed after culture of the primary and metastatic tumors. A similar ratio of mono-nucleate, multi-nucleate, and mitotic cells grew from the primary and metastatic tumors in culture, suggesting similarity of the nuclear-cytoplasmic dynamics of primary and metastatic cancer cells, further emphasizing the stochastic nature of metastasis. Our results demonstrate a similar heterogeneity of nuclear-cytoplasmic dynamics within primary tumors and metastases, which may be an important factor in the stochastic nature of metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

40 CFR 51.910 - What requirements for reasonable further progress (RFP) under sections 172(c)(2) and 182 apply...

Code of Federal Regulations, 2010 CFR

2010-07-01

...-hour ozone NAAQS? 51.910 Section 51.910 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Provisions for Implementation of 8-hour Ozone National Ambient Air Quality Standard § 51.910 What... nonattainment for the 8-hour ozone NAAQS? (a) What are the general requirements for RFP for an area classified...

40 CFR 51.910 - What requirements for reasonable further progress (RFP) under sections 172(c)(2) and 182 apply...

Code of Federal Regulations, 2011 CFR

2011-07-01

...-hour ozone NAAQS? 51.910 Section 51.910 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Provisions for Implementation of 8-hour Ozone National Ambient Air Quality Standard § 51.910 What... nonattainment for the 8-hour ozone NAAQS? (a) What are the general requirements for RFP for an area classified...

40 CFR 51.910 - What requirements for reasonable further progress (RFP) under sections 172(c)(2) and 182 apply...

Code of Federal Regulations, 2013 CFR

2013-07-01

...-hour ozone NAAQS? 51.910 Section 51.910 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Provisions for Implementation of 8-hour Ozone National Ambient Air Quality Standard § 51.910 What... nonattainment for the 8-hour ozone NAAQS? (a) What are the general requirements for RFP for an area classified...

40 CFR 51.910 - What requirements for reasonable further progress (RFP) under sections 172(c)(2) and 182 apply...

Code of Federal Regulations, 2012 CFR

2012-07-01

...-hour ozone NAAQS? 51.910 Section 51.910 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Provisions for Implementation of 8-hour Ozone National Ambient Air Quality Standard § 51.910 What... nonattainment for the 8-hour ozone NAAQS? (a) What are the general requirements for RFP for an area classified...

40 CFR 51.910 - What requirements for reasonable further progress (RFP) under sections 172(c)(2) and 182 apply...

Code of Federal Regulations, 2014 CFR

2014-07-01

...-hour ozone NAAQS? 51.910 Section 51.910 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Provisions for Implementation of 8-hour Ozone National Ambient Air Quality Standard § 51.910 What... nonattainment for the 8-hour ozone NAAQS? (a) What are the general requirements for RFP for an area classified...

In vivo tomographic imaging of deep seated cancer using fluorescence lifetime contrast

PubMed Central

Rice, William L.; Shcherbakova, Daria M; Verkusha, Vladislav V.; Kumar, Anand T.N.

2015-01-01

Preclinical cancer research would benefit from non-invasive imaging methods that allow tracking and visualization of early stage metastasis in vivo. While fluorescent proteins revolutionized intravital microscopy, two major challenges which still remain are tissue autofluorescence and hemoglobin absorption, which act to limit intravital optical techniques to large or subcutaneous tumors. Here we employ time-domain technology for the effective separation of tissue autofluorescence from extrinsic fluorophores, based on their distinct fluorescence lifetimes. Additionally, we employ cancer cells labelled with near infra-red fluorescent proteins (iRFP) to allow deep-tissue imaging. Our results demonstrate that time-domain imaging allows the detection of metastasis in deep-seated organs of living mice with a more than 20-fold increase in sensitivity compared to conventional continuous wave techniques. Furthermore, the distinct fluorescence lifetimes of each iRFP enables lifetime multiplexing of three different tumors, each expressing unique iRFP labels in the same animal. Fluorescence tomographic reconstructions reveal 3D distributions of iRFP720-expressing cancer cells in lungs and brain of live mice, allowing ready longitudinal monitoring of cancer cell fate with greater sensitivity than otherwise currently possible. PMID:25670171

Observation of Electron Bernstein Wave Heating in the MST Reversed Field Pinch

NASA Astrophysics Data System (ADS)

Seltzman, Andrew; Anderson, Jay; Dubois, Ami; Almagri, Abdulgader; Nonn, Paul; McCollam, Karsten; Chapman, Brett; Goetz, John; Forest, Cary

2016-10-01

We report the first observation of electron Bernstein wave heating in the MST RFP. Similar to a high density stellarator, the RFP is inaccessible to electromagnetic ECRH. The plasma current and |B|operating range of MST allows a 5.5 GHz RF source (100kW, 4ms pulse) to heat on the fundamental and up to 4th harmonic EC resonances. With an x-ray diagnostic most sensitive to edge electrons located +12 degrees toroidally from the antenna, the measured emission is a strong function of predicted heating inside versus outside the Bt =0 reversal layer of the RFP. Measured during a scan of plasma current, distinct edges in a plot of emissivity versus predicted deposition layer align with the deposition layers crossing of this reversal layer and confirm EBW heating on the fundamental through 4th EC harmonic. Additional confirmation of the absorption location has been demonstrated by using auxiliary poloidal current drive to reduce electron diffusion rates and sweep the location of the Bt =0 surface across a static RF absorption location in RFP discharges. In these discharges EBW enhancement of the 15-40keV x-ray energies has been observed. Work supported by USDOE.

Validation of Extended MHD Models using MST RFP Plasmas

NASA Astrophysics Data System (ADS)

Jacobson, C. M.; Chapman, B. E.; Craig, D.; McCollam, K. J.; Sovinec, C. R.

2016-10-01

Significant effort has been devoted to improvement of computational models used in fusion energy sciences. Rigorous validation of these models is necessary in order to increase confidence in their ability to predict the performance of future devices. MST is a well diagnosed reversed-field pinch (RFP) capable of operation over a wide range of parameters. In particular, the Lundquist number S, a key parameter in resistive magnetohydrodynamics (MHD), can be varied over a wide range and provide substantial overlap with MHD RFP simulations. MST RFP plasmas are simulated using both DEBS, a nonlinear single-fluid visco-resistive MHD code, and NIMROD, a nonlinear extended MHD code, with S ranging from 104 to 5 ×104 for single-fluid runs, with the magnetic Prandtl number Pm = 1 . Experiments with plasma current IP ranging from 60 kA to 500 kA result in S from 4 ×104 to 8 ×106 . Validation metric comparisons are presented, focusing on how magnetic fluctuations b scale with S. Single-fluid NIMROD results give S b - 0.21 , and experiments give S b - 0.28 for the dominant m = 1 , n = 6 mode. Preliminary two-fluid NIMROD results are also presented. Work supported by US DOE.

The rotational order-disorder structure of the reversibly photoswitchable red fluorescent protein rsTagRFP.

PubMed

Pletnev, Sergei; Subach, Fedor V; Verkhusha, Vladislav V; Dauter, Zbigniew

2014-01-01

The rotational order-disorder (OD) structure of the reversibly photoswitchable fluorescent protein rsTagRFP is discussed in detail. The structure is composed of tetramers of 222 symmetry incorporated into the lattice in two different orientations rotated 90° with respect to each other around the crystal c axis and with tetramer axes coinciding with the crystallographic twofold axes. The random distribution of alternatively oriented tetramers in the crystal creates the rotational OD structure with statistically averaged I422 symmetry. Despite order-disorder pathology, the structure of rsTagRFP has electron-density maps of good quality for both non-overlapping and overlapping parts of the model. The crystal contacts, crystal internal architecture and a possible mechanism of rotational OD crystal formation are discussed.

Numerical characterization of plasma breakdown in reversed field pinches

NASA Astrophysics Data System (ADS)

Peng, Yanli; Zhang, Ya; Mao, Wenzhe; Yang, Zhoujun; Hu, Xiwei; Jiang, Wei

2018-02-01

In the reversed field pinch, there is considerable interest in investigating the plasma breakdown. Indeed, the plasma formed during the breakdown may have an influence on the confinement and maintenance in the latter process. However, up to now there has been no related work, experimentally or in simulation, regarding plasma breakdown in reversed field pinch (RFP). In order to figure out the physical mechanism behind plasma breakdown, the effects of the toroidal and error magnetic field, as well as the loop voltage have been studied. We find that the error magnetic field cannot be neglected even though it is quite small in the short plasma breakdown phase. As the toroidal magnetic field increases, the averaged electron energy is reduced after plasma breakdown is complete, which is disadvantageous for the latter process. In addition, unlike the voltage limits in the tokamak, loop voltages can be quite high because there are no requirements for superconductivity. Volt-second consumption has a small difference under different loop voltages. The breakdown delay still exists in various loop voltage cases, but it is much shorter compared to that in the tokamak case. In all, successful breakdowns are possible in the RFP under a fairly broad range of parameters.

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

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

Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo

2014-02-21

Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesusmore » monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.« less

Recognized focused practice: Does sub-specialty designation offer value to the neurosurgeon?

PubMed

Babu, Maya A; Liau, Linda M; Meyer, Fredric B

2017-01-01

Vehicles for life-long assessment such as Maintenance of Certification tend to focus on generalist neurosurgical knowledge. However, as neurosurgeons advance in their careers, they tend to narrow their practice and increase volumes in certain specific types of operations. Failing to test the type of procedures most relevant to the practitioner is a lost opportunity to improve the knowledge and practice of the individual neurosurgeon. In this study, we assess the neurosurgical community's appetite for designations of board-recognized Recognized Focused Practice (RFP). We administered a validated, online, confidential survey to 4,899 neurosurgeons (2,435 American Board of Neurological Surgery (ABNS) Diplomates participating in MOC, 1,440 Diplomates certified prior to 1999 (grandfathered), and 1,024 retired Diplomates). We received 1,449 responses overall (30% response rate). A plurality of respondents were in practice 11-15 years (18.5%), in private practice (40%) and participate in MOC (61%). 49% of respondents felt that a RFP designation would not be helpful. For the 30% who felt that RFP would be helpful, 61.3% felt that it would support recognition by their hospital or practice, it would motivate them to stay current on medical knowledge (53.4%), or it would help attract patients (46.4%;). The most popular suggestions for RFP were Spine (56.2%), Cerebrovascular (62.9%), Pediatrics (64.1%), and Functional/Stereotactic (52%). A plurality of neurosurgeons (35.7%) felt that RFP should recognize neurosurgeons with accredited and non-accredited fellowship experience and sub-specialty experience. Ultimately, Recognized Focused Practice may provide value to individual neurosurgeons, but the neurosurgical community shows tepid interest for pursuing this designation.

Recognized focused practice: Does sub-specialty designation offer value to the neurosurgeon?

PubMed Central

Liau, Linda M.; Meyer, Fredric B.

2017-01-01

Vehicles for life-long assessment such as Maintenance of Certification tend to focus on generalist neurosurgical knowledge. However, as neurosurgeons advance in their careers, they tend to narrow their practice and increase volumes in certain specific types of operations. Failing to test the type of procedures most relevant to the practitioner is a lost opportunity to improve the knowledge and practice of the individual neurosurgeon. In this study, we assess the neurosurgical community’s appetite for designations of board-recognized Recognized Focused Practice (RFP). We administered a validated, online, confidential survey to 4,899 neurosurgeons (2,435 American Board of Neurological Surgery (ABNS) Diplomates participating in MOC, 1,440 Diplomates certified prior to 1999 (grandfathered), and 1,024 retired Diplomates). We received 1,449 responses overall (30% response rate). A plurality of respondents were in practice 11–15 years (18.5%), in private practice (40%) and participate in MOC (61%). 49% of respondents felt that a RFP designation would not be helpful. For the 30% who felt that RFP would be helpful, 61.3% felt that it would support recognition by their hospital or practice, it would motivate them to stay current on medical knowledge (53.4%), or it would help attract patients (46.4%;). The most popular suggestions for RFP were Spine (56.2%), Cerebrovascular (62.9%), Pediatrics (64.1%), and Functional/Stereotactic (52%). A plurality of neurosurgeons (35.7%) felt that RFP should recognize neurosurgeons with accredited and non-accredited fellowship experience and sub-specialty experience. Ultimately, Recognized Focused Practice may provide value to individual neurosurgeons, but the neurosurgical community shows tepid interest for pursuing this designation. PMID:29240838

Broad-host-range plasmids for red fluorescent protein labeling of gram-negative bacteria for use in the zebrafish model system.

PubMed

Singer, John T; Phennicie, Ryan T; Sullivan, Matthew J; Porter, Laura A; Shaffer, Valerie J; Kim, Carol H

2010-06-01

To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-beta-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI(q) carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.

Using Dual Fluorescence Reporting Genes to Establish an In Vivo Imaging Model of Orthotopic Lung Adenocarcinoma in Mice.

PubMed

Lai, Cheng-Wei; Chen, Hsiao-Ling; Yen, Chih-Ching; Wang, Jiun-Long; Yang, Shang-Hsun; Chen, Chuan-Mu

2016-12-01

Lung adenocarcinoma is characterized by a poor prognosis and high mortality worldwide. In this study, we purposed to use the live imaging techniques and a reporter gene that generates highly penetrative near-infrared (NIR) fluorescence to establish a preclinical animal model that allows in vivo monitoring of lung cancer development and provides a non-invasive tool for the research on lung cancer pathogenesis and therapeutic efficacy. A human lung adenocarcinoma cell line (A549), which stably expressed the dual fluorescence reporting gene (pCAG-iRFP-2A-Venus), was used to generate subcutaneous or orthotopic lung cancer in nude mice. Cancer development was evaluated by live imaging via the NIR fluorescent signals from iRFP, and the signals were verified ex vivo by the green fluorescence of Venus from the gross lung. The tumor-bearing mice received miR-16 nucleic acid therapy by intranasal administration to demonstrate therapeutic efficacy in this live imaging system. For the subcutaneous xenografts, the detection of iRFP fluorescent signals revealed delicate changes occurring during tumor growth that are not distinguishable by conventional methods of tumor measurement. For the orthotopic xenografts, the positive correlation between the in vivo iRFP signal from mice chests and the ex vivo green fluorescent signal from gross lung tumors and the results of the suppressed tumorigenesis by miR-16 treatment indicated that lung tumor size can be accurately quantified by the emission of NIR fluorescence. In addition, orthotopic lung tumor localization can be accurately visualized using iRFP fluorescence tomography in vivo, thus revealing the trafficking of lung tumor cells. We introduced a novel dual fluorescence lung cancer model that provides a non-invasive option for preclinical research via the use of NIR fluorescence in live imaging of lung.

Using molecular imaging to assess the delivery and infection of protease activated virus in animal model of myocardial infarction

NASA Astrophysics Data System (ADS)

Zhu, Banghe; Guenther, Caitlin; Kwon, Sunkuk; Sevick-Muraca, Eva M.; Suh, Junghae

2017-02-01

Cardiovascular diseases remain the greatest cause of death in the US and gene therapy has the potential to be an effective therapy. In this study, we demonstrated MMP-9 based protease-activatable virus (PAV) for selective infection of myocardial infarct (MI) that is associated with active MMP-9 expression. To test the specificity of PAV, we used expression of a far-red fluorescence protein (iRFP) delivered by the PAV together with a dual PET/NIRF imaging agent specific for active MMP-9 activity at the site of MI in a murine model. Calibrated fluorescence imaging employed a highly-sensitive intensified camera, laser diode excitation sources, and filtration schemes based upon the spectra of iRFP and the NIRF agent. One to two days after ligation of the left anterior descending artery, the PAV or WT AAV9 virus encoding for iRFP (5x1010 genomic particles) and radiolabeled MMP-9 imaging agent (3 nmol) were injected intravenously (i.v.). PET imaging showed MMP activity was associated with adverse tissue remodeling at the site of the MI. One week after, animals were again injected i.v. with the MMP-9 agent (3 nmol) and 18-24 h later, the animals were euthanized and the hearts were harvested, sliced, and imaged for congruent iRFP transgene expression and NIRF signals associated with MMP-9 tissue activity. The fluorescent margins of iRFP and NIRF contrasted tissues were quantified in terms Standard International units of mW/cm2/sr. The sensitivity, specificity, and accuracy of PAV and WT targeting to sites of MI was determined from these calibrated fluorescence measurements. The PAV demonstrated significantly higher delivery performance than that of the WT AAV9 virus.

[Experimental study of glioma stem cell-mediated immune tolerance in tumor microenvironment].

PubMed

Xie, T; Ma, J W; Liu, B; Dong, J; Huang, Q

2017-11-23

Objective: To investigate the tumor microenvironment of immune tolerance induced by glioma stem cells (GSC). Methods: Human GSC SU3 cells transfected with red fluorescent protein (SU3-RFP) gene were implanted into the brain, subcutis (armpit and foot), liver and abdominal cavity of transgenic green fluorescence protein (GFP) nude mice to establish RFP(+) /GFP(+) dual fluorescence solid tumor model. The re-cultured cells derived from implanted tumor tissues, SU3-RFP cells co-cultured with peritoneal fluid of transgenic GFP nude mice and malignant ascites of tumor-bearing mice were observed by fluorescence microscopy and real-time video image tracing to analyze the microenvironment of immune tolerance mediated by RFP(+) /GFP(+) implanted tumor. Results: Dual fluorescence labeled frozen section showed that all of cells in the tumor microenvironment were GFP(+) , while the pressed tissue-patch showed that the tumor blood vessels exhibited a RFP(+) /GFP(+) double-positioning yellow. In the GFP single fluorescence labeled tumor tissue, all of cells in the microenvironment were green, including tumor edge, necrotic foci and blood vessel. Among them, CD68(+) , F4/80(+) , CD11c(+) , CD11b(+) and CD80(+) cells were observed. In the dual fluorescence labeled co-cultured cells, the phagocytosis and fusion between green host cells and red tumor cells were also observed, and these fusion cells might transfer to the malignant dendritic cells and macrophages. Conclusions: The tumor microenvironment of immune tolerance induced by GSC is not affected by the tissue types of tumor-inoculated sites, and the immune tolerance mediated by inflammatory cells is associated with the inducible malignant transformation, which may be driven by cell fusion.

Incubation and application of transgenic green fluorescent nude mice in visualization studies on glioma tissue remodeling.

PubMed

Dong, Jun; Dai, Xing-liang; Lu, Zhao-hui; Fei, Xi-feng; Chen, Hua; Zhang, Quan-bin; Zhao, Yao-dong; Wang, Zhi-min; Wang, Ai-dong; Lan, Qing; Huang, Qiang

2012-12-01

The primary reasons for local recurrence and therapeutic failure in the treatment of malignant gliomas are the invasion and interactions of tumor cells with surrounding normal brain cells. However, these tumor cells are hard to be visualized directly in histopathological preparations, or in experimental glioma models. Therefore, we developed an experimental human dual-color in vivo glioma model, which made tracking solitary invasive glioma cells possible, for the purpose of visualizing the interactions between red fluorescence labeled human glioma cells and host brain cells. This may offer references for further studying the roles of tumor microenvironment during glioma tissue remodeling. Transgenic female C57BL/6 mice expressing enhanced green fluorescent protein (EGFP) were crossed with male Balb/c nude mice. Then sib mating was allowed to occur continuously in order to establish an inbred nude mice strain with 50% of their offspring that are EGFP positive. Human glioma cell lines U87-MG and SU3 were transfected with red fluorescent protein (RFP) gene, and a rat C6 glioma cell line was stained directly with CM-DiI, to establish three glioma cell lines emitting red fluorescence (SU3-RFP, U87-RFP, and C6-CM-DiI). Red fluorescence tumor cells were inoculated via intra-cerebral injection into caudate nucleus of the EGFP nude mice. Tumor-bearing mice were sacrificed when their clinical symptoms appeared, and the whole brain was harvested and snap frozen for further analysis. Confocal laser scanning microscopy was performed to monitor the mutual interactions between tumor cells and host brain cells. Almost all the essential tissues of the established EGFP athymic Balb/c nude mice, except hair and erythrocytes, fluoresced green under excitation using a blue light-emitting flashlight with a central peak of 470 nm, approximately 50% of the offsprings were nu/nu EGFP+. SU3-RFP, U87-RFP, and C6-CM-DiI almost 100% expressed red fluorescence under the fluorescence microscope. Under fluorescence microscopic view, RFP+ cells were observed growing wherever they arrived at, locating in the brain parenchyma, ventricles, and para-vascular region. The interactions between the transplanted tumor cells and host adjacent cells could be classified into three types: (1) interweaving; (2) mergence; and (3) fusion. Interweaving was observed in the early stage of tumor remodeling, in which both transplantable tumor cells and host cells were observed scattered in the tumor invading and spreading area without organic connections. Mergence was defined as mutual interactions between tumor cells and host stroma during tumorigenesis. Direct cell fusion between transplantable tumor cells and host cells could be observed occasionally. This study showed that self-established EGFP athymic nude mice offered the possibility of visualizing tumorigenesis of human xenograft tumor, and the dual-color xenograft glioma model was of considerable utility in studying the process of tumor remodeling. Based on this platform, mutual interactions between glioma cells and host tissues could be observed directly to further elucidate the development of tumor microenvironment.

Diesel Fuel Processing for the PAFC Process Demonstration.

DTIC Science & Technology

1986-12-15

c M-0 ca W ’ U 141 _ ____ _ _ _ __ _ _ 104 ENERGY RESEARCH CORPORATION ??~ Ŕ, E 6/17/86 Exxon Company, USA 200 Unicorn Park Woburn, Mass. 01801-3390...Sincerely, George Steinfeld Program Manager 105 , E ON COMPANY U.S.A. 200 UNICORN PARK • AOBURN, MASSACHUSE S0801-3390 M %A’KE ,DEPARTVENT T .’AQKE7N...Fort Myer Drive rTN: RFP Control-Corp. Marketing .*. Arlington, VA 22209 Bldg C2 Mail Station B199 P.O. Box 1053 El Segundo, CA 90245 Cbmmander (12

Genetic Recombination Between Stromal and Cancer Cells Results in Highly Malignant Cells Identified by Color-Coded Imaging in a Mouse Lymphoma Model.

PubMed

Nakamura, Miki; Suetsugu, Atsushi; Hasegawa, Kousuke; Matsumoto, Takuro; Aoki, Hitomi; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Hoffman, Robert M

2017-12-01

The tumor microenvironment (TME) promotes tumor growth and metastasis. We previously established the color-coded EL4 lymphoma TME model with red fluorescent protein (RFP) expressing EL4 implanted in transgenic C57BL/6 green fluorescent protein (GFP) mice. Color-coded imaging of the lymphoma TME suggested an important role of stromal cells in lymphoma progression and metastasis. In the present study, we used color-coded imaging of RFP-lymphoma cells and GFP stromal cells to identify yellow-fluorescent genetically recombinant cells appearing only during metastasis. The EL4-RFP lymphoma cells were injected subcutaneously in C57BL/6-GFP transgenic mice and formed subcutaneous tumors 14 days after cell transplantation. The subcutaneous tumors were harvested and transplanted to the abdominal cavity of nude mice. Metastases to the liver, perigastric lymph node, ascites, bone marrow, and primary tumor were imaged. In addition to EL4-RFP cells and GFP-host cells, genetically recombinant yellow-fluorescent cells, were observed only in the ascites and bone marrow. These results indicate genetic exchange between the stromal and cancer cells. Possible mechanisms of genetic exchange are discussed as well as its ramifications for metastasis. J. Cell. Biochem. 118: 4216-4221, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Color-Coded Imaging of Breast Cancer Metastatic Niche Formation in Nude Mice.

PubMed

Suetsugu, Atsushi; Momiyama, Masashi; Hiroshima, Yukihiko; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Bouvet, Michael; Hoffman, Robert M

2015-12-01

We report here a color-coded imaging model in which metastatic niches in the lung and liver of breast cancer can be identified. The transgenic green fluorescent protein (GFP)-expressing nude mouse was used as the host. The GFP nude mouse expresses GFP in all organs. However, GFP expression is dim in the liver parenchymal cells. Mouse mammary tumor cells (MMT 060562) (MMT), expressing red fluorescent protein (RFP), were injected in the tail vein of GFP nude mice to produce experimental lung metastasis and in the spleen of GFP nude mice to establish a liver metastasis model. Niche formation in the lung and liver metastasis was observed using very high resolution imaging systems. In the lung, GFP host-mouse cells accumulated around as few as a single MMT-RFP cell. In addition, GFP host cells were observed to form circle-shaped niches in the lung even without RFP cancer cells, which was possibly a niche in which future metastasis could be formed. In the liver, as with the lung, GFP host cells could form circle-shaped niches. Liver and lung metastases were removed surgically and cultured in vitro. MMT-RFP cells and GFP host cells resembling cancer-associated fibroblasts (CAFs) were observed interacting, suggesting that CAFs could serve as a metastatic niche. © 2015 Wiley Periodicals, Inc.

Observation of Electron Bernstein Wave Heating in the RFP

NASA Astrophysics Data System (ADS)

Seltzman, Andrew; Anderson, Jay; Goetz, John; Forest, Cary

2017-10-01

The first observation of RF heating in a reversed field pinch (RFP) using the electron Bernstein wave (EBW) has been demonstrated on MST. Efficient mode conversion of an outboard-launched X mode wave at 5.5 GHz leads to Doppler-shifted resonant absorption (ωrf = nωce-k||v||) for a broad range (n =1-7) of harmonics. The dynamics of EBW-heated electrons are measured using a spatial distribution of solid targets with diametrically opposed x-ray detectors. EBW heating produces a clear supra-thermal electron tail in MST. Radial deposition of the EBW is controlled with |B|and is measured using the HXR flux emitted from an insertable probe. In the thick-shelled MST RFP, the radial accessibility of EBW is limited to r/a >0.8 ( 10cm) by magnetic field error induced by the porthole necessary for the antenna. Experimental measurements show EBW propagation inward through a stochastic magnetic field. EBW-heated test electrons are used as a direct probe of edge (r/a >0.9) radial transport, showing a modest transition from `standard' to reduced-tearing RFP operation. Electron loss is too fast for collisional effects and implies a large non-collisional radial diffusivity. EBW heating has been demonstrated in reduced magnetic stochasticity plasmas with β = 15-20%. Work supported by USDOE.

A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea

PubMed Central

Ford, Kathryn L.; Baumgartner, Kendra; Henricot, Béatrice; Bailey, Andy M.; Foster, Gary D.

2016-01-01

Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. Using a yeast-adapted pCAMBIA0380 Agrobacterium vector, we have constructed a series of vectors for transformation of A. mellea, assembled using yeast-based recombination methods. These have been designed to allow easy exchange of promoters and inclusion of introns. The vectors were first tested by transformation into basidiomycete Clitopilus passeckerianus to ascertain vector functionality then used to transform A. mellea. We show that heterologous promoters from the basidiomycetes Agaricus bisporus and Phanerochaete chrysosporium that were used successfully to control the hygromycin resistance cassette were not able to support expression of mRFP or GFP in A. mellea. The endogenous A. mellea gpd promoter delivered efficient expression, and we show that inclusion of an intron was also required for transgene expression. GFP and mRFP expression was stable in mycelia and fluorescence was visible in transgenic fruiting bodies and GFP was detectable in planta. Use of these vectors has been successful in giving expression of the fluorescent proteins GFP and mRFP in A. mellea, providing an additional molecular tool for this pathogen. PMID:27384974

Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei

PubMed Central

Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

2014-01-01

Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. PMID:25044501

Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

PubMed

Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

2014-10-01

Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Navy LX(R) Amphibious Ship Program: Background and Issues for Congress

DTIC Science & Technology

2015-12-17

combined solicitation to two bidders—Ingalls Shipbuilding of Huntington Ingalls Industries (HII/Ingalls) and National Steel and Shipbuilding Company...RFP; see, for example, Sam LaGrone, “Navy Issues RFP for Oilers and LHA-8 to NASSCO, Ingalls,” USNI News, July 10, 2015; Valerie Insinna, “Navy...amphibious assault ship so that San Diego-based General Dynamics National Steel and Shipbuilding Co. (NASSCO) and the Huntington Ingalls Industries

Increased confinement and beta by inductive poloidal current drive in the RFP

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

Sarff, J.S.; Lanier, N.E.; Prager, S.C.

1996-10-01

Progress in understanding magnetic-fluctuation-induced transport in the reversed field pinch (RFP) has led to the idea of current profile control to reduce fluctuations and transport. With the addition of inductive poloidal current drive in the Madison Symmetric Torus (MST), the magnetic fluctuation amplitude is halved, leading to a four- to five-fold increase in the energy confinement time to {tau}{sub E}{approximately}5 ms as a result of both decreased plasma resistance and increased stored thermal energy. The record low fluctuation amplitude coincides with a record high electron temperature of {approximately}600 eV (for MST), and beta {beta} = 2{mu}{sub 0} / B(a){sup 2}more » increases from 6% to 8% compared with conventional MST RFP plasmas. Other improvements include increased particle confinement and impurity reduction. 19 refs., 4 figs., 1 tab.« less

Magnetohydrodynamic simulations of noninductive helicity injection in the reversed-field pinch and tokamak

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

Sovinec, Carl R.

1995-11-01

Numerical computation is used to investigate resistive magnetohydrodynamic (MHD) fluctuations in the reversed-field pinch (RFP) and in tokamak-like configurations driven solely by direct current (DC) helicity injection. A Lundquist number (S) scan of RFP turbulence without plasma pressure produces the weak scaling of S -0.18 for the root-mean-square magnetic fluctuation level for 2.5x10 3≤S≤4x10 4. The temporal behavior of fluctuations and the reversal parameter becomes more regular as S is increased, acquiring a "sawtooth" shape at the largest value of S. Simulations with plasma pressure and anisotropic thermal conduction demonstrate energy transport resulting from parallel heat fluctuations. To investigate meansmore » of improving RFP energy confinement, three forms of current profile modification are tested. Radio frequency (RF) current drive is modeled with an auxiliary electron force, and linear stability calculations are used.« less

Incorporating Software Requirements into the System RFP: Survey of RFP Language for Software by Topic, v. 2.0

DTIC Science & Technology

2009-05-01

CDRL) System - Terminal S TMOS - SEIT CL TMOS - IA/ Crypto CL Space - Payload S Space - SEIT C Space - IA/ Crypto C Role Legend: CL...strategy for maintaining the currency of the technology (through Commercial off-the-shelf software (COTS) and other reusable Non-Developmental Items (NDI...the strategy for maintaining the currency of technology (e.g., through Commercial off-the-shelf software (COTS) insertion, technology refresh

Navy LX(R) Amphibious Ship Program: Background and Issues for Congress

DTIC Science & Technology

2016-01-08

and National Steel and Shipbuilding Company of General Dynamics (GD/NASSCO)—on the grounds that these are the only two shipbuilders that have the...LaGrone, “Navy Issues RFP for Oilers and LHA-8 to NASSCO, Ingalls,” USNI News, July 10, 2015; Valerie Insinna, “Navy Quietly Issues RFP for LHA-8...amphibious assault ship so that San Diego-based General Dynamics National Steel and Shipbuilding Co. (NASSCO) and the Huntington Ingalls Industries

The effect of collisionality and diamagnetism on the plasma dynamo

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

Ji, H.; Yagi, Y.; Hattori, K.

1995-04-28

Fluctuation-induced dynamo forces are measured over a wide range of electron collisionality in the edge of TPE-1RM20 Reversed-Field Pinch (RFP). In the collisionless region the Magnetohydrodynamic (MHD) dynamo alone can sustain the parallel current, while in the collisional region a new dynamo mechanism resulting from the fluctuations in the electron diamagnetic drift becomes dominant. A comprehensive picture of the RFP dynamo emerges by combining with earlier results from MST and REPUTE RFPs.

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

PubMed Central

Mederacke, Ingmar; Komatsu, Yoshihiro; Stice, Steve; Schwabe, Robert F.; Mistretta, Charlotte M.; Mishina, Yuji; Liu, Hong-Xiang

2016-01-01

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC. PMID:26741369

Validation of a Postoperative Nomogram Predicting Recurrence in Patients with Conventional Clear Cell Renal Cell Carcinoma.

PubMed

Lee, Byron H; Feifer, Andrew; Feuerstein, Michael A; Benfante, Nicole E; Kou, Lei; Yu, Changhong; Kattan, Michael W; Russo, Paul

2018-01-01

Clear cell renal cell carcinoma (RCC) continues to be the most commonly diagnosed subtype and is associated with more aggressive behavior than papillary and chromophobe RCC. Predicting disease recurrence after surgical extirpation is important for counseling and targeting those at high risk for adjuvant therapy clinical trials. To validate a postoperative nomogram predicting 5-yr recurrence-free probability (RFP) for clinically localized clear cell RCC. We identified all patients who underwent nephrectomy for clinically localized clear cell RCC from 1990 to 2009 at Memorial Sloan Kettering Cancer Center. After excluding patients with bilateral renal masses, familial RCC syndromes, and T3c or T4 tumors due to the limited number, 1642 participants were available for analysis. Partial or radical nephrectomy. Disease recurrence was defined as any new tumor after nephrectomy or kidney cancer-specific mortality, whichever occurred first. A postoperative nomogram was used to calculate the predicted 5-yr RFP, and these values were compared with the actual 5-yr RFP. Nomogram performance was evaluated by concordance index and calibration plot. Median follow-up was 39 mo (interquartile range: 14-79 mo), and disease recurrence was observed in 50 patients. The nomogram concordance index was 0.81. The calibration curve showed that the nomogram underestimated the actual 5-yr RFP. We updated the nomogram by including the entire patient population, which maintained performance and significantly improved calibration. The updated clear cell RCC postoperative nomogram performed well in the combined cohort. Underestimation of actual 5-yr RFP by the original nomogram may be due to increased surgeon experience and other unknown variables. We updated a valuable prediction tool used for assessing the disease recurrence probability after nephrectomy for clear cell renal cell carcinoma. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate.

PubMed

Boggs, Kristin; Venkatesan, Nandakumar; Mederacke, Ingmar; Komatsu, Yoshihiro; Stice, Steve; Schwabe, Robert F; Mistretta, Charlotte M; Mishina, Yuji; Liu, Hong-Xiang

2016-01-01

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.

Distribution and characterisation of Glucagon-like peptide-1 receptor expressing cells in the mouse brain

PubMed Central

Cork, Simon C.; Richards, James E.; Holt, Marie K.; Gribble, Fiona M.; Reimann, Frank; Trapp, Stefan

2015-01-01

Objective Although Glucagon-like peptide 1 is a key regulator of energy metabolism and food intake, the precise location of GLP-1 receptors and the physiological relevance of certain populations is debatable. This study investigated the novel GLP-1R-Cre mouse as a functional tool to address this question. Methods Mice expressing Cre-recombinase under the Glp1r promoter were crossed with either a ROSA26 eYFP or tdRFP reporter strain to identify GLP-1R expressing cells. Patch-clamp recordings were performed on tdRFP-positive neurons in acute coronal brain slices from adult mice and selective targeting of GLP-1R cells in vivo was achieved using viral gene delivery. Results Large numbers of eYFP or tdRFP immunoreactive cells were found in the circumventricular organs, amygdala, hypothalamic nuclei and the ventrolateral medulla. Smaller numbers were observed in the nucleus of the solitary tract and the thalamic paraventricular nucleus. However, tdRFP positive neurons were also found in areas without preproglucagon-neuronal projections like hippocampus and cortex. GLP-1R cells were not immunoreactive for GFAP or parvalbumin although some were catecholaminergic. GLP-1R expression was confirmed in whole-cell recordings from BNST, hippocampus and PVN, where 100 nM GLP-1 elicited a reversible inward current or depolarisation. Additionally, a unilateral stereotaxic injection of a cre-dependent AAV into the PVN demonstrated that tdRFP-positive cells express cre-recombinase facilitating virally-mediated eYFP expression. Conclusions This study is a comprehensive description and phenotypic analysis of GLP-1R expression in the mouse CNS. We demonstrate the power of combining the GLP-1R-CRE mouse with a virus to generate a selective molecular handle enabling future in vivo investigation as to their physiological importance. PMID:26500843

High-Content Positional Biosensor Screening Assay for Compounds to Prevent or Disrupt Androgen Receptor and Transcriptional Intermediary Factor 2 Protein–Protein Interactions

PubMed Central

Hua, Yun; Shun, Tong Ying; Strock, Christopher J.

2014-01-01

Abstract The androgen receptor–transcriptional intermediary factor 2 (AR-TIF2) positional protein–protein interaction (PPI) biosensor assay described herein combines physiologically relevant cell-based assays with the specificity of binding assays by incorporating structural information of AR and TIF2 functional domains along with intracellular targeting sequences and fluorescent reporters. Expression of the AR-red fluorescent protein (RFP) “prey” and TIF2-green fluorescent protein (GFP) “bait” components of the biosensor was directed by recombinant adenovirus constructs that expressed the ligand binding and activation function 2 surface domains of AR fused to RFP with nuclear localization and nuclear export sequences, and three α-helical LXXLL motifs from TIF2 fused to GFP and an HIV Rev nucleolar targeting sequence. In unstimulated cells, AR-RFP was localized predominantly to the cytoplasm and TIF2-GFP was localized to nucleoli. Dihydrotestosterone (DHT) treatment induced AR-RFP translocation into the nucleus where the PPIs between AR and TIF2 resulted in the colocalization of both biosensors within the nucleolus. We adapted the translocation enhanced image analysis module to quantify the colocalization of the AR-RFP and TIF2-GFP biosensors in images acquired on the ImageXpress platform. DHT induced a concentration-dependent AR-TIF2 colocalization and produced a characteristic condensed punctate AR-RFP PPI nucleolar distribution pattern. The heat-shock protein 90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) and antiandrogens flutamide and bicalutamide inhibited DHT-induced AR-TIF2 PPI formation with 50% inhibition concentrations (IC50s) of 88.5±12.5 nM, 7.6±2.4 μM, and 1.6±0.4 μM, respectively. Images of the AR-RFP distribution phenotype allowed us to distinguish between 17-AAG and flutamide, which prevented AR translocation, and bicalutamide, which blocked AR-TIF2 PPIs. We screened the Library of Pharmacologically Active Compounds (LOPAC) set for compounds that inhibited AR-TIF2 PPI formation or disrupted preexisting complexes. Eleven modulators of steroid family nuclear receptors (NRs) and 6 non-NR ligands inhibited AR-TIF2 PPI formation, and 10 disrupted preexisting complexes. The hits appear to be either AR antagonists or nonspecific inhibitors of NR activation and trafficking. Given that the LOPAC set represents such a small and restricted biological and chemical diversity, it is anticipated that screening a much larger and more diverse compound library will be required to find AR-TIF2 PPI inhibitors/disruptors. The AR-TIF2 protein–protein interaction biosensor (PPIB) approach offers significant promise for identifying molecules with potential to modulate AR transcriptional activity in a cell-specific manner that is distinct from the existing antiandrogen drugs that target AR binding or production. Small molecules that disrupt AR signaling at the level of AR-TIF2 PPIs may also overcome the development of resistance and progression to castration-resistant prostate cancer. PMID:25181412

In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography

PubMed Central

Genevois, Coralie; Loiseau, Hugues; Couillaud, Franck

2016-01-01

Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT®). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors. PMID:27809256

In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography.

PubMed

Genevois, Coralie; Loiseau, Hugues; Couillaud, Franck

2016-10-31

Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT ® ). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors.

Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome.

PubMed

Piatkevich, Kiryl D; Subach, Fedor V; Verkhusha, Vladislav V

2013-01-01

The ability to modulate the fluorescence of optical probes can be used to enhance signal-to-noise ratios for imaging within highly autofluorescent environments, such as intact tissues and living organisms. Here, we report two bacteriophytochrome-based photoactivatable near-infrared fluorescent proteins, named PAiRFP1 and PAiRFP2. PAiRFPs utilize haem-derived biliverdin, ubiquitous in mammalian tissues, as the chromophore. Initially weakly fluorescent PAiRFPs undergo photoconversion into a highly fluorescent state with excitation/emission at 690/717 nm following a brief irradiation with far-red light. After photoactivation, PAiRFPs slowly revert back to initial state, enabling multiple photoactivation-relaxation cycles. Low-temperature optical spectroscopy reveals several intermediates involved in PAiRFP photocycles, which all differ from that of the bacteriophytochrome precursor. PAiRFPs can be photoactivated in a spatially selective manner in mouse tissues, and optical modulation of their fluorescence allows for substantial contrast enhancement, making PAiRFPs advantageous over permanently fluorescent probes for in vivo imaging conditions of high autofluorescence and low signal levels.

Electron transport in the stochastic fields of the reversed-field pinch

NASA Astrophysics Data System (ADS)

Kim, Myung-Hee; Punjabi, Alkesh

1996-08-01

We employ the Monte Carlo method for the calculation of anomalous transport developed by Punjabi and Boozer to calculate the particle diffusion coefficient for electrons in the stochastic magnetic fields of the reversed-field pinch (RFP). in the Monte Carlo calculations represented here, the transport mechanism is the loss of magnetic surfaces due to resistive perturbations. The equilibrium magnetic fields are represented by the Bessel function model for the RFP. The diffusion coefficient D is calculated as a function of a, the amplitude of the perturbation. We see three regimes as the amplitude of the tearing modes is increased: the Rechester—Rosenbluth regime where D scales as a2 the anomalous regime where D scales more rapidly than a2 and the Mynick—Krornmes regime where D scales more slowly than a2. Inclusion of the effects of loop voltage on the particle drift orbits in the RFP does not affect the intervals in the amplitude a where these regimes operate.

Simultaneous imaging of temporal changes of NF-κB activity and viable tumor cells in Huh7/NF-κB-tk-luc2/rfp tumor-bearing mice.

PubMed

Wang, Wei-Hsun; Chiang, I-Tsang; Liu, Yu-Chang; Hsu, Fei-Ting; Chen, Hong-Wen; Chen, Chuan-Lin; Lee, Yi-Jang; Lin, Wuu-Jyh; Hwang, Jeng-Jong

2013-01-01

Few studies have reported that the effect of sorafenib on advanced human hepatocellular carcinoma (HCC) is taking place via the inhibition of NF-κB signal transduction. Here we constructed a human HCC Huh7 stable clone with NF-κB-responsive element to drive dual reporter genes, herpes simplex virus thymidine kinase (tk) and firefly luciferase (luc2), and co-transfected with a third red fluorescent protein (rfp) gene, renamed as Huh7/NF-κB-tk-luc2/rfp cells, and combined with bioluminescent imaging (BLI) and red fluorescent protein imaging (RFPI) to monitor the effect of sorafenib on NF-κB activation and tumor inhibition. The results show that sorafenib could suppress the NF-κB-DNA binding activity, and the expression of downstream effector proteins. Notably, the relative photon fluxes obtained from RFPI and BLI, which represent the viable tumor cells and cells with NF-κB activation, decreased after sorafenib treatment by 50 to 65%, and 87.5 to >90%, respectively, suggesting that NF-κB activation is suppressed in viable HCC cells by sorafenib. Simultaneous molecular imaging of the temporal change of NF-κB activity and of viable cells in the same Huh7/NF-κB-tk-luc2/rfp tumors of the animal may reflect the real status of NF-κB activity and the viable tumor cells at the time of imaging.

Red-shifted aequorin-based bioluminescent reporters for in vivo imaging of Ca2 signaling.

PubMed

Curie, Thomas; Rogers, Kelly L; Colasante, Cesare; Brûlet, Philippe

2007-01-01

Real-time visualization of calcium (Ca(2+)) dynamics in the whole animal will enable important advances in understanding the complexities of cellular function. The genetically encoded bioluminescent Ca(2+) reporter green fluorescent protein-aequorin (GA) allows noninvasive detection of intracellular Ca(2+) signaling in freely moving mice. However, the emission spectrum of GA is not optimal for detection of activity from deep tissues in the whole animal. To overcome this limitation, two new reporter genes were constructed by fusing the yellow fluorescent protein (Venus) and the monomeric red fluorescent protein (mRFP1) to aequorin. Transfer of aequorin chemiluminescence energy to Venus (VA) is highly efficient and produces a 58 nm red shift in the peak emission spectrum of aequorin. This substantially improves photon transmission through tissue, such as the skin and thoracic cage. Although the Ca(2+)-induced bioluminescence spectrum of mRFP1-aequorin (RA) is similar to that of aequorin, there is also a small peak above 600 nm corresponding to the peak emission of mRFP1. Small amounts of energy transfer between aequorin and mRFP1 yield an emission spectrum with the highest percentage of total light above 600 nm compared with GA and VA. Accordingly, RA is also detected with higher sensitivity from brain areas. VA and RA will therefore improve optical access to Ca(2+) signaling events in deeper tissues, such as the heart and brain, and offer insight for engineering new hybrid molecules.

Observation of Trapped-Electron Mode Microturbulence in Improved Confinement Reversed-Field Pinch Plasmas

NASA Astrophysics Data System (ADS)

Duff, James R.

This is a dissertation for the completion of a Doctorate of Philosophy in Physics degree granted at the University of Wisconsin-Madison. Density fluctuations in the large-density-gradient region of improved confinement Madison Sym- metric Torus (MST) RFP plasmas exhibit multiple features that are characteristic of the trapped- electron mode (TEM). In fusion relevant plasmas, thermal transport is a key avenue of research in order to achieve a burning plasma. In the reversed field pinch (RFP) magnetic geometry, the dy- namics of conventional plasma discharges are primarily governed by magnetic stochasticity stem- ming from multiple long-wavelength tearing modes, that sustain the RFP discharge but have an adverse effect on the plasma confinement. Using inductive current profile control, these tearing modes are reduced, and global confinement is increased to that expected for comparable tokamak plasma. Under these conditions with certain plasma equilibria, new short-wavelength fluctuations distinct from global tearing modes appear in the spectrum at frequencies f 50 kHz that have normalized perpendicular wavenumbers k⊥rhos ≤ 0.2, and propagate in the electron diamagnetic drift direction. By adjusting the plasma current or the inductive suppression, there are observable variations in the spectral features. They exhibit a critical-gradient threshold, and the fluctuation amplitude increases with a local density gradient dependent parameter. These characteristics are consistent with the predictions of unstable TEMs based on gyrokinetic analysis using the GENE code. This thesis represents the first observation and description of TEM-like instabilities in the RFP geometry.

Development and validation of a high-content bimolecular fluorescence complementation assay for small-molecule inhibitors of HIV-1 Nef dimerization.

PubMed

Poe, Jerrod A; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E

2014-04-01

Nef is a human immunodeficiency virus 1 (HIV-1) accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to nonfluorescent, complementary fragments of yellow fluorescent protein (YFP) and coexpressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus a monomeric red fluorescent protein (mRFP) reporter were expressed from a single vector, separated by picornavirus "2A" linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type versus dimerization-defective Nef were very clearly separated, with Z factors consistently in the 0.6 to 0.7 range. A fully automated pilot screen of the National Cancer Institute Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.

In vivo near-infrared fluorescence imaging of Leishmania amazonensis expressing infrared fluorescence protein (iRFP) for real-time monitoring of cutaneous leishmaniasis in mice.

PubMed

Oliveira, Janaina Correia; da Silva, Aline Caroline; Oliveira, Renato Antonio Dos Santos; Pereira, Valéria Rêgo Alves; Gil, Laura Helena Vega Gonzales

2016-11-01

The use of Leishmania amazonensis-infected BALB/c mice is an important model for the study of experimental cutaneous leishmaniasis. Here we report the development of a non-invasive method to directly evaluate and measure parasite burden during the course of the infection, based on the near-infrared fluorescence detection of a recombinant L. amazonensis strain. So, we generated a L. amazonensis strain that stably expresses the near-infrared protein (iRFP) gene and compared the maintenance of its vitro and in vivo characteristics, such as fitness, pathogenicity and fluorescence emission. After that, we followed the disease development, as well as the parasite burden in BALB/c mice footpads infected with L. amazonensis-iRFP, by using an in vivo near-infrared fluorescence scanner. In vitro results showed a linear correlation between the fluorescence emission and the number of parasites. The in vivo study showed that the use of iRFP-transfected L. amazonensis enables the monitoring of parasite burden by measuring fluorescence signals. Therefore, this technique can be confidently used to directly monitor parasitic load and infection overtime and could be an excellent tool for in vitro and in vivo screening of anti-leishmanial drugs and vaccine efficiency. This is the first report of the use of the near-infrared fluorescence imaging technique for monitoring in vivo cutaneous leishmaniasis. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and Validation of a High-Content Bimolecular Fluorescence Complementation Assay for Small Molecule Inhibitors of HIV-1 Nef Dimerization

PubMed Central

Poe, Jerrod A.; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E.

2014-01-01

Nef is an HIV-1 accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to non-fluorescent, complementary fragments of YFP and co-expressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus an mRFP reporter were expressed from a single vector, separated by picornavirus ‘2A’ linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type vs. dimerization-defective Nef were very clearly separated, with Z-factors consistently in the 0.6–0.7 range. A fully automated pilot screen of the NIH Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function. PMID:24282155

Impact of helical boundary conditions in MHD modeling of RFP and tokamak plasmas

NASA Astrophysics Data System (ADS)

Bonfiglio, D.; Cappello, S.; Escande, D. F.; Piovesan, P.; Veranda, M.; Chacón, L.

2012-10-01

Helical boundary conditions imposed by the active control system of the RFX-mod device provide a handle to govern the plasma dynamics in both RFP and Ohmic tokamak discharges [1]. By applying an edge radial magnetic field with proper helicity, it is possible to increase the persistence of the spontaneous helical RFP states at high current,and to stimulate them also at low current or high density. Helical BCs even allow to access helical states with different helicity than the spontaneous one [2]. In Ohmic tokamak operation at q(a)<2, the presence of the 2/1 RWM reduces the sawtoothing activity of the 1/1 internal kink, which takes a stationary snake-like character instead. Many of these features are qualitatively reproduced in 3D nonlinear MHD modeling. We study the impact of helical BCs on the MHD dynamics in both RFP and tokamak with two successfully benchmarked numerical tools, SpeCyl and PIXIE3D [3]. We recover the bifurcation from a sawtooth to a snake solution when imposing a 2/1 BC in the tokamak case and we interpret this as a toroidal/nonlinear coupling effect. We show that the bifurcation is more easily stimulated with a 1/1 BC.[4pt] [1] P. Piovesan, invited talk this meeting[0pt] [2] M. Veranda et al EPS-ICPP Conference (2012) P4.004[0pt] [3] D. Bonfiglio et al Phys. Plasmas (2010)

Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

PubMed

Ganini, Douglas; Leinisch, Fabian; Kumar, Ashutosh; Jiang, JinJie; Tokar, Erik J; Malone, Christine C; Petrovich, Robert M; Mason, Ronald P

2017-08-01

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H 2 O 2 ) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H 2 O 2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O 2 •- ) and H 2 O 2 in the presence of NADH. Generation of the free radical O 2 •- and H 2 O 2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies. Published by Elsevier B.V.

Protein Degradation in a TX-TL Cell-free Expression System Using ClpXP Protease

DTIC Science & Technology

2014-07-14

function in TX-TL, as well as bacteriophage assembly [2, 6]. Circuits can also be prototyped from basic parts within 8 hours, avoiding cloning and...mRFP, and Venus and variants eGFP-ssrA, mRFP-ssrA, and Venus-ssrA, coding sequences were cloned into a T7-lacO inducible vector containing a N...12672L12677.! 6.! Shin,!J.,!P.!Jardine,!and!V.!Noireaux,!Genome(Replication,(Synthesis,(and( Assembly(of(the( Bacteriophage (T7(in(a(Single(Cell9Free

ESMD Risk Management Workshop: Systems Engineering and Integration Risks

NASA Technical Reports Server (NTRS)

Thomas, L. Dale

2005-01-01

This report has been developed by the National Aeronautics and Space Administration (NASA) Exploration Systems Mission Directorate (ESMD) Risk Management team in close coordination with the Systems Engineering Team. This document provides a point-in-time, cumulative, summary of key lessons learned derived from the SE RFP Development process. Lessons learned invariably address challenges and risks and the way in which these areas have been addressed. Accordingly the risk management thread is woven throughout the document.

A Prospective One-Year Microbiologic Survey of Combined Pneumonia and Respiratory Failure.

PubMed

Fisher, Kristen; Trupka, Tracy; Micek, Scott T; Juang, Paul; Kollef, Marin H

2017-10-01

Pneumonia and respiratory failure are common problems in the intensive care unit (ICU) setting, often occurring together. The relative prevalence of pneumonia types (community acquired, hospital acquired, ventilator associated) and causative pathogens is not well described in patients with respiratory failure. This was a prospective observational cohort study conducted in the medical ICU (34 beds) of Barnes-Jewish Hospital, an academic referral center of 1,300 beds from January 2016-December 2016. All patients who were prospectively adjudicated to have respiratory failure and pneumonia (RFP) regardless of pneumonia type were classified into one of four microbiologic categories: pathogen negative, antibiotic-susceptible pathogen (according to ceftriaxone susceptibility), antibiotic-resistant pathogen, and viruses. The primary outcomes assessed were the hospital mortality rate and inappropriate initial antibiotic therapy (IIAT) for non-viral pathogens. Among 364 consecutive patients with RFP, 63 (17.3%) had organisms that were antibiotic susceptible, 104 (28.6%) had antibiotic-resistant organisms, 118 (32.4%) were pathogen negative, and 79 (21.7%) had viral infections. For these categories, IIAT occurred in 3.2%, 21.2%, 0.8%, and 0, respectively (p < 0.001). Vasopressor-requiring shock was present in 61.9%, 72.1%, 68.6%, and 67.1%, respectively (p = 0.585), and the hospital mortality rates were 27.0%, 48.1%, 31.4%, and 36.7%, respectively (p = 0.020). Multivariable logistic regression analysis identified IIAT as an independent predictor of in-hospital death (adjusted odds ratio 5.28; 95% confidence interval 2.72-10.22; p = 0.012). Male gender, increasing Acute Physiology and Chronic Health Evaluation (APACHE) II scores, greater age, and the presence of shock also predicted death. Microbiologic categorization of patients with RFP suggests that antibiotic-resistant pathogens and viruses are associated with the highest mortality rates. Vasopressor-requiring shock was common regardless of the microbiologic categorization of RFP. Future development and use of rapid diagnostics and novel therapeutics targeting specific RFP pathogens may allow more timely administration of appropriate antimicrobial therapy and enhance antibiotic stewardship practices.

Effect of TNF-alpha blockade on coagulopathy and endothelial cell activation in xenoperfused porcine kidneys.

PubMed

Ramackers, Wolf; Klose, Johannes; Tiede, Andreas; Werwitzke, Sonja; Rataj, Dennis; Friedrich, Lars; Johanning, Kai; Vondran, Florian W R; Bergmann, Sabine; Schuettler, Wolfgang; Bockmeyer, Clemens Luitpold; Becker, Jan Ulrich; Klempnauer, Jürgen; Winkler, Michael

2015-01-01

Following pig-to-primate kidney transplantation, endothelial cell activation and xenogenic activation of the recipient's coagulation eventually leading to organ dysfunction and microthrombosis can be observed. In this study, we examined the effect of a TNF-receptor fusion protein (TNF-RFP) on endothelial cell activation and coagulopathy utilizing an appropriate ex vivo perfusion system. Using an ex vivo perfusion circuit based on C1-Inhibitor (C1-Inh) and low-dose heparin administration, we have analyzed consumptive coagulopathy following contact of human blood with porcine endothelium. Porcine kidneys were recovered following in situ cold perfusion with Histidine-tryptophan-ketoglutarate (HTK) organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled porcine or human AB blood. The experiments were performed in three individual groups: autologous perfusion (n = 5), xenogenic perfusion without any further pharmacological intervention (n = 10), or with addition of TNF-RFP (n = 5). After perfusion, tissue samples were obtained for real-time PCR and immunohistological analyses. Endothelial cell activation was assessed by measuring the expression levels of E-selectin, ICAM-1, and VCAM-1. Kidney survival during organ perfusion with human blood, C1-Inh, and heparin, but without any further pharmacological intervention was 126 ± 78 min. Coagulopathy was observed with significantly elevated concentrations of D-dimer and thrombin-antithrombin complex (TAT), resulting in the formation of multiple microthrombi. Endothelial cell activation was pronounced, as shown by increased expression of E-selectin and VCAM-1. In contrast, pharmacological intervention with TNF-RFP prolonged organ survival to 240 ± 0 min (max. perfusion time; no difference to autologous control). Formation of microthrombi was slightly reduced, although not significantly, if compared to the xenogenic control. D-dimer and TAT were elevated at similar levels to the xenogenic control experiments. In contrast, endothelial cell activation, as shown by real-time PCR, was significantly reduced in the TNF-RFP group. We conclude that although coagulopathy was not affected, TNF-RFP is able to suppress inflammation occurring after xenoperfusion in this ex vivo perfusion model. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics.

PubMed

Hontani, Yusaku; Shcherbakova, Daria M; Baloban, Mikhail; Zhu, Jingyi; Verkhusha, Vladislav V; Kennis, John T M

2016-11-18

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.

Measurements of fast ion spatial dynamics during magnetic activity in the RFP

NASA Astrophysics Data System (ADS)

Goetz, J. A.; Anderson, J. K.; Bonofiglo, P.; Kim, J.; McConnell, R.; Magee, R. M.

2017-10-01

Fast ions in the RFP are only weakly affected by a stochastic magnetic field and behave nearly classically in concentration too low to excite Alfvenic activity. At high fast ion concentration sourced by H-NBI in 300kA RFP discharges, a substantial drop in core-localized high pitch fast ions is observed during bursts of coupled EPM and IAE (magnetic island-induced Alfven eigenmode) activity (100-200kHz) through neutral particle analysis. Sourcing instead fast deuterium with NBI, the DD fusion products can measure the dynamics of the fast ion density profile. Both a collimated neutron detector and a new 3MeV fusion proton detector loaned by TriAlpha Energy measure the fast ion density profile with 5cm spatial resolution and 100 μs temporal resolution. In D-NBI, the bursting EPM is excited at slightly lower frequency and the IAE activity is nearly absent, likely due to an isotope effect and loss of wave-particle interaction. In these cases, neutral particle analysis shows little change in the core-localized high pitch fast ion content, and the fusion product profile indicates little change in the fast ion density profile, leaving unexplained the mechanism removing EPM drive. We measure a substantial redistribution of the fast ion profile due to strong lower-frequency ( 30kHz) MHD activity that accompanies the current profile relaxation in the RFP. Profile flattening is strongest in low bulk density discharges, which often occur with a total increase in global neutron flux from acceleration of the beam ions. Work supported by US DoE.

Specialized mouse embryonic stem cells for studying vascular development.

PubMed

Glaser, Drew E; Burns, Andrew B; Hatano, Rachel; Medrzycki, Magdalena; Fan, Yuhong; McCloskey, Kara E

2014-01-01

Vascular progenitor cells are desirable in a variety of therapeutic strategies; however, the lineage commitment of endothelial and smooth muscle cell from a common progenitor is not well-understood. Here, we report the generation of the first dual reporter mouse embryonic stem cell (mESC) lines designed to facilitate the study of vascular endothelial and smooth muscle development in vitro. These mESC lines express green fluorescent protein (GFP) under the endothelial promoter, Tie-2, and Discomsoma sp. red fluorescent protein (RFP) under the promoter for alpha-smooth muscle actin (α-SMA). The lines were then characterized for morphology, marker expression, and pluripotency. The mESC colonies were found to exhibit dome-shaped morphology, alkaline phosphotase activity, as well as expression of Oct 3/4 and stage-specific embryonic antigen-1. The mESC colonies were also found to display normal karyotypes and are able to generate cells from all three germ layers, verifying pluripotency. Tissue staining confirmed the coexpression of VE (vascular endothelial)-cadherin with the Tie-2 GFP+ expression on endothelial structures and smooth muscle myosin heavy chain with the α-SMA RFP+ smooth muscle cells. Lastly, it was verified that the developing mESC do express Tie-2 GFP+ and α-SMA RFP+ cells during differentiation and that the GFP+ cells colocalize with the vascular-like structures surrounded by α-SMA-RFP cells. These dual reporter vascular-specific mESC permit visualization and cell tracking of individual endothelial and smooth muscle cells over time and in multiple dimensions, a powerful new tool for studying vascular development in real time.

Illinois Clean Coal Institute 2005 annual report. Final technical report for the period September 1st, 2004, through August 31, 2005 on projects funded by the Illinois Department of Commerce and Economic Opportunity

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

NONE

2005-11-08

This final technical report contains the abstracts and executive summaries of projects funded through the Illinois Clean Coal Institute solicitation entitled 'Request for proposals No. 04-1(ICCI/RFP04-1)'. Support of these projects is by the Office of Coal Development and Department of Commerce and Economic Opportunity. The projects fall into the following categories: advanced coal mining technologies; coal preparation and coal production business practice; management of coal combustion byproducts; commercialization and technology transfer. Final project extensions are also recorded.

Advanced Development Model (ADM) Program for the Shipboard Meteorological and Oceanographic Observing System (SMOOS).

DTIC Science & Technology

1986-05-20

SOURCE DOCUMENTS 1. N62269-84- R -0309 SHOOS Request for Proposal (RFP) 2. N62269-84-C-0309, SOOS Advanced Development Model (ADM) Contract 3. SA-TS-7833...b.n . II ii t .. ~ r 5* t **v *...to, $me$ d.v.... 0", tc htlo I . .. vtod. - BT 9S.td Fig. 3.5- Susse Funtina AlloatinMtri ittnits 0~~~~~~4. vii...LSyes T ttiL e Request To r Transmit 9 Upper Air II aaDspDatalay *S Ocear Sea Depth I71 dg I Dept Data3ttu tts Data Display Data Display Controller

[Identification of the authentic quality of Longdanxiegan pill by systematic quantified fingerprint method based on three wavelength fusion chromatogram].

PubMed

Sun, Guoxiang; Zhang, Jingxian

2009-05-01

The three wavelength fusion high performance liquid chromatographic fingerprin (TWFFP) of Longdanxiegan pill (LDXGP) was established to identify the quality of LDXGP by the systematic quantified fingerprint method. The chromatographic fingerprints (CFPs) of the 12 batches of LDXGP were determined by reversed-phase high performance liquid chromatography. The technique of multi-wavelength fusion fingerprint was applied during processing the fingerprints. The TWFFPs containing 63 co-possessing peaks were obtained when choosing baicalin peak as the referential peak. The 12 batches of LDXGP were identified with hierarchical clustering analysis by using macro qualitative similarity (S(m)) as the variable. According to the results of classification, the referential fingerprint (RFP) was synthesized from 10 batches of LDXGP. Taking the RFP for the qualified model, all the 12 batches of LDXGP were evaluated by the systematic quantified fingerprint method. Among the 12 batches of LDXGP, 9 batches were completely qualified, the contents of 1 batch were obviously higher while the chemical constituents quantity and distributed proportion in 2 batches were not qualified. The systematic quantified fingerprint method based on the technique of multi-wavelength fusion fingerprint ca effectively identify the authentic quality of traditional Chinese medicine.

Towards Multiscale Interactions Between Tearing Modes and Microturbulence

NASA Astrophysics Data System (ADS)

Williams, Z. R.; Pueschel, M. J.; Terry, P. W.

2017-10-01

Work on the Madison Symmetric Torus Reversed-Field Pinch (RFP) has shown that large-scale tearing modes present in standard operation are highly detrimental to confinement. These tearing modes, even when reduced in improved confinement regimes of operation, significantly affect zonal flow activity and play a large role in setting microturbulent-induced transport levels. Previous gyrokinetic work has shown that a small but finite tearing fluctuation amplitude is necessary to produce transport values in agreement with experimental observation. This has previously been implemented via an ad-hoc, constant-in-time A∥ perturbation. This work details self-consistent modeling of tearing fluctuations in the RFP using the Gene code via the inclusion of a current gradient drive incorporated into the background distribution function. Tearing mode growth rates calculated from gyrokinetic simulations are benchmarked with results from fluid theory. Additionally, first results from multiscale Gene simulations describing tearing mode interactions with RFP microturbulence are presented. This work is supported by the U.S. Department of Energy, Grant No. DE-FG02-85ER-53121.

Sofia Science Working Group

NASA Technical Reports Server (NTRS)

Zmuidzinas, J.

1997-01-01

The purpose of this grant was to enable the Principal Investigator (P.I.) to travel to and participate in the meetings and activities of the NASA SOFIA Science Working Group (SSWG), and to spend time working on some of the associated technical issues relating to the SOFIA (Stratospheric Observatory for Infrared Astronomy) project. The SOFIA Science Working Group was established to help develop the plans and specifications for the next-generation airborne observatory ("SOFIA"), which is now under development. The P.I. was asked to serve on the SSWG due to his experience in airborne astronomy: he has developed several astronomical instruments for the Kuiper Airborne Observatory NASA's previous airborne astronomy platform (which was decommissioned in 1995 in preparation for SOFIA). SOFIA, which will be a 747 SP aircraft carrying a 2.7 meter diameter telescope, is a joint project sponsored by NASA and DLR (the German space agency), and is now under development by a consortium including Universities Space Research Association (USRA), Raytheon, Sterling Software, and United Airlines. Further details on the SOFIA project can be found on the internet at http: //sofia. arc. nasa. gov. Rather than develop the SOFIA observatory in-house, NASA decided to privatize the project by issuing a Request for Proposals (RFP). The respondents to this RFP were consortia of private organizations which together had the required facilities and expertise to be able to carry out the project; the winner was the group led by USRA. One of the main roles of the SSWG was to help develop the technical specifications for the SOFIA observatory. In particular, the SSWG provided advice to NASA on the specifications that were written into the RFP, particularly those which had an important impact on the scientific productivity of the observatory. These specifications were discussed at the meetings of the SSWG, which were held primarily at NASA/Ames (in California) and at NASA Headquarters (in Washington DC). Apart from these meetings, members of the SSWG were expected to perform more detailed analyses of the impact of certain parameters and specifications on the performance of astronomical instruments. The SSWG ended its activities with the selection of the USRA team in January 1997.

The Aluminum Falcon: a Low Cost Modern Commercial Transport

NASA Technical Reports Server (NTRS)

Bryant, Mark; Hernandez, Estela; King, Gregory; Lor, Alex Choua; Musser, Jana; Trigs, Deanne; Yee, Susan

1994-01-01

The American Institute of Aeronautics and Astronautics (AIAA) released a Request For Proposal (RFP) in the form of an undergraduate design competition for a 153 passenger jet transport with a range of 3,000 nautical miles. The primary requirement for this aircraft was low cost, both in acquisition and operation, with a technology availability date of the year 2000. This report presents the Non-Solo Design Group's response to the RFP, the Aluminum Falcon (AF-1). Non-Solo's approach to development was to take the best elements of seven individual preliminary designs, then combine and refine them. The resulting aircraft meets or exceeds all requirements of both the RFP and the Federal Aviation Administration (FAA). Highlights include a revolutionary wing planform, known as an M-wing, which offers many advantages over a conventional aft swept wing. For example, the M-wing lessens the travel in the aircraft center of gravity caused by fuel being stored in the wing. It also reduces the amount of torque imposed on the center wing box because more of the lifting load acts near the fuselage joint, rather than behind it. In essence, the M-wing offers the best of both worlds: using a forward swept wing root places the aerodynamic center of the wing further forward and allows the landing gear to be placed without the use of a yahudi. At the same time, with the outboard section swept backward the tip retains an amount of aeroelastic dampening that is lost on a completely forward swept wing. The result is a wing which has many advantages of a straight, unswept wings without the severe compressibility effects at high Mach numbers. Other highlights include judicious use of composites, giving recognition to the importance of weight and its effect on aircraft cost and performance, and an advanced passenger entertainment system which can be used as a source of revenue for the airlines. This aircraft meets the low-cost doctrine with an acquisition cost of $29 million and a direct operating cost of 3.5 cents per seat mile. The AF-1 incorporates new ideas with existing technology to result in an aircraft that will retain market viability well into the next century.

Proceedings of the Open Sessions of the Workshop on Imaging Trackers and Autonomous Acquisition Applications for Missile Guidance Held at Redstone Arsenal, Alabama on 19-20 November 1979

DTIC Science & Technology

1979-11-01

a generalized cooccurrence matrix. Describing image texture is an important problem in the design of image understanding systems . Applications as...display system design optimization and video signal processing. Based on a study by Southern Research Institute , a number of options were identified...Specification for Target Acquisition Designation System (U), RFP # AMC-DP-AAH-H4020, i2 Apr 77. 4. Terminal Homing Applications of Solid State Image

A Needs Assessment of Air Force Systems Command Buying Activity Acquisition Management Information System (AMIS) Users

DTIC Science & Technology

1989-09-01

Guidelines Generation #2 b. Electronic Submission of Commerce Business Daily ( CBD ) Notices #6 c. On-line Debarred/Suspended List #5 d. On-Line Contract...a number of years. Reality of system differs from manual. One reference - easy to follow, block by block - is needed. -Imaging and CBD electronic...milestones are tracked - and those milestones should be monitored as a natural outcome of thc process - e.g. A milestone is noted when the RFP is

Improved particle confinement in transition from multiple-helicity to quasi-single-helicity regimes of a reversed-field pinch.

PubMed

Frassinetti, L; Predebon, I; Koguchi, H; Yagi, Y; Hirano, Y; Sakakita, H; Spizzo, G; White, R B

2006-10-27

The quasi-single-helicity (QSH) state of a reversed-field pinch (RFP) plasma is a regime in which the RFP configuration can be sustained by a dynamo produced mainly by a single tearing mode and in which a helical structure with well-defined magnetic flux surfaces arises. In this Letter, we show that spontaneous transitions to the QSH regime enhance the particle confinement. This improvement is originated by the simultaneous and cooperative action of the increase of the magnetic island and the reduction of the magnetic stochasticity.

Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.

PubMed

Wallrabe, Horst; Sun, Yuansheng; Fang, Xiaolan; Periasamy, Ammasi; Bloom, George S

2015-06-01

Experiments using live cell 3-color Förster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells. © 2015 International Society for Advancement of Cytometry.

Density-Gradient-Driven trapped-electron-modes in improved-confinement RFP plasmas

NASA Astrophysics Data System (ADS)

Duff, James

2016-10-01

Short wavelength density fluctuations in improved-confinement MST plasmas exhibit multiple features characteristic of the trapped-electron-mode (TEM), strong evidence that drift wave turbulence emerges in RFP plasmas when transport associated with MHD tearing is reduced. Core transport in the RFP is normally governed by magnetic stochasticity stemming from long wavelength tearing modes that arise from current profile peaking. Using inductive control, the tearing modes are reduced and global confinement is increased to values expected for a comparable tokamak plasma. The improved confinement is associated with a large increase in the pressure gradient that can destabilize drift waves. The measured density fluctuations have frequencies >50 kHz, wavenumbers k_phi*rho_s<0.14, and propagate in the electron drift direction. Their spectral emergence coincides with a sharp decrease in fluctuations associated with global tearing modes. Their amplitude increases with the local density gradient, and they exhibit a density-gradient threshold at R/L_n 15, higher than in tokamak plasmas by R/a. the GENE code, modified for RFP equilibria, predicts the onset of microinstability for these strong-gradient plasma conditions. The density-gradient-driven TEM is the dominant instability in the region where the measured density fluctuations are largest, and the experimental threshold-gradient is close to the predicted critical gradient for linear stability. While nonlinear analysis shows a large Dimits shift associated with predicted strong zonal flows, the inclusion of residual magnetic fluctuations causes a collapse of the zonal flows and an increase in the predicted transport to a level close to the experimentally measured heat flux. Similar circumstances could occur in the edge region of tokamak plasmas when resonant magnetic perturbations are applied for the control of ELMs. Work supported by US DOE.

75 FR 33562 - Approval and Promulgation of Implementation Plans and Designations of Areas for Air Quality...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-14

...EPA is proposing to determine that the Birmingham, Alabama, nonattainment area for the 2006 24-hour fine particulate matter (PM2.5) National Ambient Air Quality Standard (NAAQS) has attained the 2006 24-hour PM2.5 NAAQS. This proposed determination is based upon complete, quality assured, quality controlled, and certified ambient air monitoring data for the years 2007-2009 showing that this area has monitored attainment of the 2006 24-hour PM2.5 NAAQS. If this proposed determination is made final, the requirement for the State of Alabama to submit an attainment demonstration and associated reasonably available control measures (RACM), reasonable further progress (RFP) plan, contingency measures, and other planning State Implementation Plans (SIPs) related to attainment of the 2006 24-hour PM2.5 standard for the Birmingham, Alabama, PM2.5 nonattainment area, shall be suspended for as long as this area continues to meet the 2006 24-hour PM2.5 NAAQS.

IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators.

PubMed

Zheng, S

2016-01-01

Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing. © 2016 Elsevier Inc. All rights reserved.

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

PubMed

Ng, Henry; Dean, Neta

2017-01-01

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

Post-mortem re-cloning of a transgenic red fluorescent protein dog.

PubMed

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

2011-12-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

Overview of MST Research

NASA Astrophysics Data System (ADS)

Sarff, J. S.; MST Team

2011-10-01

MST progress in advancing the RFP for (1) fusion plasma confinement with minimal external magnetization, (2) toroidal confinement physics, and (3) basic plasma physics is summarized. New tools and diagnostics are accessing physics barely studied in the RFP. Several diagnostic advances are important for ITER/burning plasma. A 1 MW neutral beam injector operates routinely for fast ion, heating, and transport investigations. Energetic ions are also created spontaneously by tearing mode reconnection, reminiscent of astrophysical plasmas. Classical confinement of impurity ions is measured in reduced-tearing plasmas. Fast ion slowing-down is also classical. Alfven-eigenmode-like activity occurs with NBI, but apparently not TAE. Stellarator-like helical structure appears in the core of high current plasmas, with improved confinement characteristics. FIR interferometry, Thomson scattering, and HIBP diagnostics are beginning to explore microturbulence scales, an opportunity to exploit the RFP's high beta and strong magnetic shear parameter space. A programmable power supply for the toroidal field flexibly explores scenarios from advanced inductive profile control to low current tokamak operation. A 1 MW 5.5 GHz source for electron Bernstein wave injection is nearly complete to investigate heating and current drive in over-dense plasmas. Supported by DOE and NSF.

Density-Gradient-Driven trapped-electron-modes in improved-confinement RFP plasmas

NASA Astrophysics Data System (ADS)

Duff, James; Sarff, John; Ding, Weixing; Brower, David; Parke, Eli; Chapman, Brett; Terry, Paul; Pueschel, M. J.; Williams, Zach

2017-10-01

Short wavelength density fluctuations in improved-confinement MST plasmas exhibit multiple features characteristic of the trapped-electron-mode (TEM). Core transport in the RFP is normally governed by magnetic stochasticity stemming from long wavelength tearing modes that arise from current profile peaking, which are suppressed via inductive control for this work. The improved confinement is associated with an increase in the pressure gradient that can destabilize drift waves. The measured density fluctuations have f 50 kHz, kϕρs < 0.14 , and propagate in the electron drift direction. Their spectral emergence coincides with a sharp decrease in global tearing mode associated fluctuations, their amplitude increases with local density gradient, and they exhibit a density-gradient threshold at R /Ln 15 . The GENE code, modified for the RFP, predicts the onset of density-gradient-driven TEM for these strong-gradient plasma conditions. While nonlinear analysis shows a large Dimits shift associated with predicted strong zonal flows, the inclusion of residual magnetic fluctuations, comparable to experimental magnetic fluctuations, causes a collapse of the zonal flows and an increase in the predicted transport to a level close to the experimentally measured heat flux. Work supported by US DOE.

Pharmacy benefit management contracting: an assessment from a recent public sector procurement experience.

PubMed

Bae, Jay P; Justice, Paul G

2002-01-01

In order to contain the cost of pharmaceuticals while preserving access to medically necessary drugs, Georgia state government competitively selected a single vendor in May of 2000 to manage combined pharmacy benefits under all of the state's health programs. By initiating this procedure, it intended to maximize the state's purchasing power and improve efficiency while streamlining the administrative structure. Synthesizing information from the request for proposal (RFP) and technical proposals submitted by 11 pharmacy benefit managers (PBMs) in response, we describe a model of public sector PBM contracting approach and present an assessment of the industry's service capability and performance statistics. Payers who have been using PBM services may find it interesting to compare their experience with the recent Georgia experience. Those who are considering contracting with a PBM will find the assessment of the PBM industry timely and informative.

Global modelling of plasma-wall interaction in reversed field pinches

NASA Astrophysics Data System (ADS)

Bagatin, M.; Costa, S.; Ortolani, S.

1989-04-01

The impurity production and deuterium recycling mechanisms in ETA—BETA II and RFX are firstly discussed by means of a simple model applicable to a stationary plasma interacting with the wall. This gives the time constant and the saturation values of the impurity concentration as a function of the boundary temperature and density. If the latter is sufficiently high, the impurity buildup in the main plasma becomes to some extent stabilized by the shielding effect of the edge. A self-consistent global model of the time evolution of an RFP plasma interacting with the wall is then described. The bulk and edge parameters are derived by solving the energy and particle balance equations incorporating some of the basic plasma-surface processes, such as sputtering, backscattering and desorption. The application of the model to ETA-BETA II confirms the impurity concentrations of the light and metal impurities as well as the time evolution of the average electron density found experimentally under different conditions. The model is then applied to RFX, a larger RFP experiment under construction, whose wall will be protected by a full graphite armour. The time evolution of the discharge shows that carbon sputtering could increase Zeff to ~ 4, but without affecting significantly the plasma performance.

Formaldehyde fixation is detrimental to actin cables in glucose-depleted S. cerevisiae cells

PubMed Central

Vasicova, Pavla; Rinnerthaler, Mark; Haskova, Danusa; Novakova, Lenka; Malcova, Ivana; Breitenbach, Michael; Hasek, Jiri

2016-01-01

Actin filaments form cortical patches and emanating cables in fermenting cells of Saccharomyces cerevisiae. This pattern has been shown to be depolarized in glucose-depleted cells after formaldehyde fixation and staining with rhodamine-tagged phalloidin. Loss of actin cables in mother cells was remarkable. Here we extend our knowledge on actin in live glucose-depleted cells co-expressing the marker of actin patches (Abp1-RFP) with the marker of actin cables (Abp140-GFP). Glucose depletion resulted in appearance of actin patches also in mother cells. However, even after 80 min of glucose deprivation these cells showed a clear network of actin cables labeled with Abp140-GFP in contrast to previously published data. In live cells with a mitochondrial dysfunction (rho0 cells), glucose depletion resulted in almost immediate appearance of Abp140-GFP foci partially overlapping with Abp1-RFP patches in mother cells. Residual actin cables were clustered in patch-associated bundles. A similar overlapping “patchy” pattern of both actin markers was observed upon treatment of glucose-deprived rho+ cells with FCCP (the inhibitor of oxidative phosphorylation) and upon treatment with formaldehyde. While the formaldehyde-targeted process stays unknown, our results indicate that published data on yeast actin cytoskeleton obtained from glucose-depleted cells after fixation should be considered with caution. PMID:28357356

Open system environment procurement

NASA Technical Reports Server (NTRS)

Fisher, Gary

1994-01-01

Relationships between the request for procurement (RFP) process and open system environment (OSE) standards are described. A guide was prepared to help Federal agency personnel overcome problems in writing an adequate statement of work and developing realistic evaluation criteria when transitioning to an OSE. The guide contains appropriate decision points and transition strategies for developing applications that are affordable, scalable and interoperable across a broad range of computing environments. While useful, the guide does not eliminate the requirement that agencies posses in-depth expertise in software development, communications, and database technology in order to evaluate open systems.

Alpha effect of Alfv{acute e}n waves and current drive in reversed-field pinches

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

Litwin, C.; Prager, S.C.

Circularly polarized Alfv{acute e}n waves give rise to an {alpha}-dynamo effect that can be exploited to drive parallel current. In a {open_quotes}laminar{close_quotes} magnetic the effect is weak and does not give rise to significant currents for realistic parameters (e.g., in tokamaks). However, in reversed-field pinches (RFPs) in which magnetic field in the plasma core is stochastic, a significant enhancement of the {alpha} effect occurs. Estimates of this effect show that it may be a realistic method of current generation in the present-day RFP experiments and possibly also in future RFP-based fusion reactors. {copyright} {ital 1998 American Institute of Physics.}

Post-mortem re-cloning of a transgenic red fluorescent protein dog

PubMed Central

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo

2011-01-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification. PMID:22122908

Measurement of ion velocities in the locked Single Helical Axis state in MST RFP plasmas

NASA Astrophysics Data System (ADS)

Boguski, J.; Nornberg, M. D.; Chapman, B. E.; Cianciosa, M.; den Hartog, D. J.; Craig, D.; McCollam, K. J.; Nishizawa, T.; Xing, Z. A.

2017-10-01

Charge Exchange Recombination Spectroscopy (CHERS) provides the first core-localized measurements of the 3D ion flow structure in Single Helical Axis (SHAx) plasmas. In high-current and low-density (large Lundquist number) RFP plasmas, the island associated with the innermost resonant tearing mode can grow to large amplitude and envelop the magnetic axis creating a 3D equilibrium. Measurements of the flow profile with various orientations (phases) of the helical structure relative to the CHERS diagnostic were achieved by locking the plasma with resonant magnetic perturbations. The flows persist despite mode locking, and are correlated with the amplitude and phase of the innermost resonant tearing mode. At mid-radius, a dominantly m =2 poloidal flow structure appears relative to the phase of the helical core. Near the core, non-axisymmetric flows become less pronounced, and cannot be distinguished at the innermost radii. These results place more significant constraints on the nature of the flow structure than previous line-integrated spectroscopy measurements and challenge predictions of visco-resistive MHD models of these helical RFP plasmas. This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Fusion Energy Sciences program under Award No. DE-FC02-05ER54814.

Resistivity profile effects in numerical magnetohydrodynamic simulations of the reversed-field pinch

NASA Astrophysics Data System (ADS)

Sätherblom, H.-E.; Mazur, S.; Nordlund, P.

1996-12-01

The influence of the resistivity profile on reversed-field pinch (RFP) dynamics is investigated numerically using a three-dimensional resistive magnetohydrodynamic code. This investigation is motivated by experimental observations on the EXTRAP-T1 RFP (Nordlund P et al 1994 Int. Conf. Plasma Physics and Controlled Nuclear Fusion Research IAEA-CN-60/A6/C-P-6). Two cases with profiles mainly differing in the edge region, i.e. in the region outside the reversal surface, are simulated. It is found that increasing the resistivity in this region results in a factor of two increase in magnetic fluctuation energy and an equal amount in the fluctuation-induced electric field. In spite of this, the parallel current decreases in the edge region, resulting in a factor two reduction of the field reversal ratio. The dynamics become more irregular and the characteristic timescale is reduced. The final state is characterized by a higher loop voltage, slightly lower values of the total (fluctuating plus mean part) magnetic energy and the magnetic helicity, but almost unchanged Taylor relaxation ratio. The results indicate that the edge region can be important for RFP confinement since cooling of the plasma in this region can lead to an increased fluctuation level and degraded performance.

Chaos and simple determinism in reversed field pinch plasmas: Nonlinear analysis of numerical simulation and experimental data

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

Watts, Christopher A.

In this dissertation the possibility that chaos and simple determinism are governing the dynamics of reversed field pinch (RFP) plasmas is investigated. To properly assess this possibility, data from both numerical simulations and experiment are analyzed. A large repertoire of nonlinear analysis techniques is used to identify low dimensional chaos in the data. These tools include phase portraits and Poincare sections, correlation dimension, the spectrum of Lyapunov exponents and short term predictability. In addition, nonlinear noise reduction techniques are applied to the experimental data in an attempt to extract any underlying deterministic dynamics. Two model systems are used to simulatemore » the plasma dynamics. These are the DEBS code, which models global RFP dynamics, and the dissipative trapped electron mode (DTEM) model, which models drift wave turbulence. Data from both simulations show strong indications of low dimensional chaos and simple determinism. Experimental date were obtained from the Madison Symmetric Torus RFP and consist of a wide array of both global and local diagnostic signals. None of the signals shows any indication of low dimensional chaos or low simple determinism. Moreover, most of the analysis tools indicate the experimental system is very high dimensional with properties similar to noise. Nonlinear noise reduction is unsuccessful at extracting an underlying deterministic system.« less

Fluorescence diffuse tomography for detection of RFP-expressed tumors in small animals

NASA Astrophysics Data System (ADS)

Turchin, Ilya V.; Savitsky, Alexander P.; Kamensky, Vladislav A.; Plehanov, Vladimir I.; Meerovich, Irina G.; Arslanbaeva, Lyaisan R.; Jerdeva, Viktoria V.; Orlova, Anna G.; Kleshnin, Mikhail S.; Shirmanova, Marina V.; Fiks, Ilya I.

2007-02-01

Conventional optical imaging is restricted with tumor size due to high tissue scattering. Labeling of tumors by fluorescent markers improves sensitivity of tumor detection thus increasing the value of optical imaging dramatically. Creation of tumor cell lines transfected with fluorescent proteins gives the possibility not only to detect tumor, but also to conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney HEK-293 Phoenix were transfected with DsRed-Express and TurboRFP genes. Emission of RFP in the long-wave optical range permits detection of the deeply located tumors, which is essential for whole-body imaging. Only special tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable noninvasive investigation of the internal structure of biological tissue. FDT setup for monitoring of tumor growth in small animals has been created. An animal is scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at the 532 nm wavelength. In vivo experiments were conducted immediately after the subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug responce.

Fluorescence diffuse tomography for detection of RFP-expressed tumors in small animals

NASA Astrophysics Data System (ADS)

Turchin, Ilya V.; Savitsky, Alexander P.; Kamensky, Vladislav A.; Plehanov, Vladimir I.; Orlova, Anna G.; Kleshnin, Mikhail S.; Shirmanova, Marina V.; Fix, Ilya I.; Popov, Vladimir O.

2007-07-01

Capabilities of tumor detection by different optical methods can be significantly improved by labeling of tumors with fluorescent markers. Creation of tumor cell lines transfected with fluorescent proteins provides the possibility not only to detect tumor, but also to conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney HEK-293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. Emission of RFP in the long-wave optical range permits detection of the deeply located tumors, which is essential for whole-body imaging. Only special tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable noninvasive investigation of the internal structure of biological tissue. FDT setup for monitoring of tumor growth in small animals has been created. An animal is scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at the 532 nm wavelength. An optimizing algorithm for scanning of an experimantal animal is suggested. In vivo experiments were conducted immediately after the subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug responce.

Turbulence, transport, and zonal flows in the Madison symmetric torus reversed-field pinch

NASA Astrophysics Data System (ADS)

Williams, Z. R.; Pueschel, M. J.; Terry, P. W.; Hauff, T.

2017-12-01

The robustness and the effect of zonal flows in trapped electron mode (TEM) turbulence and Ion Temperature Gradient (ITG) turbulence in the reversed-field pinch (RFP) are investigated from numerical solutions of the gyrokinetic equations with and without magnetic external perturbations introduced to model tearing modes. For simulations without external magnetic field perturbations, zonal flows produce a much larger reduction of transport for the density-gradient-driven TEM turbulence than they do for the ITG turbulence. Zonal flows are studied in detail to understand the nature of their strong excitation in the RFP and to gain insight into the key differences between the TEM- and ITG-driven regimes. The zonal flow residuals are significantly larger in the RFP than in tokamak geometry due to the low safety factor. Collisionality is seen to play a significant role in the TEM zonal flow regulation through the different responses of the linear growth rate and the size of the Dimits shift to collisionality, while affecting the ITG only minimally. A secondary instability analysis reveals that the TEM turbulence drives zonal flows at a rate that is twice that of the ITG turbulence. In addition to interfering with zonal flows, the magnetic perturbations are found to obviate an energy scaling relation for fast particles.

AAV-mediated delivery of the transcription factor XBP1s into the striatum reduces mutant Huntingtin aggregation in a mouse model of Huntington's disease

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

Zuleta, Amparo; Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, University of Chile, Santiago; Vidal, Rene L.

2012-04-13

Highlights: Black-Right-Pointing-Pointer The contribution of ER stress to HD has not been directly addressed. Black-Right-Pointing-Pointer Expression of XBP1s using AAVs decreases Huntingtin aggregation in vivo. Black-Right-Pointing-Pointer We describe a new in vivo model of HD based on the expression of a large fragment of mHtt-RFP. -- Abstract: Huntington's disease (HD) is caused by mutations that expand a polyglutamine region in the amino-terminal domain of Huntingtin (Htt), leading to the accumulation of intracellular inclusions and progressive neurodegeneration. Recent reports indicate the engagement of endoplasmic reticulum (ER) stress responses in human HD post mortem samples and animal models of the disease. Adaptationmore » to ER stress is mediated by the activation of the unfolded protein response (UPR), an integrated signal transduction pathway that attenuates protein folding stress by controlling the expression of distinct transcription factors including X-Box binding protein 1 (XBP1). Here we targeted the expression of XBP1 on a novel viral-based model of HD. We delivered an active form of XBP1 locally into the striatum of adult mice using adeno-associated vectors (AAVs) and co-expressed this factor with a large fragment of mutant Htt as a fusion protein with RFP (Htt588{sup Q95}-mRFP) to directly visualize the accumulation of Htt inclusions in the brain. Using this approach, we observed a significant reduction in the accumulation of Htt588{sup Q95}-mRFP intracellular inclusion when XBP1 was co-expressed in the striatum. These results contrast with recent findings indicating a protective effect of XBP1 deficiency in neurodegeneration using knockout mice, and suggest a potential use of gene therapy strategies to manipulate the UPR in the context of HD.« less

Recruitment of the Adaptor Protein Grb2 to EGFR Tetramers

PubMed Central

2015-01-01

Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM–FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2–mRFP) and EGFR (EGFR–eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2–mRFP with EGFR–eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR–eGFP per cluster. In contrast, the pool of EGFR–eGFP without Grb2–mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR–eGFP and Grb2–mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit. PMID:24697349

Reducing and measuring fluctuations in the MST RFP: Enhancement of energy confinement and measurement of the MHD dynamo

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

Den Hartog, D.J.; Almagri, A.F.; Cekic, M.

1996-09-01

A three- to five-fold enhancement of the energy confinement time in a reversed-field pinch (RFP) has been achieved in the Madison Symmetric Torus (MST) by reducing the amplitude of tearing mode fluctuations responsible for anomalous transport in the core of the RFP. By applying a transient poloidal inductive electric field to flatten the current density profile, the fluctuation amplitude {tilde b}/B decreases from 1.5% to 0.8%, the electron temperature T{sub e0} increases from 250 eV to 370 eV, the ohmic input power decreases from 4.5 MW to approximately 1.5 MW, the poloidal beta {beta}{sub 0} increases from 6% to 9%,more » and the energy confinement time {tau}{sub E} increases from 1 ms to {approximately}5 ms in I{sub {phi}} = 340 kA plasmas with density {tilde n} = 1 {times} 10{sup 19} m{sup -3}. Current profile control methods are being developed for the RFP in a program to eliminate transport associated with these current-gradient-driven fluctuations. In addition to controlling the amplitude of the tearing modes, we are vigorously pursuing an understanding of the physics of these fluctuations. In particular, plasma flow, both equilibrium and fluctuating, plays a critical role in a diversity of physical phenomena in MST. The key results: 1) Edge probe measurements show that the MHD dynamo is active in low collisionality plasmas, while at high collisionality a new mechanism, the `electron diamagnetic dynamo,` is observed. 2) Core spectroscopic measurements show that the toroidal velocity fluctuations of the plasma are coherent with the large-scale magnetic tearing modes; the scalar product of these two fluctuating quantities is similar to that expected for the MHD dynamo electromotive force. 3) Toroidal plasma flow in MST exhibits large radial shear and can be actively controlled, including unlocking locked discharges, by modifying E{sub r} with a robust biased probe. 24 refs.« less

Identification of choriogenin cis-regulatory elements and production of estrogen-inducible, liver-specific transgenic Medaka.

PubMed

Ueno, Tetsuro; Yasumasu, Shigeki; Hayashi, Shinji; Iuchi, Ichiro

2004-07-01

Choriogenins (chg-H, chg-L) are precursor proteins of egg envelope of medaka and synthesized in the spawning female liver in response to estrogen. We linked a gene construct chg-L1.5 kb/GFP (a 1.5 kb 5'-upstream region of the chg-L gene fused with a green fluorescence protein (GFP) gene) to another construct emgb/RFP (a cis-regulatory region of embryonic globin gene fused with an RFP gene), injected the double fusion gene construct into 1- or 2-cell-stage embryos, and selected embryos expressing the RFP in erythroid cells. From the embryos, we established two lines of chg-L1.5 kb/GFP-emgb/RFP-transgenic medaka. The 3-month-old spawning females and estradiol-17beta (E2)-exposed males displayed the liver-specific GFP expression. The E2-dependent GFP expression was detected in the differentiating liver of the stage 37-38 embryos. In addition, RT-PCR and whole-mount in situ hybridization showed that the E2-dependent chg expression was found in the liver of the stage 34 embryos of wild medaka, suggesting that such E2-dependency is achieved shortly after differentiation of the liver. Analysis using serial deletion mutants fused with GFP showed that the region -426 to -284 of the chg-L gene or the region -364 to -265 of the chg-H gene had the ability to promote the E2-dependent liver-specific GFP expression of its downstream gene. Further analyses suggested that an estrogen response element (ERE) at -309, an ERE half-site at -330 and a binding site for C/EBP at -363 of the chg-L gene played important roles in its downstream chg-L gene expression. In addition, this transgenic medaka may be useful as one of the test animals for detecting environmental estrogenic steroids.

Role of IncP-1β Plasmids pWDL7::rfp and pNB8c in Chloroaniline Catabolism as Determined by Genomic and Functional Analyses

PubMed Central

Król, J. E.; Penrod, J. T.; McCaslin, H.; Rogers, L. M.; Yano, H.; Stancik, A. D.; Dejonghe, W.; Brown, C. J.; Parales, R. E.; Wuertz, S.

2012-01-01

Broad-host-range catabolic plasmids play an important role in bacterial degradation of man-made compounds. To gain insight into the role of these plasmids in chloroaniline degradation, we determined the first complete nucleotide sequences of an IncP-1 chloroaniline degradation plasmid, pWDL7::rfp and its close relative pNB8c, as well as the expression pattern, function, and bioaugmentation potential of the putative 3-chloroaniline (3-CA) oxidation genes. Based on phylogenetic analysis of backbone proteins, both plasmids are members of a distinct clade within the IncP-1β subgroup. The plasmids are almost identical, but whereas pWDL7::rfp carries a duplicate inverted catabolic transposon, Tn6063, containing a putative 3-CA oxidation gene cluster, dcaQTA1A2BR, pNB8c contains only a single copy of the transposon. No genes for an aromatic ring cleavage pathway were detected on either plasmid, suggesting that only the upper 3-CA degradation pathway was present. The dcaA1A2B gene products expressed from a high-copy-number vector were shown to convert 3-CA to 4-chlorocatechol in Escherichia coli. Slight differences in the dca promoter region between the plasmids and lack of induction of transcription of the pNB8c dca genes by 3-CA may explain previous findings that pNB8C does not confer 3-CA transformation. Bioaugmentation of activated sludge with pWDL7::rfp accelerated removal of 3-CA, but only in the presence of an additional carbon source. Successful bioaugmentation requires complementation of the upper pathway genes with chlorocatechol cleavage genes in indigenous bacteria. The genome sequences of these plasmids thus help explain the molecular basis of their catabolic activities. PMID:22101050

40 CFR 51.491 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... different users. RFP baseline means the total of actual volatile organic compounds or nitrogen oxides..., industrial equipment, construction vehicles, off-road motorcycles, and marine vessels). National ambient air...

The drinking water treatment process as a potential source of affecting the bacterial antibiotic resistance.

PubMed

Bai, Xiaohui; Ma, Xiaolin; Xu, Fengming; Li, Jing; Zhang, Hang; Xiao, Xiang

2015-11-15

Two waterworks, with source water derived from the Huangpu or Yangtze River in Shanghai, were investigated, and the effluents were plate-screened for antibiotic-resistant bacteria (ARB) using five antibiotics: ampicillin (AMP), kanamycin (KAN), rifampicin (RFP), chloramphenicol (CM) and streptomycin (STR). The influence of water treatment procedures on the bacterial antibiotic resistance rate and the changes that bacteria underwent when exposed to the five antibiotics at concentration levels ranging from 1 to 100 μg/mL were studied. Multi-drug resistance was also analyzed using drug sensitivity tests. The results indicated that bacteria derived from water treatment plant effluent that used the Huangpu River rather than the Yangtze River as source water exhibited higher antibiotic resistance rates against AMP, STR, RFP and CM but lower antibiotic resistance rates against KAN. When the antibiotic concentration levels ranged from 1 to 10 μg/mL, the antibiotic resistance rates of the bacteria in the water increased as water treatment progressed. Biological activated carbon (BAC) filtration played a key role in increasing the antibiotic resistance rate of bacteria. Chloramine disinfection can enhance antibiotic resistance. Among the isolated ARB, 75% were resistant to multiple antibiotics. Ozone oxidation, BAC filtration and chloramine disinfection can greatly affect the relative abundance of bacteria in the community. Copyright © 2015 Elsevier B.V. All rights reserved.

Long-term fluorescence lifetime imaging of a genetically encoded sensor for caspase-3 activity in mouse tumor xenografts

NASA Astrophysics Data System (ADS)

Zherdeva, Victoria; Kazachkina, Natalia I.; Shcheslavskiy, Vladislav; Savitsky, Alexander P.

2018-03-01

Caspase-3 is known for its role in apoptosis and programmed cell death regulation. We detected caspase-3 activation in vivo in tumor xenografts via shift of mean fluorescence lifetimes of a caspase-3 sensor. We used the genetically encoded sensor TR23K based on the red fluorescent protein TagRFP and chromoprotein KFP linked by 23 amino acid residues (TagRFP-23-KFP) containing a specific caspase cleavage DEVD motif to monitor the activity of caspase-3 in tumor xenografts by means of fluorescence lifetime imaging-Forster resonance energy transfer. Apoptosis was induced by injection of paclitaxel for A549 lung adenocarcinoma and etoposide and cisplatin for HEp-2 pharynx adenocarcinoma. We observed a shift in lifetime distribution from 1.6 to 1.9 ns to 2.1 to 2.4 ns, which indicated the activation of caspase-3. Even within the same tumor, the lifetime varied presumably due to the tumor heterogeneity and the different depth of tumor invasion. Thus, processing time-resolved fluorescence images allows detection of both the cleaved and noncleaved states of the TR23K sensor in real-time mode during the course of several weeks noninvasively. This approach can be used in drug screening, facilitating the development of new anticancer agents as well as improvement of chemotherapy efficiency and its adaptation for personal treatment.

SCF increases in utero-labeled stem cells migration and improves wound healing.

PubMed

Zgheib, Carlos; Xu, Junwang; Mallette, Andrew C; Caskey, Robert C; Zhang, Liping; Hu, Junyi; Liechty, Kenneth W

2015-01-01

Diabetic skin wounds lack the ability to heal properly and constitute a major and significant complication of diabetes. Nontraumatic lower extremity amputations are the number one complication of diabetic skin wounds. The complexity of their pathophysiology requires an intervention at many levels to enhance healing and wound closure. Stem cells are a promising treatment for diabetic skin wounds as they have the ability to correct abnormal healing. Stem cell factor (SCF), a chemokine expressed in the skin, can induce stem cells migration, however the role of SCF in diabetic skin wound healing is still unknown. We hypothesize that SCF would correct the impairment and promote the healing of diabetic skin wounds. Our results show that SCF improved wound closure in diabetic mice and increased HIF-1α and vascular endothelial growth factor (VEGF) expression levels in these wounds. SCF treatment also enhanced the migration of red fluorescent protein (RFP)-labeled skin stem cells via in utero intra-amniotic injection of lenti-RFP at E8. Interestingly these RFP+ cells are present in the epidermis, stain negative for K15, and appear to be distinct from the already known hair follicle stem cells. These results demonstrate that SCF improves diabetic wound healing in part by increasing the recruitment of a unique stem cell population present in the skin. © 2015 by the Wound Healing Society.

Overview of results from the MST reversed field pinch experiment

NASA Astrophysics Data System (ADS)

Sarff, J. S.; Almagri, A. F.; Anderson, J. K.; Borchardt, M.; Carmody, D.; Caspary, K.; Chapman, B. E.; Den Hartog, D. J.; Duff, J.; Eilerman, S.; Falkowski, A.; Forest, C. B.; Goetz, J. A.; Holly, D. J.; Kim, J.-H.; King, J.; Ko, J.; Koliner, J.; Kumar, S.; Lee, J. D.; Liu, D.; Magee, R.; McCollam, K. J.; McGarry, M.; Mirnov, V. V.; Nornberg, M. D.; Nonn, P. D.; Oliva, S. P.; Parke, E.; Reusch, J. A.; Sauppe, J. P.; Seltzman, A.; Sovinec, C. R.; Stephens, H.; Stone, D.; Theucks, D.; Thomas, M.; Triana, J.; Terry, P. W.; Waksman, J.; Bergerson, W. F.; Brower, D. L.; Ding, W. X.; Lin, L.; Demers, D. R.; Fimognari, P.; Titus, J.; Auriemma, F.; Cappello, S.; Franz, P.; Innocente, P.; Lorenzini, R.; Martines, E.; Momo, B.; Piovesan, P.; Puiatti, M.; Spolaore, M.; Terranova, D.; Zanca, P.; Belykh, V.; Davydenko, V. I.; Deichuli, P.; Ivanov, A. A.; Polosatkin, S.; Stupishin, N. V.; Spong, D.; Craig, D.; Harvey, R. W.; Cianciosa, M.; Hanson, J. D.

2013-10-01

An overview of recent results from the MST programme on physics important for the advancement of the reversed field pinch (RFP) as well as for improved understanding of toroidal magnetic confinement more generally is reported. Evidence for the classical confinement of ions in the RFP is provided by analysis of impurity ions and energetic ions created by 1 MW neutral beam injection (NBI). The first appearance of energetic-particle-driven modes by NBI in a RFP plasma is described. MST plasmas robustly access the quasi-single-helicity state that has commonalities to the stellarator and ‘snake’ formation in tokamaks. In MST the dominant mode grows to 8% of the axisymmetric field strength, while the remaining modes are reduced. Predictive capability for tearing mode behaviour has been improved through nonlinear, 3D, resistive magnetohydrodynamic computation using the measured resistivity profile and Lundquist number, which reproduces the sawtooth cycle dynamics. Experimental evidence and computational analysis indicates two-fluid effects, e.g., Hall physics and gyro-viscosity, are needed to understand the coupling of parallel momentum transport and current profile relaxation. Large Reynolds and Maxwell stresses, plus separately measured kinetic stress, indicate an intricate momentum balance and a possible origin for MST's intrinsic plasma rotation. Gyrokinetic analysis indicates that micro-tearing modes can be unstable at high beta, with a critical gradient for the electron temperature that is larger than for tokamak plasmas by roughly the aspect ratio.

40 CFR 51.1000 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-07-01

.... Benchmark RFP plan means the reasonable further progress plan that requires generally linear emission... Federally enforceable national, State, or local control measure that has been approved in the SIP and that...

40 CFR 51.1000 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-07-01

.... Benchmark RFP plan means the reasonable further progress plan that requires generally linear emission... Federally enforceable national, State, or local control measure that has been approved in the SIP and that...

40 CFR 51.1000 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-07-01

.... Benchmark RFP plan means the reasonable further progress plan that requires generally linear emission... Federally enforceable national, State, or local control measure that has been approved in the SIP and that...

40 CFR 51.1000 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-07-01

.... Benchmark RFP plan means the reasonable further progress plan that requires generally linear emission... Federally enforceable national, State, or local control measure that has been approved in the SIP and that...

40 CFR 51.1000 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-07-01

.... Benchmark RFP plan means the reasonable further progress plan that requires generally linear emission... Federally enforceable national, State, or local control measure that has been approved in the SIP and that...

Self-Regulation of E×B Flow Shear via Plasma Turbulence

NASA Astrophysics Data System (ADS)

Vianello, N.; Spada, E.; Antoni, V.; Spolaore, M.; Serianni, G.; Regnoli, G.; Cavazzana, R.; Bergsåker, H.; Drake, J. R.

2005-04-01

The momentum balance has been applied to the E×B flow in the edge region of a reversed field pinch (RFP) configuration. All terms, including those involving fluctuations, have been measured in stationary condition in the edge region of the Extrap-T2R RFP experiment. It is found that the component of the Reynolds stress driven by electrostatic fluctuations is the term playing the major role in driving the shear of the E×B flow to a value marginal for turbulent suppression, so that the results are in favor of a turbulence self-regulating mechanism underlying the momentum balance at the edge. Balancing the sheared flow driving and damping terms, the plasma viscosity is found anomalous and consistent with the diffusivity due to electrostatic turbulence.

Self-regulation of E x B flow shear via plasma turbulence.

PubMed

Vianello, N; Spada, E; Antoni, V; Spolaore, M; Serianni, G; Regnoli, G; Cavazzana, R; Bergsåker, H; Drake, J R

2005-04-08

The momentum balance has been applied to the ExB flow in the edge region of a reversed field pinch (RFP) configuration. All terms, including those involving fluctuations, have been measured in stationary condition in the edge region of the Extrap-T2R RFP experiment. It is found that the component of the Reynolds stress driven by electrostatic fluctuations is the term playing the major role in driving the shear of the ExB flow to a value marginal for turbulent suppression, so that the results are in favor of a turbulence self-regulating mechanism underlying the momentum balance at the edge. Balancing the sheared flow driving and damping terms, the plasma viscosity is found anomalous and consistent with the diffusivity due to electrostatic turbulence.

Compact soft x-ray multichord camera: Design and initial operation

NASA Astrophysics Data System (ADS)

Franz, P.; Gadani, G.; Pasqualotto, R.; Marrelli, L.; Martin, P.; Spizzo, G.; Brunsell, P.; Chapman, B. E.; Paganucci, F.; Rossetti, P.; Xiao, C.

2003-03-01

A compact and low cost diagnostic for spatially resolved measurements of soft x-ray or total radiation emission has been designed and realized to be flexibly applied to different plasma physics experiments. Its reduced size (outer diameter=35 mm) makes it suited to a variety of devices. The line integrated emissivity (brightness) has been measured along up to 20 lines of sight, using an array of miniaturized silicon photodiodes. Preliminary prototypes of the diagnostic have been installed in the Madison Symmetric Torus reversed field pinch (RFP) device at University of Wisconsin and in the EXTRAP T2 RFP device at the Royal Institute of Technology in Stockholm. Application of the diagnostic to a gas-fed (argon, helium) magnetoplasma dynamic thruster (MPDT) with an external magnetic field will also be discussed.

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

Morrison, Joel

The United States has more oil and gas wells than any other country. As of December 31, 2004, there were more than half a million producing oil wells in the United States. That is more than three times the combined total for the next three leaders: China, Canada, and Russia. The Stripper Well Consortium (SWC) is a partnership that includes domestic oil and gas producers, service and supply companies, trade associations, academia, the Department of Energy’s Strategic Center for Natural Gas and Oil (SCNGO) at the National Energy Technology Laboratory (NETL), and the New York State Energy Research and Developmentmore » Authority (NYSERDA). The Consortium was established in 2000. This report serves as a final technical report for the SWC activities conducted over the May 1, 2004 to December 1, 2011 timeframe. During this timeframe, the SWC worked with 173 members in 29 states and three international countries, to focus on the development of new technologies to benefit the U.S. stripper well industry. SWC worked with NETL to develop a nationwide request-for-proposal (RFP) process to solicit proposals from the U.S. stripper well industry to develop and/or deploy new technologies that would assist small producers in improving the production performance of their stripper well operations. SWC conducted eight rounds of funding. A total of 132 proposals were received. The proposals were compiled and distributed to an industry-driven SWC executive council and program sponsors for review. Applicants were required to make a formal technical presentation to the SWC membership, executive council, and program sponsors. After reviewing the proposals and listening to the presentations, the executive council made their funding recommendations to program sponsors. A total of 64 projects were selected for funding, of which 59 were fully completed. Penn State then worked with grant awardees to issue a subcontract for their approved work. SWC organized and hosted a total of 14 meetings dedicated to technology transfer to showcase and review SWC-funded technology. The workshops were open to the stripper well industry.« less

2013 RFP Ports Initiative Supporting Documents

EPA Pesticide Factsheets

Documents include: RFPs, Project Narrative, Application fleet description (AFD), Priority County List, Model years for eligible nonroad engines (nonroad remaining useful life), Sample Drayage, Marince Engine Eligibility, FAQs, and Webinar slides

2014 RFP Ports Initiative Supporting Documents

EPA Pesticide Factsheets

Documents include: RFPs, Project Narrative, Application fleet description (AFD), Priority County List, Model years for eligible nonroad engines (nonroad remaining useful life), Sample Drayage, Marince Engine Eligibility, FAQs, and Webinar slides

77 FR 59406 - National Cancer Institute; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-27

...(s); and Budget Presentation; Reports of Special Initiatives; RFA and RFP Concept Reviews; and Scientific Presentations. Place: National Institutes of Health, Building 31, 31 Center Drive, 6th Floor...

75 FR 26761 - National Cancer Institute; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2010-05-12

... Group(s); and Budget Presentations; Reports of Special Initiatives; RFA and RFP Concept Reviews; and... campus. All visitor vehicles, including taxicabs, hotel, and airport shuttles will be inspected before...

Pathological Roles of Interleukin-22 in the Development of Recurrent Hepatitis C after Liver Transplantation

PubMed Central

Li, Jin; Cheung, Eddie; Li, Hanwei; Zhao, Jingmin; Liu, Hongling; Liu, Zhenwen; Zhang, Min

2016-01-01

Objective The aim of this study was to longitudinally evaluate and analyze the role of interleukin-22-producing CD4 positive cells (IL-22) in the pathogenesis of Hepatitis C Virus recurrence after Orthotopic Liver Transplantation (HCV-OLT). Methods 15 HCV-OLT, 15 age- and gender- matched non-HCV post-OLT (OLT) and 15 hepatitis C virus infected (HCV) patients were enrolled into our study from the liver transplantation and research center at Beijing 302 Hospital. We determined the frequencies of IL-22 using flow cytometry and expression of IL-22 mRNA using PCR in peripheral blood and liver tissue. We also divided HCV-OLT patients into rapid fibrosis progression (RFP) and slow fibrosis progression (SFP), examined IL-22 cells and analyzed the correlations between IL-22 frequencies and liver injury, fibrosis and clinical parameters. Moreover, we investigated the role of IL-22 in Human Hepatic Stellate Cells (HSCs). Results The levels of serum IL-22, frequencies of IL-22 producing cells in peripheral blood mononuclear cells, and expression of IL-22 mRNA and protein in the liver in the HCV-OLT group were significantly higher than that in the HCV and OLT groups. Furthermore, eight (53.3%) patients developed RFP after two years; another three patients were diagnosed liver cirrhosis. The frequencies of IL-22 were much higher in RFP compared with SFP, while no significant difference existed between OLT and SFP. Intrahepatic IL-22 positive cells were located in fibrotic areas and significantly correlated with α-smooth muscle actin (α-SMA) and fibrosis staging scores, not with grading scores and HCRVNA. In vitro, IL-22 administration prevented HSCs apoptosis, promoted HSCs proliferation and activation, up-regulated the expression of HSC-sourced growth factors including α-SMA, TGF-β and TIMP-1, and increased the production of liver fibrosis markers including laminin, hyaluronic acid and collagen type IV. Conclusion Peripheral and intrahepatic IL-22 is up-regulated and plays a pathological role in exacerbating liver fibrosis by activating HSCs in HCV-OLT patients, which may predict RFP and serve as an attractive target for anti-fibrotic therapy. PMID:27123854

Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26.

PubMed

Nakagawa, So; Gong, Xiang-Qun; Maeda, Shoji; Dong, Yuhua; Misumi, Yuko; Tsukihara, Tomitake; Bai, Donglin

2011-06-03

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.

Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine

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

Pletnev, Vladimir Z., E-mail: vzpletnev@gmail.com; Pletneva, Nadya V.; Lukyanov, Konstantin A.

The crystal structure of the novel red emitting fluorescent protein from lancelet Branchiostoma lanceolatum (Chordata) revealed an unusual five residues cyclic unit comprising Gly58-Tyr59-Gly60 chromophore, the following Phe61 and Tyr62 covalently bound to chromophore Tyr59. A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the ‘core’ structure, which can be extended in red-shifted proteins owing to modifications of the protein backbonemore » at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λ{sub ex}/λ{sub em} = 502/511 nm) and red laRFP (λ{sub ex}/λ{sub em} ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C{sup β} atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis.« less

Dynamically monitoring the gene expression of dual fluorophore in the cell cycle with quantitative spectrum analysis

NASA Astrophysics Data System (ADS)

Lee, Ja-Yun; Wu, Tzong-Yuan; Hsu, I.-Jen

2008-04-01

The cloning and transcription techniques on gene cloned fluorescent proteins have been widely used in many applications. They have been used as reporters of some conditions in a series of reactions. However, it is usually difficult to monitor the specific target with the exactly number of proteins during the process in turbid media, especially at micrometer scales. We successfully revealed an alternative way to monitor the cell cycle behavior and quantitatively analyzed the target cells with green and red fluorescent proteins (GFP and RFP) during different phases of the cell cycle by quantitatively analyzing its behavior and also monitoring its spatial distribution.

Autophagy maturation associated with CD38-mediated regulation of lysosome function in mouse glomerular podocytes

PubMed Central

Xiong, Jing; Xia, Min; Xu, Ming; Zhang, Yang; Abais, Justine M; Li, Guangbi; Riebling, Christopher R; Ritter, Joseph K; Boini, Krishna M; Li, Pin-Lan

2013-01-01

Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38-mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3-II, a robust marker of autophagosomes (APs), suggesting a high basal level of autophagic activity. Treatment with the mTOR inhibitor, rapamycin, increased LC3-II and the content of both APs detected by Cyto-ID Green staining and autophagolysosomes (APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin-induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co-transfection of GFP-LC3B and RFP-Lamp1 expression vectors. A colocalization of GFP-LC3B and RFP-Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. Moreover, blockade of the CD38-mediated regulation by PPADS completely abolished rapamycin-induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes. PMID:24238063

40 CFR 51.1009 - Reasonable further progress (RFP) requirements.

Code of Federal Regulations, 2011 CFR

2011-07-01

... milestone year, emissions will be at a level consistent with generally linear progress in reducing emissions... plan are derived. (6) For purposes of establishing motor vehicle emissions budgets for transportation...

40 CFR 51.1009 - Reasonable further progress (RFP) requirements.

Code of Federal Regulations, 2014 CFR

2014-07-01

... milestone year, emissions will be at a level consistent with generally linear progress in reducing emissions... plan are derived. (6) For purposes of establishing motor vehicle emissions budgets for transportation...

40 CFR 51.1009 - Reasonable further progress (RFP) requirements.

Code of Federal Regulations, 2013 CFR

2013-07-01

... milestone year, emissions will be at a level consistent with generally linear progress in reducing emissions... plan are derived. (6) For purposes of establishing motor vehicle emissions budgets for transportation...

40 CFR 51.1009 - Reasonable further progress (RFP) requirements.

Code of Federal Regulations, 2012 CFR

2012-07-01

... milestone year, emissions will be at a level consistent with generally linear progress in reducing emissions... plan are derived. (6) For purposes of establishing motor vehicle emissions budgets for transportation...

76 FR 62422 - National Cancer Institute; Notice of Meetings

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-07

... Groups; and Budget Presentations; Reports of Special Initiatives; RFA and RFP Concept Reviews; and... taxicabs, hotel, and airport shuttles will be inspected before being allowed on campus. Visitors will be...

76 FR 7575 - National Cancer Institute; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-10

... Budget Presentations; Reports of Special Initiatives; RFA and RFP Concept Reviews; and Scientific..., hotel, and airport shuttles will be inspected before being allowed on campus. Visitors will be asked to...

7 CFR 1783.9 - What are the criteria for scoring applications?

Code of Federal Regulations, 2011 CFR

2011-01-01

... thought out, comprehensive approach to accomplishing the objectives of this part, clearly defines who will.... (7) Administrator's discretion, considering such factors as creative outreach ideas for marketing RFP...

7 CFR 1783.9 - What are the criteria for scoring applications?

Code of Federal Regulations, 2014 CFR

2014-01-01

... thought out, comprehensive approach to accomplishing the objectives of this part, clearly defines who will.... (7) Administrator's discretion, considering such factors as creative outreach ideas for marketing RFP...

7 CFR 1783.9 - What are the criteria for scoring applications?

Code of Federal Regulations, 2012 CFR

2012-01-01

... thought out, comprehensive approach to accomplishing the objectives of this part, clearly defines who will.... (7) Administrator's discretion, considering such factors as creative outreach ideas for marketing RFP...

7 CFR 1783.9 - What are the criteria for scoring applications?

Code of Federal Regulations, 2013 CFR

2013-01-01

... thought out, comprehensive approach to accomplishing the objectives of this part, clearly defines who will.... (7) Administrator's discretion, considering such factors as creative outreach ideas for marketing RFP...

75 FR 63494 - National Cancer Institute; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-15

... Group(s); and Budget Presentations; Reports of Special Initiatives; RFA and RFP Concept Reviews; and... taxicabs, hotel, and airport shuttles will be inspected before being allowed on campus. Visitors will be...

78 FR 5192 - National Cancer Institute; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-24

... Review Group(s); and Budget Presentations; Reports of Special Initiatives; RFA and RFP Concept Reviews... entrance onto the NIH campus. All visitor vehicles, including taxicabs, hotel, and airport shuttles will be...

Braking due to non-resonant magnetic perturbations and comparison with neoclassical toroidal viscosity torque in EXTRAP T2R

NASA Astrophysics Data System (ADS)

Frassinetti, L.; Sun, Y.; Fridström, R.; Menmuir, S.; Olofsson, K. E. J.; Brunsell, P. R.; Khan, M. W. M.; Liang, Y.; Drake, J. R.

2015-09-01

The non-resonant magnetic perturbation (MP) braking is studied in the EXTRAP T2R reversed-field pinch (RFP) and the experimental braking torque is compared with the torque expected by the neoclassical toroidal viscosity (NTV) theory. The EXTRAP T2R active coils can apply magnetic perturbations with a single harmonic, either resonant or non-resonant. The non-resonant MP produces velocity braking with an experimental torque that affects a large part of the core region. The experimental torque is clearly related to the plasma displacement, consistent with a quadratic dependence as expected by the NTV theory. The work show a good qualitative agreement between the experimental torque in a RFP machine and NTV torque concerning both the torque density radial profile and the dependence on the non-resonant MP harmonic.

Protistan Grazing Analysis by Flow Cytometry Using Prey Labeled by In Vivo Expression of Fluorescent Proteins

PubMed Central

Fu, Yutao; O'Kelly, Charles; Sieracki, Michael; Distel, Daniel L.

2003-01-01

Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells. PMID:14602649

Prognostic value of tumor-stroma ratio combined with the immune status of tumors in invasive breast carcinoma.

PubMed

Vangangelt, K M H; van Pelt, G W; Engels, C C; Putter, H; Liefers, G J; Smit, V T H B M; Tollenaar, R A E M; Kuppen, P J K; Mesker, W E

2018-04-01

Complex interactions occur between cancer cells and cells in the tumor microenvironment. In this study, the prognostic value of the interplay between tumor-stroma ratio (TSR) and the immune status of tumors in breast cancer patients was evaluated. A cohort of 574 breast cancer patients was analyzed. The percentage of tumor stroma was visually estimated on Hematoxylin and Eosin (H&E) stained histological tumor tissue sections. Immunohistochemical staining was performed for classical human leukocyte antigen (HLA) class I, HLA-E, HLA-G, markers for regulatory T (Treg) cells, natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs). TSR (P < .001) and immune status of tumors (P < .001) were both statistically significant for recurrence free period (RFP) and both independent prognosticators (P < .001) in which tumors with a high stromal content behave more aggressively as well as tumors with a low immune status. Ten years RFP for patients with a stroma-low tumor and high immune status profile was 87% compared to 17% of patients with a stroma-high tumor combined with low immune status profile (P < .001). Classical HLA class I is the most prominent immune marker in the immune status profiles. Determination of TSR is a simple, fast and cheap method. The effect on RFP of TSR when combined with immune status of tumors or expression of classical HLA class I is even stronger. Both are promising for further prediction and achievement of tailored treatment for breast cancer patients.

Choline-Deficient-Diet-Induced Fatty Liver Is a Metastasis-Resistant Microenvironment.

PubMed

Nakamura, Miki; Suetsugu, Atsushi; Hasegawa, Kosuke; Matsumoto, Takuro; Aoki, Hitomi; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Hoffman, Robert M

2017-07-01

Fatty liver disease is increasing in the developed and developing world. Liver metastasis from malignant lymphoma in the fatty liver is poorly understood. In a previous report, we developed color-coded imaging of the tumor microenvironment (TME) of the murine EL4-RFP malignant lymphoma during metastasis, including the lung. In the present report, we investigated the potential and microenvironment of the fatty liver induced by a choline-deficient diet as a metastatic site in this mouse lymphoma model. C57BL/6-GFP transgenic mice were fed with a choline-deficient diet in order to establish a fatty liver model. EL4-RFP cells were injected in the spleen of normal mice and fatty-liver mice. Metastases in mice with fatty liver or normal liver were imaged with the Olympus SZX7 microscope and the Olympus FV1000 confocal microscope. Metastases of EL4-RFP were observed in the liver, ascites and bone marrow. Primary tumors were imaged in the spleen at the injection site. The fewest metastases were observed in the fatty liver. In addition, the fewest cancer-associated fibroblasts (CAFs) were observed in the fatty liver. The relative metastatic resistance of the fatty liver may be due to the reduced number of CAFs in the fatty livers. The mechanism of the effect of the choline-deficient diet is discussed. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Magnetic chaos healing in the helical reversed-field pinch: indications from the volume-preserving field line tracing code NEMATO

NASA Astrophysics Data System (ADS)

Bonfiglio, D.; Veranda, M.; Cappello, S.; Chacón, L.; Spizzo, G.

2010-11-01

The emergence of a self-organized reversed-field pinch (RFP) helical regime, first shown by 3D MHD numerical simulations, has been highlighted in the RFX-mod experiment at high current operation (IP above 1 MA). In fact, a quasi-stationary helical configuration spontaneously appears, characterized by strong internal electron transport barriers. In such regime electron temperature and density become, to a very good approximation, functions of the helical flux coordinate related to the dominant helical magnetic component. In addition, this regime is diagnosed to be associated with the topological transition to a single-helical-axis (SHAx) state, achieved after the expulsion of the separatrix of the dominant mode's magnetic island. The SHAx state is theoretically predicted to be resilient to the magnetic chaos induced by secondary modes. In this paper, we present initial results of the volume-preserving field line tracing code NEMATO [Finn J M and Chacón L 2005 Phys. Plasmas 12 054503] applied to study the magnetic topology resulting from 3D MHD simulations of the RFP. First, a successful 2D verification test of the code is shown, then, initial application to a systematic study of chaos healing in the helical RFP is discussed. The separatrix disappearance is confirmed to play an essential role for chaos healing. The triggering effect of a reversed magnetic shear for the formation of ordered surfaces within magnetic chaos is also diagnosed.

Magnetic chaos healing in hte helical reversed-field pinch: indications from the volume-preserving field line tracing code NEMATO

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

Bonfiglio, Daniele; Veranda, M.; Cappello, Susanna

2010-01-01

The emergence of a self-organized reversed-field pinch (RFP) helical regime, first shown by 3D MHD numerical simulations, has been highlighted in the RFX-mod experiment at high current operation (IP above 1 MA). In fact, a quasi-stationary helical configuration spontaneously appears, characterized by strong internal electron transport barriers. In such regime electron temperature and density become, to a very good approximation, functions of the helical flux coordinate related to the dominant helical magnetic component. In addition, this regime is diagnosed to be associated with the topological transition to a single-helical-axis (SHAx) state, achieved after the expulsion of the separatrix of themore » dominant mode's magnetic island. The SHAx state is theoretically predicted to be resilient to the magnetic chaos induced by secondary modes. In this paper, we present initial results of the volume-preserving field line tracing code nemato [Finn J M and Chacon L 2005 Phys. Plasmas 12 054503] applied to study the magnetic topology resulting from 3D MHD simulations of the RFP. First, a successful 2D verification test of the code is shown, then, initial application to a systematic study of chaos healing in the helical RFP is discussed. The separatrix disappearance is confirmed to play an essential role for chaos healing. The triggering effect of a reversed magnetic shear for the formation of ordered surfaces within magnetic chaos is also diagnosed.« less

Maximizing MST's inductive capability with a Bp programmable power supply

NASA Astrophysics Data System (ADS)

Chapman, B. E.; Holly, D. J.; Jacobson, C. M.; McCollam, K. J.; Morin, J. C.; Sarff, J. S.; Squitieri, A.

2016-10-01

A major goal of the MST program is the advancement of inductive control for the development of both the RFP's fusion potential and, synergistically, the predictive capability of fusion science. This entails programmable power supplies (PPS's) for the Bt and Bp circuits. A Bt PPS is already in place, allowing advanced RFP operation and the production of tokamak plasmas, and a Bp PPS prototype is under construction. To explore some of the new capabilities to be provided by the Bp PPS, the existing Bt PPS has been temporarily connected to the Bp circuit. One key result is new-found access to very low Ip (20 kA) and very low Lundquist number, S (104). At this low S, simulation of RFP plasmas with the MHD code NIMROD is readily achievable, and work toward validation of extended MHD models using NIMROD is underway with direct comparisons to these MST plasmas. The full Bp PPS will also provide higher Ip and S than presently possible, allowing MST to produce plasmas with S spanning as much as five orders of magnitude, a dramatic extension of MST's capability. In these initial tests, the PPS has also increased five-fold MST's Ip flattop duration, to about 100 ms. This, coupled with the recently demonstrated PPS ability to drive large-amplitude sinusoidal oscillations in Ip, will allow tests of extended-duration oscillating field current drive, the goal of which is ac sustainment of a quasi-dc plasma current. Work supported by US DOE.

Region 6: Texas Adequate Letter (4/7/2008)

EPA Pesticide Factsheets

This is a letter from Carl Edlund, Director, to Susana Hildebrand regarding the MVEB's for Dallas/Fort Worth 8-hour Ozone State Implementation Plan (SIP) and the Houston / Galveston / Brazoria Reasonable Further Progress (RFP) plan.

7 CFR 1783.15 - What are the terms of RFP loans?

Code of Federal Regulations, 2010 CFR

2010-01-01

..., DEPARTMENT OF AGRICULTURE (CONTINUED) REVOLVING FUNDS FOR FINANCING WATER AND WASTEWATER PROJECTS (REVOLVING...,000 or 75 percent of the total cost of a project. The total outstanding balance for all loans under...

48 CFR 2415.304 - Evaluation factors.

Code of Federal Regulations, 2011 CFR

2011-10-01

... assigned a numerical weight (except for pass-fail factors) which shall appear in the RFP. When using LPTA, each evaluation factor is applied on a “pass-fail” basis; numerical scores are not assigned. “Pass-fail...

48 CFR 2415.304 - Evaluation factors.

Code of Federal Regulations, 2012 CFR

2012-10-01

... assigned a numerical weight (except for pass-fail factors) which shall appear in the RFP. When using LPTA, each evaluation factor is applied on a “pass-fail” basis; numerical scores are not assigned. “Pass-fail...

76 FR 27290 - Approval and Promulgation of Air Quality Implementation Plans; West Virginia; Kentucky; Ohio...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-11

...EPA is proposing to make two determinations regarding the tri- state Huntington-Ashland, West Virginia-Kentucky-Ohio fine particulate matter (PM2.5) nonattainment Area (hereafter referred to as ``the Huntington-Ashland Area'' or ``Area''). First, EPA is proposing to determine that the Area has attained the 1997 annual average PM2.5 National Ambient Air Quality Standard (NAAQS). This proposed determination of attainment is based upon complete, quality- assured and certified ambient air monitoring data for the 2007-2009 period showing that the Area has attained the 1997 annual PM2.5 NAAQS, and data available to date for 2010 in EPA's Air Quality System (AQS) database that show the area continues to attain. If EPA finalizes this proposed determination of attainment, the requirements for the Area to submit attainment demonstrations and associated reasonably available control measures (RACM), a reasonable further progress (RFP) plan, contingency measures, and other planning State Implementation Plan (SIP) revisions related to attainment of the standard shall be suspended for so long as the Area continues to attain the annual PM2.5 NAAQS. Second, EPA is also proposing to determine, based on quality-assured and certified monitoring data for the 2007-2009 monitoring period, that the Area has attained the 1997 annual PM2.5 NAAQS by its applicable attainment date of April 5, 2010.

Lead (Pb) National Ambient Air Quality Standards (NAAQS) Implementation Guidance Documents

EPA Pesticide Factsheets

This page contains implementation guidance documents for the 1978 and 2008 Lead (Pb) NAAQS. Some of the topics covered are: RACM/RACT, RFP, new source review, designations, emissions inventories, attainment dates, and transition policy

Two-step cultivation for production of astaxanthin in Chlorella zofingiensis using a patented energy-free rotating floating photobioreactor (RFP).

PubMed

Zhang, Zhao; Huang, Jim Junhui; Sun, Dongzhe; Lee, Yuankun; Chen, Feng

2017-01-01

In the present study, high light and nitrogen starvation with glucose-fed to the culture was found efficient to induce astaxanthin accumulation in Chlorella zofingiensis. Therefore, a two-step cultivation strategy including high biomass yield fermentation and outdoor induction with an energy-free RFP was conducted. During the fermentation, the highest cell density of 98.4gL -1 and astaxanthin yield of 73.3mgL -1 were achieved, which were higher than those so far reported in C. zofingiensis. During the outdoor induction, astaxanthin content was further increased by 1.5-fold leading to the highest astaxanthin productivity of 5.26mgL -1 day -1 under an optimal dilution of 5-fold. Our work thus provided an effective two-step cultivation strategy for production of astaxanthin by C. zofingiensis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Experiments and modelling of active quasi-single helicity regime generation in a reversed field pinch

NASA Astrophysics Data System (ADS)

Frassinetti, L.; Brunsell, P. R.; Drake, J. R.

2009-07-01

The interaction of a static resonant magnetic perturbation (RMP) with a tearing mode (TM) is becoming a relevant topic in fusion plasma physics. RMPs can be generated by active coils and then used to affect the properties of TMs and of the corresponding magnetic islands. This paper shows how the feedback system of the EXTRAP T2R reversed field pinch (RFP) can produce a RMP that affects a rotating TM and stimulate the transition to the so-called quasi-single helicity (QSH) regime, a RFP plasma state characterized by a magnetic island surrounded by low magnetic chaos. The application of the RMP can increase the QSH probability up to 10% and enlarge the size of the corresponding island. Part of the experimental results are supported by a theoretical study that models the effect of the active coils on the magnetic island.

Comparison of confinement in resistive-shell reversed-field pinch devices with two different magnetic shell penetration times

NASA Astrophysics Data System (ADS)

Gravestijn, R. M.; Drake, J. R.; Hedqvist, A.; Rachlew, E.

2004-01-01

A loop voltage is required to sustain the reversed-field pinch (RFP) equilibrium. The configuration is characterized by redistribution of magnetic helicity but with the condition that the total helicity is maintained constant. The magnetic field shell penetration time, tgrs, has a critical role in the stability and performance of the RFP. Confinement in the EXTRAP device has been studied with two values of tgrs, first (EXTRAP-T2) with tgrs of the order of the typical relaxation cycle timescale and then (EXTRAP-T2R) with tgrs much longer than the relaxation cycle timescale, but still much shorter than the pulse length. Plasma parameters show significant improvements in confinement in EXTRAP-T2R. The typical loop voltage required to sustain comparable electron poloidal beta values is a factor of 3 lower in the EXTRAP-T2R device. The improvement is attributed to reduced magnetic turbulence.

Heat transport modelling in EXTRAP T2R

NASA Astrophysics Data System (ADS)

Frassinetti, L.; Brunsell, P. R.; Cecconello, M.; Drake, J. R.

2009-02-01

A model to estimate the heat transport in the EXTRAP T2R reversed field pinch (RFP) is described. The model, based on experimental and theoretical results, divides the RFP electron heat diffusivity χe into three regions, one in the plasma core, where χe is assumed to be determined by the tearing modes, one located around the reversal radius, where χe is assumed not dependent on the magnetic fluctuations and one in the extreme edge, where high χe is assumed. The absolute values of the core and of the reversal χe are determined by simulating the electron temperature and the soft x-ray and by comparing the simulated signals with the experimental ones. The model is used to estimate the heat diffusivity and the energy confinement time during the flat top of standard plasmas, of deep F plasmas and of plasmas obtained with the intelligent shell.

X-ray analysis of electron Bernstein wave heating in MST

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

Seltzman, A. H., E-mail: seltzman@wisc.edu; Anderson, J. K.; DuBois, A. M.

2016-11-15

A pulse height analyzing x-ray tomography system has been developed to detect x-rays from electron Bernstein wave heated electrons in the Madison symmetric torus reversed field pinch (RFP). Cadmium zinc telluride detectors are arranged in a parallel beam array with two orthogonal multi-chord detectors that may be used for tomography. In addition a repositionable 16 channel fan beam camera with a 55° field of view is used to augment data collected with the Hard X-ray array. The chord integrated signals identify target emission from RF heated electrons striking a limiter located 12° toroidally away from the RF injection port. Thismore » provides information on heated electron spectrum, transport, and diffusion. RF induced x-ray emission from absorption on harmonic electron cyclotron resonances in low current (<250 kA) RFP discharges has been observed.« less

Conversion of red fluorescent protein into a bright blue probe.

PubMed

Subach, Oksana M; Gundorov, Illia S; Yoshimura, Masami; Subach, Fedor V; Zhang, Jinghang; Grüenwald, David; Souslova, Ekaterina A; Chudakov, Dmitriy M; Verkhusha, Vladislav V

2008-10-20

We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Förster resonance energy transfer applications.

Development and Evaluation of Transgenic Nude Mice Expressing Ubiquitous Green Fluorescent Protein.

PubMed

Iyer, Srikanth; Arindkar, Shailendra; Mishra, Alaknanda; Manglani, Kapil; Kumar, Jerald Mahesh; Majumdar, Subeer S; Upadhyay, Pramod; Nagarajan, Perumal

2015-08-01

Researchers had developed and characterized transgenic green/red fluorescent protein (GFP/RFP) nude mouse with ubiquitous RFP or GFP expression, but none has evaluated the level of immune cells and expression levels of GFP in this model. The nude GFP mice were evaluated by imaging, hematological indices, and flow cytometry to compare the proportion of immune T cells. Quantitative real-time PCR (qRT-PCR) was done for evaluating the relative expression of GFP transcripts in few organs of the nude GFP mice. The hematological and immune cells of nude GFP were within the range of nude mice. However, the gene expression levels were relatively less in various tissues compared with B6 GFP mice. These findings suggest that nude GFP is an ideal model resembling normal nude mice; however, GFP expression in various tissues by fluorescence should be considered, as the expression of GFP differs in various organs.

Influence of the environment and phototoxicity of the live cell imaging system at IMP microbeam facility

NASA Astrophysics Data System (ADS)

Liu, Wenjing; Du, Guanghua; Guo, Jinlong; Wu, Ruqun; Wei, Junzhe; Chen, Hao; Li, Yaning; Zhao, Jing; Li, Xiaoyue

2017-08-01

To investigate the spatiotemporal dynamics of DNA damage and repair after the ion irradiation, an online live cell imaging system has been established based on the microbeam facility at Institute of Modern Physics (IMP). The system could provide a sterile and physiological environment by making use of heating plate and live cell imaging solution. The phototoxicity was investigated through the evaluation of DNA repair protein XRCC1 foci formed in HT1080-RFP cells during the imaging exposure. The intensity of the foci induced by phototoxicity was much lower compared with that of the foci induced by heavy ion hits. The results showed that although spontaneous foci were formed due to RFP exposure during live cell imaging, they had little impact on the analysis of the recruitment kinetics of XRCC1 in the foci induced by the ion irradiation.

Parotid adenoid cystic carcinoma: Retrospective single institute analysis.

PubMed

Mannelli, Giuditta; Cecconi, Lorenzo; Fasolati, Martina; Santoro, Roberto; Franchi, Alessandro; Gallo, Oreste

Adenoid cystic carcinoma (ACC) is a uncommon salivary malignant tumor. Our aim was to review our experience with parotid ACC, to identify clinical-pathological parameters predictive for outcome. We retrospectively reviewed 228 patients affected by parotid gland carcinomas surgically treated at our Institution. Forty-four ACC were included in this study. Multivariate analysis risk models were built to predict recurrence free probability (RFP), distant recurrence free probability (DRFP), overall survival (OS) and disease free survival (DFS). Twenty-one patients (47.7%) died from ACC and 2.3% for other causes. The 41% presented local-regional recurrence, with a regional-RFP rate of 93%, and the 34% reported distant metastases (DM). The five and ten-year OS rates were 74% and 50%, respectively. Recurrences were mainly influenced by the presence of perineural invasion and nerve paralysis, whilst female gender and age<50 were predictors for good prognosis. Copyright © 2017 Elsevier Inc. All rights reserved.

KTX circuit model and discharge waveform prediction

NASA Astrophysics Data System (ADS)

Bai, Wei; Lan, T.; Mao, W. Z.; You, W.; Li, H.; Liu, A. D.; Xie, J. L.; Wan, S. D.; Liu, W. D.; Yang, L.; Fu, P.; Xiao, C. J.; Ding, W. X.

2013-10-01

The Keda Torus eXperiment (KTX) is a constructing reversed field pinch (RFP) device in University of Science and Technology of China. The KTX power supply system includes the Ohmic heating, field shaping and toroidal power supply systems, which produce the Ohmic field, equilibrium field and toroidal field, respectively. The detailed circuit model will be introduced in this poster. Another purpose is to predict its discharge waveforms using the modified Bessel function mode (MBFM), which describes the evolution of plasma current and magnetic flux in RFP base on Taylor theory. Furthermore, the power supply requirements of external field shaping winding are also predicted in the model, which will be very helpful for the design of plasma equilibrium controlling system. Supported by ITER-China program (No. 2011GB106000), NNSFC (Nos. 10990210, 10990211, 10335060 and 10905057), CPSF (No. 20080440104), YIF (No. WK2030040019) and KIPCAS (No. kjcx-yw-n28).

Neutron and Gamma-ray Detection in Reversed-Field Pinch Deuterium Plasmas in the RFX-mod Device

NASA Astrophysics Data System (ADS)

Zuin, Matteo; Stevanato, Luca; Martines, Emilio; Gonzalez, Winder; Cavazzana, Roberto; Cester, Davide; Nebbia, G.; Sajo-Bohus, Laszlo; Viesti, Giuseppe

2014-10-01

An experimental analysis of neutron and gamma-ray fluxes exiting purely ohmically heated plasmas in reversed-field pinch (RFP) configuration is presented. The diagnostic system, installed in the RFX-mod, is made of 2 scintillators (EJ-301 liquid and NaI(Tl)) coupled to flat-panel photomultipliers, which can be operated under magnetic fields. The production of neutrons and gamma rays in Deuterium plasmas is found to be strongly dependent on the Ohmic input power, with a threshold value of about 1.2 MA in terms of plasma current level, below which low levels of gamma rays and almost no neutrons are detected. Neutron and gamma production is characterized by a bursty behavior, correlated to the spontaneous magnetic reconnection events, occurring almost cyclically in the RFP plasmas. The role of ion heating and particle acceleration during such events is discussed.

Three-Dimensional Reconstruction of Three-Way FRET Microscopy Improves Imaging of Multiple Protein-Protein Interactions.

PubMed

Scott, Brandon L; Hoppe, Adam D

2016-01-01

Fluorescence resonance energy transfer (FRET) microscopy is a powerful tool for imaging the interactions between fluorescently tagged proteins in two-dimensions. For FRET microscopy to reach its full potential, it must be able to image more than one pair of interacting molecules and image degradation from out-of-focus light must be reduced. Here we extend our previous work on the application of maximum likelihood methods to the 3-dimensional reconstruction of 3-way FRET interactions within cells. We validated the new method (3D-3Way FRET) by simulation and fluorescent protein test constructs expressed in cells. In addition, we improved the computational methods to create a 2-log reduction in computation time over our previous method (3DFSR). We applied 3D-3Way FRET to image the 3D subcellular distributions of HIV Gag assembly. Gag fused to three different FPs (CFP, YFP, and RFP), assembled into viral-like particles and created punctate FRET signals that become visible on the cell surface when 3D-3Way FRET was applied to the data. Control experiments in which YFP-Gag, RFP-Gag and free CFP were expressed, demonstrated localized FRET between YFP and RFP at sites of viral assembly that were not associated with CFP. 3D-3Way FRET provides the first approach for quantifying multiple FRET interactions while improving the 3D resolution of FRET microscopy data without introducing bias into the reconstructed estimates. This method should allow improvement of widefield, confocal and superresolution FRET microscopy data.

The Revolving Fund Pharmacy Model: backing up the Ministry of Health supply chain in western Kenya.

PubMed

Manji, Imran; Manyara, Simon M; Jakait, Beatrice; Ogallo, William; Hagedorn, Isabel C; Lukas, Stephanie; Kosgei, Eunice J; Pastakia, Sonak D

2016-10-01

A pressing challenge in low and middle-income countries (LMIC) is inadequate access to essential medicines, especially for chronic diseases. The Revolving Fund Pharmacy (RFP) model is an initiative to provide high-quality medications consistently to patients, using revenues generated from the sale of medications to sustainably resupply medications. This article describes the utilization of RFPs developed by the Academic Model Providing Access to Healthcare (AMPATH) with the aim of stimulating the implementation of similar models elsewhere to ensure sustainable access to quality and affordable medications in similar LMIC settings. The service evaluation of three pilot RFPs started between April 2011 and January 2012 in select government facilities is described. The evaluation assessed cross-sectional availability of essential medicines before and after implementation of the RFPs, number of patient encounters and the impact of community awareness activities. Availability of essential medicines in the three pilot RFPs increased from 40%, 36% and <10% to 90%, 94% and 91% respectively. After the first year of operation, the pilot RFPs had a total of 33 714 patient encounters. As of February 2014, almost 3 years after starting up the first RFP, the RFPs had a total of 115 991 patient encounters. In the Eldoret RFP, community awareness activities led to a 51% increase in sales. With proper oversight and stakeholder involvement, this model is a potential solution to improve availability of essential medicines in LMICs. These pilots exemplify the feasibility of implementing and scaling up this model in other locations. © 2016 Royal Pharmaceutical Society.

Systematic evaluation of markers used for the identification of human induced pluripotent stem cells

PubMed Central

Bharathan, Sumitha Prameela; Manian, Kannan Vrindavan; Aalam, Syed Mohammed Musheer; Palani, Dhavapriya; Deshpande, Prashant Ajit; Pratheesh, Mankuzhy Damodaran; Srivastava, Alok

2017-01-01

ABSTRACT Low efficiency of somatic cell reprogramming and heterogeneity among human induced pluripotent stem cells (hiPSCs) demand extensive characterization of isolated clones before their use in downstream applications. By monitoring human fibroblasts undergoing reprogramming for their morphological changes and expression of fibroblast (CD13), pluripotency markers (SSEA-4 and TRA-1-60) and a retrovirally expressed red fluorescent protein (RV-RFP), we compared the efficiency of these features to identify bona fide hiPSC colonies. The co-expression kinetics of fibroblast and pluripotency markers in the cells being reprogrammed and the emerging colonies revealed the heterogeneity within SSEA-4+ and TRA-1-60+ cells, and the inadequacy of these commonly used pluripotency markers for the identification of bona fide hiPSC colonies. The characteristic morphological changes in the emerging hiPSC colonies derived from fibroblasts expressing RV-RFP showed a good correlation between hiPSC morphology acquisition and silencing of RV-RFP and facilitated the easy identification of hiPSCs. The kinetics of retroviral silencing and pluripotency marker expression in emerging colonies suggested that combining both these markers could demarcate the stages of reprogramming with better precision than with pluripotency markers alone. Our results clearly demonstrate that the pluripotency markers that are routinely analyzed for the characterization of established iPSC colonies are not suitable for the isolation of pluripotent cells in the early stages of reprogramming, and silencing of retrovirally expressed reporter genes helps in the identification of colonies that have attained a pluripotent state and the morphology of human embryonic stem cells (hESCs). PMID:28089995

Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

PubMed Central

Lichtnekert, Julia; Kaverina, Natalya V.; Eng, Diana G.; Gross, Kenneth W.; Kutz, J. Nathan; Pippin, Jeffrey W.

2016-01-01

Because adult podocytes cannot proliferate and are therefore unable to self-renew, replacement of these cells depends on stem/progenitor cells. Although podocyte number is higher after renin-angiotensin-aldosterone system (RAAS) inhibition in glomerular diseases, the events explaining this increase are unclear. Cells of renin lineage (CoRL) have marked plasticity, including the ability to acquire a podocyte phenotype. To test the hypothesis that RAAS inhibition partially replenishes adult podocytes by increasing CoRL number, migration, and/or transdifferentiation, we administered tamoxifen to Ren1cCreERxRs-tdTomato-R CoRL reporter mice to induce permanent labeling of CoRL with red fluorescent protein variant tdTomato. We then induced experimental FSGS, typified by abrupt podocyte depletion, with a cytopathic antipodocyte antibody. RAAS inhibition by enalapril (angiotensin-converting enzyme inhibitor) or losartan (angiotensin-receptor blocker) in FSGS mice stimulated the proliferation of CoRL, increasing the reservoir of these cells in the juxtaglomerular compartment (JGC). Compared with water or hydralazine, RAAS inhibition significantly increased the migration of CoRL from the JGC to the intraglomerular compartment (IGC), with more glomeruli containing RFP+CoRL and, within these glomeruli, more RFP+CoRL. Moreover, RAAS inhibition in FSGS mice increased RFP+CoRL transdifferentiation in the IGC to phenotypes, consistent with those of podocytes (coexpression of synaptopodin and Wilms tumor protein), parietal epithelial cells (PAX 8), and mesangial cells (α8 integrin). These results show that in the context of podocyte depletion in FSGS, RAAS inhibition augments CoRL proliferation and plasticity toward three different glomerular cell lineages. PMID:27080979

Railroad classification yard technology : computer system methodology : case study : Potomac Yard

DOT National Transportation Integrated Search

1981-08-01

This report documents the application of the railroad classification yard computer system methodology to Potomac Yard of the Richmond, Fredericksburg, and Potomac Railroad Company (RF&P). This case study entailed evaluation of the yard traffic capaci...

RAPID INCIDENT RESPONSE FRAMEWORK

EPA Science Inventory

Will discuss WERF Contract (RFP# 03-HHE-5PP), Protocols for the Timely Investigation of Potential Health Incidents Associated with Biosolids Land Application, as a member of the project advisory committee. The contractor, University of North Carolina, started work in early June, ...

Color-coded Live Imaging of Heterokaryon Formation and Nuclear Fusion of Hybridizing Cancer Cells.

PubMed

Suetsugu, Atsushi; Matsumoto, Takuro; Hasegawa, Kosuke; Nakamura, Miki; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Bouvet, Michael; Hoffman, Robert M

2016-08-01

Fusion of cancer cells has been studied for over half a century. However, the steps involved after initial fusion between cells, such as heterokaryon formation and nuclear fusion, have been difficult to observe in real time. In order to be able to visualize these steps, we have established cancer-cell sublines from the human HT-1080 fibrosarcoma, one expressing green fluorescent protein (GFP) linked to histone H2B in the nucleus and a red fluorescent protein (RFP) in the cytoplasm and the other subline expressing RFP in the nucleus (mCherry) linked to histone H2B and GFP in the cytoplasm. The two reciprocal color-coded sublines of HT-1080 cells were fused using the Sendai virus. The fused cells were cultured on plastic and observed using an Olympus FV1000 confocal microscope. Multi-nucleate (heterokaryotic) cancer cells, in addition to hybrid cancer cells with single-or multiple-fused nuclei, including fused mitotic nuclei, were observed among the fused cells. Heterokaryons with red, green, orange and yellow nuclei were observed by confocal imaging, even in single hybrid cells. The orange and yellow nuclei indicate nuclear fusion. Red and green nuclei remained unfused. Cell fusion with heterokaryon formation and subsequent nuclear fusion resulting in hybridization may be an important natural phenomenon between cancer cells that may make them more malignant. The ability to image the complex processes following cell fusion using reciprocal color-coded cancer cells will allow greater understanding of the genetic basis of malignancy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Dumbo heavy lifter aircraft

NASA Technical Reports Server (NTRS)

Riester, Peter; Ellis, Colleen; Wagner, Michael; Orren, Scott; Smith, Byron; Skelly, Michael; Zgraggen, Craig; Webber, Matt

1992-01-01

The world is rapidly changing from one with two military superpowers, with which most countries were aligned, to one with many smaller military powers. In this environment, the United States cannot depend on the availability of operating bases from which to respond to crises requiring military intervention. Several studies (e.g. the SAB Global Reach, Global Power Study) have indicated an increased need to be able to rapidly transport large numbers of troops and equipment from the continental United States to potential trouble spots throughout the world. To this end, a request for proposals (RFP) for the concept design of a large aircraft capable of 'projecting' a significant military force without reliance on surface transportation was developed. These design requirements are: minimum payload of 400,000 pounds at 2.5 g maneuver load factor; minimum unfueled range of 6,000 nautical miles; and aircraft must operate from existing domestic air bases and use existing airbases or sites of opportunity at the destination.

Region 4: South Carolina Adequate Letter and Response to Comments (6/21/2012)

EPA Pesticide Factsheets

This May 25, 2012 letter from EPA approves South Carolina's reasonable further progress (RFP) plan including the motor vehicle emission budget (MVEB) for volatile organic compounds (VOC) and EPA's response to comments received on the adequacy notification

Consortium for Petroleum & Natural Gas Stripper Wells PART 3 OF 3

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

Morrison, Joel

The United States has more oil and gas wells than any other country. As of December 31, 2004, there were more than half a million producing oil wells in the United States. That is more than three times the combined total for the next three leaders: China, Canada, and Russia. The Stripper Well Consortium (SWC) is a partnership that includes domestic oil and gas producers, service and supply companies, trade associations, academia, the Department of Energy’s Strategic Center for Natural Gas and Oil (SCNGO) at the National Energy Technology Laboratory (NETL), and the New York State Energy Research and Developmentmore » Authority (NYSERDA). The Consortium was established in 2000. This report serves as a final technical report for the SWC activities conducted over the May 1, 2004 to December 1, 2011 timeframe. During this timeframe, the SWC worked with 173 members in 29 states and three international countries, to focus on the development of new technologies to benefit the U.S. stripper well industry. SWC worked with NETL to develop a nationwide request-for-proposal (RFP) process to solicit proposals from the U.S. stripper well industry to develop and/or deploy new technologies that would assist small producers in improving the production performance of their stripper well operations. SWC conducted eight rounds of funding. A total of 132 proposals were received. The proposals were compiled and distributed to an industrydriven SWC executive council and program sponsors for review. Applicants were required to make a formal technical presentation to the SWC membership, executive council, and program sponsors. After reviewing the proposals and listening to the presentations, the executive council made their funding recommendations to program sponsors. A total of 64 projects were selected for funding, of which 59 were fully completed. Penn State then worked with grant awardees to issue a subcontract for their approved work. SWC organized and hosted a total of 14 meetings dedicated to technology transfer to showcase and review SWC-funded technology. The workshops were open to the stripper well industry.« less

Consortium for Petroleum & Natural Gas Stripper Wells PART 2 OF 3

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

Morrison, Joel

2011-12-01

The United States has more oil and gas wells than any other country. As of December 31, 2004, there were more than half a million producing oil wells in the United States. That is more than three times the combined total for the next three leaders: China, Canada, and Russia. The Stripper Well Consortium (SWC) is a partnership that includes domestic oil and gas producers, service and supply companies, trade associations, academia, the Department of Energy’s Strategic Center for Natural Gas and Oil (SCNGO) at the National Energy Technology Laboratory (NETL), and the New York State Energy Research and Developmentmore » Authority (NYSERDA). The Consortium was established in 2000. This report serves as a final technical report for the SWC activities conducted over the May 1, 2004 to December 1, 2011 timeframe. During this timeframe, the SWC worked with 173 members in 29 states and three international countries, to focus on the development of new technologies to benefit the U.S. stripper well industry. SWC worked with NETL to develop a nationwide request-for-proposal (RFP) process to solicit proposals from the U.S. stripper well industry to develop and/or deploy new technologies that would assist small producers in improving the production performance of their stripper well operations. SWC conducted eight rounds of funding. A total of 132 proposals were received. The proposals were compiled and distributed to an industrydriven SWC executive council and program sponsors for review. Applicants were required to make a formal technical presentation to the SWC membership, executive council, and program sponsors. After reviewing the proposals and listening to the presentations, the executive council made their funding recommendations to program sponsors. A total of 64 projects were selected for funding, of which 59 were fully completed. Penn State then worked with grant awardees to issue a subcontract for their approved work. SWC organized and hosted a total of 14 meetings dedicated to technology transfer to showcase and review SWC-funded technology. The workshops were open to the stripper well industry.« less

Role of a continuous MHD dynamo in the formation of 3D equilibria in fusion plasmas

NASA Astrophysics Data System (ADS)

Piovesan, P.; Bonfiglio, D.; Cianciosa, M.; Luce, T. C.; Taylor, N. Z.; Terranova, D.; Turco, F.; Wilcox, R. S.; Wingen, A.; Cappello, S.; Chrystal, C.; Escande, D. F.; Holcomb, C. T.; Marrelli, L.; Paz-Soldan, C.; Piron, L.; Predebon, I.; Zaniol, B.; DIII-D, The; RFX-Mod Teams

2017-07-01

Stationary 3D equilibria can form in fusion plasmas via saturation of magnetohydrodynamic (MHD) instabilities or stimulated by external 3D fields. In these cases the current profile is anomalously broad due to magnetic flux pumping produced by the MHD modes. Flux pumping plays an important role in hybrid tokamak plasmas, maintaining the minimum safety factor above unity and thus removing sawteeth. It also enables steady-state hybrid operation, by redistributing non-inductive current driven near the center by electron cyclotron waves. A validated flux pumping model is not yet available, but it would be necessary to extrapolate hybrid operation to future devices. In this work flux pumping physics is investigated for helical core equilibria stimulated by external 3D fields in DIII-D hybrid plasmas. We show that flux pumping can be produced in a continuous way by an MHD dynamo emf. The same effect maintains helical equilibria in reversed-field pinch (RFP) plasmas. The effective MHD dynamo loop voltage is calculated for experimental 3D equilibrium reconstructions, by balancing Ohm’s law over helical flux surfaces, and is consistent with the expected current redistribution. Similar results are also obtained with more sophisticated nonlinear MHD simulations. The same modelling approach is applied to helical RFP states forming spontaneously in RFX-mod as the plasma current is raised above 0.8-1 MA. This comparison allows to identify the underlying physics common to tokamak and RFP: a helical core displacement modulates parallel current density along flux tubes, which requires a helical electrostatic potential to build up, giving rise to a helical MHD dynamo flow.

Relative Accuracy of Nucleic Acid Amplification Tests and Culture in Detecting Chlamydia in Asymptomatic Men

PubMed Central

Cheng, Hong; Macaluso, Maurizio; Vermund, Sten H.; Hook, Edward W.

2001-01-01

Published estimates of the sensitivity and specificity of PCR and ligase chain reaction (LCR) for detecting Chlamydia trachomatis are potentially biased because of study design limitations (confirmation of test results was limited to subjects who were PCR or LCR positive but culture negative). Relative measures of test accuracy are less prone to bias in incomplete study designs. We estimated the relative sensitivity (RSN) and relative false-positive rate (RFP) for PCR and LCR versus cell culture among 1,138 asymptomatic men and evaluated the potential bias of RSN and RFP estimates. PCR and LCR testing in urine were compared to culture of urethral specimens. Discordant results (PCR or LCR positive, but culture negative) were confirmed by using a sequence including the other DNA amplification test, direct fluorescent antibody testing, and a DNA amplification test to detect chlamydial major outer membrane protein. The RSN estimates for PCR and LCR were 1.45 (95% confidence interval [CI] = 1.3 to 1.7) and 1.49 (95% CI = 1.3 to 1.7), respectively, indicating that both methods are more sensitive than culture. Very few false-positive results were found, indicating that the specificity levels of PCR, LCR, and culture are high. The potential bias in RSN and RFP estimates were <5 and <20%, respectively. The estimation of bias is based on the most likely and probably conservative parameter settings. If the sensitivity of culture is between 60 and 65%, then the true sensitivity of PCR and LCR is between 90 and 97%. Our findings indicate that PCR and LCR are significantly more sensitive than culture, while the three tests have similar specificities. PMID:11682509

Lower hybrid accessibility in a large, hot reversed field pinch

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

Dziubek, R.A.

1995-02-01

Recent theoretical and experimental results indicate that driving a current in the outer radius of an RPF suppresses sawtooth activity and increases particle and energy confinement times. One candidate for a form of steady state current drive is the slow wave at the lower hybrid frequency. Here, the accessibility of such a wave in an RFP plasma is investigated theoretically, with focus on the RFX machine of Padua, Italy. To drive current, the slow wave with frequency between 1.0--1.5 GHz is considered where optimal Landau damping is desired at r/a {approximately} 0.7. By numerically determining the values of the wave`smore » perpendicular index of refraction which satisfy the hot plasma dispersion relation, regions of propagation and evanescence can be found. The path of the wave can then be traced over a contour map of these regions so that accessibility can be clearly seen. The possibility of mode conversion events can be ascertained by plotting the values of the perpendicular index of refraction for the fast and slow wave and observing convergence points. To locate regions of maximum Landau damping, a technique developed by Stix was adapted for use with the slow wave in an RFP plasma. Results show that the slow wave is accessible to the target region without mode conversion so long as the value of the parallel index of refraction is correctly chosen at the edge of the plasma. Landau damping can also be optimized with this method. In an RFP, 2--20% of the electron population consists of fast electrons. Because this species alters the total electron distribution function and raises the effective temperature in the outer regions of the plasma, its presence is expected to shift the location of ideal Landau damping.« less

Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications

PubMed Central

Herfst, Sander; Bestebroer, Theo M.; Vaes, Vincent P.; van der Hoeven, Barbara; Koster, Abraham J.; Kremers, Gert-Jan; Scott, Dana P.; Gultyaev, Alexander P.; Sorell, Erin M.; de Graaf, Miranda; Bárcena, Montserrat; Rimmelzwaan, Guus F.; Fouchier, Ron A.

2015-01-01

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses. PMID:26241861

Color-coded Imaging Enables Fluorescence-guided Surgery to Resect the Tumor Along with the Tumor Microenvironment in a Syngeneic Mouse Model of EL-4 Lymphoma.

PubMed

Hasegawa, Kosuke; Suetsugu, Atsushi; Nakamura, Miki; Matsumoto, Takuro; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Bouvet, Michael; Hoffman, Robert M

2016-09-01

Fluorescence-guided surgery (FGS) of cancer is an emerging technology. We have previously shown the importance of resecting both the tumor and the tumor microenvironment (TME) for curative FGS. We also previously developed a syngeneic model using the mouse lymphoma cell line EL-4, expressing red fluorescent protein (EL-4-RFP), growing in green fluorescent protein (GFP) transgenic mice, which we have used in the present report to develop FGS of the tumor microenvironment. EL-4-RFP lymphoma cells were injected subcutaneously in C57/BL6 GFP transgenic mice. EL-4-RFP cells subsequently formed tumors by 35 days after cell transplantation. Using the portable hand-held Dino-Lite digital imaging system, subcutaneous tumors were resected by FGS. Resected tumor tissues were visualized with the Olympus FV1000 confocal microscope. Using the Dino-Lite, subcutaneous tumors and the tumor microenvironment were clearly visualized and resected. In the resected tumor, host stromal cells, including adipocyte-like cells and blood vessels with lymphocytes, were observed by confocal microscopy in addition to cancer cells by color-coded confocal imaging. The cancer cells and stromal cells in the TME were deeply intermingled in a highly-complex pattern. Color-coded FGS is an effective method to completely resect cancer cells along with the stromal cells in the TME which interact in a highly-complex pattern. Microscopically, cancer cells invade the TME and vice versa. To prevent tumor recurrence, it is necessary to resect the TME along with the tumor. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells.

PubMed

von Einem, Bjoern; Weber, Petra; Wagner, Michael; Malnar, Martina; Kosicek, Marko; Hecimovic, Silva; Arnim, Christine A F von; Schneckenburger, Herbert

2012-11-26

Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer's disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1-/-), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1-/- cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1-/- cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1-/- cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1-/-. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.

In vivo imaging and quantitative monitoring of autophagic flux in tobacco BY-2 cells.

PubMed

Hanamata, Shigeru; Kurusu, Takamitsu; Okada, Masaaki; Suda, Akiko; Kawamura, Koki; Tsukada, Emi; Kuchitsu, Kazuyuki

2013-01-01

Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli.

In vivo imaging and quantitative monitoring of autophagic flux in tobacco BY-2 cells

PubMed Central

Hanamata, Shigeru; Kurusu, Takamitsu; Okada, Masaaki; Suda, Akiko; Kawamura, Koki; Tsukada, Emi; Kuchitsu, Kazuyuki

2013-01-01

Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli. PMID:23123450

Recloned dogs derived from adipose stem cells of a transgenic cloned beagle.

PubMed

Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Hong, So Gun; Ra, Jeong Chan; Jo, Jung Youn; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2011-04-15

A number of studies have postulated that efficiency in mammalian cloning is inversely correlated with donor cell differentiation status and may be increased by using undifferentiated cells as nuclear donors. Here, we attempted the recloning of dogs by nuclear transfer of canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) from a transgenic cloned beagle to determine if cAd-MSCs can be a suitable donor cell type. In order to isolate cAd-MSCs, adipose tissues were collected from a transgenic cloned beagle produced by somatic cell nuclear transfer (SCNT) of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. The cAd-MSCs expressed the RFP gene and cell-surface marker characteristics of MSCs including CD29, CD44 and thy1.1. Furthermore, cAd-MSCs underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. In order to investigate the developmental potential of cAd-MSCs, we carried out SCNT. Fused-couplets (82/109, 75.2%) were chemically activated and transferred into the uterine tube of five naturally estrus-synchronized surrogates. One of them (20%) maintained pregnancy and subsequently gave birth to two healthy cloned pups. The present study demonstrated for the first time the successful production of cloned beagles by nuclear transfer of cAd-MSCs. Another important outcome of the present study is the successful recloning of RFP-expressing transgenic cloned beagle pups by nuclear transfer of cells derived from a transgenic cloned beagle. In conclusion, the present study demonstrates that adipose stem cells can be a good nuclear donor source for dog cloning. Copyright © 2011 Elsevier Inc. All rights reserved.

Controlling Protein Surface Orientation by Strategic Placement of Oligo-Histidine Tags

PubMed Central

2017-01-01

We report oriented immobilization of proteins using the standard hexahistidine (His6)-Ni2+:NTA (nitrilotriacetic acid) methodology, which we systematically tuned to give control of surface coverage. Fluorescence microscopy and surface plasmon resonance measurements of self-assembled monolayers (SAMs) of red fluorescent proteins (TagRFP) showed that binding strength increased by 1 order of magnitude for each additional His6-tag on the TagRFP proteins. All TagRFP variants with His6-tags located on only one side of the barrel-shaped protein yielded a 1.5 times higher surface coverage compared to variants with His6-tags on opposite sides of the so-called β-barrel. Time-resolved fluorescence anisotropy measurements supported by polarized infrared spectroscopy verified that the orientation (and thus coverage and functionality) of proteins on surfaces can be controlled by strategic placement of a His6-tag on the protein. Molecular dynamics simulations show how the differently tagged proteins reside at the surface in “end-on” and “side-on” orientations with each His6-tag contributing to binding. Also, not every dihistidine subunit in a given His6-tag forms a full coordination bond with the Ni2+:NTA SAMs, which varied with the position of the His6-tag on the protein. At equal valency but different tag positions on the protein, differences in binding were caused by probing for Ni2+:NTA moieties and by additional electrostatic interactions between different fractions of the β-barrel structure and charged NTA moieties. Potential of mean force calculations indicate there is no specific single-protein interaction mode that provides a clear preferential surface orientation, suggesting that the experimentally measured preference for the end-on orientation is a supra-protein, not a single-protein, effect. PMID:28850777

Immunization with antigenic extracts of Leishmania associated with Montanide ISA 763 adjuvant induces partial protection in BALB/c mice against Leishmania (Leishmania) amazonensis infection.

PubMed

Cargnelutti, Diego Esteban; Salomón, María Cristina; Celedon, Verónica; García Bustos, María Fernanda; Morea, Gastón; Cuello-Carrión, Fernando Darío; Scodeller, Eduardo Alberto

2016-02-01

A proper adjuvant has a relevant role in vaccine formulations to generate an effective immune response. In this study, total Leishmania antigen (TLA) formulated with Montanide ISA 763 or R848 as adjuvants were evaluated as a first generation Leishmania vaccine in a murine model. Immunization protocols were tested in BALB/c mice with a subcutaneous prime/boost regimen with an interval of 3 weeks. Mice immunized with unadjuvanted TLA and phosphate-buffered saline (PBS) served as control groups. On Day 21 and Day 36 of the protocol, we evaluated the humoral immune response induced by each formulation. Fifteen days after the boost, the immunized mice were challenged with 1 × 10(5) promastigotes of Leishmania (Leishmania) amazonensis in the right footpad (RFP). The progress of the infection was followed for 10 weeks; at the end of this period, histopathological studies were performed in the RFP. Vaccines formulated with Montanide ISA 763 generated an increase in the production of immunoglobulin G (IgG; p < 0.05) compared with the control group. There were no statistically significant differences in IgG1 production between the study groups. However, immunization with TLA-Montanide ISA 763 resulted in an increase in IgG2a compared to the unadjuvanted control (p < 0.001). Also noteworthy was the fact that a significant reduction in swelling and histopathological damage of the RFP was recorded with the Montanide ISA 763 formulation. We conclude that the immunization of BALB/c mice with a vaccine formulated with TLA and Montanide ISA 763 generated a protective immune response against L. (L.) amazonensis, characterized by an intense production of IgG2a. Copyright © 2014. Published by Elsevier B.V.

Effect of lipophilicity on the bioavailability of drugs after percutaneous administration by dissolving microneedles.

PubMed

Ito, Yukako; Yoshimura, Masahiro; Tanaka, Tsutomu; Takada, Kanji

2012-03-01

To elucidate drug lipophilicity effects on the bioavailability (BA) of drugs from skin after administration by dissolving microneedles, nine compounds with different lipophilicity indexes (log p value) were formulated into two-layered dissolving microneedles and administered percutaneously to rat skin: desmopressin (DDAVP), sumatriptan (ST), fluorescein (FL), granisetron (GRN), pindolol (PDL), pravastatin (PRV), rhodamine 123 (Rho), rifampicin (RFP), and salmeterol (SLM). Plasma drug concentrations were measured using liquid chromatography-tandem mass spectrometry and spectrofluorometry. In vivo dissolution and diffusion in both horizontal and vertical directions of FL and RH in the skin were studied using fluorescence microscopy. Respective BAs were 95.1 ± 7.9% (DDAVP), 84.2 ± 2.7% (ST), 82.3 ± 7.2% (FL), 82.7 ± 6.7% (GRN), 71.6 ± 3.8% (PDL), 63.6 ± 7.5% (PRV), 53.7 ± 8.3% (Rho), 46.2 ± 6.1% (RFP), and 38.4 ± 2.7% (SM). BA decreased as the lipophilicity index, log p value, of the drug increased from-1.95 to 1.73. The respective remaining percentages in skin tissue were 1.4 ± 0.7% (DDAVP), 0.9 ± 0.1% (ST), 1.0 ± 0.2% (FL), 3.4 ± 1.2% (GRN), 14.5 ± 3.7% (PDL), 23.4 ± 5.2% (PRV), 32.2 ± 6.0% (Rho), 40.7 ± 4.9% (RFP), and 40.6 ± 5.1% (SLM), dependent on log p. Fluorescence microscopy showed no FL or Rho in skin tissue within 4 and 24 h after administration, respectively. The BA of drugs delivered by dissolving microneedles depends on the drug solubility in the skin epidermis and dermis. Copyright © 2011 Wiley Periodicals, Inc.

Role of a continuous MHD dynamo in the formation of 3D equilibria in fusion plasmas

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

Piovesan, P.; Bonfiglio, D.; Cianciosa, M.

Stationary 3D equilibria can form in fusion plasmas via saturation of magnetohydrodynamic (MHD) instabilities or stimulated by external 3D fields. In these cases the current profile is anomalously broad due to magnetic flux pumping produced by the MHD modes. Flux pumping plays an important role in hybrid tokamak plasmas, maintaining the minimum safety factor above unity and thus removing sawteeth. It also enables steady-state hybrid operation, by redistributing non-inductive current driven near the center by electron cyclotron waves. A validated flux pumping model is not yet available, but it would be necessary to extrapolate hybrid operation to future devices. Inmore » this work flux pumping physics is investigated for helical core equilibria stimulated by external 3D fields in DIII-D hybrid plasmas. We show that flux pumping can be produced in a continuous way by an MHD dynamo emf. The same effect maintains helical equilibria in reversed-field pinch (RFP) plasmas. The effective MHD dynamo loop voltage is calculated for experimental 3D equilibrium reconstructions, by balancing Ohm’s law over helical flux surfaces, and is consistent with the expected current redistribution. Similar results are also obtained with more sophisticated nonlinear MHD simulations. The same modelling approach is applied to helical RFP states forming spontaneously in RFX-mod as the plasma current is raised above 0.8–1 MA. This comparison allows to identify the underlying physics common to tokamak and RFP: a helical core displacement modulates parallel current density along flux tubes, which requires a helical electrostatic potential to build up, giving rise to a helical MHD dynamo flow.« less

Role of a continuous MHD dynamo in the formation of 3D equilibria in fusion plasmas

DOE PAGES

Piovesan, P.; Bonfiglio, D.; Cianciosa, M.; ...

2017-04-28

Stationary 3D equilibria can form in fusion plasmas via saturation of magnetohydrodynamic (MHD) instabilities or stimulated by external 3D fields. In these cases the current profile is anomalously broad due to magnetic flux pumping produced by the MHD modes. Flux pumping plays an important role in hybrid tokamak plasmas, maintaining the minimum safety factor above unity and thus removing sawteeth. It also enables steady-state hybrid operation, by redistributing non-inductive current driven near the center by electron cyclotron waves. A validated flux pumping model is not yet available, but it would be necessary to extrapolate hybrid operation to future devices. Inmore » this work flux pumping physics is investigated for helical core equilibria stimulated by external 3D fields in DIII-D hybrid plasmas. We show that flux pumping can be produced in a continuous way by an MHD dynamo emf. The same effect maintains helical equilibria in reversed-field pinch (RFP) plasmas. The effective MHD dynamo loop voltage is calculated for experimental 3D equilibrium reconstructions, by balancing Ohm’s law over helical flux surfaces, and is consistent with the expected current redistribution. Similar results are also obtained with more sophisticated nonlinear MHD simulations. The same modelling approach is applied to helical RFP states forming spontaneously in RFX-mod as the plasma current is raised above 0.8–1 MA. This comparison allows to identify the underlying physics common to tokamak and RFP: a helical core displacement modulates parallel current density along flux tubes, which requires a helical electrostatic potential to build up, giving rise to a helical MHD dynamo flow.« less

Measurement of magnetic fluctuation-induced heat transport in tokamaks and RFP

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

Fiksel, G.; Hartog, D.D.; Cekic, M.

1996-08-01

It has long been recognized that fluctuations in the magnetic field are a potent mechanism for the anomalous transport of energy in confined plasmas. The energy transport process originates from particle motion along magnetic fields, which have a fluctuating component in the radial direction (perpendicular to the confining equilibrium magnetic surfaces). A key feature is that the transport can be large even if the fluctuation amplitude is small. If the fluctuations are resonant with the equilibrium magnetic field (i.e., the fluctuation amplitude is constant along an equilibrium field line) then a small fluctuation can introduce stochasticity to the field linemore » trajectories. Particles following the chaotically wandering field lines can rapidly carry energy across the plasma.« less

Impurity profiles and radial transport in the EXTRAP-T2 reversed field pinch

NASA Astrophysics Data System (ADS)

Sallander, J.

1999-05-01

Radially resolved spectroscopy has been used to measure the radial distribution of impurity ions (O III-O V and C III-CVI) in the EXTRAP-T2 reversed field pinch (RFP). The radial profile of the emission is reconstructed from line emission measured along five lines of sight. The ion density profile is the fitted quantity in the reconstruction of the brightness profile and is thus obtained directly in this process. These measurements are then used to adjust the parameters in transport calculations in order to obtain consistency with the observed ion density profiles. Comparison between model and measurements show that a radial dependence in the diffusion is needed to explain the measured ion densities.

76 FR 17103 - Submission for OMB Review; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2011-03-28

.... Description of Respondents: Business or other for-profit; Not-for- profit institutions. Number of Respondents... Government loan security. Description of Respondents: Not-for-profit institutions; Business or other for... communities with water or wastewater systems. Qualified private non-profit organizations will receive RFP...

75 FR 33172 - Approval and Promulgation of Air Quality Implementation Plans; Maryland; Reasonable Further...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-11

... Emission Inventory, Contingency Measures, Reasonably Available Control Measures, and Transportation... inventory, RFP contingency measure, and reasonably available control measure (RACM) requirements of the...) associated with this revision. EPA is approving the SIP revision because it satisfies the emission inventory...

7 CFR 4284.11 - Award requirements.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Agriculture Regulations of the Department of Agriculture (Continued) RURAL BUSINESS-COOPERATIVE SERVICE AND... RFP; (b) Submit a feasibility study and business plan showing the viability of the venture, if any...; (d) Maintain a financial management system that is acceptable to the Agency; and (e) Collect and...

40 CFR 52.2239 - Original Identification of plan section.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Carbon Monoxide Reasonable Further Progress (RFP) curve adopted on May 8, 1985. (ii) Other material. (A...) Permit for battery receiving and breaking operation for Refined Metals Corporation which became effective... Efficiency. (35) Rule 1200-3-18-.46 Test Method for Determination of Solvent...

40 CFR 52.2239 - Original Identification of plan section.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Carbon Monoxide Reasonable Further Progress (RFP) curve adopted on May 8, 1985. (ii) Other material. (A...) Permit for battery receiving and breaking operation for Refined Metals Corporation which became effective... Efficiency. (35) Rule 1200-3-18-.46 Test Method for Determination of Solvent...

SITE TECHNOLOGY CAPSULE: FILTER FLOW TECHNOLOGY, INC. - COLLOID POLISHING FILTER METHOD

EPA Science Inventory

The Filter Flow Technology, Inc. (FFT) Coloid Polishing Filter Method (CPFM) was demonstrated at the U.S Department of Energy's (DOE) Rock Flats Plant (RFP) as part of the U.S. Environmental Protection Agency's (EPA) Superfund and Innovative Technology Evaluation (SITE) program. ...

Current profile redistribution driven by neutral beam injection in a reversed-field pinch

NASA Astrophysics Data System (ADS)

Parke, E.; Anderson, J. K.; Brower, D. L.; Den Hartog, D. J.; Ding, W. X.; Johnson, C. A.; Lin, L.

2016-05-01

Neutral beam injection in reversed-field pinch (RFP) plasmas on the Madison Symmetric Torus [Dexter et al., Fusion Sci. Technol. 19, 131 (1991)] drives current redistribution with increased on-axis current density but negligible net current drive. Internal fluctuations correlated with tearing modes are observed on multiple diagnostics; the behavior of tearing mode correlated structures is consistent with flattening of the safety factor profile. The first application of a parametrized model for island flattening to temperature fluctuations in an RFP allows inferrence of rational surface locations for multiple tearing modes. The m = 1, n = 6 mode is observed to shift inward by 1.1 ± 0.6 cm with neutral beam injection. Tearing mode rational surface measurements provide a strong constraint for equilibrium reconstruction, with an estimated reduction of q0 by 5% and an increase in on-axis current density of 8% ± 5%. The inferred on-axis current drive is consistent with estimates of fast ion density using TRANSP [Goldston et al., J. Comput. Phys. 43, 61 (1981)].

Observation of trapped-electron-mode microturbulence in reversed field pinch plasmas

NASA Astrophysics Data System (ADS)

Duff, J. R.; Williams, Z. R.; Brower, D. L.; Chapman, B. E.; Ding, W. X.; Pueschel, M. J.; Sarff, J. S.; Terry, P. W.

2018-01-01

Density fluctuations in the large-density-gradient region of improved confinement Madison Symmetric Torus reversed field pinch (RFP) plasmas exhibit multiple features that are characteristic of the trapped-electron mode (TEM). Core transport in conventional RFP plasmas is governed by magnetic stochasticity stemming from multiple long-wavelength tearing modes. Using inductive current profile control, these tearing modes are reduced, and global confinement is increased to that expected for comparable tokamak plasmas. Under these conditions, new short-wavelength fluctuations distinct from global tearing modes appear in the spectrum at a frequency of f ˜ 50 kHz, which have normalized perpendicular wavenumbers k⊥ρs≲ 0.2 and propagate in the electron diamagnetic drift direction. They exhibit a critical-gradient threshold, and the fluctuation amplitude increases with the local electron density gradient. These characteristics are consistent with predictions from gyrokinetic analysis using the Gene code, including increased TEM turbulence and transport from the interaction of remnant tearing magnetic fluctuations and zonal flow.

In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein.

PubMed

Kolostova, Katarina; Zhang, Yong; Hoffman, Robert M; Bobek, Vladimir

2014-09-01

In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.

PubMed

Koo, Ok Jae; Park, Sol Ji; Lee, Choongil; Kang, Jung Taek; Kim, Sujin; Moon, Joon Ho; Choi, Ji Yei; Kim, Hyojin; Jang, Goo; Kim, Jin-Soo; Kim, Seokjoong; Lee, Byeong-Chun

2014-03-01

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP(+)/eGFP(+)) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

Comparing nonlinear MHD simulations of low-aspect-ratio RFPs to RELAX experiments

NASA Astrophysics Data System (ADS)

McCollam, K. J.; den Hartog, D. J.; Jacobson, C. M.; Sovinec, C. R.; Masamune, S.; Sanpei, A.

2016-10-01

Standard reversed-field pinch (RFP) plasmas provide a nonlinear dynamical system as a validation domain for numerical MHD simulation codes, with applications in general toroidal confinement scenarios including tokamaks. Using the NIMROD code, we simulate the nonlinear evolution of RFP plasmas similar to those in the RELAX experiment. The experiment's modest Lundquist numbers S (as low as a few times 104) make closely matching MHD simulations tractable given present computing resources. Its low aspect ratio ( 2) motivates a comparison study using cylindrical and toroidal geometries in NIMROD. We present initial results from nonlinear single-fluid runs at S =104 for both geometries and a range of equilibrium parameters, which preliminarily show that the magnetic fluctuations are roughly similar between the two geometries and between simulation and experiment, though there appear to be some qualitative differences in their temporal evolution. Runs at higher S are planned. This work is supported by the U.S. DOE and by the Japan Society for the Promotion of Science.

PACS strategy for imaging centers.

PubMed

Bedel, Victoria; Zdanowicz, Mark

2004-01-01

Picture archiving and communications systems (PACS) have been available in imaging centers for many years, but they often were less functional, were not well integrated into patient information systems, and lacked the network backbone to implement a system. As modalities are replaced and technology improves, the ability and time for an imaging center to acquire, integrate, and utilize PACS has arrived. However, each imaging center must determine why it should invest in PACS. A business plan is the fundamental need. Each imaging center must understand its target market, growth rate, and staffing plans. Additional considerations lie in current and future modality availability, the need for offsite delivery of images and reports, and the potential need for remote transmission of images. These issues must be identified and prioritized. A multidisciplinary team is essential. The most successful PACS implementation begins with complete involvement from all levels. The team should be comprised of people with complementary skills who are committed to a common purpose, set of performance goals, and approach for which they hold themselves mutually accountable. The team must jointly decide on the project's objectives. These objectives fall under 4 categories: clinical, service, financial, and performance. PACS must be considered a tool to help accomplish each objective. The imaging center must determine its top priorities, then translate them into a technology "wish list." The center can then list those pieces of technology that are most important and prioritize them. There are even more considerations for connecting multiple imaging centers. The team must create a comprehensive request for proposal (RFP) and determine the vendors that will receive the document. Once the RFP responses have been received and the vendor has been selected, an effective training plan must be executed. Training plans should be competency-based, ensuring comfort and competency among all staff. Upon the project's completion, it is essential for the imaging center to evaluate PACS' effect upon its services and relationships with patients, staff, and referring physicians. Marketing and demonstrating the latest technology can positively impact all these areas.

Creating Student Engagement Through Immersion: Exploring the MT6 Shipwreck in Puget Sound via Submersible, Using a Team Challenge Format

NASA Astrophysics Data System (ADS)

Sarason, C. P.; Hartzler, R.; Anderson, A.

2016-02-01

Educational literature has many stories describing the "aha" moment that teachers are privileged to observe: a student who suddenly grasps a concept (ding!), dives into an activity (whee!) or works tirelessly to complete a complicated project (wow!). Designing moments like these for students can be one of the great joys of teaching. Experiencing such a moment can have a lasting impact on student engagement and motivation, and is the underlying rationale for the importance of creating a wide array of outreach efforts, from field work to telepresence activities.During the spring of 2015, OceanGate Foundation and Seattle Central College partnered on a pilot program to design a program that had this kind of impact and created the Open The Oceans Challenge. Student teams responded to our Request for Proposal (RFP), which was centered around the exploration of a shipwreck in 150 meters of water just off the Seattle waterfront, the MT-6. Students spent a significant amount of time crafting their proposals and reported that they enjoyed the process of writing the proposal as a team. The two winning teams accompanied us on a series of submersible dives performed by OceanGate, Inc. and worked up the results afterward. In addition to researching the MT-6 wreck and documenting what they found, students were able to do basic observational research on the wreck, providing sonar and photographic data that will help form the basis of future expeditions to this site.We report on lessons learned from this pilot — the impact for the small set of students involved in this project was profound, but how can we expand the reach of such activities? Results from this experience suggest that providing a highly engaging topic and expedition may not be enough to inspire an "aha" moment that creates lasting engagement; the scaffolding and mentorship that surrounds a rich experience is also critical.

A Laboratory Exercise for Visible Gel Filtration Chromatography Using Fluorescent Proteins

ERIC Educational Resources Information Center

Zhang, Wenqiang; Cao, Yibin; Xu, Lishan; Gong, Jufang; Sun, Meihao

2015-01-01

Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified,…

48 CFR 2415.304 - Evaluation factors.

Code of Federal Regulations, 2010 CFR

2010-10-01

... DEVELOPMENT CONTRACTING METHODS AND CONTRACTING TYPES CONTRACTING BY NEGOTIATION Source Selection 2415.304... assigned a numerical weight (except for pass-fail factors) which shall appear in the RFP. When using LPTA, each evaluation factor is applied on a “pass-fail” basis; numerical scores are not assigned. “Pass-fail...

Navy LX(R) Amphibious Ship Program: Background and Issues for Congress

DTIC Science & Technology

2016-05-27

and LHA-8 to NASSCO, Ingalls,” USNI News, July 10, 2015; Valerie Insinna, “Navy Quietly Issues RFP for LHA-8, TAO(X),” (continued...) Navy LX(R...to two bidders—Huntington Ingalls Industries’ Ingalls Shipbuilding (HII/Ingalls) and General Dynamics’ National Steel and Shipbuilding Company—on

76 FR 70091 - Approval and Promulgation of Implementation Plans and Designation of Areas for Air Quality...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-10

... reasonable further progress (RFP), reasonable available control measures, and contingency measures under... request. The General Preamble sets forth EPA's view of applicable requirements for purposes of evaluating...) contingency measures is not applicable for purposes of redesignation. Id. Section 172(c)(3) requires...

77 FR 6467 - Approval and Promulgation of Implementation Plans; Alabama, Georgia, and Tennessee: Chattanooga...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-08

...; Particulate Matter 2002 Base Year Emissions Inventory AGENCY: Environmental Protection Agency (EPA). ACTION... 2.5 ) 2002 base year emissions inventory portion of the State Implementation Plan (SIP) revisions... control measures (RACM), reasonable further progress (RFP) plans, contingency measures, a 2002 base year...

77 FR 31262 - Approval and Promulgation of Implementation Plans; Kentucky; Louisville; Fine Particulate Matter...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-25

... Promulgation of Implementation Plans; Kentucky; Louisville; Fine Particulate Matter 2002 Base Year Emissions... approve the fine particulate matter (PM 2.5 ) 2002 base year emissions inventory, portion of the State...), a reasonable further progress (RFP) plan, contingency measures, a 2002 base year emissions inventory...

The Art of the RFP

ERIC Educational Resources Information Center

Villano, Matt

2006-01-01

By this time of the year, springtime rituals are blossoming like begonias, and that's true for higher education, too. Students move inexorably toward the end of another year; professors get ready for summer session; and in campus technology departments, CIOs and other decision-makers furiously set their plans to purchase hardware and software for…

Enzymatic Synthesis of Self-assembled Dicer Substrate RNA Nanostructures for Programmable Gene Silencing.

PubMed

Jang, Bora; Kim, Boyoung; Kim, Hyunsook; Kwon, Hyokyoung; Kim, Minjeong; Seo, Yunmi; Colas, Marion; Jeong, Hansaem; Jeong, Eun Hye; Lee, Kyuri; Lee, Hyukjin

2018-06-08

Enzymatic synthesis of RNA nanostructures is achieved by isothermal rolling circle transcription (RCT). Each arm of RNA nanostructures provides a functional role of Dicer substrate RNA inducing sequence specific RNA interference (RNAi). Three different RNAi sequences (GFP, RFP, and BFP) are incorporated within the three-arm junction RNA nanostructures (Y-RNA). The template and helper DNA strands are designed for the large-scale in vitro synthesis of RNA strands to prepare self-assembled Y-RNA. Interestingly, Dicer processing of Y-RNA is highly influenced by its physical structure and different gene silencing activity is achieved depending on its arm length and overhang. In addition, enzymatic synthesis allows the preparation of various Y-RNA structures using a single DNA template offering on demand regulation of multiple target genes.

Red fluorescent proteins: advanced imaging applications and future design.

PubMed

Shcherbakova, Daria M; Subach, Oksana M; Verkhusha, Vladislav V

2012-10-22

In the past few years a large series of the advanced red-shifted fluorescent proteins (RFPs) has been developed. These enhanced RFPs provide new possibilities to study biological processes at the levels ranging from single molecules to whole organisms. Herein the relationship between the properties of the RFPs of different phenotypes and their applications to various imaging techniques are described. Existing and emerging imaging approaches are discussed for conventional RFPs, far-red FPs, RFPs with a large Stokes shift, fluorescent timers, irreversibly photoactivatable and reversibly photoswitchable RFPs. Advantages and limitations of specific RFPs for each technique are presented. Recent progress in understanding the chemical transformations of red chromophores allows the future RFP phenotypes and their respective novel imaging applications to be foreseen. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

40 CFR 52.1426 - Original identification of plan section.

Code of Federal Regulations, 2013 CFR

2013-07-01

... paragraph (c)(8) above. Approval action was taken on the deletion of these requirements except as they... 3, 1985. Action was also taken to delete review requirements for complex sources of air pollution in Omaha; see paragraph (c)(32) of this section. (i) Incorporation by reference. (A) An RFP curve from page...

76 FR 55541 - Approval and Promulgation of Air Quality Implementation Plans; West Virginia: Kentucky; Ohio...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-09-07

... Cabell and Wayne Counties in their entireties and a portion of Mason County (Graham Tax District) in West... reasonable further progress (RFP) plan, contingency measures, and other planning State Implementation Plan... Regulatory Development Section, Air Planning Branch, Air, Pesticides and Toxics Management Division, U.S...

23 CFR 636.114 - What factors should be considered in risk allocation?

Code of Federal Regulations, 2010 CFR

2010-04-01

... 23 Highways 1 2010-04-01 2010-04-01 false What factors should be considered in risk allocation... ENGINEERING AND TRAFFIC OPERATIONS DESIGN-BUILD CONTRACTING General § 636.114 What factors should be considered in risk allocation? (a) You may consider, identify, and allocate the risks in the RFP document and...

23 CFR 636.114 - What factors should be considered in risk allocation?

Code of Federal Regulations, 2011 CFR

2011-04-01

... 23 Highways 1 2011-04-01 2011-04-01 false What factors should be considered in risk allocation... ENGINEERING AND TRAFFIC OPERATIONS DESIGN-BUILD CONTRACTING General § 636.114 What factors should be considered in risk allocation? (a) You may consider, identify, and allocate the risks in the RFP document and...

Improving Project Management of Complex or Major Systems

DTIC Science & Technology

2016-05-05

funding available through vehicle Mod-lines --- shared across various motor transport programs PB-17 $M (including OCO) Program ACAT FYDP (FY17...Requirements Description (CARD) ▪ ▪ ▪ ▪ ▪ ▪ DOT&E REPORT ON INITIAL OPERATIONAL TEST AND EVALUATION ( IOT &E) ▪ Development RFP Release Cost Assessment

77 FR 62479 - Approval and Promulgation of Implementation Plans; Portion of York County, South Carolina Within...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-15

... vehicle emissions budgets (MVEB) for volatile organic compounds that were included in South Carolina's RFP..., Pesticides and Toxics Management Division, U.S. Environmental Protection Agency, Region 4, 61 Forsyth Street... Development Section, Air Planning Branch, Air, Pesticides and Toxics Management Division, U.S. Environmental...

78 FR 45152 - Approval and Promulgation of Implementation Plans and Designation of Areas; North Carolina...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-26

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RFP tags for labeling secretory pathway proteins

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

Han, Liyang; Zhao, Yanhua; Zhang, Xi

2014-05-09

Highlights: • Membrane protein Orai1 can be used to report the fusion properties of RFPs. • Artificial puncta are affected by dissociation constant as well as pKa of RFPs. • Among tested RFPs mOrange2 is the best choice for secretory protein labeling. - Abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to anmore » environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.« less

RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences.

PubMed

Brule, C E; Dean, K M; Grayhack, E J

2016-01-01

The identification and analysis of sequences that regulate gene expression is critical because regulated gene expression underlies biology. RNA-ID is an efficient and sensitive method to discover and investigate regulatory sequences in the yeast Saccharomyces cerevisiae, using fluorescence-based assays to detect green fluorescent protein (GFP) relative to a red fluorescent protein (RFP) control in individual cells. Putative regulatory sequences can be inserted either in-frame or upstream of a superfolder GFP fusion protein whose expression, like that of RFP, is driven by the bidirectional GAL1,10 promoter. In this chapter, we describe the methodology to identify and study cis-regulatory sequences in the RNA-ID system, explaining features and variations of the RNA-ID reporter, as well as some applications of this system. We describe in detail the methods to analyze a single regulatory sequence, from construction of a single GFP variant to assay of variants by flow cytometry, as well as modifications required to screen libraries of different strains simultaneously. We also describe subsequent analyses of regulatory sequences. © 2016 Elsevier Inc. All rights reserved.

A novel feedback algorithm for simulating controlled dynamics and confinement in the advanced reversed-field pinch

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

Dahlin, J.-E.; Scheffel, J.

2005-06-15

In the advanced reversed-field pinch (RFP), the current density profile is externally controlled to diminish tearing instabilities. Thus the scaling of energy confinement time with plasma current and density is improved substantially as compared to the conventional RFP. This may be numerically simulated by introducing an ad hoc electric field, adjusted to generate a tearing mode stable parallel current density profile. In the present work a current profile control algorithm, based on feedback of the fluctuating electric field in Ohm's law, is introduced into the resistive magnetohydrodynamic code DEBSP [D. D. Schnack and D. C. Baxter, J. Comput. Phys. 55,more » 485 (1984); D. D. Schnack, D. C. Barnes, Z. Mikic, D. S. Marneal, E. J. Caramana, and R. A. Nebel, Comput. Phys. Commun. 43, 17 (1986)]. The resulting radial magnetic field is decreased considerably, causing an increase in energy confinement time and poloidal {beta}. It is found that the parallel current density profile spontaneously becomes hollow, and that a formation, being related to persisting resistive g modes, appears close to the reversal surface.« less

Current profile redistribution driven by neutral beam injection in a reversed-field pinch

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

Parke, E.; Department of Physics, University of Wisconsin-Madison 1150 University Ave., Madison, Wisconsin 53706; Anderson, J. K.

2016-05-15

Neutral beam injection in reversed-field pinch (RFP) plasmas on the Madison Symmetric Torus [Dexter et al., Fusion Sci. Technol. 19, 131 (1991)] drives current redistribution with increased on-axis current density but negligible net current drive. Internal fluctuations correlated with tearing modes are observed on multiple diagnostics; the behavior of tearing mode correlated structures is consistent with flattening of the safety factor profile. The first application of a parametrized model for island flattening to temperature fluctuations in an RFP allows inferrence of rational surface locations for multiple tearing modes. The m = 1, n = 6 mode is observed to shift inward by 1.1 ± 0.6 cm withmore » neutral beam injection. Tearing mode rational surface measurements provide a strong constraint for equilibrium reconstruction, with an estimated reduction of q{sub 0} by 5% and an increase in on-axis current density of 8% ± 5%. The inferred on-axis current drive is consistent with estimates of fast ion density using TRANSP [Goldston et al., J. Comput. Phys. 43, 61 (1981)].« less

The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-Terminal yellow fluorescent protein tag.

PubMed

Swulius, Matthew T; Jensen, Grant J

2012-12-01

Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFP(SW)) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, "patchy" localization patterns of MreB-RFP(SW), even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered.

Ion Heating Anisotropy during Dynamo Activity in the MST RFP

NASA Astrophysics Data System (ADS)

den Hartog, D. J.; Chapman, J. T.; Craig, D.; Fiksel, G.; Fontana, P. W.

1999-11-01

MHD dynamo activity is large in the MST Reversed-Field Pinch during sawtooth crashes, and small otherwise. During a sawtooth crash, ion temperature increases rapidly to a level several times as high as the temperature between sawteeth, which itself can be larger than the electron temperature. Several theories have been developed to explain this ion heating, some indicating a possible asymmetry in perpendicular to parallel heating [C. G. Gimblett, Europhys. Lett. 11, 541 (1990); Z. Yoshida, Nucl. Fusion 31, 386 (1991); N. Mattor, P. W. Terry, and S. C. Prager, Comments Plasma Phys. Controlled Fusion 15, 65 (1992)]. In standard MST discharges, impurity ion temperature measured perpendicular to the magnetic field (T_⊥) is higher than impurity ion temperature parallel to the magnetic field (T_allel) during a sawtooth crash. Throughout the rest of the sawtooth cycle, T_⊥ <= T_allel. This is in contrast to results obtained on the EXTRAP-T2 RFP which showed T_⊥ < T_allel throughout the discharge [K. Sasaki et al., Plasma Phys. Control. Fusion 39, 333 (1997)

Self-Organisation and Intermittent Coherent Oscillations in the EXTRAP T2 Reversed Field Pinch

NASA Astrophysics Data System (ADS)

Cecconello, M.; Malmberg, J.-A.; Sallander, E.; Drake, J. R.

Many reversed-field pinch (RFP) experiments exhibit a coherent oscillatory behaviour that is characteristic of discrete dynamo events and is associated with intermittent current profile self-organisation phenomena. However, in the vast majority of the discharges in the resistive shell RFP experiment EXTRAP T2, the dynamo activity does not show global, coherent oscillatory behaviour. The internally resonant tearing modes are phase-aligned and wall-locked resulting in a large localised magnetic perturbation. Equilibrium and plasma parameters have a level of high frequency fluctuations but the average values are quasi-steady. For some discharges, however, the equilibrium parameters exhibit the oscillatory behaviour characteristic of the discrete dynamo events. For these discharges, the trend observed in the tearing mode spectra, associated with the onset of the discrete relaxation event behaviour, is a relative higher amplitude of m = 0 mode activity and relative lower amplitude of the m = 1 mode activity compared with their average values. Global plasma parameters and model profile calculations for sample discharges representing the two types of relaxation dynamics are presented.

Magnetic and velocity fluctuations from nonlinearly coupled tearing modes in the reversed field pinch with and without the reversal surface

NASA Astrophysics Data System (ADS)

Craig, D.; Martin, D.; Den Hartog, D. J.; Nornberg, M. D.; Reusch, J. A.

2017-08-01

We investigate the role of poloidal mode number m = 0 fluctuations on m = 1 velocity and magnetic field fluctuations in the Reversed Field Pinch (RFP). Removing the m = 0 resonant surface in the Madison Symmetric Torus (MST), results in suppressed m = 0 activity without a reduction in m = 1 magnetic activity. However, the m = 1 velocity fluctuations and fluctuation-induced mean emf are reduced as m = 0 modes are suppressed. Velocity fluctuations are measured directly using fast Doppler spectroscopy. Similar results are seen in visco-resistive MHD simulation with the DEBS code. An artificial line-averaged velocity diagnostic is developed for DEBS simulations to facilitate direct comparisons with experimental measurements. The sensitivity of the m = 1 velocity fluctuations and corresponding emf to changes in m = 0 mode activity is a feature of tearing modes in the nonlinear regime with a spectrum of interacting modes. These results have implications for RFP sustainment strategies and inform our understanding of the role of magnetic turbulence in astrophysical contexts.

Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism.

PubMed

Zhang, Zhenyu; Zhao, Wei; Li, Deshan; Yang, Jinlong; Zsak, Laszlo; Yu, Qingzhong

2015-08-01

In the present study, we developed a novel approach for foreign gene expression by Newcastle disease virus (NDV) from a second ORF through an internal ribosomal entry site (IRES). Six NDV LaSota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein (RFP) gene behind the nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) or large polymerase (L) gene ORF were generated using reverse genetics technology. The insertion of the second ORF slightly attenuated virus pathogenicity, but did not affect ability of the virus to grow. Quantitative measurements of RFP expression in virus-infected DF-1 cells revealed that the abundance of viral mRNAs and red fluorescence intensity were positively correlated with the gene order of NDV, 3'-NP-P-M-F-HN-L-5', proving the sequential transcription mechanism for NDV. The results herein suggest that the level of foreign gene expression could be regulated by selecting the second ORF insertion site to maximize the efficacy of vaccine and gene therapy.

Three dimensional equilibrium solutions for a current-carrying reversed-field pinch plasma with a close-fitting conducting shell

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

Koliner, J. J.; Boguski, J., E-mail: boguski@wisc.edu; Anderson, J. K.

2016-03-15

In order to characterize the Madison Symmetric Torus (MST) reversed-field pinch (RFP) plasmas that bifurcate to a helical equilibrium, the V3FIT equilibrium reconstruction code was modified to include a conducting boundary. RFP plasmas become helical at a high plasma current, which induces large eddy currents in MST's thick aluminum shell. The V3FIT conducting boundary accounts for the contribution from these eddy currents to external magnetic diagnostic coil signals. This implementation of V3FIT was benchmarked against MSTFit, a 2D Grad-Shafranov solver, for axisymmetric plasmas. The two codes both fit B{sub θ} measurement loops around the plasma minor diameter with qualitative agreementmore » between each other and the measured field. Fits in the 3D case converge well, with q-profile and plasma shape agreement between two distinct toroidal locking phases. Greater than 60% of the measured n = 5 component of B{sub θ} at r = a is due to eddy currents in the shell, as calculated by the conducting boundary model.« less

Garlic virus X 11-kDa protein granules move within the cytoplasm and traffic a host protein normally found in the nucleolus.

PubMed

Lu, Yuwen; Yan, Fei; Guo, Wei; Zheng, Hongying; Lin, Lin; Peng, Jiejun; Adams, Michael J; Chen, Jianping

2011-09-01

The subcellular localization of the 11-kDa protein (p11) encoded by ORF3 of Garlic virus X (GarVX; genus Allexivirus, family Alphaflexiviridae) was examined by confocal microscopy. Granules with intense fluorescence were visible on the endoplasmic reticulum when p11 fused with green or red fluorescent protein (GFP or RFP) was expressed in epidermal cells of Nicotiana benthamiana. Moreover, the p11-RFP granules moved in the cytoplasm, along the cell periphery and through the cell membranes to adjacent cells. A 17-kDa protein (p17) of garlic interacting with p11 was identified by yeast two-hybridization and bimolecular fluorescence complementation assay. When p17 fused to GFP was expressed in epidermal cells of N. benthamiana, it localized to the nucleolus. However, in the presence of GarVX p11, the distribution of p17 changed to that of p11, but did not appear to affect the pattern of movement of p11. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD. NO CLAIM TO ORIGINAL US GOVERNMENT WORKS.

Evaluating foodservice software: a suggested approach.

PubMed

Fowler, K D

1986-09-01

In an era of cost containment, the computer has become a viable management tool. Its use in health care has demonstrated accelerated growth in recent years, and a literature review supports an increased trend in this direction. Foodservice, which is a major cost center, is no exception to this predicted trend. Because software has proliferated, foodservice managers and dietitians are experiencing growing concern about how to evaluate the numerous software packages from which to choose. A suggested approach to evaluating software is offered to dietitians and managers alike to lessen the confusion in software selection and to improve the system satisfaction level post-purchase. Steps of the software evaluatory approach include: delineation of goals, assessment of needs, assignment of value weight factors, development of a vendor checklist, survey of vendors by means of the vendor checklist and elimination of inappropriate systems, thorough development of the request for proposal (RFP) for submission to the selected vendors, an analysis of the returned RFPs in terms of system features and cost factors, and selection of the system(s) for implementation.

Locoregional Confinement and Major Clinical Benefit of 188Re-Loaded CXCR4-Targeted Nanocarriers in an Orthotopic Human to Mouse Model of Glioblastoma.

PubMed

Séhédic, Delphine; Chourpa, Igor; Tétaud, Clément; Griveau, Audrey; Loussouarn, Claire; Avril, Sylvie; Legendre, Claire; Lepareur, Nicolas; Wion, Didier; Hindré, François; Davodeau, François; Garcion, Emmanuel

2017-01-01

Gold standard beam radiation for glioblastoma (GBM) treatment is challenged by resistance phenomena occurring in cellular populations well prepared to survive or to repair damage caused by radiation. Among signals that have been linked with radio-resistance, the SDF1/CXCR4 axis, associated with cancer stem-like cell, may be an opportune target. To avoid the problem of systemic toxicity and blood-brain barrier crossing, the relevance and efficacy of an original system of local brain internal radiation therapy combining a radiopharmaceutical with an immuno-nanoparticle was investigated. The nanocarrier combined lipophilic thiobenzoate complexes of rhenium-188 loaded in the core of a lipid nanocapsule (LNC 188 Re) with a function-blocking antibody, 12G5 directed at the CXCR4, on its surface. The efficiency of 12G5-LNC 188 Re was investigated in an orthotopic and xenogenic GBM model of CXCR4-positive U87MG cells implanted in the striatum of Scid mice. We demonstrated that 12G5-LNC 188 Re single infusion treatment by convection-enhanced delivery resulted in a major clinical improvement in median survival that was accompanied by locoregional effects on tumor development including hypovascularization and stimulation of the recruitment of bone marrow derived CD11b- or CD68-positive cells as confirmed by immunohistochemistry analysis. Interestingly, thorough analysis by spectral imaging in a chimeric U87MG GBM model containing CXCR4-positive/red fluorescent protein (RFP)-positive- and CXCR4-negative/RFP-negative-GBM cells revealed greater confinement of DiD-labeled 12G5-LNCs than control IgG2a-LNCs in RFP compartments. Main conclusion: These findings on locoregional impact and targeting of disseminated cancer cells in tumor margins suggest that intracerebral active targeting of nanocarriers loaded with radiopharmaceuticals may have considerable benefits in clinical applications.

Fluctuation reduction and enhanced confinement in the MST reversed-field pinch

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

Chapman, Brett Edward

1997-10-01

Plasmas with a factor of ≥3 improvement in energy confinement have been achieved in the MST reversed-field pinch (RFP). These plasmas occur spontaneously, following sawtooth crashes, subject to constraints on, eg, toroidal magnetic field reversal and wall conditioning. Possible contributors to the improved confinement include a reduction of core-resonant, global magnetic fluctuations and a reduction of electrostatic fluctuations over the entire plasma edge. One feature of these plasmas is a region of strong ExB flow shear in the edge. Never before observed in conjunction with enhanced confinement in the RFP, such shear is common in enhanced confinement discharges in tokamaks and stellarators. Another feature of these plasmas is a new type of discrete dynamo event. Like sawtooth crashes, a common form of discrete dynamo, these events correspond to bursts of edge parallel current. The reduction of electrostatic fluctuations in these plasmas occurs within and beyond the region of strong ExB flow shear, similar to what is observed in tokamaks and stellarators. However, the reductions in the MST include fluctuations whose correlation lengths are larger than the width of the shear region. The reduction of the global magnetic fluctuations is most likely due to flattening of the μ=μ 0more » $$\\vec{J}$$∙$$\\vec{B}$$/B 2 profile. Flattening can occur, eg, due to the new type of discrete dynamo event and reduced edge resistivity. Enhanced confinement plasmas are also achieved in the MST when auxiliary current is applied to flatten the μ profile and reduce magnetic fluctuations. Unexpectedly, these plasmas also exhibit a region (broader than in the case above) of strong ExB flow shear in the edge, an edge-wide reduction of electrostatic fluctuations, and the new type of discrete dynamo event. Auxiliary current drive has historically been viewed as the principal route to fusion reactor viability for the RFP.« less

Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus.

PubMed

Scobey, Trevor; Yount, Boyd L; Sims, Amy C; Donaldson, Eric F; Agnihothram, Sudhakar S; Menachery, Vineet D; Graham, Rachel L; Swanstrom, Jesica; Bove, Peter F; Kim, Jeeho D; Grego, Sonia; Randell, Scott H; Baric, Ralph S

2013-10-01

Severe acute respiratory syndrome with high mortality rates (~50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ~0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3-5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.

Effects of multiple resistive shells and transient electromagnetic torque on the dynamics of mode locking in reversed field pinch plasmas

NASA Astrophysics Data System (ADS)

Guo, S. C.; Chu, M. S.

2002-11-01

The effects of multiple resistive shells and transient electromagnetic torque on the dynamics of mode locking in the reversed field pinch (RFP) plasmas are studied. Most RFP machines are equipped with one or more metal shells outside of the vacuum vessel. These shells have finite resistivities. The eddy currents induced in each of the shells contribute to the braking electromagnetic (EM) torque which slows down the plasma rotation. In this work we study the electromagnetic torque acting on the plasma (tearing) modes produced by a system of resistive shells. These shells may consist of several nested thin shells or several thin shells enclosed within a thick shell. The dynamics of the plasma mode is investigated by balancing the EM torque from the resistive shells with the plasma viscous torque. Both the steady state theory and the time-dependent theory are developed. The steady state theory is shown to provide an accurate account of the resultant EM torque if (dω/dt)ω-2≪1 and the time scale of interest is much longer than the response (L/R) time of the shell. Otherwise, the transient theory should be adopted. As applications, the steady state theory is used to evaluate the changes of the EM torque response from the resistive shells in two variants of two RFP machines: (1) modification from Reversed Field Experiment (RFX) [Gnesotto et al., Fusion Eng. Des. 25, 335 (1995)] to the modified RFX: both of them are equipped with one thin shell plus one thick shell; (2) modification from Extrap T2 to Extrap T2R [Brunsell et al., Plasma Phys. Controlled Fusion 43, 1457 (2001)]: both of them are equipped with two thin shells. The transient theory has been applied numerically to study the time evolution of the EM torque during the unlocking of a locked tearing mode in the modified RFX.

Biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged m protein allows dynamic imaging of m protein and virus uncoating in infected cells.

PubMed

Das, Subash C; Panda, Debasis; Nayak, Debasis; Pattnaik, Asit K

2009-03-01

A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-DeltaM-Mtc and VSV-DeltaM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-DeltaM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-DeltaM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

Diagnosing the pathophysiologic mechanisms of nocturnal polyuria.

PubMed

Goessaert, An-Sofie; Krott, Louise; Hoebeke, Piet; Vande Walle, Johan; Everaert, Karel

2015-02-01

Diagnosis of nocturnal polyuria (NP) is based on a bladder diary. Addition of a renal function profile (RFP) for analysis of concentrating and solute-conserving capacity allows differentiation of NP pathophysiology and could facilitate individualized treatment. To map circadian rhythms of water and solute diuresis by comparing participants with and without NP. This prospective observational study was carried out in Ghent University Hospital between 2011 and 2013. Participants with and without NP completed a 72-h bladder dairy. RFP, free water clearance (FWC), and creatinine, solute, sodium, and urea clearance were measured for all participants. The study participants were divided into those with (n=77) and those without (n=35) NP. The mean age was 57 yr (SD 16 yr) and 41% of the participants were female. Compared to participants without NP, the NP group exhibited a higher diuresis rate throughout the night (p=0.015); higher FWC (p=0.013) and lower osmolality (p=0.030) at the start of the night; and persistently higher sodium clearance during the night (p<0.001). The pathophysiologic mechanism of NP was identified as water diuresis alone in 22%, sodium diuresis alone in 19%, and a combination of water and sodium diuresis in 47% of the NP group. RFP measurement in first-line NP screening to discriminate between water and solute diuresis as pathophysiologic mechanisms complements the bladder diary and could facilitate optimal individualized treatment of patients with NP. We evaluated eight urine samples collected over 24h to detect the underlying problem in NP. We found that NP can be attributed to water or sodium diuresis or a combination of both. This urinalysis can be used to adapt treatment according to the underlying mechanism in patients with bothersome consequences of NP, such as nocturia and urinary incontinence. Copyright © 2014 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Dual Infection and Superinfection Inhibition of Epithelial Skin Cells by Two Alphaherpesviruses Co-Occur in the Natural Host

PubMed Central

Jarosinski, Keith W.

2012-01-01

Hosts can be infected with multiple herpesviruses, known as superinfection; however, superinfection of cells is rare due to the phenomenon known as superinfection inhibition. It is believed that dual infection of cells occurs in nature, based on studies examining genetic exchange between homologous alphaherpesviruses in the host, but to date, this has not been directly shown in a natural model. In this report, gallid herpesvirus 2 (GaHV-2), better known as Marek’s disease virus (MDV), was used in its natural host, the chicken, to determine whether two homologous alphaherpesviruses can infect the same cells in vivo. MDV shares close similarities with the human alphaherpesvirus, varicella zoster virus (VZV), with respect to replication in the skin and exit from the host. Recombinant MDVs were generated that express either the enhanced GFP (eGFP) or monomeric RFP (mRFP) fused to the UL47 (VP13/14) herpesvirus tegument protein. These viruses exhibited no alteration in pathogenic potential and expressed abundant UL47-eGFP or -mRFP in feather follicle epithelial cells in vivo. Using laser scanning confocal microscopy, it was evident that these two similar, but distinguishable, viruses were able to replicate within the same cells of their natural host. Evidence of superinfection inhibition was also observed. These results have important implications for two reasons. First, these results show that during natural infection, both dual infection of cells and superinfection inhibition can co-occur at the cellular level. Secondly, vaccination against MDV with homologous alphaherpesvirus like attenuated GaHV-2, or non-oncogenic GaHV-3 or meleagrid herpesvirus (MeHV-1) has driven the virus to greater virulence and these results implicate the potential for genetic exchange between homologous avian alphaherpesviruses that could drive increased virulence. Because the live attenuated varicella vaccine is currently being administered to children, who in turn could be superinfected by wild-type VZV, this could potentiate recombination events of VZV as well. PMID:22629393

Extended MHD Modeling of Tearing-Driven Magnetic Relaxation

NASA Astrophysics Data System (ADS)

Sauppe, Joshua

2016-10-01

Driven plasma pinch configurations are characterized by the gradual accumulation and episodic release of free energy in discrete relaxation events. The hallmark of this relaxation in a reversed-field pinch (RFP) plasma is flattening of the parallel current density profile effected by a fluctuation-induced dynamo emf in Ohm's law. Nonlinear two-fluid modeling of macroscopic RFP dynamics has shown appreciable coupling of magnetic relaxation and the evolution of plasma flow. Accurate modeling of RFP dynamics requires the Hall effect in Ohm's law as well as first order ion finite Larmor radius (FLR) effects, represented by the Braginskii ion gyroviscous stress tensor. New results find that the Hall dynamo effect from < J × B > / ne can counter the MHD effect from - < V × B > in some of the relaxation events. The MHD effect dominates these events and relaxes the current profile toward the Taylor state, but the opposition of the two dynamos generates plasma flow in the direction of equilibrium current density, consistent with experimental measurements. Detailed experimental measurements of the MHD and Hall emf terms are compared to these extended MHD predictions. Tracking the evolution of magnetic energy, helicity, and hybrid helicity during relaxation identifies the most important contributions in single-fluid and two-fluid models. Magnetic helicity is well conserved relative to the magnetic energy during relaxation. The hybrid helicity is dominated by magnetic helicity in realistic low-beta pinch conditions and is also well conserved. Differences of less than 1 % between magnetic helicity and hybrid helicity are observed with two-fluid modeling and result from cross helicity evolution through ion FLR effects, which have not been included in contemporary relaxation theories. The kinetic energy driven by relaxation in the computations is dominated by velocity components perpendicular to the magnetic field, an effect that had not been predicted. Work performed at University of Wisconsin-Madison. LA-UR-16-24727.

Locoregional Confinement and Major Clinical Benefit of 188Re-Loaded CXCR4-Targeted Nanocarriers in an Orthotopic Human to Mouse Model of Glioblastoma

PubMed Central

Séhédic, Delphine; Chourpa, Igor; Tétaud, Clément; Griveau, Audrey; Loussouarn, Claire; Avril, Sylvie; Legendre, Claire; Lepareur, Nicolas; Wion, Didier; Hindré, François; Davodeau, François; Garcion, Emmanuel

2017-01-01

Purpose: Gold standard beam radiation for glioblastoma (GBM) treatment is challenged by resistance phenomena occurring in cellular populations well prepared to survive or to repair damage caused by radiation. Among signals that have been linked with radio-resistance, the SDF1/CXCR4 axis, associated with cancer stem-like cell, may be an opportune target. To avoid the problem of systemic toxicity and blood-brain barrier crossing, the relevance and efficacy of an original system of local brain internal radiation therapy combining a radiopharmaceutical with an immuno-nanoparticle was investigated. Experiment design: The nanocarrier combined lipophilic thiobenzoate complexes of rhenium-188 loaded in the core of a lipid nanocapsule (LNC188Re) with a function-blocking antibody, 12G5 directed at the CXCR4, on its surface. The efficiency of 12G5-LNC188Re was investigated in an orthotopic and xenogenic GBM model of CXCR4-positive U87MG cells implanted in the striatum of Scid mice. Results: We demonstrated that 12G5-LNC188Re single infusion treatment by convection-enhanced delivery resulted in a major clinical improvement in median survival that was accompanied by locoregional effects on tumor development including hypovascularization and stimulation of the recruitment of bone marrow derived CD11b- or CD68-positive cells as confirmed by immunohistochemistry analysis. Interestingly, thorough analysis by spectral imaging in a chimeric U87MG GBM model containing CXCR4-positive/red fluorescent protein (RFP)-positive- and CXCR4-negative/RFP-negative-GBM cells revealed greater confinement of DiD-labeled 12G5-LNCs than control IgG2a-LNCs in RFP compartments. Main conclusion: These findings on locoregional impact and targeting of disseminated cancer cells in tumor margins suggest that intracerebral active targeting of nanocarriers loaded with radiopharmaceuticals may have considerable benefits in clinical applications. PMID:29158842

IDH1 R132H Mutation Is Accompanied with Malignant Progression of Paired Primary-Recurrent Astrocytic Tumours.

PubMed

Mu, Luyan; Xu, Wanzhen; Li, Qingla; Ge, Haitao; Bao, Hongbo; Xia, Songsong; Ji, Jingjing; Jiang, Jie; Song, Yuwen; Gao, Qiang

2017-01-01

IDH1 R132H mutation is an important marker of survival in patients with gliomas. Although there are many changes of genes in tumour malignant progression, IDH1 R132H mutation status in glioma progression remained unclear. Here, an in-depth characterization of IDH1 R132H mutations were assessed by immunohistochemistry in 55 paired primary-recurrent astrocytomas tissues, including 5 paired primary pilocytic astrocytoma (pPA, WHO grade I), 35 paired primary low grade astrocytoma (pLGA, WHO grade II and III) and 15 paired primary high grade astrocytoma (pHGA/ Glioblastoma, WHO grade IV). Meanwhile, the DNA was isolated from paired samples, and PCR amplification was used for IDH1 exon4 sequencing. Nonparametric test, KM and Cox models were used to examine the statistical difference and survival function. We found that the percent of IDH1 R132H mutation was 68.6% (24/35) in pLGA group, but no IDH1 mutation was found in pPA and pHGA groups. Meanwhile, the results from immunohistochemistry and DNA sequencing showed that, compared with primary astrocytoma, there was no change of IDH1 status in recurrent astrocytoma whatever tumour pathological grade raise or indolent. The pPA group has the longest recurrence-free period (RFP) and overall survival (OS) in three groups ( p<0.01 ), while the pHGA group has the shortest ones ( p<0.01 ). In pLGA group, the IDH1 R132H mutation subgroup has longer RFP than IDH1 wild type subgroup ( p<0.01 ), but the OS has no statistical difference between two subgroups ( p>0.6 ). Additionally, IDH1 R132H mutation independently predicted a long RFP in patients with pLGA (HR 1.073, 95% CI 0.151-0.775, p<0.01 ).

IDH1 R132H Mutation Is Accompanied with Malignant Progression of Paired Primary-Recurrent Astrocytic Tumours

PubMed Central

Mu, Luyan; Xu, Wanzhen; Li, Qingla; Ge, Haitao; Bao, Hongbo; Xia, Songsong; Ji, Jingjing; Jiang, Jie; Song, Yuwen; Gao, Qiang

2017-01-01

IDH1 R132H mutation is an important marker of survival in patients with gliomas. Although there are many changes of genes in tumour malignant progression, IDH1 R132H mutation status in glioma progression remained unclear. Here, an in-depth characterization of IDH1 R132H mutations were assessed by immunohistochemistry in 55 paired primary-recurrent astrocytomas tissues, including 5 paired primary pilocytic astrocytoma (pPA, WHO grade I), 35 paired primary low grade astrocytoma (pLGA, WHO grade II and III) and 15 paired primary high grade astrocytoma (pHGA/ Glioblastoma, WHO grade IV). Meanwhile, the DNA was isolated from paired samples, and PCR amplification was used for IDH1 exon4 sequencing. Nonparametric test, KM and Cox models were used to examine the statistical difference and survival function. We found that the percent of IDH1 R132H mutation was 68.6% (24/35) in pLGA group, but no IDH1 mutation was found in pPA and pHGA groups. Meanwhile, the results from immunohistochemistry and DNA sequencing showed that, compared with primary astrocytoma, there was no change of IDH1 status in recurrent astrocytoma whatever tumour pathological grade raise or indolent. The pPA group has the longest recurrence-free period (RFP) and overall survival (OS) in three groups (p<0.01), while the pHGA group has the shortest ones (p<0.01). In pLGA group, the IDH1 R132H mutation subgroup has longer RFP than IDH1 wild type subgroup (p<0.01), but the OS has no statistical difference between two subgroups (p>0.6). Additionally, IDH1 R132H mutation independently predicted a long RFP in patients with pLGA (HR 1.073, 95% CI 0.151-0.775, p<0.01). PMID:28928859

[Management of adverse effects with antituberculosis chemotherapy].

PubMed

Tsuyuguchi, Kazunari; Wada, Masako

2011-02-01

Tuberculosis has now become a curable disease with chemotherapy. So it is natural that the present issues in tuberculosis management are focused on how to complete standard chemotherapy. In this context, management of adverse effects constitutes an essential part of antituberculosis chemotherapy, as well as directly observed therapy. In this symposium, discussions were held about three major subjects on this issue. First, hepatotoxicity develops frequently and has sometimes fatal outcome, which makes it the most problematic adverse effect. "Management of hepatotoxicity during antituberculosis chemotherapy" was published by the Japanese Society for Tuberculosis (JST) in 2006. Dr. Shinsho Yoshiba evaluated this recommendation and pointed out that the criteria for discontinuation of drug based on AST, ALT and bilirubin levels is too sensitive and the concept of predicting fulminant hepatic failure (FHF) is lacking. He stressed the importance of monitoring serum prothrombin time for predicting FHF. Next, allergic drug reaction such as fever or skin rash often causes distress, although rarely fatal. As isoniazid (INH) and rifampicin (RFP) are key drugs for the cure, readministration of these drugs is often attempted by desensitization therapy. "Recommendation about desensitization therapy of antituberculosis drugs" was also published by JST in 1997. Dr. Yoshihiro Kobashi reported high success rates of 79 percent for INH and 75 percent for RFP according to this recommendation. He also reported correlated factor with the success, such as the longer period from the discontinuation to the desensitization therapy and lower doses of drugs at starting desensitization. Finally, we sometimes experience transient worsening of radiographical findings and general symptoms during antituberculosis chemotherapy. This is presumed to be due to allergic reaction to dead bacilli without requiring discontinuation of the drug. Differential diagnosis includes drug-induced pneumonia requring discontinuation and true worsening of pulmonary tuberculosis due to drug resistance requiring change in therapy. Dr. Masanori Akira reported that presence of ground-glass attenuation and/or consolidation by HRCT suggests transient worsening or drug-induced pneumonia, whereas presence of centrilobular nodules and/or tree-in bud suggests true worsening. We believe that these findings from the symposium will add useful information for management of adverse effects and be helpful for implementation of antituberculosis chemotherapy. (1) Hepatotoxicity of antituberculosis drugs: Shinsho YOSHIBA (Sempo Tokyo Takanawa Hospital) Antituberculosis drugs are sometimes hepatotoxic. Doctors who are responsible for the treatment of patients with tuberculosis should always be aware of their hepatotoxicity, because it seldom leads to fulminant hepatic failure. The Japanese Society for Tuberculosis proposed criteria based on the levels of AST, ALT and bilirubin for the prevention of such grave hepatic injury in 2006. In recent years attempts have been made to predict fulminant hepatic failure (FHF) before patients develop coma. Yoshiba's formula using prothrombin time, etiology, cholinesterase and bilirubin is widely accepted as useful to predict FHF. Introduction of the formula to this area is recommended. (2) Desensitization therapy for allergic reactions of antituberculous drugs: Yoshihiro KOBASHI, Mikio OKA (Division of Respiratory Diseases, Department of Medicine, Kawasaki Medical School) We evaluated the usefulness of desensitization therapy for patients showing allergic reactions of INH and RFP according to the guideline proposed by the Japanese Society for Tuberculosis. Adverse reactions were 22 patients with drug eruption, 22 with drug fever and 6 with drug fever plus eruption. The clinical effect of desensitization therapy was good in 27 out of 36 patients for RFP (75%), and in 19 out of 24 patients for INH (79%). The comparative study between patient group with success desensitization therapy and that with failure desensitization therapy was not a significant difference except for initiation period of desensitization therapy. (3) The imaging features of early transient radiographic progression, true worsening of TB, and drug induced pneumonitis during TB treatment: Masanori AKIRA (Department of Radiology, NHO Kinki-chuo Chest Medical Center) HRCT findings of the new lesions in the early transient radiographic progression are enlargement or confluence of the original lesions, development of areas of ground-glass attenuation and/or consolidation ipsilateral to the original lesion, and development of areas of ground-glass attenuation and/or consolidation in the subpleural region contralateral to the lesion. These CT findings may suggest a local hypersensitivity reaction to drug or massive dead tubercle bacilli per se. In contrast, CT findings of patients with multiple drug-resistant tuberculosis and true progression are centrilobular nodules, tree-in-bud appearance, nodules, and cavitation. These CT findings may suggest a bronchogenic spread from the original tuberculous lesions.

Turbulent energy transfer in electromagnetic turbulence: hints from a Reversed Field Pinch plasma

NASA Astrophysics Data System (ADS)

Vianello, N.; Bergsaker, H.

2005-10-01

The relationship between electromagnetic turbulence and sheared plasma flow in a Reversed Field Pinch is addressed. ExB sheared flows and turbulence at the edge tends to organize themeselves near marginal stability, suggesting an underlying energy exchange process between turbulence and mean flow. In MHD this process is well described through the quantity P which represents the energy transfer (per mass and time unit) from turbulence to mean fields. In the edge region of RFP configuration, where magnetic field is mainly poloidal and the mean ExB is consequently toroidal, the quantity P results: P =[ -ρμ0 + ]Vφr where Vφ is the mean ExB toroidal flow, ρ the mean mass density and b and v the fluctuations of velocity and magnetic field respectively. Both the radial profiles and the temporal evolution of P have been measured in the edge region of Extrap-T2R Reversed Field Pinch experiment. The results support the existence of oscillating energy exchange process between fluctuations and mean flow.

FluBreaks: early epidemic detection from Google flu trends.

PubMed

Pervaiz, Fahad; Pervaiz, Mansoor; Abdur Rehman, Nabeel; Saif, Umar

2012-10-04

The Google Flu Trends service was launched in 2008 to track changes in the volume of online search queries related to flu-like symptoms. Over the last few years, the trend data produced by this service has shown a consistent relationship with the actual number of flu reports collected by the US Centers for Disease Control and Prevention (CDC), often identifying increases in flu cases weeks in advance of CDC records. However, contrary to popular belief, Google Flu Trends is not an early epidemic detection system. Instead, it is designed as a baseline indicator of the trend, or changes, in the number of disease cases. To evaluate whether these trends can be used as a basis for an early warning system for epidemics. We present the first detailed algorithmic analysis of how Google Flu Trends can be used as a basis for building a fully automated system for early warning of epidemics in advance of methods used by the CDC. Based on our work, we present a novel early epidemic detection system, called FluBreaks (dritte.org/flubreaks), based on Google Flu Trends data. We compared the accuracy and practicality of three types of algorithms: normal distribution algorithms, Poisson distribution algorithms, and negative binomial distribution algorithms. We explored the relative merits of these methods, and related our findings to changes in Internet penetration and population size for the regions in Google Flu Trends providing data. Across our performance metrics of percentage true-positives (RTP), percentage false-positives (RFP), percentage overlap (OT), and percentage early alarms (EA), Poisson- and negative binomial-based algorithms performed better in all except RFP. Poisson-based algorithms had average values of 99%, 28%, 71%, and 76% for RTP, RFP, OT, and EA, respectively, whereas negative binomial-based algorithms had average values of 97.8%, 17.8%, 60%, and 55% for RTP, RFP, OT, and EA, respectively. Moreover, the EA was also affected by the region's population size. Regions with larger populations (regions 4 and 6) had higher values of EA than region 10 (which had the smallest population) for negative binomial- and Poisson-based algorithms. The difference was 12.5% and 13.5% on average in negative binomial- and Poisson-based algorithms, respectively. We present the first detailed comparative analysis of popular early epidemic detection algorithms on Google Flu Trends data. We note that realizing this opportunity requires moving beyond the cumulative sum and historical limits method-based normal distribution approaches, traditionally employed by the CDC, to negative binomial- and Poisson-based algorithms to deal with potentially noisy search query data from regions with varying population and Internet penetrations. Based on our work, we have developed FluBreaks, an early warning system for flu epidemics using Google Flu Trends.

A Droplet Microfluidic Platform for Automating Genetic Engineering.

PubMed

Gach, Philip C; Shih, Steve C C; Sustarich, Jess; Keasling, Jay D; Hillson, Nathan J; Adams, Paul D; Singh, Anup K

2016-05-20

We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. The chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (∼5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as Saccharomyces cerevisiae and Aspergillus niger. Duration and temperatures of the microfluidic heat-shock procedures were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.

Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

ERIC Educational Resources Information Center

Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

2008-01-01

We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

77 FR 60904 - Air Quality Implementation Plans; Alabama; Attainment Plan for the Alabama Portion of the...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-05

... available control technology (RACT) and reasonably available control measures (RACM); reasonable further... determination for PM 2.5 and nitrogen oxides (NO X ) for the mobile source contribution to ambient PM 2.5 levels..., an RFP plan, contingency measures, and an insignificance determination for mobile direct PM 2.5 and...

Alternative Fuels Data Center: Memorandums of Understanding-Broadening

Science.gov Websites

to more affordable NGVs for government fleets. In Oklahoma, for example, the post-RFP cost of a Dodge commented. In another example, the Oklahoma Secretary of Energy and Environment held a series of town hall releasing NGV models and providing additional options for consumers and fleets alike. For example, General

77 FR 62454 - Approval and Promulgation of Implementation Plans; Portion of York County, South Carolina Within...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-15

... ambient air quality standards (NAAQS) for the portion of York County, South Carolina that is within the bi... ``bi-state Charlotte Area'') is comprised of Cabarrus, Gaston, Lincoln, Mecklenburg, Rowan, Union and a... North Carolina's RFP plan for its portion of the bi-state Charlotte Area, in a separate action. DATES...

A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea

USDA-ARS?s Scientific Manuscript database

Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. Using a yeast-adapted pCAMBIA0380 Agrobacterium vector, we have constructed a series of vectors for transformation of A. mellea, assembled u...

78 FR 32135 - Approval and Promulgation of Implementation Plans; Atlanta, Georgia 1997 8-Hour Ozone...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-29

... allowed by 40 CFR 51.918 for the Atlanta Area.\\5\\ Subsequently, EPA approved Georgia's SIP revisions... CFR 51.910. Specifically, in ozone nonattainment areas with air quality classified as ``moderate'' or... CFR 51.910(b)(2). For an area like Atlanta, the RFP SIP revision must provide for a 15 percent...

Young Children's Reading for Pleasure with Digital Books: Six Key Facets of Engagement

ERIC Educational Resources Information Center

Kucirkova, Natalia; Littleton, Karen; Cremin, Teresa

2017-01-01

This paper offers a new characterisation of young children's (2-8 years) reading for pleasure (RfP) with digital books. This characterisation is rooted in a re-contextualisation of Anna Craft's conceptualisation of twenty-first century childhoods in "Creativity and education futures" (Stoke on Trent, Trentham, 2011) and a review of the…

75 FR 12090 - Approval and Promulgation of Implementation Plans and Designation of Areas for Air Quality...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-12

... Ozone Standard in Lake and Porter Counties? 4. What Is the Contingency Plan for Lake and Porter Counties... your concerns, and suggest alternatives. 7. Explain your views as clearly as possible, avoiding the use... CAA as contingencies for areas not making Reasonable Further Progress (RFP) toward attainment of the...

75 FR 57221 - Approval and Promulgation of Air Quality Implementation Plans; Connecticut, Massachusetts, and...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-20

... emissions properly accounted for within these RFP plans? F. What are the pollution control programs that... combined with, VOC control in order to maximize the reduction in ozone pollution. The most current such... these revised estimates are reflected in Table 3e above. F. What are the pollution control programs that...

Electrocatalysis of Oxygen Using Water Soluble Metal Porphyrins and Chemically Modified Porphyrin Electrodes.

DTIC Science & Technology

1983-11-01

NUMBER(’s Theodore Kuwana Grant No. ,AFOSR-78-36729 9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELMENJT. PROJECT. TASK AREA &WORK UNIT...RFP. (e) sample (d) following post plasma treatment with a reducing agent, NaAlH2- (OCH2CH20CH3 )2, (Vitride, Hexcell Corp., Zeeland. itch.) at SOIC

The Function of Neuroendocrine Cells in Prostate Cancer

DTIC Science & Technology

2014-04-01

cells were sorted based on HLA + RFP+ GFP+ and further divided into CD56+/- fractions and transplanted into recipient mice. (C) Tumors formed...in Prostate Cancer PRINCIPAL INVESTIGATOR: Jiaoti Huang CONTRACTING ORGANIZATION : REPORT DATE: April 2014 TYPE OF REPORT... ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER University of California Los Angeles Los Angeles, CA 90095

Methods for the Detection of Autophagy in Mammalian Cells

PubMed Central

Zhang, Ziyan; Singh, Rajat; Aschner, Michael

2016-01-01

Macroautophagy (hereafter referred to as autophagy) is a degradation pathway that delivers cytoplasmic materials to lysosomes via double-membraned vesicles designated autophagosomes. Cytoplasmic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes, where the cargo is degraded. Autophagy is a crucial mechanism involved in many aspects of cell function, including cellular metabolism and energy balance; and alterations in autophagy have been linked to various human pathological processes. Thus, methods that accurately measure autophagic activity are necessary. In this unit, we introduce several approaches to analyze autophagy in mammalian cells, including immunoblotting analysis of LC3 and p62, detection of autophagosome formation by fluorescence microscopy, and monitoring autophagosome maturation by tandem mRFP-GFP fluorescence microscopy. Overall, we recommend a combined use of multiple methods to accurately assess the autophagic activity in any given biological setting. PMID:27479363

How NASA Expanded its Innovation Framework to Find New Solutions to Old Problems

NASA Technical Reports Server (NTRS)

Davis, Jeffrey R.

2010-01-01

A radio frequency engineer from rural New Hampshire contributed the best solution to a public challenge issued by NASA's Space Life Sciences Directorate. This is a clear example of what Aneesh Chopra, the US Federal Chief Technology Officer, describes as the notion that in our society, knowledge is widely dispersed. And if it s widely dispersed, how do we capture the insights from the American people?" Chopra later said, to a live audience at the 2010 Rethinking Government event: "A semi-retired radio frequency engineer was able to share his idea about how to solve this problem, and it so blew away other ideas that NASA said it exceeded their requirements! No complicated RFP, the need for lobbyists, some convoluted processes, etc. Just a smart person who was paid a modest fee for his insight."

The Outer Planetary Mission Design Project

NASA Astrophysics Data System (ADS)

Benfield, Michael; Turner, M. W.

2010-10-01

With the recent focus from the planetary science community on the outer planets of the solar system, The University of Alabama in Huntsville Integrated Product Team program is embarking on a new challenge to develop an outer planetary mission for the academic year 2010-2011. Currently four bodies are of interest for this mission: Titan, Europa, Triton, and Enceledus, with one body being chosen by the instructors by the beginning of the fall semester. This project will use the 2010 Discovery Announcement of Opportunity as its Request for Proposal (RFP). All of the teams competing in this project will use the AO to respond with a proposal to the instructors for their proposed mission and spacecraft concept. The project employs the two-semester design sequence of the IPT program to provide a framework for the development of this mission. This sequence is divided into four phases. Phase 1 - Requirements Development - focuses on the development of both the scientific and engineering requirements of the mission. During this phase the teams work very closely with the PI organization, represented by the College of Charleston. Phase 2 - Team Formation and Architecture Development - concentrates on the assessment of the overall mission architecture from the launch vehicle to the ground operations of the proposed spacecraft. Phase 3 - System Definition - provides for spacecraft subsystem trade studies and further refinement of the specific spacecraft to meet the scientific requirements and objectives developed in Phase 1. Phase 4 - Design - is the phase where the engineers provide the spacecraft design that is required for the mission of interest. At the conclusion of Phases 2 and 4, an external review board evaluates the proposed designs and chooses one winner of the competition.

Experimental demonstration of the possible role of Acanthamoeba polyphaga in the infection and disease progression in Buruli Ulcer (BU) using ICR mice

PubMed Central

Azumah, Bright K.; Addo, Phyllis G.; Dodoo, Alfred; Awandare, Gordon; Mosi, Lydia; Boakye, Daniel A.

2017-01-01

The transmission of Buruli ulcer (BU), caused by Mycobacterium ulcerans (MU), remains puzzling although a number of hypothesis including through bites of infected aquatic insects have been proposed. We report the results of experiments using ICR mice that give credence to our hypothesis that Acanthamoeba species may play a role in BU transmission. We cocultured MU N2 and MU 1615 which expresses red fluorescent protein (RFP) and Acanthamoeba polyphaga (AP), and confirmed infected AP by Ziehl-Neelsen (ZN) staining. We tested for viability of MU inside AP and observed strong RFP signals inside both trophozoites and cysts after 3 and 42 days of coculturing respectively. ICR mice were topically treated, either on shaved intact or shaved pinpricked rumps, with one of the following; MU N2 only (2.25 x 106 colony forming units [CFU] / ml), MU N2:AP coculture (2.96 x 104 CFU: 1.6 x 106 cells/ml), AP only (1.6 x 106 cells/ml), PYG medium and sterile distilled water. Both MU N2 only and MU N2:AP elicited reddening on day (D) 31; edema on D 45 and D 44 respectively, and ulcers on D 49 at pinpricked sites only. To ascertain infectivity and pathogenicity of MU N2 only and MU N2:AP, and compare their virulence, the standard mouse footpad inoculation method was used. MU N2:AP elicited reddening in footpads by D 3 compared to D 14 with MU N2 only of the same dose of MU N2 (2.96 x 104 CFU). ZN-stained MU were observed in both thin sectioned and homogenized lesions, and aspirates from infected sites. Viable MU N2 were recovered from cultures of the homogenates and aspirates. This study demonstrates in ICR mice MU transmission via passive infection, and shows that punctures in the skin are prerequisite for infection, and that coculturing of MU with AP enhances pathogenesis. PMID:28329001

Development of PCR-RFP and DNA barcoding plastic markers for yellow toadflax and Dalmatian toadflax

Treesearch

Andrew Boswell

2013-01-01

Yellow toadflax and Dalmatian toadflax are problematic invasive plant species in North America. Yellow toadflax was introduced multiple times to the United States from Europe, beginning in the late 1600s. Dalmatian toadflax has similarly been repeatedly introduced to the United States, starting in 1874. Both species are known to inhabit disturbed areas, competing for...

77 FR 31200 - Revision to the South Coast Air Quality Management District Portion of the California State...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-25

.... Comment 1: CSE's first comment provides an overview of the reasonable further progress (RFP) and base year... year (i.e. pre-1997) emissions credits for particulate matter of 10 microns or less (PM 10 ) and sulfur... base- year requirement.'') [Footnote omitted] CSE's basis for concluding the 2003 AQMP is not ``valid...

LETTER TO THE EDITOR: Anisotropy of ion temperature in a reversed-field-pinch plasma

NASA Astrophysics Data System (ADS)

Sasaki, K.; Hörling, P.; Fall, T.; Brzozowski, J. H.; Brunsell, P.; Hokin, S.; Tennfors, E.; Sallander, J.; Drake, J. R.; Inoue, N.; Morikawa, J.; Ogawa, Y.; Yoshida, Z.

1997-03-01

Anomalous heating of ions has been observed in the EXTRAP-T2 reversed-field-pinch (RFP) plasma. Ions are heated primarily in the parallel direction (with respect to the magnetic field), resulting in an appreciable anisotropy of the ion temperature. This observation suggests that the magnetohydrodynamic fluctuations are dissipated primarily by the ion viscosity.

Measuring Outcomes for Young Children and Their Families. Outcome Indicators for Everyday Kids, Everyday Lives: A Vision for Pennsylvania's Children

ERIC Educational Resources Information Center

Trivette, Carol M.; Dunst, Carl J.

2011-01-01

This monograph includes the final report for a project funded by the Pennsylvania Developmental Disabilities Council for "Measuring Outcomes for Children" (2008 RFP). The goal of the project was to "develop and demonstrate the effectiveness of an instrument designed to measure life outcomes of children with disabilities being…

78 FR 57573 - Approval and Promulgation of Air Quality Implementation Plans; Delaware; Attainment Plan for the...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-19

...) emissions inventory requirements; (3) modeling; (4) RACM and RACT; (5) RFP; (6) contingency measures; and (7... there are multiple precursors to PM 10 , and in particular to PM 2.5 .\\20\\ PM 2.5 chemical precursors... Philadelphia Area. D. Modeling As required, Delaware submitted modeling as part of the attainment plan for the...

Chaos in plasma simulation and experiment

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

Watts, C.; Newman, D.E.; Sprott, J.C.

1993-09-01

We investigate the possibility that chaos and simple determinism are governing the dynamics of reversed field pinch (RFP) plasmas using data from both numerical simulations and experiment. A large repertoire of nonlinear analysis techniques is used to identify low dimensional chaos. These tools include phase portraits and Poincard sections, correlation dimension, the spectrum of Lyapunov exponents and short term predictability. In addition, nonlinear noise reduction techniques are applied to the experimental data in an attempt to extract any underlying deterministic dynamics. Two model systems are used to simulate the plasma dynamics. These are -the DEBS code, which models global RFPmore » dynamics, and the dissipative trapped electron mode (DTEM) model, which models drift wave turbulence. Data from both simulations show strong indications of low,dimensional chaos and simple determinism. Experimental data were obtained from the Madison Symmetric Torus RFP and consist of a wide array of both global and local diagnostic signals. None of the signals shows any indication of low dimensional chaos or other simple determinism. Moreover, most of the analysis tools indicate the experimental system is very high dimensional with properties similar to noise. Nonlinear noise reduction is unsuccessful at extracting an underlying deterministic system.« less

New MHD feedback control schemes using the MARTe framework in RFX-mod

NASA Astrophysics Data System (ADS)

Piron, Chiara; Manduchi, Gabriele; Marrelli, Lionello; Piovesan, Paolo; Zanca, Paolo

2013-10-01

Real-time feedback control of MHD instabilities is a topic of major interest in magnetic thermonuclear fusion, since it allows to optimize a device performance even beyond its stability bounds. The stability properties of different magnetic configurations are important test benches for real-time control systems. RFX-mod, a Reversed Field Pinch experiment that can also operate as a tokamak, is a well suited device to investigate this topic. It is equipped with a sophisticated magnetic feedback system that controls MHD instabilities and error fields by means of 192 active coils and a corresponding grid of sensors. In addition, the RFX-mod control system has recently gained new potentialities thanks to the introduction of the MARTe framework and of a new CPU architecture. These capabilities allow to study new feedback algorithms relevant to both RFP and tokamak operation and to contribute to the debate on the optimal feedback strategy. This work focuses on the design of new feedback schemes. For this purpose new magnetic sensors have been explored, together with new algorithms that refine the de-aliasing computation of the radial sideband harmonics. The comparison of different sensor and feedback strategy performance is described in both RFP and tokamak experiments.

[Efficacy of regimens containing INH, RFP with varied chemotherapy courses on retreated culture positive pneumoconio-tuberculosis].

PubMed

Jiang, W; Chen, H; Gao, J; Huaibei, M

1998-11-01

To evaluate the short and long-term effects of regimens containing INH, RFP with varied chemotherapy courses on culture-positive pneumoconio-tuberculosis. 79 patients with culture-positive pneumoconio-tuberculosis were divided into three groups according to chemotherapy duration: 9-months group (M9: 2SHRZ/7HRE) 28 cases, 12-month group (2SHRZ/10HRE) 25 cases, 18-month group (M18: 2SHRZ/10HRE/6HR) 26 cases. Evaluating the efficacy of regimens depended predominantly on sputum bacteriological conversion, and the patients who completed the regimens were followed up for 5-8 years. Sputum negative conversion rates of three groups at the end of chemotherapy were 83%, 96%, 95%, and their recurrent rates in follow-up period 41%, 4% and 5% respectively. Of all patients who completed the regimens bacteriological relapse rates from the first to fourth year are 6%, 8%, 2%, 2% in the follow-up period. There was no bacteriological relapse from fifth to eighth year. It is effective for SHRZ/HRE combination with 12-months course to retreated tubercle bacillus positive pneumoconio-tuberculosis, and it is appropriate for such patients to be followed up for 4-5 years.

Guide to red fluorescent proteins and biosensors for flow cytometry.

PubMed

Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-01-01

Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Ion energy balance in enhanced-confinement reversed-field pinch plasmas

NASA Astrophysics Data System (ADS)

Xing, Z. A.; Nornberg, M. D.; Boguski, J.; Craig, D.; den Hartog, D. J.; McCollam, K.

2017-10-01

Testing the applicability of collisional ion transport theory using tearing suppressed RFP plasma in MST achieved through Pulsed Poloidal Current Drive (PPCD), we find that the ion temperature dynamics in the core to be well-predicted by classical and collisional terms. Prior work demonstrated that impurity ion particle transport in PPCD plasmas is classical. Neoclassical effects on ions in the RFP are small and the stochastic transport is greatly suppressed during PPCD. Recent neutral modelling with DEGAS2 suggests higher core neutral temperatures than expected due to the preferential penetration of higher temperature neutrals generated by charge exchange. Further, investigations through equilibrium reconstruction point to the existence of an inward pinch flow associated with ExB drift. The heat balance model pulls together a wide range of diagnostic data to forward model Ti evolution in PPCD, which is then compared to charge exchange spectroscopy measurements of Ti. Ion power balance is mostly driven by classical effects including compressional heating, electron collisional heating, and charge exchange transport. This understanding provides a good baseline for investigations of anomalous heating in plasmas with tearing mode activity. This work is supported by US DOE.

Comparing magnetic fluctuation dynamics in nonlinear MHD simulations of low-aspect-ratio RFPs to RELAX experiments

NASA Astrophysics Data System (ADS)

McCollam, K. J.; den Hartog, D. J.; Jacobson, C. M.; Sovinec, C. R.; Masamune, S.; Sanpei, A.

2017-10-01

We present comparisons of magnetic tearing fluctuation activity between RFP experiments on the low-aspect-ratio RELAX device (R / a 2) and nonlinear simulations of zero-beta, single-fluid MHD using the NIMROD code in both cylindrical and toroidal geometries at a Lundquist number of S =104 , nearly as high as experimental values. Time-average fluctuation amplitudes observed in the simulations are similar to those from the experiments, but more rigorous comparisons versus spectral mode numbers are in progress. We also focus on how the spatiotemporal dynamics of the fluctuations vary with RFP equilibrium parameters. Interestingly, at shallow reversal, cylindrical simulations show a relatively uncoupled spectrum of nearly quiescent modes periodically varying in time, whereas the corresponding toroidal cases show a fully chaotic spectrum of strongly nonlinearly interacting modes. We ascribe this to the geometric m = 1 coupling present in the toroidal but not the cylindrical case. We present initial results from convergence studies with increased spatial resolution for both geometries. Simulations at higher S are planned. This work is supported by the U.S. DOE and by the Japan Society for the Promotion of Science.

Overview of MST Research

NASA Astrophysics Data System (ADS)

Sarff, J. S.

2016-10-01

MST progress in advancing the RFP for (1) fusion plasma confinement with ohmic heating and minimal external magnetization, (2) predictive capability in toroidal confinement physics, and (3) basic plasma physics is summarized. Validation of key plasma models is a program priority. Programmable power supplies (PPS) are being developed to maximize inductive capability. Well-controlled flattops with current as low as 0.02 MA are produced with an existing PPS, and Ip <= 0.8 MA is anticipated with a second PPS under construction. The Lundquist number spans S =10(4 - 9) for 0.02-0.8 MA, allowing nonlinear MHD validation using NIMROD and DEBS at low S to be connected to highest S experiments. The PPS also enables MST tokamak operation for studying transients and runaway electron suppression with RMPs. Gyrokinetic modeling with GENE predicts unstable TEM in improved-confinement plasmas. Fluctuations are measured with TEM properties including a density-gradient threshold larger than for tokamak plasmas. Probe measurements hint that drift waves are also excited via the turbulent cascade in standard RFP plasmas. Turbulent energization of an electron tail occurs during sawtooth reconnection. New diagnostics are being developed to measure the energetic ion profile and transport from EP instabilities with NBI. Supported by US DoE and NSF.

Subcellular real-time in vivo imaging of intralymphatic and intravascular cancer-cell trafficking

NASA Astrophysics Data System (ADS)

McElroy, M.; Hayashi, K.; Kaushal, S.; Bouvet, M.; Hoffman, Robert M.

2008-02-01

With the use of fluorescent cells labeled with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm and a highly sensitive small animal imaging system with both macro-optics and micro-optics, we have developed subcellular real-time imaging of cancer cell trafficking in live mice. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 small animal imaging system with a sensitive CCD camera and four objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. We have also developed real-time imaging of cancer cell trafficking in lymphatic vessels. Cancer cells labeled with GFP and/or RFP were injected into the inguinal lymph node of nude mice. The labeled cancer cells trafficked through lymphatic vessels where they were imaged via a skin flap in real-time at the cellular level until they entered the axillary lymph node. The bright dual-color fluorescence of the cancer cells and the real-time microscopic imaging capability of the Olympus OV100 enabled imaging the trafficking cancer cells in both blood vessels and lymphatics. With the dual-color cancer cells and the highly sensitive imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time.

Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation

PubMed Central

Hartmann, Michael; Gas-Pascual, Elisabet; Hemmerlin, Andrea; Rohmer, Michel; Bach, Thomas J.

2015-01-01

In a preceding study we have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was there demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, in this initial study complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established now new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system. PMID:26309725

Collaborative Project: Building improved optimized parameter estimation algorithms to improve methane and nitrogen fluxes in a climate model

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

Mahowald, Natalie

Soils in natural and managed ecosystems and wetlands are well known sources of methane, nitrous oxides, and reactive nitrogen gases, but the magnitudes of gas flux to the atmosphere are still poorly constrained. Thus, the reasons for the large increases in atmospheric concentrations of methane and nitrous oxide since the preindustrial time period are not well understood. The low atmospheric concentrations of methane and nitrous oxide, despite being more potent greenhouse gases than carbon dioxide, complicate empirical studies to provide explanations. In addition to climate concerns, the emissions of reactive nitrogen gases from soils are important to the changing nitrogenmore » balance in the earth system, subject to human management, and may change substantially in the future. Thus improved modeling of the emission fluxes of these species from the land surface is important. Currently, there are emission modules for methane and some nitrogen species in the Community Earth System Model’s Community Land Model (CLM-ME/N); however, there are large uncertainties and problems in the simulations, resulting in coarse estimates. In this proposal, we seek to improve these emission modules by combining state-of-the-art process modules for emissions, available data, and new optimization methods. In earth science problems, we often have substantial data and knowledge of processes in disparate systems, and thus we need to combine data and a general process level understanding into a model for projections of future climate that are as accurate as possible. The best methodologies for optimization of parameters in earth system models are still being developed. In this proposal we will develop and apply surrogate algorithms that a) were especially developed for computationally expensive simulations like CLM-ME/N models; b) were (in the earlier surrogate optimization Stochastic RBF) demonstrated to perform very well on computationally expensive complex partial differential equations in earth science with limited numbers of simulations; and, c) will be (as part of the proposed research) significantly improved both by adding asynchronous parallelism, early truncation of unsuccessful simulations, and the improvement of both serial and parallel performance by the use of derivative and sensitivity information from global and local surrogate approximations S(x). The algorithm development and testing will be focused on the CLM-ME/N model application, but the methods are general and are expected to also perform well on optimization for parameter estimation of other climate models and other classes of continuous multimodal optimization problems arising from complex simulation models. In addition, this proposal will compile available datasets of emissions of methane, nitrous oxides and reactive nitrogen species and develop protocols for site level comparisons with the CLM-ME/N. Once the model parameters are optimized against site level data, the model will be simulated at the global level and compared to atmospheric concentration measurements for the current climate, and future emissions will be estimated using climate change as simulated by the CESM. This proposal combines experts in earth system modeling, optimization, computer science, and process level understanding of soil gas emissions in an interdisciplinary team in order to improve the modeling of methane and nitrogen gas emissions. This proposal thus meets the requirements of the SciDAC RFP, by integrating state-of-the-art computer science and earth system to build an improved earth system model.« less

An improved monomeric infrared fluorescent protein for neuronal and tumour brain imaging.

PubMed

Yu, Dan; Gustafson, William Clay; Han, Chun; Lafaye, Céline; Noirclerc-Savoye, Marjolaine; Ge, Woo-Ping; Thayer, Desiree A; Huang, Hai; Kornberg, Thomas B; Royant, Antoine; Jan, Lily Yeh; Jan, Yuh Nung; Weiss, William A; Shu, Xiaokun

2014-05-15

Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging, and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the haeme oxygenase responsible for BV biosynthesis and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared with monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labelling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumours in whole mice. Our work shows promise in the application of IFPs for protein labelling and in vivo imaging.

RFP Patterns and Techniques for Successful Agile Contracting

DTIC Science & Technology

2016-11-01

2016-SR-025 SOFTWARE ENGINEERING INSTITUTE | CARNEGIE MELLON UNIVERSITY [Distribution Statement A] This material has been approved for public...release and unlimited distribution. Please see Copyright notice for non-U.S. Government use and distribution. Copyright 2016 Carnegie Mellon University...This material is based upon work funded and supported by the Department of Defense under Contract No. FA8721-05-C-0003 with Carnegie Mellon

KAPSE Interface Team (KIT) Public Report. Volume 7

DTIC Science & Technology

1989-10-01

e E&V STATUS - Ray Szymanski (Wriaht-Patterson) was announced as the new chairman of the Evaluation and Validation Team. Two RFP’s are coming, for...are language interoperability problems (implementations using multiple languages where interoperability problems are experienced such as transferring...visitors were introduced. Ray Szymanski , the Evaluation & Validation Team Leader, is replacing Jinny Castor from Wright-Patterson Air Force Base. Dr

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells.

PubMed

Xie, Li; Zhu, Dan; Dolai, Subhankar; Liang, Tao; Qin, Tairan; Kang, Youhou; Xie, Huanli; Huang, Ya-Chi; Gaisano, Herbert Y

2015-06-01

Of the four exocytotic syntaxins (Syns), much is now known about the role of Syn-1A (pre-docked secretory granules [SGs]) and Syn-3 (newcomer SGs) in insulin exocytosis. Some work was reported on Syn-4's role in biphasic glucose-stimulated insulin secretion (GSIS), but its precise role in insulin SG exocytosis remains unclear. In this paper we examine this role in human beta cells. Endogenous function of Syn-4 in human islets was assessed by knocking down its expression with lentiviral single hairpin RNA (lenti-shRNA)-RFP. Biphasic GSIS was determined by islet perifusion assay. Single-cell analysis of exocytosis of red fluorescent protein (RFP)-positive beta cells (exhibiting near-total depletion of Syn-4) was by patch clamp capacitance measurements (Cm) and total internal reflection fluorescence microscopy (TIRFM), the latter to further assess single SG behaviour. Co-immunoprecipitations were conducted on INS-1 cells to assess exocytotic complexes. Syn-4 knockdown (KD) of 77% in human islets caused a concomitant reduction in cognate Munc18c expression (46%) without affecting expression of other exocytotic proteins; this resulted in reduction of GSIS in the first phase (by 42%) and the second phase (by 40%). Cm of RFP-tagged Syn-4-KD beta cells showed severe inhibition in the readily releasable pool (by 71%) and mobilisation from reserve pools (by 63%). TIRFM showed that Syn-4-KD-induced inhibition of first-phase GSIS was attributed to reduction in exocytosis of both pre-docked and newcomer SGs (which undergo minimal residence or docking time at the plasma membrane before fusion). Second-phase inhibition was attributed to reduction in newcomer SGs. Stx-4 co-immunoprecipitated Munc18c, VAMP2 and VAMP8, suggesting that these exocytotic complexes may be involved in exocytosis of pre-docked and newcomer SGs. Syn-4 is involved in distinct molecular machineries that influence exocytosis of both pre-docked and newcomer SGs in a manner functionally redundant to Syn-1A and Syn-3, respectively; this underlies Syn-4's role in mediating portions of first-phase and second-phase GSIS.

Density and beta limits in the Madison Symmetric Torus Reversed-Field Pinch

NASA Astrophysics Data System (ADS)

Caspary, Kyle Jonathan

Operational limits and the underlying physics are explored on the Madison Symmetric Torus (MST) Reversed-Field Pinch (RFP) using deuterium pellet fueling. The injection of a fast pellet provides a large source of fuel in the plasma edge upon impact with the vessel wall, capable of triggering density limit terminations for the full range of plasma current, up to 600 kA. As the pellet size and plasma density increase, approaching the empirical Greenwald limit, plasma degradation is observed in the form of current decay, increased magnetic activity in the edge and core, increased radiation and plasma cooling. The complete termination of the plasma is consistent with the Greenwald limit; however, a slightly smaller maximum density is observed in discharges without toroidal field reversal. The plasma beta is the ratio of the plasma pressure to the confining magnetic pressure. Beta limits are known to constrain other magnetic confinement devices, but no beta limit has yet been established on the RFP. On MST, the highest beta values are obtained in improved confinement discharges with pellet fueling. By using pellet injection to scan the plasma density during PPCD, we also achieve a scan of Ohmic input power due to the increase in plasma resistivity. We observe a factor of 3 or more increase in Ohmic power as we increase the density from 1*1019 to 3*10 19 m-3. Despite this increased Ohmic power, the electron contribution to beta is constant, suggesting a confinement limited beta for the RFP. The electrons and ions are classically well coupled in these cold, dense pellet fueled plasmas, so the increase in total beta at higher density is primarily due to the increased ion contribution. The interaction of pellet fueling and NBI heating is explored. Modeling of MST's neutral heating beam suggests an optimal density for beam power deposition of 2-3*1019 m-3. Low current, NBI heated discharges show evidence of an increased electron beta in this density range. Additionally, the fast ion population can enhance ablation as well as cause pellet deflection. Other exploratory experiments with the pellet injection system explore additional injection scenarios and expand the injector capabilities.

Survival of mature mouse olfactory sensory neurons labeled genetically perinatally.

PubMed

Holl, Anna-Maria

2018-04-01

The main olfactory epithelium (MOE) of an adult mouse harbors a few million mature olfactory sensory neurons (OSNs), which are traditionally defined as mature by their expression of the olfactory marker protein (OMP). Mature OSNs differentiate in situ from stem cells at the base of the MOE. The consensus view is that mature OSNs have a defined lifespan and then undergo programmed cell death, and that the adult MOE maintains homeostasis by generating new mature OSNs from stem cells. But there is also evidence for mature OSNs that are long-lived. Thus far modern genetic tools have not been applied to quantify survival of a population of OSNs that are mature at a given point in time. Here, a genetic strategy was developed to label irreversibly OMP-expressing OSNs in mice. A gene-targeted OMP-CreERT2 strain was generated in which mature OSNs express an enzymatically inactive version of the Cre recombinase. The fusion protein CreERT2 becomes transiently active when exposed to tamoxifen, and in the presence of a Cre reporter in the genome such as tdRFP, CreERT2-expressing cells become irreversibly labeled. A cohort of mice was generated with the same day of birth by in vitro fertilization and embryo transfer, and injected tamoxifen in their mothers at E18.5 of gestation. I counted RFP immunoreactive cells in the MOE and vomeronasal organ of 36 tamoxifen-exposed OMP-CreERT2 × tdRFP mice from 7 age groups: postnatal day (PD)1.5, PD3.5, PD6.5, 3 weeks, 9 weeks, 6 months, and 12 months. Approximately 7.8% of perinatally labeled cells remain at 12 months, confirming that some mature OSNs are indeed long-lived. The survival curve of the population of perinatally labeled MOE cells can be modeled with a mean half-life of 26 days for the population as a whole, excluding the long-lived cells. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

Inhibition of Growth and Metastasis of Tumor in Nude Mice after Intraperitoneal Injection of Bevacizumab.

PubMed

Zhao, Ze-Xue; Li, Xiang; Liu, Wei-Dong; Liu, Xiao-Zhou; Wu, Su-Jia; Hu, Xiao-Hui

2016-05-01

To explore the inhibitory effect of bevacizumab, a vascular endothelial growth factor antibody, on angiogenesis in human osteosarcoma of nude mice. Twenty-one nude mice were inoculated with red fluorescent protein (RFP)-labeled human osteosarcoma cell line 143B-RFP, that is, clones that expressed RFP in the cytoplasm, and randomly assigned to one of three groups: G1 (Control group, injected with saline solution); G2 (intraperitoneal bevacizumab 2 mg/kg twice per week) and G3 (intraperitoneal bevacizumab 5 mg/kg, twice per week). The tumor-bearing mice were examined in a fluorescence light box that was illuminated periodically. The primary tumors were measured by fluorescence imaging weekly and their volumes calculated. The mean tumor volumes were significantly smaller in the G3 (186.4 ± 100.8 mm(3) ) than the control group (587.0 ± 406.8 mm(3) ) (P < 0.05) on Day 31, and again significantly smaller in the G3 (677.3 ± 461.9 mm(3) ) than the control group (3162.6 ± 1529.2 mm(3) ) on Day 38 (P < 0.01). The average tumor volume in the G2 group was 493.5 ± 425.4 mm(3) on Day 31 and 1870.1 ± 1524.8 mm(3) on Day 38. The effect on tumor volume was greater in the G3 than the G2 group. Three mice in the G2 group, four in the G3 group and four in the control group developed lung metastases that were confirmed by pathological examination; these differences were not statistically significant (P < 0.05). Bevacizumab exhibits strong antiangiogenesis activity in experimental osteosarcoma in a nude mouse model but does not influence the incidence of lung metastasis. Our findings may have considerable potential for the treatment of osteosarcoma. © 2016 The Authors. Orthopaedic Surgery Published by John Wiley & Sons Australia, Ltd and Chinese Orthopaedic Association.

An adaptive, conservative 0D-2V multispecies Rosenbluth–Fokker–Planck solver for arbitrarily disparate mass and temperature regimes

DOE PAGES

Taitano, William; Chacon, Luis; Simakov, Andrei Nikolaevich

2016-04-25

In this paper, we propose an adaptive velocity-space discretization scheme for the multi-species, multidimensional Rosenbluth–Fokker–Planck (RFP) equation, which is exactly mass-, momentum-, and energy-conserving. Unlike most earlier studies, our approach normalizes the velocity-space coordinate to the temporally varying individual plasma species' local thermal velocity, v th (t), and explicitly considers the resulting inertial terms in the Fokker–Planck equation. Our conservation strategy employs nonlinear constraints to enforce discretely the conservation properties of these inertial terms and the Fokker–Planck collision operator. To deal with situations of extreme thermal velocity disparities among different species, we employ an asymptotic v th -ratio-based expansion ofmore » the Rosenbluth potentials that only requires the computation of several velocity-space integrals. Numerical examples demonstrate the favorable efficiency and accuracy properties of the scheme. Specifically, we show that the combined use of the velocity-grid adaptivity and asymptotic expansions delivers many orders-of-magnitude savings in mesh resolution requirements compared to a single, static uniform mesh.« less

78 FR 21599 - Defense Acquisition University Board of Visitors; Notice of Federal Advisory Committee Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-11

... 9:00 a.m. RFP Team-learning 9:30 a.m. Better Buying Power 2.0 10:30 a.m. Acker Knowledge Repository...: Pursuant to 5 U.S.C. 552b and 41 CFR 102-3.140 through 102-3.165, and the availability of space, this meeting is open to the public. However, because of space limitations, allocation of seating will be made...

Axon-Sorting Multifunctional Nerve Guides: Accelerating Restoration of Nerve Function

DTIC Science & Technology

2014-10-01

factor (singly & in selected combinations) in the organotypic model system for preferential sensory or motor axon extension. Use confocal microscopy to...track axon extension of labeled sensory or motor neurons from spinal cord slices (motor) or dorsal root ganglia ( DRG ) (sensory). 20 Thy1-YFP mice...RESEARCH ACCOMPLISHMENTS: • Established a system of color-coded mixed nerve tracking using GFP and RFP expressing motor and sensory neurons (Figure 1

Quarrelsome Committees in U.S. Defense Acquisition: The KC-X Case

DTIC Science & Technology

2014-04-30

close to its original specifications in the RfP published on February 24, 2010. NG-EADS supporters were decidedly negative. Sen. Sessions ( R -AL...party. Thus, for example, the Boeing congressional delegation included Rep. Todd Tiahrt ( R -KS), along with the largely Democratic Washington State...delegation. The congressional EADS supporters included Sen. Jeff Sessions ( R -AL). And political figures stepped up to claim a major role in changing

Reliability Programs for Nonelectronic Designs. Volume 2

DTIC Science & Technology

1983-04-01

afforded. Differ- ences between critical and minor failures must be defined in the RFP so that the test need not be stopped for minor failures. However...not be afforded. Specialized test plans must be developed for nonelectronic equipment. First, differences between critical and minor failures must be...determined prior to initiating the test program so that the test need not be stopped for minor failures. Second, although the test must be interrupted

Defense Attache Saigon: RVNAF Quarterly Assessment, 3rd Quarter FY74.

DTIC Science & Technology

1974-05-01

1,600 * Includes one SAiK Regiment ** Includes 12.7, 14.5, 23, 37, 40, 57, 85, and 100MM weapons. 7. (S) ENEM 1Y THREAT AND INTENTIONS BY REGION: a. DAO...there is validity in field reporting, the kill ratio between RF/P? units and enem .’ forces has been running more than 3 to i in favor of the RF/F. g

New integrative modules for multicolor-protein labeling and live-cell imaging in Saccharomyces cerevisiae.

PubMed

Malcova, Ivana; Farkasovsky, Marian; Senohrabkova, Lenka; Vasicova, Pavla; Hasek, Jiri

2016-05-01

Live-imaging analysis is performed in many laboratories all over the world. Various tools have been developed to enable protein labeling either in plasmid or genomic context in live yeast cells. Here, we introduce a set of nine integrative modules for the C-terminal gene tagging that combines three fluorescent proteins (FPs)-ymTagBFP, mCherry and yTagRFP-T with three dominant selection markers: geneticin, nourseothricin and hygromycin. In addition, the construction of two episomal modules for Saccharomyces cerevisiae with photostable yTagRFP-T is also referred to. Our cassettes with orange, red and blue FPs can be combined with other fluorescent probes like green fluorescent protein to prepare double- or triple-labeled strains for multicolor live-cell imaging. Primers for PCR amplification of the cassettes were designed in such a way as to be fully compatible with the existing PCR toolbox representing over 50 various integrative modules and also with deletion cassettes either for single or repeated usage to enable a cost-effective and an easy exchange of tags. New modules can also be used for biochemical analysis since antibodies are available for all three fluorescent probes. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Rocky Flats CAAS System Recalibrated, Retested, and Analyzed to Install in the Criticality Experiments Facility at the Nevada Test Site

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

Kim, S; Heinrichs, D; Biswas, D

2009-05-27

Neutron detectors and control panels transferred from the Rocky Flats Plant (RFP) were recalibrated and retested for redeployment to the CEF. Testing and calibration were successful with no failure to any equipment. Detector sensitivity was tested at a TRIGA reactor, and the response to thermal neutron flux was satisfactory. MCNP calculated minimum fission yield ({approx} 2 x 10{sup 15} fissions) was applied to determine the thermal flux at selected detector positions at the CEF. Thermal flux levels were greater than 6.39 x 10{sup 6} (n/cm{sup 2}-sec), which was about four orders of magnitude greater than the minimum alarm flux. Calculationsmore » of detector survivable distances indicate that, to be out of lethal area, a detector needs to be placed greater than 15 ft away from a maximum credible source. MCNP calculated flux/dose results were independently verified by COG. CAAS calibration and the testing confirmed that the RFP CAAS system is performing its functions as expected. New criteria for the CAAS detector placement and 12-rad zone boundaries at the CEF are established. All of the CAAS related documents and hardware have been transferred from LLNL to NSTec for installation at the CEF high bay areas.« less

Role of renal urothelium in the development and progression of kidney disease.

PubMed

Carpenter, Ashley R; McHugh, Kirk M

2017-04-01

The clinical and financial impact of chronic kidney disease (CKD) is significant, while its progression and prognosis is variable and often poor. Studies using the megabladder (mgb -/- ) model of CKD show that renal urothelium plays a key role in modulating early injury responses following the development of congenital obstruction. The aim of this review is to examine the role that urothelium has in normal urinary tract development and pathogenesis. We discuss normal morphology of renal urothelium and then examine the role that uroplakins (Upks) play in its development. Histologic, biochemical, and molecular characterization of Upk1b RFP/RFP mice indicated Upk1b expression is essential for normal urinary tract development, apical plaque/asymmetric membrane unit (AUM) formation, and differentiation and functional integrity of the renal urothelium. Our studies provide the first evidence that Upk1b is directly associated with the development of congenital anomalies of the urinary tract (CAKUT), spontaneous age-dependent hydronephrosis, and dysplastic urothelia. These observations demonstrate the importance of proper urothelial differentiation in normal development and pathogenesis of the urinary tract and provide a unique working model to test the hypothesis that the complex etiology associated with CKD is dependent upon predetermined genetic susceptibilities that establish pathogenic thresholds for disease initiation and progression.

Role of Renal Urothelium in the Development and Progression of Kidney Disease

PubMed Central

Carpenter, Ashley R.; McHugh, Kirk M.

2016-01-01

The clinical and financial impact of chronic kidney disease (CKD) is significant, while the progression and prognosis of CKD is variable and often poor. Studies using the megabladder (mgb−/−) model of CKD have shown that renal urothelium plays a key role in modulating the early injury responses following the development of congenital obstruction. The aim of this review is to examine the role that urothelium has in normal urinary tract development and pathogenesis. We discuss normal morphology of renal urothelium and then examine the role that uroplakins (Upks) play in its development. Histologic, biochemical and molecular characterization of Upk1bRFP/RFP mice indicated Upk1b expression is essential for normal urinary tract development, apical plaque/AUM formation and differentiation and functional integrity of the renal urothelium. Our studies provide the first evidence Upk1b is directly associated with the development of congenital anomalies of the urinary tract (CAKUT), spontaneous age-dependent hydronephrosis and dysplastic urothelia. These observations demonstrate the importance of proper urothelial differentiation in the normal development and pathogenesis of the urinary tract, and provide a unique working model to test the hypothesis that the complex etiology associated with CKD is dependent upon predetermined genetic susceptibilities that establish pathogenic thresholds for disease initiation and progression. PMID:27115886

Electron Thermal Transport due to Magnetic Diffusion in the MST RFP

NASA Astrophysics Data System (ADS)

Reusch, J. A.; Anderson, J. K.; den Hartog, D. J.; Forest, C. B.; Kasten, C. P.; Schnack, D. D.; Stephens, H. D.

2011-10-01

Comparison of measurements made in the MST RFP to the results from extensive nonlinear resistive MHD simulations has provided two key observations. First, trapped particles reduce electron thermal diffusion; inclusion of this effect is required for quantitative agreement of simulation to measurement. Second, the structure and evolution of long-wavelength temperature fluctuations measured in MST shows remarkable qualitative similarity to fluctuations appearing in a finite-pressure simulation. These simulations were run at parameters matching those of 400 kA discharges in MST (S ~ 4 ×106). In a zero β simulation, the measured χe is compared to the thermal diffusion due to parallel losses along diffusing magnetic field lines, χst =v∥Dmag . Agreement is only found if the reduction in χst due to trapped particles is taken into account. In a second simulation, the pressure field was evolved self consistently assuming Ohmic heating and anisotropic thermal conduction. Fluctuations in the simulated temperature are very similar in character and time evolution to temperature fluctuations measured in MST. This includes m = 1 , n = 6 fluctuations that flatten the temperature profile as well as m = 1 , n = 5 fluctuations that generate hot island structures near the core shortly after sawtooth crashes. This work supported by the US DOE and NSF.

Energy and Pitch Distribution of Spontaneously-generated High-energy Bulk Ions in the RFP

NASA Astrophysics Data System (ADS)

Kim, Jungha; Anderson, Jay; Reusch, Joshua; Eilerman, Scott; Capecchi, William

2014-10-01

Magnetic reconnection events in the reversed field pinch (RFP) are known to heat bulk and impurity ions. Runaway due to a parallel electric field has recently been confirmed as an important acceleration mechanism for high energy test ions supplied by a neutral beam. This effect does not, however, explain the change in distribution of nearly Maxwellian bulk ions at a reconnection event. By operating MST near maximum current and low electron density, significant fusion neutron flux can be generated without neutral beam injection. The bulk ion distribution created in these plasmas is well-confined, non-Maxwellian, and can be measured by the Advanced Neutral Particle Analyzer (ANPA) placed at a radial or tangential porthole. Data show a high energy tail up to 25 keV with a relatively higher signal in the low energy channels (8-15 keV) at the radial port following a reconnection event. Analysis of the energy dependence of trapped orbits sampled by the ANPA at the radial view implies an abundance of lower energy particles in regions of higher neutral density. This mandates a careful deconvolution of the measured ANPA signal to compute the fast ion distribution. This work is supported by the US DOE and NSF.

A Dual-Intein Autoprocessing Domain that Directs Synchronized Protein Co-Expression in Both Prokaryotes and Eukaryotes

PubMed Central

Zhang, Bei; Rapolu, Madhusudhan; Liang, Zhibin; Han, Zhenlin; Williams, Philip G.; Su, Wei Wen

2015-01-01

Being able to coordinate co-expression of multiple proteins is necessary for a variety of important applications such as assembly of protein complexes, trait stacking, and metabolic engineering. Currently only few options are available for multiple recombinant protein co-expression, and most of them are not applicable to both prokaryotic and eukaryotic hosts. Here, we report a new polyprotein vector system that is based on a pair of self-excising mini-inteins fused in tandem, termed the dual-intein (DI) domain, to achieve synchronized co-expression of multiple proteins. The DI domain comprises an Ssp DnaE mini-intein N159A mutant and an Ssp DnaB mini-intein C1A mutant connected in tandem by a peptide linker to mediate efficient release of the flanking proteins via autocatalytic cleavage. Essentially complete release of constituent proteins, GFP and RFP (mCherry), from a polyprotein precursor, in bacterial, mammalian, and plant hosts was demonstrated. In addition, successful co-expression of GFP with chloramphenicol acetyltransferase, and thioredoxin with RFP, respectively, further substantiates the general applicability of the DI polyprotein system. Collectively, our results demonstrate the DI-based polyprotein technology as a highly valuable addition to the molecular toolbox for multi-protein co-expression which finds vast applications in biotechnology, biosciences, and biomedicine. PMID:25712612

Tracing the Mass of Early-type Galaxies using Planetary Nebulae

NASA Astrophysics Data System (ADS)

Sluis, A. P. N.; William, T. B.

2002-12-01

We report on observations of two ellipticals (NGC 3379 and NGC 1549) and two S0s (NGC 3384 and NGC 4636) performed with the Rutgers Fabry-Perot (RFP). The observations are part of a larger project to study the distribution of mass in the outer regions of early-type galaxies. Efforts to determine this distribution are generally hampered by the scarcity of useful tracers of the potential at large radii. Ellipticals and S0s have steep surface brightness profiles that make absorption line spectroscopy of the stellar population practically impossible beyond a few kpc from the center. Also, their gas content is low and does not extend far beyond the nucleus. Planetary Nebulae (PNe) offer a way around these problems: as remants of intermediate mass stars we expect them to follow the stellar light distribution and be numerous enough to be an effective tracer. PNe radiate hundreds of solar luminosities in a few emission lines (mostly [OIII] 5007 Å), making it possible to detect them over extragalactic distances and at the same time measure their line of sight velocities using the RFP. We present the photometry and the kinematics of the PN systems as well as some simple dynamical mass models for the four galaxies mentioned above.

Application of targeted proteomics to metabolically engineered Escherichia coli.

PubMed

Singh, Pragya; Batth, Tanveer S; Juminaga, Darmawi; Dahl, Robert H; Keasling, Jay D; Adams, Paul D; Petzold, Christopher J

2012-04-01

As synthetic biology matures to compete with chemical transformation of commodity and high-value compounds, a wide variety of well-characterized biological parts are needed to facilitate system design. Protein quantification based on selected-reaction monitoring (SRM) mass spectrometry compliments metabolite and transcript analysis for system characterization and optimizing flux through engineered pathways. By using SRM quantification, we assayed red fluorescent protein (RFP) expressed from plasmids containing several inducible and constitutive promoters and subsequently assessed protein production from the same promoters driving expression of eight mevalonate pathway proteins in Escherichia coli. For each of the promoter systems, the protein level for the first gene in the operon followed that of RFP, however, the levels of proteins produced from genes farther from the promoter were much less consistent. Second, we used targeted proteomics to characterize tyrosine biosynthesis pathway proteins after removal of native regulation. The changes were not expected to cause significant impact on protein levels, yet significant variation in protein abundance was observed and tyrosine production for these strains spanned a range from less than 1 mg/L to greater than 250 mg/L. Overall, our results underscore the importance of targeted proteomics for determining accurate protein levels in engineered systems and fine-tuning metabolic pathways. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Measuring viscosity with a resonant magnetic perturbation in the MST RFP

NASA Astrophysics Data System (ADS)

Fridström, Richard; Munaretto, Stefano; Frassinetti, Lorenzo; Chapman, Brett; Brunsell, Per; Sarff, John; MST Team

2016-10-01

Application of an m = 1 resonant magnetic perturbation (RMP) causes braking and locking of naturally rotating m = 1 tearing modes (TMs) in the MST RFP. The experimental TM dynamics are replicated by a theoretical model including the interaction between the RMP and multiple TMs [Fridström PoP 23, 062504 (2016)]. The viscosity is the only free parameter in the model, and it is chosen such that model TM velocity evolution matches that of the experiment. The model does not depend on the means by which the natural rotation is generated. The chosen value of the viscosity, about 40 m2/s, is consistent with separate measurements in MST using a biased probe to temporarily spin up the plasma. This viscosity is about 100 times larger than the classical prediction, likely due to magnetic stochasticity in the core of these plasmas. Viscosity is a key parameter in visco-resistive MHD codes like NIMROD. The validation of these codes requires measurement of the viscosity over a broad parameter range, which will now be possible with the RMP technique that, unlike the biased probe, is not limited to low-energy-density plasmas. Estimation with the RMP technique of the viscosity in several MST discharges suggests that the viscosity decreases as the electron beta increases. Work supported by USDOE.