48 CFR 1852.211-70 - Packaging, handling, and transportation.

Code of Federal Regulations, 2010 CFR

2010-10-01

... transportation. 1852.211-70 Section 1852.211-70 Federal Acquisition Regulations System NATIONAL AERONAUTICS AND... and Clauses 1852.211-70 Packaging, handling, and transportation. As prescribed in 1811.404-70, insert the following clause: Packaging, Handling, and Transportation (SEPT 2005) (a) The Contractor shall...

48 CFR 1852.211-70 - Packaging, handling, and transportation.

Code of Federal Regulations, 2012 CFR

2012-10-01

... transportation. 1852.211-70 Section 1852.211-70 Federal Acquisition Regulations System NATIONAL AERONAUTICS AND... and Clauses 1852.211-70 Packaging, handling, and transportation. As prescribed in 1811.404-70, insert the following clause: Packaging, Handling, and Transportation (SEP 2005) (a) The Contractor shall...

48 CFR 1852.211-70 - Packaging, handling, and transportation.

Code of Federal Regulations, 2013 CFR

2013-10-01

... transportation. 1852.211-70 Section 1852.211-70 Federal Acquisition Regulations System NATIONAL AERONAUTICS AND... and Clauses 1852.211-70 Packaging, handling, and transportation. As prescribed in 1811.404-70, insert the following clause: Packaging, Handling, and Transportation (SEP 2005) (a) The Contractor shall...

48 CFR 1852.211-70 - Packaging, handling, and transportation.

Code of Federal Regulations, 2014 CFR

2014-10-01

... transportation. 1852.211-70 Section 1852.211-70 Federal Acquisition Regulations System NATIONAL AERONAUTICS AND... and Clauses 1852.211-70 Packaging, handling, and transportation. As prescribed in 1811.404-70, insert the following clause: Packaging, Handling, and Transportation (SEP 2005) (a) The Contractor shall...

21 CFR 516.165 - Records and reports.

Code of Federal Regulations, 2014 CFR

2014-04-01

... within 60 days thereafter. The report must contain data and information for the full reporting period..., include a summary of those changes and the holder's and distributor's current package labeling, including any package inserts. For large-size package labeling or large shipping cartons, submit a...

21 CFR 516.165 - Records and reports.

Code of Federal Regulations, 2012 CFR

2012-04-01

... within 60 days thereafter. The report must contain data and information for the full reporting period..., include a summary of those changes and the holder's and distributor's current package labeling, including any package inserts. For large-size package labeling or large shipping cartons, submit a...

Real Time Sensing and Discrimination of Single Chemicals Using Channel of Phi29 DNA Packaging Nanomotor

PubMed Central

Haque, Farzin; Lunn, Jennifer; Fang, Huaming; Smithrud, David; Guo, Peixuan

2012-01-01

A highly sensitive and reliable method to sense and identify a single chemical at extremely low concentrations and high contamination is important for environmental surveillance, homeland security, athlete drug monitoring, toxin/drug screening, and earlier disease diagnosis. This manuscript reports a method for precise detection of single chemicals. The hub of the bacteriophage phi29 DNA packaging motor is a connector consisting of twelve protein subunits encircled into a 3.6-nm channel as a path for dsDNA to enter during packaging and to exit during infection. The connector has previously been inserted into a lipid bilayer to serve as a membrane-embedded channel. Herein we report the modification of the phi29 channel to develop a class of sensors to detect single chemicals. The Lysine-234 of each protein subunit was mutated to cysteine, generating 12-SH ring lining the channel wall. Chemicals passing through this robust channel and interactions with the SH-group generated extremely reliable, precise, and sensitive current signatures as revealed by single channel conductance assays. Ethane (57 Daltons), thymine (167 Daltons), and benzene (105 Daltons) with reactive thioester moieties were clearly discriminated upon interaction with the available set of cysteine residues. The covalent attachment of each analyte induced discrete step-wise blockage in current signature with a corresponding decrease in conductance due to the physical blocking of the channel. Transient binding of the chemicals also produced characteristic fingerprints that were deduced from the unique blockage amplitude and pattern of the signals. This study shows that the phi29 connector can be used to sense chemicals with reactive thioesters or maleimide using single channel conduction assays based on their distinct fingerprints. The results demonstrated that this channel system could be further developed into very sensitive sensing devices. PMID:22458779

Real-time sensing and discrimination of single chemicals using the channel of phi29 DNA packaging nanomotor.

PubMed

Haque, Farzin; Lunn, Jennifer; Fang, Huaming; Smithrud, David; Guo, Peixuan

2012-04-24

A highly sensitive and reliable method to sense and identify a single chemical at extremely low concentrations and high contamination is important for environmental surveillance, homeland security, athlete drug monitoring, toxin/drug screening, and earlier disease diagnosis. This article reports a method for precise detection of single chemicals. The hub of the bacteriophage phi29 DNA packaging motor is a connector consisting of 12 protein subunits encircled into a 3.6 nm channel as a path for dsDNA to enter during packaging and to exit during infection. The connector has previously been inserted into a lipid bilayer to serve as a membrane-embedded channel. Herein we report the modification of the phi29 channel to develop a class of sensors to detect single chemicals. The lysine-234 of each protein subunit was mutated to cysteine, generating 12-SH ring lining the channel wall. Chemicals passing through this robust channel and interactions with the SH group generated extremely reliable, precise, and sensitive current signatures as revealed by single channel conductance assays. Ethane (57 Da), thymine (167 Da), and benzene (105 Da) with reactive thioester moieties were clearly discriminated upon interaction with the available set of cysteine residues. The covalent attachment of each analyte induced discrete stepwise blockage in current signature with a corresponding decrease in conductance due to the physical blocking of the channel. Transient binding of the chemicals also produced characteristic fingerprints that were deduced from the unique blockage amplitude and pattern of the signals. This study shows that the phi29 connector can be used to sense chemicals with reactive thioesters or maleimide using single channel conduction assays based on their distinct fingerprints. The results demonstrated that this channel system could be further developed into very sensitive sensing devices.

The NASA Electronic Parts and Packaging (NEPP) Program: Insertion of New Electronics Technologies

NASA Technical Reports Server (NTRS)

LaBel, Kenneth A.; Sampson, Michael J.

2007-01-01

This viewgraph presentation gives an overview of NASA Electronic Parts and Packaging (NEPP) Program's new electronics technology trends. The topics include: 1) The Changing World of Radiation Testing of Memories; 2) Even Application-Specific Tests are Costly!; 3) Hypothetical New Technology Part Qualification Cost; 4) Where we are; 5) Approaching FPGAs as a More Than a "Part" for Reliability; 6) FPGAs Beget Novel Radiation Test Setups; 7) Understanding the Complex Radiation Data; 8) Tracking Packaging Complexity and Reliability for FPGAs; 9) Devices Supporting the FPGA Need to be Considered; 10) Summary of the New Electronic Technologies and Insertion into Flight Programs Workshop; and 11) Highlights of Panel Notes and Comments

Evaluation of a microarray-based genotyping assay for the rapid detection of cytochrome P450 2C19 *2 and *3 polymorphisms from whole blood using nanoparticle probes.

PubMed

Buchan, Blake W; Peterson, Jess F; Cogbill, Christopher H; Anderson, Dennis K; Ledford, Joellen S; White, Mary N; Quigley, Neil B; Jannetto, Paul J; Ledeboer, Nathan A

2011-10-01

Numerous drugs such as clopidogrel have been developed to reduce coagulation or inhibit platelet function. The hepatic cytochrome P450 (CYP) pathway is involved in the conversion of clopidogrel to its active metabolite. A recent black-box warning was included in the clopidogrel package insert indicating a significant clinical link between specific CYP2C19 genetic variants and poor metabolism of clopidogrel. Of these variants, *2 and *3 are the most common and are associated with complete loss of enzyme activity. In patients who are carriers of a CYP2C19 *2 or *3 allele, the conversion of clopidogrel to its active metabolite may be reduced, which can lead to ischemic events and negative consequence for the patient. We examined the ability of the Verigene CLO assay (Nanosphere, Northbrook, IL) to identify CYP2C19 *2 and *3 polymorphisms in 1,286 unique whole blood samples. The Verigene CLO assay accurately identified homozygous and heterozygous *2 and *3 phenotypes with a specificity of 100% and a final call rate of 99.7%. The assay is fully automated and can produce a result in approximately 3.5 hours.

ProtSqueeze: simple and effective automated tool for setting up membrane protein simulations.

PubMed

Yesylevskyy, Semen O

2007-01-01

The major challenge in setting up membrane protein simulations is embedding the protein into the pre-equilibrated lipid bilayer. Several techniques were proposed to achieve optimal packing of the lipid molecules around the protein. However, all of them possess serious disadvantages, which limit their applicability and discourage the users of simulation packages from using them. In the present work, we analyzed existing approaches and proposed a new procedure of protein insertion into the lipid bilayer, which is implemented in the ProtSqueeze software. The advantages of ProtSqueeze are as follows: (1) the insertion algorithm is simple, understandable, and controllable; (2) the software can work with virtually any simulation package on virtually any platform; (3) no modification of the source code of the simulation package is needed; (4) the procedure of insertion is as automated as possible; (5) ProtSqueeze is distributed for free under a general public license. In this work, we present the architecture and the algorithm of ProtSqueeze and demonstrate its usage in case studies.

48 CFR 552.211-89 - Non-manufactured wood packaging material for export.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 48 Federal Acquisition Regulations System 4 2012-10-01 2012-10-01 false Non-manufactured wood... and Clauses 552.211-89 Non-manufactured wood packaging material for export. As prescribed in 511.204(b)(4), insert the following clause: Non-Manufactured Wood Packaging Material for Export (JAN 2010) (a...

48 CFR 552.211-89 - Non-manufactured wood packaging material for export.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 48 Federal Acquisition Regulations System 4 2014-10-01 2014-10-01 false Non-manufactured wood... and Clauses 552.211-89 Non-manufactured wood packaging material for export. As prescribed in 511.204(b)(4), insert the following clause: Non-Manufactured Wood Packaging Material for Export (JAN 2010) (a...

48 CFR 552.211-89 - Non-manufactured wood packaging material for export.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 48 Federal Acquisition Regulations System 4 2013-10-01 2013-10-01 false Non-manufactured wood... and Clauses 552.211-89 Non-manufactured wood packaging material for export. As prescribed in 511.204(b)(4), insert the following clause: Non-Manufactured Wood Packaging Material for Export (JAN 2010) (a...

48 CFR 552.211-89 - Non-manufactured wood packaging material for export.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 48 Federal Acquisition Regulations System 4 2011-10-01 2011-10-01 false Non-manufactured wood... and Clauses 552.211-89 Non-manufactured wood packaging material for export. As prescribed in 511.204(b)(4), insert the following clause: Non-Manufactured Wood Packaging Material for Export (JAN 2010) (a...

48 CFR 552.211-89 - Non-manufactured wood packaging material for export.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Non-manufactured wood... and Clauses 552.211-89 Non-manufactured wood packaging material for export. As prescribed in 511.204(b)(4), insert the following clause: Non-Manufactured Wood Packaging Material for Export (JAN 2010) (a...

[Incremental effect and mechanism of cyclosporine on blood concentration of statins and statin package insert information in Japan].

PubMed

Hirata-Koizumi, Mutsuko; Saito, Mitsuo; Miyake, Shinji; Hasegawa, Ryuichi

2005-01-01

Cyclosporine is an indispensable immunosuppressant used in organ transplant patients, who frequently manifest hyperlipidemia. Statins, which are cholesterol-lowering agents, are often combined with cyclosporine in the treatment of hyperlipidemia of organ transplant patients. Since cyclosporine is a substrate and inhibitor of CYP3A4, researchers suspect that the immunosuppressant inhibits CYP3A4-mediated metabolism of statins, leading to an increase in statin plasma concentration and infrequently resulting in rhabdomyolysis. However, a number of clinical trials have shown cyclosporine to increase the plasma concentration of all developed statins, including those not metabolized by CYP3A4. Furthemore, recent mechanistic studies have shown organic anion transporting peptides (OATP) C to mediate the uptake of some statins and cyclosporine has been shown to inhibit the uptake via OATP-C in cultured cells. Therefore, the inhibition of hepatic uptake of statins is considered to be one of the mechanisms by which cyclosporine incrementally increases statin blood concentration. However, most current Japanese package inserts of statins give no information on change in pharmacokinetic parameters such as AUC and Cmax in the combined medication with cyclosporine. Furthermore, in the Japanese package inserts, it is either stated that cyclosporine inhibits CYP3A4-mediated metabolism or no comment is made on the mechanism. The package insert should properly provide available quantitative information on the change of pharmacokinetic parameters and the probable mechanism of action.

Low-profile fiber connector for co-packaged optics

NASA Astrophysics Data System (ADS)

Brusberg, Lars; DeJong, Michael; Butler, Douglas L.; Clark, Jeffrey S.; Sutton, Clifford G.

2018-02-01

We developed a small form factor connector that can be assembled on all four sides of a high-data switch package for fiber connectivity. This paper discusses a novel connector approach that has the potential to meet all co-packaging requirements including solder-reflow-compatibility, de-mateability, low insertion loss and state-of-the art FAU attach. The connector was attached to the PIC for performance evaluation. The average insertion loss across all eight fibers of the assembly was 1.77 dB, including the three optical interfaces: (1) MT-to-MT between connector and receptacle, (2) receptacle-to-PLC and (3) PIC-to-FAU. Also included is the propagation loss of the PIC waveguide. Optical return loss was measured to be -55 dB or lower.

Cigarette package inserts can promote efficacy beliefs and sustained smoking cessation attempts: A longitudinal assessment of an innovative policy in Canada

PubMed Central

Thrasher, James F.; Swayampakala, Kamala; Cummings, K. Michael; Hammond, David; Anshari, Dien; Krugman, Dean M.; Hardin, James W.

2016-01-01

Background In June 2012, Canada implemented new pictorial warnings on cigarette packages, along with package inserts with messages to promote response efficacy (i.e., perceived quitting benefits) and self-efficacy (i.e., confidence to quit). This study assessed smokers’ attention towards warnings and inserts and its relationship with efficacy beliefs, risk perceptions and cessation at follow-up. Methods Data were analysed in 2015 from a prospective online consumer panel of adult Canadian smokers surveyed every four months between September 2012 and September 2014. Generalized Estimating Equation models assessed associations between reading inserts, reading warnings and efficacy beliefs (self-efficacy, response efficacy), risk perceptions, quit attempts of any length, and sustained quit attempts (i.e., 30 days or more) at follow-up. Models adjusted for socio-demographics, smoking-related variables, and time-in-sample effects. Results Over the study period, reading warnings significantly decreased (p<0.0001) while reading inserts increased (p=0.004). More frequent reading of warnings was associated independently with stronger response efficacy (Boften/very often vs never=0.28, 95% CI: 0.11–0.46) and risk perceptions at follow-up (Boften/very often vs never=0.31, 95% CI: 0.06–0.56). More frequent reading of inserts was associated independently with stronger self-efficacy to quit at follow-up (Btwice or more vs none=0.30, 95% CI: 0.14–0.47), quit attempts (ORtwice or more vs none= 1.68, 95% CI: 1.28–2.19), and quit attempts lasting 30 days or longer (ORtwice or more vs none=1.48, 95% CI: 1.01 – 2.17). Conclusions More frequent reading of inserts was associated with self-efficacy to quit, quit attempts, and sustained quitting at follow-up, suggesting that inserts complement pictorial HWLs. PMID:26970037

40 CFR 60.431 - Definitions and notations.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 6 2011-07-01 2011-07-01 false Definitions and notations. 60.431 Section 60.431 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS..., package inserts, book jackets, market circulars, magazine inserts, and shopping news, Newspapers, magazine...

40 CFR 60.431 - Definitions and notations.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 7 2012-07-01 2012-07-01 false Definitions and notations. 60.431 Section 60.431 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS..., package inserts, book jackets, market circulars, magazine inserts, and shopping news, Newspapers, magazine...

40 CFR 60.431 - Definitions and notations.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 7 2014-07-01 2014-07-01 false Definitions and notations. 60.431 Section 60.431 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS..., package inserts, book jackets, market circulars, magazine inserts, and shopping news, Newspapers, magazine...

40 CFR 60.431 - Definitions and notations.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 6 2010-07-01 2010-07-01 false Definitions and notations. 60.431 Section 60.431 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS..., package inserts, book jackets, market circulars, magazine inserts, and shopping news, Newspapers, magazine...

40 CFR 60.431 - Definitions and notations.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 7 2013-07-01 2013-07-01 false Definitions and notations. 60.431 Section 60.431 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS..., package inserts, book jackets, market circulars, magazine inserts, and shopping news, Newspapers, magazine...

Do laboratories follow heart failure recommendations and guidelines and did we improve? The CARdiac MArker Guideline Uptake in Europe (CARMAGUE).

PubMed

Hammerer-Lercher, Angelika; Collinson, Paul; van Dieijen-Visser, Marja P; Pulkki, Kari; Suvisaari, Janne; Ravkilde, Jan; Stavljenic-Rukavina, Ana; Baum, Hannsjörg; Laitinen, Päivi

2013-06-01

Natriuretic peptides (NP) are well-established markers of heart failure (HF). During the past 5 years, analytical and clinical recommendations for measurement of these biomarkers have been published in guidelines. The aim of this follow-up survey was to investigate how well these guidelines for measurement of NP have been implemented in laboratory practice in Europe. Member societies of the European Federation of Clinical Chemistry and Laboratory Medicine were invited in 2009 to participate in a web-based audit questionnaire. The questionnaire requested information on type of tests performed, decision limits for HF, turn-around time and frequency of testing. There was a moderate increase (12%) of laboratories measuring NP compared to the initial survey in 2006. The most frequently used HF decision limits for B-type NP (BNP) and N-terminal BNP (NT-proBNP) were, respectively, 100 ng/L and 125 ng/L, derived from the package inserts in 55%. Fifty laboratories used a second decision limit. Age or gender dependent decision limits were applied in 10% (8.5% in 2006). The vast majority of laboratories (80%) did not have any criteria regarding frequency of testing, compared to 33% in 2006. The implementation of NP measurement for HF management was a slow process between 2006 and 2009 at a time when guidelines had just been established. The decision limits were derived from package insert information and literature. There was great uncertainty concerning frequency of testing which may reflect the debate about the biological variability which was not published for most of the assays in 2009.

Conformity of package inserts information to regulatory requirements among selected branded and generic medicinal products circulating on the East African market.

PubMed

Sillo, Hiiti B; Masota, Nelson E; Kisoma, Sunday; Rago, Lembit; Mgoyela, Veronica; Kaale, Eliangiringa A

2018-01-01

Availability of correct and adequate information about medicines is an important aspect in ensuring rational use of medicines and hence facilitating safety and expected efficacy of medicines during therapy. Package inserts have proven to be a good source of information to the prescribers and patients whereby they have been useful in highlighting important information pertaining proper use and handling of the medicines. The present study was aimed at establishing the extent to which package inserts of medicines circulating on the markets of the East African Community (EAC) Partner States conform to medicines information requirements as established in the harmonized guidelines as well as national guidelines. A total of 99 package inserts from six (6) types of medicines namely Albendazole, Artemether/Lumefantrine (ALu), Ciprofloxacin, Paracetamol, Amoxicillin and Metronidazole were purposefully collected from three EAC Partner States: Kenya, Tanzania and Uganda. The medicines were selected based on their indications as first line treatments, high rates of utilization within the medicines supply system and their positions in treatment of diseases of public importance across EAC Partner States. The inserts were evaluated on the availability of information regarding fifteen (15) parameters as extracted from the EAC harmonized guidelines for registration of medicines. Moreover, comparisons were made between the percentage conformity of the branded versus generic products, markets from which the samples were collected, origin of the manufacturer and type of medicine. Majority (93.9-100%) of the medicines' package inserts highly conformed to the inclusion of the information regarding the description and composition of the medications, indications, dosage and methods of administration, warnings and precautions, contraindications and storage conditions. However, the information on handling and disposal, container package description, excipients used, clinical pharmacology of the medicines, and directions regarding overdose ranked the least in conformance with conformity ranging from 13.1-52.5%. The parameter with the lowest observed percentage conformity among the branded products scored 50% as compared to 10.8% among the generic products. Moreover, there was no significant difference (P<0.05) in the percentage conformity of the package inserts collected from each of the three Partner States as compared to the average from studied medicines. A generally good conformity was observed among medicines manufactured by European based manufacturers as compared to those based in Asia and EAC Partner States. In addition, PIs of Albendazole, Ciprofloxacin, Amoxicillin and Artemether/Lumefantrine did show overall high conformity across most of the product information requirements. Our study revealed the existence of a significant number of medicinal products circulating on the markets of EAC Partner States without necessary compliance with all product information requirements. We therefore recommend that NMRAs ensure thorough pre-market assessment of product information as well as strengthening their post marketing surveillance to ensure that medicines circulating on the market comply to medicines information requirements at all times. Emphasis should also be given to manufacturers on the importance of inclusion of appropriate and adequate product information for the safety of patients, including advocating for inclusion of patient-friendly and easy to understand medicines information.

Malaria rapid diagnostic kits: quality of packaging, design and labelling of boxes and components and readability and accuracy of information inserts.

PubMed

Gillet, Philippe; Maltha, Jessica; Hermans, Veerle; Ravinetto, Raffaella; Bruggeman, Cathrien; Jacobs, Jan

2011-02-13

The present study assessed malaria RDT kits for adequate and correct packaging, design and labelling of boxes and components. Information inserts were studied for readability and accuracy of information. Criteria for packaging, design, labelling and information were compiled from Directive 98/79 of the European Community (EC), relevant World Health Organization (WHO) documents and studies on end-users' performance of RDTs. Typography and readability level (Flesch-Kincaid grade level) were assessed. Forty-two RDT kits from 22 manufacturers were assessed, 35 of which had evidence of good manufacturing practice according to available information (i.e. CE-label affixed or inclusion in the WHO list of ISO13485:2003 certified manufacturers). Shortcomings in devices were (i) insufficient place for writing sample identification (n=40) and (ii) ambiguous labelling of the reading window (n=6). Buffer vial labels were lacking essential information (n=24) or were of poor quality (n=16). Information inserts had elevated readability levels (median Flesch Kincaid grade 8.9, range 7.1-12.9) and user-unfriendly typography (median font size 8, range 5-10). Inadequacies included (i) no referral to biosafety (n=18), (ii) critical differences between depicted and real devices (n=8), (iii) figures with unrealistic colours (n=4), (iv) incomplete information about RDT line interpretations (n=31) and no data on test characteristics (n=8). Other problems included (i) kit names that referred to Plasmodium vivax although targeting a pan-species Plasmodium antigen (n=4), (ii) not stating the identity of the pan-species antigen (n=2) and (iii) slight but numerous differences in names displayed on boxes, device packages and information inserts. Three CE labelled RDT kits produced outside the EC had no authorized representative affixed and the shape and relative dimensions of the CE symbol affixed did not comply with the Directive 98/79/EC. Overall, RDTs with evidence of GMP scored better compared to those without but inadequacies were observed in both groups. Overall, malaria RDTs showed shortcomings in quality of construction, design and labelling of boxes, device packages, devices and buffers. Information inserts were difficult to read and lacked relevant information.

The loss-of-allele assay for ES cell screening and mouse genotyping.

PubMed

Frendewey, David; Chernomorsky, Rostislav; Esau, Lakeisha; Om, Jinsop; Xue, Yingzi; Murphy, Andrew J; Yancopoulos, George D; Valenzuela, David M

2010-01-01

Targeting vectors used to create directed mutations in mouse embryonic stem (ES) cells consist, in their simplest form, of a gene for drug selection flanked by mouse genomic sequences, the so-called homology arms that promote site-directed homologous recombination between the vector and the target gene. The VelociGene method for the creation of targeted mutations in ES cells employs targeting vectors, called BACVecs, that are based on bacterial artificial chromosomes. Compared with conventional short targeting vectors, BacVecs provide two major advantages: (1) their much larger homology arms promote high targeting efficiencies without the need for isogenicity or negative selection strategies; and (2) they enable deletions and insertions of up to 100kb in a single targeting event, making possible gene-ablating definitive null alleles and other large-scale genomic modifications. Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus. To exploit the advantages of BACVecs for gene targeting, we inverted the conventional screening logic in developing the loss-of-allele (LOA) assay, which quantifies the number of copies of the native locus to which the mutation was directed. In a correctly targeted ES cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector. The LOA assay reveals a correctly targeted clone as having lost one copy of the native target gene and gained one copy of the drug resistance gene or other inserted marker. The combination of these quantitative assays makes LOA genotyping unequivocal and amenable to automated scoring. We use the quantitative polymerase chain reaction (qPCR) as our method of allele quantification, but any method that can reliably distinguish the difference between one and two copies of the target gene can be used to develop an LOA assay. We have designed qPCR LOA assays for deletions, insertions, point mutations, domain swaps, conditional, and humanized alleles and have used the insert assays to quantify the copy number of random insertion BAC transgenics. Because of its quantitative precision, specificity, and compatibility with high throughput robotic operations, the LOA assay eliminates bottlenecks in ES cell screening and mouse genotyping and facilitates maximal speed and throughput for knockout mouse production. Copyright (c) 2010 Elsevier Inc. All rights reserved.

48 CFR 52.247-65 - F.o.b. Origin, Prepaid Freight-Small Package Shipments.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 2 2010-10-01 2010-10-01 false F.o.b. Origin, Prepaid... Text of Provisions and Clauses 52.247-65 F.o.b. Origin, Prepaid Freight—Small Package Shipments. As prescribed in 47.303-17(f), insert the following clause: F.o.b. Origin Prepaid Freight—Small Package...

Packaging double-helical DNA into viral capsids.

PubMed

LaMarque, Jaclyn C; Le, Thuc-Vy L; Harvey, Stephen C

2004-02-15

DNA packaging in bacteriophage P4 has been examined using a molecular mechanics model with a reduced representation containing one pseudoatom per turn of the double helix. The model is a discretized version of an elastic continuum model. The DNA is inserted piecewise into the model capsid, with the structure being reoptimized after each piece is inserted. Various optimization protocols were investigated, and it was found that molecular dynamics at a very low temperature (0.3 K) produces the optimal packaged structure. This structure is a concentric spool, rather than the coaxial spool that has been commonly accepted for so many years. This geometry, which was originally suggested by Hall and Schellman in 1982 (Biopolymers Vol. 21, pp. 2011-2031), produces a lower overall elastic energy than coaxial spooling. Copyright 2003 Wiley Periodicals, Inc.

Investigating the Correlation Between Pharmacy Student Performance on the Health Science Reasoning Test and a Critical Thinking Assignment.

PubMed

Nornoo, Adwoa O; Jackson, Jonathan; Axtell, Samantha

2017-03-25

Objective. To determine whether there is a correlation between pharmacy students' scores on the Health Science Reasoning Test (HSRT) and their grade on a package insert assignment designed to assess critical thinking. Methods. The HSRT was administered to first-year pharmacy students during a critical-thinking course in the spring semester. In the same semester, a required package insert assignment was completed in a pharmacokinetics course. To determine whether there was a relationship between HSRT scores and grades on the assignment, a Spearman's rho correlation test was performed. Results. A very weak but significant positive correlation was found between students' grades on the assignment and their overall HSRT score (r=0.19, p <0.05), as well as deduction (a scale score of the HSRT; r=0.26, p <0.01). Conclusion. Based on a very weak but significant correlation to HSRT scores, this study demonstrated the potential of a package insert assignment to be used as one of the components to measure critical-thinking skills in pharmacy students.

Investigating the Correlation Between Pharmacy Student Performance on the Health Science Reasoning Test and a Critical Thinking Assignment

PubMed Central

Jackson, Jonathan; Axtell, Samantha

2017-01-01

Objective. To determine whether there is a correlation between pharmacy students’ scores on the Health Science Reasoning Test (HSRT) and their grade on a package insert assignment designed to assess critical thinking. Methods. The HSRT was administered to first-year pharmacy students during a critical-thinking course in the spring semester. In the same semester, a required package insert assignment was completed in a pharmacokinetics course. To determine whether there was a relationship between HSRT scores and grades on the assignment, a Spearman’s rho correlation test was performed. Results. A very weak but significant positive correlation was found between students’ grades on the assignment and their overall HSRT score (r=0.19, p<0.05), as well as deduction (a scale score of the HSRT; r=0.26, p<0.01). Conclusion. Based on a very weak but significant correlation to HSRT scores, this study demonstrated the potential of a package insert assignment to be used as one of the components to measure critical-thinking skills in pharmacy students. PMID:28381884

Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions

PubMed Central

Tanti, N.C.; Jones, L.; Sheardown, H.

2010-01-01

Purpose Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model. Methods An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2-hydroxyethylmethacrylate  (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis. Results Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of β1 and α3 integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed at 24 h. These latter results suggest that in vitro exposure to low concentration of borate/boric acid results in cell dysfunction, leading to necrosis rather than apoptosis. Conclusions Borate-buffered packaging solutions were shown to adversely affect the viability and integrin expression of HCECs in vitro. When used in ophthalmic packaging solutions, the antimicrobial properties of borate buffer may be outweighed by its relatively cytotoxic effects on cells. PMID:20169012

Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions.

PubMed

Gorbet, M B; Tanti, N C; Jones, L; Sheardown, H

2010-02-19

Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model. An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2-hydroxyethylmethacrylate (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis. Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of beta(1) and alpha(3) integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed at 24 h. These latter results suggest that in vitro exposure to low concentration of borate/boric acid results in cell dysfunction, leading to necrosis rather than apoptosis. Borate-buffered packaging solutions were shown to adversely affect the viability and integrin expression of HCECs in vitro. When used in ophthalmic packaging solutions, the antimicrobial properties of borate buffer may be outweighed by its relatively cytotoxic effects on cells.

Pilot Fullerton uses water dispenser kit gun to rehydrate food package

NASA Image and Video Library

1982-03-30

STS003-26-254 (30 March 1982) --- Astronaut Gordon Fullerton, STS-3 pilot, wearing communications kit assembly (ASSY) mini-headset (HDST), inserts the JSC water dispenser kit water gun in rehydratable plastic food (cereal) package to fill it with hot water. Photo credit: NASA

Health Instruction Packages: Specific Nursing Skills.

ERIC Educational Resources Information Center

Bates, Clarice; And Others

Text, illustrations, and exercises are utilized in a set of five learning modules designed to instruct nursing students in a variety of clinical skills. The first module, "Down the Tube: Insertion of a Nasogastric Tube" by Clarice Bates, describes materials and procedures used to insert a nasogastric tube through the nose and esophagus…

Dual Use of Packaging on the Moon: Logistics-2-Living

NASA Technical Reports Server (NTRS)

Howe, A. Scott; Howard, Robert

2010-01-01

This paper describes a modular packaging system for logistics that can be reconfigured into internal outfitting for a lunar outpost, including desks, chairs, partitions, cabinets, and radiation shielding. Logistics include clothes, equipment, food, and other consumables needed to sustain the crew for the duration of the mission. A significant mass penalty is required for the packaging and handling of logistics for re-supply of short to long-term space missions that must be brought out of the gravity well on a launch vehicle. Once the supplies have been exhausted, the packaging material is typically of no further use and is discarded. If a scheme can be developed that reuses the logistics packaging, the mass penalty can be reduced. In this research, a modular packaging system has been devised as a kit-of-parts that can be used for both handling logistics supplies, and then reconfigured into desks, chairs, partitions, cabinets, and radiation shielding. The system is derived from a standard International Space Station (ISS)-type Cargo Transfer Bag (CTB), using soft, unfoldable box-like containers with stiff metal inserts. The empty hydrogen-impregnated CTBs can be used as-is for cabinets, opened up for use as partitions, or draped over the habitat as layers of radiation shielding. Stiff metal inserts can be reconfigured into desks and other useful outfitting.

Reliability Technology to Achieve Insertion of Advanced Packaging (RELTECH) program

NASA Astrophysics Data System (ADS)

Fayette, Daniel F.; Speicher, Patricia; Stoklosa, Mark J.; Evans, Jillian V.; Evans, John W.; Gentile, Mike; Pagel, Chuck A.; Hakim, Edward

1993-08-01

A joint military-commercial effort to evaluate multichip module (MCM) structures is discussed. The program, Reliability Technology to Achieve Insertion of Advanced Packaging (RELTECH), has been designed to identify the failure mechanisms that are possible in MCM structures. The RELTECH test vehicles, technical assessment task, product evaluation plan, reliability modeling task, accelerated and environmental testing, and post-test physical analysis and failure analysis are described. The information obtained through RELTECH can be used to address standardization issues, through development of cost effective qualification and appropriate screening criteria, for inclusion into a commercial specification and the MIL-H-38534 general specification for hybrid microcircuits.

Current oral contraceptive use instructions: an analysis of patient package inserts.

PubMed

Williams-Deane, M; Potter, L S

1992-01-01

Oral contraceptive use instructions contained in manufacturers' patient package inserts (PPIs) are often inconsistent or conflicting, both among manufacturers and among different brands and regimens from the same manufacturer. Instructions on what to do about missed pills are often incomplete or inadequate, as are instructions on backup contraceptive use when pills are missed. The format of many PPIs is confusing and makes instructions difficult to find and read. Comprehending the PPIs requires the user to read at a 10th-12th-grade level, far higher than the generally accepted 5th-6th grade level considered standard for health education materials.

Reliability Technology to Achieve Insertion of Advanced Packaging (RELTECH) program

NASA Technical Reports Server (NTRS)

Fayette, Daniel F.; Speicher, Patricia; Stoklosa, Mark J.; Evans, Jillian V.; Evans, John W.; Gentile, Mike; Pagel, Chuck A.; Hakim, Edward

1993-01-01

A joint military-commercial effort to evaluate multichip module (MCM) structures is discussed. The program, Reliability Technology to Achieve Insertion of Advanced Packaging (RELTECH), has been designed to identify the failure mechanisms that are possible in MCM structures. The RELTECH test vehicles, technical assessment task, product evaluation plan, reliability modeling task, accelerated and environmental testing, and post-test physical analysis and failure analysis are described. The information obtained through RELTECH can be used to address standardization issues, through development of cost effective qualification and appropriate screening criteria, for inclusion into a commercial specification and the MIL-H-38534 general specification for hybrid microcircuits.

Malaria rapid diagnostic kits: quality of packaging, design and labelling of boxes and components and readability and accuracy of information inserts

PubMed Central

2011-01-01

Background The present study assessed malaria RDT kits for adequate and correct packaging, design and labelling of boxes and components. Information inserts were studied for readability and accuracy of information. Methods Criteria for packaging, design, labelling and information were compiled from Directive 98/79 of the European Community (EC), relevant World Health Organization (WHO) documents and studies on end-users' performance of RDTs. Typography and readability level (Flesch-Kincaid grade level) were assessed. Results Forty-two RDT kits from 22 manufacturers were assessed, 35 of which had evidence of good manufacturing practice according to available information (i.e. CE-label affixed or inclusion in the WHO list of ISO13485:2003 certified manufacturers). Shortcomings in devices were (i) insufficient place for writing sample identification (n = 40) and (ii) ambiguous labelling of the reading window (n = 6). Buffer vial labels were lacking essential information (n = 24) or were of poor quality (n = 16). Information inserts had elevated readability levels (median Flesch Kincaid grade 8.9, range 7.1 - 12.9) and user-unfriendly typography (median font size 8, range 5 - 10). Inadequacies included (i) no referral to biosafety (n = 18), (ii) critical differences between depicted and real devices (n = 8), (iii) figures with unrealistic colours (n = 4), (iv) incomplete information about RDT line interpretations (n = 31) and no data on test characteristics (n = 8). Other problems included (i) kit names that referred to Plasmodium vivax although targeting a pan-species Plasmodium antigen (n = 4), (ii) not stating the identity of the pan-species antigen (n = 2) and (iii) slight but numerous differences in names displayed on boxes, device packages and information inserts. Three CE labelled RDT kits produced outside the EC had no authorized representative affixed and the shape and relative dimensions of the CE symbol affixed did not comply with the Directive 98/79/EC. Overall, RDTs with evidence of GMP scored better compared to those without but inadequacies were observed in both groups. Conclusion Overall, malaria RDTs showed shortcomings in quality of construction, design and labelling of boxes, device packages, devices and buffers. Information inserts were difficult to read and lacked relevant information. PMID:21314992

Packaging of microwave integrated circuits operating beyond 100 GHz

NASA Technical Reports Server (NTRS)

Samoska, L.; Daniel, E.; Sokolov, V.; Sommerfeldt, S.; Bublitz, J.; Olson, K.; Gilbert, B.; Chow, D.

2002-01-01

Several methods of packaging high speed (75-330 GHz) InP HEMT MMIC devices are discussed. Coplanar wirebonding is presented with measured insertion loss of less than 0.5dB and return loss better than -17 dB from DC to 110 GHz. A motherboard/daughterboard packaging scheme is presented which supports minimum loss chains of MMICs using this coplanar wirebonding method. Split waveguide block packaging approaches are presented in G-band (140-220 GHz) with two types of MMIC-waveguide transitions: E-plane probe andantipodal finline.

Method of fabricating a microelectronic device package with an integral window

DOEpatents

Peterson, Kenneth A.; Watson, Robert D.

2003-01-01

A method of fabricating a microelectronic device package with an integral window for providing optical access through an aperture in the package. The package is made of a multilayered insulating material, e.g., a low-temperature cofired ceramic (LTCC) or high-temperature cofired ceramic (HTCC). The window is inserted in-between personalized layers of ceramic green tape during stackup and registration. Then, during baking and firing, the integral window is simultaneously bonded to the sintered ceramic layers of the densified package. Next, the microelectronic device is flip-chip bonded to cofired thick-film metallized traces on the package, where the light-sensitive side is optically accessible through the window. Finally, a cover lid is attached to the opposite side of the package. The result is a compact, low-profile package, flip-chip bonded, hermetically-sealed package having an integral window.

Testing Cessation Messages for Cigarette Package Inserts: Findings from a Best/Worst Discrete Choice Experiment

PubMed Central

Thrasher, James F.; Davis, Rachel E.; Popova, Lucy; Cho, Yoo Jin; Salloum, Ramzi G.; Louviere, Jordan; Hammond, David

2018-01-01

This study assessed smokers’ responses to different smoking cessation topics and imagery for cigarette package inserts. Adult smokers from Canada (n = 1000) participated in three discrete choice experiments (DCEs): DCE 1 assessed five cessation benefit topics and five imagery types; DCE 2 assessed five messages with tips to improve cessation success and five imagery types; DCE 3 assessed four reproductive health benefits of cessation topics and four imagery types. In each DCE, participants evaluated four or five sets of four inserts, selecting the most and least motivating (DCEs 1 & 3) or helpful (DCE 2) for quitting. Linear mixed models regressed choices on insert and smoker characteristics. For DCE 1, the most motivating messages involved novel disease topics and imagery of younger women. For DCE 2, topics of social support, stress reduction and nicotine replacement therapy were selected as most helpful, with no differences by imagery type. For DCE 3, imagery influenced choices more than topic, with imagery of a family or a mom and baby selected as most motivating. Statistically significant interactions for all three experiments indicated that the influence of imagery type on choices depended on the message topic. Messages to promote smoking cessation through cigarette pack inserts should consider specific combinations of message topic and imagery. PMID:29415523

A Review of Patents for the Smart Packaging of Meat and Muscle-based Food Products.

PubMed

Holman, Benjamin; Kerry, Joseph P; Hopkins, David L

2017-10-31

Meat packaging once acted primarily as an inert barrier to protect its contents against contamination and this function has shifted. Packaging now includes complementary functions that improve product quality, longevity and customer/retail appeal. The devices and methods applied to achieve these functions may be categorised as smart packaging, which includes intelligent packaging, devised to monitor and communicate packaged content status, and active packaging, to provide passive adjustment of in-pack conditions from its interactions with the packaged meat. Smart packaging examples already available from recent patents include antimicrobial and antioxidant packaging coatings and inserts; sensors or indicators that identify spoilage and freshness; functional engineering customisations; improvements to packaging integrity; leak or tamper detectors; and, environmentally sustainable options. Together, these inventions respond to industry and customer demands for meat packaging and are therefore the focus of this review, in which we discuss their applications and limitations in meat packaging. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

"rsed" is an R package that contains tools for stream editing: manipulating text files by making insertions, replacements, deletions, substitutions, or commenting. It hails from the powerful Unix command, "sed". While the "rsed" package is not nearly as powerful as "see", it is much simpler to use. R programmers often write scripts that may require simple manipulation of text files. "rsed" addresses that need.

Microstructure-Evolution and Reliability Assessment Tool for Lead-Free Component Insertion in Army Electronics

DTIC Science & Technology

2008-10-01

provide adequate means for thermal heat dissipation and cooling. Thus electronic packaging has four main functions [1]: • Signal distribution which... dissipation , involving structural and materials consideration. • Mechanical, chemical and electromagnetic protection of components and... nature when compared to phenomenological models. Microelectronic packaging industry spends typically several months building and reliability

A Novel Assay for the Identification of NOTCH1 PEST Domain Mutations in Chronic Lymphocytic Leukemia

PubMed Central

Petroni, Roberta Cardoso; Muto, Nair Hideko; Sitnik, Roberta; de Carvalho, Flavia Pereira; Bacal, Nydia Strachman; Velloso, Elvira Deolinda Rodrigues Pereira; Oliveira, Gislaine Borba; Pinho, João Renato Rebello; Torres, Davi Coe; Mansur, Marcela Braga; Hassan, Rocio; Lorand-Metze, Irene Gyongyvér Heidemarie; Chiattone, Carlos Sérgio; Hamerschlak, Nelson; Mangueira, Cristovão Luis Pitangueira

2016-01-01

Aims. To develop a fast and robust DNA-based assay to detect insertions and deletions mutations in exon 34 that encodes the PEST domain of NOTCH1 in order to evaluate patients with chronic lymphocytic leukemia (CLL). Methods. We designed a multiplexed allele-specific polymerase chain reaction (PCR) combined with a fragment analysis assay to detect specifically the mutation c.7544_7545delCT and possibly other insertions and deletions in exon 34 of NOTCH1. Results. We evaluated our assay in peripheral blood samples from two cohorts of patients with CLL. The frequency of NOTCH1 mutations was 8.4% in the first cohort of 71 unselected CLL patients. We then evaluated a second cohort of 26 CLL patients with known cytogenetic abnormalities that were enriched for patients with trisomy 12. NOTCH1 mutations were detected in 43.7% of the patients with trisomy 12. Conclusions. We have developed a fast and robust assay combining allele-specific PCR and fragment analysis able to detect NOTCH1 PEST domain insertions and deletions. PMID:28074183

Do package inserts reflect symptoms experienced in practice?: assessment using an automated phone pharmacovigilance system with varenicline and zolpidem in a primary care setting.

PubMed

Haas, Jennifer S; Amato, Mary; Marinacci, Lucas; Orav, E John; Schiff, Gordon D; Bates, David W

2012-08-01

While the US FDA maintains a voluntary reporting system, postmarketing adverse drug events (ADEs) are underreported, and this case report-based system does not allow accurate determination of incidence. The aim of the study was to assess the usefulness of an automated phone pharmacovigilance system for ambulatory patients by comparing systematically collected, patient-reported symptoms to reflect possible ADEs with those reported on the package inserts of two drugs with postmarketing safety concerns, varenicline and zolpidem. English-speaking adults who received a prescription for zolpidem (n = 370) or varenicline (n = 107) from a primary care physician at one of 11 participating clinics, and who participated in the pharmacovigilance system during 2008-2010, were included in the study. Patients were called approximately 4 weeks following their visit and asked to complete a standard script that asked about adherence and pre-specified symptoms. The main outcome measures were elicited rates of pre-specified symptoms or possible ADEs. Compared with the package insert, patients taking zolpidem were significantly (p < 0.001) more likely to report fatigue (9.0% vs 1.0%), itching (4.5% vs 1.0%) and muscle aches (5.6% vs 1.0%). Elicited rates of depression and hallucination were similar to those reported in the package insert. Patients taking varenicline were significantly more likely to report confusion (1.7% vs 0.1%), depression (3.4% vs 0.1%), fatigue (6.0% vs 1.0%), hallucinations (1.7% vs 0.1%), muscle aches (6.0% vs 1.0%) and sexual dysfunction (4.3% vs 0.1%). Automated phone pharmacovigilance can provide estimates of possible ADEs in clinical practice. In the case of varenicline, these data support some of the safety concerns that have come to light postmarketing, while others such as depression and hallucination related to zolpidem were not detected. These data highlight the potential value of, and innovative ways of collecting, information about possible ADEs directly from patients.

Technical Note: Computer-Manufactured Inserts for Prosthetic Sockets

PubMed Central

Sanders, Joan E.; McLean, Jake B.; Cagle, John C.; Gardner, David W.; Allyn, Katheryn J.

2016-01-01

The objective of this research was to use computer-aided design software and a tabletop 3-D additive manufacturing system to design and fabricate custom plastic inserts for trans-tibial prosthesis users. Shape quality of inserts was tested right after they were inserted into participant’s test sockets and again after four weeks of wear. Inserts remained properly positioned and intact throughout testing. Right after insertion the inserts caused the socket to be slightly under-sized, by a mean of 0.11 mm, approximately 55% of the thickness of a nylon sheath. After four weeks of wear the under-sizing was less, averaging 0.03 mm, approximately 15% of the thickness of a nylon sheath. Thus the inserts settled into the sockets over time. If existing prosthetic design software packages were enhanced to conduct insert design and to automatically generate fabrication files for manufacturing, then computer manufactured inserts may offer advantages over traditional methods in terms of speed of fabrication, ease of design, modification, and record keeping. PMID:27212209

Membrane insertion for the detection of lipopolysaccharides: Exploring the dynamics of amphiphile-in-lipid assays

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

Stromberg, Loreen R.; Hengartner, Nicolas W.; Swingle, Kirstie L.

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assaysmore » to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. In addition, we also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures.« less

Membrane insertion for the detection of lipopolysaccharides: Exploring the dynamics of amphiphile-in-lipid assays

DOE PAGES

Stromberg, Loreen R.; Hengartner, Nicolas W.; Swingle, Kirstie L.; ...

2016-05-26

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assaysmore » to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. In addition, we also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures.« less

Reading about over-the-counter medications.

PubMed

Nabors, Laura A; Lehmkuhl, Heather D; Parkins, Irina S; Drury, Anna M

2004-01-01

Many adolescents and young adults purchase and use over-the-counter (OTC) medications, and some may take these medications without reading about how to use them. Most do read package inserts and labels to learn about the medication, but studies examining what influences label reading for youth are needed. This study assessed factors related to label reading for young people, including demographic variables (gender, health status) and the types of information they were seeking about the medication. Eight hundred and seventy-six high school and college students participated, and most reported reading labels or package inserts to learn about medications. Participants experiencing pain were more likely to read labels, except for those experiencing headaches who reported being less likely to read labels. When reading labels, participants were interested in information about side effects, ingredients, dosage instructions, and symptoms treated by the medication. Future research should examine whether youth take medications as directed and what factors make labels and inserts easier to read and understand.

Jig Aligns Shadow Mask On CCD

NASA Technical Reports Server (NTRS)

Matus, Carlos V.

1989-01-01

Alignment viewed through microscope. Alignment jig positions shadow mask on charge-coupled device (CCD) so metal film deposited on it precisely. Allows CCD package to be inserted and removed without disturbing alignment of mask. Holds CCD packages securely and isolates it electrostatically while providing electrical contact to each of its pins. When alignment jig assembled with CCD, used to move mask under micrometer control.

Development of a functional cell-based assay that probes the specific interaction between influenza A virus NP and its packaging signal sequence RNA.

PubMed

Woo, Jiwon; Yu, Kyung Lee; Lee, Sun Hee; You, Ji Chang

2015-02-06

Although cis-acting packaging signal RNA sequences for the influenza virus NP encoding vRNA have been identified recently though genetic studies, little is known about the interaction between NP and the vRNA packaging signals either in vivo or in vitro. Here, we provide evidence that NP is able to interact specifically with the vRNA packaging sequence RNA within living cells and that the specific RNA binding activity of NP in vivo requires both the N-terminal and central region of the protein. This assay established would be a valuable tool for further detailed studies of the NP-packaging signal RNA interaction in living cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Using PATIMDB to Create Bacterial Transposon Insertion Mutant Libraries

PubMed Central

Urbach, Jonathan M.; Wei, Tao; Liberati, Nicole; Grenfell-Lee, Daniel; Villanueva, Jacinto; Wu, Gang; Ausubel, Frederick M.

2015-01-01

PATIMDB is a software package for facilitating the generation of transposon mutant insertion libraries. The software has two main functions: process tracking and automated sequence analysis. The process tracking function specifically includes recording the status and fates of multiwell plates and samples in various stages of library construction. Automated sequence analysis refers specifically to the pipeline of sequence analysis starting with ABI files from a sequencing facility and ending with insertion location identifications. The protocols in this unit describe installation and use of PATIMDB software. PMID:19343706

A Pre- and Post-Evaluation of Integrating Sustainability Curriculum by Inserting Okala Modules into an Interior Design Materials and Methods Course

ERIC Educational Resources Information Center

Schneiderman, Deborah; Freihoefer, Kara

2012-01-01

Purpose: The purpose of this paper is to examine the integration of Okala curriculum into Interior Design coursework. Okala, as a teaching package, is utilized extensively in industrial design education. However, this study examines the expansion and insertion of Okala modules in an existing interior design curriculum. The Okala modules included…

Mental models in risk assessment: informing people about drugs.

PubMed

Jungermann, H; Schütz, H; Thüring, M

1988-03-01

One way to communicate about the risks of drugs is through the use of package inserts. The problems associated with this medium of informing patients have been investigated by several researchers who found that people require information about drugs they are using, including extensive risk information, and that they are willing to take this information into account in their usage of drugs. But empirical results also show that people easily misinterpret the information given. A conceptual framework is proposed that might be used for better understanding the cognitive processes involved in such a type of risk assessment and communication. It is based on the idea that people develop, through experience, a mental model of how a drug works, which effects it might produce, that contraindications have to be considered, etc. This mental model is "run" when a specific package insert has been read and a specific question arises such as, for example, whether certain symptoms can be explained as normal or whether they require special attention and action. We argue that the mental model approach offers a useful perspective for examining how people understand package inserts, and consequently for improving their content and design. The approach promises to be equally useful for other aspects of risk analysis that are dependent upon human judgment and decision making, e.g., threat diagnosis and human reliability analysis.

Technical note: Computer-manufactured inserts for prosthetic sockets.

PubMed

Sanders, Joan E; McLean, Jake B; Cagle, John C; Gardner, David W; Allyn, Katheryn J

2016-08-01

The objective of this research was to use computer-aided design software and a tabletop 3-D additive manufacturing system to design and fabricate custom plastic inserts for trans-tibial prosthesis users. Shape quality of inserts was tested right after they were inserted into participant's test sockets and again after four weeks of wear. Inserts remained properly positioned and intact throughout testing. Right after insertion the inserts caused the socket to be slightly under-sized, by a mean of 0.11mm, approximately 55% of the thickness of a nylon sheath. After four weeks of wear the under-sizing was less, averaging 0.03mm, approximately 15% of the thickness of a nylon sheath. Thus the inserts settled into the sockets over time. If existing prosthetic design software packages were enhanced to conduct insert design and to automatically generate fabrication files for manufacturing, then computer manufactured inserts may offer advantages over traditional methods in terms of speed of fabrication, ease of design, modification, and record keeping. Copyright © 2016 IPEM. Published by Elsevier Ltd. All rights reserved.

Young adult smokers' perceptions of plain packs, numbered packs and pack inserts in Turkey: a focus group study.

PubMed

Mucan, Burcu; Moodie, Crawford

2017-11-09

The Turkish Government's 'National Tobacco Control Program 2015-2018' included plans to introduce plain packaging and also a ban on brand names on cigarette packs, allowing only assigned numbers on packs. We explored perceptions of these proposed measures, and also pack inserts with cessation messages, another novel way of using the packaging to communicate with consumers. Eight focus groups were conducted with 47 young adult smokers in Manisa and Kutahya (Turkey) in December 2016. Participants were shown three straight-edged plain cigarette packs, as required in Australia, and then three bevelled-edged plain packs, as permitted in the UK. They were then shown plain packs with numbers rather than brand names, and finally three pack inserts with messages encouraging quitting or offering tips on how to do so. Participants were asked about their perceptions of each. Plain packs were considered unappealing and off-putting, although the bevelled-edged packs were viewed more favourably than the straight-edged packs. Numbered packs were thought by some to diminish the appeal created by the brand name and potentially decrease interest among never smokers and newer smokers. Pack inserts were thought to have less of an impact than the on-pack warnings, but could potentially help discourage initiation and encourage cessation. That bevelled-edged plain packs were perceived more positively than straight-edged plain packs is relevant to countries planning to introduce plain packaging. The study provides a first insight into smokers' perceptions of a ban on brand names, which was perceived to reduce appeal among young people. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Evaluation of strategies to communicate harmful and potentially harmful constituent (HPHC) information through cigarette package inserts: a discrete choice experiment.

PubMed

Salloum, Ramzi G; Louviere, Jordan J; Getz, Kayla R; Islam, Farahnaz; Anshari, Dien; Cho, Yoojin; O'Connor, Richard J; Hammond, David; Thrasher, James F

2017-07-13

The US Food and Drug Administration (FDA) has regulatory authority to use inserts to communicate with consumers about harmful and potentially harmful constituents (HPHCs) in tobacco products; however, little is known about the most effective manner for presenting HPHC information. In a discrete choice experiment, participants evaluated eight choice sets, each of which showed two cigarette packages from four different brands and tar levels (high vs low), accompanied by an insert that included between-subject manipulations (ie, listing of HPHCs vs grouping by disease outcome and numeric values ascribed to HPHCs vs no numbers) and within-subject manipulations (ie, 1 of 4 warning topics; statement linking an HPHC with disease vs statement with no HPHC link). For each choice set, participants were asked: (1) which package is more harmful and (2) which motivates them to not smoke; each with a 'no difference' option. Alternative-specific logit models regressed choice on attribute levels. 1212 participants were recruited from an online consumer panel (725 18-29-year-old smokers and susceptible non-smokers and 487 30-64-year-old smokers). Participants were more likely to endorse high-tar products as more harmful than low-tar products, with a greater effect when numeric HPHC information was present. Compared with a simple warning statement, the statement linking HPHCs with disease encouraged quit motivation. Numeric HPHC information on inserts appears to produce misunderstandings that some cigarettes are less harmful than others. Furthermore, brief narratives that link HPHCs to smoking-related disease may promote cessation versus communications that do not explicitly link HPHCs to disease. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Copy number determination of genetically-modified hematopoietic stem cells.

PubMed

Schuesler, Todd; Reeves, Lilith; Kalle, Christof von; Grassman, Elke

2009-01-01

Human gene transfer with gammaretroviral, murine leukemia virus (MLV) based vectors has been shown to effectively insert and express transgene sequences at a level of therapeutic benefit. However, there are numerous reports of disruption of the normal cellular processes caused by the viral insertion, even of replication deficient gammaretroviral vectors. Current gammaretroviral and lentiviral vectors do not control the site of insertion into the genome, hence, the possibility of disruption of the target cell genome. Risk related to viral insertions is linked to the number of insertions of the transgene into the cellular DNA, as has been demonstrated for replication competent and replication deficient retroviruses in experiments. At high number of insertions per cell, cell transformation due to vector induced activation of proto-oncogenes is more likely to occur, in particular since more than one transforming event is needed for oncogenesis. Thus, determination of the vector copy number in bulk transduced populations, individual colony forming units, and tissue from the recipient of the transduced cells is an increasingly important safety assay and has become a standard, though not straightforward assay, since the inception of quantitative PCR.

Developing targets for radiation transport experiments at the Omega laser facility

DOE PAGES

Capelli, Deanna; Charsley-Groffman, C. A.; Randolph, Randall Blaine; ...

2017-07-13

Targets have been developed to measure supersonic radiation transport in aerogel foams using absorption spectroscopy. The target consists of an aerogel foam uniformly doped with either titanium or scandium inserted into an undoped aerogel foam package. This creates a localized doped foam region to provide spatial resolution for the measurement. Development and characterization of the foams is a key challenge in addition to machining and assembling the two foams so they mate without gaps. The foam package is inserted into a beryllium sleeve and mounted on a gold hohlraum. The target is mounted to a holder created using additive manufacturingmore » and mounted on a stalk. As a result, the manufacturing of the components, along with assembly and metrology of the target are described here.« less

Developing targets for radiation transport experiments at the Omega laser facility

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

Capelli, Deanna; Charsley-Groffman, C. A.; Randolph, Randall Blaine

Targets have been developed to measure supersonic radiation transport in aerogel foams using absorption spectroscopy. The target consists of an aerogel foam uniformly doped with either titanium or scandium inserted into an undoped aerogel foam package. This creates a localized doped foam region to provide spatial resolution for the measurement. Development and characterization of the foams is a key challenge in addition to machining and assembling the two foams so they mate without gaps. The foam package is inserted into a beryllium sleeve and mounted on a gold hohlraum. The target is mounted to a holder created using additive manufacturingmore » and mounted on a stalk. As a result, the manufacturing of the components, along with assembly and metrology of the target are described here.« less

Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

PubMed Central

Johnson, Reed F.; McCarthy, Sarah E.; Godlewski, Peter J.; Harty, Ronald N.

2006-01-01

The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions. PMID:16698994

Pilot Fullerton prepares meal on middeck

NASA Image and Video Library

1982-03-30

STS003-26-253 (30 March 1982) --- Astronaut Gordon Fullerton, STS-3 pilot, wearing communications kit assembly (assy) mini-headset (HDST), prepares meal on middeck. Fullerton clips corner of rehydratable food (cereal) package with scissors. The opening will allow Fullerton to insert JSC water dispenser kit water gun in order to heat contents with hot water. Meal tray assembly is secured to forward middeck locker and holds additional food packages and beverage containers. Photo credit: NASA

European multicenter analytical evaluation of the Abbott ARCHITECT STAT high sensitive troponin I immunoassay.

PubMed

Krintus, Magdalena; Kozinski, Marek; Boudry, Pascal; Capell, Nuria Estañ; Köller, Ursula; Lackner, Karl; Lefèvre, Guillaume; Lennartz, Lieselotte; Lotz, Johannes; Herranz, Antonio Mora; Nybo, Mads; Plebani, Mario; Sandberg, Maria B; Schratzberger, Wolfgang; Shih, Jessie; Skadberg, Øyvind; Chargui, Ahmed Taoufik; Zaninotto, Martina; Sypniewska, Grazyna

2014-11-01

International recommendations highlight the superior value of cardiac troponins (cTns) for early diagnosis of myocardial infarction along with analytical requirements of improved precision and detectability. In this multicenter study, we investigated the analytical performance of a new high sensitive cardiac troponin I (hs-cTnI) assay and its 99th percentile upper reference limit (URL). Laboratories from nine European countries evaluated the ARCHITECT STAT high sensitive troponin I (hs-TnI) immunoassay on the ARCHITECT i2000SR/i1000SR immunoanalyzers. Imprecision, limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ) linearity of dilution, interferences, sample type, method comparisons, and 99th percentile URLs were evaluated in this study. Total imprecision of 3.3%-8.9%, 2.0%-3.5% and 1.5%-5.2% was determined for the low, medium and high controls, respectively. The lowest cTnI concentration corresponding to a total CV of 10% was 5.6 ng/L. Common interferences, sample dilution and carryover did not affect the hs-cTnI results. Slight, but statistically significant, differences with sample type were found. Concordance between the investigated hs-cTnI assay and contemporary cTnI assay at 99th percentile cut-off was found to be 95%. TnI was detectable in 75% and 57% of the apparently healthy population using the lower (1.1 ng/L) and upper (1.9 ng/L) limit of the LoD range provided by the ARCHITECT STAT hs-TnI package insert, respectively. The 99th percentile values were gender dependent. The new ARCHITECT STAT hs-TnI assay with improved analytical features meets the criteria of high sensitive Tn test and will be a valuable diagnostic tool.

Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

PubMed

Niescierowicz, Katarzyna; Caro, Lydia; Cherezov, Vadim; Vivaudou, Michel; Moreau, Christophe J

2014-01-07

Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that ion channel-coupled receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain. Copyright © 2014 Elsevier Ltd. All rights reserved.

21 CFR 864.7455 - Fetal hemoglobin assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Fetal hemoglobin assay. 864.7455 Section 864.7455...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7455 Fetal hemoglobin assay. (a) Identification. A fetal hemoglobin assay is a device that is used to determine the presence...

21 CFR 864.7455 - Fetal hemoglobin assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Fetal hemoglobin assay. 864.7455 Section 864.7455...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7455 Fetal hemoglobin assay. (a) Identification. A fetal hemoglobin assay is a device that is used to determine the presence...

Application of Au-Sn eutectic bonding in hermetic radio-frequency microelectromechanical system wafer level packaging

NASA Astrophysics Data System (ADS)

Wang, Qian; Choa, Sung-Hoon; Kim, Woonbae; Hwang, Junsik; Ham, Sukjin; Moon, Changyoul

2006-03-01

Development of packaging is one of the critical issues toward realizing commercialization of radio-frequency-microelectromechanical system (RF-MEMS) devices. The RF-MEMS package should be designed to have small size, hermetic protection, good RF performance, and high reliability. In addition, packaging should be conducted at sufficiently low temperature. In this paper, a low-temperature hermetic wafer level packaging scheme for the RF-MEMS devices is presented. For hermetic sealing, Au-Sn eutectic bonding technology at temperatures below 300°C is used. Au-Sn multilayer metallization with a square loop of 70 µm in width is performed. The electrical feed-through is achieved by the vertical through-hole via filling with electroplated Cu. The size of the MEMS package is 1 mm × 1 mm × 700 µm. The shear strength and hermeticity of the package satisfies the requirements of MIL-STD-883F. Any organic gases or contamination are not observed inside the package. The total insertion loss for the packaging is 0.075 dB at 2 GHz. Furthermore, the robustness of the package is demonstrated by observing no performance degradation and physical damage of the package after several reliability tests.

Development of Multifunctional Active Film and Its Application in Modified Atmosphere Packaging of Shiitake Mushrooms.

PubMed

Wang, Hong Jiang; An, Duck Soon; Lee, Dong Sun

2016-09-01

Agar-based films with multiple functions (CO 2 absorption, water vapor absorption, and antimicrobial activity) were developed, tested for their properties, and then applied to the packaging of fresh shiitake mushrooms as an insert label. The films were cast from an agar-based aqueous solution containing a dissolving plasticizer (glycerol), a CO 2 absorbent (sodium carbonate [SC] alone or a combination of SC and sodium glycinate [SC-SG]), and a volatile antimicrobial agent (carvacrol [CRV]). The agar of the film matrix is designed to serve as a water vapor absorbent. The multifunctional films tended to have poor mechanical properties, with a hard texture and an opaque and yellowish color. The CO 2 absorbent, either SC alone or SC-SG, affected CRV retention and release along with the CO 2 and water vapor absorption behavior. Both films (SC-CRV and SC-SG-CRV films) showed good inhibitory effects against Pseudomonas fluorescens and Saccharomyces cerevisiae . SC-CRV film had a higher and faster CO 2 absorption property, higher retention and extended release of CRV, and lower and slower water vapor absorption and was assessed to be better suited for use in shiitake mushroom packaging. The packaging in which the SC-CRV film with an appropriate amount of CRV was used as an insert label was able to generate the desired atmosphere and less moisture condensation inside the package, producing the best preservation of quality in terms of mushroom color, firmness, flavor score, and microbial counts after 6 days of storage at 10°C. A tailored modified atmosphere packaging system using multifunctional film would be useful in the preservation of CO 2 -sensitive fresh commodities.

Single Pore Translocation of Folded, Double-Stranded, and Tetra-stranded DNA through Channel of Bacteriophage Phi29 DNA Packaging Motor

PubMed Central

Haque, Farzin; Wang, Shaoying; Stites, Chris; Chen, Li; Wang, Chi; Guo, Peixuan

2015-01-01

The elegant architecture of the channel of bacteriophage phi29 DNA packaging motor has inspired the development of biomimetics for biophysical and nanobiomedical applications. The reengineered channel inserted into a lipid membrane exhibits robust electrophysiological properties ideal for precise sensing and fingerprinting of dsDNA at the single-molecule level. Herein, we used single channel conduction assays to quantitatively evaluate the translocation dynamics of dsDNA as a function of the length and conformation of dsDNA. We extracted the speed of dsDNA translocation from the dwell time distribution and estimated the various forces involved in the translocation process. A ~35-fold slower speed of translocation per base pair was observed for long dsDNA, a significant contrast to the speed of dsDNA crossing synthetic pores. It was found that the channel could translocate both dsDNA with ~32% of channel current blockage and ~64% for tetra-stranded DNA (two parallel dsDNA). The calculation of both cross-sectional areas of the dsDNA and tetra-stranded DNA suggested that the blockage was purely proportional to the physical space of the channel lumen and the size of the DNA substrate. Folded dsDNA configuration was clearly reflected in their characteristic current signatures. The finding of translocation of tetra-stranded DNA with 64% blockage is in consent with the recently elucidated mechanism of viral DNA packaging via a revolution mode that requires a channel larger than the dsDNA diameter of 2 nm to provide room for viral DNA revolving without rotation. The understanding of the dynamics of dsDNA translocation in the phi29 system will enable us to design more sophisticated single pore DNA translocation devices for future applications in nanotechnology and personal medicine. PMID:25890769

9 CFR 381.123 - Official inspection mark; official establishment number.

Code of Federal Regulations, 2011 CFR

2011-01-01

... label of a canned product, or on other packaging or labeling in the container, e.g., on aluminum pans... insert label placed under a transparent covering if clearly visible and legible and accompanied by the...

9 CFR 381.123 - Official inspection mark; official establishment number.

Code of Federal Regulations, 2010 CFR

2010-01-01

... label of a canned product, or on other packaging or labeling in the container, e.g., on aluminum pans... insert label placed under a transparent covering if clearly visible and legible and accompanied by the...

Microbial viability in preparations packaged for single use.

PubMed

Obayashi, Akiko; Oie, Shigeharu; Kamiya, Akira

2003-05-01

We evaluated microbial viability in preparations packaged for single use only which mandate that residual solution be discarded such as albumin and globulin preparations as blood products, preparations containing albumin (such as urokinase and interferon), fat emulsions, and a preparation containing fat emulsions (propofol). In most preparations, Serratia marcescens and Burkholderia cepacia proliferated rapidly at 30 degrees C. However, in globulin preparations containing 1-2.25% glycine to prevent protein degradation (Gamma-Venin P, Venilon-I, Globulin Injection, and Ahlbulin), no growth of S. marcescens and B. cepacia was detected over 24 h at 30 degrees C. For globulin preparations containing 1-2.25% glycine, the injunction to "Discard residual solution after the package has been used" in the package inserts can be revised to "It is possible to use residual solution within 24 h after the package has been used with storage in a cool place."

QuantWorm: a comprehensive software package for Caenorhabditis elegans phenotypic assays.

PubMed

Jung, Sang-Kyu; Aleman-Meza, Boanerges; Riepe, Celeste; Zhong, Weiwei

2014-01-01

Phenotypic assays are crucial in genetics; however, traditional methods that rely on human observation are unsuitable for quantitative, large-scale experiments. Furthermore, there is an increasing need for comprehensive analyses of multiple phenotypes to provide multidimensional information. Here we developed an automated, high-throughput computer imaging system for quantifying multiple Caenorhabditis elegans phenotypes. Our imaging system is composed of a microscope equipped with a digital camera and a motorized stage connected to a computer running the QuantWorm software package. Currently, the software package contains one data acquisition module and four image analysis programs: WormLifespan, WormLocomotion, WormLength, and WormEgg. The data acquisition module collects images and videos. The WormLifespan software counts the number of moving worms by using two time-lapse images; the WormLocomotion software computes the velocity of moving worms; the WormLength software measures worm body size; and the WormEgg software counts the number of eggs. To evaluate the performance of our software, we compared the results of our software with manual measurements. We then demonstrated the application of the QuantWorm software in a drug assay and a genetic assay. Overall, the QuantWorm software provided accurate measurements at a high speed. Software source code, executable programs, and sample images are available at www.quantworm.org. Our software package has several advantages over current imaging systems for C. elegans. It is an all-in-one package for quantifying multiple phenotypes. The QuantWorm software is written in Java and its source code is freely available, so it does not require use of commercial software or libraries. It can be run on multiple platforms and easily customized to cope with new methods and requirements.

Method of forming a package for MEMS-based fuel cell

DOEpatents

Morse, Jeffrey D; Jankowski, Alan F

2013-05-21

A MEMS-based fuel cell package and method thereof is disclosed. The fuel cell package comprises seven layers: (1) a sub-package fuel reservoir interface layer, (2) an anode manifold support layer, (3) a fuel/anode manifold and resistive heater layer, (4) a Thick Film Microporous Flow Host Structure layer containing a fuel cell, (5) an air manifold layer, (6) a cathode manifold support structure layer, and (7) a cap. Fuel cell packages with more than one fuel cell are formed by positioning stacks of these layers in series and/or parallel. The fuel cell package materials such as a molded plastic or a ceramic green tape material can be patterned, aligned and stacked to form three dimensional microfluidic channels that provide electrical feedthroughs from various layers which are bonded together and mechanically support a MEMS-based miniature fuel cell. The package incorporates resistive heating elements to control the temperature of the fuel cell stack. The package is fired to form a bond between the layers and one or more microporous flow host structures containing fuel cells are inserted within the Thick Film Microporous Flow Host Structure layer of the package.

Method of forming a package for mems-based fuel cell

DOEpatents

Morse, Jeffrey D.; Jankowski, Alan F.

2004-11-23

A MEMS-based fuel cell package and method thereof is disclosed. The fuel cell package comprises seven layers: (1) a sub-package fuel reservoir interface layer, (2) an anode manifold support layer, (3) a fuel/anode manifold and resistive heater layer, (4) a Thick Film Microporous Flow Host Structure layer containing a fuel cell, (5) an air manifold layer, (6) a cathode manifold support structure layer, and (7) a cap. Fuel cell packages with more than one fuel cell are formed by positioning stacks of these layers in series and/or parallel. The fuel cell package materials such as a molded plastic or a ceramic green tape material can be patterned, aligned and stacked to form three dimensional microfluidic channels that provide electrical feedthroughs from various layers which are bonded together and mechanically support a MEMOS-based miniature fuel cell. The package incorporates resistive heating elements to control the temperature of the fuel cell stack. The package is fired to form a bond between the layers and one or more microporous flow host structures containing fuel cells are inserted within the Thick Film Microporous Flow Host Structure layer of the package.

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in...

21 CFR 864.7400 - Hemoglobin A2 assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Hemoglobin A2 assay. 864.7400 Section 864.7400...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7400 Hemoglobin A2 assay. (a) Identification. A hemoglobin A2 assay is a device used to determine the hemoglobin A2 content...

21 CFR 864.7400 - Hemoglobin A2 assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Hemoglobin A2 assay. 864.7400 Section 864.7400...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7400 Hemoglobin A2 assay. (a) Identification. A hemoglobin A2 assay is a device used to determine the hemoglobin A2 content...

Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

PubMed

Coleman, John W; Wright, Kevin J; Wallace, Olivia L; Sharma, Palka; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Zamb, Timothy P; Zhang, Xinsheng; Parks, Christopher L

2015-03-01

Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert. Copyright © 2014 Elsevier B.V. All rights reserved.

rSalvador: An R Package for the Fluctuation Experiment

PubMed Central

Zheng, Qi

2017-01-01

The past few years have seen a surge of novel applications of the Luria-Delbrück fluctuation assay protocol in bacterial research. Appropriate analysis of fluctuation assay data often requires computational methods that are unavailable in the popular web tool FALCOR. This paper introduces an R package named rSalvador to bring improvements to the field. The paper focuses on rSalvador’s capabilities to alleviate three kinds of problems found in recent investigations: (i) resorting to partial plating without properly accounting for the effects of partial plating; (ii) conducting attendant fitness assays without incorporating mutants’ relative fitness in subsequent data analysis; and (iii) comparing mutation rates using methods that are in general inapplicable to fluctuation assay data. In addition, the paper touches on rSalvador’s capabilities to estimate sample size and the difficulties related to parameter nonidentifiability. PMID:29084818

21 CFR 864.7525 - Heparin assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

21 CFR 864.7525 - Heparin assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

21 CFR 864.7525 - Heparin assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

21 CFR 864.7525 - Heparin assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

21 CFR 864.7525 - Heparin assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

SINE Retrotransposition: Evaluation of Alu Activity and Recovery of De Novo Inserts.

PubMed

Ade, Catherine; Roy-Engel, Astrid M

2016-01-01

Mobile element activity is of great interest due to its impact on genomes. However, the types of mobile elements that inhabit any given genome are remarkably varied. Among the different varieties of mobile elements, the Short Interspersed Elements (SINEs) populate many genomes, including many mammalian species. Although SINEs are parasites of Long Interspersed Elements (LINEs), SINEs have been highly successful in both the primate and rodent genomes. When comparing copy numbers in mammals, SINEs have been vastly more successful than other nonautonomous elements, such as the retropseudogenes and SVA. Interestingly, in the human genome the copy number of Alu (a primate SINE) outnumbers LINE-1 (L1) copies 2 to 1. Estimates suggest that the retrotransposition rate for Alu is tenfold higher than LINE-1 with about 1 insert in every twenty births. Furthermore, Alu-induced mutagenesis is responsible for the majority of the documented instances of human retroelement insertion-induced disease. However, little is known on what contributes to these observed differences between SINEs and LINEs. The development of an assay to monitor SINE retrotransposition in culture has become an important tool for the elucidation of some of these differences. In this chapter, we present details of the SINE retrotransposition assay and the recovery of de novo inserts. We also focus on the nuances that are unique to the SINE assay.

Body of Knowledge (BOK) for Leadless Quad Flat No-Lead/bottom Termination Components (QFN/BTC) Package Trends and Reliability

NASA Technical Reports Server (NTRS)

Ghaffarian, Reza

2014-01-01

Bottom terminated components and quad flat no-lead (BTC/QFN) packages have been extensively used by commercial industry for more than a decade. Cost and performance advantages and the closeness of the packages to the boards make them especially unique for radio frequency (RF) applications. A number of high-reliability parts are now available in this style of package configuration. This report presents a summary of literature surveyed and provides a body of knowledge (BOK) gathered on the status of BTC/QFN and their advanced versions of multi-row QFN (MRQFN) packaging technologies. The report provides a comprehensive review of packaging trends and specifications on design, assembly, and reliability. Emphasis is placed on assembly reliability and associated key design and process parameters because they show lower life than standard leaded package assembly under thermal cycling exposures. Inspection of hidden solder joints for assuring quality is challenging and is similar to ball grid arrays (BGAs). Understanding the key BTC/QFN technology trends, applications, processing parameters, workmanship defects, and reliability behavior is important when judicially selecting and narrowing the follow-on packages for evaluation and testing, as well as for the low risk insertion in high-reliability applications.

Antimicrobial, antioxidant and sensory features of eugenol, carvacrol and trans-anethole in active packaging for organic ready-to-eat iceberg lettuce.

PubMed

Wieczyńska, Justyna; Cavoski, Ivana

2018-09-01

In this study, bio-based emitting sachets containing eugenol (EUG), carvacrol (CAR) and trans-anethole (ANT) were inserted into cellulose (CE) and polypropylene (PP) pillow packages of organic ready-to-eat (RTE) iceberg lettuce to investigate their functional features. EUG, CAR and ANT sachets in CE; and CAR in PP packages showed antimicrobial activities against coliforms (Δlog CFU g -1 of -1.38, -0.91, -0.93 and -0.93, respectively). EUG and ANT sachets in both packages reduced discoloration (ΔE of 9.5, 1.8, 9.4 and 5.6, respectively). ANT in both, and EUG only in PP packages induced biosynthesis of caffeoyl derivatives (C a T A , D i C a T A , D i C a Q A ), total phenolics and antioxidant activity (FRAP). Also, ANT and EUG in both packages improved overall freshness and odor. Principal component analysis separated ANT and EUG from CAR in both packages. The Pearson correlation confirmed that overall quality improvements were more pronounced by ANT inside the packages in comparison to EUG and CAR. Copyright © 2018 Elsevier Ltd. All rights reserved.

Body of Knowledge (BOK) for Leadless Quad Flat No-Lead/Bottom Termination Components (QFN/BTC) Package Trends and Reliability

NASA Technical Reports Server (NTRS)

Ghaffarian, Reza

2014-01-01

Bottom terminated components and quad flat no-lead (BTC/QFN) packages have been extensively used by commercial industry for more than a decade. Cost and performance advantages and the closeness of the packages to the boards make them especially unique for radio frequency (RF) applications. A number of high-reliability parts are now available in this style of package configuration. This report presents a summary of literature surveyed and provides a body of knowledge (BOK) gathered on the status of BTC/QFN and their advanced versions of multi-row QFN (MRQFN) packaging technologies. The report provides a comprehensive review of packaging trends and specifications on design, assembly, and reliability. Emphasis is placed on assembly reliability and associated key design and process parameters because they show lower life than standard leaded package assembly under thermal cycling exposures. Inspection of hidden solder joints for assuring quality is challenging and is similar to ball grid arrays (BGAs). Understanding the key BTC/QFN technology trends, applications, processing parameters, workmanship defects, and reliability behavior is important when judicially selecting and narrowing the follow-on packages for evaluation and testing, as well as for the low risk insertion in high-reliability applications.

Fuzzy based attitude controller for flexible spacecraft with on/off thrusters

NASA Astrophysics Data System (ADS)

Knapp, Roger G.; Adams, Neil J.

A fuzzy-based attitude controller is designed for attitude control of a generic spacecraft with on/off thrusters. The controller is comprised of packages of rules dedicated to addressing different objectives (e.g., disturbance rejection, low fuel consumption, avoiding the excitation of flexible appendages, etc.). These rule packages can be inserted or removed depending on the requirements of the particular spacecraft and are parameterized based on vehicle parameters such as inertia or operational parameters such as the maneuvering rate. Individual rule packages can be 'weighted' relative to each other to emphasize the importance of one objective relative to another. Finally, the fuzzy controller and rule packages are demonstrated using the high-fidelity Space Shuttle Interactive On-Orbit Simulator (IOS) while performing typical on-orbit operations and are subsequently compared with the existing shuttle flight control system performance.

Fuzzy based attitude controller for flexible spacecraft with on/off thrusters

NASA Astrophysics Data System (ADS)

Knapp, Roger Glenn

1993-05-01

A fuzzy-based attitude controller is designed for attitude control of a generic spacecraft with on/off thrusters. The controller is comprised of packages of rules dedicated to addressing different objectives (e.g., disturbance rejection, low fuel consumption, avoiding the excitation of flexible appendages, etc.). These rule packages can be inserted or removed depending on the requirements of the particular spacecraft and are parameterized based on vehicle parameters such as inertia or operational parameters such as the maneuvering rate. Individual rule packages can be 'weighted' relative to each other to emphasize the importance of one objective relative to another. Finally, the fuzzy controller and rule packages are demonstrated using the high-fidelity Space Shuttle Interactive On-Orbit Simulator (IOS) while performing typical on-orbit operations and are subsequently compared with the existing shuttle flight control system performance.

Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging.

PubMed

Mellado-García, P; Maisanaba, S; Puerto, M; Llana-Ruiz-Cabello, M; Prieto, A I; Marcos, R; Pichardo, S; Cameán, A M

2015-12-01

Essential oils from onion (Allium cepa L.), garlic (Allium sativum L.), and their main components, such as propyl thiosulfinate oxide (PTSO) are being intended for active packaging with the purpose of maintaining and extending food product quality and shelf life. The present work aims to assess for the first time the potential mutagenicity/genotoxicity of PTSO (0-50 µM) using the following battery of genotoxicity tests: (1) the bacterial reverse-mutation assay in Salmonella typhimurium (Ames test, OECD 471); (2) the micronucleus test (OECD 487) (MN) and (3) the mouse lymphoma thymidine-kinase assay (OECD 476) (MLA) on L5178YTk(+/-), cells; and (4) the comet assay (with and without Endo III and FPG enzymes) on Caco-2 cells. The results revealed that PTSO was not mutagenic in the Ames test, however it was mutagenic in the MLA assay after 24 h of treatment (2.5-20 µM). The parent compound did not induce MN on mammalian cells; however, its metabolites (in the presence S9) produced positive results (from 15 µM). Data from the comet assay indicated that PTSO did not induce DNA breaks or oxidative DNA damage. Further in vivo genotoxicity tests are needed to confirm its safety before it is used as active additive in food packaging. Copyright © 2015 Elsevier Ltd. All rights reserved.

21 CFR 660.25 - Potency tests without reference preparations.

Code of Federal Regulations, 2012 CFR

2012-04-01

... manufacturer's package insert using red blood cells showing heterozygous or diminished expression of the... SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.25 Potency tests without reference preparations. Products for which Reference Blood...

21 CFR 660.25 - Potency tests without reference preparations.

Code of Federal Regulations, 2010 CFR

2010-04-01

... manufacturer's package insert using red blood cells showing heterozygous or diminished expression of the... SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.25 Potency tests without reference preparations. Products for which Reference Blood...

21 CFR 660.25 - Potency tests without reference preparations.

Code of Federal Regulations, 2014 CFR

2014-04-01

... manufacturer's package insert using red blood cells showing heterozygous or diminished expression of the... SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.25 Potency tests without reference preparations. Products for which Reference Blood...

21 CFR 660.25 - Potency tests without reference preparations.

Code of Federal Regulations, 2011 CFR

2011-04-01

... manufacturer's package insert using red blood cells showing heterozygous or diminished expression of the... SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.25 Potency tests without reference preparations. Products for which Reference Blood...

21 CFR 660.25 - Potency tests without reference preparations.

Code of Federal Regulations, 2013 CFR

2013-04-01

... manufacturer's package insert using red blood cells showing heterozygous or diminished expression of the... SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.25 Potency tests without reference preparations. Products for which Reference Blood...

Comparative assay of antioxidant packages for dimer of estolide esters

USDA-ARS?s Scientific Manuscript database

A series of 26 different antioxidants and commercial antioxidant packages, containing both natural and synthetic-based materials, were evaluated with dimeric coconut-oleic estolide 2-ethylhexyl ester. The different antioxidants were broken down into different classes of materials: phenolic, aminic, ...

A Tale of Two Citizens: A State Attorney General and a Hematologist Facilitate Translation of Research Into US Food and Drug Administration Actions—A SONAR Report

PubMed Central

Chen, Brian; Restaino, John; Norris, LeAnn; Xirasagar, Sudha; Qureshi, Zaina P.; McKoy, June M.; Lopez, Isaac S.; Trenery, Alyssa; Murday, Alanna; Kahn, Adam; Mattison, Donald R.; Ray, Paul; Sartor, Oliver; Bennett, Charles L.

2012-01-01

Purpose: Pharmaceutical safety is a public health issue. In 2005, the Connecticut Attorney General (AG) raised concerns over adverse drug reactions in off-label settings, noting that thalidomide was approved to treat a rare illness, but more than 90% of its use was off label. A hematologist had reported thalidomide with doxorubicin or dexamethasone was associated with venous thromboembolism (VTE) rates of 25%. We review US Food and Drug Administration (FDA) and manufacturer responses to a citizen petition filed to address these thalidomide safety issues. Methods: Case study. Results: The AG petitioned the FDA requesting thalidomide-related safety actions. Coincidentally, the manufacturer submitted a supplemental New Drug Approval (sNDA), requesting approval to treat multiple myeloma with thalidomide-dexamethasone. FDA safety officers reviewed the petition and the literature and noted that VTE risks with thalidomide were not appropriately addressed in the existing package insert. In the sNDA application, the manufacturer reported thalidomide-associated toxicities for multiple myeloma were primarily somnolence and neurotoxicity, and a proposed package insert did not focus on VTE risks. In October, the FDA informed the Oncology Drug Division that VTE risks with thalidomide were poorly addressed in the existing label. After reviewing this memorandum, an Oncology Drug Division reviewer informed the manufacturer that approval of the sNDA would be delayed until several thalidomide-associated VTE safety actions, including revisions of the package insert, were implemented. The manufacturer and FDA agreed on these actions, and the sNDA was approved. Conclusion: New approaches addressing off-label safety are needed. The conditions that facilitated the successful response to this citizen petition are uncommon. PMID:23598851

A tale of two citizens: a State Attorney General and a hematologist facilitate translation of research into US Food and Drug Administration actions--a SONAR report.

PubMed

Chen, Brian; Restaino, John; Norris, LeAnn; Xirasagar, Sudha; Qureshi, Zaina P; McKoy, June M; Lopez, Isaac S; Trenery, Alyssa; Murday, Alanna; Kahn, Adam; Mattison, Donald R; Ray, Paul; Sartor, Oliver; Bennett, Charles L

2012-11-01

Pharmaceutical safety is a public health issue. In 2005, the Connecticut Attorney General (AG) raised concerns over adverse drug reactions in off-label settings, noting that thalidomide was approved to treat a rare illness, but more than 90% of its use was off label. A hematologist had reported thalidomide with doxorubicin or dexamethasone was associated with venous thromboembolism (VTE) rates of 25%. We review US Food and Drug Administration (FDA) and manufacturer responses to a citizen petition filed to address these thalidomide safety issues. Case study. The AG petitioned the FDA requesting thalidomide-related safety actions. Coincidentally, the manufacturer submitted a supplemental New Drug Approval (sNDA), requesting approval to treat multiple myeloma with thalidomide-dexamethasone. FDA safety officers reviewed the petition and the literature and noted that VTE risks with thalidomide were not appropriately addressed in the existing package insert. In the sNDA application, the manufacturer reported thalidomide-associated toxicities for multiple myeloma were primarily somnolence and neurotoxicity, and a proposed package insert did not focus on VTE risks. In October, the FDA informed the Oncology Drug Division that VTE risks with thalidomide were poorly addressed in the existing label. After reviewing this memorandum, an Oncology Drug Division reviewer informed the manufacturer that approval of the sNDA would be delayed until several thalidomide-associated VTE safety actions, including revisions of the package insert, were implemented. The manufacturer and FDA agreed on these actions, and the sNDA was approved. New approaches addressing off-label safety are needed. The conditions that facilitated the successful response to this citizen petition are uncommon.

Student-Designed Enzyme-Linked Metabolite Assay Kits

ERIC Educational Resources Information Center

Hancock, D.; Johnston, J.; Dimauro, J.; Denyer, G.

2004-01-01

The extensive use of commercial kits in molecular biology and biochemistry has prompted us to design a series of practical sessions to help students become familiar with the uses and limitations of pre-packaged assay systems. To facilitate an understanding of these assay systems and to promote reflection on their appropriate use, students…

A Web-based searchable system to confirm magnetic resonance compatibility of implantable medical devices in Japan: a preliminary study.

PubMed

Fujiwara, Yasuhiro; Fujioka, Hitoshi; Watanabe, Tomoko; Sekiguchi, Maiko; Murakami, Ryuji

2017-09-01

Confirmation of the magnetic resonance (MR) compatibility of implanted medical devices (IMDs) is mandatory before conducting magnetic resonance imaging (MRI) examinations. In Japan, few such confirmation methods are in use, and they are time-consuming. This study aimed to develop a Web-based searchable MR safety information system to confirm IMD compatibility and to evaluate the usefulness of the system. First, MR safety information for intravascular stents and stent grafts sold in Japan was gathered by interviewing 20 manufacturers. These IMDs were categorized based on the descriptions available on medical package inserts as: "MR Safe," "MR Conditional," "MR Unsafe," "Unknown," and "No Medical Package Insert Available". An MR safety information database for implants was created based on previously proposed item lists. Finally, a Web-based searchable system was developed using this database. A questionnaire was given to health-care personnel in Japan to evaluate the usefulness of this system. Seventy-nine datasets were collected using information provided by 12 manufacturers and by investigating the medical packaging of the IMDs. Although the datasets must be updated by collecting data from other manufacturers, this system facilitates the easy and rapid acquisition of MR safety information for IMDs, thereby improving the safety of MRI examinations.

Women's preferences for vaginal antimicrobial contraceptives. III. Choice of a formulation, applicator, and packaging.

PubMed

Hardy, E; Jiménez, A L; de Pádua, K S; Zaneveld, L J

1998-10-01

Novel vaginal formulations are under development to combat the increasing incidence of sexually transmitted diseases, including AIDS, and also unplanned pregnancies. A study was performed to determine women's preferences for different dosage forms (gel, cream, ovule/suppository, film, foam, tablet), width, length, and color of an applicator, and various types of packages. The study was conducted in Campinas, Brazil. A total of 635 women were interviewed, including both adolescents and adults and low and middle-high socioeconomic groups. The large majority of the women preferred a gel over a cream; both were preferred over the other methods. When asked which method they would not use, the film was most frequently identified, followed by the tablet and ovule. The primary reasons for selecting a particular dosage form were ease of use, absence of odor or the presence of a pleasant one, absence of color, and insertion with an applicator. The major reasons for not using a method were discomfort, "plastic" appearance, distrust of effectiveness, difficulty with insertion, messiness, and rigidity/hardness. The majority of the women liked the applicator shown. The prefilled single dose applicator was by far the preferred packaging. This information should aid in the development of consumer-friendly, vaginal formulations.

Public judgments of information in a diazepam patient package insert.

PubMed

Fisher, S; Mansbridge, B; Lankford, D A

1982-06-01

As part of a larger study of the effects of giving patients written take-home information with prescription medications, a "patient package insert" (PPI) for diazepam was prepared based on content determined by "experts." This report compares the experts' judgments of what information should be included with judgments obtained from the public. Information judged to be most important for inclusion in a PPI was identified by having subjects sort cards containing facts about diazepam. Subjects who had previously used diazepam were no different in their judgments than inexperienced subjects. In general, there was a high degree of concordance between public and expert judgments and also a remarkably strong consensus across very different demographic samples. In those few instances of disagreement, the public attached even greater importance to warnings and "bad news" about diazepam than to information providing reassurances, benign general education, and "good news." To what extent patients would effectively use this information--whether conveyed by PPIs or alternative educational routes--must await empirical evaluation.

I-125 seed calibration using the SeedSelectron® afterloader: a practical solution to fulfill AAPM-ESTRO recommendations

PubMed Central

Perez-Calatayud, Jose; Richart, Jose; Guirado, Damián; Pérez-García, Jordi; Rodríguez, Silvia; Santos, Manuel

2012-01-01

Purpose SeedSelectron® v1.26b (Nucletron BV, The Netherlands) is an afterloader system used in prostate interstitial permanent brachytherapy with I-125 selectSeed seeds. It contains a diode array to assay all implanted seeds. Only one or two seeds can be extracted during the surgical procedure and assayed using a well chamber to check the manufacturer air-kerma strength (SK) and to calibrate the diode array. Therefore, it is not feasible to assay 5–10% seeds as required by the AAPM-ESTRO. In this study, we present a practical solution of the SeedSelectron® users to fulfill the AAPM- ESTRO recommendations. Material and methods The method is based on: a) the SourceCheck® well ionization chamber (PTW, Germany) provided with a PTW insert; b) n = 10 selectSeed from the same batch and class as the seeds for the implant; c) the Nucletron insert to accommodate the n = 10 seeds on the SourceCheck® and to measure their averaged SK. Results for 56 implants have been studied comparing the SK value from the manufacturer with the one obtained with the n = 10 seeds using the Nucletron insert prior to the implant and with the SK of just one seed measured with the PTW insert during the implant. Results We are faced with SK deviation for individual seeds up to 7.8%. However, in the majority of cases SK is in agreement with the manufacturer value. With the method proposed using the Nucletron insert, the large deviations of SK are reduced and for 56 implants studied no deviation outside the range of the class were found. Conclusions The new Nucletron insert and the proposed procedure allow to evaluate the SK of the n = 10 seeds prior to the implant, fulfilling the AAPM-ESTRO recommendations. It has been adopted by Nucletron to be extended to seedSelectron® users under request. PMID:23346136

Genome Editing-Enabled HTS Assays Expand Drug Target Pathways for Charcot–Marie–Tooth Disease

PubMed Central

2015-01-01

Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot–Marie–Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin. PMID:25188731

Device-packaging method and apparatus for optoelectronic circuits

DOEpatents

Zortman, William A.; Henry, Michael David; Jarecki, Jr., Robert L.

2017-04-25

An optoelectronic device package and a method for its fabrication are provided. The device package includes a lid die and an active die that is sealed or sealable to the lid die and in which one or more optical waveguides are integrally defined. The active die includes one or more active device regions, i.e. integral optoelectronic devices or etched cavities for placement of discrete optoelectronic devices. Optical waveguides terminate at active device regions so that they can be coupled to them. Slots are defined in peripheral parts of the active dies. At least some of the slots are aligned with the ends of integral optical waveguides so that optical fibers or optoelectronic devices inserted in the slots can optically couple to the waveguides.

Fuzzy based attitude controller for flexible spacecraft with on/off thrusters. M.S. Thesis - M.I.T., 1993

NASA Technical Reports Server (NTRS)

Knapp, Roger Glenn

1993-01-01

A fuzzy-based attitude controller is designed for attitude control of a generic spacecraft with on/off thrusters. The controller is comprised of packages of rules dedicated to addressing different objectives (e.g., disturbance rejection, low fuel consumption, avoiding the excitation of flexible appendages, etc.). These rule packages can be inserted or removed depending on the requirements of the particular spacecraft and are parameterized based on vehicle parameters such as inertia or operational parameters such as the maneuvering rate. Individual rule packages can be 'weighted' relative to each other to emphasize the importance of one objective relative to another. Finally, the fuzzy controller and rule packages are demonstrated using the high-fidelity Space Shuttle Interactive On-Orbit Simulator (IOS) while performing typical on-orbit operations and are subsequently compared with the existing shuttle flight control system performance.

Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93.

PubMed

Köppen-Rung, Pánja; Dittmer, Alexandra; Bogner, Elke

2016-07-01

DNA packaging into procapsids is a common multistep process during viral maturation in herpesviruses. In human cytomegalovirus (HCMV), the proteins involved in this process are terminase subunits pUL56 and pUL89, which are responsible for site-specific cleavage and insertion of the DNA into the procapsid via portal protein pUL104. However, additional viral proteins are required for the DNA packaging process. We have shown previously that the plasmid that encodes capsid-associated pUL77 encodes another potential player during capsid maturation. Pulse-chase experiments revealed that pUL77 is stably expressed during HCMV infection. Time course analysis demonstrated that pUL77 is expressed in the early late part of the infectious cycle. The sequence of pUL77 was analyzed to find nuclear localization sequences (NLSs), revealing monopartite NLSm at the N terminus and bipartite NLSb in the middle of pUL77. The potential NLSs were inserted into plasmid pHM829, which encodes a chimeric protein with β-galactosidase and green fluorescent protein. In contrast to pUL56, neither NLSm nor NLSb was sufficient for nuclear import. Furthermore, we investigated by coimmunoprecipitation whether packaging proteins, as well as pUL93, the homologue protein of herpes simplex virus 1 pUL17, are interaction partners of pUL77. The interactions between pUL77 and packaging proteins, as well as pUL93, were verified. We showed that the capsid-associated pUL77 is another potential player during capsid maturation of HCMV. Protein UL77 (pUL77) is a conserved core protein of HCMV. This study demonstrates for the first time that pUL77 has early-late expression kinetics during the infectious cycle and an intrinsic potential for nuclear translocation. According to its proposed functions in stabilization of the capsid and anchoring of the encapsidated DNA during packaging, interaction with further DNA packaging proteins is required. We identified physical interactions with terminase subunits pUL56 and pUL89 and another postulated packaging protein, pUL93, in infected, as well as transfected, cells. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

22 CFR 125.6 - Certification requirements for exemptions.

Code of Federal Regulations, 2010 CFR

2010-04-01

... LICENSES FOR THE EXPORT OF TECHNICAL DATA AND CLASSIFIED DEFENSE ARTICLES § 125.6 Certification requirements for exemptions. (a) To claim an exemption for the export of technical data under the provisions of.... Certifications consist of clearly marking the package or letter containing the technical data “22 CFR [insert...

22 CFR 125.6 - Certification requirements for exemptions.

Code of Federal Regulations, 2011 CFR

2011-04-01

.... Certifications consist of clearly marking the package or letter containing the technical data “22 CFR [insert... LICENSES FOR THE EXPORT OF TECHNICAL DATA AND CLASSIFIED DEFENSE ARTICLES § 125.6 Certification requirements for exemptions. (a) To claim an exemption for the export of technical data under the provisions of...

22 CFR 125.6 - Certification requirements for exemptions.

Code of Federal Regulations, 2013 CFR

2013-04-01

.... Certifications consist of clearly marking the package or letter containing the technical data “22 CFR [insert... LICENSES FOR THE EXPORT OF TECHNICAL DATA AND CLASSIFIED DEFENSE ARTICLES § 125.6 Certification requirements for exemptions. (a) To claim an exemption for the export of technical data under the provisions of...

22 CFR 125.6 - Certification requirements for exemptions.

Code of Federal Regulations, 2014 CFR

2014-04-01

.... Certifications consist of clearly marking the package or letter containing the technical data “22 CFR [insert... LICENSES FOR THE EXPORT OF TECHNICAL DATA AND CLASSIFIED DEFENSE ARTICLES § 125.6 Certification requirements for exemptions. (a) To claim an exemption for the export of technical data under the provisions of...

22 CFR 125.6 - Certification requirements for exemptions.

Code of Federal Regulations, 2012 CFR

2012-04-01

.... Certifications consist of clearly marking the package or letter containing the technical data “22 CFR [insert... LICENSES FOR THE EXPORT OF TECHNICAL DATA AND CLASSIFIED DEFENSE ARTICLES § 125.6 Certification requirements for exemptions. (a) To claim an exemption for the export of technical data under the provisions of...

An Assessment of the Influence of the Industry Distribution Chain on the Oxygen Levels in Commercial Modified Atmosphere Packaged Cheddar Cheese Using Non-Destructive Oxygen Sensor Technology.

PubMed

O' Callaghan, Karen A M; Papkovsky, Dmitri B; Kerry, Joseph P

2016-06-20

The establishment and control of oxygen levels in packs of oxygen-sensitive food products such as cheese is imperative in order to maintain product quality over a determined shelf life. Oxygen sensors quantify oxygen concentrations within packaging using a reversible optical measurement process, and this non-destructive nature ensures the entire supply chain can be monitored and can assist in pinpointing negative issues pertaining to product packaging. This study was carried out in a commercial cheese packaging plant and involved the insertion of 768 sensors into 384 flow-wrapped cheese packs (two sensors per pack) that were flushed with 100% carbon dioxide prior to sealing. The cheese blocks were randomly assigned to two different storage groups to assess the effects of package quality, packaging process efficiency, and handling and distribution on package containment. Results demonstrated that oxygen levels increased in both experimental groups examined over the 30-day assessment period. The group subjected to a simulated industrial distribution route and handling procedures of commercial retailed cheese exhibited the highest level of oxygen detected on every day examined and experienced the highest rate of package failure. The study concluded that fluctuating storage conditions, product movement associated with distribution activities, and the possible presence of cheese-derived contaminants such as calcium lactate crystals were chief contributors to package failure.

An Assessment of the Influence of the Industry Distribution Chain on the Oxygen Levels in Commercial Modified Atmosphere Packaged Cheddar Cheese Using Non-Destructive Oxygen Sensor Technology

PubMed Central

O’ Callaghan, Karen A.M.; Papkovsky, Dmitri B.; Kerry, Joseph P.

2016-01-01

The establishment and control of oxygen levels in packs of oxygen-sensitive food products such as cheese is imperative in order to maintain product quality over a determined shelf life. Oxygen sensors quantify oxygen concentrations within packaging using a reversible optical measurement process, and this non-destructive nature ensures the entire supply chain can be monitored and can assist in pinpointing negative issues pertaining to product packaging. This study was carried out in a commercial cheese packaging plant and involved the insertion of 768 sensors into 384 flow-wrapped cheese packs (two sensors per pack) that were flushed with 100% carbon dioxide prior to sealing. The cheese blocks were randomly assigned to two different storage groups to assess the effects of package quality, packaging process efficiency, and handling and distribution on package containment. Results demonstrated that oxygen levels increased in both experimental groups examined over the 30-day assessment period. The group subjected to a simulated industrial distribution route and handling procedures of commercial retailed cheese exhibited the highest level of oxygen detected on every day examined and experienced the highest rate of package failure. The study concluded that fluctuating storage conditions, product movement associated with distribution activities, and the possible presence of cheese-derived contaminants such as calcium lactate crystals were chief contributors to package failure. PMID:27331815

Cross-reactivity of steroid hormone immunoassays: clinical significance and two-dimensional molecular similarity prediction

PubMed Central

2014-01-01

Background Immunoassays are widely used in clinical laboratories for measurement of plasma/serum concentrations of steroid hormones such as cortisol and testosterone. Immunoassays can be performed on a variety of standard clinical chemistry analyzers, thus allowing even small clinical laboratories to do analysis on-site. One limitation of steroid hormone immunoassays is interference caused by compounds with structural similarity to the target steroid of the assay. Interfering molecules include structurally related endogenous compounds and their metabolites as well as drugs such as anabolic steroids and synthetic glucocorticoids. Methods Cross-reactivity of a structurally diverse set of compounds were determined for the Roche Diagnostics Elecsys assays for cortisol, dehydroepiandrosterone (DHEA) sulfate, estradiol, progesterone, and testosterone. These data were compared and contrasted to package insert data and published cross-reactivity studies for other marketed steroid hormone immunoassays. Cross-reactivity was computationally predicted using the technique of two-dimensional molecular similarity. Results The Roche Elecsys Cortisol and Testosterone II assays showed a wider range of cross-reactivity than the DHEA sulfate, Estradiol II, and Progesterone II assays. 6-Methylprednisolone and prednisolone showed high cross-reactivity for the cortisol assay, with high likelihood of clinically significant effect for patients administered these drugs. In addition, 21-deoxycortisol likely produces clinically relevant cross-reactivity for cortisol in patients with 21-hydroxylase deficiency, while 11-deoxycortisol may produce clinically relevant cross-reactivity in 11β-hydroxylase deficiency or following metyrapone challenge. Several anabolic steroids may produce clinically significant false positives on the testosterone assay, although interpretation is limited by sparse pharmacokinetic data for some of these drugs. Norethindrone therapy may impact immunoassay measurement of testosterone in women. Using two-dimensional similarity calculations, all compounds with high cross-reactivity also showed a high degree of similarity to the target molecule of the immunoassay. Conclusions Compounds producing cross-reactivity in steroid hormone immunoassays generally have a high degree of structural similarity to the target hormone. Clinically significant interactions can occur with structurally similar drugs (e.g., prednisolone and cortisol immunoassays; methyltestosterone and testosterone immunoassays) or with endogenous compounds such as 21-deoxycortisol that can accumulate to very high concentrations in certain disease conditions. Simple similarity calculations can help triage compounds for future testing of assay cross-reactivity. PMID:25071417

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity

PubMed Central

Traverse, Charles C.

2017-01-01

ABSTRACT Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola, which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. PMID:28851848

High-Tc superconductor coplanar waveguide filter

NASA Technical Reports Server (NTRS)

Chew, Wilbert; Bajuk, Louis J.; Cooley, Thomas W.; Foote, Marc C.; Hunt, Brian D.; Rascoe, Daniel L.; Riley, A. L.

1991-01-01

Coplanar waveguide (CPW) low-pass filters made of YBa2Cu3O(7-delta) (YBCO) on LaAlO3 substrates, with dimensions suited for integrated circuits, were fabricated and packaged. A complete filter gives a true idea of the advantages and difficulties in replacing thin-film metal with a high-temperature superconductor in a practical circuit. Measured insertion losses in liquid nitrogen were superior to the loss of a similar thin-film copper filter throughout the 0- to 9.5-GHz passband. These results demonstrate the performance of fully patterned YBCO in a practical CPW structure after sealing in a hermetic package.

Apparatus and Method for Packaging and Integrating Microphotonic Devices

NASA Technical Reports Server (NTRS)

Nguyen, Hung (Inventor)

2008-01-01

An apparatus is disclosed that includes a carrier structure and an optical coupling arrangement. The carrier structure is made of a silicon material and allows for the packaging and integrating of microphotonic devices onto a single chip. The optical coupling mechanism enables laser light to be coupled into and out of a microphotonic resonant disk integrated on the carrier. The carrier provides first, second and third cavities that are dimensioned so as to accommodate the insertion and snug fitting of the microphotonic resonant disk and first and second prisms that are implemented by the optical coupling arrangement to accommodate the laser coupling.

Agony of the ecstasy: report of five cases of MDMA smuggling.

PubMed

Low, V H S; Dillon, E K

2005-10-01

The international smuggling of illicit drugs by the ingestion or rectal insertion of drug-filled packages is recognized in the trafficking of heroin and cocaine. Customs authorities, with suspicion of such activities, presented five subjects. The legally allowed radiological examination comprising one supine abdominal radiograph was performed. Radiographic findings demonstrated the presence of multiple enteric oval, capsule-shaped packages of soft tissue density. This was confirmed following supervised evacuation of bowel contents induced by the administration of laxatives. Analysis of the concealed material identified ecstasy (methylenedioxymethamphetamine (MDMA)), a substance not previously reported as transported by this route.

21 CFR 864.7490 - Sulfhemoglobin assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

21 CFR 864.7490 - Sulfhemoglobin assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

21 CFR 864.7425 - Carboxyhemoglobin assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin...

21 CFR 864.7490 - Sulfhemoglobin assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

21 CFR 864.7425 - Carboxyhemoglobin assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin...

21 CFR 864.7490 - Sulfhemoglobin assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

21 CFR 864.7425 - Carboxyhemoglobin assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin...

21 CFR 864.7490 - Sulfhemoglobin assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

21 CFR 864.7425 - Carboxyhemoglobin assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin...

SynergyFinder: a web application for analyzing drug combination dose-response matrix data.

PubMed

Ianevski, Aleksandr; He, Liye; Aittokallio, Tero; Tang, Jing

2017-08-01

Rational design of drug combinations has become a promising strategy to tackle the drug sensitivity and resistance problem in cancer treatment. To systematically evaluate the pre-clinical significance of pairwise drug combinations, functional screening assays that probe combination effects in a dose-response matrix assay are commonly used. To facilitate the analysis of such drug combination experiments, we implemented a web application that uses key functions of R-package SynergyFinder, and provides not only the flexibility of using multiple synergy scoring models, but also a user-friendly interface for visualizing the drug combination landscapes in an interactive manner. The SynergyFinder web application is freely accessible at https://synergyfinder.fimm.fi ; The R-package and its source-code are freely available at http://bioconductor.org/packages/release/bioc/html/synergyfinder.html . jing.tang@helsinki.fi. © The Author(s) 2017. Published by Oxford University Press.

Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

PubMed

Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

2016-01-29

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

Applications Where Snap is BPM for Radioactive Waste Assay

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

Miller, T.J.

2008-07-01

Historically, the Atomic Weapons Establishment (AWE) at Aldermaston in the United Kingdom (UK), has used a variety of assay techniques to measure the radioactive content of a diverse range of waste packages from decommissioning, operational and legacy sources. The regulator, the Environment Agency in the UK, places conditions and limits on AWE through an authorisation within the Radioactive Substances Act (RSA93). The conditions and limits require Best Practical Means (BPM) measurements to be used to demonstrate compliance with the authorisation. Hence, the assay technique employed needs to achieve a balance between risk of exposure, environmental considerations, technological considerations, health andmore » safety considerations and cost effectiveness, without being grossly disproportionate in terms of money, time or trouble. Recently published work has concluded that the Spectral Non-destructive Assay Platform (SNAP) assay system is BPM for Depleted Uranium (DU) waste assay at AWE (1) and low level plutonium in soft drummed waste, HEPA filters and soils (2-4). The purpose of this paper is to highlight other applications where SNAP represents BPM for radioactive waste assay. This has been done by intercomparison studies of SNAP with other assay techniques, such as Segmented Gamma Scanner (SGS) and Passive Neutron Coincidence Counter (PNCC). It has been concluded that, for a large range of waste packages encountered at AWE, SNAP is BPM. (author)« less

Oxidation promotes insertion of the CLIC1 chloride intracellular channel into the membrane.

PubMed

Goodchild, Sophia C; Howell, Michael W; Cordina, Nicole M; Littler, Dene R; Breit, Samuel N; Curmi, Paul M G; Brown, Louise Jennifer

2009-12-01

Members of the chloride intracellular channel (CLIC) family exist primarily as soluble proteins but can also auto-insert into cellular membranes to form ion channels. While little is known about the process of CLIC membrane insertion, a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural metamorphosis between a monomeric glutathione-S-transferase homolog and an all-helical dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis facilitates CLIC1 membrane insertion is unclear. In this work, we have sought to characterise the role of oxidation in the process of CLIC1 membrane insertion. We examined how redox conditions modify the ability of CLIC1 to associate with and insert into the membrane using fluorescence quenching studies and a sucrose-loaded vesicle sedimentation assay to measure membrane binding. Our results suggest that oxidation of monomeric CLIC1, in the presence of membranes, promotes insertion into the bilayer more effectively than the oxidised CLIC1 dimer.

21 CFR 864.7415 - Abnormal hemoglobin assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Abnormal hemoglobin assay. 864.7415 Section 864.7415 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7415 Abnormal...

21 CFR 864.7375 - Glutathione reductase assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

21 CFR 864.7375 - Glutathione reductase assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

21 CFR 864.7375 - Glutathione reductase assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

21 CFR 864.7415 - Abnormal hemoglobin assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Abnormal hemoglobin assay. 864.7415 Section 864.7415 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7415 Abnormal...

21 CFR 864.7375 - Glutathione reductase assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

21 CFR 864.7375 - Glutathione reductase assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

21 CFR 864.7415 - Abnormal hemoglobin assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Abnormal hemoglobin assay. 864.7415 Section 864.7415 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7415 Abnormal...

21 CFR 357.152 - Package inserts for anthelmintic drug products.

Code of Federal Regulations, 2013 CFR

2013-04-01

... discussion of the symptoms suggestive of pinworm infestation, including a statement that pinworms must be... the pinworm. (c) A commentary on the life cycle of the pinworm. (d) A commentary on the ways in which pinworms may be spread from person to person and hygienic procedures to follow to avoid such spreading. (e...

21 CFR 357.152 - Package inserts for anthelmintic drug products.

Code of Federal Regulations, 2010 CFR

2010-04-01

... discussion of the symptoms suggestive of pinworm infestation, including a statement that pinworms must be... the pinworm. (c) A commentary on the life cycle of the pinworm. (d) A commentary on the ways in which pinworms may be spread from person to person and hygienic procedures to follow to avoid such spreading. (e...

21 CFR 357.152 - Package inserts for anthelmintic drug products.

Code of Federal Regulations, 2011 CFR

2011-04-01

... discussion of the symptoms suggestive of pinworm infestation, including a statement that pinworms must be... the pinworm. (c) A commentary on the life cycle of the pinworm. (d) A commentary on the ways in which pinworms may be spread from person to person and hygienic procedures to follow to avoid such spreading. (e...

21 CFR 357.152 - Package inserts for anthelmintic drug products.

Code of Federal Regulations, 2012 CFR

2012-04-01

... discussion of the symptoms suggestive of pinworm infestation, including a statement that pinworms must be... the pinworm. (c) A commentary on the life cycle of the pinworm. (d) A commentary on the ways in which pinworms may be spread from person to person and hygienic procedures to follow to avoid such spreading. (e...

21 CFR 357.152 - Package inserts for anthelmintic drug products.

Code of Federal Regulations, 2014 CFR

2014-04-01

... discussion of the symptoms suggestive of pinworm infestation, including a statement that pinworms must be... the pinworm. (c) A commentary on the life cycle of the pinworm. (d) A commentary on the ways in which pinworms may be spread from person to person and hygienic procedures to follow to avoid such spreading. (e...

Isolating USB connections in medical equipment.

PubMed

Broeders, Jan-Hein

2009-01-01

Although offering several benefits, the universal serial bus (USB) port has not been rapidly adopted for connecting medical equipment. This is because it could affect safety procedures, with equipment not operating isolated from the mains. To overcome this, a single package isolation device has been developed that can be inserted directly into the USB signal path.

Standard Hardware Acquisition and Reliability Program's (SHARP's) efforts in incorporating fiber optic interconnects into standard electronic module (SEM) connectors

NASA Astrophysics Data System (ADS)

Riggs, William R.

1994-05-01

SHARP is a Navy wide logistics technology development effort aimed at reducing the acquisition costs, support costs, and risks of military electronic weapon systems while increasing the performance capability, reliability, maintainability, and readiness of these systems. Lower life cycle costs for electronic hardware are achieved through technology transition, standardization, and reliability enhancement to improve system affordability and availability as well as enhancing fleet modernization. Advanced technology is transferred into the fleet through hardware specifications for weapon system building blocks of standard electronic modules, standard power systems, and standard electronic systems. The product lines are all defined with respect to their size, weight, I/O, environmental performance, and operational performance. This method of defining the standard is very conducive to inserting new technologies into systems using the standard hardware. This is the approach taken thus far in inserting photonic technologies into SHARP hardware. All of the efforts have been related to module packaging; i.e. interconnects, component packaging, and module developments. Fiber optic interconnects are discussed in this paper.

[Safety profile of heterologous serum produced by the Butantan Institute, in São Paulo-SP, Brazil, from 2012 to 2015].

PubMed

Gattás, Vera Lúcia; Braga, Patrícia Emília; Koike, Marcelo Eiji; Lucchesi, Maria Beatriz; Precioso, Alexander Roberto

2017-01-01

to describe the safety profile of the heterologous serum produced by the Butantan Institute (BI) of São Paulo-SP, Brazil. a descriptive study of adverse events (AEs) post-exposure to serum produced by the BI, encoded in the medical terminology of the Medical Dictionary for Regulatory Activities (MedDRA), and spontaneously reported to BI from 2012 to 2015. 52 individuals reported AEs, mainly related to Bothrops antivenom (n=11), diphtheria antitoxin (n=9) and unspecified snakebite serum (n=9); a mean of 3.2 AEs per individual was observed; among the total of 173 AEs, 63.0% were expected considering that they were described in the package insert; most of them were classified as skin and subcutaneous tissue disorders (30.6%); there were six deaths temporally related to the use of serum, but this association was discarded. in the studied period, the serum produced by the BI had no changes in their safety profiles, considering that the AEs were expected, according to the information previously described in the package insert.

21 CFR 864.7060 - Antithrombin III assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Antithrombin III assay. 864.7060 Section 864.7060 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7060 Antithrombin III...

21 CFR 864.7400 - Hemoglobin A 2 assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Hemoglobin A 2 assay. 864.7400 Section 864.7400 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7400 Hemoglobin A 2...

21 CFR 864.7060 - Antithrombin III assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Antithrombin III assay. 864.7060 Section 864.7060 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7060 Antithrombin III...

21 CFR 864.7060 - Antithrombin III assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Antithrombin III assay. 864.7060 Section 864.7060 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7060 Antithrombin III...

21 CFR 864.7400 - Hemoglobin A2 assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Hemoglobin A2 assay. 864.7400 Section 864.7400 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7400 Hemoglobin A2...

21 CFR 864.7060 - Antithrombin III assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Antithrombin III assay. 864.7060 Section 864.7060 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7060 Antithrombin III...

21 CFR 864.7400 - Hemoglobin A 2 assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Hemoglobin A 2 assay. 864.7400 Section 864.7400 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7400 Hemoglobin A 2...

21 CFR 864.7455 - Fetal hemoglobin assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Fetal hemoglobin assay. 864.7455 Section 864.7455 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7455 Fetal hemoglobin...

21 CFR 864.7060 - Antithrombin III assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antithrombin III assay. 864.7060 Section 864.7060 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7060 Antithrombin III...

21 CFR 864.7455 - Fetal hemoglobin assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Fetal hemoglobin assay. 864.7455 Section 864.7455 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7455 Fetal hemoglobin...

21 CFR 864.7455 - Fetal hemoglobin assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Fetal hemoglobin assay. 864.7455 Section 864.7455 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7455 Fetal hemoglobin...

Blood leak alarm interference by hydoxocobalamin is hemodialysis machine dependent.

PubMed

Sutter, M E; Clarke, M E; Cobb, J; Daubert, G P; Rathore, V S; Aston, L S; Poppenga, R H; Ford, J B; Owen, K P; Albertson, T E

2012-12-01

Hydroxocobalamin has been reported to interfere with the blood leak alarm on hemodialysis machines making it difficult to use this treatment modality after hydroxocobalamin infusion. The objective was to determine if this interference with hydroxocobalamin occurs across hemodialysis machines by different manufacturers. Additionally, we aimed to see if this represented a colorimetric interference alone or if it is the optical properties of hydroxocobalamin. Hydroxocobalamin was reconstituted per package insert. Food coloring was added to 0.9% saline to create the colors of the visual spectrum. Optical properties of absorbance and transmittance were measured. Hydroxocobalamin and the saline solutions were infused into the Fresenius 2008K™ and the Gambro Phoenix X36™ machines. Times were recorded from the start of the machine until the solution finished or the alarm triggered. When evaluating the Gambro Phoenix X36™ machine and dialysis circuit; the alarm did not trigger. In contrast, the blood leak alarm on the Fresenius 2008K™ machine was tripped by both the red solution and hydoxocobalamin infused per the package insert. The alarm stopped the machine between 128 and 132 seconds for the red solution and between 30 and 35 seconds with the hydroxocobalamin. Membranes of the circuits where the alarm tripped were examined and remained intact without blood. Results were validated on different machines with new circuits. Hydroxocobalamin infusion per package insert and the red saline solution prepared with Red Dye 40 both triggered the blood leak alarm and stopped the Fresenius 2008K™ machine. However, this was not true for the Gambro Phoenix X36™ machine as the alarm never triggered. The interference with the Fresenius 2008K™ appears colorimetric due to normal saline with Red Dye 40 triggering the alarm. We alert physicians to become familiar with the properties of individual dialysis machines prior to use of hydroxocobalamin. When facing difficulties with hemodialysis after the administration of hydroxocobalamin, consider attempting with a different manufactures machine or model if available or contact the manufacturer directly.

Direct interaction of the bacteriophage SPP1 packaging ATPase with the portal protein.

PubMed

Oliveira, Leonor; Cuervo, Ana; Tavares, Paulo

2010-03-05

DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.

SLDAssay: A software package and web tool for analyzing limiting dilution assays.

PubMed

Trumble, Ilana M; Allmon, Andrew G; Archin, Nancie M; Rigdon, Joseph; Francis, Owen; Baldoni, Pedro L; Hudgens, Michael G

2017-11-01

Serial limiting dilution (SLD) assays are used in many areas of infectious disease related research. This paper presents SLDAssay, a free and publicly available R software package and web tool for analyzing data from SLD assays. SLDAssay computes the maximum likelihood estimate (MLE) for the concentration of target cells, with corresponding exact and asymptotic confidence intervals. Exact and asymptotic goodness of fit p-values, and a bias-corrected (BC) MLE are also provided. No other publicly available software currently implements the BC MLE or the exact methods. For validation of SLDAssay, results from Myers et al. (1994) are replicated. Simulations demonstrate the BC MLE is less biased than the MLE. Additionally, simulations demonstrate that exact methods tend to give better confidence interval coverage and goodness-of-fit tests with lower type I error than the asymptotic methods. Additional advantages of using exact methods are also discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Hensley, D.C.

Nuclear Fuel Services sent more than 800 drums of nuclear waste to Oak Ridge National Laboratory, with the majority of the waste packaged into five different waste matrix types. A thorough and complete assay of the waste was performed at both NFS and at ORNL. A detailed comparing of the two assay sets provides valuable. insights into problems encountered in typical assay campaigns, particularly as there is, for the most part, excellent agreement between these two campaigns.

Telomere Chromatin Condensation Assay (TCCA): a novel approach to study structural telomere integrity.

PubMed

Gonzalez-Vasconcellos, Iria; Alonso-Rodríguez, Silvia; López-Baltar, Isidoro; Fernández, José Luis

2015-01-01

Telomeres, the DNA-protein complexes located at the end of linear eukaryotic chromosomes are essential for genome stability. Improper higher-order chromatin organization at the chromosome ends can give rise to telomeric recombination and genomic instability. We report the development of an assay to quantify differences in the condensation of telomeric chromatin, thereby offering new opportunities to study telomere biology and stability. We have combined a DNA nuclease digestion with a quantitative PCR (qPCR) assay of telomeric DNA, which we term the Telomere Chromatin Condensation Assay (TCCA). By quantifying the relative quantities of telomeric DNA that are progressively digested with the exonuclease Bal 31 the method can discriminate between different levels of telomeric chromatin condensation. The structural chromatin packaging at telomeres shielded against exonuclease digestion delivered an estimate, which we term Chromatin Protection Factor (CPF) that ranged from 1.7 to 2.3 fold greater than that present in unpacked DNA. The CPF was significantly decreased when cell cultures were incubated with the DNA hypomethylating agent 5-azacytidine, demonstrating the ability of the TCCA assay to discriminate between packaging levels of telomeric DNA. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression of human argininosuccinate synthetase after retroviral-mediated gene transfer.

PubMed

Wood, P A; Partridge, C A; O'Brien, W E; Beaudet, A L

1986-09-01

The cDNA sequence for human argininosuccinate synthetase (AS) was introduced into plasmid expression vectors with an SV40 promoter or Rous sarcoma virus promoter to construct pSV2-AS and pRSV-AS, respectively, and human enzyme was synthesized after gene transfer into Chinese hamster cells. The functional cDNA was inserted into the retroviral vectors pZIP-NeoSV(X) and pZIP-NeoSV(B). Ecotropic AS retrovirus was produced after calcium-phosphate-mediated gene transfer of these constructions into the packaging cell line psi-2, and viral titers up to 10(5) CFU/ml were obtained. Recombinant AS retrovirus was evaluated by detecting G-418-resistant colonies after infection of the rodent cells, XC, NRK, and 3T3. Colonies were also obtained when infected XC cells were selected in citrulline medium for expression of AS activity. Southern blot analysis of infected cells demonstrated that the recombinant retroviral genome was not altered grossly after infecting some rodent cells, while other cells showed evidence of rearrangement. A rapid assay for detecting AS retrovirus was developed based on the incorporation of [14C]citrulline into protein by intact 3T3 cells or XC cells.

Fluidics platform and method for sample preparation and analysis

DOEpatents

Benner, W. Henry; Dzenitis, John M.; Bennet, William J.; Baker, Brian R.

2014-08-19

Herein provided are fluidics platform and method for sample preparation and analysis. The fluidics platform is capable of analyzing DNA from blood samples using amplification assays such as polymerase-chain-reaction assays and loop-mediated-isothermal-amplification assays. The fluidics platform can also be used for other types of assays and analyzes. In some embodiments, a sample in a sealed tube can be inserted directly. The following isolation, detection, and analyzes can be performed without a user's intervention. The disclosed platform may also comprises a sample preparation system with a magnetic actuator, a heater, and an air-drying mechanism, and fluid manipulation processes for extraction, washing, elution, assay assembly, assay detection, and cleaning after reactions and between samples.

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity

PubMed Central

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-01-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3’untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3’ UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3’UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3’UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3’ UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity. PMID:26646790

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

PubMed

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-12-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity.

Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

PubMed

Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

2017-01-01

Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

Prospective clinical evaluation of the Polyperf® Safe, a safety Huber needle, in cancer patients.

PubMed

Goossens, Godelieve A; Moons, Philip; Jérôme, Martine; Stas, Marguerite

2011-01-01

Evaluation of the Polyperf® Safe (PPS) needle on safety and user-friendliness, as experienced by first-time and non-first-time users of the device. A prospective, descriptive study was carried out at the University Hospitals Leuven, Belgium. Five hundred PPS needles were individually evaluated in cancer patients. Different aspects of the PPS were assessed: packaging, needle insertion, and needle removal. Nurses were asked whether they had previously inserted or removed this type of needle. We compared the PPS needle with the standard Gripper® needle in terms of safety, ease of use, and ease of training. Three hundred sixty-six evaluation forms were available for analysis (73.2%). Packaging and access evaluations were scored positively, except for two aspects: (1) needle stability, and (2) ease of dressing. Ease of removal was scored unsatisfactory in up to 22.4% of the registrations. Pain at insertion was reported in about 20% registrations, and blood contact was reported by 2.5% of non-first-time users. Safety was scored as good, although ease of use and ease in training scored 25.4% and 43.8%, respectively, lower than the Gripper®. In general, nurses evaluated the PPS positively, with the exception of needle stability, ease of dressing, and ease of removal. No needlestick accidents were recorded. Aspects of ease of use and ease of training for PPS needles scored less than those for the Gripper® needles in up to one-third of the registrations.

Genetic Control of Plant Root Colonization by the Biocontrol agent, Pseudomonas fluorescens

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

Cole, Benjamin J.; Fletcher, Meghan; Waters, Jordan

Plant growth promoting rhizobacteria (PGPR) are a critical component of plant root ecosystems. PGPR promote plant growth by solubilizing inaccessible minerals, suppressing pathogenic microorganisms in the soil, and directly stimulating growth through hormone synthesis. Pseudomonas fluorescens is a well-established PGPR isolated from wheat roots that can also colonize the root system of the model plant, Arabidopsis thaliana. We have created barcoded transposon insertion mutant libraries suitable for genome-wide transposon-mediated mutagenesis followed by sequencing (TnSeq). These libraries consist of over 105 independent insertions, collectively providing loss-of-function mutants for nearly all genes in the P.fluorescens genome. Each insertion mutant can be unambiguouslymore » identified by a randomized 20 nucleotide sequence (barcode) engineered into the transposon sequence. We used these libraries in a gnotobiotic assay to examine the colonization ability of P.fluorescens on A.thaliana roots. Taking advantage of the ability to distinguish individual colonization events using barcode sequences, we assessed the timing and microbial concentration dependence of colonization of the rhizoplane niche. These data provide direct insight into the dynamics of plant root colonization in an in vivo system and define baseline parameters for the systematic identification of the bacterial genes and molecular pathways using TnSeq assays. Having determined parameters that facilitate potential colonization of roots by thousands of independent insertion mutants in a single assay, we are currently establishing a genome-wide functional map of genes required for root colonization in P.fluorescens. Importantly, the approach developed and optimized here for P.fluorescens>A.thaliana colonization will be applicable to a wide range of plant-microbe interactions, including biofuel feedstock plants and microbes known or hypothesized to impact on biofuel-relevant traits including biomass productivity and pathogen resistance.« less

78 FR 63226 - GlaxoSmithKline LLC; Withdrawal of Approval of the Indication for Treatment of Patients With...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-10-23

... have not received prior rituximab, for BEXXAR (tositumomab and iodine I 131 tositumomab) Injection held... withdraw the rituximab-na[iuml]ve indication for BEXXAR (tositumomab and iodine I 131 tositumomab... indication for BEXXAR (tositumomab and iodine I 131 tositumomab) Injection from the package insert. In the...

21 CFR 201.323 - Aluminum in large and small volume parenterals used in total parenteral nutrition.

Code of Federal Regulations, 2011 CFR

2011-04-01

... used in total parenteral nutrition. 201.323 Section 201.323 Food and Drugs FOOD AND DRUG ADMINISTRATION... parenteral nutrition. (a) The aluminum content of large volume parenteral (LVP) drug products used in total parenteral nutrition (TPN) therapy must not exceed 25 micrograms per liter (µg/L). (b) The package insert of...

21 CFR 201.323 - Aluminum in large and small volume parenterals used in total parenteral nutrition.

Code of Federal Regulations, 2012 CFR

2012-04-01

... used in total parenteral nutrition. 201.323 Section 201.323 Food and Drugs FOOD AND DRUG ADMINISTRATION... parenteral nutrition. (a) The aluminum content of large volume parenteral (LVP) drug products used in total parenteral nutrition (TPN) therapy must not exceed 25 micrograms per liter (µg/L). (b) The package insert of...

21 CFR 201.323 - Aluminum in large and small volume parenterals used in total parenteral nutrition.

Code of Federal Regulations, 2013 CFR

2013-04-01

... used in total parenteral nutrition. 201.323 Section 201.323 Food and Drugs FOOD AND DRUG ADMINISTRATION... parenteral nutrition. (a) The aluminum content of large volume parenteral (LVP) drug products used in total parenteral nutrition (TPN) therapy must not exceed 25 micrograms per liter (µg/L). (b) The package insert of...

21 CFR 201.323 - Aluminum in large and small volume parenterals used in total parenteral nutrition.

Code of Federal Regulations, 2010 CFR

2010-04-01

... used in total parenteral nutrition. 201.323 Section 201.323 Food and Drugs FOOD AND DRUG ADMINISTRATION... parenteral nutrition. (a) The aluminum content of large volume parenteral (LVP) drug products used in total parenteral nutrition (TPN) therapy must not exceed 25 micrograms per liter (µg/L). (b) The package insert of...

21 CFR 201.323 - Aluminum in large and small volume parenterals used in total parenteral nutrition.

Code of Federal Regulations, 2014 CFR

2014-04-01

... used in total parenteral nutrition. 201.323 Section 201.323 Food and Drugs FOOD AND DRUG ADMINISTRATION... parenteral nutrition. (a) The aluminum content of large volume parenteral (LVP) drug products used in total parenteral nutrition (TPN) therapy must not exceed 25 micrograms per liter (µg/L). (b) The package insert of...

GSFC Cutting Edge Avionics Technologies for Spacecraft

NASA Technical Reports Server (NTRS)

Luers, Philip J.; Culver, Harry L.; Plante, Jeannette

1998-01-01

With the launch of NASA's first fiber optic bus on SAMPEX in 1992, GSFC has ushered in an era of new technology development and insertion into flight programs. Predating such programs the Lewis and Clark missions and the New Millenium Program, GSFC has spearheaded the drive to use cutting edge technologies on spacecraft for three reasons: to enable next generation Space and Earth Science, to shorten spacecraft development schedules, and to reduce the cost of NASA missions. The technologies developed have addressed three focus areas: standard interface components, high performance processing, and high-density packaging techniques enabling lower cost systems. To realize the benefits of standard interface components GSFC has developed and utilized radiation hardened/tolerant devices such as PCI target ASICs, Parallel Fiber Optic Data Bus terminals, MIL-STD-1773 and AS1773 transceivers, and Essential Services Node. High performance processing has been the focus of the Mongoose I and Mongoose V rad-hard 32-bit processor programs as well as the SMEX-Lite Computation Hub. High-density packaging techniques have resulted in 3-D stack DRAM packages and Chip-On-Board processes. Lower cost systems have been demonstrated by judiciously using all of our technology developments to enable "plug and play" scalable architectures. The paper will present a survey of development and insertion experiences for the above technologies, as well as future plans to enable more "better, faster, cheaper" spacecraft. Details of ongoing GSFC programs such as Ultra-Low Power electronics, Rad-Hard FPGAs, PCI master ASICs, and Next Generation Mongoose processors.

A High-Throughput Arabidopsis Reverse Genetics System

PubMed Central

Sessions, Allen; Burke, Ellen; Presting, Gernot; Aux, George; McElver, John; Patton, David; Dietrich, Bob; Ho, Patrick; Bacwaden, Johana; Ko, Cynthia; Clarke, Joseph D.; Cotton, David; Bullis, David; Snell, Jennifer; Miguel, Trini; Hutchison, Don; Kimmerly, Bill; Mitzel, Theresa; Katagiri, Fumiaki; Glazebrook, Jane; Law, Marc; Goff, Stephen A.

2002-01-01

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from ∼100,000 transformed lines. A total of 85,108 TAIL-PCR products from 52,964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org. PMID:12468722

Cytotoxicity and mutagenicity studies on migration extracts from nanocomposites with potential use in food packaging.

PubMed

Maisanaba, Sara; Pichardo, Silvia; Jordá-Beneyto, María; Aucejo, Susana; Cameán, Ana M; Jos, Ángeles

2014-04-01

Clays are used in the food packaging industry to obtain nanocomposites. The use of these new materials is a concern, because they could reach consumers by oral exposure through possible migration, and potential toxic effects could be derived. In the present study, several in vitro basal cytotoxicity and mutagenicity tests on migration extracts obtained from a nanocomposite material with poly (lactic) acid (PLA) and two modified clays, Clay1 and Clay2, are shown. Migration extracts in distilled water showed values of 0.1 ± 0.2mg/dm(2) in all samples. Also, the content of characteristic metals of the clays structure (Al, Ca, Mg, Fe, Si) was studied and no statistical differences were observed. For the cytotoxicity assays, the human intestinal Caco-2 and human liver HepG2 cells were selected. Cells were exposed to concentrations between 2.5% and 100% extracts determining three different biomarkers of cellular viability. No significant differences were observed in the cytotoxicity assays. Finally, mutagenicity was evaluated by the Ames test and resulted in the absence of mutagenic response at all the concentrations assayed. Taking in account all above mentioned, these new materials show a good profile for their use in food packaging although further research is still needed. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mistaken identity of an open reading frame proposed for PCR-based identification of Mycoplasma bovis and the effect of polymorphisms and insertions on assay performance

USDA-ARS?s Scientific Manuscript database

Mycoplasma bovis is an important cause of disease in cattle and bison. Because the bacterium requires specialized growth conditions many diagnostic laboratories routinely use PCR to replace or complement conventional isolation and identification methods. A frequently used target of such assays is th...

RNA secondary structures of the bacteriophage phi6 packaging regions.

PubMed

Pirttimaa, M J; Bamford, D H

2000-06-01

Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models for the pac sites of phi6 single-stranded RNA segments. Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons. Previously reported pac site deletion studies are also discussed. Each pac site possesses a unique architecture, that, however, contains common structural elements.

Structural constraints in the packaging of bluetongue virus genomic segments

PubMed Central

Burkhardt, Christiane; Sung, Po-Yu; Celma, Cristina C.

2014-01-01

The mechanism used by bluetongue virus (BTV) to ensure the sorting and packaging of its 10 genomic segments is still poorly understood. In this study, we investigated the packaging constraints for two BTV genomic segments from two different serotypes. Segment 4 (S4) of BTV serotype 9 was mutated sequentially and packaging of mutant ssRNAs was investigated by two newly developed RNA packaging assay systems, one in vivo and the other in vitro. Modelling of the mutated ssRNA followed by biochemical data analysis suggested that a conformational motif formed by interaction of the 5′ and 3′ ends of the molecule was necessary and sufficient for packaging. A similar structural signal was also identified in S8 of BTV serotype 1. Furthermore, the same conformational analysis of secondary structures for positive-sense ssRNAs was used to generate a chimeric segment that maintained the putative packaging motif but contained unrelated internal sequences. This chimeric segment was packaged successfully, confirming that the motif identified directs the correct packaging of the segment. PMID:24980574

Circular permutant GFP insertion folding reporters

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2008-06-24

Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

Circular permutant GFP insertion folding reporters

DOEpatents

Waldo, Geoffrey S; Cabantous, Stephanie

2013-02-12

Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

Circular permutant GFP insertion folding reporters

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2011-06-14

Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

Circular permutant GFP insertion folding reporters

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2013-04-16

Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

MEMS Direct Chip Attach Packaging Methodologies and Apparatuses for Harsh Environments

NASA Technical Reports Server (NTRS)

Okojie, Robert S. (Inventor)

2009-01-01

Methods of bulk manufacturing high temperature sensor subassembly packages are disclosed and claimed. Sensors are sandwiched between a top cover and a bottom cover so as to enable the peripheries of the top covers, sensors and bottom covers to be sealed and bound securely together are disclosed and claimed. Sensors are placed on the bottom covers leaving the periphery of the bottom cover exposed. Likewise, top covers are placed on the sensors leaving the periphery of the sensor exposed. Individual sensor sub-assemblies are inserted into final packaging elements which are also disclosed and claimed. Methods of directly attaching wires or pins to contact pads on the sensors are disclosed and claimed. Sensors, such as pressure sensors and accelerometers, and headers made out of silicon carbide and aluminum nitride are disclosed and claimed. Reference cavities are formed in some embodiments disclosed and claimed herein where top covers are not employed.

MEMS Direct Chip Attach Packaging Methodologies and Apparatuses for Harsh Environments

NASA Technical Reports Server (NTRS)

Okojie, Robert S. (Inventor)

2005-01-01

Methods of bulk manufacturing high temperature sensor sub-assembly packages are disclosed and claimed. Sensors are sandwiched between a top cover and a bottom cover so as to enable the peripheries of the top covers, sensors and bottom covers to be sealed and bound securely together are disclosed and claimed. Sensors are placed on the bottom covers leaving the periphery of the bottom cover exposed. Likewise, top covers are placed on the sensors leaving the periphery of the sensor exposed. Individual sensor sub- assemblies are inserted into final packaging elements which are also disclosed and claimed. Methods of directly attach- ing wires or pins to contact pads on the sensors are disclosed and claimed. Sensors, such as pressure sensors and accelerometers, and headers made out of silicon carbide and aluminum nitride are disclosed and claimed. Reference cavities are formed in some embodiments disclosed and claimed herein where top covers are not employed.

The combined effects of storage temperature and packaging on the sensory, chemical, and physical properties of a Cabernet Sauvignon wine.

PubMed

Hopfer, Helene; Buffon, Peter A; Ebeler, Susan E; Heymann, Hildegarde

2013-04-03

A Californian Cabernet Sauvignon was stored for 6 months at three different constant temperatures to study the combined effects of storage temperature and packaging configuration. Glass bottles with natural cork, synthetic cork, and screw cap closure, as well as two Bag-in-Box treatments, were used in the experiment. A trained sensory panel was able to detect significant changes in aroma, flavor, taste, mouthfeel, and color attributes among the samples, differences that were found also with various chemical and physical measurements (volatile profile, polyphenol pattern, enological parameters, color space). Additionally, two commonly used polyphenol assays were compared to each other in terms of their ability to detect the changes in the polyphenol profile. Generally, sample changes were more pronounced due to the different storage temperatures, with 30 sensory attributes differing significantly among the three different storage temperatures, while only 17 sensory attributes showed a significant packaging effect. With increasing storage temperature the packaging effect became more pronounced, resulting in the largest changes in the Bag-in-Box samples stored at the highest temperature of 40 °C. At the highest storage temperature, all wines showed oxidized characters, independent of the wine packaging configurations, but to a varying degree. Generally, wines that received highest oxygen amounts and storage temperatures were much lighter, less red, and more brown-yellow at the end of the 6-month storage period, compared to their counterparts stored at 10 °C. These changes in color and polyphenols, respectively, were also detected with the two spectrophotometric assays. With increasing storage temperature both assays measured reduced concentrations in total phenols and total anthocyanins, while total tannins, degree of ionized anthocyanins, and color density increased. Various volatile compounds differed significantly among the samples, with largest relative concentration changes in acetates, organic acids, and alcohols, in good agreement with previous literature reports, with some being well correlated to specific sensory attributes too; for example, various acetates correlated to cherry and fruit aromas and flavors. The study shows that storage at elevated temperatures could be a valuable tool for wine packaging screening and testing new and improved wine packaging types under the worst conditions, which are unfortunately not unrealistic.

High-energy metal air batteries

DOEpatents

Zhang, Ji-Guang; Xiao, Jie; Xu, Wu; Wang, Deyu; Williford, Ralph E.; Liu, Jun

2014-07-01

Disclosed herein are embodiments of lithium/air batteries and methods of making and using the same. Certain embodiments are pouch-cell batteries encased within an oxygen-permeable membrane packaging material that is less than 2% of the total battery weight. Some embodiments include a hybrid air electrode comprising carbon and an ion insertion material, wherein the mass ratio of ion insertion material to carbon is 0.2 to 0.8. The air electrode may include hydrophobic, porous fibers. In particular embodiments, the air electrode is soaked with an electrolyte comprising one or more solvents including dimethyl ether, and the dimethyl ether subsequently is evacuated from the soaked electrode. In other embodiments, the electrolyte comprises 10-20% crown ether by weight.

High-energy metal air batteries

DOEpatents

Zhang, Ji-Guang; Xiao, Jie; Xu, Wu; Wang, Deyu; Williford, Ralph E.; Liu, Jun

2013-07-09

Disclosed herein are embodiments of lithium/air batteries and methods of making and using the same. Certain embodiments are pouch-cell batteries encased within an oxygen-permeable membrane packaging material that is less than 2% of the total battery weight. Some embodiments include a hybrid air electrode comprising carbon and an ion insertion material, wherein the mass ratio of ion insertion material to carbon is 0.2 to 0.8. The air electrode may include hydrophobic, porous fibers. In particular embodiments, the air electrode is soaked with an electrolyte comprising one or more solvents including dimethyl ether, and the dimethyl ether subsequently is evacuated from the soaked electrode. In other embodiments, the electrolyte comprises 10-20% crown ether by weight.

Botulism challenge studies of a modified atmosphere package for fresh mussels: inoculated pack studies.

PubMed

Newell, C R; Ma, Li; Doyle, Michael

2012-06-01

A series of botulism challenge studies were performed to determine the possibility of production of botulinum toxin in mussels (Mytilus edulis) held under a commercial high-oxygen (60 to 65% O(2)), modified atmosphere packaging (MAP) condition. Spore mixtures of six strains of nonproteolytic Clostridium botulinum were introduced into mussel MAP packages receiving different packaging buffers with or without the addition of lactic acid bacteria. Dye studies and package flipping trials were conducted to ensure internalization of spores by packed mussels. Inoculated mussel packages were stored at normal (4°C) and abusive (12°C) temperatures for 21 and 13 days, respectively, which were beyond the packaged mussels' intended shelf life. Microbiological and chemical analyses were conducted at predetermined intervals (a total of five sampling times at each temperature), including total aerobic plate counts, C. botulinum counts, lactic acid bacterial counts, package headspace gas composition, pH of packaging buffer and mussel meat, and botulinum toxin assays of packaging buffer and mussel meat. Results revealed that C. botulinum inoculated in fresh mussels packed under MAP packaging did not produce toxin, even at an abusive storage temperature and when held beyond their shelf life. No evidence was found that packaging buffers or gas composition influenced the lack of botulinum toxin production in packed mussels.

LDRD final report on microencapsulated immunoreagents for development of one-step ELISA

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

Henderson, C.C.; Singh, A.K.

1997-08-01

Microencapsulation of biological macromolecules was investigated as a method for incorporating the necessary immunoreagents into an improved enzyme-linked immunosorbant assay (ELISA) package that would self-develop. This self-contained ELISA package would eliminate the need for a trained technician to perform multiple additions of immunoreagent to the assay. Microencapsulation by insolution drying was selected from the many available microencapsulation methods, and two satisfactory procedures for microencapsulation of proteins were established. The stability and potential for rapid release of protein from these microencapsulates was then evaluated. The results suggest that the chosen method for protein entrapment produces microcapsules with a considerable amount ofmore » protein in the walls making these particular microcapsules unsuitable for their intended use.« less

MEMS Reliability Assurance Guidelines for Space Applications

NASA Technical Reports Server (NTRS)

Stark, Brian (Editor)

1999-01-01

This guide is a reference for understanding the various aspects of microelectromechanical systems, or MEMS, with an emphasis on device reliability. Material properties, failure mechanisms, processing techniques, device structures, and packaging techniques common to MEMS are addressed in detail. Design and qualification methodologies provide the reader with the means to develop suitable qualification plans for the insertion of MEMS into the space environment.

ATACseqQC: a Bioconductor package for post-alignment quality assessment of ATAC-seq data.

PubMed

Ou, Jianhong; Liu, Haibo; Yu, Jun; Kelliher, Michelle A; Castilla, Lucio H; Lawson, Nathan D; Zhu, Lihua Julie

2018-03-01

ATAC-seq (Assays for Transposase-Accessible Chromatin using sequencing) is a recently developed technique for genome-wide analysis of chromatin accessibility. Compared to earlier methods for assaying chromatin accessibility, ATAC-seq is faster and easier to perform, does not require cross-linking, has higher signal to noise ratio, and can be performed on small cell numbers. However, to ensure a successful ATAC-seq experiment, step-by-step quality assurance processes, including both wet lab quality control and in silico quality assessment, are essential. While several tools have been developed or adopted for assessing read quality, identifying nucleosome occupancy and accessible regions from ATAC-seq data, none of the tools provide a comprehensive set of functionalities for preprocessing and quality assessment of aligned ATAC-seq datasets. We have developed a Bioconductor package, ATACseqQC, for easily generating various diagnostic plots to help researchers quickly assess the quality of their ATAC-seq data. In addition, this package contains functions to preprocess aligned ATAC-seq data for subsequent peak calling. Here we demonstrate the utilities of our package using 25 publicly available ATAC-seq datasets from four studies. We also provide guidelines on what the diagnostic plots should look like for an ideal ATAC-seq dataset. This software package has been used successfully for preprocessing and assessing several in-house and public ATAC-seq datasets. Diagnostic plots generated by this package will facilitate the quality assessment of ATAC-seq data, and help researchers to evaluate their own ATAC-seq experiments as well as select high-quality ATAC-seq datasets from public repositories such as GEO to avoid generating hypotheses or drawing conclusions from low-quality ATAC-seq experiments. The software, source code, and documentation are freely available as a Bioconductor package at https://bioconductor.org/packages/release/bioc/html/ATACseqQC.html .

Rationale for patient package inserts.

PubMed

Morris, L A

1978-02-01

The need for patient package inserts (PPIs) is discussed in terms of the "consumers' right-to-know" and "patient health education" themes, as well as the premise that verbal means of communication are often inadequate. Some of the reasons for the frequent failure of verbal messages are reviewed by detailing the five stages through which any communication must be processed in order to be effective. Verbal information may be inadequate because: (1) patients are not exposed to necessary material, (2) they may not pay attention to the information, (3) they may not understand it, (4) the information may not be accepted as valid, and (5) patients may not remember the material. Written communication which must be processed through the same five stages also may be frequently inadequate. Therefore, PPIs must be viewed as reinforcing and augmenting verbal consultation, and both written and verbal forms are necessary. Consumer surveys and demonstration projects have shown that written communications are a desired and effective means for conveying additional prescription drug information to patients. The wide implementation of PPIs is apt to cause many problems for the pharmacists; however, it may also serve as a means to enhance the pharmacist's clinical role.

Graphical content of medicinal package inserts: an exploratory study to evaluate potential legibility issues.

PubMed

Pires, Carla; Vigário, Marina; Cavaco, Afonso

2016-06-01

The graphical content of the Medicines Package Inserts (MPIs), such as illustrations and typographic features should be legible and appropriate, as required by international pharmaceutical regulations. To study: (1) the frequency and type of MPIs' key graphic elements, (2) their compliance with regulations and (3) how educated people understand them. Descriptive study: characterisation of the graphical content of 651 MPIs. Usability study: illustrations and tables (purposively selected) were evaluated with questionnaires in three groups of humanities undergraduates (illustrations only, illustrations plus text and text only). Descriptive study: illustrations and tables were respectively identified in 6.3% and 11.8% of the MPIs. The illustrations were mainly related to how to take/use the medicine. Non-recommended graphical representations were found (e.g. italic or underline). Usability test: legibility issues were identified, especially for the group of isolated illustrations. The scarce use of illustrations and tables possibly affected the legibility of the MPIs. Compulsory legibility tests are needed to guarantee the MPIs' proper use, thus contributing to a safe use of medicines. Overall, this study highlighted the need to carefully revise/assess the MPIs' design and probably increase health information experts' awareness on this issue. © 2015 Health Libraries Group.

Sex-specific 99th percentiles derived from the AACC Universal Sample Bank for the Roche Gen 5 cTnT assay: Comorbidities and statistical methods influence derivation of reference limits.

PubMed

Gunsolus, Ian L; Jaffe, Allan S; Sexter, Anne; Schulz, Karen; Ler, Ranka; Lindgren, Brittany; Saenger, Amy K; Love, Sara A; Apple, Fred S

2017-12-01

Our purpose was to determine a) overall and sex-specific 99th percentile upper reference limits (URL) and b) influences of statistical methods and comorbidities on the URLs. Heparin plasma from 838 normal subjects (423 men, 415 women) were obtained from the AACC (Universal Sample Bank). The cobas e602 measured cTnT (Roche Gen 5 assay); limit of detection (LoD), 3ng/L. Hemoglobin A1c (URL 6.5%), NT-proBNP (URL 125ng/L) and eGFR (60mL/min/1.73m 2 ) were measured, along with identification of statin use, to better define normality. 99th percentile URLs were determined by the non-parametric (NP), Harrell-Davis Estimator (HDE) and Robust (R) methods. 355 men and 339 women remained after exclusions. Overall<50% of subjects had measureable concentrations ≥ LoD: 45.6% no exclusion, 43.5% after exclusion; compared to men: 68.1% no exclusion, 65.1% post exclusion; women: 22.7% no exclusion, 20.9% post exclusion. The statistical method used influenced URLs as follows: pre/post exclusion overall, NP 16/16ng/L, HDE 17/17ng/L, R not available; men NP 18/16ng/L, HDE 21/19ng/L, R 16/11ng/L; women NP 13/10ng/L, HDE 14/14ng/L, R not available. We demonstrated that a) the Gen 5 cTnT assay does not meet the IFCC guideline for high-sensitivity assays, b) surrogate biomarkers significantly lowers the URLs and c) statistical methods used impact URLs. Our data suggest lower sex-specific cTnT 99th percentiles than reported in the FDA approved package insert. We emphasize the importance of detailing the criteria used to include and exclude subjects for defining a healthy population and the statistical method used to calculate 99th percentiles and identify outliers. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Sequence variations of the partially dominant DELLA gene Rht-B1c in wheat and their functional impacts

PubMed Central

Ma, Zhengqiang

2013-01-01

Rht-B1c, allelic to the DELLA protein-encoding gene Rht-B1a, is a natural mutation documented in common wheat (Triticum aestivum). It confers variation to a number of traits related to cell and plant morphology, seed dormancy, and photosynthesis. The present study was conducted to examine the sequence variations of Rht-B1c and their functional impacts. The results showed that Rht-B1c was partially dominant or co-dominant for plant height, and exhibited an increased dwarfing effect. At the sequence level, Rht-B1c differed from Rht-B1a by one 2kb Veju retrotransposon insertion, three coding region single nucleotide polymorphisms (SNPs), one 197bp insertion, and four SNPs in the 1kb upstream sequence. Haplotype investigations, association analyses, transient expression assays, and expression profiling showed that the Veju insertion was primarily responsible for the extreme dwarfing effect. It was found that the Veju insertion changed processing of the Rht-B1c transcripts and resulted in DELLA motif primary structure disruption. Expression assays showed that Rht-B1c caused reduction of total Rht-1 transcript levels, and up-regulation of GATA-like transcription factors and genes positively regulated by these factors, suggesting that one way in which Rht-1 proteins affect plant growth and development is through GATA-like transcription factor regulation. PMID:23918966

Software for the Integration of Multiomics Experiments in Bioconductor.

PubMed

Ramos, Marcel; Schiffer, Lucas; Re, Angela; Azhar, Rimsha; Basunia, Azfar; Rodriguez, Carmen; Chan, Tiffany; Chapman, Phil; Davis, Sean R; Gomez-Cabrero, David; Culhane, Aedin C; Haibe-Kains, Benjamin; Hansen, Kasper D; Kodali, Hanish; Louis, Marie S; Mer, Arvind S; Riester, Markus; Morgan, Martin; Carey, Vince; Waldron, Levi

2017-11-01

Multiomics experiments are increasingly commonplace in biomedical research and add layers of complexity to experimental design, data integration, and analysis. R and Bioconductor provide a generic framework for statistical analysis and visualization, as well as specialized data classes for a variety of high-throughput data types, but methods are lacking for integrative analysis of multiomics experiments. The MultiAssayExperiment software package, implemented in R and leveraging Bioconductor software and design principles, provides for the coordinated representation of, storage of, and operation on multiple diverse genomics data. We provide the unrestricted multiple 'omics data for each cancer tissue in The Cancer Genome Atlas as ready-to-analyze MultiAssayExperiment objects and demonstrate in these and other datasets how the software simplifies data representation, statistical analysis, and visualization. The MultiAssayExperiment Bioconductor package reduces major obstacles to efficient, scalable, and reproducible statistical analysis of multiomics data and enhances data science applications of multiple omics datasets. Cancer Res; 77(21); e39-42. ©2017 AACR . ©2017 American Association for Cancer Research.

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

PubMed

Torrent, C; Gabus, C; Darlix, J L

1994-02-01

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.

A fully wafer-level packaged RF MEMS switch with low actuation voltage using a piezoelectric actuator

NASA Astrophysics Data System (ADS)

Park, Jae-Hyoung; Lee, Hee-Chul; Park, Yong-Hee; Kim, Yong-Dae; Ji, Chang-Hyeon; Bu, Jonguk; Nam, Hyo-Jin

2006-11-01

In this paper, a fully wafer-level packaged RF MEMS switch has been demonstrated, which has low operation voltage, using a piezoelectric actuator. The piezoelectric actuator was designed to operate at low actuation voltage for application to advanced mobile handsets. The dc contact type RF switch was packaged using the wafer-level bonding process. The CPW transmission lines and piezoelectric actuators have been fabricated on separate wafers and assembled together by the wafer-level eutectic bonding process. A gold and tin composite was used for eutectic bonding at a low temperature of 300 °C. Via holes interconnecting the electrical contact pads through the wafer were filled completely with electroplated copper. The fully wafer-level packaged RF MEMS switch showed an insertion loss of 0.63 dB and an isolation of 26.4 dB at 5 GHz. The actuation voltage of the switch was 5 V. The resonant frequency of the piezoelectric actuator was 38.4 kHz and the spring constant of the actuator was calculated to be 9.6 N m-1. The size of the packaged SPST (single-pole single-through) switch was 1.2 mm × 1.2 mm including the packaging sealing rim. The effect of the proposed package structure on the RF performance was characterized with a device having CPW through lines and vertical feed lines excluding the RF switches. The measured packaging loss was 0.2 dB and the return loss was 33.6 dB at 5 GHz.

Differences in the Language and Design of Four PPIs for Valium. Technical Assistance Report No. 1.

ERIC Educational Resources Information Center

Redish, Janice C.

As part of the evaluation of four different versions of a patient package insert (PPI) for diazepam (Valium) created by the Food and Drug Administration (FDA), the content, organization, language, and design of the PPIs were compared. One PPI was a short prose piece with clear organization that did not particularly highlight warnings or, indeed,…

Development of an iron chelating polyethylene film for active packaging applications.

PubMed

Tian, Fang; Decker, Eric A; Goddard, Julie M

2012-02-29

Metal-promoted oxidation reactions are a major cause of food quality deterioration. Active packaging offers novel approaches to controlling such oxidation for the purpose of extending shelf life. Herein, we report modification of the surface of polyethylene (PE) films to possess metal chelating activity. Metal chelating carboxylic acids were introduced to the film surface using cross-linking agents [polyethylenimine (PEI) or ethylenediamine (ED)] to increase the number of available carboxylic acids. ATR-FTIR, contact angle, dye assay, and iron chelating assay were used to characterize changes in surface chemistry after each functionalization step. The chelator poly(acrylic acid) (PAA) was attached to the surface at a density of 9.12 ± 0.71 nmol carboxyl groups/cm², and exhibited an iron chelating activity. The results indicate that PAA-modified PE films might have a higher affinity to Fe³⁺ than Fe²⁺ with the optimum binding pH at 5.0. Such inexpensive active packaging materials are promising in food industry for the preservation of liquid and semiliquid food products and have application in heavy metal chelation therapy for biomedical materials as well.

Comparison between Roche and Xpert in HIV-1 RNA quantitation: A high concordance between the two techniques except for a CRF02_AG subtype variant with high viral load titters detected by Roche but undetected by Xpert.

PubMed

Avidor, Boaz; Matus, Natalia; Girshengorn, Shirley; Achsanov, Svetlana; Gielman, Simona; Zeldis, Irene; Schweitzer, Inbal; Adler, Amos; Turner, Dan

2017-08-01

HIV-1 viral load (VL) testing is important to predict viral progression and to monitor the response to antiretroviral therapy. New HIV-1 VL tests are continuously introduced to the market. Their performance is usually compared to Abbott and/or Roche HIV-1 VL assays, as reference. The Xpert HIV-1 VL test was recently introduced, but its performance compared to Roche has not been sufficiently studied. To compare the Xpert assay with Roche and to assess its use in the HIV clinical laboratory. A total of 383 plasma samples of HIV-1 infected patients previously tested by Roche, were retrospectively tested by Xpert to determine concordance between the two assays. Samples included a diversity of HIV-1 subtypes and a wide range of VLs. There was a high concordance between the two assays, except for a CRF02_AG subtype variant with high VL titters, that was detected by Roche but undetected by Xpert. The 5' long terminal repeat gene region of this virus, targeted by the Xpert assay, was amplified and sequenced. A 25 nucleotide insert was identified, but was unmatched to any known sequences of HIV-1. This particular insert, however could not explain the false-negativity by the Xpert assay. This study underlines the challenge to routine VL testing due to the high genetic diversity of HIV-1. Clinicians should, therefore be advised that a negative VL in cases where the clinical picture does not match the laboratory report, might in fact be, a false-negative result of the VL assay. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of ROP18 alleles in human toxoplasmosis.

PubMed

Sánchez, Víctor; de-la-Torre, Alejandra; Gómez-Marín, Jorge Enrique

2014-04-01

The role of the virulent gene ROP18 polymorphisms is not known in human toxoplasmosis. A total of 320 clinical samples were analyzed. In samples positive for ROP18 gene, we determined by an allele specific PCR, if patients got the upstream insertion positive ROP18 sequence Toxoplasma strain (mouse avirulent strain) or the upstream insertion negative ROP18 sequence Toxoplasma strain (mouse virulent strain). We designed an ELISA assay for antibodies against ROP18 derived peptides from the three major clonal lineages of Toxoplasma. 20 clinical samples were of quality for ROP18 allele analysis. In patients with ocular toxoplasmosis, a higher inflammatory reaction on eye was associated to a PCR negative result for the upstream region of ROP18. 23.3%, 33% and 16.6% of serums from individuals with ocular toxoplasmosis were positive for type I, type II and type III ROP18 derived peptides, respectively but this assay was affected by cross reaction. The absence of Toxoplasma ROP18 promoter insertion sequence in ocular toxoplasmosis was correlated with severe ocular inflammatory response. Determination of antibodies against ROP18 protein was not useful for serotyping in human toxoplasmosis. © 2013.

Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

PubMed Central

Cousins, Matthew M.; Donnell, Deborah; Eshleman, Susan H.

2013-01-01

We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology. PMID:23178437

Influence of the Thermal Conductivity of Thermally Conductive Plastics on the Thermal Distribution of an Light-Emitting Diode Headlight for Vehicles.

PubMed

Lee, Dong Kyu; Lee, Jae Min; Cho, Moon Uk; Park, Hyun Jung; Cha, Yu-Jung; Kim, Hyeong Jin; Kwak, Joon Seop

2018-09-01

This paper investigates the thermal distribution of an LED headlight for vehicles based on the thermal conductivity of thermally conductive plastics (TCP). In general, heat dissipation structures used for LED headlights are made from metallic materials. However, headlight structures made from TCP have not been investigated. The headlights made from TCP having a various thermal conductivity were fabricated by injection molding with and without a metal plate insert. The temperature characteristics were compared and analyzed using thermal simulations and measurement. The inserted metal in TCP greatly reduced the temperature at solder point, indicating that the fast heat dissipation from the high power LED package to TCP though the inserted metal is essential. The measured temperature at solder points decreased as the thermal conductivity of TCP increased, which is well matched to the simulation results. The measured temperature at the solder point was lower than 150 °C when the thermal conductivity of the TCP was 10 W/mK.

An alphavirus temperature-sensitive capsid mutant reveals stages of nucleocapsid assembly

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

Zheng, Yan, E-mail: yzheng15@students.kgi.edu; Kielian, Margaret, E-mail: margaret.kielian@einstein.yu.edu

2015-10-15

Alphaviruses have a nucleocapsid core composed of the RNA genome surrounded by an icosahedral lattice of capsid protein. An insertion after position 186 in the capsid protein produced a strongly temperature-sensitive growth phenotype. Even when the structural proteins were synthesized at the permissive temperature (28 °C), subsequent incubation of the cells at the non-permissive temperature (37 °C) dramatically decreased mutant capsid protein stability and particle assembly. Electron microscopy confirmed the presence of cytoplasmic nucleocapsids in mutant-infected cells cultured at the permissive temperature, but these nucleocapsids were not stable to sucrose gradient separation. In contrast, nucleocapsids isolated from mutant virus particlesmore » had similar stability to that of wildtype virus. Our data support a model in which cytoplasmic nucleocapsids go through a maturation step during packaging into virus particles. The insertion site lies in the interface between capsid proteins in the assembled nucleocapsid, suggesting the region where such a stabilizing transition occurs. - Highlights: • We characterize an alphavirus capsid insertion mutation. • These capsid mutants are highly temperature sensitive for growth. • The insertion affects nucleocapsid stability. • Results suggest that the nucleocapsid is stabilized during virus budding.« less

MIRA: An R package for DNA methylation-based inference of regulatory activity.

PubMed

Lawson, John T; Tomazou, Eleni M; Bock, Christoph; Sheffield, Nathan C

2018-03-01

DNA methylation contains information about the regulatory state of the cell. MIRA aggregates genome-scale DNA methylation data into a DNA methylation profile for independent region sets with shared biological annotation. Using this profile, MIRA infers and scores the collective regulatory activity for each region set. MIRA facilitates regulatory analysis in situations where classical regulatory assays would be difficult and allows public sources of open chromatin and protein binding regions to be leveraged for novel insight into the regulatory state of DNA methylation datasets. R package available on Bioconductor: http://bioconductor.org/packages/release/bioc/html/MIRA.html. nsheffield@virginia.edu.

Gas chromatography with mass spectrometry for the determination of phthalates preconcentrated by microextraction based on an ionic liquid.

PubMed

Cacho, Juan Ignacio; Campillo, Natalia; Viñas, Pilar; Hernández-Córdoba, Manuel

2017-03-01

A new procedure is proposed for the analysis of migration test solutions obtained from plastic bottles used in the packaging of edible oils. Ultrasound-assisted emulsification microextraction with ionic liquids was applied for the preconcentration of six phthalate esters: dimethylphthalate, diethylphthalate, di-n-butylphthalate, n-butylbenzylphthalate, di-2-ethylhexylphthalate, and di-n-octylphthalate. The enriched ionic liquid was directly analyzed by gas chromatography and mass spectrometry using direct insert microvial thermal desorption. The different factors affecting the microextraction efficiency, such as volume of the extracting phase (30 μL of the ionic liquid) and ultrasound application time (25 s), and the thermal desorption step, such as desorption temperature and time, and gas flow rate, were studied. Under the selected conditions, detection limits for the analytes were in the 0.012-0.18 μg/L range, while recovery assays provided values ranging from 80 to 112%. The use of butyl benzoate as internal standard increased the reproducibility of the analytical procedure. When the release of the six phthalate esters from the tested plastic bottles to liquid simulants was monitored using the optimized procedure, analyte concentrations of between 1.0 and 273 μg/L were detected. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A powerful enhancement to the DMAP alter capability

NASA Technical Reports Server (NTRS)

Pamidi, P. R.

1989-01-01

A powerful enhancement to the DMAP alter capability was developed and is available on all RPK-supported versions of COSMIC/NASTRAN. This enhancement involves the addition of two alter control cards, called INSERT and DELETE, to the Executive Control Deck. These cards allow for DMAP alters to be made by referencing DMAP statements by their module names rather than by their statement numbers in the rigid format DMAP sequence. This allows for increased user convenience and flexibility and makes alters more meaningful to the user. In addition, DMAP alter packages employing the alter control cards will be much less susceptible to future changes in rigid format DMAPs than alter packages employing the standard ALTER control cards. The usage of the cards is illustrated by examples.

A Knock-in Reporter for a Novel AR-Targeted Therapy

DTIC Science & Technology

2016-05-01

of this research is to explore a possibility whether the CRISPR -Cas9 technology, an emerging genome-editing approach, could be applied to develop a...in this report that the CRISPR -Cas9 system could indeed mediate high-efficient insertion of a selection gene into a site immediately downstream of...inhibitory for AR expression. 15. SUBJECT TERMS Androgen receptor, high-throughput drug screening assay, reporter gene assay, CRISPR -Cas9, genome editing

High-throughput microtitre plate-based assay for DNA topoisomerases.

PubMed

Taylor, James A; Burton, Nicolas P; Maxwell, Anthony

2012-01-01

We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of DNA topoisomerases. The assay utilizes intermolecular triplex formation between an immobilized triplex-forming oligo (TFO) and a triplex-forming region inserted into the plasmid substrate (pNO1), and capitalizes on the observation that supercoiled DNA forms triplexes more readily than relaxed DNA. Thus, supercoiled DNA is preferentially retained by the TFO under triplex-forming conditions while relaxed DNA can be washed away. Due to its high speed of sample analysis and reduced sample handling over conventional gel-based techniques, this assay can be used to screen chemical libraries for novel inhibitors of topoisomerases.

Unlocking Short Read Sequencing for Metagenomics

DOE PAGES

Rodrigue, Sébastien; Materna, Arne C.; Timberlake, Sonia C.; ...

2010-07-28

We describe an experimental and computational pipeline yielding millions of reads that can exceed 200 bp with quality scores approaching that of traditional Sanger sequencing. The method combines an automatable gel-less library construction step with paired-end sequencing on a short-read instrument. With appropriately sized library inserts, mate-pair sequences can overlap, and we describe the SHERA software package that joins them to form a longer composite read.

Consumer and health literacy: The need to better design tobacco-cessation product packaging, labels, and inserts.

PubMed

Weiss, Stephanie M; Smith-Simone, Stephanie Y

2010-03-01

Tobacco-cessation product packaging and instruction materials may not be appropriate for some smokers and may contribute to the underuse and misuse of evidence-based treatments. The dual goals of this project are to analyze literacy levels of Food and Drug Administration (FDA)-approved and non-approved tobacco-cessation product packaging, directions, and claims, and to identify and categorize claims found on product packaging. The Campaign for Tobacco Free Kids (CTFK) maintains the Quitting and Reducing Tobacco Use Inventory of Products (QuiTIP) database, which catalogs products marketed and sold to consumers to reduce or quit use of tobacco products. It also includes all medications approved by the FDA for tobacco cessation as well as a sample of non-approved products such as homeopathic, herbal, nutritional, or dietary supplements commonly marketed as either cessation aids or alternative tobacco/nicotine products. This paper assesses the reading levels required to understand product packaging, labeling, and instructions using the Simple Measure of Gobbledygook (SMOG) and identifies claims on the product package labels using standard qualitative methods. Key findings show that the average reading levels needed to understand instructions for both FDA-approved and non-approved cessation products are above the reading levels recommended to ensure maximum comprehension. Improving the packaging and directions of evidence-based tobacco-cessation products so that they are preferably at or below a fifth-grade reading level, along with using consumer-based design principles to develop packaging, may help smokers take advantage of and correctly use products that will greatly increase their chances of successful quitting. 2010 American Journal of Preventive Medicine. Published by Elsevier Inc. All rights reserved.

Reliability of CGA/LGA/HDI Package Board/Assembly (Final Report)

NASA Technical Reports Server (NTRS)

Ghaffaroam. Reza

2014-01-01

Package manufacturers are now offering commercial-off-the-shelf column grid array (COTS CGA) packaging technologies in high-reliability versions. Understanding the process and quality assurance (QA) indicators for reliability are important for low-risk insertion of these advanced electronics packages. The previous reports, released in January of 2012 and January of 2013, presented package test data, assembly information, and reliability evaluation by thermal cycling for CGA packages with 1752, 1517, 1509, and 1272 inputs/outputs (I/Os) and 1-mm pitch. It presented the thermal cycling (-55C either 100C or 125C) test results for up to 200 cycles. This report presents up to 500 thermal cycles with quality assurance and failure analysis evaluation represented by optical photomicrographs, 2D real time X-ray images, dye-and-pry photomicrographs, and optical/scanning electron Microscopy (SEM) cross-sectional images. The report also presents assembly challenge using reflowing by either vapor phase or rework station of CGA and land grid array (LGA) versions of three high I/O packages both ceramic and plastic configuration. A new test vehicle was designed having high density interconnect (HDI) printed circuit board (PCB) with microvia-in-pad to accommodate both LGA packages as well as a large number of fine pitch ball grid arrays (BGAs). The LGAs either were assembled onto HDI PCB as an LGA or were solder paste print and reflow first to form solder dome on pads before assembly. Both plastic BGAs with 1156 I/O and ceramic LGAs were assembled. It also presented the X-ray inspection results as well as failures due to 200 thermal cycles. Lessons learned on assembly of ceramic LGAs are also presented.

beachmat: A Bioconductor C++ API for accessing high-throughput biological data from a variety of R matrix types

PubMed Central

Pagès, Hervé

2018-01-01

Biological experiments involving genomics or other high-throughput assays typically yield a data matrix that can be explored and analyzed using the R programming language with packages from the Bioconductor project. Improvements in the throughput of these assays have resulted in an explosion of data even from routine experiments, which poses a challenge to the existing computational infrastructure for statistical data analysis. For example, single-cell RNA sequencing (scRNA-seq) experiments frequently generate large matrices containing expression values for each gene in each cell, requiring sparse or file-backed representations for memory-efficient manipulation in R. These alternative representations are not easily compatible with high-performance C++ code used for computationally intensive tasks in existing R/Bioconductor packages. Here, we describe a C++ interface named beachmat, which enables agnostic data access from various matrix representations. This allows package developers to write efficient C++ code that is interoperable with dense, sparse and file-backed matrices, amongst others. We evaluated the performance of beachmat for accessing data from each matrix representation using both simulated and real scRNA-seq data, and defined a clear memory/speed trade-off to motivate the choice of an appropriate representation. We also demonstrate how beachmat can be incorporated into the code of other packages to drive analyses of a very large scRNA-seq data set. PMID:29723188

beachmat: A Bioconductor C++ API for accessing high-throughput biological data from a variety of R matrix types.

PubMed

Lun, Aaron T L; Pagès, Hervé; Smith, Mike L

2018-05-01

Biological experiments involving genomics or other high-throughput assays typically yield a data matrix that can be explored and analyzed using the R programming language with packages from the Bioconductor project. Improvements in the throughput of these assays have resulted in an explosion of data even from routine experiments, which poses a challenge to the existing computational infrastructure for statistical data analysis. For example, single-cell RNA sequencing (scRNA-seq) experiments frequently generate large matrices containing expression values for each gene in each cell, requiring sparse or file-backed representations for memory-efficient manipulation in R. These alternative representations are not easily compatible with high-performance C++ code used for computationally intensive tasks in existing R/Bioconductor packages. Here, we describe a C++ interface named beachmat, which enables agnostic data access from various matrix representations. This allows package developers to write efficient C++ code that is interoperable with dense, sparse and file-backed matrices, amongst others. We evaluated the performance of beachmat for accessing data from each matrix representation using both simulated and real scRNA-seq data, and defined a clear memory/speed trade-off to motivate the choice of an appropriate representation. We also demonstrate how beachmat can be incorporated into the code of other packages to drive analyses of a very large scRNA-seq data set.

Variation in Prescribing Patterns and Therapeutic Drug Monitoring of Intravenous Busulfan in Pediatric Hematopoietic Cell Transplant Recipients

PubMed Central

McCune, Jeannine S.; Baker, K. Scott; Blough, David K.; Gamis, Alan; Bemer, Meagan J.; Kelton-Rehkopf, Megan C.; Winter, Laura; Barrett, Jeffrey S.

2016-01-01

Personalizing intravenous (IV) busulfan doses in children using therapeutic drug monitoring (TDM) is an integral component of hematopoietic cell transplant. The authors sought to characterize initial dosing and TDM of IV busulfan, along with factors associated with busulfan clearance, in 729 children who underwent busulfan TDM from December 2005 to December 2008. The initial IV busulfan dose in children weighing ≤12 kg ranged 4.8-fold, with only 19% prescribed the package insert dose of 1.1 mg/kg. In those children weighing >12 kg, the initial dose ranged 5.4-fold, and 79% were prescribed the package insert dose. The initial busulfan dose achieved the target exposure in only 24.3% of children. A wide range of busulfan exposures were targeted for children with the same disease (eg, 39 target busulfan exposures for the 264 children diagnosed with acute myeloid leukemia). Considerable heterogeneity exists regarding when TDM is conducted and the number of pharmacokinetic samples obtained. Busulfan clearance varied by age and dosing frequency but not by underlying disease. The authors’ group is currently evaluating how using population pharmacokinetics to optimize initial busulfan dose and TDM (eg, limited sampling schedule in conjunction with maximum a posteriori Bayesian estimation) may affect clinical outcomes in children. PMID:23444282

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

PubMed Central

Torrent, C; Gabus, C; Darlix, J L

1994-01-01

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer. Images PMID:8289369

Genes involved in Listeria monocytogenes biofilm formation at a simulated food processing plant temperature of 15 °C.

PubMed

Piercey, Marta J; Hingston, Patricia A; Truelstrup Hansen, Lisbeth

2016-04-16

Listeria monocytogenes is a pathogenic foodborne bacterium whose persistence in food processing environments is in part attributed to its biofilm formation. Most biofilm studies have been carried out at 30-37 °C rather than at temperatures found in the food processing plants (i.e., 10-20 °C). The objective of the present study was to mine for novel genes that contribute to L. monocytogenes biofilm formation at 15 °C using the random insertional mutagenesis approach. A library of 11,024 L. monocytogenes 568 (serotype 1/2a) Himar1 insertional mutants was created. Mutants with reduced or enhanced biofilm formation at 15 °C were detected in microtiter plate assays with crystal violet and safranin staining. Fourteen mutants expressed enhanced biofilm phenotypes, and harbored transposon insertions in genes encoding cell wall biosynthesis, motility, metabolism, stress response, and cell surface associated proteins. Deficient mutants (n=5) contained interruptions in genes related to peptidoglycan, teichoic acid, or lipoproteins. Enhanced mutants produced significantly (p<0.05) higher cell densities in biofilm formed on stainless steel (SS) coupons at 15 °C (48 h) than deficient mutants, which were also more sensitive to benzalkonium chloride. All biofilm deficient mutants and four enhanced mutants in the microtiter plate assay (flaA, cheR, lmo2563 and lmo2488) formed no biofilm in a peg lid assay (Calgary biofilm device) while insertions in lmo1224 and lmo0543 led to excess biofilm in all assays. Two enhanced biofilm formers were more resistant to enzymatic removal with DNase, proteinase K or pectinase than the parent strain. Scanning electron microscopy of individual biofilms made by five mutants and the parent on SS surfaces showed formation of heterogeneous biofilm with dense zones by immotile mutants, while deficient mutants exhibited sparse growth. In conclusion, interruptions of 9 genes not previously linked to biofilm formation in L. monocytogenes (lmo2572, lmo2488 (uvrA), lmo1224, lmo0434 (inlB), lmo0263 (inlH), lmo0543, lmo0057 (EsaA), lmo2563, lmo0453), caused enhanced biofilm formation in the bacterium at 15 °C. The remaining mutants harbored interruptions in 10 genetic loci previously associated with biofilm formation at higher temperatures, indicating some temperature driven differences in the formation of biofilm by L. monocytogenes. Copyright © 2016 Elsevier B.V. All rights reserved.

High-Throughput Incubation and Quantification of Agglutination Assays in a Microfluidic System.

PubMed

Castro, David; Conchouso, David; Kodzius, Rimantas; Arevalo, Arpys; Foulds, Ian G

2018-06-04

In this paper, we present a two-phase microfluidic system capable of incubating and quantifying microbead-based agglutination assays. The microfluidic system is based on a simple fabrication solution, which requires only laboratory tubing filled with carrier oil, driven by negative pressure using a syringe pump. We provide a user-friendly interface, in which a pipette is used to insert single droplets of a 1.25-µL volume into a system that is continuously running and therefore works entirely on demand without the need for stopping, resetting or washing the system. These assays are incubated by highly efficient passive mixing with a sample-to-answer time of 2.5 min, a 5⁻10-fold improvement over traditional agglutination assays. We study system parameters such as channel length, incubation time and flow speed to select optimal assay conditions, using the streptavidin-biotin interaction as a model analyte quantified using optical image processing. We then investigate the effect of changing the concentration of both analyte and microbead concentrations, with a minimum detection limit of 100 ng/mL. The system can be both low- and high-throughput, depending on the rate at which assays are inserted. In our experiments, we were able to easily produce throughputs of 360 assays per hour by simple manual pipetting, which could be increased even further by automation and parallelization. Agglutination assays are a versatile tool, capable of detecting an ever-growing catalog of infectious diseases, proteins and metabolites. A system such as this one is a step towards being able to produce high-throughput microfluidic diagnostic solutions with widespread adoption. The development of analytical techniques in the microfluidic format, such as the one presented in this work, is an important step in being able to continuously monitor the performance and microfluidic outputs of organ-on-chip devices.

A Novel Locomotion-based Validation Assay for Candidate Drugs Using Drosophila DYT1 Disease Model

DTIC Science & Technology

2014-06-01

rescue the locomotion defects of Drosophila larvae caused by the expression of human torsinAΔE. These results demonstrated that human torsinA can... Drosophila dtorsin∆D transgenic lines dtorsin∆E and dtorsin∆D cDNA constructs were made from the wild type dtorsin cDNA using QuikChange II XL Site...After confirming mutated sequences , the insert was again cut out with EcoRI and NotI and inserted between EcoRI and NotI sites of pUAST [2] to produce

Linked Tumor-Selective Virus Replication and Transgene Expression from E3-Containing Oncolytic Adenoviruses†

PubMed Central

Zhu, Mingzhu; Bristol, J. Andrew; Xie, Yuefeng; Mina, Mervat; Ji, Hong; Forry-Schaudies, Suzanne; Ennist, David L.

2005-01-01

Historically, the adenoviral E3 region was found to be nonessential for viral replication in vitro. In addition, adenoviruses whose genome was more than approximately 105% the size of the native genome were inefficiently packaged. These profound observations were used experimentally to insert transgenes into the adenoviral backbone. More recently, however, the reintroduction of the E3 region into oncolytic adenoviruses has been found to positively influence antitumor efficacy in preclinical models and clinical trials. In the studies reported here, the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA sequence has been substituted for the E3-gp19 gene in oncolytic adenoviruses that otherwise retained the E3 region. Five viruses that differed slightly in the method of transgene insertion were generated and compared to Ar6pAE2fGmF (E2F/GM/ΔE3), a previously described E3-deleted oncolytic adenovirus encoding GM-CSF. In all of the viruses, the human E2F-1 promoter regulated E1A expression and GM-CSF expression was under the control of the adenoviral E3 promoter and the packaging signal was relocated immediately upstream from the right terminal repeat. The E3-gp19-deleted viruses had similar cytolytic properties, as measured in vitro by cytotoxicity assays, but differed markedly in their capacity to express and secrete GM-CSF. Ar15pAE2fGmF (E2F/GM/E3b), the virus that produced the highest levels of GM-CSF and retained the native GM-CSF leader sequence, was selected for further analysis. The E2F/GM/E3b and E2F/GM/ΔE3 viruses exhibited similar cytotoxic activity and GM-CSF production in several tumor cell lines in vitro. However, when compared in vivo in nude mouse xenograft tumor models, E2F/GM/E3b spread through tumors to a greater extent, resulted in higher peak GM-CSF and total exposure levels in both tumor and serum, and was more efficacious than the E3-deleted virus. Using the matched WI-38 (parental) and WI-38-VA13 (simian virus 40 large T antigen transformed) cell pair, GM-CSF was shown to be selectively produced in cells expressing high levels of E2F, indicating that the tumor-selective E2F promoter controlled E1A and GM-CSF expression. PMID:15827160

Surface acoustic wave resonators

NASA Astrophysics Data System (ADS)

Avitabile, Gianfranco; Roselli, Luca; Atzeni, Carlo; Manes, Gianfranco

1991-10-01

The development of surface acoustic wave (SAW) resonators is reviewed with attention given to the design of a simulation package for CAD-assisted SAW resonator design. Basic design configurations and operation parameters are set forth for the SAW resonators including the phase of the reflection factor, evaluation of the stopband center frequency, stopband width, and the free propagation speed. The use of synchronous designs is shown to reduce device sensitivity to variations in the technological process but generate higher insertion losses. The existence of transverse modes and propagation losses is shown to affect the rejection of spurious modes and the achievement of low insertion losses. Several SAW resonators are designed and fabricated with the CAD process, and the resonators in the VHF-UHF bands perform in a manner predicted by simulated results.

Applications of nanotechnology in food packaging and food safety: barrier materials, antimicrobials and sensors.

PubMed

Duncan, Timothy V

2011-11-01

In this article, several applications of nanomaterials in food packaging and food safety are reviewed, including: polymer/clay nanocomposites as high barrier packaging materials, silver nanoparticles as potent antimicrobial agents, and nanosensors and nanomaterial-based assays for the detection of food-relevant analytes (gasses, small organic molecules and food-borne pathogens). In addition to covering the technical aspects of these topics, the current commercial status and understanding of health implications of these technologies are also discussed. These applications were chosen because they do not involve direct addition of nanoparticles to consumed foods, and thus are more likely to be marketed to the public in the short term. Published by Elsevier Inc.

Quantitative multiplex detection of pathogen biomarkers

DOEpatents

Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I.; Martinez, Jennifer; Grace, Wynne K.

2016-02-09

The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

Quantitative multiplex detection of pathogen biomarkers

DOEpatents

Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I; Martinez, Jennifer; Grace, Wynne K

2014-10-14

The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

Does implant coating with antibacterial-loaded hydrogel reduce bacterial colonization and biofilm formation in vitro?

PubMed

Drago, Lorenzo; Boot, Willemijn; Dimas, Kostantinos; Malizos, Kostantinos; Hänsch, Gertrud M; Stuyck, Jos; Gawlitta, Debby; Romanò, Carlo L

2014-11-01

Implant-related infections represent one of the most severe complications in orthopaedics. A fast-resorbable, antibacterial-loaded hydrogel may reduce or prevent bacterial colonization and biofilm formation of implanted biomaterials. We asked: (1) Is a fast-resorbable hydrogel able to deliver antibacterial compounds in vitro? (2) Can a hydrogel (alone or antibacterial-loaded) coating on implants reduce bacterial colonization? And (3) is intraoperative coating feasible and resistant to press-fit implant insertion? We tested the ability of Disposable Antibacterial Coating (DAC) hydrogel (Novagenit Srl, Mezzolombardo, Italy) to deliver antibacterial agents using spectrophotometry and a microbiologic assay. Antibacterial and antibiofilm activity were determined by broth microdilution and a crystal violet assay, respectively. Coating resistance to press-fit insertion was tested in rabbit tibias and human femurs. Complete release of all tested antibacterial compounds was observed in less than 96 hours. Bactericidal and antibiofilm effect of DAC hydrogel in combination with various antibacterials was shown in vitro. Approximately 80% of the hydrogel coating was retrieved on the implant after press-fit insertion. Implant coating with an antibacterial-loaded hydrogel reduces bacterial colonization and biofilm formation in vitro. A fast-resorbable, antibacterial-loaded hydrogel coating may help prevent implant-related infections in orthopaedics. However, further validation in animal models and properly controlled human studies is required.

Control of lipid oxidation by nonmigratory active packaging films prepared by photoinitiated graft polymerization.

PubMed

Tian, Fang; Decker, Eric A; Goddard, Julie M

2012-08-08

Transition metal-promoted oxidation impacts the quality, shelf life, and nutrition of many packaged foods. Metal-chelating active packaging therefore offers a means to protect foods against oxidation. Herein, we report the development and characterization of nonmigratory metal-chelating active packaging. To prepare the films, carboxylic acids were grafted onto the surfaces of polypropylene films by photoinitiated graft polymerization of acrylic acid. Attenuated total reflectance/Fourier transform infrared spectroscopy, contact angle, scanning electron microscopy, and iron-chelating assay were used to characterize film properties. Graft polymerization yielded a carboxylic acid density of 68.67 ± 9.99 nmol per cm(2) film, with ferrous iron-chelating activity of 71.07 ± 12.95 nmol per cm(2). The functionalized films extended the lag phase of lipid oxidation in a soybean oil-in-water emulsion system from 2 to 9 days. The application of such nonmigratory active packaging films represents a promising approach to reduce additive use while maintaining food quality.

KSC-97PC1277

NASA Image and Video Library

1997-08-22

In the Payload Hazardous Servicing Facility (PHSF), Dan Maynard, a Jet Propulsion Laboratory technician, inserts the Digital Video Disk (DVD) into a shallow cavity between two pieces of aluminum that will protect it from micrometeoroid impacts. The package will be mounted to the side of the two-story-tall spacecraft beneath a pallet carrying cameras and other space instruments that will be used to study the Saturnian system. A specially designed, multicolored patch of thermal blanket material will be installed over the disk package. Along with the spacecraft, the disk will reside in Saturn's orbit centuries after the primary mission is completed in July 2008. The Cassini mission is managed for NASA's Office of Space Science, Washington, D.C., by the Jet Propulsion Laboratory, a division of the California Institute of Technology

Keeping waived tests simple.

PubMed

2004-01-01

Laboratories performing waived testing must follow the manufacturer's instructions as well as good laboratory practices to ensure that test results are reliable. Four things to concentrate on to maximize the performance and reliability of waived tests are to: 1. Read and follow the information found in the package inserts. 2. Follow the manufacturer's recommendations for running quality control. 3. Train staff members to perform tests correctly. 4. Follow established policies and procedures for patient testing in the practice.

Field Level Computer Exploitation Package

DTIC Science & Technology

2007-03-01

to take advantage of the data retrieved from the computer. Major Barge explained that if a tool could be designed that nearly anyone could use...the study of network forensics. This has become a necessity because of the constantly growing eCommerce industry and the stiff competition between...Security. One big advantage that Insert has is the fact that it is quite small compared to most bootable CDs. At only 60 megabytes it can be burned

Agrobacterium tumefaciens mutants affected in attachment to plant cells.

PubMed Central

Douglas, C J; Halperin, W; Nester, E W

1982-01-01

An analysis of Agrobacterium tumefaciens mutants with Tn5 insertions in chromosomal DNA showed that the chromosome of A. tumefaciens codes for a specific ability of this bacterium to attach to plant cells. This ability is associated with tumorigenesis by A. tumefaciens, the ability of avirulent A. tumefaciens to inhibit tumorigenesis, and the ability to adsorb certain phages. A second class of chromosomal mutations affects tumorigenesis without altering the ability to attach to plant cells. The attachment of A. tumefaciens to plant cells was assayed by mixing radiolabeled bacteria with suspensions of tobacco tissue culture cells or freshly isolated Zinnia leaf mesophyll cells. Under the conditions of this assay, an avirulent Ti plasmid-cured strain attached to the same extent as the same strain containing pTiB6806. Six of eight avirulent mutants with Tn5 insertions in chromosomal DNA showed defective attachment, whereas two retained wild-type attachment ability. In contrast to the strains showing wild-type attachment, the attachment-defective mutants failed to inhibit tumorigenesis when inoculated onto Jerusalem artichoke slices before inoculation of a virulent strain and also showed a loss of sensitivity to two Agrobacterium phages. The loss of phage sensitivity appeared to be due to a loss of ability to adsorb the phages. Staining with Calcofluor indicated that the mutants retained the ability to synthesize cellulose fibrils, which have been implicated in the attachment process. Southern filter hybridizations demonstrated that each mutant contained a single Tn5 insertion, and genetic linkage between the Tn5 insertion in one mutant and the attachment phenotype has also been demonstrated. Images PMID:6292165

Use of the dye stain assay and ultraviolet light test for assessing vaginal insertion of placebo-filled applicators before and after sex.

PubMed

Keller, Marla J; Buckley, Niall; Katzen, Lauren L; Walsh, Jennifer; Friedland, Barbara; Littlefield, Sarah; Lin, Juan; Xue, Xiaonan; Cornelison, Terri; Herold, Betsy C; Einstein, Mark H

2013-12-01

Applicator dye staining and ultraviolet (UV) light have been used in trials to measure adherence, but not in the setting of before and after sex gel dosing (BAT-24). This study was designed to determine if semen or presex gel dosing impacts the sensitivity and specificity of a dye stain assay (DSA) for measuring vaginal insertion of placebo-filled applicators with BAT-24 dosing. Healthy monogamous couples received Microlax-type applicators (Tectubes, Åstorp, Sweden) filled with hydroxyethylcelluose placebo gel. Women were instructed to vaginally insert 1 dose of gel before and a second dose after sex and to return applicators within 48 hours after sex. Applicators were stained to detect semen, followed by UV then DSA, and scored by 2 readers. Positive and negative controls were randomly included in applicator batches. Fifteen couples completed the study. Each woman returned at least 6 applicators over a 30-day period. The sensitivity for insertion of postsex applicators was higher for UV (97%) compared with DSA (90%), and the specificity was similar (≥96%). For presex applicators, the sensitivity and specificity were higher for DSA (100%) compared with UV testing (87% sensitivity, 96% specificity). Among returned postsex applicators, 95% tested positive by UV compared with 87% by DSA. Agreement between readers was significantly better on the presex applicators for DSA than for UV, and for postsex readings, agreement was less than half that for UV, although the results were not statistically significant. Applicator tests are feasible for measuring adherence in trials with gel dosing before and after sex.

The use of a newspaper insertion to promote DIY testing of vision in India.

PubMed

Murthy, G V; Gupta, S K; Dada, V K; Pant, T D; Savita, C; Sanga, L; Neena, J

2001-08-01

The mass media have the potential to motivate people to participate in self appraisal of their own health status. An innovative communication package was designed to help people to examine vision at home. The impact of publishing the "do it yourself" (DIY) kit in Indian newspapers was evaluated. A pretested bilingual vision testing kit was published in three newspapers. The kit comprised four tumbling Es corresponding to 6/12 line of Snellen's optotypes. Directions on using the kit were enclosed. 3 -7 days after publication of the kit, a telephone survey of newspaper readers was undertaken to evaluate the impact and cost effectiveness. 603 people were contacted over the telephone. 125 (20.73%) subscribed to the newspaper carrying the DIY insertion. 43.2% (54) noticed the insertion of which 88.89% (48) read the enclosed instructions carefully. 58.33% respondents felt sufficiently motivated to contact an ophthalmologist. Graduates had a 3.83 times higher probability of reading the communication insertion compared with others. Differences in relation to other demographic variables were not statistically significant. Newspapers are an excellent medium for communicating self appraisal kits for vision testing. The medium is cost effective and has significant reach in the urban agglomerates of India.

The use of a newspaper insertion to promote DIY testing of vision in India

PubMed Central

Murthy, G; Gupta, S. K.; Dada, V. K.; Pant, T. D.; Savita, C.; Sanga, L.; Neena, J.

2001-01-01

BACKGROUND—The mass media have the potential to motivate people to participate in self appraisal of their own health status. An innovative communication package was designed to help people to examine vision at home. The impact of publishing the "do it yourself" (DIY) kit in Indian newspapers was evaluated.
METHODS—A pretested bilingual vision testing kit was published in three newspapers. The kit comprised four tumbling Es corresponding to 6/12 line of Snellen's optotypes. Directions on using the kit were enclosed. 3 -7 days after publication of the kit, a telephone survey of newspaper readers was undertaken to evaluate the impact and cost effectiveness.
RESULTS—603 people were contacted over the telephone. 125 (20.73%) subscribed to the newspaper carrying the DIY insertion. 43.2% (54) noticed the insertion of which 88.89% (48) read the enclosed instructions carefully. 58.33% respondents felt sufficiently motivated to contact an ophthalmologist. Graduates had a 3.83 times higher probability of reading the communication insertion compared with others. Differences in relation to other demographic variables were not statistically significant.
CONCLUSIONS—Newspapers are an excellent medium for communicating self appraisal kits for vision testing. The medium is cost effective and has significant reach in the urban agglomerates of India.

 PMID:11466254

Fissile material detector

DOEpatents

Ivanov, Alexander I.; Lushchikov, Vladislav I.; Shabalin, Eugeny P.; Maznyy, Nikita G.; Khvastunov, Michael M.; Rowland, Mark

2002-01-01

A detector for fissile materials which provides for integrity monitoring of fissile materials and can be used for nondestructive assay to confirm the presence of a stable content of fissile material in items. The detector has a sample cavity large enough to enable assay of large items of arbitrary configuration, utilizes neutron sources fabricated in spatially extended shapes mounted on the endcaps of the sample cavity, incorporates a thermal neutron filter insert with reflector properties, and the electronics module includes a neutron multiplicity coincidence counter.

BoolFilter: an R package for estimation and identification of partially-observed Boolean dynamical systems.

PubMed

Mcclenny, Levi D; Imani, Mahdi; Braga-Neto, Ulisses M

2017-11-25

Gene regulatory networks govern the function of key cellular processes, such as control of the cell cycle, response to stress, DNA repair mechanisms, and more. Boolean networks have been used successfully in modeling gene regulatory networks. In the Boolean network model, the transcriptional state of each gene is represented by 0 (inactive) or 1 (active), and the relationship among genes is represented by logical gates updated at discrete time points. However, the Boolean gene states are never observed directly, but only indirectly and incompletely through noisy measurements based on expression technologies such as cDNA microarrays, RNA-Seq, and cell imaging-based assays. The Partially-Observed Boolean Dynamical System (POBDS) signal model is distinct from other deterministic and stochastic Boolean network models in removing the requirement of a directly observable Boolean state vector and allowing uncertainty in the measurement process, addressing the scenario encountered in practice in transcriptomic analysis. BoolFilter is an R package that implements the POBDS model and associated algorithms for state and parameter estimation. It allows the user to estimate the Boolean states, network topology, and measurement parameters from time series of transcriptomic data using exact and approximated (particle) filters, as well as simulate the transcriptomic data for a given Boolean network model. Some of its infrastructure, such as the network interface, is the same as in the previously published R package for Boolean Networks BoolNet, which enhances compatibility and user accessibility to the new package. We introduce the R package BoolFilter for Partially-Observed Boolean Dynamical Systems (POBDS). The BoolFilter package provides a useful toolbox for the bioinformatics community, with state-of-the-art algorithms for simulation of time series transcriptomic data as well as the inverse process of system identification from data obtained with various expression technologies such as cDNA microarrays, RNA-Seq, and cell imaging-based assays.

Readability of medicinal package leaflets: a systematic review.

PubMed

Pires, Carla; Vigário, Marina; Cavaco, Afonso

2015-01-01

OBJECTIVE To review studies on the readability of package leaflets of medicinal products for human use. METHODS We conducted a systematic literature review between 2008 and 2013 using the keywords "Readability and Package Leaflet" and "Readability and Package Insert" in the academic search engine Biblioteca do Conhecimento Online, comprising different bibliographic resources/databases. The preferred reporting items for systematic reviews and meta-analyses criteria were applied to prepare the draft of the report. Quantitative and qualitative original studies were included. Opinion or review studies not written in English, Portuguese, Italian, French, or Spanish were excluded. RESULTS We identified 202 studies, of which 180 were excluded and 22 were enrolled [two enrolling healthcare professionals, 10 enrolling other type of participants (including patients), three focused on adverse reactions, and 7 descriptive studies]. The package leaflets presented various readability problems, such as complex and difficult to understand texts, small font size, or few illustrations. The main methods to assess the readability of the package leaflet were usability tests or legibility formulae. Limitations with these methods included reduced number of participants; lack of readability formulas specifically validated for specific languages (e.g., Portuguese); and absence of an assessment on patients literacy, health knowledge, cognitive skills, levels of satisfaction, and opinions. CONCLUSIONS Overall, the package leaflets presented various readability problems. In this review, some methodological limitations were identified, including the participation of a limited number of patients and healthcare professionals, the absence of prior assessments of participant literacy, humor or sense of satisfaction, or the predominance of studies not based on role-plays about the use of medicines. These limitations should be avoided in future studies and be considered when interpreting the results.

Humidity data for 9975 shipping packages with cane fiberboard

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

Daugherty, W. L.

The 9975 surveillance program is developing a technical basis to support extending the storage period of 9975 packages in K-Area Complex beyond the currently approved 15 years. A key element of this effort is developing a better understanding of degradation of the fiberboard assembly under storage conditions. This degradation is influenced greatly by the moisture content of the fiberboard, which is not well characterized on an individual package basis. Direct measurements of humidity and fiberboard moisture content have been made on two test packages with cane fiberboard and varying internal heat levels from 0 up to 19W. With an internalmore » heat load, a temperature gradient in the fiberboard assembly leads to varying relative humidity in the air around the fiberboard. However, the absolute humidity tends to remain approximately constant throughout the package. The moisture content of fiberboard varies under the influence of several phenomena. Changes in local fiberboard temperature (from an internal heat load) can cause fiberboard moisture changes through absorption or evaporation. Fiberboard degradation at elevated temperature will produce water as a byproduct. And the moisture level within the package is constantly seeking equilibrium with that of the surrounding room air, which varies on a daily and seasonal basis. One indicator of the moisture condition within a 9975 package might be obtained by measuring the relative humidity in the upper air space, by inserting a humidity probe through a caplug hole. However, the data indicate that for the higher internal heat loads (15 and 19 watts), a large variation in internal moisture conditions produces little or no variation in the air space relative humidity. Therefore, this approach does not appear to be sensitive to fiberboard moisture variations at the higher heat loads which are of most interest to maintaining fiberboard integrity.« less

Antimicrobial effectiveness of bioactive packaging materials from edible chitosan and casein polymers: assessment on carrot, cheese, and salami.

PubMed

Moreira, Maria del Rosario; Pereda, Mariana; Marcovich, Norma E; Roura, Sara I

2011-01-01

Antimicrobial packaging is one of the most promising active packaging systems for controlling spoilage and pathogenic microorganisms. In this work, the intrinsic antimicrobial properties of chitosan (CH) were combined with the excellent thermoplastic and film-forming properties of sodium caseinate (SC) to prepare SC/CH film-forming solutions and films. The antimicrobial effectiveness of SC, CH, and SC/CH coatings on the native microfloras of cheese, salami, and carrots was evaluated. In vitro assays through the test tube assay indicated that the most significant antimicrobial effect was achieved by CH and SC/CH solutions on carrot and cheese native microfloras. SC film-forming solutions did not exert antimicrobial activity on any of the native microflora studied. SC, CH, and SC/CH films stored in controlled environments showed that the retention of the antimicrobial action was observed until 5-d storage, at 65% relative humidity in both temperatures (10 °C and 20 °C). In vivo assays were also performed with SC, CH, and SC/CH applied as coatings or wrappers on the 3 food substrates. CH and SC/CH applied at both immersion and wrapper exerted a significant bactericidal action on mesophilic, psychrotrophic, and yeasts and molds counts, showing the 3 microbial populations analyzed a significant reduction (2.0 to 4.5 log CFU/g). An improvement of the bactericidal properties of the CH/SC blend respect to those of the neat CH film is reported. The ionic interaction between both macromolecules enhances its antimicrobial properties. Practical Application: The continuous consumer interest in high quality and food safety, combined with environmental concerns has stimulated the development and study of biodegradable coatings that avoid the use of synthetic materials. Among them, edible coatings, obtained from generally recognized as safe (GRAS) materials, have the potential to reduce weight loss, respiration rate, and improve food appearance and integrity. They can be used in combination with other food preservation techniques in order to extend the effectiveness of the food preservation chain. Moreover, antimicrobial films and coatings have innovated the concept of active packaging and have been developed to reduce, inhibit, or delay the growth of microorganisms on the surface of food in contact with the package. The use of antimicrobials packaging films to control the growth of microorganisms in food can have a significant impact on shelf-life extension and food safety. In addition, antimicrobial films can be prepared by the combination of inherent antimicrobial materials (that is, CH), with good film-forming protein-based ones (that is, SC). Therefore, the objective of this work is to study the performance of 2 biodegradable and edible biopolymers and their combination as natural packages for selected food products.

APPARATUS AND TECHNIC FOR THE ADMINISTRATION OF INTRACAVITARY RADIOACTIVE ISOTOPES

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

Michaud, N.J.; Liegner, L.M.

1961-08-01

The method of administration of radioactive isotopes in the treatment of pleural effusions and ascites associated with cancer will vary according to the therapeutic technic. A procedure with a suitable apparatus that utilizes an economical and sterile disposable package is described. The radioactive isotope, whether colloidal chromic phosphate (P/sup 32/) or colloidal gold (Au/sup 198/), can be obtained in th e exact amount prescribed. The entire apparatus is assembled within a few minutes under sterile conditions. Before the hypodermic needles are inserted into the radioactive isotope vial, the air is removed from the tubing by the flow of saline inmore » each segment. Each section is then clamped. The shielded radioactive isotope is then placed on a table or stand and the rubber seal of the vial is swabbed with alcohol or iodine. The inflow needle is inserted just through the rubber stopper and the outflow needle is inserted to the bottom of the vial. This procedure is carried out without removing the vial from the lead container. (auth)« less

Strategies to improve electrode positioning and safety in cochlear implants.

PubMed

Rebscher, S J; Heilmann, M; Bruszewski, W; Talbot, N H; Snyder, R L; Merzenich, M M

1999-03-01

An injection-molded internal supporting rib has been produced to control the flexibility of silicone rubber encapsulated electrodes designed to electrically stimulate the auditory nerve in human subjects with severe to profound hearing loss. The rib molding dies, and molds for silicone rubber encapsulation of the electrode, were designed and machined using AutoCad and MasterCam software packages in a PC environment. After molding, the prototype plastic ribs were iteratively modified based on observations of the performance of the rib/silicone composite insert in a clear plastic model of the human scala tympani cavity. The rib-based electrodes were reliably inserted farther into these models, required less insertion force and were positioned closer to the target auditory neural elements than currently available cochlear implant electrodes. With further design improvements the injection-molded rib may also function to accurately support metal stimulating contacts and wire leads during assembly to significantly increase the manufacturing efficiency of these devices. This method to reliably control the mechanical properties of miniature implantable devices with multiple electrical leads may be valuable in other areas of biomedical device design.

“Agrolistic” transformation of plant cells: Integration of T-strands generated in planta

PubMed Central

Hansen, Geneviève; Chilton, Mary-Dell

1996-01-01

We describe a novel plant transformation technique, termed “agrolistic,” that combines the advantages of the Agrobacterium transformation system with the high efficiency of biolistic DNA delivery. Agrolistic transformation allows integration of the gene of interest without undesired vector sequence. The virulence genes virD1 and virD2 from Agrobacterium tumefaciens that are required in bacteria for excision of T-strands from the tumor-inducing plasmid were placed under the control of the CaMV35S promoter and codelivered with a target plasmid containing border sequences flanking the gene of interest. Transient expression assays in tobacco and in maize cells indicated that vir gene products caused strand-specific nicking in planta at the right border sequence, similar to VirD1/VirD2-catalyzed T-strand excision observed in Agrobacterium. Agrolistically transformed tobacco calli were obtained after codelivery of virD1 and virD2 genes together with a selectable marker flanked by border sequences. Some inserts exhibited right junctions with plant DNA that corresponded precisely to the sequence expected for T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion events. We designate these as “agrolistic” inserts, as distinguished from “biolistic” inserts. Both types of inserts were found in some transformed lines. The frequency of agrolistic inserts was 20% that of biolistic inserts. PMID:8962167

Preparation and screening of an arrayed human genomic library generated with the P1 cloning system.

PubMed Central

Shepherd, N S; Pfrogner, B D; Coulby, J N; Ackerman, S L; Vaidyanathan, G; Sauer, R H; Balkenhol, T C; Sternberg, N

1994-01-01

We describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophage P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. The inserts were cloned into the pAd10sacBII vector and packaged in vitro into P1 phage. These were used to generate recombinant bacterial clones, each of which was picked robotically from an agar plate into a well of a 96-well microtiter dish, grown overnight, and stored at -70 degrees C. The resulting library, designated DMPC-HFF#1 series A, consists of approximately 130,000-140,000 recombinant clones that were stored in 1500 microtiter dishes. To screen the library, clones were combined in a pooling strategy and specific loci were identified by PCR analysis. On average, the library contains two or three different clones for each locus screened. To date we have identified a total of 17 clones containing the hypoxanthine-guanine phosphoribosyltransferase, human serum albumin-human alpha-fetoprotein, p53, cyclooxygenase I, human apurinic endonuclease, beta-polymerase, and DNA ligase I genes. The cloned inserts average 80 kb in size and range from 70 to 95 kb, with one 49-kb insert and one 62-kb insert. Images PMID:8146166

The Ontology of Vaccine Adverse Events (OVAE) and its usage in representing and analyzing adverse events associated with US-licensed human vaccines.

PubMed

Marcos, Erica; Zhao, Bin; He, Yongqun

2013-11-26

Licensed human vaccines can induce various adverse events (AE) in vaccinated patients. Due to the involvement of the whole immune system and complex immunological reactions after vaccination, it is difficult to identify the relations among vaccines, adverse events, and human populations in different age groups. Many known vaccine adverse events (VAEs) have been recorded in the package inserts of US-licensed commercial vaccine products. To better represent and analyze VAEs, we developed the Ontology of Vaccine Adverse Events (OVAE) as an extension of the Ontology of Adverse Events (OAE) and the Vaccine Ontology (VO). Like OAE and VO, OVAE is aligned with the Basic Formal Ontology (BFO). The commercial vaccines and adverse events in OVAE are imported from VO and OAE, respectively. A new population term 'human vaccinee population' is generated and used to define VAE occurrence. An OVAE design pattern is developed to link vaccine, adverse event, vaccinee population, age range, and VAE occurrence. OVAE has been used to represent and classify the adverse events recorded in package insert documents of commercial vaccines licensed by the USA Food and Drug Administration (FDA). OVAE currently includes over 1,300 terms, including 87 distinct types of VAEs associated with 63 human vaccines licensed in the USA. For each vaccine, occurrence rates for every VAE in different age groups have been logically represented in OVAE. SPARQL scripts were developed to query and analyze the OVAE knowledge base data. To demonstrate the usage of OVAE, the top 10 vaccines accompanying with the highest numbers of VAEs and the top 10 VAEs most frequently observed among vaccines were identified and analyzed. Asserted and inferred ontology hierarchies classify VAEs in different levels of AE groups. Different VAE occurrences in different age groups were also analyzed. The ontology-based data representation and integration using the FDA-approved information from the vaccine package insert documents enables the identification of adverse events from vaccination in relation to predefined parts of the population (age groups) and certain groups of vaccines. The resulting ontology-based VAE knowledge base classifies vaccine-specific VAEs and supports better VAE understanding and future rational AE prevention and treatment.

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.

PubMed

Traverse, Charles C; Ochman, Howard

2017-08-29

Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola , which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. IMPORTANCE The high level of mistakes generated during transcription can result in the accumulation of malfunctioning and misfolded proteins which can alter global gene regulation and in the expenditure of energy to degrade these nonfunctional proteins. The transcriptome-wide occurrence of base substitutions has been elucidated in bacteria, but information on transcription insertions and deletions-errors that potentially have more dire effects on protein function-is limited to reporter gene constructs. Here, we capture the transcriptome-wide spectrum of insertions and deletions in Escherichia coli and Buchnera aphidicola and show that they occur at rates approaching those of base substitutions. Knowledge of the full extent of sequences subject to transcription indels supports a new model of bacterial transcription slippage, one that relies on the number of complementary bases between the transcript and the DNA template to which it slipped. Copyright © 2017 Traverse and Ochman.

Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

NASA Astrophysics Data System (ADS)

Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

1990-09-01

Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

Detection of drugs in the urine of body-packers.

PubMed

Gherardi, R K; Baud, F J; Leporc, P; Marc, B; Dupeyron, J P; Diamant-Berger, O

1988-05-14

The presence of opiates and benzoylecgonine, the major metabolite of cocaine, in the urine was detected by means of enzyme immunoassay in a series of 120 smugglers who had either ingested or inserted into their rectum cocaine or heroin packaged for transportation. There was a striking relation between the presence of drugs in the urine and swallowing of drug-filled bundles (cocaine 49 of 50 cases, heroin 9 of 10). The proportion of positive results was also high in cases of rectal insertion (cocaine 2 of 2, heroin 35 of 58). In 30 cases of cocaine-packet ingestion, serial measurements showed that the accuracy of the test progressively decreased with respect to the detection of residual packets in the body. Drug detection in the urine of suspected body-packers seems to be a useful test, positive results justifying subsequent radiological investigations.

ROBOCAL: Gamma-ray isotopic hardware/software interface

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

Hurd, J.R.; Bonner, C.A.; Ostenak, C.A.

1989-01-01

ROBOCAL, presently being developed at the Los Alamos National Laboratory, is a full-scale prototypical robotic system for remotely performing calorimetric and gamma-ray isotopics measurements of nuclear materials. It features a fully automated vertical stacker-retriever for storing and retrieving packaged nuclear materials from a multi-drawer system, and a fully automated, uniquely integrated gantry robot for programmable selection and transfer of nuclear materials to calorimetric and gamma-ray isotopic measurement stations. Since ROBOCAL is to require almost no operator intervention, a mechanical control system is required in addition to a totally automated assay system. The assay system must be a completely integrated datamore » acquisition and isotopic analysis package fully capable of performing state-of-the-art homogeneous and heterogeneous analyses on many varied matrices. The TRIFID assay system being discussed at this conference by J. G. Fleissner of the Rocky Flats Plant has been adopted because of its many automated features. These include: MCA/ADC setup and acquisition; spectral storage and analysis utilizing an expert system formalism; report generation with internal measurement control printout; user friendly screens and menus. The mechanical control portion consists primarily of two detector platforms and a sample platform, each with independent movement. Some minor modifications and additions are needed with TRIFID to interface the assay and mechanical portions with the CimRoc 4000 software controlling the robot. 6 refs., 5 figs., 3 tabs.« less

Construction and production of oncotropic vectors, derived from MVM(p), that share reduced sequence homology with helper plasmids.

PubMed

Clément, Nathalie; Velu, Thierry; Brandenburger, Annick

2002-09-01

The production of currently available vectors derived from autonomous parvoviruses requires the expression of capsid proteins in trans, from helper sequences. Cotransfection of a helper plasmid always generates significant amounts of replication-competent virus (RCV) that can be reduced by the integration of helper sequences into a packaging cell line. Although stocks of minute virus of mice (MVM)-based vectors with no detectable RCV could be produced by transfection into packaging cells; the latter appear after one or two rounds of replication, precluding further amplification of the vector stock. Indeed, once RCVs become detectable, they are efficiently amplified and rapidly take over the culture. Theoretically RCV-free vector stocks could be produced if all homology between vector and helper DNA is eliminated, thus preventing homologous recombination. We constructed new vectors based on the structure of spontaneously occurring defective particles of MVM. Based on published observations related to the size of vectors and the sequence of the viral origin of replication, these vectors were modified by the insertion of foreign DNA sequences downstream of the transgene and by the introduction of a consensus NS-1 nick site near the origin of replication to optimize their production. In one of the vectors the inserted fragment of mouse genomic DNA had a synergistic effect with the modified origin of replication in increasing vector production.

Evaluation of package inserts of Ayurveda drug formulations from Mumbai city.

PubMed

Shirolkar, Sudatta; Tripathi, Raakhi K; Potey, Anirudha V

2015-01-01

Package insert (PI) is a vital document accompanying a prescribed medication to provide information to the prescriber and end-user at a glance. Studies regarding PIs of Ayurvedic medicines in accordance with standard guidelines are lacking. Present study was undertaken to evaluate PI of Ayurveda drugs. PIs of Ayurveda drugs were obtained from five randomly selected Ayurveda medical shops located in three main zones of Mumbai. From each medical shop, a range of 15-20 PI was planned to be collected for different formulations. It was decided to collect a minimum fifty PIs/group for equitable distribution of various formulations in period of January-June2013. Checklist was prepared, and content validity was achieved. Final validated checklist contained a total of 13 items, and the presence or absence of information pertaining to these items on the PI was evaluated. Any other additional information present on PI was also noted. Each item was analyzed and expressed as percentages. The information on 258 PIs included: Name of ingredients (67%), quantity of ingredients (47.27%), route of administration (86.8%), dosage form (86.8%), indications (18%), dose (18%), contraindications (18%), side effects (9%), shelf life (5.81%), storage conditions (11%), and manufacturers name with contact details (34%). PIs accompanying Ayurveda medicinal products in India are deficient in information required to be furnished by them.

Recommendations for the use of medications with continuous enteral nutrition.

PubMed

Wohlt, Paul D; Zheng, Lan; Gunderson, Shelly; Balzar, Sarah A; Johnson, Benjamin D; Fish, Jeffrey T

2009-08-15

Recommendations for the use of medications with continuous enteral nutrition are provided. A literature review was conducted to identify primary literature reporting medication interactions with continuous enteral nutrition. For medications without supporting literature, manufacturers were contacted for information. Package inserts for specific medications were also investigated for any information to help guide recommendations. If no specific recommendations were made by the pharmaceutical manufacturer or the package insert concerning administration of products with continuous enteral nutrition, a tertiary database was consulted. Recommendations were generated by a consensus of clinicians for those medications that lacked specific recommendations in the primary literature or from the pharmaceutical manufacturer. Documentation of medication interactions with continuous enteral nutrition and food was then collated along with specific recommendations on how to administer the medication with regard to continuous enteral nutrition. Recommendations were classified as strong (grade 1) or weak (grade 2). The quality of evidence was classified as high (grade A), moderate (grade B), or low (grade C). Forty-six medications commonly given to hospitalized patients were evaluated. Twenty-four medications had recommendations based on available data, and the remaining 22 medications had recommendations based on a consensus of clinicians. There was a lack of published data regarding drug-nutrient interactions for a majority of the drugs commonly administered to patients receiving continuous enteral nutrition. Clinicians should recognize potential drug-nutrient interactions and use available evidence to optimize patients' drug therapy.

Microplitis demolitor bracovirus genome segments vary in abundance and are individually packaged in virions

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

Beck, Markus H.; Inman, Ross B.; Strand, Michael R.

2007-03-01

Polydnaviruses (PDVs) are distinguished by their unique association with parasitoid wasps and their segmented, double-stranded (ds) DNA genomes that are non-equimolar in abundance. Relatively little is actually known, however, about genome packaging or segment abundance of these viruses. Here, we conducted electron microscopy (EM) and real-time polymerase chain reaction (PCR) studies to characterize packaging and segment abundance of Microplitis demolitor bracovirus (MdBV). Like other PDVs, MdBV replicates in the ovaries of females where virions accumulate to form a suspension called calyx fluid. Wasps then inject a quantity of calyx fluid when ovipositing into hosts. The MdBV genome consists of 15more » segments that range from 3.6 (segment A) to 34.3 kb (segment O). EM analysis indicated that MdBV virions contain a single nucleocapsid that encapsidates one circular DNA of variable size. We developed a semi-quantitative real-time PCR assay using SYBR Green I. This assay indicated that five (J, O, H, N and B) segments of the MdBV genome accounted for more than 60% of the viral DNAs in calyx fluid. Estimates of relative segment abundance using our real-time PCR assay were also very similar to DNA size distributions determined from micrographs. Analysis of parasitized Pseudoplusia includens larvae indicated that copy number of MdBV segments C, B and J varied between hosts but their relative abundance within a host was virtually identical to their abundance in calyx fluid. Among-tissue assays indicated that each viral segment was most abundant in hemocytes and least abundant in salivary glands. However, the relative abundance of each segment to one another was similar in all tissues. We also found no clear relationship between MdBV segment and transcript abundance in hemocytes and fat body.« less

Magnetic thermometry in the aseptic processing of foods containing particulates (abstract)

NASA Astrophysics Data System (ADS)

Ghiron, Kenneth; Litchfield, Bruce

1997-04-01

Aseptic processing of foods has many advantages over canning, including higher efficiency, lighter packaging, better taste, and higher nutritional value. Aseptic processing is different from canning where the food and container are sterilized together. Instead, a thin stream of food is heated and the packaging is independently sterilized before the food is placed in the package. However, no aseptic processes have been successfully filed with the FDA for foods containing sizable solid particles because of uncertainties in the thermal sterilization of the particles (e.g., soup). We have demonstrated that by inserting small paramagnetic particles in the interior of the simulated and real food particles, the local temperature can be measured. With this information, any questions about the adequate sterilization of the particles can be resolved. The measurements were done by directing the food stream through a magnetic field and sensing the voltages induced in a pickup coil by the motion of the magnetized particles. Details of the equipment design and data analysis will be discussed along with an introduction to the aseptic processing of foods.

From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

PubMed Central

Ferrer, Mireia; Henriet, Simon; Chamontin, Célia; Lainé, Sébastien; Mougel, Marylène

2016-01-01

In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community. PMID:27556480

Microelectronic DNA assay for the detection of BRCA1 gene mutations

NASA Technical Reports Server (NTRS)

Chen, Hua; Han, Jie; Li, Jun; Meyyappan, Meyya

2004-01-01

Mutations in BRCA1 are characterized by predisposition to breast cancer, ovarian cancer and prostate cancer as well as colon cancer. Prognosis for this cancer survival depends upon the stage at which cancer is diagnosed. Reliable and rapid mutation detection is crucial for the early diagnosis and treatment. We developed an electronic assay for the detection of a representative single nucleotide polymorphism (SNP), deletion and insertion in BRCA1 gene by the microelectronics microarray instrumentation. The assay is rapid, and it takes 30 minutes for the immobilization of target DNA samples, hybridization, washing and readout. The assay is multiplexing since it is carried out at the same temperature and buffer conditions for each step. The assay is also highly specific, as the signal-to-noise ratio is much larger than recommended value (72.86 to 321.05 vs. 5) for homozygotes genotyping, and signal ratio close to the perfect value 1 for heterozygotes genotyping (1.04).

International ring trial for the validation of an event-specific Golden Rice 2 quantitative real-time polymerase chain reaction method.

PubMed

Jacchia, Sara; Nardini, Elena; Bassani, Niccolò; Savini, Christian; Shim, Jung-Hyun; Trijatmiko, Kurniawan; Kreysa, Joachim; Mazzara, Marco

2015-05-27

This article describes the international validation of the quantitative real-time polymerase chain reaction (PCR) detection method for Golden Rice 2. The method consists of a taxon-specific assay amplifying a fragment of rice Phospholipase D α2 gene, and an event-specific assay designed on the 3' junction between transgenic insert and plant DNA. We validated the two assays independently, with absolute quantification, and in combination, with relative quantification, on DNA samples prepared in haploid genome equivalents. We assessed trueness, precision, efficiency, and linearity of the two assays, and the results demonstrate that both the assays independently assessed and the entire method fulfill European and international requirements for methods for genetically modified organism (GMO) testing, within the dynamic range tested. The homogeneity of the results of the collaborative trial between Europe and Asia is a good indicator of the robustness of the method.

Pathology consultation on anticoagulation monitoring: factor X-related assays.

PubMed

Wool, Geoffrey D; Lu, Chuanyi M

2013-11-01

To review various anticoagulation therapies and related laboratory monitoring issues, with a focus on factor X-related chromogenic assays. A case-based approach is used to review pertinent published literatures and product inserts of anticoagulation drugs and to look back on clinical use of factor X-related chromogenic assays. The number of anticoagulants available to clinicians has increased greatly in the past decade. Whether and how these anticoagulants should be monitored are areas of uncertainty for clinicians, which can lead to misuse of laboratory assays and suboptimal patient management. Factor X-related assays are of particular concern because of the similar and often confusing test names. Based on a common clinical case scenario and literature review regarding anticoagulant monitoring, an up-to-date discussion and review of the various factor X-related assays are provided, focusing on the differences in test designs and clinical utilities between the chromogenic anti-Xa and chromogenic factor X activity assays. Anticoagulation therapy and related laboratory monitoring are rapidly evolving areas of clinical practices. A good knowledge of relevant laboratory assays and their clinical applications is necessary to help optimize patient care.

Testing implications of varying targets for Bordetella pertussis: comparison of the FilmArray Respiratory Panel and the Focus B. pertussis PCR assay

PubMed Central

Jerris, Robert C; Williams, Sally R; MacDonald, Heather J; Ingebrigtsen, Danielle R; Westblade, Lars F; Rogers, Beverly B

2015-01-01

Background The FilmArray Respiratory Panel (RP) detects multiple pathogens, including Bordetella pertussis. The multiplex PCR system is appropriate for a core laboratory or point of care due to ease of use. The purpose of this study is to compare the analytical sensitivity of the FilmArray RP, which targets the promoter region of the B. pertussis toxin gene, with the Focus real-time PCR assay, which targets the insertion sequence IS481. Methods Seventy-one specimens from patients aged 1 month to 18 years, which had tested positive for B. pertussis using the Focus assay, were analysed using the FilmArray RP. Results Forty-six specimens were positive for B. pertussis by both the Focus and the FilmArray RP assays. Twenty-five specimens were negative for B. pertussis using the FilmArray RP assay, but positive using the Focus assay. Conclusions The FilmArray RP assays will detect approximately 1/3 less cases of B. pertussis than the Focus assay. PMID:25742911

GEMINI-TITAN (GT)-XI - MISC. EXPERIMENTS - MSC

NASA Image and Video Library

1966-03-22

S66-05515 (2 June 1966) --- Gemini-11 Experiment S-9 Nuclear Emulsion. This experiment will be used to study the cosmic radiation incident on Earth's atmosphere, to obtain detailed chemical composition of the heavy primary nuclei and to search for rare particles. Equipment is a rectangular package eight and a half by six by three inches weighing 15 pounds and including the nuclear emulsion film stack, motor to advance the emulsion and a timer to regulate the motor. The package is mounted atop the spacecraft retro adapter section prior to launch, is activated at insertion, and is retrieved by the EVA pilot. The experiment is conducted with the spacecraft in plus or minus 15 degrees of Earth's average magnetic field vector. Sponsors are NASA's Office of Space Science and Applications and the U.S. Naval Research Laboratories. Photo credit: NASA

Autonomous microexplosives subsurface tracing system final report.

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

Engler, Bruce Phillip; Nogan, John; Melof, Brian Matthew

The objective of the autonomous micro-explosive subsurface tracing system is to image the location and geometry of hydraulically induced fractures in subsurface petroleum reservoirs. This system is based on the insertion of a swarm of autonomous micro-explosive packages during the fracturing process, with subsequent triggering of the energetic material to create an array of micro-seismic sources that can be detected and analyzed using existing seismic receiver arrays and analysis software. The project included investigations of energetic mixtures, triggering systems, package size and shape, and seismic output. Given the current absence of any technology capable of such high resolution mapping ofmore » subsurface structures, this technology has the potential for major impact on petroleum industry, which spends approximately $1 billion dollar per year on hydraulic fracturing operations in the United States alone.« less

The effectiveness of triclosan-incorporated plastic against bacteria on beef surfaces.

PubMed

Cutter, C N

1999-05-01

Triclosan is a nonionic, broad-spectrum, antimicrobial agent that has been incorporated into a variety of personal hygiene products, including hand soaps, deodorants, shower gels, mouthwashes, and toothpastes. In this study, plastic containing 1,500 ppm of triclosan was evaluated in plate overlay assays and meat experiments as a means of reducing populations of bacteria. Plate overlay assays indicated that the triclosan-incorporated plastic (TIP) inhibited the following organisms: Brochothrix thermosphacta ATCC 11509, Salmonella Typhimurium ATCC 14028, Staphylococcus aureus ATCC 12598, Bacillus subtilis ATCC 6051, Shigella flexneri ATCC 12022, Escherichia coli ATCC 25922, and several strains of E. coli O157:H7. In meat experiment 1, irradiated, lean beef surfaces inoculated with B. thermosphacta, Salmonella Typhimurium, E. coli O157:H7, or B. subtilis were covered with TIP, vacuum packaged, and stored for 24 h at 4 degrees C. Of the organisms tested, only populations of B. thermosphacta were slightly reduced. In meat experiment 2, prerigor beef surfaces were inoculated with E. coli O157: H7, Salmonella Typhimurium, or B. thermosphacta incubated at 4 degrees C for 24 h, wrapped in TIP or control plastic, vacuum packaged, and stored at 4 degrees C for up to 14 days. There was a slight reduction in the population of the organisms after initial application with TIP. However, bacterial populations following long-term, refrigerated (4 degrees C), vacuum-packaged storage up to 14 days were not statistically (P< or =0.05) or numerically different than controls. In meat experiment 3, even TIP-wrapped, vacuum-packaged beef samples that were temperature abused at 12 degrees C did not exhibit significant (P< or =0.05) or sustainable reductions after 14 days of 4 degrees C storage. Another study indicated that populations of E. coli O157:H7 or B. thermosphacta added directly to TIP were not affected after 2 h of refrigerated storage or that the antimicrobial activity could be extracted from the plastic. Additional experiments suggest that presence of fatty acids or adipose may diminish the antimicrobial activity of TIP on meat surfaces. This study demonstrates that while antimicrobial activity is detected against bacterial cultures in antimicrobial plate assays, plastic containing 1,500 ppm of triclosan does not effectively reduce bacterial populations on refrigerated, vacuum-packaged meat surfaces.

Fluorophore Labeled Kinase Detects Ligands That Bind within the MAPK Insert of p38α Kinase

PubMed Central

Termathe, Martin; Grütter, Christian; Rabiller, Matthias; van Otterlo, Willem A. L.; Rauh, Daniel

2012-01-01

The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway. PMID:22768308

Use of the Dye Stain Assay and Ultraviolet Light Test for Assessing Vaginal Insertion of Placebo-filled Applicators Before and After Sex

PubMed Central

Keller, Marla J.; Buckley, Niall; Katzen, Lauren L.; Walsh, Jennifer; Friedland, Barbara; Littlefield, Sarah; Lin, Juan; Xue, Xiaonan; Cornelison, Terri; Herold, Betsy C.; Einstein, Mark H.

2014-01-01

Background Applicator dye staining and ultraviolet (UV) light have been used in trials to measure adherence, but not in the setting of before and after sex gel dosing (BAT-24). This study was designed to determine if semen or pre-sex gel dosing impacts the sensitivity and specificity of a dye stain assay (DSA) for measuring vaginal insertion of placebo-filled applicators with BAT-24 dosing. Methods Healthy monogamous couples received Microlax®-type applicators filled with hydroxyethylcelluose placebo gel. Women were instructed to vaginally insert one dose of gel before and a second dose after sex and to return applicators within 48 hours after sex. Applicators were stained to detect semen followed by UV then DSA and scored by two readers. Positive and negative controls were randomly included in applicator batches. Results Fifteen couples completed the study. Each female returned at least six applicators over a 30-day period. The sensitivity for insertion of post-sex applicators was higher for UV (97%) compared to DSA (90%) and the specificity was similar (≥96%). For pre-sex applicators, the sensitivity and specificity were higher for DSA (100%) compared to UV testing (87% sensitivity, 96% specificity). Among returned post-sex applicators, 95% tested positive by UV compared to 87% by DSA. Agreement between readers was significantly better on the pre-sex applicators for DSA than for UV and for post-sex readings agreement was less than half that for UV, although the results were not statistically significant. Conclusions Applicator tests are feasible for measuring adherence in trials with gel dosing before and after sex. PMID:24220355

Characterization of the exogenous insert and development of event-specific PCR detection methods for genetically modified Huanong No. 1 papaya.

PubMed

Guo, Jinchao; Yang, Litao; Liu, Xin; Guan, Xiaoyan; Jiang, Lingxi; Zhang, Dabing

2009-08-26

Genetically modified (GM) papaya (Carica papaya L.), Huanong No. 1, was approved for commercialization in Guangdong province, China in 2006, and the development of the Huanong No. 1 papaya detection method is necessary for implementing genetically modified organism (GMO) labeling regulations. In this study, we reported the characterization of the exogenous integration of GM Huanong No. 1 papaya by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. The results suggested that one intact copy of the initial construction was integrated in the papaya genome and which probably resulted in one deletion (38 bp in size) of the host genomic DNA. Also, one unintended insertion of a 92 bp truncated NptII fragment was observed at the 5' end of the exogenous insert. Furthermore, we revealed its 5' and 3' flanking sequences between the insert DNA and the papaya genomic DNA, and developed the event-specific qualitative and quantitative PCR assays for GM Huanong No. 1 papaya based on the 5' integration flanking sequence. The relative limit of detection (LOD) of the qualitative PCR assay was about 0.01% in 100 ng of total papaya genomic DNA, corresponding to about 25 copies of papaya haploid genome. In the quantitative PCR, the limits of detection and quantification (LOD and LOQ) were as low as 12.5 and 25 copies of papaya haploid genome, respectively. In practical sample quantification, the quantified biases between the test and true values of three samples ranged from 0.44% to 4.41%. Collectively, we proposed that all of these results are useful for the identification and quantification of Huanong No. 1 papaya and its derivates.

A novel (A)γδβ(0)-thalassemia caused by DNA deletion-inversion-insertion of the β-globin gene cluster and five olfactory receptor genes: Genetic interactions, hematological phenotypes and molecular characterization.

PubMed

Singha, Kritsada; Fucharoen, Goonnapa; Hama, Abdulloh; Fucharoen, Supan

2015-07-01

To report the phenotypes and genetic basis of a novel (A)γδβ(0)-thalassemia found in Thai individuals with several forms of thalassemia. An initial study was done in an adult Thai woman who had hypochromic microcytic red cells with unusually 100% Hb F. Extended study was carried out on her parents and another 17 unrelated individuals with elevated Hb F. Hb analysis was performed by capillary electrophoresis and DNA analysis was done using PCR. A novel diagnostic method based on multiplex PCR assays was developed. DNA analysis of the proband revealed the homozygosity for a novel deletion of 118.3 kb, removing the entire (A)γ, ψβ, δ-, β-globin and five olfactory receptor (OR) genes with an insertion of a 179 bp inverted DNA sequence located behind the OR52A5 gene located downstream and an insertion of 7 orphan nucleotides. Her parents were both carriers of this mutation. Further screening in suspected cases in our series unexpectedly led to identification of an additional 17 cases with this mutation in different genotypes including plain heterozygote, homozygote, compound heterozygote with Hb E, and double heterozygote with several forms of α-thalassemia. Hematological features associated with these genetic interactions are presented. Haplotype analysis indicated a single origin of this novel deletion-inversion-insertion (A)γδβ(0)-thalassemia in the Thai population. Differentiation of this mutation and other high Hb F determinants documented previously could be done by using a developed multiplex PCR assay. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Smooth muscle myosin isoform expression and LC20 phosphorylation in innate rat airway hyperresponsiveness.

PubMed

Gil, Fulvio R; Zitouni, Nedjma B; Azoulay, Eric; Maghni, Karim; Lauzon, Anne-Marie

2006-11-01

Four smooth muscle myosin heavy chain (SMMHC) isoforms are generated by alternative mRNA splicing of a single gene. Two of these isoforms differ by the presence [(+)insert] or absence [(-)insert] of a 7-amino acid insert in the motor domain. The rate of actin filament propulsion of the (+)insert SMMHC isoform, as measured in the in vitro motility assay, is twofold greater than that of the (-)insert isoform. We hypothesized that a greater expression of the (+)insert SMMHC isoform and greater regulatory light chain (LC(20)) phosphorylation contribute to airway hyperresponsiveness. We measured airway responsiveness to methacholine in Fischer hyperresponsive and Lewis normoresponsive rats and determined SMMHC isoform mRNA and protein expression, as well as essential light chain (LC(17)) isoforms, h-caldesmon, and alpha-actin protein expression in their tracheae. We also measured tracheal muscle strip contractility in response to methacholine and corresponding LC(20) phosphorylation. We found Fischer rats have more (+)insert mRNA (69.4 +/- 2.0%) (mean +/- SE) than Lewis rats (53.0 +/- 2.4%; P < 0.05) and a 44% greater content of (+)insert isoform relative to total myosin protein. No difference was found for LC(17) isoform, h-caldesmon, and alpha-actin expression. The contractility experiments revealed a greater isometric force for Fischer trachealis segments (4.2 +/- 0.8 mN) than Lewis (1.9 +/- 0.4 mN; P < 0.05) and greater LC(20) phosphorylation level in Fischer (55.1 +/- 6.4) than in Lewis (41.4 +/- 6.1; P < 0.05) rats. These results further support the contention that innate airway hyperresponsiveness is a multifactorial disorder in which increased expression of the fast (+)insert SMMHC isoform and greater activation of LC(20) lead to smooth muscle hypercontractility.

Thermodynamics of membrane insertion and refolding of the diphtheria toxin T-domain

PubMed Central

Vargas-Uribe, Mauricio; Rodnin, Mykola V.; Öjemalm, Karin; Holgado, Aurora; Kyrychenko, Alexander; Nilsson, IngMarie; Posokhov, Yevgen O.; Makhatadze, George; von Heijne, Gunnar; Ladokhin, Alexey S.

2014-01-01

The diphtheria toxin translocation (T) domain inserts into the endosomal membrane in response to the endosomal acidification and enables the delivery of the catalytic domain into the cell. The insertion pathway consists of a series of conformational changes that occur in solution and in the membrane and leads to the conversion of a water-soluble state into a transmembrane state. In this work, we utilize various biophysical techniques to characterize the insertion pathway from the thermodynamic perspective. Thermal and chemical unfolding measured by differential scanning calorimetry, circular dichroism and tryptophan fluorescence reveal that the free energy of unfolding of the T-domain at neutral and mildly acidic pH differ by 3–5 kcal/mol, depending on the experimental conditions. Fluorescence correlation spectroscopy measurements show that the free energy change from the membrane-competent state to the interfacial state is approximately −8 kcal/mol and is pH-independent, while that from the membrane-competent state to the transmembrane state ranges between −9.5 to −12 kcal/mol, depending on the membrane lipid composition and pH. Finally, the thermodynamics of transmembrane insertion of individual helices was tested using an in vitro assay that measures the translocon-assisted integration of test sequences into the microsomal membrane. These experiments suggest that even the most hydrophobic helix TH8 has only a small favorable free energy of insertion. The free energy for the insertion of the consensus insertion unit TH8-TH9 is slightly more favorable, yet less favorable than that measured for the entire protein, suggesting a cooperative effect for the membrane insertion of the helices of the T-domain. PMID:25281329

Real-time PCR Detection of Brucella Abortus: A Comparative Study of SYBR Green I, 5'-exonuclease, and Hybridization Probe Assays

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

Newby, Deborah Trishelle; Hadfield, Ted; Roberto, Francisco Figueroa

Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and themore » IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.« less

The 3'-end region of the human PDGFR-β core promoter nuclease hypersensitive element forms a mixture of two unique end-insertion G-quadruplexes.

PubMed

Onel, Buket; Carver, Megan; Agrawal, Prashansa; Hurley, Laurence H; Yang, Danzhou

2018-04-01

While the most stable G-quadruplex formed in the human PDGFR-β promoter nuclease hypersensitive element (NHE) is the 5'-mid G-quadruplex, the 3'-end sequence that contains a 3'-GGA run forms a less stable G-quadruplex. Recently, the 3'-end G-quadruplex was found to be a transcriptional repressor and can be selectively targeted by a small molecule for PDGFR-β downregulation. We use 1D and 2D high-field NMR, in combination with Dimethylsulfate Footprinting, Circular Dichroism Spectroscopy, and Electrophoretic Mobility Shift Assay. We determine that the PDGFR-β extended 3'-end NHE sequence forms two novel end-insertion intramolecular G-quadruplexes that co-exist in equilibrium under physiological salt conditions. One G-quadruplex has a 3'-non-adjacent flanking guanine inserted into the 3'-external tetrad (3'-insertion-G4), and another has a 5'-non-adjacent flanking guanine inserted into the 5'-external tetrad (5'-insertion-G4). The two guanines in the GGA-run move up or down within the G-quadruplex to accommodate the inserted guanine. Each end-insertion G-quadruplex has a low thermal stability as compared to the 5'-mid G-quadruplex, but the selective stabilization of GSA1129 shifts the equilibrium toward the 3'-end G-quadruplex in the PDGFR-β NHE. An equilibrium mixture of two unique end-insertion intramolecular G-quadruplexes forms in the PDGFR-β NHE 3'-end sequence that contains a GGA-run and non-adjacent guanines in both the 3'- and 5'- flanking segments; the novel end-insertion structures of the 3'-end G-quadruplex are selectively stabilized by GSA1129. We show for the first time that an equilibrium mixture of two unusual end-insertion G-quadruplexes forms in a native promoter sequence and appears to be the molecular recognition for PDGFR-β downregulation. Copyright © 2017 Elsevier B.V. All rights reserved.

Software management tools: Lessons learned from use

NASA Technical Reports Server (NTRS)

Reifer, D. J.; Valett, J.; Knight, J.; Wenneson, G.

1985-01-01

Experience in inserting software project planning tools into more than 100 projects producing mission critical software are discussed. The problems the software project manager faces are listed along with methods and tools available to handle them. Experience is reported with the Project Manager's Workstation (PMW) and the SoftCost-R cost estimating package. Finally, the results of a survey, which looked at what could be done in the future to overcome the problems experienced and build a set of truly useful tools, are presented.

Numerical investigation of heat transfer and friction factor characteristics in a circular tube fitted with V-cut twisted tape inserts.

PubMed

Salman, Sami D; Kadhum, Abdul Amir H; Takriff, Mohd S; Mohamad, Abu Bakar

2013-01-01

Numerical investigation of the heat transfer and friction factor characteristics of a circular fitted with V-cut twisted tape (VCT) insert with twist ratio (y = 2.93) and different cut depths (w = 0.5, 1, and 1.5 cm) were studied for laminar flow using CFD package (FLUENT-6.3.26). The data obtained from plain tube were verified with the literature correlation to ensure the validation of simulation results. Classical twisted tape (CTT) with different twist ratios (y = 2.93, 3.91, 4.89) were also studied for comparison. The results show that the enhancement of heat transfer rate induced by the classical and V-cut twisted tape inserts increases with the Reynolds number and decreases with twist ratio. The results also revealed that the V-cut twisted tape with twist ratio y = 2.93 and cut depth w = 0.5 cm offered higher heat transfer rate with significant increases in friction factor than other tapes. In addition the results of V-cut twist tape compared with experimental and simulated data of right-left helical tape inserts (RLT), it is found that the V-cut twist tape offered better thermal contact between the surface and the fluid which ultimately leads to a high heat transfer coefficient. Consequently, 107% of maximum heat transfer was obtained by using this configuration.

Numerical Investigation of Heat Transfer and Friction Factor Characteristics in a Circular Tube Fitted with V-Cut Twisted Tape Inserts

PubMed Central

Salman, Sami D.; Kadhum, Abdul Amir H.; Takriff, Mohd S.; Mohamad, Abu Bakar

2013-01-01

Numerical investigation of the heat transfer and friction factor characteristics of a circular fitted with V-cut twisted tape (VCT) insert with twist ratio (y = 2.93) and different cut depths (w = 0.5, 1, and 1.5 cm) were studied for laminar flow using CFD package (FLUENT-6.3.26). The data obtained from plain tube were verified with the literature correlation to ensure the validation of simulation results. Classical twisted tape (CTT) with different twist ratios (y = 2.93, 3.91, 4.89) were also studied for comparison. The results show that the enhancement of heat transfer rate induced by the classical and V-cut twisted tape inserts increases with the Reynolds number and decreases with twist ratio. The results also revealed that the V-cut twisted tape with twist ratio y = 2.93 and cut depth w = 0.5 cm offered higher heat transfer rate with significant increases in friction factor than other tapes. In addition the results of V-cut twist tape compared with experimental and simulated data of right-left helical tape inserts (RLT), it is found that the V-cut twist tape offered better thermal contact between the surface and the fluid which ultimately leads to a high heat transfer coefficient. Consequently, 107% of maximum heat transfer was obtained by using this configuration. PMID:24078795

viRome: an R package for the visualization and analysis of viral small RNA sequence datasets.

PubMed

Watson, Mick; Schnettler, Esther; Kohl, Alain

2013-08-01

RNA interference (RNAi) is known to play an important part in defence against viruses in a range of species. Second-generation sequencing technologies allow us to assay these systems and the small RNAs that play a key role with unprecedented depth. However, scientists need access to tools that can condense, analyse and display the resulting data. Here, we present viRome, a package for R that takes aligned sequence data and produces a range of essential plots and reports. viRome is released under the BSD license as a package for R available for both Windows and Linux http://virome.sf.net. Additional information and a tutorial is available on the ARK-Genomics website: http://www.ark-genomics.org/bioinformatics/virome. mick.watson@roslin.ed.ac.uk.

An Immunoassay for Quantification of Contamination by Raw Meat Juice on Food Contact Surfaces.

PubMed

Chen, Fur-Chi; Godwin, Sandria; Chambers, Edgar

2016-11-01

Raw chicken products often are contaminated with Salmonella and Campylobacter , which can be transmitted from packages to contact surfaces. Raw meat juices from these packages also provide potential media for cross-contamination. There are limited quantitative data on the levels of consumer exposure to raw meat juice during shopping for and handling of chicken products. An exposure assessment is needed to quantify the levels of transmission and to assess the risk. An enzyme-linked immunosorbent assay (ELISA) was developed and validated for quantitative detection of raw meat juice on hands and various food contact surfaces. Analytical procedures were designed to maximize the recovery of raw meat juice from various surfaces: hands, plastic, wood, stainless steel, laminated countertops, glass, and ceramics. The ELISA was based on the detection of a soluble muscle protein, troponin I (TnI), in the raw meat juice. The assay can detect levels as low as 1.25 ng of TnI, which is equivalent to less than 1 μl of the raw meat juice. The concentrations of TnI in the raw meat juices from 10 retail chicken packages, as determined by ELISA, were between 0.46 and 3.56 ng/μl, with an average of 1.69 ng/μl. The analytical procedures, which include swabbing, extraction, and concentration, enable the detection of TnI from various surfaces. The recoveries of raw meat juice from surfaces of hands were 92%, and recoveries from other tested surfaces were from 55% on plastic cutting boards to 75% on laminated countertops. The ELISA developed has been used for monitoring the transfer of raw meat juice during shopping for and handling of raw chicken products in our studies. The assay also can be applied to other raw meat products, such as pork and beef.

Mass Spec Studio for Integrative Structural Biology

PubMed Central

Rey, Martial; Sarpe, Vladimir; Burns, Kyle; Buse, Joshua; Baker, Charles A.H.; van Dijk, Marc; Wordeman, Linda; Bonvin, Alexandre M.J.J.; Schriemer, David C.

2015-01-01

SUMMARY The integration of biophysical data from multiple sources is critical for developing accurate structural models of large multiprotein systems and their regulators. Mass spectrometry (MS) can be used to measure the insertion location for a wide range of topographically sensitive chemical probes, and such insertion data provide a rich, but disparate set of modeling restraints. We have developed a software platform that integrates the analysis of label-based MS data with protein modeling activities (Mass Spec Studio). Analysis packages can mine any labeling data from any mass spectrometer in a proteomics-grade manner, and link labeling methods with data-directed protein interaction modeling using HADDOCK. Support is provided for hydrogen/ deuterium exchange (HX) and covalent labeling chemistries, including novel acquisition strategies such as targeted HX-tandem MS (MS2) and data-independent HX-MS2. The latter permits the modeling of highly complex systems, which we demonstrate by the analysis of microtubule interactions. PMID:25242457

BACCardI--a tool for the validation of genomic assemblies, assisting genome finishing and intergenome comparison.

PubMed

Bartels, Daniela; Kespohl, Sebastian; Albaum, Stefan; Drüke, Tanja; Goesmann, Alexander; Herold, Julia; Kaiser, Olaf; Pühler, Alfred; Pfeiffer, Friedhelm; Raddatz, Günter; Stoye, Jens; Meyer, Folker; Schuster, Stephan C

2005-04-01

We provide the graphical tool BACCardI for the construction of virtual clone maps from standard assembler output files or BLAST based sequence comparisons. This new tool has been applied to numerous genome projects to solve various problems including (a) validation of whole genome shotgun assemblies, (b) support for contig ordering in the finishing phase of a genome project, and (c) intergenome comparison between related strains when only one of the strains has been sequenced and a large insert library is available for the other. The BACCardI software can seamlessly interact with various sequence assembly packages. Genomic assemblies generated from sequence information need to be validated by independent methods such as physical maps. The time-consuming task of building physical maps can be circumvented by virtual clone maps derived from read pair information of large insert libraries.

A new FPGA architecture suitable for DSP applications

NASA Astrophysics Data System (ADS)

Liyun, Wang; Jinmei, Lai; Jiarong, Tong; Pushan, Tang; Xing, Chen; Xueyan, Duan; Liguang, Chen; Jian, Wang; Yuan, Wang

2011-05-01

A new FPGA architecture suitable for digital signal processing applications is presented. DSP modules can be inserted into FPGA conveniently with the proposed architecture, which is much faster when used in the field of digital signal processing compared with traditional FPGAs. An advanced 2-level MUX (multiplexer) is also proposed. With the added SLEEP MODE PASS to traditional 2-level MUX, static leakage is reduced. Furthermore, buffers are inserted at early returns of long lines. With this kind of buffer, the delay of the long line is improved by 9.8% while the area increases by 4.37%. The layout of this architecture has been taped out in standard 0.13 μm CMOS technology successfully. The die size is 6.3 × 4.5 mm2 with the QFP208 package. Test results show that performances of presented classical DSP cases are improved by 28.6%-302% compared with traditional FPGAs.

Total internal reflection-evanescent coupler for fiber-to-waveguide integration of planar optoelectric devices.

PubMed

Lu, Zhaolin; Prather, Dennis W

2004-08-01

We present a method for parallel coupling from a single-mode fiber, or fiber ribbon, into a silicon-on-insulator waveguide for integration with silicon optoelectronic circuits. The coupler incorporates the advantages of the vertically tapered waveguides and prism couplers, yet offers the flexibility of planar integration. The coupler can be fabricated by use of either wafer polishing technology or gray-scale photolithography. When optimal coupling is achieved in our experimental setup, the coupler can be packaged by epoxy bonding to form a fiber-waveguide parallel coupler or connector. Two-dimensional electromagnetic calculation predicts a coupling efficiency of 77% (- 1.14-dB insertion loss) for a silicon-to-silicon coupler with a uniform tunnel layer. The coupling efficiency is experimentally achieved to be 46% (-3.4-dB insertion loss), excluding the loss in silicon and the reflections from the input surface and the output facet.

Drugs dispensed at the Division of Neonatology at University Hospital in Río Cuarto, Córdoba, Argentina.

PubMed

Fungo, Marta Stella Maris; Vega, Elena María

2013-04-01

The objective was to analyze the number of drugs dispensed by the Pharmacy Department to the Neonatology Division, to find out if the use of these drugs is described on the package insert approved by the Administración Nacional de Medicamentos, Alimentos y Tecnología, ANMAT (Drug, Food and Technology Administration of Argentina) and to compare such information with that provided by Medical Associations and Commissions. Analytical, observational and retrospective study in which drugs were analyzed based on dosage units, costs and relevance in the 2011 annual budget. We analyzed the information found in ANMAT-approved label inserts, in the Neonatal Pharmacopeia of the Sociedad Argentina de Pediatría and in the Formularies of the Confederación Médica Argentina and the Comisión Nacional del Medicamento (National Medication Commission). A total of 102 drugs (91 drug substances) were dispensed throughout 2011. Drugs most commonly supplied were: antiinfective agents for systemic use (24.51%), agents for the blood and blood forming organs, cardiovascular system, and nervous system (12.72% each). The total expenditure was ARS 263,285.52. Only 21 drugs accounted for 90.73% of the cost. Out of the 14 drugs in this group, only 1 had information related to its use in neonatology in all its labels (package inserts), only 4 in some of their product information and there was no information at all in any of the remaining 9 drugs. The Neonatal Pharmacopeia reported on 12 of the 14 drugs, while the Formularies made a reference to 9 of the 14 drugs. The most widely used drugs were antiinfectives for systemic use. A total of 21 drugs accounted for 90.73% of the annual cost in drugs. Out of 14, only 1 had information of its use in neonatology in all its labels and 9 corresponded to off-label use.

System for Packaging Planetary Samples for Return to Earth

NASA Technical Reports Server (NTRS)

Badescu, Mircea; Bar-Cohen, Yoseph; Backes, paul G.; Sherrit, Stewart; Bao, Xiaoqi; Scott, James S.

2010-01-01

A system is proposed for packaging material samples on a remote planet (especially Mars) in sealed sample tubes in preparation for later return to Earth. The sample tubes (Figure 1) would comprise (1) tubes initially having open tops and closed bottoms; (2) small, bellows-like collapsible bodies inside the tubes at their bottoms; and (3) plugs to be eventually used to close the tops of the tubes. The top inner surface of each tube would be coated with solder. The side of each plug, which would fit snugly into a tube, would feature a solder-filled ring groove. The system would include equipment for storing, manipulating, filling, and sealing the tubes. The containerization system (see Figure 2) will be organized in stations and will include: the storage station, the loading station, and the heating station. These stations can be structured in circular or linear pattern to minimize the manipulator complexity, allowing for compact design and mass efficiency. The manipulation of the sample tube between stations is done by a simple manipulator arm. The storage station contains the unloaded sample tubes and the plugs before sealing as well as the sealed sample tubes with samples after loading and sealing. The chambers at the storage station also allow for plug insertion into the sample tube. At the loading station the sample is poured or inserted into the sample tube and then the tube is topped off. At the heating station the plug is heated so the solder ring melts and seals the plug to the sample tube. The process is performed as follows: Each tube is filled or slightly overfilled with sample material and the excess sample material is wiped off the top. Then, the plug is inserted into the top section of the tube packing the sample material against the collapsible bellowslike body allowing the accommodation of the sample volume. The plug and the top of the tube are heated momentarily to melt the solder in order to seal the tube.

Read count-based method for high-throughput allelic genotyping of transposable elements and structural variants.

PubMed

Kuhn, Alexandre; Ong, Yao Min; Quake, Stephen R; Burkholder, William F

2015-07-08

Like other structural variants, transposable element insertions can be highly polymorphic across individuals. Their functional impact, however, remains poorly understood. Current genome-wide approaches for genotyping insertion-site polymorphisms based on targeted or whole-genome sequencing remain very expensive and can lack accuracy, hence new large-scale genotyping methods are needed. We describe a high-throughput method for genotyping transposable element insertions and other types of structural variants that can be assayed by breakpoint PCR. The method relies on next-generation sequencing of multiplex, site-specific PCR amplification products and read count-based genotype calls. We show that this method is flexible, efficient (it does not require rounds of optimization), cost-effective and highly accurate. This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.

Transcriptional Enhancers Induce Insertional Gene Deregulation Independently From the Vector Type and Design

PubMed Central

Maruggi, Giulietta; Porcellini, Simona; Facchini, Giulia; Perna, Serena K; Cattoglio, Claudia; Sartori, Daniela; Ambrosi, Alessandro; Schambach, Axel; Baum, Christopher; Bonini, Chiara; Bovolenta, Chiara; Mavilio, Fulvio; Recchia, Alessandra

2009-01-01

The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) γ-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase—PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a γ-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design. PMID:19293778

R classes and methods for SNP array data.

PubMed

Scharpf, Robert B; Ruczinski, Ingo

2010-01-01

The Bioconductor project is an "open source and open development software project for the analysis and comprehension of genomic data" (1), primarily based on the R programming language. Infrastructure packages, such as Biobase, are maintained by Bioconductor core developers and serve several key roles to the broader community of Bioconductor software developers and users. In particular, Biobase introduces an S4 class, the eSet, for high-dimensional assay data. Encapsulating the assay data as well as meta-data on the samples, features, and experiment in the eSet class definition ensures propagation of the relevant sample and feature meta-data throughout an analysis. Extending the eSet class promotes code reuse through inheritance as well as interoperability with other R packages and is less error-prone. Recently proposed class definitions for high-throughput SNP arrays extend the eSet class. This chapter highlights the advantages of adopting and extending Biobase class definitions through a working example of one implementation of classes for the analysis of high-throughput SNP arrays.

Image analysis to evaluate the browning degree of banana (Musa spp.) peel.

PubMed

Cho, Jeong-Seok; Lee, Hyeon-Jeong; Park, Jung-Hoon; Sung, Jun-Hyung; Choi, Ji-Young; Moon, Kwang-Deog

2016-03-01

Image analysis was applied to examine banana peel browning. The banana samples were divided into 3 treatment groups: no treatment and normal packaging (Cont); CO2 gas exchange packaging (CO); normal packaging with an ethylene generator (ET). We confirmed that the browning of banana peels developed more quickly in the CO group than the other groups based on sensory test and enzyme assay. The G (green) and CIE L(∗), a(∗), and b(∗) values obtained from the image analysis sharply increased or decreased in the CO group. And these colour values showed high correlation coefficients (>0.9) with the sensory test results. CIE L(∗)a(∗)b(∗) values using a colorimeter also showed high correlation coefficients but comparatively lower than those of image analysis. Based on this analysis, browning of the banana occurred more quickly for CO2 gas exchange packaging, and image analysis can be used to evaluate the browning of banana peels. Copyright © 2015 Elsevier Ltd. All rights reserved.

Three-step Channel Conformational Changes Common to DNA Packaging Motors of Bacterial Viruses T3, T4, SPP1, and Phi29

PubMed Central

Wang, Shaoying; Ji, Zhouxiang; Yan, Erfu; Haque, Farzin; Guo, Peixuan

2016-01-01

The DNA packaging motor of dsDNA bacterial viruses contains a head-tail connector with a channel for genome to enter during assembly and to exit during host infection. The DNA packaging motor of bacterial virus phi29 was recently reported to use the “One-way Revolution” mechanism for DNA packaging. This raises a question of how dsDNA is ejected during infection if the channel acts as a one-way inward valve. Here we report a three step conformational change of the portal channel that is common among DNA translocation motors of bacterial viruses T3, T4, SPP1, and phi29. The channels of these motors exercise three discrete steps of gating, as revealed by electrophysiological assays. It is proposed that the three step channel conformational changes occur during DNA entry process, resulting in a structural transition in preparation of DNA movement in the reverse direction during ejection. PMID:27181501

Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays

PubMed Central

2009-01-01

Background Identical blood samples tested using different kits can give markedly different hepatitis B virus (HBV) DNA levels, which can cause difficulty in the interpretation of viral load. A universal double-stranded DNA control or standard that can be used in all commercial HBV DNA nucleic acid amplification assay kits is urgently needed. By aligning all HBV genotypes (A-H), we found that the surface antigen gene and precore-core gene regions of HBV are the most conserved regions among the different HBV genotypes. We constructed a chimeric fragment by overlapping extension polymerase chain reaction and obtained a 1,349-bp HBVC+S fragment. We then packaged the fragment into lambda phages using a traditional lambda phage cloning procedure. Results The obtained armored DNA was resistant to DNase I digestion and was stable, noninfectious to humans, and could be easily extracted using commercial kits. More importantly, the armored DNA may be used with all HBV DNA nucleic acid amplification assay kits. Conclusions The lambda phage packaging system can be used as an excellent expression platform for armored DNA. The obtained armored DNA possessed all characteristics of an excellent positive control or standard. In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits. Thus, the constructed armored DNA can probably be used as a universal positive control or standard in HBV DNA assays. PMID:20025781

AOP: An R Package For Sufficient Causal Analysis in Pathway ...

EPA Pesticide Factsheets

Summary: How can I quickly find the key events in a pathway that I need to monitor to predict that a/an beneficial/adverse event/outcome will occur? This is a key question when using signaling pathways for drug/chemical screening in pharma-cology, toxicology and risk assessment. By identifying these sufficient causal key events, we have fewer events to monitor for a pathway, thereby decreasing assay costs and time, while maximizing the value of the information. I have developed the “aop” package which uses backdoor analysis of causal net-works to identify these minimal sets of key events that are suf-ficient for making causal predictions. Availability and Implementation: The source and binary are available online through the Bioconductor project (http://www.bioconductor.org/) as an R package titled “aop”. The R/Bioconductor package runs within the R statistical envi-ronment. The package has functions that can take pathways (as directed graphs) formatted as a Cytoscape JSON file as input, or pathways can be represented as directed graphs us-ing the R/Bioconductor “graph” package. The “aop” package has functions that can perform backdoor analysis to identify the minimal set of key events for making causal predictions.Contact: burgoon.lyle@epa.gov This paper describes an R/Bioconductor package that was developed to facilitate the identification of key events within an AOP that are the minimal set of sufficient key events that need to be tested/monit

PIMMS (Pragmatic Insertional Mutation Mapping System) Laboratory Methodology a Readily Accessible Tool for Identification of Essential Genes in Streptococcus

PubMed Central

Blanchard, Adam M.; Egan, Sharon A.; Emes, Richard D.; Warry, Andrew; Leigh, James A.

2016-01-01

The Pragmatic Insertional Mutation Mapping (PIMMS) laboratory protocol was developed alongside various bioinformatics packages (Blanchard et al., 2015) to enable detection of essential and conditionally essential genes in Streptococcus and related bacteria. This extended the methodology commonly used to locate insertional mutations in individual mutants to the analysis of mutations in populations of bacteria. In Streptococcus uberis, a pyogenic Streptococcus associated with intramammary infection and mastitis in ruminants, the mutagen pGhost9:ISS1 was shown to integrate across the entire genome. Analysis of >80,000 mutations revealed 196 coding sequences, which were not be mutated and a further 67 where mutation only occurred beyond the 90th percentile of the coding sequence. These sequences showed good concordance with sequences within the database of essential genes and typically matched sequences known to be associated with basic cellular functions. Due to the broad utility of this mutagen and the simplicity of the methodology it is anticipated that PIMMS will be of value to a wide range of laboratories in functional genomic analysis of a wide range of Gram positive bacteria (Streptococcus, Enterococcus, and Lactococcus) of medical, veterinary, and industrial significance. PMID:27826289

An investigation of nonuniform dose deposition from an electron beam

NASA Astrophysics Data System (ADS)

Lilley, William; Luu, Kieu X.

1994-08-01

In a search for an explanation of nonuniform electron-beam dose deposition, the integrated tiger series (ITS) of coupled electron/photon Monte Carlo transport codes was used to calculate energy deposition in the package materials of an application-specific integrated circuit (ASIC) while the thicknesses of some of the materials were varied. The thicknesses of three materials that were in the path of an electron-beam pulse were varied independently so that analysis could determine how the radiation dose measurements using thermoluminescent dosimeters (TLD's) would be affected. The three materials were chosen because they could vary during insertion of the die into the package or during the process of taking dose measurements. The materials were aluminum, HIPEC (a plastic), and silver epoxy. The calculations showed that with very small variations in thickness, the silver epoxy had a large effect on the dose uniformity over the area of the die.

Commercial processed food may have endocrine-disrupting potential: soy-based ingredients making the difference.

PubMed

Omoruyi, Iyekhoetin Matthew; Kabiersch, Grit; Pohjanvirta, Raimo

2013-01-01

Processed and packaged food items as well as ready-to-eat snacks are neglected and poorly characterised sources of human exposure to endocrine-disrupting chemicals (EDCs). In this study we investigated the presence of xenoestrogens in commercially processed and packaged Finnish foods, arising from substances deliberately added or inadvertently contaminating the food, substances formed as a result of food processing, or substances leaching from food packaging materials. Samples were obtained in three separate batches of equivalent products from both a supermarket and a local representative of a global chain of hamburger restaurants and extracted by a solid-phase extraction method. Their endocrine-disrupting potential was determined by yeast bioluminescent assay, using two recombinant yeast strains Saccharomyces cerevisiae BMAEREluc/ERα and S. cerevisiae BMA64/luc. In this test system, the majority of samples (both foodstuffs and wrappers) analysed proved negative. However, all batches of industrially prepared hamburgers (but not those obtained from a hamburger restaurant) as well as pepper salami significantly induced luciferase activity in the BMAEREluc/ERα yeast strain indicating the presence of xenoestrogens, with estradiol equivalents of these products ranging from 0.2 to 443 pg g(-1). All three products contained soy-based ingredients, which apparently accounted for, or at least contributed to, their high estrogenic activity, since no signal in the assay was observed with extracts of the packaging material, while two different soy sauces tested yielded an intense signal (28 and 54 pg ml(-1) estradiol-equivalent). These findings imply that by and large chemicals arising in the processing or packaging of foodstuffs in Finland constitute an insignificant source of xenoestrogens to consumers. However, soy-derived ingredients in certain food items might render the entire products highly estrogenic. The estrogenic activity of soy is attributed to isoflavones whose health effects - though widely considered beneficial - are controversial. As hamburgers are a popular type of food among children, the findings are noteworthy and possibly of concern.

RNA packaging device of double-stranded RNA bacteriophages, possibly as simple as hexamer of P4 protein.

PubMed

Kainov, Denis E; Pirttimaa, Markus; Tuma, Roman; Butcher, Sarah J; Thomas, George J; Bamford, Dennis H; Makeyev, Eugene V

2003-11-28

Genomes of complex viruses have been demonstrated, in many cases, to be packaged into preformed empty capsids (procapsids). This reaction is performed by molecular motors translocating nucleic acid against the concentration gradient at the expense of NTP hydrolysis. At present, the molecular mechanisms of packaging remain elusive due to the complex nature of packaging motors. In the case of the double-stranded RNA bacteriophage phi 6 from the Cystoviridae family, packaging of single-stranded genomic precursors requires a hexameric NTPase, P4. In the present study, the purified P4 proteins from two other cystoviruses, phi 8 and phi 13, were characterized and compared with phi 6 P4. All three proteins are hexameric, single-stranded RNA-stimulated NTPases with alpha/beta folds. Using a direct motor assay, we found that phi 8 and phi 13 P4 hexamers translocate 5' to 3' along ssRNA, whereas the analogous activity of phi 6 P4 requires association with the procapsid. This difference is explained by the intrinsically high affinity of phi 8 and phi 13 P4s for nucleic acids. The unidirectional translocation results in RNA helicase activity. Thus, P4 proteins of Cystoviridae exhibit extensive similarity to hexameric helicases and are simple models for studying viral packaging motor mechanisms.

Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery.

PubMed

Adelman, K; Salmon, B; Baines, J D

2001-03-13

The product of the herpes simplex virus type 1 U(L)28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of U(L)28 in this process, purified U(L)28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for U(L)28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by U(L)28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by U(L)28 protein. To determine the relevance of the observed U(L)28 protein-pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of U(L)28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by U(L)28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the U(L)28 protein-pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the U(L)28-encoded component of the herpesvirus cleavage and packaging machinery.

PrimerSuite: A High-Throughput Web-Based Primer Design Program for Multiplex Bisulfite PCR.

PubMed

Lu, Jennifer; Johnston, Andrew; Berichon, Philippe; Ru, Ke-Lin; Korbie, Darren; Trau, Matt

2017-01-24

The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its regulatory role in numerous biological processes, but the requisite need for assays which amplify bisulfite-converted DNA represents a major bottleneck due to the unique design constraints imposed on bisulfite-PCR primers. Moreover, a review of the literature indicated no available software solutions which accommodated both high-throughput primer design, support for multiplex amplification assays, and primer-dimer prediction. In response, the tri-modular software package PrimerSuite was developed to support bisulfite multiplex PCR applications. This software was constructed to (i) design bisulfite primers against multiple regions simultaneously (PrimerSuite), (ii) screen for primer-primer dimerizing artefacts (PrimerDimer), and (iii) support multiplex PCR assays (PrimerPlex). Moreover, a major focus in the development of this software package was the emphasis on extensive empirical validation, and over 1300 unique primer pairs have been successfully designed and screened, with over 94% of them producing amplicons of the expected size, and an average mapping efficiency of 93% when screened using bisulfite multiplex resequencing. The potential use of the software in other bisulfite-based applications such as methylation-specific PCR is under consideration for future updates. This resource is freely available for use at PrimerSuite website (www.primer-suite.com).

Single-pipetting microfluidic assay device for rapid detection of Salmonella from poultry package.

PubMed

Fronczek, Christopher F; You, David J; Yoon, Jeong-Yeol

2013-02-15

A direct, sensitive, near-real-time, handheld optical immunoassay device was developed to detect Salmonella typhimurium in the naturally occurring liquid from fresh poultry packages (hereafter "chicken matrix"), with just single pipetting of sample (i.e., no filtration, culturing and/or isolation, thus reducing the assay time and the error associated with them). Carboxylated, polystyrene microparticles were covalently conjugated with anti-Salmonella, and the immunoagglutination due to the presence of Salmonella was detected by reading the Mie scatter signals from the microfluidic channels using a handheld device. The presence of chicken matrix did not affect the light scatter signal, since the optical parameters (particle size d, wavelength of incident light λ and scatter angle θ) were optimized to minimize the effect of sample matrix (animal tissues and blood proteins, etc.). The sample was loaded into a microfluidic chip that was split into two channels, one pre-loaded with vacuum-dried, antibody-conjugated particles and the other with vacuum-dried, bovine serum albumin-conjugated particles. This eliminated the need for a separate negative control, effectively minimizing chip-to-chip and sample-to-sample variations. Particles and the sample were diffused in-channel through chemical agitation by Tween 80, also vacuum-dried within the microchannels. Sequential mixing of the sample to the reagents under a strict laminar flow condition synergistically improved the reproducibility and linearity of the assay. In addition, dried particles were shown to successfully detect lower Salmonella concentrations for up to 8 weeks. The handheld device contains simplified circuitry eliminating unnecessary adjustment stages, providing a stable signal, thus maximizing sensitivity. Total assay time was 10 min, and the detection limit 10 CFU mL(-1) was observed in all matrices, demonstrating the suitability of this device for field assays. Copyright © 2012 Elsevier B.V. All rights reserved.

Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)

PubMed Central

2013-01-01

Background Persistent infection of Penaeus stylirostris densovirus (PstDNV) (also called IHHNV) and its non-infectious inserts in the black tiger shrimp, Penaeus monodon (P. monodon) genome are commonly found without apparent disease. Here, we introduced the method of multiplex PCR in order to differentiate shrimp with viral inserts from ones with the infectious virus. The method allowed us to study the effect of pre-infection of IHHNV, in comparison to IHHNV inserts, on WSSV resistance in P. monodon. Results A multiplex PCR system was developed to amplify the entire IHHNV genome, ensuring the accurate diagnosis. Field samples containing IHHNV DNA templates as low as 20 pg or equivalent 150 viral copies can be detected by this method. By challenging the two groups of diagnosed shrimp with WSSV, we found that shrimp with IHHNV infection and those with viral inserts responded to WSSV differently. Considering cumulative mortality, average time to death of shrimp in IHHNV-infected group (day 14) was significantly delayed relative to that (day 10) of IHHNV-inserted group. Real-time PCR analysis of WSSV copy number indicated the lower amount of WSSV in the IHHNV-infected group than the virus-inserted group. The ratio of IHHNV: WSSV copy number in all determined IHHNV-infected samples ranged from approximately 4 to 300-fold. Conclusion The multiplex PCR assay developed herein proved optimal for convenient differentiation of shrimp specimens with real IHHNV infection and those with insert types. Diagnosed shrimp were also found to exhibit different WSSV tolerance. After exposed to WSSV, the naturally pre-infected IHHNV P. monodon were less susceptible to WSSV and, consequently, survived longer than the IHHNV-inserted shrimp. PMID:23414329

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe.

PubMed

Chen, Bo-Ruei; Hale, Devin C; Ciolek, Peter J; Runge, Kurt W

2012-05-03

Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches.

Gluten content of medications.

PubMed

Cruz, Joseph E; Cocchio, Craig; Lai, Pak Tsun; Hermes-DeSantis, Evelyn

2015-01-01

The establishment of a database for the identification of the presence of gluten in excipients of prescription medications is described. While resources are available to ascertain the gluten content of a given medication, these resources are incomplete and often do not contain a source and date of contact. The drug information service (DIS) at Robert Wood Johnson University Hospital (RWJUH) determined that directly contacting the manufacturer of a product is the best method to determine the gluten content of medications. The DIS sought to establish a resource for use within the institution and create directions for obtaining this information from manufacturers to ensure uniformity of the data collected. To determine the gluten content of a medication, the DIS analyzed the manufacturer's package insert to identify any statement indicating that the product contained gluten or inactive ingredients from known sources of gluten. If there was any question about the source of an inactive ingredient or if no information about gluten content appeared in the package insert, the manufacturer of the particular formulation of the queried medication was contacted to provide clarification. Manufacturers' responses were collected, and medications were categorized as "gluten free," "contains gluten," or "possibly contains gluten." To date, the DIS at RWJUH has received queries about 84 medications and has cataloged their gluten content. The DIS at RWJUH developed a database that categorizes the gluten status of medications, allowing clinicians to easily identify drugs that are safe for patients with celiac disease. Copyright © 2015 by the American Society of Health-System Pharmacists, Inc. All rights reserved.

Import of honeybee prepromelittin into the endoplasmic reticulum: structural basis for independence of SRP and docking protein.

PubMed Central

Müller, G; Zimmermann, R

1987-01-01

Honeybee prepromelittin is correctly processed and imported by dog pancreas microsomes. Insertion of prepromelittin into microsomal membranes, as assayed by signal sequence removal, does not depend on signal recognition particle (SRP) and docking protein. We addressed the question as to how prepromelittin bypasses the SRP/docking protein system. Hybrid proteins between prepromelittin, or carboxy-terminally truncated derivatives, and the cytoplasmic protein dihydrofolate reductase from mouse were constructed. These hybrid proteins were analysed for membrane insertion and sequestration into microsomes. The results suggest the following: (i) The signal sequence of prepromelittin is capable of interacting with the SRP/docking protein system, but this interaction is not mandatory for membrane insertion; this is related to the small size of prepromelittin. (ii) In prepromelittin a cluster of negatively charged amino acids must be balanced by a cluster of positively charged amino acids in order to allow membrane insertion. (iii) In general, a signal sequence can be sufficient to mediate membrane insertion independently of SRP and docking protein in the case of short precursor proteins; however, the presence and distribution of charged amino acids within the mature part of these precursors can play distinct roles. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:2820722

Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

PubMed

Sangesland, Maya; Atwood-Moore, Angela; Rai, Sudhir K; Levin, Henry L

2016-01-01

Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells.

PubMed

Weyer, U; Possee, R D

1991-12-01

A baculovirus transfer vector, pAcUW3, was developed to facilitate the insertion of two influenza virus genes, those encoding the haemagglutinin (HA) and neuraminidase (NA) membrane glycoproteins, into the Autographa californica nuclear polyhedrosis virus genome in a single cotransfection experiment. The NA gene was inserted in place of the polyhedrin coding sequences under the control of the polyhedrin promoter, whereas the HA gene was placed under the control of a copy of the p10 promoter at a site upstream of and in opposite orientation to the polyhedrin promoter. After infection of Spodoptera frugiperda cells with the recombinant virus, AcUW3HANA, both HA and NA were expressed in the very late phase of infection and were shown to be functional in appropriate assays. Immunofluorescence assays demonstrated their localization at the surface of infected insect cells. The expression of both foreign genes in the recombinant virus was found to be stable for at least 12 passages in cell culture.

High-performance and high-reliability SOT-6 packaged diplexer based on advanced IPD fabrication techniques

NASA Astrophysics Data System (ADS)

Qiang, Tian; Wang, Cong; Kim, Nam-Young

2017-08-01

A diplexer offering the advantages of compact size, high performance, and high reliability is proposed on the basis of advanced integrated passive device (IPD) fabrication techniques. The proposed diplexer is developed by combining a third-order low-pass filter (LPF) and a third-order high-pass filter (HPF), which are designed on the basis of the elliptic function prototype low-pass filter. Primary components, such as inductors and capacitors, are designed and fabricated with high Q-factor and appropriate values, and they are subsequently used to construct a compact diplexer having a chip area of 900 μm × 1100 μm (0.009 λ0 × 0.011 λ0, where λ0 is the guided wavelength). In addition, a small-outline transistor (SOT-6) packaging method is adopted, and reliability tests (including temperature, humidity, vibration, and pressure) are conducted to guarantee long-term stability and commercial success. The packaged measurement results indicate excellent RF performance with insertion losses of 1.39 dB and 0.75 dB at operation bands of 0.9 GHz and 1.8 GHz, respectively. The return loss is lower than 10 dB from 0.5 GHz to 4.0 GHz, while the isolation is higher than 15 dB from 0.5 GHz to 3.0 GHz. Thus, it can be concluded that the proposed SOT-6 packaged diplexer is a promising candidate for GSM/CDMA applications. Synthetic solution of diplexer design, RF performance optimization, fabrication process, packaging, RF response measurement, and reliability test is particularly explained and analyzed in this work.

A Mutation in UL15 of Herpes Simplex Virus 1 That Reduces Packaging of Cleaved Genomes▿

PubMed Central

Yang, Kui; Wills, Elizabeth G.; Baines, Joel D.

2011-01-01

Herpesvirus genomic DNA is cleaved from concatemers that accumulate in infected cell nuclei. Genomic DNA is inserted into preassembled capsids through a unique portal vertex. Extensive analyses of viral mutants have indicated that intact capsids, the portal vertex, and all components of a tripartite terminase enzyme are required to both cleave and package viral DNA, suggesting that DNA cleavage and packaging are inextricably linked. Because the processes have not been functionally separable, it has been difficult to parse the roles of individual proteins in the DNA cleavage/packaging reaction. In the present study, a virus bearing the deletion of codons 400 to 420 of UL15, encoding a terminase component, was analyzed. This virus, designated vJB27, failed to replicate on noncomplementing cells but cleaved concatemeric DNA to ca. 35 to 98% of wild-type levels. No DNA cleavage was detected in cells infected with a UL15-null virus or a virus lacking UL15 codons 383 to 385, comprising a motif proposed to couple ATP hydrolysis to DNA translocation. The amount of vJB27 DNA protected from DNase I digestion was reduced compared to the wild-type virus by 6.5- to 200-fold, depending on the DNA fragment analyzed, thus indicating a profound defect in DNA packaging. Capsids containing viral DNA were not detected in vJB27-infected cells, as determined by electron microscopy. These data suggest that pUL15 plays an essential role in DNA translocation into the capsid and indicate that this function is separable from its role in DNA cleavage. PMID:21880766

Computational methods for evaluation of cell-based data assessment--Bioconductor.

PubMed

Le Meur, Nolwenn

2013-02-01

Recent advances in miniaturization and automation of technologies have enabled cell-based assay high-throughput screening, bringing along new challenges in data analysis. Automation, standardization, reproducibility have become requirements for qualitative research. The Bioconductor community has worked in that direction proposing several R packages to handle high-throughput data including flow cytometry (FCM) experiment. Altogether, these packages cover the main steps of a FCM analysis workflow, that is, data management, quality assessment, normalization, outlier detection, automated gating, cluster labeling, and feature extraction. Additionally, the open-source philosophy of R and Bioconductor, which offers room for new development, continuously drives research and improvement of theses analysis methods, especially in the field of clustering and data mining. This review presents the principal FCM packages currently available in R and Bioconductor, their advantages and their limits. Copyright © 2012 Elsevier Ltd. All rights reserved.

OptoZIF Drive: a 3D printed implant and assembly tool package for neural recording and optical stimulation in freely moving mice

NASA Astrophysics Data System (ADS)

Freedman, David S.; Schroeder, Joseph B.; Telian, Gregory I.; Zhang, Zhengyang; Sunil, Smrithi; Ritt, Jason T.

2016-12-01

Objective. Behavioral neuroscience studies in freely moving rodents require small, light-weight implants to facilitate neural recording and stimulation. Our goal was to develop an integrated package of 3D printed parts and assembly aids for labs to rapidly fabricate, with minimal training, an implant that combines individually positionable microelectrodes, an optical fiber, zero insertion force (ZIF-clip) headstage connection, and secondary recording electrodes, e.g. for electromyography (EMG). Approach. Starting from previous implant designs that position recording electrodes using a control screw, we developed an implant where the main drive body, protective shell, and non-metal components of the microdrives are 3D printed in parallel. We compared alternative shapes and orientations of circuit boards for electrode connection to the headstage, in terms of their size, weight, and ease of wire insertion. We iteratively refined assembly methods, and integrated additional assembly aids into the 3D printed casing. Main results. We demonstrate the effectiveness of the OptoZIF Drive by performing real time optogenetic feedback in behaving mice. A novel feature of the OptoZIF Drive is its vertical circuit board, which facilities direct ZIF-clip connection. This feature requires angled insertion of an optical fiber that still can exit the drive from the center of a ring of recording electrodes. We designed an innovative 2-part protective shell that can be installed during the implant surgery to facilitate making additional connections to the circuit board. We use this feature to show that facial EMG in mice can be used as a control signal to lock stimulation to the animal’s motion, with stable EMG signal over several months. To decrease assembly time, reduce assembly errors, and improve repeatability, we fabricate assembly aids including a drive holder, a drill guide, an implant fixture for microelectode ‘pinning’, and a gold plating fixture. Significance. The expanding capability of optogenetic tools motivates continuing development of small optoelectric devices for stimulation and recording in freely moving mice. The OptoZIF Drive is the first to natively support ZIF-clip connection to recording hardware, which further supports a decrease in implant cross-section. The integrated 3D printed package of drive components and assembly tools facilities implant construction. The easy interfacing and installation of auxiliary electrodes makes the OptoZIF Drive especially attractive for real time feedback stimulation experiments.

Development of antioxidant food packaging materials containing eugenol for extending display life of fresh beef.

PubMed

Navikaite-Snipaitiene, Vesta; Ivanauskas, Liudas; Jakstas, Valdas; Rüegg, Nadine; Rutkaite, Ramune; Wolfram, Evelyn; Yildirim, Selçuk

2018-05-23

In this study, clove essential oil (CL) or eugenol (EU) containing cellulose acetate (CA) or acrylic component/hydrophobically modified starch (AC/S) coatings on corona treated oriented polypropylene film (OPP) were designed and investigated for their possible applications as antioxidant packaging materials for fresh meat. The antioxidant properties of the coatings were investigated by Vapour Phase-DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. The CA coatings containing CL or EU showed 43-92% and 43-94% inhibition against DPPH free radicals through the vapour phase, respectively, whereas AC/S/CL and AC/S/EU coatings resulted in DPPH inhibition of 21-65% and 25-84%, respectively. AC/S/EU and CA/EU coatings on OPP containing from 0.32 ± 0.03 to 6.40 ± 0.14 g/m 2 of EU were used to prepare packaging for fresh beef (Longissimus thoracis). After 14 days, the lipid oxidation in beef steaks kept in control and antioxidant packages was 3.33 and 1.00-1.22 mg of malondialdehyde per kilogram of meat, respectively. Moreover, red colour of beef in antioxidant packages was retained. Copyright © 2018 Elsevier Ltd. All rights reserved.

ProbeDesigner: for the design of probesets for branched DNA (bDNA) signal amplification assays.

PubMed

Bushnell, S; Budde, J; Catino, T; Cole, J; Derti, A; Kelso, R; Collins, M L; Molino, G; Sheridan, P; Monahan, J; Urdea, M

1999-05-01

The sensitivity and specificity of branched DNA (bDNA) assays are derived in part through the judicious design of the capture and label extender probes. To minimize non-specific hybridization (NSH) events, which elevate assay background, candidate probes must be computer screened for complementarity with generic sequences present in the assay. We present a software application which allows for rapid and flexible design of bDNA probesets for novel targets. It includes an algorithm for estimating the magnitude of NSH contribution to background, a mechanism for removing probes with elevated contributions, a methodology for the simultaneous design of probesets for multiple targets, and a graphical user interface which guides the user through the design steps. The program is available as a commercial package through the Pharmaceutical Drug Discovery program at Chiron Diagnostics.

TaqMan 5′-Nuclease Human Immunodeficiency Virus Type 1 PCR Assay with Phage-Packaged Competitive Internal Control for High-Throughput Blood Donor Screening

PubMed Central

Drosten, C.; Seifried, E.; Roth, W. K.

2001-01-01

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5′-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5′-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5′-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a ≥95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1. PMID:11724836

A Look to Future Directions in Gene Therapy Research for Monogenic Diseases

PubMed Central

Porteus, Matthew H; Connelly, Jon P; Pruett, Shondra M

2006-01-01

The concept of gene therapy has long appealed to biomedical researchers and clinicians because it promised to treat certain diseases at their origins. In the last several years, there have been several trials in which patients have benefited from gene therapy protocols. This progress, however, has revealed important problems, including the problem of insertional oncogenesis. In this review, which focuses on monogenic diseases, we discuss the problem of insertional oncogenesis and identify areas for future research, such as developing more quantitative assays for risk and efficacy, and ways of minimizing the genotoxic effects of gene therapy protocols, which will be important if gene therapy is to fulfill its conceptual promise. PMID:17009872

Progress in performance enhancement methods for capacitive silicon resonators

NASA Astrophysics Data System (ADS)

Van Toan, Nguyen; Ono, Takahito

2017-11-01

In this paper, we review the progress in recent studies on the performance enhancement methods for capacitive silicon resonators. We provide information on various fabrication technologies and design considerations that can be employed to improve the performance of capacitive silicon resonators, including low motional resistance, small insertion loss, and high quality factor (Q). This paper contains an overview of device structures and working principles, fabrication technologies consisting of hermetic packaging, deep reactive-ion etching and neutral beam etching, and design considerations including mechanically coupled, movable electrode structures and piezoresistive heat engines.

Board-level optical clock signal distribution using Si CMOS-compatible polyimide-based 1- to 48-fanout H-tree

NASA Astrophysics Data System (ADS)

Wu, Linghui; Bihari, Bipin; Gan, Jianhua; Chen, Ray T.; Tang, Suning

1998-08-01

Si-CMOS compatible polymer-based waveguides for optoelectronic interconnects and packaging have been fabricated and characterized. A 1-to-48 fanout optoelectronic interconnection layer (OIL) structure based on Ultradel 9120/9020 for the high-speed massive clock signal distribution for a Cray T-90 supercomputer board has been constructed. The OIL employs multimode polymeric channel waveguides in conjunction with surface-normal waveguide output coupler and 1-to-2 splitter. A total insertion loss of 7.98 dB at 850 nm was measured experimentally.

Apollo 17

NASA Technical Reports Server (NTRS)

Garrett, David

1972-01-01

This is the Press Kit that was given to the various media outlets that were interested in covering the Apollo 17 mission. It includes information about the moon, lunar science, concentrating on the planned mission. The kit includes information about the flight, and the trajectory, planned orbit insertion maneuvers, the extravehicular mission events, a comparison with the Apollo 16, a map of the lunar surface, and the surface activity, information about the Taurus-Littrow landing site, the planned science experiments, the power source for the experiment package and diagrams of some of the instrumentation that was used to perform the experiments.

Superlattice structure modeling and simulation of High Electron Mobility Transistor for improved performance

NASA Astrophysics Data System (ADS)

Munusami, Ravindiran; Yakkala, Bhaskar Rao; Prabhakar, Shankar

2013-12-01

Magnetic tunnel junction were made by inserting the magnetic materials between the source, channel and the drain of the High Electron Mobility Transistor (HEMT) to enhance the performance. Material studio software package was used to design the superlattice layers. Different cases were analyzed to optimize the performance of the device by placing the magnetic material at different positions of the device. Simulation results based on conductivity reveals that the device has a very good electron transport due to the magnetic materials and will amplify very low frequency signals.

Comparative evaluation of commercially available point-of-care heartworm antigen tests using well-characterized canine plasma samples.

PubMed

Starkey, Lindsay A; Bowles, Joy V; Payton, Mark E; Blagburn, Byron L

2017-11-09

Dirofilaria immitis is a worldwide parasite that is endemic in many parts of the United States. There are many commercial assays available for the detection of D. immitis antigen, one of which was modified and has reentered the market. Our objective was to compare the recently reintroduced Witness® Heartworm (HW) Antigen test Kit (Zoetis, Florham Park, NJ) and the SNAP® Heartworm RT (IDEXX Laboratories, Inc., Westbrook, ME) to the well-based ELISA DiroChek® Heartworm Antigen Test Kit (Zoetis, Florham Park, NJ). Canine plasma samples were either received at the Auburn Diagnostic Parasitology Laboratory from veterinarians submitting samples for additional heartworm testing (n = 100) from 2008 to 2016 or purchased from purpose-bred beagles (n = 50, presumed negative) in 2016. Samples were categorized as "positive," "borderline" or "negative" using our established spectrophotometric cutoff value with the DiroChek® assay when a sample was initially received and processed. Three commercially available heartworm antigen tests (DiroChek®, Witness® HW, and SNAP® RT) were utilized for simultaneous testing of the 150 samples in random order as per their package insert with the addition of spectrophotometric optical density (OD) readings of the DiroChek® assay. Any samples yielding discordant test results between assays were further evaluated by heat treatment of plasma and retesting. Chi-square tests for the equality of proportions were utilized for statistical analyses. Concordant results occurred in 140/150 (93.3%) samples. Discrepant results occurred in 10/150 samples tested (6.6%): 9/10 occurring in the borderline heartworm (HW) category and 1/10 occurring in the negative HW category. The sensitivity and specificity of each test compared to the DiroChek® read by spectrophotometer was similar to what has been reported previously (Witness®: sensitivity 97.0% [94.1-99.4%], specificity 96.4% [95.5-100.0%]; SNAP® RT: sensitivity 90.9% [78.0-100.0%], specificity 98.8% [96.0-100.0%]). There were significant differences detected when comparing the sensitivities of the SNAP® RT and the Witness® HW to the DiroChek® among the 150 total samples (p = 0.003) and the 50 "borderline" samples (p = 0.001). In this study, the sensitivity of the Witness® HW was higher than the sensitivity of the SNAP® RT when compared with the DiroChek® test results prior to heat treatment of samples.

Cooperative heteroassembly of the adenoviral L4-22K and IVa2 proteins onto the viral packaging sequence DNA.

PubMed

Yang, Teng-Chieh; Maluf, Nasib Karl

2012-02-21

Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus. Viral DNA packaging refers to the process whereby the viral genome becomes encapsulated by the viral particle. In Ad, activation of the DNA packaging reaction requires at least three viral components: the IVa2 and L4-22K proteins and a section of DNA within the viral genome, called the packaging sequence. Previous studies have shown that the IVa2 and L4-22K proteins specifically bind to conserved elements within the packaging sequence and that these interactions are absolutely required for the observation of DNA packaging. However, the equilibrium mechanism for assembly of IVa2 and L4-22K onto the packaging sequence has not been determined. Here we characterize the assembly of the IVa2 and L4-22K proteins onto truncated packaging sequence DNA by analytical sedimentation velocity and equilibrium methods. At limiting concentrations of L4-22K, we observe a species with two IVa2 monomers and one L4-22K monomer bound to the DNA. In this species, the L4-22K monomer is promoting positive cooperative interactions between the two bound IVa2 monomers. As L4-22K levels are increased, we observe a species with one IVa2 monomer and three L4-22K monomers bound to the DNA. To explain this result, we propose a model in which L4-22K self-assembly on the DNA competes with IVa2 for positive heterocooperative interactions, destabilizing binding of the second IVa2 monomer. Thus, we propose that L4-22K levels control the extent of cooperativity observed between adjacently bound IVa2 monomers. We have also determined the hydrodynamic properties of all observed stoichiometric species; we observe that species with three L4-22K monomers bound have more extended conformations than species with a single L4-22K bound. We suggest this might reflect a molecular switch that controls insertion of the viral DNA into the capsid.

MS2 bacteriophage as a delivery vessel of porphyrins for photodynamic therapy

NASA Astrophysics Data System (ADS)

Cohen, Brian A.; Kaloyeros, Alain E.; Bergkvist, Magnus

2011-02-01

Challenges associated with photodynamic therapy (PDT) include the packaging and site-specific delivery of therapeutic agents to the tissue of interest. Nanoscale encapsulation of PDT agents inside targeted virus capsids is a novel concept for packaging and site-specific targeting. The icosahedral MS2 bacteriophage is one potential candidate for such a packaging-system. MS2 has a porous capsid with an exterior diameter of ~28 nm where the pores allow small molecules access to the capsid interior. Furthermore, MS2 presents suitable residues on the exterior capsid for conjugation of targeting ligands. Initial work by the present investigators has successfully demonstrated RNA-based self-packaging of a heterocyclic PDT agent (meso-tetrakis(para-N-trimethylanilinium)porphine, TMAP) into the MS2 capsid. Packaging photoactive compounds in confined spaces could result in energy transfer between the molecules upon photoactivation, which could in turn reduce the production of radical oxygen species (ROS). ROS are key components in photodynamic therapy, and a reduced production could negatively impact the efficacy of PDT treatment. Here, findings are presented from an investigation of ROS generation of TMAP encapsulated within the MS2 capsid compared to free TMAP in solution. Monitoring of ROS production upon photoactivation via a specific singlet oxygen assay revealed the impact on ROS generation between packaged porphyrins as compared to free porphyrin in an aqueous solution. Follow on work will study the ability of MS2-packaged porphyrins to generate ROS in vitro and subsequent cytotoxic effects on cells in culture.

[Methods of hygromycin B phosphotransferase activity assay in transgenic plant].

PubMed

Zhuo, Qin; Yang, Xiaoguang

2004-07-01

Hygromycin B phosphotransferase (HPT) is a widely used selectable marker protein of transgenic plant. Detection of its activity is critical to studies on the development of various transgenic plants, silence of inserted gene, marker-free system development and safety assessment of transgenic food. In this paper, several methods for detecting the activity of this enzyme were reviewed.

Evaluation of a multitarget real-time PCR assay for detection of Bordetella species during a pertussis outbreak in New Hampshire in 2011.

PubMed

Gao, Fengxiang; Mahoney, Jennifer C; Daly, Elizabeth R; Lamothe, Wendy; Tullo, Daniel; Bean, Christine

2014-01-01

A multitarget real-time PCR assay with three targets, including insertion sequence 481 (IS481), IS1001, and an IS1001-like element, as well as pertussis toxin subunit S1 (ptxS1), for the detection of Bordetella species was evaluated during a pertussis outbreak. The sensitivity and specificity were 77 and 88% (PCR) and 66 and 100% (culture), respectively. All patients with an IS481 C(T) of <30 also tested positive by ptxS1 assay and were clinical pertussis cases. No patients with IS481 C(T) values of ≥40 tested positive by culture. Therefore, we recommend that culture be performed only for specimens with IS481 C(T) values of 30 ≤ CT <40.

Evaluation of a Multitarget Real-Time PCR Assay for Detection of Bordetella Species during a Pertussis Outbreak in New Hampshire in 2011

PubMed Central

Mahoney, Jennifer C.; Daly, Elizabeth R.; Lamothe, Wendy; Tullo, Daniel; Bean, Christine

2014-01-01

A multitarget real-time PCR assay with three targets, including insertion sequence 481 (IS481), IS1001, and an IS1001-like element, as well as pertussis toxin subunit S1 (ptxS1), for the detection of Bordetella species was evaluated during a pertussis outbreak. The sensitivity and specificity were 77 and 88% (PCR) and 66 and 100% (culture), respectively. All patients with an IS481 CT of <30 also tested positive by ptxS1 assay and were clinical pertussis cases. No patients with IS481 CT values of ≥40 tested positive by culture. Therefore, we recommend that culture be performed only for specimens with IS481 CT values of 30 ≤ CT <40. PMID:24131698

Idarucizumab (Praxbind) Formulary Review.

PubMed

Buchheit, Jessica; Reddy, Prabashni; Connors, Jean M

2016-09-01

Idarucizumab (Praxbind), a humanized monoclonal antibody fragment was granted accelerated approval from the Food and Drug Administration in October 2015 as the first agent to reverse the effects of a novel oral anticoagulant. The drug is indicated for dabigatran reversal in patients requiring emergency surgery/urgent procedures or with life-threatening or uncontrolled bleeding. In a randomized study with healthy volunteers, compared with placebo, idarucizumab reduced the clotting times for all tests assays (assessed pre-, end of-, and 24 hours after infusion), while the results for the placebo group remained unchanged. Another randomized clinical trial assessed the safety and efficacy of idarucizumab in patients with either overt bleeding or undergoing emergency surgery where hemostasis was required. This study is ongoing, but preliminary results showed reversal efficacy demonstrated a reasonable safety profile from the time of the infusion to 90 days after. The wholesale acquisition cost of two 2.5 g vials of idarucizumab is currently $3482.50. To treat 10 or 20 patients per year with a single 5 g dose is estimated to cost $34,825 and $69,650, respectively. In the clinical trial described above, approximately 20% of patients required a second dose, which would further increase the cost of use. In this formulary review for a health system's pharmacy and therapeutics committee, idarucizumab clinical trials and medication package insert were summarized and, after consulting with clinical experts from our institutions, practical recommendations for use were generated to ensure appropriate and safe use of this agent.

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

Serwer, Philip, E-mail: serwer@uthscsa.edu; Wright, Elena T.; Liu, Zheng

DNA packaging of phages phi29, T3 and T7 sometimes produces incompletely packaged DNA with quantized lengths, based on gel electrophoretic band formation. We discover here a packaging ATPase-free, in vitro model for packaged DNA length quantization. We use directed evolution to isolate a five-site T3 point mutant that hyper-produces tail-free capsids with mature DNA (heads). Three tail gene mutations, but no head gene mutations, are present. A variable-length DNA segment leaks from some mutant heads, based on DNase I-protection assay and electron microscopy. The protected DNA segment has quantized lengths, based on restriction endonuclease analysis: six sharp bands of DNAmore » missing 3.7–12.3% of the last end packaged. Native gel electrophoresis confirms quantized DNA expulsion and, after removal of external DNA, provides evidence that capsid radius is the quantization-ruler. Capsid-based DNA length quantization possibly evolved via selection for stalling that provides time for feedback control during DNA packaging and injection. - Graphical abstract: Highlights: • We implement directed evolution- and DNA-sequencing-based phage assembly genetics. • We purify stable, mutant phage heads with a partially leaked mature DNA molecule. • Native gels and DNase-protection show leaked DNA segments to have quantized lengths. • Native gels after DNase I-removal of leaked DNA reveal the capsids to vary in radius. • Thus, we hypothesize leaked DNA quantization via variably quantized capsid radius.« less

[Efficient packaging retrovirus and construction of transgenic chicken technical platform].

PubMed

Man, Chaolai; Zhang, Qing; Chen, Yan; Zhu, Dahai

2007-10-01

Transgenic chicken and oviduct bioreactor are growing to be one of the hotspot of scientific study in the field of biology. The most successful method of producing transgenic chicken is pseudotyped retrovirus vector system, but no one has reported the production of transgenic chicken by retrovirus system recently in our country. In order to accelerate our study in this field, we introduced the relevant technical methods such as packaging retrovirus and vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped retrovirus, optimizing the conditions of packaging retrovirus, concentrating VSV-G pseudotyped retrovirus, helper virus assays, and microinjection of retrovirus. Furthermore, we successfully conducted in vivo study for detecting the marker gene EGFP of chicken embryo as well as in vitro study for detecting that gene of chicken embryo myoblast (CFM), thus we have provided an applied technical platform for studies of transgenic chicken in the future.

Detection of bisphenol A in food packaging based on fluorescent conjugated polymer PPESO3 and enzyme system.

PubMed

Huang, Hui; Li, Yongxin; Liu, Jintong; Tong, Jin; Su, Xingguang

2015-10-15

Bisphenol A (BPA) is a kind of carcinogen, which can interfere with the body's endocrine system. In this paper, a new kind of fluorescent sensor for BPA detection was established based on the fluorescent conjugated polymer PPESO3. The oxidative product of BPA is able to quench PPESO3 in the presence of HRP and H2O2, and the quenched PL intensity of PPESO3 was proportionally to the concentration of BPA in the range of 1-100 μmol/L with a detection limit of 4 × 10(-7) mol/L. The proposed method has been applied to detect BPA in eight food packaging samples with satisfactory results. The proposed method has the potential for the assay of BPA in food or food packaging samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

Packaging of Human Chromosome 19-Specific Adeno-Associated Virus (AAV) Integration Sites in AAV Virions during AAV Wild-Type and Recombinant AAV Vector Production

PubMed Central

Hüser, Daniela; Weger, Stefan; Heilbronn, Regine

2003-01-01

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought. PMID:12663794

Evolution and Diversity of the Antimicrobial Resistance Associated Mobilome in Streptococcus suis: A Probable Mobile Genetic Elements Reservoir for Other Streptococci.

PubMed

Huang, Jinhu; Ma, Jiale; Shang, Kexin; Hu, Xiao; Liang, Yuan; Li, Daiwei; Wu, Zuowei; Dai, Lei; Chen, Li; Wang, Liping

2016-01-01

Streptococcus suis is a previously neglected, newly emerging multidrug-resistant zoonotic pathogen. Mobile genetic elements (MGEs) play a key role in intra- and interspecies horizontal transfer of antimicrobial resistance (AMR) determinants. Although, previous studies showed the presence of several MGEs, a comprehensive analysis of AMR-associated mobilome as well as their interaction and evolution has not been performed. In this study, we presented the AMR-associated mobilome and their insertion hotspots in S. suis . Integrative conjugative elements (ICEs), prophages and tandem MGEs were located at different insertion sites, while 86% of the AMR-associated MGEs were inserted at rplL and rum loci. Comprehensive analysis of insertions at rplL and rum loci among four pathogenic Streptococcus species ( Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes , and S. suis ) revealed the existence of different groups of MGEs, including Tn5252, ICE Sp 1108, and TnGBS2 groups ICEs, Φm46.1 group prophage, ICE_ICE and ICE_prophage tandem MGEs. Comparative ICE genomics of ICE Sa 2603 family revealed that module exchange and acquisition/deletion were the main mechanisms in MGEs' expansion and evolution. Furthermore, the observation of tandem MGEs reflected a novel mechanism for MGE diversity. Moreover, an in vitro competition assay showed no visible fitness cost was observed between different MGE-carrying isolates and a conjugation assay revealed the transferability of ICE Sa 2603 family of ICEs. Our statistics further indicated that the prevalence and diversity of MGEs in S. suis is much greater than in other three species which prompted our hypothesis that S. suis is probably a MGEs reservoir for other streptococci. In conclusion, our results showed that acquisition of MGEs confers S. suis not only its capability as a multidrug resistance pathogen, but also represents a paradigm to study the modular evolution and matryoshkas of MGEs.

Evolution and Diversity of the Antimicrobial Resistance Associated Mobilome in Streptococcus suis: A Probable Mobile Genetic Elements Reservoir for Other Streptococci

PubMed Central

Huang, Jinhu; Ma, Jiale; Shang, Kexin; Hu, Xiao; Liang, Yuan; Li, Daiwei; Wu, Zuowei; Dai, Lei; Chen, Li; Wang, Liping

2016-01-01

Streptococcus suis is a previously neglected, newly emerging multidrug-resistant zoonotic pathogen. Mobile genetic elements (MGEs) play a key role in intra- and interspecies horizontal transfer of antimicrobial resistance (AMR) determinants. Although, previous studies showed the presence of several MGEs, a comprehensive analysis of AMR-associated mobilome as well as their interaction and evolution has not been performed. In this study, we presented the AMR-associated mobilome and their insertion hotspots in S. suis. Integrative conjugative elements (ICEs), prophages and tandem MGEs were located at different insertion sites, while 86% of the AMR-associated MGEs were inserted at rplL and rum loci. Comprehensive analysis of insertions at rplL and rum loci among four pathogenic Streptococcus species (Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and S. suis) revealed the existence of different groups of MGEs, including Tn5252, ICESp1108, and TnGBS2 groups ICEs, Φm46.1 group prophage, ICE_ICE and ICE_prophage tandem MGEs. Comparative ICE genomics of ICESa2603 family revealed that module exchange and acquisition/deletion were the main mechanisms in MGEs' expansion and evolution. Furthermore, the observation of tandem MGEs reflected a novel mechanism for MGE diversity. Moreover, an in vitro competition assay showed no visible fitness cost was observed between different MGE-carrying isolates and a conjugation assay revealed the transferability of ICESa2603 family of ICEs. Our statistics further indicated that the prevalence and diversity of MGEs in S. suis is much greater than in other three species which prompted our hypothesis that S. suis is probably a MGEs reservoir for other streptococci. In conclusion, our results showed that acquisition of MGEs confers S. suis not only its capability as a multidrug resistance pathogen, but also represents a paradigm to study the modular evolution and matryoshkas of MGEs. PMID:27774436

The comet moment as a measure of DNA damage in the comet assay.

PubMed

Kent, C R; Eady, J J; Ross, G M; Steel, G G

1995-06-01

The development of rapid assays of radiation-induced DNA damage requires the definition of reliable parameters for the evaluation of dose-response relationships to compare with cellular endpoints. We have used the single-cell gel electrophoresis (SCGE) or 'comet' assay to measure DNA damage in individual cells after irradiation. Both the alkaline and neutral protocols were used. In both cases, DNA was stained with ethidium bromide and viewed using a fluorescence microscope at 516-560 nm. Images of comets were stored as 512 x 512 pixel images using OPTIMAS, an image analysis software package. Using this software we tested various parameters for measuring DNA damage. We have developed a method of analysis that rigorously conforms to the mathematical definition of the moment of inertia of a plane figure. This parameter does not require the identification of separate head and tail regions, but rather calculates a moment of the whole comet image. We have termed this parameter 'comet moment'. This method is simple to calculate and can be performed using most image analysis software packages that support macro facilities. In experiments on CHO-K1 cells, tail length was found to increase linearly with dose, but plateaued at higher doses. Comet moment also increased linearly with dose, but over a larger dose range than tail length and had no tendency to plateau.

RD2-MolPack-Chim3, a packaging cell line for stable production of lentiviral vectors for anti-HIV gene therapy.

PubMed

Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

2013-08-01

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

PubMed

Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G

2008-12-01

With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

Life cycle assessment of EPS and CPB inserts: design considerations and end of life scenarios.

PubMed

Tan, Reginald B H; Khoo, Hsien H

2005-02-01

Expanded polystyrene (EPS) and corrugated paperboard (CPB) are used in many industrial applications, such as containers, shock absorbers or simply as inserts. Both materials pose two different types of environmental problems. The first is the pollution and resource consumption that occur during the production of these materials; the second is the growing landfills that arise out of the excessive disposal of these packaging materials. Life cycle assessment or LCA will be introduced in this paper as a useful tool to compare the environmental performance of both EPS and CPB throughout their life cycle stages. This paper is divided into two main parts. The first part investigates the environmental impacts of the production of EPS and CPB from 'cradle-to-gate', comparing two inserts--both the original and proposed new designs. In the second part, LCA is applied to investigate various end-of-life cases for the same materials. The study will evaluate the environmental impacts of the present waste management practices in Singapore. Several 'what-if' cases are also discussed, including various percentages of landfilling and incineration. The SimaPro LCA Version 5.0 software's Eco-indicator 99 method is used to investigate the following five environmental impact categories: climate change, acidification/eutrophication, ecotoxicity, fossil fuels and respiratory inorganics.

The R package 'icosa' for coarse resolution global triangular and penta-hexagonal gridding

NASA Astrophysics Data System (ADS)

Kocsis, Adam T.

2017-04-01

With the development of the internet and the computational power of personal computers, open source programming environments have become indispensable for science in the past decade. This includes the increase of the GIS capacity of the free R environment, which was originally developed for statistical analyses. The flexibility of R made it a preferred programming tool in a multitude of disciplines from the area of the biological and geological sciences. Many of these subdisciplines operate with incidence (occurrence) data that are in a large number of cases to be grained before further analyses can be conducted. This graining is executed mostly by gridding data to cells of a Gaussian grid of various resolutions to increase the density of data in a single unit of the analyses. This method has obvious shortcomings despite the ease of its application: well-known systematic biases are induced to cell sizes and shapes that can interfere with the results of statistical procedures, especially if the number of incidence points influences the metrics in question. The 'icosa' package employs a common method to overcome this obstacle by implementing grids with roughly equal cell sizes and shapes that are based on tessellated icosahedra. These grid objects are essentially polyhedra with xyz Cartesian vertex data that are linked to tables of faces and edges. At its current developmental stage, the package uses a single method of tessellation which balances grid cell size and shape distortions, but its structure allows the implementation of various other types of tessellation algorithms. The resolution of the grids can be set by the number of breakpoints inserted into a segment forming an edge of the original icosahedron. Both the triangular and their inverted penta-hexagonal grids are available for creation with the package. The package also incorporates functions to look up coordinates in the grid very effectively and data containers to link data to the grid structure. The classes defined in the package are communicating with classes of the 'sp' and 'raster' packages and functions are supplied that allow resolution change and type conversions. Three-dimensional rendering is made available with the 'rgl' package and two-dimensional projections can be calculated using 'sp' and 'rgdal'. The package was developed as part of a project funded by the Deutsche Forschungsgemeinschaft (KO - 5382/1-1).

An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation.

PubMed

Ingemarsdotter, Carin K; Zeng, Jingwei; Long, Ziqi; Lever, Andrew M L; Kenyon, Julia C

2018-03-14

NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays. Treatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect. NSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594 the flexibility of SL3 appears to be a unique requirement for genome encapsidation and identifies this process as a highly specific drug target. This study is proof of principle that development of a new class of antiretroviral drugs that specifically target viral packaging by binding to the viral genomic RNA is achievable.

Evaluation of Risk Versus Benefit Information in Direct-To-Consumer (DTC) Prescription Drug Television Advertisements.

PubMed

Fahim, Germin; Toscani, Michael; Barone, Joseph A; Wang, Chris; Gandhi, Shivani

2018-01-01

The FDA's Presenting Risk Information draft guidance from May 2009 states that the time of risk versus benefit is a factor taken into consideration when evaluating audio and video direct-to-consumer (DTC) broadcasts. The objective of the study is to evaluate the proportion of risk narration on television (TV) advertisements in comparison to the actual proportion of serious adverse effects findings across select therapeutic areas. The study reviews prescription drug TV advertisements between the years 2010 and 2015 separated by therapeutic class. Indicators to assess risk versus benefit are as follows: total benefit time, total risk time, total ad time, percentage proportion of risk, and number of serious adverse effects (SAEs) listed in the package insert. The objective is establishing proportion of risk-to-benefit narration across therapeutic areas and the proportion of risk narration compared to the number of SAEs in the package insert. These outcomes will reflect whether TV advertisements abide by the "fair balance" rule and if the time spent on risk narrations is proportional to the number of SAEs across therapeutic areas. An analysis of risk versus benefit showed that there was a vast range of percentage differences in risk versus benefit narration across the products selected. The majority of the products narrated showed a 40% to 60% risk-to-benefit ratio. Six out of the 10 products evaluated communicated applicable black box warnings. There was variability among the SAE percentages presented between products. Lack of consistency exists between risks versus benefit proportions among different drug products.

The increase in prescriptions of bisphosphonates and the incidence proportion of osteonecrosis of the jaw after risk communication activities in Japan: a hospital-based cohort study†

PubMed Central

Sumi, Eriko; Yamazaki, Toru; Tanaka, Shiro; Yamamoto, Keiichi; Nakayama, Takeo; Bessho, Kazuhisa; Yokode, Masayuki

2014-01-01

Purpose The purpose of this study was to investigate the impact of risk communication about bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) on the number of reported cases to the Drug Adverse Reactions Reporting System and on the incidence proportion of ONJ in a hospital-based cohort study in Japan. Method We conducted a survey of the safety information on BP-related ONJ available from regulatory authorities, pharmaceutical manufacturers and academic associations. We also performed a trend analysis of a dataset from the Drug Adverse Reactions Reporting System and a sub-analysis, using previously constructed data from a retrospective cohort study. Results Risk communication from pharmaceutical manufacturers and academic associations began within 1 year after revisions were made to the package inserts, in October 2006. Twenty times more cases of ONJ have been reported to regulatory authority since 2007, compared with the period before 2007. In our cohort, the incidence proportion of ONJ during and after 2009 was four times greater than before 2009. During this period, BPs were frequently prescribed, whereas there was no increase in the use of alternative agents, such as selective estrogen receptor modulators. Conclusion ONJ was increasingly diagnosed after risk communication efforts, but the impact of the communications was not clear. Safety notifications were diligently disseminated after the package insert was revised. However, there was no surveillance for ONJ before the revision. © 2014 The Authors. Pharmacoepidemiology and Drug Safety published by John Wiley & Sons, Ltd. PMID:24399628

Factors influencing subjects' comprehension of a set of medicine package inserts.

PubMed

Pires, Carla; Vigário, Marina; Cavaco, Afonso

2016-08-01

Background Package inserts (PIs) should promote the safe and effective use of medicines. The comprehension of PIs is related to socio-demographic features, such as education. Objectives To evaluate the participants' comprehension of a sample of PIs and to build an explanatory model of subjects' understanding of the content of these documents. Setting The data were collected from municipalities, city halls, firefighters, the military, schools and charities from two Portuguese regions. Methods Cross-sectional descriptive survey: 503 participants, homogeneously distributed by education and gender. The self-administered tool comprised questions on socio-demographic data, literacy tasks and comprehension evaluation of 12 purposively selected PIs. A logistic regression analysis was used. Main outcome measures Scores of numeracy tasks and comprehension. Results The average comprehension score for the PIs was 63 % (±32 %), with 48 % (n = 239) of the participants scoring <75 %. The most important predictors in explaining a comprehension score ≥75 % were having >12 years of education and correctly performing a numeracy task [respectively, OR 49.6 (CI 95 %: 22.8-108) and OR 2.48 (CI 95 %: 1.5-4.2)]. Conclusion An explanatory model of subjects' knowledge about the content of the tested PIs was built. Given that a high level of education and literacy were found to be the most relevant predictors for acceptable comprehension rates, PIs should be clearly written to assure that they are understood by all potential users, including the less educated. The evaluated PIs may thus need to be simplified.

Medication Safety During Pregnancy: Improving Evidence-Based Practice.

PubMed

Sinclair, Susan M; Miller, Richard K; Chambers, Christina; Cooper, Elizabeth M

2016-01-01

Nearly 90% of women in the United States have taken medications during pregnancy. Medication exposures during pregnancy can result in adverse pregnancy and neonatal outcomes including birth defects, fetal loss, intrauterine growth restriction, prematurity, and longer-term neurodevelopmental outcomes. Advising pregnant women about the safety of medication use during pregnancy is complicated by a lack of data necessary to engage the woman in an informed discussion. Routinely, health care providers turn to the package insert, yet this information can be incomplete and can be based entirely on animal studies. Often, adequate safety data are not available. In a busy clinical setting, health care providers need to be able to quickly locate the most up-to-date information in order to counsel pregnant women concerned about medication exposure. Deciding where to locate the best available information is difficult, particularly when the needed information does not exist. Pregnancy registries are initiated to obtain more data about the safety of specific medication exposures during pregnancy; however, these studies are slow to produce meaningful information, and when they do, the information may not be readily available in a published form. Health care providers have valuable data in their everyday practice that can expand the knowledge base about medication safety during pregnancy. This review aims to discuss the limitations of the package insert regarding medication safety during pregnancy, highlight additional resources available to health care providers to inform practice, and communicate the importance of pregnancy registries for expanding knowledge about medication safety during pregnancy. © 2016 by the American College of Nurse-Midwives.

Unsuccessfully Treated Hypertension: A Major Public Health Problem With a Potential Solution.

PubMed

Furberg, Curt D; Sealey, Jean E; Blumenfeld, Jon D

2017-09-01

About one-half of all hypertensive adults do not have their blood pressure controlled. They are often prescribed medications that conform to national guidelines but they continue to have elevated blood pressure. This public health problem might be improved by applying plasma renin guided therapy. A contributor to the public health problem of unsuccessfully treated hypertension is that the circulating renin-angiotensin system (RAS) is not recognized in treatment guidelines as clinically relevant for the treatment of hypertension or as important as the body salt status for determining blood pressure levels. Another contributor to the problem is the lack of specificity in the package inserts for antihypertensive drugs. They do not specifically state under the heading "Indications" that RAS blockers are primarily most effective in hypertensive subjects with medium and high plasma renin levels; by contrast, natriuretic drugs are most effective in those with low plasma renin levels. Literature review. To address the problem of unsuccessfully treated hypertension, we recommend that the "Indications" section of package inserts for antihypertensive drugs be more specific. The primary indication for RAS blockers ought to be hypertension with medium and high plasma renin levels, and natriuretic agents for those with low plasma renin levels. Similar language ought to be added to treatment guidelines. Additionally, 3 other reasons for lack of blood pressure control also need to be addressed-failure to prescribe antihypertensive drugs to hypertensive subjects, failure of patients to fill prescriptions, and low drug adherence. © American Journal of Hypertension, Ltd 2017. All rights reserved. For Permissions, please email: journals.permissions@oup.com

An evaluation of the completeness of drug-drug interaction-related information in package inserts.

PubMed

Ng, Giok Qin; Sklar, Grant Edward; Chng, Hui Ting

2017-02-01

The project aimed to evaluate the completeness of drug-drug interaction (DDI)-related information in package inserts (PIs) and develop a systematic approach to conduct the evaluation. DDI-related information in the branded PIs of statins, macrolides, protease inhibitors and selected drugs of narrow therapeutic index (DNTI) were evaluated against the criteria distilled from the Food and Drug Administration (FDA) labelling recommendation guidance document. Decision trees were crafted and employed in the evaluation process. Scores were computed to give each PI an overall completeness score and individual criterion completeness score. The Kruskal-Wallis test and Dunn's multiple comparison test were used to assess the differences in the completeness scores. The mean overall completeness score of the 21 PIs was 35.7 ± 13.4 % (range 12.2-62 %). Eight out of the 11 individual evaluation criterion had a mean completeness score below 50 %. A subclass analysis conducted revealed that PIs from the different drug classes differed in the type of DDI-related information, such that they are more complete or less complete. The completeness score of DDI-related information in the PIs varied extensively amongst and within drug classes. A consensus between the authorities and drug companies on the type and quality of DDI-related information to be included could improve their completeness in PIs and make PIs a valuable source of DDI reference. Decision trees, albeit not validated yet, lay the groundwork for a valuable tool to evaluate DDI-related or other drug information.

A manpower scheduling heuristic for aircraft maintenance application

NASA Astrophysics Data System (ADS)

Sze, San-Nah; Sze, Jeeu-Fong; Chiew, Kang-Leng

2012-09-01

This research studies a manpower scheduling for aircraft maintenance, focusing on in-flight food loading operation. A group of loading teams with flexible shifts is required to deliver and upload packaged meals from the ground kitchen to aircrafts in multiple trips. All aircrafts must be served within predefined time windows. The scheduling process takes into account of various constraints such as meal break allocation, multi-trip traveling and food exposure time limit. Considering the aircrafts movement and predefined maximum working hours for each loading team, the main objective of this study is to form an efficient roster by assigning a minimum number of loading teams to the aircrafts. We proposed an insertion based heuristic to generate the solutions in a short period of time for large instances. This proposed algorithm is implemented in various stages for constructing trips due to the presence of numerous constraints. The robustness and efficiency of the algorithm is demonstrated in computational results. The results show that the insertion heuristic more efficiently outperforms the company's current practice.

Materials and structures technology insertion into spacecraft systems: Successes and challenges

NASA Astrophysics Data System (ADS)

Rawal, Suraj

2018-05-01

Over the last 30 years, significant advancements have led to the use of multifunctional materials and structures technologies in spacecraft systems. This includes the integration of adaptive structures, advanced composites, nanotechnology, and additive manufacturing technologies. Development of multifunctional structures has been directly influenced by the implementation of processes and tools for adaptive structures pioneered by Prof. Paolo Santini. Multifunctional materials and structures incorporating non-structural engineering functions such as thermal, electrical, radiation shielding, power, and sensors have been investigated. The result has been an integrated structure that offers reduced mass, packaging volume, and ease of integration for spacecraft systems. Current technology development efforts are being conducted to develop innovative multifunctional materials and structures designs incorporating advanced composites, nanotechnology, and additive manufacturing. However, these efforts offer significant challenges in the qualification and acceptance into spacecraft systems. This paper presents a brief overview of the technology development and successful insertion of advanced material technologies into spacecraft structures. Finally, opportunities and challenges to develop and mature next generation advanced materials and structures are presented.

SU-8 microprobe with microelectrodes for monitoring electrical impedance in living tissues.

PubMed

Tijero, M; Gabriel, G; Caro, J; Altuna, A; Hernández, R; Villa, R; Berganzo, J; Blanco, F J; Salido, R; Fernández, L J

2009-04-15

This paper presents a minimally invasive needle-shaped probe capable of monitoring the electrical impedance of living tissues. This microprobe consists of a 160 microm thick SU-8 substrate containing four planar platinum (Pt) microelectrodes. We design the probe to minimize damage to the surrounding tissue and to be stiff enough to be inserted in living tissues. The proposed batch fabrication process is low cost and low time consuming. The microelectrodes obtained with this process are strongly adhered to the SU-8 substrate and their impedance does not depend on frequency variation. In vitro experiments are compared with previously developed Si and SiC based microprobes and results suggest that it is preferable to use the SU-8 based microprobes due to their flexibility and low cost. The microprobe is assembled on a flexible printed circuit FPC with a conductive glue, packaged with epoxy and wired to the external instrumentation. This flexible probe is inserted into a rat kidney without fracturing and succeeds in demonstrating the ischemia monitoring.

Gamma inert sterilization: a solution to polyethylene oxidation?

PubMed

Medel, Francisco J; Kurtz, Steven M; Hozack, William J; Parvizi, Javad; Purtill, James J; Sharkey, Peter F; MacDonald, Daniel; Kraay, Matthew J; Goldberg, Victor; Rimnac, Clare M

2009-04-01

In the 1990s, oxidation was found to occur in ultra-high molecular weight polyethylene total joint replacement components following gamma irradiation and prolonged shelf aging in air. Orthopaedic manufacturers developed barrier packaging to reduce oxidation during and after radiation sterilization. The present study explores the hypothesis that polyethylene components sterilized in a low-oxygen environment undergo similar in vivo oxidative mechanisms as inserts sterilized in air. In addition, the potential influence of the different sterilization processes on the wear performance of the polyethylene components was examined. An analysis of oxidation, wear, and surface damage was performed for forty-eight acetabular liners and 123 tibial inserts. The mean implantation time was 12.3+/-3.7 years for thirty-one acetabular liners that had been gamma sterilized in air and 4.0+/-2.5 years for the seventeen acetabular liners that had been gamma sterilized in inert gas. The mean implantation time was 11.0+/-3.2 years for the twenty-six tibial inserts that had been sterilized in air and 2.8+/-2.2 years for the ninety-seven tibial inserts that had been gamma sterilized in inert gas. Oxidation and hydroperoxide levels were characterized in loaded and unloaded regions of the inserts. Measurable oxidation and oxidation potential were observed in all cohorts. The oxidation and hydroperoxide levels were regional. Surfaces with access to body fluids were more heavily oxidized than protected bearing surfaces were. This variation appeared to be greater in historical (gamma-in-air-sterilized) components. Regarding wear performance, historical and conventional acetabular liners showed similar wear penetration rates, whereas a low incidence of delamination was confirmed for the conventional tibial inserts in the first decade of implantation. The present study explores the impact of industry-wide changes in sterilization practices for polyethylene. We found lower oxidation and oxidation potential in the conventional acetabular liners and tibial inserts that had been gamma sterilized in inert gas as compared with the historical components that had been gamma sterilized in air. However, we also found strong evidence that conventional components undergo mechanisms of in vivo oxidation similar to those observed following gamma irradiation in air. In addition, gamma sterilization in inert gas did not provide polyethylene with a significant improvement in terms of wear resistance as compared with gamma sterilization in air, except for a lower incidence of delamination in the first decade of implantation for tibial inserts.

Stability of Ribavirin for Inhalation Packaged in Syringes or Glass Vials when Stored Frozen, Refrigerated, and at Room Temperature.

PubMed

Larson, Bayli; Bushman, Lane R; Casciano, Matthew L; Oldland, Alan R; Kiser, Jennifer J; Kiser, Tyree H

2016-01-01

The primary aim of this study was to investigate ribavirin solution for inhalation stability under three different conditions (frozen, refrigerated, room temperature) over a 45-day period. A ribavirin 6000-mg vial was reconstituted with 90 mL of Sterile Water for Injection per the package insert to yield a concentration of approximately 67 mg/mL. The solution was then placed in either syringes or empty glass vials and stored in the freezer (-20°C), in the refrigerator (~0°C to 4°C), or at room temperature (~20°C to 25°C). Original concentrations were measured on day 0 and subsequent concentrations were measured on day 2, 14, and 45 utilizing a validated liquid chromatography with tandem mass spectrometry assay. All analyses were performed in triplicate for each storage condition. Additionally, at each time point the physical stability was evaluated and the pH of solution was measured. The solution was considered stable if =90% of the original concentration was retained over the study period. A validated liquid chromatography with tandem mass spectrometry analysis demonstrated that >95% of the original ribavirin concentration was preserved over the 45-day period for all study conditions. The ribavirin concentration remained within the United States Pharmacopeia (USP)-required range of 95% to 105% of the original labeled product amount throughout the entire study period for all study conditions. Precipitation of ribavirin was noted during the thawing cycle for frozen samples, but the drug went back into solution once the thawing process was completed. No changes in color or turbidity were observed in any of the prepared solutions. Values for pH remained stable over the study period and ranged from 4.1 to 5.3. Ribavirin for inhalation solution is physically and chemically stable for at least 45 days when frozen, refrigerated, or kept at room temperature after reconstitution to a concentration of approximately 67 mg/mL and placed in syringes or glass vials. Copyright© by International Journal of Pharmaceutical Compounding, Inc.

msgbsR: An R package for analysing methylation-sensitive restriction enzyme sequencing data.

PubMed

Mayne, Benjamin T; Leemaqz, Shalem Y; Buckberry, Sam; Rodriguez Lopez, Carlos M; Roberts, Claire T; Bianco-Miotto, Tina; Breen, James

2018-02-01

Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high diversity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between samples. To fill this computational need, we present msgbsR, an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the individual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package ( https://bioconductor.org/packages/release/bioc/html/msgbsR.html ).

BamA β16C strand and periplasmic turns are critical for outer membrane protein insertion and assembly.

PubMed

Gu, Yinghong; Zeng, Yi; Wang, Zhongshan; Dong, Changjiang

2017-11-21

Outer membrane (OM) β-barrel proteins play important roles in importing nutrients, exporting wastes and conducting signals in Gram-negative bacteria, mitochondria and chloroplasts. The outer membrane proteins (OMPs) are inserted and assembled into the OM by OMP85 family proteins. In Escherichia coli , the β-barrel assembly machinery (BAM) contains four lipoproteins such as BamB, BamC, BamD and BamE, and one OMP BamA, forming a 'top hat'-like structure. Structural and functional studies of the E. coli BAM machinery have revealed that the rotation of periplasmic ring may trigger the barrel β1C-β6C scissor-like movement that promote the unfolded OMP insertion without using ATP. Here, we report the BamA C-terminal barrel structure of Salmonella enterica Typhimurium str. LT2 and functional assays, which reveal that the BamA's C-terminal residue Trp, the β16C strand of the barrel and the periplasmic turns are critical for the functionality of BamA. These findings indicate that the unique β16C strand and the periplasmic turns of BamA are important for the outer membrane insertion and assembly. The periplasmic turns might mediate the rotation of the periplasmic ring to the scissor-like movement of BamA β1C-β6C, triggering the OMP insertion. These results are important for understanding the OMP insertion in Gram-negative bacteria, as well as in mitochondria and chloroplasts. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing.

PubMed

Gilling, Damian H; Luna, Vicki Ann; Pflugradt, Cori

2014-01-01

The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target the IS407A-fliP interface in B. mallei and the fliP gene specifically at the insertion point in B. pseudomallei. We then examined DNA from ten B. mallei, ten B. pseudomallei, 14 B. cepacia, eight other Burkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either the fliP gene with the IS407A interruption or the fully intact fliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new species B. mallei from B. pseudomallei and may point to new targets that can be exploited in the future.

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

PubMed Central

2012-01-01

Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches. PMID:22554201

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

PubMed Central

Kalloush, Rawan M.; Vivet-Boudou, Valérie; Ali, Lizna M.; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A.

2016-01-01

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2′hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5′ region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. PMID:27095024

Remote down-hole well telemetry

DOEpatents

Briles, Scott D [Los Alamos, NM; Neagley, Daniel L [Albuquerque, NM; Coates, Don M [Santa Fe, NM; Freund, Samuel M [Los Alamos, NM

2004-07-20

The present invention includes an apparatus and method for telemetry communication with oil-well monitoring and recording instruments located in the vicinity of the bottom of gas or oil recovery pipes. Such instruments are currently monitored using electrical cabling that is inserted into the pipes; cabling has a short life in this environment, and requires periodic replacement with the concomitant, costly shutdown of the well. Modulated reflectance, a wireless communication method that does not require signal transmission power from the telemetry package will provide a long-lived and reliable way to monitor down-hole conditions. Normal wireless technology is not practical since batteries and capacitors have to frequently be replaced or recharged, again with the well being removed from service. RF energy generated above ground can also be received, converted and stored down-hole without the use of wires, for actuating down-hole valves, as one example. Although modulated reflectance reduces or eliminates the loss of energy at the sensor package because energy is not consumed, during the transmission process, additional stored extra energy down-hole is needed.

Are condom instructions readable? Results of a readability study.

PubMed Central

Richwald, G A; Wamsley, M A; Coulson, A H; Morisky, D E

1988-01-01

The use of condoms has assumed a central position in the current strategy to prevent sexual transmission of the human immunodeficiency virus. The effectiveness of condoms in disease prophylaxis is dependent, to a degree, on their correct use. Condom manufacturers routinely include information on condom use either printed on the actual package or in an enclosed package insert. With the use of three readability formulas, the reading grade level was determined for 14 different sets of instructions included with 25 brands of condoms manufactured by 7 domestic and 1 overseas manufacturer. The readability formulas, when applied to instructions for condom use, estimated that, conservatively, 8 of the 14 instructions required at least reading at the level of a high school graduate and none required less than a 10th grade level. Clearly written instructions and simple concepts could assist current and future condom users in the correct use of condoms and improve the effectiveness of condoms in the prevention of AIDS and other sexually transmitted diseases. PMID:3136494

Variable Depth Bragg Peak Method for Single Event Effects Testing

NASA Technical Reports Server (NTRS)

Buchner, S.; Kanyogoro, N.; Foster, C.; O'Neill, P.

2011-01-01

Traditionally, accelerator SEE testing is accomplished by removing the tops of packages so that the IC chips are accessible to heavy ions. However, ICs in some advanced packages cannot be de-lidded so a different approach is used that involves grinding and/or chemically etching away part of the package and the chip from the back side. The parts are then tested from the back side with ions having sufficient range to reach the sensitive volume. More recently, the entire silicon substrate in an SOI/SRAM was removed, making it possible to use low-energy ions with shorter ranges. Where removal of part of the package is not possible, facilities at Michigan State, NASA Space Radiation Laboratory, GANIL (France) and GSI (Germany) offer high-energy heavy ions with long ranges so that the ions can reach the devices' sensitive volumes without much change in the LET. Unfortunately, a run will typically involve only one ion species having a single energy and LET due to the long time it takes to tune a new energy. The Variable Depth Bragg Peak (VDBP) method is similar to the above method in that it involves the use of high-energy heavy ions that are able to pass through the packaging material and reach the device, obviating the need to remove the package. However, the method provides a broad range of LETs from a single ion by inserting degraders in the beam that modify the ion energy and, therefore, the LET. The crux of the method involves establishing a fiduciary point for degrader thickness, i.e., where the Bragg peak is located precisely at the sensitive volume in the device, for which the measured SEU cross-section and the ion LET are both also maxima and can be calculated using a Monte-Carlo program, TRIM. Once the fiduciary point has been established, calibrated high density polyethylene (HDPE) degraders are inserted into or removed from the beam to vary the ion LET at the device in a known manner. After each change of degrader thickness, the SEU cross-section is measured and the corresponding LET calculated from the change in degrader thickness. That information is used to generate a plot of cross-section as a function of ion LET. The advantages of this approach are that the part does not have to be de-lidded and a broad range of LETs is available from a single heavy ion without having to go to non-normal angles of incidence to change the "effective" LET. As we will show, it is possible to obtain an entire curve of cross-section versus LET using just two or three ions. Fig. 1 shows curves of cross-section vs LET for a Freescale 4 Mbit SOI/SRAM measured at the 88" Cyclotron at Berkeley and at NSRL. The open symbols are the data obtained from Berkeley for top-side and back-side irradiation. The solid data points are for the data obtained at NSRL using a device for which the package was intact. The data are for Iron and Gold and cover a range of LETs from 4 MeV.cm2/mg to 84 MeV.cm2/mg. The agreement between the data obtained from Berkeley and from NSRL is excellent, demonstrating that the VDBP method is capable of providing accurate values of cross-section versus LET, at least for the 4 Mbit SRAM. Details of the technique will be included in the final presentation.

A high-throughput assay format for determination of nitrate reductase and nitrite reductase enzyme activities

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

McNally, N.; Liu, Xiang Yang; Choudary, P.V.

1997-01-01

The authors describe a microplate-based high-throughput procedure for rapid assay of the enzyme activities of nitrate reductase and nitrite reductase, using extremely small volumes of reagents. The new procedure offers the advantages of rapidity, small sample size-nanoliter volumes, low cost, and a dramatic increase in the throughput sample number that can be analyzed simultaneously. Additional advantages can be accessed by using microplate reader application software packages that permit assigning a group type to the wells, recording of the data on exportable data files and exercising the option of using the kinetic or endpoint reading modes. The assay can also bemore » used independently for detecting nitrite residues/contamination in environmental/food samples. 10 refs., 2 figs.« less

Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay.

PubMed

Jacchia, Sara; Nardini, Elena; Savini, Christian; Petrillo, Mauro; Angers-Loustau, Alexandre; Shim, Jung-Hyun; Trijatmiko, Kurniawan; Kreysa, Joachim; Mazzara, Marco

2015-02-18

In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.

Measurement of radionuclides in waste packages

DOEpatents

Brodzinski, R.L.; Perkins, R.W.; Rieck, H.G.; Wogman, N.A.

1984-09-12

A method is described for non-destructively assaying the radionuclide content of solid waste in a sealed container by analysis of the waste's gamma-ray spectrum and neutron emissions. Some radionuclides are measured by characteristic photopeaks in the gamma-ray spectrum; transuranic nuclides are measured by neutron emission rate; other radionuclides are measured by correlation with those already measured.

Measurement of radionuclides in waste packages

DOEpatents

Brodzinski, Ronald L.; Perkins, Richard W.; Rieck, Henry G.; Wogman, Ned A.

1986-01-01

A method is described for non-destructively assaying the radionuclide content of solid waste in a sealed container by analysis of the waste's gamma-ray spectrum and neutron emissions. Some radionuclides are measured by characteristic photopeaks in the gamma-ray spectrum; transuranic nuclides are measured by neutron emission rate; other radionuclides are measured by correlation with those already measured.

A physical interaction between viral replicase and capsid protein is required for genome-packaging specificity in an RNA virus.

PubMed

Seo, Jang-Kyun; Kwon, Sun-Jung; Rao, A L N

2012-06-01

Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.

A Physical Interaction between Viral Replicase and Capsid Protein Is Required for Genome-Packaging Specificity in an RNA Virus

PubMed Central

Seo, Jang-Kyun; Kwon, Sun-Jung

2012-01-01

Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity. PMID:22438552

Stochastic approach for radionuclides quantification

NASA Astrophysics Data System (ADS)

Clement, A.; Saurel, N.; Perrin, G.

2018-01-01

Gamma spectrometry is a passive non-destructive assay used to quantify radionuclides present in more or less complex objects. Basic methods using empirical calibration with a standard in order to quantify the activity of nuclear materials by determining the calibration coefficient are useless on non-reproducible, complex and single nuclear objects such as waste packages. Package specifications as composition or geometry change from one package to another and involve a high variability of objects. Current quantification process uses numerical modelling of the measured scene with few available data such as geometry or composition. These data are density, material, screen, geometric shape, matrix composition, matrix and source distribution. Some of them are strongly dependent on package data knowledge and operator backgrounds. The French Commissariat à l'Energie Atomique (CEA) is developing a new methodology to quantify nuclear materials in waste packages and waste drums without operator adjustment and internal package configuration knowledge. This method suggests combining a global stochastic approach which uses, among others, surrogate models available to simulate the gamma attenuation behaviour, a Bayesian approach which considers conditional probability densities of problem inputs, and Markov Chains Monte Carlo algorithms (MCMC) which solve inverse problems, with gamma ray emission radionuclide spectrum, and outside dimensions of interest objects. The methodology is testing to quantify actinide activity in different kind of matrix, composition, and configuration of sources standard in terms of actinide masses, locations and distributions. Activity uncertainties are taken into account by this adjustment methodology.

Optical isolators for 2-micron fibre lasers

NASA Astrophysics Data System (ADS)

Stevens, Gary; Legg, Thomas H.; Shardlow, Peter

2015-02-01

We report on the development and testing of optical isolators for use in 2-micron fiber laser systems. A variety of potential Faraday rotator materials were characterised to identify the most suitable materials for use in the 1700-2100nm wavelength range. Isolators based on the three best performing materials were then developed and packaged as fiber-in, fiber-out and fiber-in, beam-out devices. The isolators were then tested in CW, pulsed and ultrafast laser systems. The three different designs produced different performance characteristics, but all designs demonstrated isolation >25dB and insertion losses of <1.2 dB.

Visualization Techniques for Assessing Design Factors That Affect the Interaction between Pharmaceutical Vials and Stoppers.

PubMed

Lam, Philippe; Stern, Al

2010-01-01

We developed several techniques for visualizing the fit between a stopper and a vial in the critical flange area, a location typically hidden from view. Using these tools, it is possible to identify surfaces involved in forming the initial seal immediately after stopper insertion. We present examples illustrating important design elements that can contribute to forming a robust primary package. These techniques can also be used for component screening by facilitating the identification of combinations that do not fit well together so that they can be eliminated early in the selection process.

Solar Mesosphere Explorer

NASA Technical Reports Server (NTRS)

1981-01-01

Five experiments and the instruments packages designed for use in studying reactions between sunlight, ozone, and other chemicals in the atmosphere as well as for determining how ozone concentrations are transported in the mesosphere are described. The spin-stabilized satellite carrying the experiments consists of an observatory module and a spacecraft bus. Powered by a solar array which charges the nickel-cadmium batteries, the satellite is to be inserted into a sun synchronous orbit by a two stage Delta 2310 launch vehicle. The mission objectives, spacecraft configurations, and various subsystems are described as well as the ground support and prelaunch operations.

Fiber-optic coupling based on nonimaging expanded-beam optics.

PubMed

Moslehi, B; Ng, J; Kasimoff, I; Jannson, T

1989-12-01

We have fabricated and experimentally tested low-cost and mass-producible multimode fiber-optic couplers and connectors based on nonimaging beam-expanding optics and Liouville's theorem. Analysis indicates that a pair coupling loss of -0.25 dB can be achieved. Experimentally, we measured insertion losses as low as -0.38 dB. The beam expanders can be mass produced owing to the use of plastic injection-molding fabrication techniques and packaged in standard connector housings. This design is compatible with the fiber geometry and can yield highly stable coupling owing to its high tolerance for misalignments.

High-Voltage Leak Detection of a Parenteral Proteinaceous Solution Product Packaged in Form-Fill-Seal Plastic Laminate Bags. Part 3. Chemical Stability and Visual Appearance of a Protein-Based Aqueous Solution for Injection as a Function of HVLD Exposure.

PubMed

Rasmussen, Mats; Damgaard, Rasmus; Buus, Peter; Guazzo, Dana Morton

2013-01-01

This Part 3 of this three-part research series reports the impact of high-voltage leak detection (HVLD) exposure on the physico-chemical stability of the packaged product. The product, intended for human administration by injection, is an aqueous solution formulation of the rapid acting insulin analogue, insulin aspart (NovoRapid®/NovoLog®) by Novo Nordisk A/S, Bagsværd, Denmark. The package is a small-volume form-fill-seal plastic laminate bag. Product-packages exposed to HVLD were compared to unexposed product after storage for 9 months at recommended storage conditions of 5 ± 3 °C. No differences in active ingredient or degradation products assays were noted. No changes in any other stability indicating parameter results were observed. This report concludes this three-part series. Part 1 documented HVLD method development and validation work. Part 2 explored the impact of various package material, package temperature, and package storage conditions on HVLD test results. Detection of leaks in the bag seal area was investigated. In conclusion, HVLD is reported to be a validatable leak test method suitable for rapid, nondestructive container-closure integrity evaluation of the subject product-package. In Part 1 of this three-part series, a leak test method based on electrical conductivity and capacitance, also called high-voltage leak detection (HVLD), was proven to find hole leaks in small plastic bags filled with a solution of insulin aspart intended for human injection (NovoRapid®/NovoLog® by Novo Nordisk A/S, Bagsværd, Denmark). In Part 2, the ability of the HVLD method to find other types of package leaks was tested, and the impact of package material and product storage temperature on HVLD results was explored. This final Part 3 checked how well the packaged protein drug solution maintained its potency after HVLD exposure over 9 months of storage under long-term stability conditions. Results showed that HVLD caused no harm to the product.

The Wnt-1 (int-1) oncogene promoter and its mechanism of activation by insertion of proviral DNA of the mouse mammary tumor virus.

PubMed Central

Nusse, R; Theunissen, H; Wagenaar, E; Rijsewijk, F; Gennissen, A; Otte, A; Schuuring, E; van Ooyen, A

1990-01-01

Wnt-1 (int-1) is a cellular oncogene often activated by insertion of proviral DNA of the mouse mammary tumor virus. We have mapped the 5' end and the promoter area of the Wnt-1 gene by nuclease protection and primer extension assays. In differentiating P19 embryonal carcinoma cells, in which Wnt-1 is naturally expressed, two start sites of transcription were found, one preceded by two TATA boxes and one preceded by several GC boxes. In P19 cells, a 1-kilobase upstream sequence of Wnt-1 was able to confer differentiation-specific expression on a heterologous gene. We have investigated how Wnt-1 transcription was affected by mouse mammary tumor virus proviral integrations in various configurations near the promoters of the gene. One provirus has been inserted in the 5' nontranslated part of Wnt-1, in the same transcriptional orientation, and has functionally replaced the Wnt-1 promoters. Wnt-1 transcription in this tumor starts in the right long terminal repeat of the provirus, with considerable readthrough transcription from the left long terminal repeat. Another provirus has been inserted in the orientation opposite that of Wnt-1 into a GC box, disrupting the first Wnt-1 transcription start site but not the downstream start site. Most insertions have not structurally altered the Wnt-1 transcripts and have enhanced the activity of the normal two promoters. Images PMID:1695322

[Serum immunoglobulin levels in women fitted with a copper T intrauterine contraceptive device (author's transl)].

PubMed

Tatra, G; Beck, A; Prunner, Ch; Breitenecker, G

1975-10-31

Serum concentrations of the immunoglobulins IgG, IgM and IgA were assayed by single radial immunodiffusion in 37 women before insertion of Copper T IUD and 6 to 12 months subsequently. There was a tendency towards an increase in immuno-globulin levels with the IUD in situ, but the results were not of statistical significance.

Development of lattice-inserted 5-Fluorouracil-hydroxyapatite nanoparticles as a chemotherapeutic delivery system.

PubMed

Tseng, Ching-Li; Chen, Jung-Chih; Wu, Yu-Chun; Fang, Hsu-Wei; Lin, Feng-Huei; Tang, Tzu-Piao

2015-10-01

Developing an effective vehicle for cancer treatment, hydroxyapatite nanoparticles were fabricated for drug delivery. When 5-Fluorouracil, a major chemoagent, is combined with hydroxyapatite nanocarriers by interclay insertion, the modified hydroxyapatite nanoparticles have superior lysosomal degradation profiles, which could be leveraged as controlled drug release. The decomposition of the hydroxyapatite nanocarriers facilitates the release of 5-Fluorouracil into the cytoplasm causing cell death. Hydroxyapatite nanoparticles with/without 5-Fluorouracil were synthesized and analyzed in this study. Their crystallization properties and chemical composition were examined by X-ray diffraction and Fourier transforms infrared spectroscopy. The 5-Fluorouracil release rate was determined by UV spectroscopy. The biocompatibility of hydroxyapatite-5-Fluorouracil extraction solution was assessed using 3T3 cells via a WST-8 assay. The effect of hydroxyapatite-5-Fluorouracil particles which directly work on the human lung adenocarcinoma (A549) cells was evaluated by a lactate dehydrogenase assay via contact cultivation. A 5-Fluorouracil-absorbed hydroxyapatite particles were also tested. Overall, hydroxyapatite-5-Fluorouracils were prepared using a co-precipitation method wherein 5-Fluorouracil was intercalated in the hydroxyapatite lattice as determined by X-ray diffraction. Energy dispersive scanning examination showed the 5-Fluorouracil content was higher in hydroxyapatite-5-Fluorouracil than in a prepared absorption formulation. With 5-Fluorouracil insertion in the lattice, the widths of the a and c axial constants of the hydroxyapatite crystal increased. The extraction solution of hydroxyapatite-5-Fluorouracil was nontoxic to 3T3 cells, in which 5-Fluorouracil was not released in a neutral phosphate buffer solution. In contrast, at a lower pH value (2.5), 5-Fluorouracil was released by the acidic decomposition of hydroxyapatite. Finally, the results of the lactate dehydrogenase assay revealed that 5-Fluorouracil-hydroxyapatite was highly toxic to A549 cells through direct culture, this phenomenon may result from lysosomal decomposition of particles causing 5-Fluorouracil releasing. The pH-responsive hydroxyapatite-5-Fluorouracil nanoparticles have the potential to be part of a selective drug-delivery system in chemotherapy for cancer treatment. © The Author(s) 2015.

Development of the EpiOcular(TM) eye irritation test for hazard identification and labelling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation.

PubMed

Kaluzhny, Yulia; Kandárová, Helena; Hayden, Patrick; Kubilus, Joseph; d'Argembeau-Thornton, Laurence; Klausner, Mitchell

2011-09-01

The recently implemented 7th Amendment to the EU Cosmetics Directive and the EU REACH legislation have heightened the need for in vitro ocular test methods. To address this need, the EpiOcular(TM) eye irritation test (EpiOcular-EIT), which utilises the normal (non-transformed) human cell-based EpiOcular tissue model, has been developed. The EpiOcular-EIT prediction model is based on an initial training set of 39 liquid and 21 solid test substances and uses a single exposure period and a single cut-off in tissue viability, as determined by the MTT assay. A chemical is classified as an irritant (GHS Category 1 or 2), if the tissue viability is ≤ 60%, and as a non-irritant (GHS unclassified), if the viability is > 60%. EpiOcular-EIT results for the training set, along with results for an additional 52 substances, which included a range of alcohols, hydrocarbons, amines, esters, and ketones, discriminated between ocular irritants and non-irritants with 98.1% sensitivity, 72.9% specificity, and 84.8% accuracy. To ensure the long-term commercial viability of the assay, EpiOcular tissues produced by using three alternative cell culture inserts were evaluated in the EpiOcular-EIT with 94 chemicals. The assay results obtained with the initial insert and the three alternative inserts were very similar, as judged by correlation coefficients (r²) that ranged from 0.82 to 0.96. The EpiOcular-EIT was pre-validated in 2007/2008, and is currently involved in a formal, multi-laboratory validation study sponsored by the European Cosmetics Association (COLIPA) under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The EpiOcular-EIT, together with EpiOcular's long history of reproducibility and proven utility for ultra-mildness testing, make EpiOcular a useful model for addressing current legislation related to animal use in the testing of potential ocular irritants. 2011 FRAME.

Experiences with the in vivo and in vitro comet assay in regulatory testing.

PubMed

Frötschl, Roland

2015-01-01

The in vivo comet assay has recently been implemented into regulatory genotoxicity testing of pharmaceuticals with inclusion into the ICH S2R1 guidance. Regulatory genotoxicity testing aims to detect DNA alterations in form of gene mutations, larger scale chromosomal damage and recombination and aneuploidy. The ICH S2R1 guideline offers two options of standard batteries of tests for the detection of these endpoints. Both options start with an AMES assay and option 1 includes an in vitro mammalian cell assay and an in vivo micronucleus assay in rodent, whereas option 2 includes an in vivo micronucleus assay in bone marrow in rodent and a second in vivo assay in a second tissue with a second endpoint. The test recommended as second in vivo test is the comet assay in rat liver. The in vivo comet assay is considered as mature enough to ensure reliable detection of relevant in vivo genotoxicants in combination with the micronucleus test in bone marrow and the AMES assay. Although lots of research papers have been published using the in vitro comet assay, the in vitro version has not been implemented into official regulatory testing guidelines. A survey of the years 1999-2014 revealed 27 in vivo comet assays submitted to BfArM with market authorisation procedures, European and national advice procedures and clinical trial applications. In three procedures, in vitro comet assays had been submitted within the genetic toxicology packages. © The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Development of an in vitro PIG-A gene mutation assay in human cells

PubMed Central

Rees, Benjamin J; Tate, Matthew; Lynch, Anthony M; Thornton, Catherine A; Jenkins, Gareth J; Walmsley, Richard M; Johnson, George E

2017-01-01

Abstract Mutagens can be carcinogens, and traditionally, they have been identified in vitro using the Salmonella ‘Ames’ reverse mutation assay. However, prokaryotic DNA packaging, replication and repair systems are mechanistically very different to those in the humans we inevitably seek to protect. Therefore, for many years, mammalian cell line genotoxicity assays that can detect eukaryotic mutagens as well as clastogens and aneugens have been used. The apparent lack of specificity in these largely rodent systems, due partly to their mutant p53 status, has contributed to the use of animal studies to resolve data conflicts. Recently, silencing mutations at the PIG-A locus have been demonstrated to prevent glycophosphatidylinositol (GPI) anchor synthesis and consequentially result in loss of GPI-anchored proteins from the cell’s extracellular surface. The successful exploitation of this mutant phenotype in animal studies has triggered interest in the development of an analogous in vitro PIG-A mutation screening assay. This article describes the development of a robust assay design using metabolically active human cells. The assay includes viability and cell membrane integrity assessment and conforms to the future ideas of the 21st-century toxicology testing. PMID:28057708

[Effects of ezrin silencing on pancreatic cancer cell line Panc-1].

PubMed

Meng, Yun-xiao; Yu, Shuang-ni; Lu, Zhao-hui; Chen, Jie

2012-12-01

To explore the effects of ezrin silencing on pancreatic cancer cell line Panc-1. Pancreatic cancer cell line Panc-1 was transfected with ezrin silencing plasmid. The proliferation and the cell cycle status were determined by CCK-8 assay and flow cytometry analysis, respectively. Cellular membrane protrusions/microvilli formation were visualized by scanning election microscopy. Colony formation assay was used to determine the cell anchor-independent growth ability in vitro. Trans-filter migration and invasion assays were performed with 8 µm pore inserts in a 24-well BioCoat chamber with/without Matrigel. Ezrin silencing decreased cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion, but had no effects on cell proliferation in vitro and cell cycle, in pancreatic cancer cell line Panc-1. Ezrin expression affects the cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion in pancreatic cancer cell line Panc-1.

Minimal Residual Disease Monitoring of Acute Myeloid Leukemia by Massively Multiplex Digital PCR in Patients with NPM1 Mutations.

PubMed

Mencia-Trinchant, Nuria; Hu, Yang; Alas, Maria Antonina; Ali, Fatima; Wouters, Bas J; Lee, Sangmin; Ritchie, Ellen K; Desai, Pinkal; Guzman, Monica L; Roboz, Gail J; Hassane, Duane C

2017-07-01

The presence of minimal residual disease (MRD) is widely recognized as a powerful predictor of therapeutic outcome in acute myeloid leukemia (AML), but methods of measurement and quantification of MRD in AML are not yet standardized in clinical practice. There is an urgent, unmet need for robust and sensitive assays that can be readily adopted as real-time tools for disease monitoring. NPM1 frameshift mutations are an established MRD marker present in half of patients with cytogenetically normal AML. However, detection is complicated by the existence of hundreds of potential frameshift insertions, clonal heterogeneity, and absence of sequence information when the NPM1 mutation is identified using capillary electrophoresis. Thus, some patients are ineligible for NPM1 MRD monitoring. Furthermore, a subset of patients with NPM1-mutated AML will have false-negative MRD results because of clonal evolution. To simplify and improve MRD testing for NPM1, we present a novel digital PCR technique composed of massively multiplex pools of insertion-specific primers that selectively detect mutated but not wild-type NPM1. By measuring reaction end points using digital PCR technology, the resulting single assay enables sensitive and specific quantification of most NPM1 exon 12 mutations in a manner that is robust to clonal heterogeneity, does not require NPM1 sequence information, and obviates the need for maintenance of hundreds of type-specific assays and associated plasmid standards. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Fitness costs of various mobile genetic elements in Enterococcus faecium and Enterococcus faecalis

PubMed Central

Starikova, Irina; Al-Haroni, Mohammed; Werner, Guido; Roberts, Adam P.; Sørum, Vidar; Nielsen, Kaare M.; Johnsen, Pål J.

2013-01-01

Objectives To determine the fitness effects of various mobile genetic elements (MGEs) in Enterococcus faecium and Enterococcus faecalis when newly acquired. We also tested the hypothesis that the biological cost of vancomycin resistance plasmids could be mitigated during continuous growth in the laboratory. Methods Different MGEs, including two conjugative transposons (CTns) of the Tn916 family (18 and 33 kb), a pathogenicity island (PAI) of 200 kb and vancomycin-resistance (vanA) plasmids (80–200 kb) of various origins and classes, were transferred into common ancestral E. faecium and E. faecalis strains by conjugation assays and experimentally evolved (vanA plasmids only). Transconjugants were characterized by PFGE, S1 nuclease assays and Southern blotting hybridization analyses. Single specific primer PCR was performed to determine the target sites for the insertion of the CTns. The fitness costs of various MGEs in E. faecium and E. faecalis were estimated in head-to-head competition experiments, and evolved populations were generated in serial transfer assays. Results The biological cost of a newly acquired PAI and two CTns were both host- and insertion-locus-dependent. Newly acquired vanA plasmids may severely reduce host fitness (25%–27%), but these costs were rapidly mitigated after only 400 generations of continuous growth in the absence of antibiotic selection. Conclusions Newly acquired MGEs may impose an immediate biological cost in E. faecium. However, as demonstrated for vanA plasmids, the initial costs of MGE carriage may be mitigated during growth and beneficial plasmid–host association can rapidly emerge. PMID:23833178

Validation of a dye stain assay for vaginally inserted HEC-filled microbicide applicators

PubMed Central

Katzen, Lauren L.; Fernández-Romero, José A.; Sarna, Avina; Murugavel, Kailapuri G.; Gawarecki, Daniel; Zydowsky, Thomas M.; Mensch, Barbara S.

2011-01-01

Background The reliability and validity of self-reports of vaginal microbicide use are questionable given the explicit understanding that participants are expected to comply with study protocols. Our objective was to optimize the Population Council's previously validated dye stain assay (DSA) and related procedures, and establish predictive values for the DSA's ability to identify vaginally inserted single-use, low-density polyethylene microbicide applicators filled with hydroxyethylcellulose gel. Methods Applicators, inserted by 252 female sex workers enrolled in a microbicide feasibility study in Southern India, served as positive controls for optimization and validation experiments. Prior to validation, optimal dye concentration and staining time were ascertained. Three validation experiments were conducted to determine sensitivity, specificity, negative predictive values and positive predictive values. Results The dye concentration of 0.05% (w/v) FD&C Blue No. 1 Granular Food Dye and staining time of five seconds were determined to be optimal and were used for the three validation experiments. There were a total of 1,848 possible applicator readings across validation experiments; 1,703 (92.2%) applicator readings were correct. On average, the DSA performed with 90.6% sensitivity, 93.9% specificity, and had a negative predictive value of 93.8% and a positive predictive value of 91.0%. No statistically significant differences between experiments were noted. Conclusions The DSA was optimized and successfully validated for use with single-use, low-density polyethylene applicators filled with hydroxyethylcellulose (HEC) gel. We recommend including the DSA in future microbicide trials involving vaginal gels in order to identify participants who have low adherence to dosing regimens. In doing so, we can develop strategies to improve adherence as well as investigate the association between product use and efficacy. PMID:21992983

Usefulness of immunohistochemistry for the detection of the BRAF V600E mutation in Japanese lung adenocarcinoma.

PubMed

Sasaki, Hidefumi; Shimizu, Shigeki; Tani, Yoichi; Shitara, Masayuki; Okuda, Katsuhiro; Hikosaka, Yu; Moriyama, Satoru; Yano, Motoki; Fujii, Yoshitaka

2013-10-01

Mutations in components of the mitogen-activated protein kinase (MAPK) cascade may be a new candidate for target for lung cancer. The usefulness of immunohistochemistry (IHC) as a new approach for the detection of BRAF V600E in cancer patients has been recently reported. To increase the sensitivity, we modified BRAF V600E expression detection assay by IHC using mutation specific antibody. From the screening step, we found a novel 599 insertion T BRAF mutation in lung adenocarcinoma. In this study included 26 surgically removed cases with EGFR, Kras, erbB2, EML4-ALK and KIF5B-RET wild-type (wt) lung adenocarcinomas, including 7 BRAF mutants (5 V600E, 1 N581I, and 1 novel 599 insertion T mutation) analyzed by DNA sequencing. Detection of the BRAF V600E mutation was carried out by the Dako EnVision™ FLEX detection system using the VE1 clone antibody and compared with the results of direct sequencing. The autostainer IHC VE1 assay was positive in 5 of 5 (100%) BRAF V600E-mutated tumors and negative in 20 of 21 (95.2%) BRAF non-V600E tumors, except for a novel 599 insertion T case. IHC using the VE1 clone and FLEX linker is a specific method for the detection BRAF V600E and may be an alternative to molecular biology for the detection of mutations in lung adenocarcinomas. This method might be useful for screening to use molecular target therapy for lung adenocarcinomas. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Use of an internal control in a nested-PCR assay for Mycoplasma hyopneumoniae detection and quantification in tracheobronchiolar washings from pigs.

PubMed

Verdin, E; Kobisch, M; Bové, J M; Garnier, M; Saillard, C

2000-12-01

We have previously reported a nested PCR assay for the detection of Mycoplasma hyopneumoniae directly in tracheobronchiolar washings from living pigs in field conditions. Here, we describe the construction and use of an internal control to monitor the presence of PCR inhibitors. A PCR modified target DNA was constructed by insertion of a small DNA fragment into the M. hyopneumoniae specific DNA target. We have demonstrated that the internal control failed to be amplified in only three tracheobronchiolar washings samples out of the 362 tested. This control molecule was inserted in a Spiroplasma citri derived plasmid vector and introduced into S. citri cells by electroporation. After a few passages we ensured that the recombinant plasmid became inserted into the genome of S. citri. PCR amplification of the DNA of this transformed S. citri strain using nested PCR primers led to amplification of a 900-bp fragment which can be discriminated from the M. hyopneumoniae PCR product 700 bp. The S. citri transformants with the integrated internal control were added to the tracheobronchiolar washings prior to PCR and used as an internal control to check the efficiency of sample processing, and to demonstrate the presence of inhibitors. Furthermore, we have been able to estimate the number of mycoplasma cells in the tracheobronchiolar washings. Quantitation was performed by comparing the PCR signal intensity of the specific M. hyopneumoniae template with known concentrations of the S. citri competitor. The titer in tracheobronchiolar washings ranged approximatively from 10(4)to 10(8)M. hyopneumoniae cells per ml of clinical specimen. Quantitative PCR can be a useful tool for monitoring the progression of M. hyopneumoniae in the disease process. Copyright 2000 Academic Press.

Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging.

PubMed

Sun, Meng; Grigsby, Iwen F; Gorelick, Robert J; Mansky, Louis M; Musier-Forsyth, Karin

2014-01-01

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences.

PubMed

Kalloush, Rawan M; Vivet-Boudou, Valérie; Ali, Lizna M; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A

2016-06-01

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2'hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5' region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. © 2016 Kalloush et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Evaluation of diminished microbial contamination in handling of a novel daily disposable flat pack contact lens.

PubMed

Nomachi, Miya; Sakanishi, Kotaro; Ichijima, Hideji; Cavanagh, H Dwight

2013-05-01

To evaluate the efficacy of a novel daily disposable (DD) flat package in regard to microbial contamination on the anterior and posterior surfaces of a contact lens (CL) during handling of the lens for insertion. Four kinds of commercially available general blister-packed daily disposable contact lenses (DD CLs) as controls and a novel Magic 1-day Menicon Flat Pack as a test lens were used for this in vitro study. Lenses were removed from their packages using fingers coated with fluorescein 3 to 5 μm beads or an approximately 7×10(2) to 2×10(3) colony-forming unit (CFU)/mL Staphylococcus aureus suspension. The transfer of fluorescein beads to the surface of the lenses was then observed by fluorescence microscopy. Microbial contamination on the lenses was observed by light microscopy after a 2-day incubation period; and, the number of colonies isolated from the contaminated lenses was determined after 4 days of incubation. The number of fluorescein beads on the Magic lens was significantly less (p<0.05) than that of the general blister-packed control lenses. Adherence of microbial colonies was observed on both inner and outer surfaces of general blister-packed lenses, whereas no colony formation was found on the inner surface of the Magic lens, and the lowest bacterial adherence was observed for the Magic lens. The data demonstrated that placement of the Magic DD lens onto the eye is accompanied by diminished microbial contamination compared with general blister-packed DD CLs. Eye care professionals; however, should instruct patients to comply with intended use of DD CLs to prevent CL-associated microbial keratitis. In all cases, hand washing is mandated prelens insertion.

Ka-Band MMIC Subarray Technology Program (Ka-Mist)

NASA Technical Reports Server (NTRS)

Pottinger, W.

1995-01-01

Ka-band monolithic microwave integrated circuit (MMIC) arrays have been considered as having high potential for increasing the capability of space, aircraft, and land mobile communication systems in terms of scan performance, data rate, link margin, and flexibility while offering a significant reduction in size, weight, and power consumption. Insertion of MMIC technology into antenna systems, particularly at millimeter wave frequencies using low power and low noise amplifiers in closed proximity to the radiating elements, offers a significant improvement in the array transmit efficiency, receive system noise figure, and overall array reliability. Application of active array technology also leads to the use of advanced beamforming techniques that can improve beam agility, diversity, and adaptivity to complex signal environments. The objective of this program was to demonstrate the technical feasibility of the 'tile' array packaging architecture at EHF via the insertion of 1990 MMIC technology into a functional tile array or subarray module. The means test of this objective was to demonstrate and deliver to NASA a minimum of two 4 x 4 (16 radiating element) subarray modules operating in a transmit mode at 29.6 GHz. Available (1990) MMIC technology was chosen to focus the program effort on the novel interconnect schemes and packaging requirements rather than focusing on MMIC development. Major technical achievements of this program include the successful integration of two 4 x 4 subarray modules into a single antenna array. This 32 element array demonstrates a transmit EIRP of over 300 watts yielding an effective directive power gain in excess of 55 dB at 29.63 GHz. The array has been actively used as the transmit link in airborne/terrestrial mobile communication experiments accomplished via the ACTS satellite launched in August 1993.

Effects Of Environmental And Operational Stresses On RF MEMS Switch Technologies For Space Applications

NASA Technical Reports Server (NTRS)

Jah, Muzar; Simon, Eric; Sharma, Ashok

2003-01-01

Micro Electro Mechanical Systems (MEMS) have been heralded for their ability to provide tremendous advantages in electronic systems through increased electrical performance, reduced power consumption, and higher levels of device integration with a reduction of board real estate. RF MEMS switch technology offers advantages such as low insertion loss (0.1- 0.5 dB), wide bandwidth (1 GHz-100 GHz), and compatibility with many different process technologies (quartz, high resistivity Si, GaAs) which can replace the use of traditional electronic switches, such as GaAs FETS and PIN Diodes, in microwave systems for low signal power (x < 500 mW) applications. Although the electrical characteristics of RF MEMS switches far surpass any existing technologies, the unknown reliability, due to the lack of information concerning failure modes and mechanisms inherent to MEMS devices, create an obstacle to insertion of MEMS technology into high reliability applications. All MEMS devices are sensitive to moisture and contaminants, issues easily resolved by hermetic or near-hermetic packaging. Two well-known failure modes of RF MEMS switches are charging in the dielectric layer of capacitive membrane switches and contact interface stiction of metal-metal switches. Determining the integrity of MEMS devices when subjected to the shock, vibration, temperature extremes, and radiation of the space environment is necessary to facilitate integration into space systems. This paper will explore the effects of different environmental stresses, operational life cycling, temperature, mechanical shock, and vibration on the first commercially available RF MEMS switches to identify relevant failure modes and mechanisms inherent to these device and packaging schemes for space applications. This paper will also describe RF MEMS Switch technology under development at NASA GSFC.

The US Food and Drug Administration's Risk Evaluation and Mitigation Strategy (REMS) Program - Current Status and Future Direction.

PubMed

Wu, Jasmanda; Juhaeri, Juhaeri

2016-12-01

The US Food and Drug Administration (FDA) Amendments Act of 2007 granted the FDA new authorities to enhance drug safety by requiring application holders to submit a proposed Risk Evaluation and Mitigation Strategy (REMS). A REMS is a required risk management plan that uses tools beyond the package insert. REMS elements may include a medication guide and patient package insert for patients and a communication plan focused on health care professionals. Elements to assure safe use (ETASUs) are put in place to mitigate a specific known serious risk when other less restrictive elements of a REMS are not sufficient to mitigate such risk. An implementation system is required for an REMS that includes the ETASUs. With approximately eight years of experience with REMS programs, many health care settings have created systems to manage REMS and also to integrate REMS into their practice settings. At the same time, there are issues associated with the development and implementation of REMS. In 2011, FDA created the REMS Integration Initiative to develop guidance on how to apply statutory criteria to determine when a REMS is required, to improve standardization and assessment of REMS, and to improve integration of REMS into the existing healthcare system. A key component of the REMS Integration Initiative is stakeholder outreach to better understand how existing REMS programs are working and to identify opportunities for improvement. This review attempts to share our company's experience with the REMS program, and to provide updates on FDA's efforts to improve REMS communication, to standardize REMS process, to reduce REMS program burdens and to build a common REMS platform. Copyright © 2016 Elsevier HS Journals, Inc. All rights reserved.

A review of digital microfluidics as portable platforms for lab-on a-chip applications.

PubMed

Samiei, Ehsan; Tabrizian, Maryam; Hoorfar, Mina

2016-07-07

Following the development of microfluidic systems, there has been a high tendency towards developing lab-on-a-chip devices for biochemical applications. A great deal of effort has been devoted to improve and advance these devices with the goal of performing complete sets of biochemical assays on the device and possibly developing portable platforms for point of care applications. Among the different microfluidic systems used for such a purpose, digital microfluidics (DMF) shows high flexibility and capability of performing multiplex and parallel biochemical operations, and hence, has been considered as a suitable candidate for lab-on-a-chip applications. In this review, we discuss the most recent advances in the DMF platforms, and evaluate the feasibility of developing multifunctional packages for performing complete sets of processes of biochemical assays, particularly for point-of-care applications. The progress in the development of DMF systems is reviewed from eight different aspects, including device fabrication, basic fluidic operations, automation, manipulation of biological samples, advanced operations, detection, biological applications, and finally, packaging and portability of the DMF devices. Success in developing the lab-on-a-chip DMF devices will be concluded based on the advances achieved in each of these aspects.

Detection of M. tuberculosis using DNA chips combined with an image analysis system.

PubMed

Huang, T-S; Liu, Y-C; Bair, C-H; Sy, C-L; Chen, Y-S; Tu, H-Z; Chen, B-C

2008-01-01

To develop a packaged DNA chip assay (the DR. MTBC Screen assay) for direct detection of the Mycobacterium tuberculosis complex. We described a DNA chip assay based on the IS6110 gene that can be used for the detection of M. tuberculosis complex. Probes were spotted onto the polystyrene strips in the wells of 96-well microtitre plates and used for hybridisation with biotin-labelled amplicon to yield a pattern of visualised positive spots. The plate image was scanned, analysed and interpreted automatically. The results corresponded well with those obtained by conventional culture as well as clinical diagnosis, with sensitivity and specificity rates of respectively 83.8% and 94.2%, and 84.6% and 96.3%. We conclude that the DR. MTBC Screen assay can detect M. tuberculosis complex rapidly in respiratory specimens, readily adapts to routine work and provides a flexible choice to meet different cost-effectiveness and automation needs in TB-endemic countries. The cost for reagents is around US$10 per sample.

A multiparametric assay for quantitative nerve regeneration evaluation.

PubMed

Weyn, B; van Remoortere, M; Nuydens, R; Meert, T; van de Wouwer, G

2005-08-01

We introduce an assay for the semi-automated quantification of nerve regeneration by image analysis. Digital images of histological sections of regenerated nerves are recorded using an automated inverted microscope and merged into high-resolution mosaic images representing the entire nerve. These are analysed by a dedicated image-processing package that computes nerve-specific features (e.g. nerve area, fibre count, myelinated area) and fibre-specific features (area, perimeter, myelin sheet thickness). The assay's performance and correlation of the automatically computed data with visually obtained data are determined on a set of 140 semithin sections from the distal part of a rat tibial nerve from four different experimental treatment groups (control, sham, sutured, cut) taken at seven different time points after surgery. Results show a high correlation between the manually and automatically derived data, and a high discriminative power towards treatment. Extra value is added by the large feature set. In conclusion, the assay is fast and offers data that currently can be obtained only by a combination of laborious and time-consuming tests.

Cell-free formation and interactome analysis of caveolae.

PubMed

Jung, WooRam; Sierecki, Emma; Bastiani, Michele; O'Carroll, Ailis; Alexandrov, Kirill; Rae, James; Johnston, Wayne; Hunter, Dominic J B; Ferguson, Charles; Gambin, Yann; Ariotti, Nicholas; Parton, Robert G

2018-06-04

Caveolae have been linked to the regulation of signaling pathways in eukaryotic cells through direct interactions with caveolins. Here, we describe a cell-free system based on Leishmania tarentolae ( Lt ) extracts for the biogenesis of caveolae and show its use for single-molecule interaction studies. Insertion of expressed caveolin-1 (CAV1) into Lt membranes was analogous to that of caveolin in native membranes. Electron tomography showed that caveolins generate domains of precise size and curvature. Cell-free caveolae were used in quantitative assays to test the interaction of membrane-inserted caveolin with signaling proteins and to determine the stoichiometry of interactions. Binding of membrane-inserted CAV1 to several proposed binding partners, including endothelial nitric-oxide synthase, was negligible, but a small number of proteins, including TRAF2, interacted with CAV1 in a phosphorylation-(CAV1 Y14 )-stimulated manner. In cells subjected to oxidative stress, phosphorylated CAV1 recruited TRAF2 to the early endosome forming a novel signaling platform. These findings lead to a novel model for cellular stress signaling by CAV1. © 2018 Jung et al.

Arginine insertion and loss of N-linked glycosylation site in HIV-1 envelope V3 region confer CXCR4-tropism

PubMed Central

Tsuchiya, Kiyoto; Ode, Hirotaka; Hayashida, Tsunefusa; Kakizawa, Junko; Sato, Hironori; Oka, Shinichi; Gatanaga, Hiroyuki

2013-01-01

The third variable region (V3) of HIV-1 envelope glycoprotein gp120 plays a key role in determination of viral coreceptor usage (tropism). However, which combinations of mutations in V3 confer a tropism shift is still unclear. A unique pattern of mutations in antiretroviral therapy-naive HIV-1 patient was observed associated with the HIV-1 tropism shift CCR5 to CXCR4. The insertion of arginine at position 11 and the loss of the N-linked glycosylation site were indispensable for acquiring pure CXCR4-tropism, which were confirmed by cell-cell fusion assay and phenotype analysis of recombinant HIV-1 variants. The same pattern of mutations in V3 and the associated tropism shift were identified in two of 53 other patients (3.8%) with CD4+ cell count <200/mm3. The combination of arginine insertion and loss of N-linked glycosylation site usually confers CXCR4-tropism. Awareness of this rule will help to confirm the tropism prediction from V3 sequences by conventional rules. PMID:23925152

Lambda Red Mediated Gap Repair Utilizes a Novel Replicative Intermediate in Escherichia coli

PubMed Central

Reddy, Thimma R.; Fevat, Léna M. S.; Munson, Sarah E.; Stewart, A. Francis; Cowley, Shaun M.

2015-01-01

The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination. PMID:25803509

Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag.

PubMed

Rizvi, Tahir A; Kenyon, Julia C; Ali, Jahabar; Aktar, Suriya J; Phillip, Pretty S; Ghazawi, Akela; Mustafa, Farah; Lever, Andrew M L

2010-10-15

The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ. Copyright © 2010 Elsevier Ltd. All rights reserved.

Development of a new microtiter plate format for clinically relevant assays.

PubMed

Piletska, Elena V; Piletsky, Stanislav S; Whitcombe, Michael J; Chianella, Iva; Piletsky, Sergey A

2012-02-21

A new format for the microtiter plate-based assays was proposed. The novelty involves the use of disk-shaped inserts for immobilization of biological and chemical reagents. The internal opening of the disks allows measurements of the reactions by standard microtiter plate readers without any additional steps involving liquid handling. Ideally the plate end-users just have to add the sample and take the measurement without any need of multiple reagent additions or transfer of the liquid to a different plate. The novel assay format also allows handling of reagents which are not soluble in an aqueous environment. As a proof of concept we describe here several model reactions which are compatible with microtiter plate format, such as monitoring enzymatic reactions catalyzed by glucose oxidase (GOx) and urease, measurements of proteins by BCA assay, analysis of pH, and concentration of antioxidants. The "mix and match" approach in the disk-shape format allows multiplexing and could be particularly useful for high throughput screening. One of the potential application areas for this novel assay format could be in a multianalyte system for measurement of clinically relevant analytes in primary care.

A Novel Locomotion-based Validation Assay for Candidate Drugs Using Drosophila DYT1 Disease Model

DTIC Science & Technology

2013-11-01

the genome using the same parental fly line, minimizing the effect of surrounding sequences and genetic variations on the ...locomotion and GTPC cyclrohydolase protein levels; (3) supplementation of dopamine can partially rescue the locomotion defects of Drosophila larvae...8217- GCGAACAACCAAAAAATCATTGAGATAATAAACTCCTCCATTAG-3’) to make dtorsin cDNA that lacks GAC (D307) (Fig. 1) respectively. After confirming mutated sequences , the insert was again

The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing

PubMed Central

Gilling, Damian H.; Luna, Vicki Ann; Pflugradt, Cori

2014-01-01

The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target the IS407A-fliP interface in B. mallei and the fliP gene specifically at the insertion point in B. pseudomallei. We then examined DNA from ten B. mallei, ten B. pseudomallei, 14 B. cepacia, eight other Burkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either the fliP gene with the IS407A interruption or the fully intact fliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new species B. mallei from B. pseudomallei and may point to new targets that can be exploited in the future. PMID:27350960

CXCR4-using HIV variants in a cohort of Black men who have sex with men: HIV Prevention Trials Network 061.

PubMed

Chen, Iris; Huang, Wei; Connor, Matthew B; Frantzell, Arne; Cummings, Vanessa; Beauchamp, Geetha G; Griffith, Sam; Fields, Sheldon D; Scott, Hyman M; Shoptaw, Steven; Del Rio, Carlos; Magnus, Manya; Mannheimer, Sharon; Tieu, Hong-Van; Wheeler, Darrell P; Mayer, Kenneth H; Koblin, Beryl A; Eshleman, Susan H

2016-07-01

To evaluate factors associated with HIV tropism among Black men who have sex with men (MSM) in the United States enrolled in a clinical study (HIV Prevention Trials Network 061). HIV tropism was analyzed using a phenotypic assay (Trofile assay, Monogram Biosciences). Samples were analyzed from 43 men who were HIV infected at enrollment and reported either exclusive insertive intercourse or exclusive receptive intercourse; samples were also analyzed from 20 men who were HIV uninfected at enrollment and seroconverted during the study. Clonal analysis of individual viral variants was performed for seroconverters who had dual/mixed (DM) viruses. DM viruses were detected in samples from 11 (26%) of the 43 HIV-infected men analyzed at the enrollment visit; HIV tropism did not differ between those reporting exclusive insertive vs receptive intercourse. DM viruses were also detected in five (25%) of the 20 seroconverters. DM viruses were associated with lower CD4 cell counts. Seroconverters with DM viruses had dual-tropic viruses only or mixed populations of CCR5- and dual-tropic viruses. DM viruses were frequently detected among Black MSM in this study, including seroconverters. Further studies are needed to understand factors driving transmission and selection of CXCR4- and dual-tropic viruses among Black MSM.

Hepatitis B Virus Core Gene Mutations Which Block Nucleocapsid Envelopment

PubMed Central

Koschel, Matthias; Oed, Daniela; Gerelsaikhan, Tudevdagwa; Thomssen, Reiner; Bruss, Volker

2000-01-01

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment. PMID:10590084

A noise control package for vibrating screens1),2)

PubMed Central

Lowe, M. Jenae; Yantek, David S.; Yang, Junyi; Schuster, Kevin C.; Mechling, Jessie J.

2015-01-01

Hearing loss was the second-most common illness reported to the Mine Safety and Health Administration (MSHA) in 2009. Furthermore, between 2000 and 2010, 30% of all noise-related injury complaints reported to MSHA were for coal preparation plant employees. Previous National Institute for Occupational Safety and Health (NIOSH) studies have shown that vibrating screens are key noise sources to address in order to reduce coal preparation plant noise. In response, NIOSH researchers have developed a suite of noise controls for vibrating screens consisting of constrained layer damping (CLD) treatments, a tuned mechanism suspension, an acoustic enclosure, and spring inserts. Laboratory testing demonstrates that this noise control suite reduces the A-weighted sound power level of the vibrating screen by 6 dB. To provide a comparison to laboratory results and prove durability, field testing of two noise controls was performed on a vibrating screen in a working coal preparation plant. The spring inserts and CLD treatments were selected due to their ease of installation and practicability. Field testing of these controls yielded reductions that were comparable to laboratory results. PMID:26257468

Confined disclinations: exterior versus material constraints in developable thin elastic sheets.

PubMed

Efrati, Efi; Pocivavsek, Luka; Meza, Ruben; Lee, Ka Yee C; Witten, Thomas A

2015-02-01

We examine the shape change of a thin disk with an inserted wedge of material when it is pushed against a plane, using analytical, numerical, and experimental methods. Such sheets occur in packaging, surgery, and nanotechnology. We approximate the sheet as having vanishing strain, so that it takes a conical form in which straight generators converge to a disclination singularity. Then, its shape is that which minimizes elastic bending energy alone. Real sheets are expected to approach this limiting shape as their thickness approaches zero. The planar constraint forces a sector of the sheet to buckle into the third dimension. We find that the unbuckled sector is precisely semicircular, independent of the angle δ of the inserted wedge. We generalize the analysis to include conical as well as planar constraints and thereby establish a law of corresponding states for shallow cones of slope ε and thin wedges. In this regime, the single parameter δ/ε^{2} determines the shape. We discuss the singular limit in which the cone becomes a plane, and the unexpected slow convergence to the semicircular buckling observed in real sheets.

Confined disclinations: Exterior versus material constraints in developable thin elastic sheets

NASA Astrophysics Data System (ADS)

Efrati, Efi; Pocivavsek, Luka; Meza, Ruben; Lee, Ka Yee C.; Witten, Thomas A.

2015-02-01

We examine the shape change of a thin disk with an inserted wedge of material when it is pushed against a plane, using analytical, numerical, and experimental methods. Such sheets occur in packaging, surgery, and nanotechnology. We approximate the sheet as having vanishing strain, so that it takes a conical form in which straight generators converge to a disclination singularity. Then, its shape is that which minimizes elastic bending energy alone. Real sheets are expected to approach this limiting shape as their thickness approaches zero. The planar constraint forces a sector of the sheet to buckle into the third dimension. We find that the unbuckled sector is precisely semicircular, independent of the angle δ of the inserted wedge. We generalize the analysis to include conical as well as planar constraints and thereby establish a law of corresponding states for shallow cones of slope ɛ and thin wedges. In this regime, the single parameter δ /ɛ2 determines the shape. We discuss the singular limit in which the cone becomes a plane, and the unexpected slow convergence to the semicircular buckling observed in real sheets.

Aqueous Rechargeable Alkaline CoxNi2-xS2/TiO2 Battery.

PubMed

Liu, Jilei; Wang, Jin; Ku, Zhiliang; Wang, Huanhuan; Chen, Shi; Zhang, Lili; Lin, Jianyi; Shen, Ze Xiang

2016-01-26

An electrochemical energy storage system with high energy density, stringent safety, and reliability is highly desirable for next-generation energy storage devices. Here an aqueous rechargeable alkaline CoxNi2-xS2 // TiO2 battery system is designed by integrating two reversible electrode processes associated with OH(-) insertion/extraction in the cathode part and Li ion insertion/extraction in the anode part, respectively. The prototype CoxNi2-xS2 // TiO2 battery is able to deliver high energy/power densities of 83.7 Wh/kg at 609 W/kg (based on the total mass of active materials) and good cycling stabilities (capacity retention 75.2% after 1000 charge/discharge cycles). A maximum volumetric energy density of 21 Wh/l (based on the whole packaged cell) has been achieved, which is comparable to that of a thin-film battery and better than that of typical commercial supercapacitors, benefiting from the unique battery and hierarchical electrode design. This hybrid system would enrich the existing aqueous rechargeable LIB chemistry and be a promising battery technology for large-scale energy storage.

Replication Competent Molecular Clones of HIV-1 Expressing Renilla Luciferase Facilitate the Analysis of Antibody Inhibition in PBMC

PubMed Central

Edmonds, Tara G.; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S.; Conway, Joan A.; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T.; Montefiori, David C.; Kappes, John C.; Ochsenbauer, Christina

2010-01-01

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. PMID:20863545

Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC.

PubMed

Edmonds, Tara G; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S; Conway, Joan A; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T; Montefiori, David C; Kappes, John C; Ochsenbauer, Christina

2010-12-05

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. Copyright © 2010 Elsevier Inc. All rights reserved.

Transmembrane helices containing a charged arginine are thermodynamically stable.

PubMed

Ulmschneider, Martin B; Ulmschneider, Jakob P; Freites, J Alfredo; von Heijne, Gunnar; Tobias, Douglas J; White, Stephen H

2017-10-01

Hydrophobic amino acids are abundant in transmembrane (TM) helices of membrane proteins. Charged residues are sparse, apparently due to the unfavorable energetic cost of partitioning charges into nonpolar phases. Nevertheless, conserved arginine residues within TM helices regulate vital functions, such as ion channel voltage gating and integrin receptor inactivation. The energetic cost of arginine in various positions along hydrophobic helices has been controversial. Potential of mean force (PMF) calculations from atomistic molecular dynamics simulations predict very large energetic penalties, while in vitro experiments with Sec61 translocons indicate much smaller penalties, even for arginine in the center of hydrophobic TM helices. Resolution of this conflict has proved difficult, because the in vitro assay utilizes the complex Sec61 translocon, while the PMF calculations rely on the choice of simulation system and reaction coordinate. Here we present the results of computational and experimental studies that permit direct comparison with the Sec61 translocon results. We find that the Sec61 translocon mediates less efficient membrane insertion of Arg-containing TM helices compared with our computational and experimental bilayer-insertion results. In the simulations, a combination of arginine snorkeling, bilayer deformation, and peptide tilting is sufficient to lower the penalty of Arg insertion to an extent such that a hydrophobic TM helix with a central Arg residue readily inserts into a model membrane. Less favorable insertion by the translocon may be due to the decreased fluidity of the endoplasmic reticulum (ER) membrane compared with pure palmitoyloleoyl-phosphocholine (POPC). Nevertheless, our results provide an explanation for the differences between PMF- and experiment-based penalties for Arg burial.

Instrumentation of Java Bytecode for Runtime Analysis

NASA Technical Reports Server (NTRS)

Goldberg, Allen; Haveland, Klaus

2003-01-01

This paper describes JSpy, a system for high-level instrumentation of Java bytecode and its use with JPaX, OUT system for runtime analysis of Java programs. JPaX monitors the execution of temporal logic formulas and performs predicative analysis of deadlocks and data races. JSpy s input is an instrumentation specification, which consists of a collection of rules, where a rule is a predicate/action pair The predicate is a conjunction of syntactic constraints on a Java statement, and the action is a description of logging information to be inserted in the bytecode corresponding to the statement. JSpy is built using JTrek an instrumentation package at a lower level of abstraction.

Method and means of packaging nuclear fuel rods for handling

DOEpatents

Adam, Milton F.

1979-01-01

Nuclear fuel rods, especially spent nuclear fuel rods that may show physical distortion, are encased within a metallic enclosing structure by forming a tube about the fuel rod. The tube has previously been rolled to form an overlapping tubular structure and then unrolled and coiled about an axis perpendicular to the tube. The fuel rod is inserted into the tube as the rolled tube is removed from a coiled strip and allowed to reassume its tubular shape about the fuel rod. Rollers support the coiled strip in an open position as the coiled strip is uncoiled and allowed to roll about the fuel rod.

Implementation Status of a Ultra-Wideband Receiver Package for the next-generation Very Large Array

NASA Astrophysics Data System (ADS)

Lazio, T. Joseph W.; Velazco, Jose; Soriano, Melissa; Hoppe, Daniel; Russell, Damon; D'Addario, Larry; Long, Ezra; Bowen, James; Samoska, Lorene; Janzen, Andrew

2017-01-01

The next-generation Very Large Array (ngVLA) is a concept for a radio astronomical interferometric array operating in the frequency range 1.2 GHz to 116 GHz and designed to provide substantial improvements in sensitivity, angular resolution, and frequency coverage above the current Very Large Array (VLA). As notional design goals, it would have a continuous frequency coverage of 1.2 GHz to 48 GHz and be 10 times more sensitive than the VLA (and 25 times more sensitive than a 34 m diameter antenna of the Deep Space Network [DSN]). One of the key goals for the ngVLA is to reduce the operating costs without sacrificing performance. We are designing an ultra-wideband receiver package designed to operate across the 8 to 48 GHz frequency range, which can be contrasted to the current VLA, which covers this frequency range with five receiver packages. Reducing the number of receiving systems required to cover the full frequency range would reduce operating costs, and the objective of this work is to develop a prototype integrated feed-receiver package with a sensitivity performance comparable to current narrower band systems on radio telescopes and the DSN, but with a design that meets the requirement of low long-term operational costs. The ultra-wideband receiver package consists of a feed horn, low-noise amplifier (LNA), and down-converters to analog intermediate frequencies. Key features of this design are a quad-ridge feed horn with dielectric loading and a cryogenic receiver with a noise temperature of no more than 30 K at the low end of the band. We will report on the status of this receiver package development including the feed design and LNA implementation. We will present simulation studies of the feed horn including the insertion of dielectric components for improved illumination efficiencies across the band of interest. In addition, we will show experimental results of low-noise 35nm InP HEMT amplifier testing performed across the 8-50 GHz frequency range.Part of this research was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration.

A Vignette (User’s Guide) for “An R Package for Statistical Analysis of Chemistry, Histopathology, and Reproduction Endpoints Including Repeated Measures and Multi-Generation Studies (StatCharrms).”

EPA Science Inventory

StatCharrms is a graphical user front-end for ease of use in analyzing data generated from OCSPP 890.2200, Medaka Extended One Generation Reproduction Test (MEOGRT) and OCSPP 890.2300, Larval Amphibian Gonad Development Assay (LAGDA). The analyses StatCharrms is capable of perfor...

Structure-Based Mutagenesis of Sulfolobus Turreted Icosahedral Virus B204 Reveals Essential Residues in the Virion-Associated DNA-Packaging ATPase.

PubMed

Dellas, Nikki; Snyder, Jamie C; Dills, Michael; Nicolay, Sheena J; Kerchner, Keshia M; Brumfield, Susan K; Lawrence, C Martin; Young, Mark J

2015-12-23

Sulfolobus turreted icosahedral virus (STIV), an archaeal virus that infects the hyperthermoacidophile Sulfolobus solfataricus, is one of the most well-studied viruses of the domain Archaea. STIV shares structural, morphological, and sequence similarities with viruses from other domains of life, all of which are thought to belong to the same viral lineage. Several of these common features include a conserved coat protein fold, an internal lipid membrane, and a DNA-packaging ATPase. B204 is the ATPase encoded by STIV and is thought to drive packaging of viral DNA during the replication process. Here, we report the crystal structure of B204 along with the biochemical analysis of B204 mutants chosen based on structural information and sequence conservation patterns observed among members of the same viral lineage and the larger FtsK/HerA superfamily to which B204 belongs. Both in vitro ATPase activity assays and transfection assays with mutant forms of B204 confirmed the essentiality of conserved and nonconserved positions. We also have identified two distinct particle morphologies during an STIV infection that differ in the presence or absence of the B204 protein. The biochemical and structural data presented here are not only informative for the STIV replication process but also can be useful in deciphering DNA-packaging mechanisms for other viruses belonging to this lineage. STIV is a virus that infects a host from the domain Archaea that replicates in high-temperature, acidic environments. While STIV has many unique features, there exist several striking similarities between this virus and others that replicate in different environments and infect a broad range of hosts from Bacteria and Eukarya. Aside from structural features shared by viruses from this lineage, there exists a significant level of sequence similarity between the ATPase genes carried by these different viruses; this gene encodes an enzyme thought to provide energy that drives DNA packaging into the virion during infection. The experiments described here highlight the elements of this enzyme that are essential for proper function and also provide supporting evidence that B204 is present in the mature STIV virion. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

NASA Astrophysics Data System (ADS)

Borjigin, Jimo; Nathans, Jeremy

1993-01-01

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

PubMed Central

Zhao, Wei; Jardine, Paul J.

2015-01-01

ABSTRACT During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. IMPORTANCE During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs. PMID:26423956

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor.

PubMed

Zhao, Wei; Jardine, Paul J; Grimes, Shelley

2015-12-01

During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

In cell mutational interference mapping experiment (in cell MIME) identifies the 5' polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging.

PubMed

Smyth, Redmond P; Smith, Maureen R; Jousset, Anne-Caroline; Despons, Laurence; Laumond, Géraldine; Decoville, Thomas; Cattenoz, Pierre; Moog, Christiane; Jossinet, Fabrice; Mougel, Marylène; Paillart, Jean-Christophe; von Kleist, Max; Marquet, Roland

2018-05-18

Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5' region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5' PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.

Characterization and antimicrobial properties of food packaging methylcellulose films containing stem extract of Ginja cherry.

PubMed

Campos, Débora; Piccirillo, Clara; Pullar, Robert C; Castro, Paula Ml; Pintado, Maria M E

2014-08-01

Food contamination and spoilage is a problem causing growing concern. To avoid it, the use of food packaging with appropriate characteristics is essential; ideally, the packaging should protect food from external contamination and exhibit antibacterial properties. With this aim, methylcellulose (MC) films containing natural extracts from the stems of Ginja cherry, an agricultural by-product, were developed and characterized. The antibacterial activity of films was screened by the disc diffusion method and quantified using the viable cell count assay. The films inhibited the growth of both Gram-positive and Gram-negative strains (Listeria innocua, methicillin-sensitive Staphylococcus aureus, methicillin-resistant S. aureus, Salmonella Enteritidis, Escherichia coli). For the films with lower extract content, effectiveness against the microorganisms depended on the inoculum concentration. Scanning electron microscope images of the films showed that those containing the extracts had a smooth and continuous structure. UV-visible spectroscopy showed that these materials do not transmit light in the UV. This study shows that MC films containing agricultural by-products, in this case Ginja cherry stem extract, could be used to prevent food contamination by relevant bacterial strains and degradation by UV light. Using such materials in food packaging, the shelf life of food products could be extended while utilizing an otherwise wasted by-product. © 2013 Society of Chemical Industry.

In cell mutational interference mapping experiment (in cell MIME) identifies the 5′ polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging

PubMed Central

Smith, Maureen R; Jousset, Anne-Caroline; Despons, Laurence; Laumond, Géraldine; Decoville, Thomas; Cattenoz, Pierre; Moog, Christiane; Jossinet, Fabrice; Mougel, Marylène; Paillart, Jean-Christophe

2018-01-01

Abstract Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5′ region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5′ PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production. PMID:29514260

Further characterisation of rotavirus cores: Ss(+)RNAs can be packaged in vitro but packaging lacks sequence specificity☆

PubMed Central

Desselberger, Ulrich; Richards, James; Tchertanov, Luba; Lepault, Jean; Lever, Andrew; Burrone, Oscar; Cohen, Jean

2013-01-01

Rotavirus (RV) cores were released from double-layered particles (DLPs) by high concentrations of CaCl2, purified and ‘opened’ by treatment with EDTA or EGTA. Under appropriate in vitro conditions DLPs have been shown to have transcriptase and ‘open cores’ replicase activity. Furthermore, it has been demonstrated that transcriptase activity and infectivity of native cores can be restored by transcapsidation with VP6, VP7 and VP4. The missing link for particle reconstitution in vitro has been the manipulation of ‘open cores’ to become functionally active cores again. The experiments described here were undertaken with the aim of exploring packaging of RV RNAs into opened cores in vitro. Rotavirus cores were opened by approximately 200 μM EGTA, leading to the release of genomic dsRNA. Conversely, RV cores were found to be stable in the presence of minimum concentrations of Ca2+, Mg2+, spermidine3+ and cobalthexamine3+ of between 40 and 300 μM. Aggregates of purified cores were resolved in the presence of 0.3 mM deoxycholate (minimum concentration). Core shells opened with EGTA were reconstituted by the addition of di- or trivalent cations within 2 min of the opening procedure. Addition of purified, baculovirus recombinant-expressed VP6 to native and reconstituted cores led to the formation of DLPs or DLP-like particles, which upon transfection into MA104 cells were infectious. The rescued infectivity likely originated in part from unopened and in part from reconstituted cores. Radiolabelled RV (+) ssRNAs could be packaged into reconstituted cores and DLPs, as indicated by resistance to RNase I digestion. The packaging reaction was, however, not RV RNA sequence-specific, since unrelated ssRNAs, such as those transcribed from HIV-2 cDNAs, were also packaged. The kinetics of packaging of homologous and heterologous RNAs were similar, as evidenced by competitive packaging assays. None of the packaged in vitro engineered RNA segments has so far been rescued into infectious virus. PMID:24091366

Numerical modeling and experimental validation of the acoustic transmission of aircraft's double-wall structures including sound package

NASA Astrophysics Data System (ADS)

Rhazi, Dilal

In the field of aeronautics, reducing the harmful effects of acoustics constitutes a major concern at the international level and justifies the call for further research, particularly in Canada where aeronautics is a key economic sector, which operates in a context of global competition. Aircraft sidewall structure is usually of a double wall construction with a curved ribbed metallic skin and a lightweight composite or sandwich trim separated by a cavity filled with a noise control treatment. The latter is of a great importance in the transport industry, and continues to be of interest in many engineering applications. However, the insertion loss noise control treatment depends on the excitation of the supporting structure. In particular, Turbulent Boundary Layer is of interest to several industries. This excitation is difficult to simulate in laboratory conditions, given the prohibiting costs and difficulties associated with wind tunnel and in-flight tests. Numerical simulation is the only practical way to predict the response to such excitations and to analyze effects of design changes to the response to such excitation. Another kinds of excitations encountered in industrial are monopole, rain on the Roof and diffuse acoustic field. Deterministic methods can calculate in each point the spectral response of the system. Most known are numerical methods such as finite elements and boundary elements methods. These methods generally apply to the low frequency where modal behavior of the structure dominates. However, the high limit of calculation in frequency of these methods cannot be defined in a strict way because it is related to the capacity of data processing and to the nature of the studied mechanical system. With these challenges in mind, and with limitations of the main numerical codes on the market, the manufacturers have expressed the need for simple models immediately available as early as the stage of preliminary drafts. This thesis represents an attempt to address this need. A numerical tool based on two approaches (Wave and Modal) is developed. It allows a fast computation of the vibroacoustic response for multilayer structures over full frequency spectrum and for various kinds of excitations (monople, rain on the roof, diffuse acoustic filed, turbulent boundary layer) . A comparison between results obtained by the developed model, experimental tests and the finite element method is given and discussed. The results are very promising with respect to the potential of such a model for industrial use as a prediction tool, and even for design. The code can be also integrated within an SEA (Statistical Energy Analysis) strategy in order to model a full vehicle by computing in particular the insertion loss and the equivalent damping added by the sound package. Keywords: Transfer Matrix Method, Wave Approach,Turbulent Boundary Layer, Rain on the Roof, Monopole, Insertion loss, Double-wall, Sound Package.

OpenComet: An automated tool for comet assay image analysis

PubMed Central

Gyori, Benjamin M.; Venkatachalam, Gireedhar; Thiagarajan, P.S.; Hsu, David; Clement, Marie-Veronique

2014-01-01

Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time. PMID:24624335

OpenComet: an automated tool for comet assay image analysis.

PubMed

Gyori, Benjamin M; Venkatachalam, Gireedhar; Thiagarajan, P S; Hsu, David; Clement, Marie-Veronique

2014-01-01

Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time.

Contamination of injectable solutions with 2-mercaptobenzothiazole leached from rubber closures.

PubMed

Reepmeyer, J C; Juhl, Y H

1983-11-01

An impurity, discovered in a sample of digoxin injectable solution commercially packaged in a syringe for single-dose delivery, was found to originate from the rubber closure of the syringe and was identified as 2-mercaptobenzothiazole, a common accelerator for rubber vulcanization. Several similarly packaged injectable solutions of a variety of drugs from various manufacturers were examined and over half contained 2-mercaptobenzothiazole. The compound was identified by UV spectrophotometry (including a pH-dependent shift in its absorbance maximum), by mass spectrometry, and by comparison with standard 2-mercaptobenzothiazole using silica gel and reverse-phase high-performance liquid chromatography (HPLC). The presence of this impurity in injectable solutions may have implications with regard to toxicity and may interfere with the assay of digoxin injectable solution by HPLC.

Ferric plasmonic nanoparticles, aptamers, and magnetofluidic chips: toward the development of diagnostic surface-enhanced Raman spectroscopy assays

NASA Astrophysics Data System (ADS)

Marks, Haley; Huang, Po-Jung; Mabbott, Samuel; Graham, Duncan; Kameoka, Jun; Coté, Gerard

2016-12-01

Conjugation of aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound nanoclusters that cause a measurable change in the colloid's optical properties. The optimization of a surface-enhanced Raman spectroscopy (SERS) competitive binding assay utilizing plasmonic "target" and magnetic "probe" nanoparticles for the detection of the toxin bisphenol-A (BPA) is presented. These assay nanoclusters were housed inside three types of optofluidic chips patterned with magnetically activated nickel pads, in either a straight or array pattern. Both Fe2O3 and Fe2CoO4 were compared as potential magnetic cores for the silver-coated probe nanoparticles. We found that the Ag@Fe2O3 particles were, on average, more uniform in size and more stable than Ag@Fe2CoO4, whereas the addition of cobalt significantly improved the collection time of particles. Using Raman mapping of the assay housed within the magnetofluidic chips, it was determined that a 1×5 array of 50 μm square nickel pads provided the most uniform SERS enhancement of the assay (coefficient of variation ˜25%) within the magnetofluidic chip. Additionally, the packaged assay demonstrated the desired response to BPA, verifying the technology's potential to translate magnetic nanoparticle assays into a user-free optical analysis platform.

Ferric plasmonic nanoparticles, aptamers, and magnetofluidic chips: toward the development of diagnostic surface-enhanced Raman spectroscopy assays

PubMed Central

Marks, Haley; Huang, Po-Jung; Mabbott, Samuel; Graham, Duncan; Kameoka, Jun; Coté, Gerard

2016-01-01

Abstract. Conjugation of aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound nanoclusters that cause a measurable change in the colloid’s optical properties. The optimization of a surface-enhanced Raman spectroscopy (SERS) competitive binding assay utilizing plasmonic “target” and magnetic “probe” nanoparticles for the detection of the toxin bisphenol-A (BPA) is presented. These assay nanoclusters were housed inside three types of optofluidic chips patterned with magnetically activated nickel pads, in either a straight or array pattern. Both Fe2O3 and Fe2CoO4 were compared as potential magnetic cores for the silver-coated probe nanoparticles. We found that the Ag@Fe2O3 particles were, on average, more uniform in size and more stable than Ag@Fe2CoO4, whereas the addition of cobalt significantly improved the collection time of particles. Using Raman mapping of the assay housed within the magnetofluidic chips, it was determined that a 1×5 array of 50  μm square nickel pads provided the most uniform SERS enhancement of the assay (coefficient of variation ∼25%) within the magnetofluidic chip. Additionally, the packaged assay demonstrated the desired response to BPA, verifying the technology’s potential to translate magnetic nanoparticle assays into a user-free optical analysis platform. PMID:27997017

Methodological requirements for valid tissue-based biomarker studies that can be used in clinical practice.

PubMed

True, Lawrence D

2014-03-01

Paralleling the growth of ever more cost efficient methods to sequence the whole genome in minute fragments of tissue has been the identification of increasingly numerous molecular abnormalities in cancers--mutations, amplifications, insertions and deletions of genes, and patterns of differential gene expression, i.e., overexpression of growth factors and underexpression of tumor suppressor genes. These abnormalities can be translated into assays to be used in clinical decision making. In general terms, the result of such an assay is subject to a large number of variables regarding the characteristics of the available sample, particularities of the used assay, and the interpretation of the results. This review discusses the effects of these variables on assays of tissue-based biomarkers, classified by macromolecule--DNA, RNA (including micro RNA, messenger RNA, long noncoding RNA, protein, and phosphoprotein). Since the majority of clinically applicable biomarkers are immunohistochemically detectable proteins this review focuses on protein biomarkers. However, the principles outlined are mostly applicable to any other analyte. A variety of preanalytical variables impacts on the results obtained, including analyte stability (which is different for different analytes, i.e., DNA, RNA, or protein), period of warm and of cold ischemia, fixation time, tissue processing, sample storage time, and storage conditions. In addition, assay variables play an important role, including reagent specificity (notably but not uniquely an issue concerning antibodies used in immunohistochemistry), technical components of the assay, quantitation, and assay interpretation. Finally, appropriateness of an assay for clinical application is an important issue. Reference is made to publicly available guidelines to improve on biomarker development in general and requirements for clinical use in particular. Strategic goals are formulated in order to improve on the quality of biomarker reporting, including issues of analyte quality, experimental detail, assay efficiency and precision, and assay appropriateness.

Trick Simulation Environment 07

NASA Technical Reports Server (NTRS)

Lin, Alexander S.; Penn, John M.

2012-01-01

The Trick Simulation Environment is a generic simulation toolkit used for constructing and running simulations. This release includes a Monte Carlo analysis simulation framework and a data analysis package. It produces all auto documentation in XML. Also, the software is capable of inserting a malfunction at any point during the simulation. Trick 07 adds variable server output options and error messaging and is capable of using and manipulating wide characters for international support. Wide character strings are available as a fundamental type for variables processed by Trick. A Trick Monte Carlo simulation uses a statistically generated, or predetermined, set of inputs to iteratively drive the simulation. Also, there is a framework in place for optimization and solution finding where developers may iteratively modify the inputs per run based on some analysis of the outputs. The data analysis package is capable of reading data from external simulation packages such as MATLAB and Octave, as well as the common comma-separated values (CSV) format used by Excel, without the use of external converters. The file formats for MATLAB and Octave were obtained from their documentation sets, and Trick maintains generic file readers for each format. XML tags store the fields in the Trick header comments. For header files, XML tags for structures and enumerations, and the members within are stored in the auto documentation. For source code files, XML tags for each function and the calling arguments are stored in the auto documentation. When a simulation is built, a top level XML file, which includes all of the header and source code XML auto documentation files, is created in the simulation directory. Trick 07 provides an XML to TeX converter. The converter reads in header and source code XML documentation files and converts the data to TeX labels and tables suitable for inclusion in TeX documents. A malfunction insertion capability allows users to override the value of any simulation variable, or call a malfunction job, at any time during the simulation. Users may specify conditions, use the return value of a malfunction trigger job, or manually activate a malfunction. The malfunction action may consist of executing a block of input file statements in an action block, setting simulation variable values, call a malfunction job, or turn on/off simulation jobs.

integIRTy: a method to identify genes altered in cancer by accounting for multiple mechanisms of regulation using item response theory.

PubMed

Tong, Pan; Coombes, Kevin R

2012-11-15

Identifying genes altered in cancer plays a crucial role in both understanding the mechanism of carcinogenesis and developing novel therapeutics. It is known that there are various mechanisms of regulation that can lead to gene dysfunction, including copy number change, methylation, abnormal expression, mutation and so on. Nowadays, all these types of alterations can be simultaneously interrogated by different types of assays. Although many methods have been proposed to identify altered genes from a single assay, there is no method that can deal with multiple assays accounting for different alteration types systematically. In this article, we propose a novel method, integration using item response theory (integIRTy), to identify altered genes by using item response theory that allows integrated analysis of multiple high-throughput assays. When applied to a single assay, the proposed method is more robust and reliable than conventional methods such as Student's t-test or the Wilcoxon rank-sum test. When used to integrate multiple assays, integIRTy can identify novel-altered genes that cannot be found by looking at individual assay separately. We applied integIRTy to three public cancer datasets (ovarian carcinoma, breast cancer, glioblastoma) for cross-assay type integration which all show encouraging results. The R package integIRTy is available at the web site http://bioinformatics.mdanderson.org/main/OOMPA:Overview. kcoombes@mdanderson.org. Supplementary data are available at Bioinformatics online.

Development and characterization of bioactive edible films from spider crab (Maja crispata) chitosan incorporated with Spirulina extract.

PubMed

Balti, Rafik; Mansour, Mohamed Ben; Sayari, Nadhem; Yacoubi, Lamia; Rabaoui, Lotfi; Brodu, Nicolas; Massé, Anthony

2017-12-01

Active food packaging films based on crab chitosan and Spirulina extract (SE) were developed. The effects of the SE incorporation at different levels on physical (color, opacity water vapor and oxygen permeability) and mechanical (tensile strength and elongation at break) properties of chitosan films were investigated. FTIR was carried out to observe the potential modifications of the chitosan films when incorporated with SE. The obtained results suggested that incorporation of SE into chitosan films improved mechanical and barrier properties. The antioxidant activity of the chitosan/SE films was characterized by means of three different analytical assays (DPPH, FRAP and FIC). Crab chitosan edible films containing SE showed higher antioxidant activity, regardless concentrations and methods assayed. Furthermore, the antioxidant activity occurred in a concentration-dependent manner. The agar disc diffusion method was used to determine the antibacterial activities of chitosan edible films against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, Bacillus subtilis and Bacillus cereus. The chitosan/SE films were more effective (p<0.05) against five of the seven tested bacteria. The obtained crab chitosan edible films incorporated with SE showed great potential to be used for active food packaging due to its excellent antioxidant and antibacterial activities. Copyright © 2017 Elsevier B.V. All rights reserved.

Anti-listerial activity of a polymeric film coated with hybrid coatings doped with Enterocin 416K1 for use as bioactive food packaging.

PubMed

Iseppi, Ramona; Pilati, Francesco; Marini, Michele; Toselli, Maurizio; de Niederhäusern, Simona; Guerrieri, Elisa; Messi, Patrizia; Sabia, Carla; Manicardi, Giuliano; Anacarso, Immacolata; Bondi, Moreno

2008-04-30

In this study, Enterocin 416K1, a bacteriocin produced by Enterococcus casseliflavus IM 416K1, was entrapped in an organic-inorganic hybrid coating applied to a LDPE (low-density polyethylene) film for its potential use in the active food packaging field. The antibacterial activity of the coated film was evaluated against Listeria monocytogenes NCTC 10888 by qualitative modified agar diffusion assay, quantitative determination in listeria saline solution suspension and direct contact with artificially contaminated food samples (frankfurters and fresh cheeses) stored at room and refrigeration temperatures. All investigations demonstrated that enterocin-activated coatings have a good anti-listeria activity. Qualitative tests showed a clear zone of inhibition in the indicator lawn in contact with and around the coated film. During the quantitative antibacterial evaluation the L. monocytogenes viable counts decreased to 1.5 log units compared to the control. The inhibitory capability was confirmed also in food-contact assays. In all food samples packed with coated films we observed a significant decrease in L. monocytogenes viable counts in the first 24 h compared to the control. This difference was generally maintained up to the seventh day and then decreased, with the exception of the cheese samples stored at refrigeration temperature.

Kinetics of apoptotic markers in exogeneously induced apoptosis of EL4 cells.

PubMed

Jessel, Robert; Haertel, Steffen; Socaciu, Carmen; Tykhonova, Svetlana; Diehl, Horst A

2002-01-01

We investigated the time-dependence of apoptotic events in EL4 cells by monitoring plasma membrane changes in correlation to DNA fragmentation and cell shrinkage. We applied three apoptosis inducers (staurosporine, tubericidine and X-rays) and we looked at various markers to follow the early-to-late apoptotic events: phospholipid translocation (identified through annexin V-fluorescein assay and propidium iodide), lipid package (via merocyanine assay), membrane fluidity and anisotropy (via fluorescent measurements), DNA fragmentation by the fluorescence-labeling test and cell size measurements. The different apoptotic inducers caused different reactions of the cells: staurosporine induced apoptosis most rapidly in a high number of cells, tubercidine triggered apoptosis only in the S phase cells, while X-rays caused a G2/M arrest and subsequently apoptosis. Loss of lipid asymmetry is promptly detectable after one hour of incubation time. The phosphatidylserine translocation, decrease of lipid package and anisotropy, and the increase of membrane fluidity appeared to be based on the same process of lipid asymmetry loss. Therefore, the DNA fragmentation and the cell shrinkage appear to be parallel and independent processes running on different time scales but which are kinetically inter-related. The results indicate different signal steps to apoptosis dependent on inducer characteristics but the kinetics of "early-to-late" apoptosis appears to be a fixed program.

A subchronic 90-day oral toxicity study of Origanum vulgare essential oil in rats.

PubMed

Llana-Ruiz-Cabello, M; Maisanaba, S; Puerto, M; Pichardo, S; Jos, A; Moyano, R; Cameán, A M

2017-03-01

Oregano essential oil (Origanum vulgare L. virens) (OEO) is being used in the food industry due to its useful properties to develop new active packaging systems. In this concern, the safety assessment of this natural extract is of great interest before being commercialized. The European Food Safety Authority requests different in vivo assays to ensure the safety of food contact materials. One of these studies is a 90 days repeated-dose oral assay in rodents. In the present work, 40 male and 40 female Wistar rats were orally exposed to 50, 100 and 200 mg/kg body weight (b.w.) OEO during 90 days following the OECD guideline 408. Data revealed no mortality and no treatment-related adverse effects of the OEO in food/water consumption, body weight, haematology, biochemistry, necropsy, organ weight and histopathology. These findings suggest that the oral no-observed-adverse-effect level (NOAEL) of this OEO is 200 mg/kg b.w. in Wistar rats, the highest dose tested. In conclusion, the use of this OEO in food packaging appears to be safe based on the lack of toxicity during the subchronic study at doses 330-fold higher than those expected to be in contact consumers in the worst scenario of exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expanded polyglutamine embedded in the endoplasmic reticulum causes membrane distortion and coincides with Bax insertion

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

Ueda, Masashi; Li, Shimo; Itoh, Masanori

The endoplasmic reticulum (ER) is important in various cellular functions, such as secretary and membrane protein biosynthesis, lipid synthesis, and calcium storage. ER stress, including membrane distortion, is associated with many diseases such as Huntington's disease. In particular, nuclear envelope distortion is related to neuronal cell death associated with polyglutamine. However, the mechanism by which polyglutamine causes ER membrane distortion remains unclear. We used electron microscopy, fluorescence protease protection assay, and alkaline treatment to analyze the localization of polyglutamine in cells. We characterized polyglutamine embedded in the ER membrane and noted an effect on morphology, including the dilation of ERmore » luminal space and elongation of ER-mitochondria contact sites, in addition to the distortion of the nuclear envelope. The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. These results demonstrated that the ER membrane may be a target of polyglutamine, which triggers cell death through Bax. -- Highlights: •We characterized polyglutamine embedded in the ER membrane. •The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. •The ER membrane may be a target of polyglutamine, which triggers cell death.« less

Human-urine diabetes assay and in vivo rat bladder assay using a fluorine-doped carbon nanotube catheter sensor.

PubMed

Ly, Suw Young; Lee, Jin Hui

2009-10-01

The creation of a novel biosensor consisting of a fluorine-doped carbon nanotube (FCN) was explored for use in cyclic voltammetric (CV) and square-wave stripping voltammetric (SW) glucose assay. In the experiment that was carried out in this study, analytical optimum conditions were attained at the low detection limit (S/N3) of 0.6 microg/L (3.3 x 10(-9) M). In the 0.1 mg/L spike, the relative standard deviation of 0.607 (n = 15) was obtained. This was used for the diagnosis of the urine of patients with diabetes. Moreover, the catheter-type electrode (CE) can be inserted into a rat bladder through the rat's organs. Thus, it can be connected with an electrochemical analyzer that can be fitted with an interface for the real-time in vivo analysis of metabolic glucose. The developed system can be used for organ treatment, biological analysis, and in vivo control.

A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity

PubMed Central

Wahba, Alexander S.; Esmaeili, Abbasali; Damha, Masad J.; Hudson, Robert H. E.

2010-01-01

6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H. PMID:19933258

Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.

PubMed

Paradh, A D; Hill, A E; Mitchell, W J

2014-01-01

DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories. © 2013 Elsevier B.V. All rights reserved.

Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η

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

Patra, Amritaj; Zhang, Qianqian; Lei, Li

2015-02-09

The most prevalent lesion in DNA is an abasic site resulting from glycolytic cleavage of a base. In a number of cellular studies, abasic sites preferentially code for dATP insertion (the “A rule”). In some cases frameshifts are also common. X-ray structures with abasic sites in oligonucleotides have been reported for several microbial and human DNA polymerases (pols), e.g. Dpo4, RB69, KlenTaq, yeast pol ι, human (h) pol ι, and human pol β. We reported previously that hpol η is a major pol involved in abasic site bypass (Choi, J.-Y., Lim, S., Kim, E. J., Jo, A., and Guengerich, F.more » P. (2010 J. Mol. Biol. 404, 34–44). hpol η inserted all four dNTPs in steady-state and pre-steady-state assays, preferentially inserting A and G. In LC-MS analysis of primer-template pairs, A and G were inserted but little C or T was inserted. Frameshifts were observed when an appropriate pyrimidine was positioned 5' to the abasic site in the template. In x-ray structures of hpol η with a non-hydrolyzable analog of dATP or dGTP opposite an abasic site, H-bonding was observed between the phosphate 5' to the abasic site and water H-bonded to N1 and N6 of A and N1 and O6 of G nucleoside triphosphate analogs, offering an explanation for what appears to be a “purine rule.” A structure was also obtained for an A inserted and bonded in the primer opposite the abasic site, but it did not pair with a 5' T in the template. Finally, we conclude that hpol η, a major copying enzyme with abasic sites, follows a purine rule, which can also lead to frameshifts. The phenomenon can be explained with H-bonds.« less

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage

PubMed Central

Zygiel, Emily M.; Noren, Karen A.; Adamkiewicz, Marta A.; Aprile, Richard J.; Bowditch, Heather K.; Carroll, Christine L.; Cerezo, Maria Abigail S.; Dagher, Adelle M.; Hebert, Courtney R.; Hebert, Lauren E.; Mahame, Gloria M.; Milne, Stephanie C.; Silvestri, Kelly M.; Sutherland, Sara E.; Sylvia, Alexandria M.; Taveira, Caitlyn N.; VanValkenburgh, David J.; Noren, Christopher J.

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5’-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5’-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries. PMID:28445507

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage.

PubMed

Zygiel, Emily M; Noren, Karen A; Adamkiewicz, Marta A; Aprile, Richard J; Bowditch, Heather K; Carroll, Christine L; Cerezo, Maria Abigail S; Dagher, Adelle M; Hebert, Courtney R; Hebert, Lauren E; Mahame, Gloria M; Milne, Stephanie C; Silvestri, Kelly M; Sutherland, Sara E; Sylvia, Alexandria M; Taveira, Caitlyn N; VanValkenburgh, David J; Noren, Christopher J; Hall, Marilena Fitzsimons

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5'-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5'-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries.

In vitro expressed GPCR inserted in polymersome membranes for ligand-binding studies.

PubMed

May, Sylvia; Andreasson-Ochsner, Mirjam; Fu, Zhikang; Low, Ying Xiu; Tan, Darren; de Hoog, Hans-Peter M; Ritz, Sandra; Nallani, Madhavan; Sinner, Eva-Kathrin

2013-01-07

The dopamine receptor D2 (DRD2), a G-protein coupled receptor is expressed into PBd(22)-PEO(13) and PMOXA(20)-PDMS(54)-PMOXA(20) block copolymer vesicles. The conformational integrity of the receptor is confirmed by antibody- and ligand-binding assays. Replacement of bound dopamine is demonstrated on surface-immobilized polymersomes, thus making this a promising platform for drug screening. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CXCR4-using HIV variants in a cohort of Black men who have sex with men: HIV Prevention Trials Network 061

PubMed Central

Chen, Iris; Huang, Wei; Connor, Matthew B.; Frantzell, Arne; Cummings, Vanessa; Beauchamp, Geetha G.; Griffith, Sam; Fields, Sheldon D.; Scott, Hyman M.; Shoptaw, Steven; del Rio, Carlos; Magnus, Manya; Mannheimer, Sharon; Tieu, Hong-Van; Wheeler, Darrell P.; Mayer, Kenneth H.; Koblin, Beryl A.; Eshleman, Susan H.

2016-01-01

Objective To evaluate factors associated with HIV tropism among Black men who have sex with men (MSM) in the United States enrolled in a clinical study (HIV Prevention Trials Network 061). Methods HIV tropism was analyzed using a phenotypic assay (Trofile assay, Monogram Biosciences). Samples were analyzed from 43 men who were HIV infected at enrollment and reported either exclusive insertive intercourse or exclusive receptive intercourse; samples were also analyzed from 20 men who were HIV uninfected at enrollment and seroconverted during the study. Clonal analysis of individual viral variants was performed for seroconverters who had dual/mixed viruses. Results Dual/mixed viruses were detected in samples from 11 (26%) of the 43 HIV-infected men analyzed at the enrollment visit; HIV tropism did not differ between those reporting exclusive insertive vs. receptive intercourse. Dual/mixed viruses were also detected in five (25%) of the 20 seroconverters. Dual/mixed viruses were associated with lower CD4 cell counts. Seroconverters with dual/mixed viruses had dual-tropic viruses only or mixed populations of CCR5− and dual-tropic viruses. Conclusions Dual/mixed viruses were frequently detected among Black MSM in this study, including seroconverters. Further studies are needed to understand factors driving transmission and selection of CXCR4− and dual-tropic viruses among Black MSM. PMID:27300696

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

Lin, Qingqing; Li, Huilin; Wang, Tong

Perfringolysin O (PFO) is a transmembrane (TM) β-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes, PFO assembles into pore-forming oligomers containing 30–50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. In this study, we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO, and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakagemore » assay. In contrast, PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM β-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.« less

Effects of PEO-PPO-PEO Triblock Copolymers on Phospholipid Membrane Integrity under Osmotic Stress

PubMed Central

Wang, Jia-Yu; Chin, Jaemin; Marks, Jeremy D.; Lee, Ka Yee C.

2010-01-01

The effects of PEO-PPO-PEO triblock copolymers, mainly Poloxamer 188, on phospholipid membrane integrity under osmotic gradients were explored using giant unilamellar vesicles (GUVs). Fluorescence leakage assays showed two opposing effects of P188 on the structural integrity of GUVs depending on the duration of their incubation time. A two-state transition mechanism of interaction between the triblock copolymers and the phospholipid membrane is proposed: an adsorption (I) and an insertion (II) state. While the triblock copolymer in state I acts to moderately retard the leakage, their insertion in state II perturbs the lipid packing, thus increasing the membrane permeability. Our results suggest that the biomedical application of PEO-PPO-PEO triblock copolymers, either as cell membrane resealing agents or as accelerators for drug delivery, is directed by the delicate balance between these two states. PMID:20666423

Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases.

PubMed

Nerys-Junior, Arildo; Costa, Lendel C; Braga-Dias, Luciene P; Oliveira, Márcia; Rossi, Atila D; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S; Tanuri, Amilcar

2014-03-01

Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

Reaction-based Indicator displacement Assay (RIA) for the selective colorimetric and fluorometric detection of peroxynitrite.

PubMed

Sun, Xiaolong; Lacina, Karel; Ramsamy, Elena C; Flower, Stephen E; Fossey, John S; Qian, Xuhong; Anslyn, Eric V; Bull, Steven D; James, Tony D

2015-05-01

Using the self-assembly of aromatic boronic acids with Alizarin Red S (ARS), we developed a new chemosensor for the selective detection of peroxynitrite. Phenylboronic acid (PBA), benzoboroxole (BBA) and 2-( N , N -dimethylaminomethyl)phenylboronic acid (NBA) were employed to bind with ARS to form the complex probes. In particular, the ARS-NBA system with a high binding affinity can preferably react with peroxynitrite over hydrogen peroxide and other ROS/RNS due to the protection of the boron via the solvent-insertion B-N interaction. Our simple system produces a visible colorimetric change and on-off fluorescence response towards peroxynitrite. By coupling a chemical reaction that leads to an indicator displacement, we have developed a new sensing strategy, referred to herein as RIA (Reaction-based Indicator displacement Assay).

Hepatitis E Virus (HEV) Detection and Quantification by a Real-Time Reverse Transcription-PCR Assay Calibrated to the World Health Organization Standard for HEV RNA

PubMed Central

Germer, Jeffrey J.; Ankoudinova, Irina; Belousov, Yevgeniy S.; Mahoney, Walt; Dong, Chen; Meng, Jihong; Mandrekar, Jayawant N.

2017-01-01

ABSTRACT Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA. PMID:28228493

Development of a new DHPLC assay for genotyping UGT1A (TA)n polymorphism associated with Gilbert's syndrome.

PubMed

Mlakar, Simona Jurkovic; Ostanek, Barbara

2011-01-01

Gilbert's syndrome is the most common hereditary disorder of bilirubin metabolism. The causative mutation in Caucasians is almost exclusively a (TA) dinucleotide insertion in the UGT1A1 promoter. Affected individuals are homozygous for the variant promoter and have 7 TA repeats instead of 6. Promoters with 5 and 8 TA repeats also exist but are extremely rare in Caucasians. The aim of our study was to develop denaturing high-performance liquid chromatography (DHPLC) assay for genotyping UGT1A1(TA)n polymorphism and to compare it with a previously described single-strand conformation polymorphism (SSCP) assay. Fifty DNA samples with common genotypes ((TA)6/6, (TA)6/7, (TA)7/7) as well as 7 samples with one of the following rare genotypes- (TA)5/6, (TA)5/7, (TA)6/8 or (TA)7/8 were amplified by polymerase chain reaction (PCR) and genotyped by DHPLC using sizing mode. All samples were previously genotyped by SSCP assay which was validated by sequencing analysis. All samples with either common or rare genotypes showed completely concordant results between DHPLC and SSCP assays. Our results show that sizing DHPLC assay is more efficient compared to classical SSCP assay due to shorter time of genotyping analysis, ability of genotyping increased number of samples per day, higher robustness, reproducibility and cost-effectiveness with no loss of accuracy in detection of all UGT1A1(TA)n genotypes. We developed a new DHPLC assay which is suitable for accurate, automated, highthroughput, robust genotyping of all UGT1A1(TA)n polymorphism variants, compared to a labour intensive and time-consuming SSCP assay.

Preparation of crotaline F-ab antivenom (CroFab) with automated mixing methods: in vitro observations.

PubMed

Vohra, Rais; Kelner, Michael; Clark, Richard F

2009-01-01

Crotaline Polyvalent Ovine Fab antivenom (CroFab, Savage Laboratories and Protherics Inc., Brentwood, TN, USA) preparation requires that the lyophilized powder be manually reconstituted before use. We compared automated methods for driving the product into solution with the standard manual method of reconstitution, and the effect of repeated rinsing of the product vial, on the per-vial availability of antivenom. Normal saline (NS, 10 mL) was added to 12 vials of expired CroFab. Vials were assigned in pairs to each of six mixing methods, including one pair mixed manually as recommended by the product package insert. Each vial's contents were diluted to a final volume of 75 mL of normal saline. Protein concentration was measured with a colorimetric assay. The fluid left in each vial was removed and the vial was washed with 10 mL NS. Total protein yield from each step was calculated. There was no significant change in protein yield among three of five automated mixing methods when compared to manual reconstitution. Repeat rinsing of the product vial with an additional 10 mLs of fluid added to the protein yield regardless of the mixing method used. We found slightly higher protein yields with all automated methods compared to manual mixing, but only two of five comparisons with the standard mixing method demonstrated statistical significance. However, for all methods tested, the addition of a second rinsing and recovery step increased the amount of protein recovered considerably, presumably by allowing solution of protein trapped in the foamy residues. Automated mixing methods and repeat rinsing of the product vial may allow higher protein yields in the preparation of CroFab antivenom.

The association between drospirenone and hyperkalemia: a comparative-safety study

PubMed Central

2011-01-01

Background Drospirenone/ethinyl-estradiol is an oral contraceptive (OC) that possesses unique antimineralocorticoid activity. It is conjectured that drospirenone, taken alone or concomitantly with spironolactone, may be associated with an increased risk of hyperkalemia. Methods A retrospective cohort study was conducted evaluating women between 18-46 years of age in the Lifelink™ Health Plan Claims Database. The study was restricted to new users of OCs between 1997-2009. Cox proportional hazards models were used to estimate the time to first occurrence of hyperkalemia diagnosis. The main analysis compared OCs containing drospirenone with OCs containing levonorgestrel, a second generation OC not known to impact potassium homeostasis. Logistic regression evaluated concomitant prescribing of drospirenone and spironolactone Results The cohort included 1,148,183 women, averaging 28.8 years of age and 280 days of OC therapy. 2325 cases of hyperkalemia were identified. The adjusted hazard ratio (HR) for hyperkalemia with drospirenone compared to levonorgestrel was 1.10 (95%CI 0.95-1.26). There was an increased risk of hyperkalemia with norethindrone HR 1.15 (95%CI: 1.00-1.33) and norgestimate HR 1.27 (95%CI: 1.11-1.46). Other OCs were unassociated with hyperkalemia. The odds of receiving spironolactone while taking drospirenone were 2.66 (95%CI 2.53-2.80) times higher than the odds of receiving spironolactone and levonorgestrel. Only 6.5% of patients taking drospirenone and spironolactone had a serum potassium assay within 180 days of starting concomitant therapy. Conclusions A clinically significant signal for hyperkalemia with drospirenone was not demonstrated in the current study. Despite the bolded warning for hyperkalemia with joint drospirenone and spironolactone administration, physicians are actually using them together preferentially, and are not following the recommended potassium monitoring requirements in the package insert. PMID:22208934

Solution stability of Captisol-stabilized melphalan (Evomela) versus Propylene glycol-based melphalan hydrochloride injection.

PubMed

Singh, Ramsharan; Chen, Jin; Miller, Teresa; Bergren, Michael; Mallik, Rangan

2016-12-14

The objective of this study was to compare the stability of recently approved Captisol-stabilized propylene glycol-free melphalan injection (Evomela™) against currently marketed propylene glycol-based melphalan injection. The products were compared as reconstituted solutions in vials as well as admixture solutions prepared from normal saline in infusion bags. Evomela and propylene glycol-based melphalan injection were reconstituted in normal saline and organic custom diluent, respectively, according to their package insert instructions. The reconstituted solutions were diluted in normal saline to obtain drug admixture solutions at specific drug concentrations. Stability of the solutions was studied at room temperature by assay of melphalan and determination of melphalan-related impurities. Results show that based on the increase in total impurities in propylene glycol-based melphalan injection at 0.45 mg/mL, Evomela admixture solutions are about 5, 9, 15 and 29 times more stable at concentrations of 0.45, 1.0, 2.0 and 5.0 mg/mL, respectively. Results confirmed that reconstituted Evomela solution can be stored in the vial for up to 1 h at RT or for up to 24 h at refrigerated temperature (2-8 °C) with no significant degradation. After storage in the vial, it remains stable for an additional 3-29 h after preparation of admixture solution in infusion bags at concentrations of 0.25-5.0 mg/mL, respectively. In addition, Evomela solution in saline, at concentration of 5.0 mg/mL melphalan was bacteriostatic through 72 h storage at 2-8 °C. Formulation of melphalan with Captisol technology significantly improved stability compared to melphalan hydrochloride reconstituted with propylene-glycol based diluents.

The association between drospirenone and hyperkalemia: a comparative-safety study.

PubMed

Bird, Steven T; Pepe, Salvatore R; Etminan, Mahyar; Liu, Xinyue; Brophy, James M; Delaney, Joseph Ac

2011-12-30

Drospirenone/ethinyl-estradiol is an oral contraceptive (OC) that possesses unique antimineralocorticoid activity. It is conjectured that drospirenone, taken alone or concomitantly with spironolactone, may be associated with an increased risk of hyperkalemia. A retrospective cohort study was conducted evaluating women between 18-46 years of age in the Lifelink™ Health Plan Claims Database. The study was restricted to new users of OCs between 1997-2009. Cox proportional hazards models were used to estimate the time to first occurrence of hyperkalemia diagnosis. The main analysis compared OCs containing drospirenone with OCs containing levonorgestrel, a second generation OC not known to impact potassium homeostasis. Logistic regression evaluated concomitant prescribing of drospirenone and spironolactone The cohort included 1,148,183 women, averaging 28.8 years of age and 280 days of OC therapy. 2325 cases of hyperkalemia were identified. The adjusted hazard ratio (HR) for hyperkalemia with drospirenone compared to levonorgestrel was 1.10 (95%CI 0.95-1.26). There was an increased risk of hyperkalemia with norethindrone HR 1.15 (95%CI: 1.00-1.33) and norgestimate HR 1.27 (95%CI: 1.11-1.46). Other OCs were unassociated with hyperkalemia. The odds of receiving spironolactone while taking drospirenone were 2.66 (95%CI 2.53-2.80) times higher than the odds of receiving spironolactone and levonorgestrel. Only 6.5% of patients taking drospirenone and spironolactone had a serum potassium assay within 180 days of starting concomitant therapy. A clinically significant signal for hyperkalemia with drospirenone was not demonstrated in the current study. Despite the bolded warning for hyperkalemia with joint drospirenone and spironolactone administration, physicians are actually using them together preferentially, and are not following the recommended potassium monitoring requirements in the package insert.

Vero/BC-F: an efficient packaging cell line stably expressing F protein to generate single round-infectious human parainfluenza virus type 2 vector.

PubMed

Ohtsuka, J; Fukumura, M; Tsurudome, M; Hara, K; Nishio, M; Kawano, M; Nosaka, T

2014-08-01

A stable packaging cell line (Vero/BC-F) constitutively expressing fusion (F) protein of the human parainfluenza virus type 2 (hPIV2) was established for production of the F-defective and single round-infectious hPIV2 vector in a strategy for recombinant vaccine development. The F gene expression has not evoked cytostatic or cytotoxic effects on the Vero/BC-F cells and the F protein was physiologically active to induce syncytial formation with giant polykaryocytes when transfected with a plasmid expressing hPIV2 hemagglutinin-neuraminidase (HN). Transduction of the F-defective replicon RNA into the Vero/BC-F cells led to the release of the infectious particles that packaged the replicon RNA (named as hPIV2ΔF) without detectable mutations, limiting the infectivity to a single round. The maximal titer of the hPIV2ΔF was 6.0 × 10(8) median tissue culture infections dose per ml. The influenza A virus M2 gene was inserted into hPIV2ΔF, and the M2 protein was found to be highly expressed in a human lung cancer cell line after transduction. Furthermore, in vivo airway infection experiments revealed that the hPIV2ΔF was capable of delivering transgenes to hamster tracheal cells. Thus, non-transmissible or single round-infectious hPIV2 vector will be potentially applicable to human gene therapy or recombinant vaccine development.

[Cocaine smuggling in the gastrointestinal tract--the case report with the review of literature].

PubMed

Ciszowski, Krzysztof; Hydzik, Piotr; Waldman, Wojciech; Sein Anand, Jacek

2005-01-01

Body-packing is the way of psychoactive substances smuggling by swallowing of carefully prepared packages with drugs into the gastrointestinal tract or by insertion them into the vagina or the rectum, especially in order to avoid finding them by the custom service. Cocaine, as well as opiates, is the one of the most often smuggled drugs by so called body-packers. In the present study the first case of the body-packer from Malopolska region in Poland, who was observed in the Toxicology Department of the Collegium Medicum UJ, was described. The 29-year-old man swallowed 60 packages containing cocaine with a total net weight of about 500 grams. The plain abdominal radiography revealed multiple shadows of foreign bodies in the gastrointestinal tract, but the results of blood and urine cocaine measurements were negative. During the 37-hours stay in our department the patient was monitored (blood pressure, heart rate, temperature), laxatives and oral fluids were administered. All the packages were evacuated through the natural way and it was followed up by the control abdominal radiography. No symptoms of acute cocaine intoxication or any other complications were observed. According to the case there is also the thorough review of literature presented including the kinds of body-packing, the diagnostic methods used in recognizing of body-packers and the ways of their treatment taking into consideration the conservative management as well as surgical methods.

Method of using sacrificial materials for fabricating internal cavities in laminated dielectric structures

DOEpatents

Peterson, Kenneth A [Albuquerque, NM

2009-02-24

A method of using sacrificial materials for fabricating internal cavities and channels in laminated dielectric structures, which can be used as dielectric substrates and package mounts for microelectronic and microfluidic devices. A sacrificial mandrel is placed in-between two or more sheets of a deformable dielectric material (e.g., unfired LTCC glass/ceramic dielectric), wherein the sacrificial mandrel is not inserted into a cutout made in any of the sheets. The stack of sheets is laminated together, which deforms the sheet or sheets around the sacrificial mandrel. After lamination, the mandrel is removed, (e.g., during LTCC burnout), thereby creating a hollow internal cavity in the monolithic ceramic structure.

Preliminary design report, Large Space Telescope OTA/SI Phase B study: High speed area photometer. [systems analysis

NASA Technical Reports Server (NTRS)

1975-01-01

A photometer is examined which combines several features from separate instruments into a single package. The design presented has both point and area photometry capability with provision for inserting filters to provide spectral discrimination. The electronics provide for photon counting mode for the point detectors and both photon counting and analog modes for the area detector. The area detector also serves as a target locating device for the point detectors. Topics discussed include: (1) electronic equipment requirements, (2) optical properties, (3) structural housing for the instrument, (4) motors and other mechanical components, (5) ground support equipment, and (6) environment control for the instrument. Engineering drawings and block diagrams are shown.

Tasimelteon (Hetlioz™): A New Melatonin Receptor Agonist for the Treatment of Non-24-Hour Sleep-Wake Disorder.

PubMed

Bonacci, Janene M; Venci, Jineane V; Gandhi, Mona A

2015-10-01

In January 2014, the US Food and Drug Administration approved tasimelteon (Hetlioz™), a melatonin-receptor agonist for the treatment of non-24-hour sleep-wake disorder. This article provides an overview of the mechanism of action, pharmacokinetic properties, as well as the clinical efficacy, safety, and tolerability of tasimelteon. Relevant information was identified through a comprehensive literature search of several databases using the key words tasimelteon, Non-24-hour Sleep-Wake disorder, Non-24, and melatonin. Further information was obtained from the tasimelteon package insert, fda.gov, clinicaltrials.gov, briefing materials provided by Vanda Pharmaceuticals, and posters from scientific meetings. © The Author(s) 2014.

Aqueous extracts of Tribulus terrestris protects against oxidized low-density lipoprotein-induced endothelial dysfunction.

PubMed

Jiang, Yue-hua; Yang, Chuan-hua; Li, Wei; Wu, Sai; Meng, Xian-qing; Li, Dong-na

2016-03-01

To investigate the role of aqueous extracts of Tribulus terrestris (TT) against oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) dysfunction in vitro. HUVECs were pre-incubated for 60 min with TT (30 and 3 μg/mL respectively) or 10(-5) mol/L valsartan (as positive controls) and then the injured endothelium model was established by applying 100 μg/mL ox-LDL for 24 h. Cell viability of HUVECs was observed by real-time cell electronic sensing assay and apoptosis rate by Annexin V/PI staining. The cell migration assay was performed with a transwell insert system. Cytoskeleton remodeling was observed by immunofluorescence assay. The content of endothelial nitric oxide synthase (eNOS) was measured by enzyme-linked immunosorbent assay. Intracellular reactive oxygen species (ROS) generation was assessed by immunofluorescence and flow cytometer. Key genes associated with the metabolism of ox-LDL were chosen for quantitative real-time polymerase chain reaction to explore the possible mechanism of TT against oxidized LDL-induced endothelial dysfunction. TT suppressed ox-LDL-induced HUVEC proliferation and apoptosis rates significantly (41.1% and 43.5% after treatment for 3 and 38 h, respectively; P<0.05). It also prolonged the HUVEC survival time and postponed the cell's decaying stage (from the 69th h to over 100 h). According to the immunofluorescence and transwell insert system assay, TT improved the endothelial cytoskeletal network, and vinculin expression and increased cell migration. Additionally, TT regulated of the synthesis of endothelial nitric oxide synthase and generation of intracellular reactive oxygen species (P<0.05). Both 30 and 3 μg/mL TT demonstrated similar efficacy to valsartan. TT normalized the increased mRNA expression of PI3Kα and Socs3. It also decreased mRNA expression of Akt1, AMPKα1, JAK2, LepR and STAT3 induced by ox-LDL. The most notable changes were JAK2, LepR, PI3Kα, Socs3 and STAT3. TT demonstrated potential lowering lipid benefits, anti-hypertension and endothelial protective effects. It also suggested that the JAK2/STAT3 and/or PI3K/AKT pathway might be a very important pathway which was involved in the pharmacological mechanism of TT as the vascular protective agent.

Transcriptome-wide effects of inverted SINEs on gene expression and their impact on RNA polymerase II activity.

PubMed

Tajaddod, Mansoureh; Tanzer, Andrea; Licht, Konstantin; Wolfinger, Michael T; Badelt, Stefan; Huber, Florian; Pusch, Oliver; Schopoff, Sandy; Janisiw, Michael; Hofacker, Ivo; Jantsch, Michael F

2016-10-25

Short interspersed elements (SINEs) represent the most abundant group of non-long-terminal repeat transposable elements in mammalian genomes. In primates, Alu elements are the most prominent and homogenous representatives of SINEs. Due to their frequent insertion within or close to coding regions, SINEs have been suggested to play a crucial role during genome evolution. Moreover, Alu elements within mRNAs have also been reported to control gene expression at different levels. Here, we undertake a genome-wide analysis of insertion patterns of human Alus within transcribed portions of the genome. Multiple, nearby insertions of SINEs within one transcript are more abundant in tandem orientation than in inverted orientation. Indeed, analysis of transcriptome-wide expression levels of 15 ENCODE cell lines suggests a cis-repressive effect of inverted Alu elements on gene expression. Using reporter assays, we show that the negative effect of inverted SINEs on gene expression is independent of known sensors of double-stranded RNAs. Instead, transcriptional elongation seems impaired, leading to reduced mRNA levels. Our study suggests that there is a bias against multiple SINE insertions that can promote intramolecular base pairing within a transcript. Moreover, at a genome-wide level, mRNAs harboring inverted SINEs are less expressed than mRNAs harboring single or tandemly arranged SINEs. Finally, we demonstrate a novel mechanism by which inverted SINEs can impact on gene expression by interfering with RNA polymerase II.

Expression of Wheat High Molecular Weight Glutenin Subunit 1Bx Is Affected by Large Insertions and Deletions Located in the Upstream Flanking Sequences

PubMed Central

Hao, Chenyang; Tang, Saijun; Zhang, Xueyong; Li, Tian

2014-01-01

To better understand the transcriptional regulation of high molecular weight glutenin subunit (HMW-GS) expression, we isolated four Glu-1Bx promoters from six wheat cultivars exhibiting diverse protein expression levels. The activities of the diverse Glu-1Bx promoters were tested and compared with β-glucuronidase (GUS) reporter fusions. Although all the full-length Glu-1Bx promoters showed endosperm-specific activities, the strongest GUS activity was observed with the 1Bx7OE promoter in both transient expression assays and stable transgenic rice lines. A 43 bp insertion in the 1Bx7OE promoter, which is absent in the 1Bx7 promoter, led to enhanced expression. Analysis of promoter deletion constructs confirmed that a 185 bp MITE (miniature inverted-repeat transposable element) in the 1Bx14 promoter had a weak positive effect on Glu-1Bx expression, and a 54 bp deletion in the 1Bx13 promoter reduced endosperm-specific activity. To investigate the effect of the 43 bp insertion in the 1Bx7OE promoter, a functional marker was developed to screen 505 Chinese varieties and 160 European varieties, and only 1Bx7-type varieties harboring the 43 bp insertion in their promoters showed similar overexpression patterns. Hence, the 1Bx7OE promoter should be important tool in crop genetic engineering as well as in molecular assisted breeding. PMID:25133580

The Risa R/Bioconductor package: integrative data analysis from experimental metadata and back again.

PubMed

González-Beltrán, Alejandra; Neumann, Steffen; Maguire, Eamonn; Sansone, Susanna-Assunta; Rocca-Serra, Philippe

2014-01-01

The ISA-Tab format and software suite have been developed to break the silo effect induced by technology-specific formats for a variety of data types and to better support experimental metadata tracking. Experimentalists seldom use a single technique to monitor biological signals. Providing a multi-purpose, pragmatic and accessible format that abstracts away common constructs for describing Investigations, Studies and Assays, ISA is increasingly popular. To attract further interest towards the format and extend support to ensure reproducible research and reusable data, we present the Risa package, which delivers a central component to support the ISA format by enabling effortless integration with R, the popular, open source data crunching environment. The Risa package bridges the gap between the metadata collection and curation in an ISA-compliant way and the data analysis using the widely used statistical computing environment R. The package offers functionality for: i) parsing ISA-Tab datasets into R objects, ii) augmenting annotation with extra metadata not explicitly stated in the ISA syntax; iii) interfacing with domain specific R packages iv) suggesting potentially useful R packages available in Bioconductor for subsequent processing of the experimental data described in the ISA format; and finally v) saving back to ISA-Tab files augmented with analysis specific metadata from R. We demonstrate these features by presenting use cases for mass spectrometry data and DNA microarray data. The Risa package is open source (with LGPL license) and freely available through Bioconductor. By making Risa available, we aim to facilitate the task of processing experimental data, encouraging a uniform representation of experimental information and results while delivering tools for ensuring traceability and provenance tracking. The Risa package is available since Bioconductor 2.11 (version 1.0.0) and version 1.2.1 appeared in Bioconductor 2.12, both along with documentation and examples. The latest version of the code is at the development branch in Bioconductor and can also be accessed from GitHub https://github.com/ISA-tools/Risa, where the issue tracker allows users to report bugs or feature requests.

The Risa R/Bioconductor package: integrative data analysis from experimental metadata and back again

PubMed Central

2014-01-01

Background The ISA-Tab format and software suite have been developed to break the silo effect induced by technology-specific formats for a variety of data types and to better support experimental metadata tracking. Experimentalists seldom use a single technique to monitor biological signals. Providing a multi-purpose, pragmatic and accessible format that abstracts away common constructs for describing Investigations, Studies and Assays, ISA is increasingly popular. To attract further interest towards the format and extend support to ensure reproducible research and reusable data, we present the Risa package, which delivers a central component to support the ISA format by enabling effortless integration with R, the popular, open source data crunching environment. Results The Risa package bridges the gap between the metadata collection and curation in an ISA-compliant way and the data analysis using the widely used statistical computing environment R. The package offers functionality for: i) parsing ISA-Tab datasets into R objects, ii) augmenting annotation with extra metadata not explicitly stated in the ISA syntax; iii) interfacing with domain specific R packages iv) suggesting potentially useful R packages available in Bioconductor for subsequent processing of the experimental data described in the ISA format; and finally v) saving back to ISA-Tab files augmented with analysis specific metadata from R. We demonstrate these features by presenting use cases for mass spectrometry data and DNA microarray data. Conclusions The Risa package is open source (with LGPL license) and freely available through Bioconductor. By making Risa available, we aim to facilitate the task of processing experimental data, encouraging a uniform representation of experimental information and results while delivering tools for ensuring traceability and provenance tracking. Software availability The Risa package is available since Bioconductor 2.11 (version 1.0.0) and version 1.2.1 appeared in Bioconductor 2.12, both along with documentation and examples. The latest version of the code is at the development branch in Bioconductor and can also be accessed from GitHub https://github.com/ISA-tools/Risa, where the issue tracker allows users to report bugs or feature requests. PMID:24564732

Randomization and In Vivo Selection Reveal a GGRG Motif Essential for Packaging Human Immunodeficiency Virus Type 2 RNA ▿ †

PubMed Central

Baig, Tayyba T.; Lanchy, Jean-Marc; Lodmell, J. Stephen

2009-01-01

The packaging signal (ψ) of human immunodeficiency virus type 2 (HIV-2) is present in the 5′ noncoding region of RNA and contains a 10-nucleotide palindrome (pal; 5′-392-GGAGUGCUCC) located upstream of the dimerization signal stem-loop 1 (SL1). pal has been shown to be functionally important in vitro and in vivo. We previously showed that the 3′ side of pal (GCUCC-3′) is involved in base-pairing interactions with a sequence downstream of SL1 to make an extended SL1, which is important for replication in vivo and the regulation of dimerization in vitro. However, the role of the 5′ side of pal (5′-GGAGU) was less clear. Here, we characterized this role using an in vivo SELEX approach. We produced a population of HIV-2 DNA genomes with random sequences within the 5′ side of pal and transfected these into COS-7 cells. Viruses from COS-7 cells were used to infect C8166 permissive cells. After several weeks of serial passage in C8166 cells, surviving viruses were sequenced. On the 5′ side of pal there was a striking convergence toward a GGRGN consensus sequence. Individual clones with consensus and nonconsensus sequences were tested in infectivity and packaging assays. Analysis of individuals that diverged from the consensus sequence showed normal viral RNA and protein synthesis but had replication defects and impaired RNA packaging. These findings clearly indicate that the GGRG motif is essential for viral replication and genomic RNA packaging. PMID:18971263

Cell Communication during Aggregation and Development of the Cellular Slime Mould Distyostelium discoideum.

DTIC Science & Technology

1985-01-01

of actin protein xg relative centrifugal force glorin N-propionyl- Y -L-glutawyl-L-ornithine- S- lactam ethyl ester [3 H]FA [7,9,3’,5 ’-3H]folic acid...solubilize the pellet and radioactivity was measured on a LKB Rack Beta scintillation counter. cAMP Binding to Whole Cells. This assay followed the well...inserts, pre-filled with 4ml of Unisolve I scintillant, and radioactivity measured on a LKB Rack Beta scintillation counter. Controls included: a) no

Understanding risk evaluation and mitigation strategies in organ transplantation.

PubMed

Gabardi, Steven

2011-07-01

The United States Food and Drug Administration (FDA) Amendments Act of 2007 mandated that Risk Evaluation and Mitigation Strategies (REMS) be required of manufacturers. These REMS are strategies implemented to manage known or potential risks associated with drugs and to ensure ongoing pharmacovigilance throughout the life of a pharmaceutical product, including once the product becomes available as generic. The elements of an individual REMS program consist of three levels: medication guide or patient package insert, communication plan, and elements to assure safe use (ETASU). A medication guide or patient package insert is used to help prevent serious adverse events, aid in patient decision making, and enhance drug adherence. Communication plans are used to educate health care providers and to encourage their compliance with REMS. The ETASU is a restrictive process that is implemented when it is deemed necessary to ensure that patients have safe access to products with known serious risks that would otherwise be unavailable. To review the components of REMS and specifically assess their impact on health care providers practicing within the organ transplantation arena, a literature search of the MEDLINE database (January 2007-December 2010) was performed, and published materials from the FDA and its Web site were also reviewed. In transplantation, REMS programs exist for both everolimus (medication guide and communication plan) and sirolimus (medication guide). The FDA has stated that all mycophenolic acid derivatives will be subject to a proposed REMS that has not yet been approved; however, both branded mycophenolic acid agents already have approved medication guides. The REMS are a permanent fixture in the development and marketing of pharmaceutical agents, and their further implementation in solid organ transplantation is inevitable. Transplantation providers should take a proactive role in patient education and implementation of REMS within the therapeutic area. It is imperative for health care providers to realize that the ultimate goals of REMS are to reduce the potential for serious risks and to make outcomes from the treatment of disease more predictable.

Pharmacist, general practitioner and consumer use of written medicine information in Australia: are they on the same page?

PubMed

Hamrosi, Kim K; Raynor, David K; Aslani, Parisa

2014-01-01

Providing written medicine information to consumers enables them to make informed decisions about their medicines, playing an important role in educating and improving health literacy. In Australia, standardized written medicine information called Consumer Medicine Information (CMI) is available for medicines as package inserts, computer prints, or leaflets. Consumers want and read CMI, but may not always ask for it. General practitioners (GPs) and pharmacists are an important source of written medicine information, yet may not always provide CMI in their practice. To examine and compare the awareness, use and provision of CMI by consumers, pharmacists and general practitioners (GPs). Based on previous studies, structured questionnaires were developed and administered to a national sample of consumers (phone survey); community pharmacists and GPs (postal surveys) about utilization of CMI. Descriptive, comparative and logistic regression analyses were conducted. The respondents comprised of 349 pharmacists, 181 GPs and 1000 consumers. Two-thirds of consumers, nearly all (99%) pharmacists and 90% of GPs were aware of CMI. About 88% of consumers reported receiving CMI as a package insert, however most pharmacists (99%) and GPs (56%) reported providing computer-generated CMI. GPs' and pharmacists' main reason for providing CMI was on patient request. Reasons for not providing were predominantly because consumers were already taking the medicine, concerns regarding difficulty understanding the information, or potential non-adherence. Of the 691 consumers reportedly reading CMI, 35% indicated concerns after reading. Factors associated with reading included gender, type of CMI received and frequency of provision. Consumers want and read information about their medicines, especially when received from their GP or pharmacist. Healthcare professionals report usually discussing CMI when providing it to patients, although continued improvements in dissemination rates are desirable. Regular use of CMI remains a challenge, and ongoing strategies to promote CMI use are necessary to improve uptake of CMI in Australia. Copyright © 2014 Elsevier Inc. All rights reserved.

Computational Materials Program for Alloy Design

NASA Technical Reports Server (NTRS)

Bozzolo, Guillermo

2005-01-01

The research program sponsored by this grant, "Computational Materials Program for Alloy Design", covers a period of time of enormous change in the emerging field of computational materials science. The computational materials program started with the development of the BFS method for alloys, a quantum approximate method for atomistic analysis of alloys specifically tailored to effectively deal with the current challenges in the area of atomistic modeling and to support modern experimental programs. During the grant period, the program benefited from steady growth which, as detailed below, far exceeds its original set of goals and objectives. Not surprisingly, by the end of this grant, the methodology and the computational materials program became an established force in the materials communitiy, with substantial impact in several areas. Major achievements during the duration of the grant include the completion of a Level 1 Milestone for the HITEMP program at NASA Glenn, consisting of the planning, development and organization of an international conference held at the Ohio Aerospace Institute in August of 2002, finalizing a period of rapid insertion of the methodology in the research community worlwide. The conference, attended by citizens of 17 countries representing various fields of the research community, resulted in a special issue of the leading journal in the area of applied surface science. Another element of the Level 1 Milestone was the presentation of the first version of the Alloy Design Workbench software package, currently known as "adwTools". This software package constitutes the first PC-based piece of software for atomistic simulations for both solid alloys and surfaces in the market.Dissemination of results and insertion in the materials community worldwide was a primary focus during this period. As a result, the P.I. was responsible for presenting 37 contributed talks, 19 invited talks, and publishing 71 articles in peer-reviewed journals, as detailed later in this Report.

Design, Fabrication, and Packaging of Mach-Zehnder Interferometers for Biological Sensing Applications

NASA Astrophysics Data System (ADS)

Novak, Joseph

Optical biological sensors are widely used in the fields of medical testing, water treatment and safety, gene identification, and many others due to advances in nanofabrication technology. This work focuses on the design of fiber-coupled Mach-Zehnder Interferometer (MZI) based biosensors fabricated on silicon-on-insulator (SOI) wafer. Silicon waveguide sensors are designed with multimode and single-mode dimensions. Input coupling efficiency is investigated by design of various taper structures. Integration processing and packaging is performed for fiber attachment and enhancement of input coupling efficiency. Optical guided-wave sensors rely on single-mode operation to extract an induced phase-shift from the output signal. A silicon waveguide MZI sensor designed and fabricated for both multimode and single-mode dimensions. Sensitivity of the sensors is analyzed for waveguide dimensions and materials. An s-bend structure is designed for the multimode waveguide to eliminate higher-order mode power as an alternative to single-mode confinement. Single-mode confinement is experimentally demonstrated through near field imaging of waveguide output. Y-junctions are designed for 3dB power splitting to the MZI arms and for power recombination after sensing to utilize the interferometric function of the MZI. Ultra-short 10microm taper structures with curved geometries are designed to improve insertion loss from fiber-to-chip without significantly increasing device area and show potential for applications requiring misalignment tolerance. An novel v-groove process is developed for self-aligned integration of fiber grooves for attachment to sensor chips. Thermal oxidation at temperatures from 1050-1150°C during groove processing creates an SiO2 layer on the waveguide end facet to protect the waveguide facet during integration etch processing without additional e-beam lithography processing. Experimental results show improvement of insertion loss compared to dicing preparation and Focused Ion Beam methods using the thermal oxidation process.

Comparative analysis of package inserts of local and imported antihypertensive medications in Palestine.

PubMed

Qatmosh, Sandra A; Koni, Amer A; Qeeno, Baraa G; Arandy, Dina A; Abu-Hashia, Maysa W; Al-Hroub, Bahaa M; Zyoud, Sa'ed H

2017-09-25

Package inserts (PIs) as a reliable reference for patients and health care providers should provide accurate, complete and up-to-date information. The purpose of the current study is to assess and compare the PIs of antihypertensive agents locally produced in Palestine and their imported counterparts. Thirty-five PIs were assessed for the presence of 31 information statements using a scoring method. Word counting of 20 headings and subheadings was used to evaluate and compare local and imported PIs for information quantity. None of the analysed PIs fulfilled the criteria. All of them included the brand name, active ingredients, indications, directions for use, adverse drug reactions, drug-drug interactions, pregnancy and lactation considerations, and storage. Whereas none of them, either local or imported PIs, included the shelf life and instructions to convert tablets or capsules into liquid forms. Additionally, only one (5%) imported and no (0%) local PIs mentioned the duration of therapy. Moreover, 93.4% of local PIs were deficient in areas regarding the inactive ingredients and date of last revision, and 86.7% did not mention the drug dose and possibility of tablet splitting. Furthermore, the maximum dose was not indicated in 90% of imported and 86.7% of local PIs. In general, imported PIs contained more detailed information than their local counterparts, where the range of differences in medians between the local and imported PIs was from 1.5-fold for pregnancy considerations to >42.00-fold for the effect on the ability to drive and use machines. The findings of this study revealed the superiority of imported over local PIs in both quality and quantity of information provided. This emphasises the need for appropriate measures to be taken by the Ministry of Health and local manufacturers to ensure efficiency of local PIs in providing accurate, complete and up-to-date information.

High-throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE.

PubMed

Fan, Jun; Crooks, Casey; Lamb, Chris

2008-01-01

Bioluminescent strains of the Arabidopsis thaliana pathogens Pseudomonas syringae pathovar (pv.) tomato and pv. maculicola were made by insertion of the luxCDABE operon from Photorhabdus luminescens into the P. syringae chromosome under the control of a constitutive promoter. Stable integration of luxCDABE did not affect bacterial fitness, growth in planta or disease outcome. Luminescence accurately and reliably reported bacterial growth in infected Arabidopsis leaves both with a fixed inoculum followed over time and with varying inocula assayed at a single time point. Furthermore, the bioluminescence assay could detect a small (1.3-fold) difference in bacterial growth between different plant genotypes with a precision comparable to that of the standard plate assay. Luminescence of luxCDABE-tagged P. syringae allows rapid and convenient quantification of bacterial growth without the tissue extraction, serial dilution, plating and manual scoring involved in standard assays of bacterial growth by colony formation in plate culture of samples from infected tissue. The utility of the bioluminescence assay was illustrated by surveying the 500-fold variation in growth of the universally virulent P. syringae pv. maculicola ES4326 among more than 100 Arabidopsis ecotypes and identification of two quantitative trait loci accounting for 48% and 16%, respectively, of the variance of basal resistance to P. syringae pv. tomato DC3000 in the Col-0 x Fl-1 F(2) population. Luminescence assay of bacteria chromosomally tagged with luxCDABE should greatly facilitate the genetic dissection of quantitative differences in gene-for-gene, basal and acquired disease resistance and other aspects of plant interactions with bacterial pathogens requiring high-throughput assays or large-scale quantitative screens.

Contrasting influences of Drosophila white/mini-white on ethanol sensitivity in two different behavioral assays

PubMed Central

Chan, Robin F.; Lewellyn, Lara; DeLoyht, Jacqueline M.; Sennett, Kristyn; Coffman, Scarlett; Hewitt, Matthew; Bettinger, Jill C.; Warrick, John M.; Grotewiel, Mike

2014-01-01

Background The fruit fly Drosophila melanogaster has been used extensively to investigate genetic mechanisms of ethanol-related behaviors. Many past studies in flies, including studies from our laboratory, have manipulated gene expression using transposons carrying the genetic-phenotypic marker mini-white, a derivative of the endogenous gene white. Whether the mini-white transgenic marker or the endogenous white gene influence behavioral responses to acute ethanol exposure in flies has not been systematically investigated. Methods We manipulated mini-white and white expression via (i) transposons marked with mini-white, (ii) RNAi against mini-white and white and (iii) a null allele of white. We assessed ethanol sensitivity and tolerance using a previously described eRING assay (based on climbing in the presence of ethanol) and an assay based on ethanol-induced sedation. Results In eRING assays, ethanol-induced impairment of climbing correlated inversely with expression of the mini-white marker from a series of transposon insertions. Additionally, flies harboring a null allele of white or flies with RNAi-mediated knockdown of mini-white were significantly more sensitive to ethanol in eRING assays than controls expressing endogenous white or the mini-white marker. In contrast, ethanol sensitivity and rapid tolerance measured in the ethanol sedation assay were not affected by decreased expression of mini-white or endogenous white in flies. Conclusions Ethanol sensitivity measured in the eRING assay is noticeably influenced by white and mini-white, making eRING problematic for studies on ethanol-related behavior in Drosophila using transgenes marked with mini-white. In contrast, the ethanol sedation assay described here is a suitable behavioral paradigm for studies on ethanol sedation and rapid tolerance in Drosophila including those that use widely available transgenes marked with mini-white. PMID:24890118

Utility of coupling nonlinear optimization methods with numerical modeling software

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

Murphy, M.J.

1996-08-05

Results of using GLO (Global Local Optimizer), a general purpose nonlinear optimization software package for investigating multi-parameter problems in science and engineering is discussed. The package consists of the modular optimization control system (GLO), a graphical user interface (GLO-GUI), a pre-processor (GLO-PUT), a post-processor (GLO-GET), and nonlinear optimization software modules, GLOBAL & LOCAL. GLO is designed for controlling and easy coupling to any scientific software application. GLO runs the optimization module and scientific software application in an iterative loop. At each iteration, the optimization module defines new values for the set of parameters being optimized. GLO-PUT inserts the new parametermore » values into the input file of the scientific application. GLO runs the application with the new parameter values. GLO-GET determines the value of the objective function by extracting the results of the analysis and comparing to the desired result. GLO continues to run the scientific application over and over until it finds the ``best`` set of parameters by minimizing (or maximizing) the objective function. An example problem showing the optimization of material model is presented (Taylor cylinder impact test).« less

How important are internal temperature gradients in french straws during freezing of bovine sperm in nitrogen vapor?

PubMed

Santos, M V; Sansinena, M; Zaritzky, N; Chirife, J

2013-01-01

The subject of present work was to predict internal temperature gradients developed during freezing of bovine sperm diluted in extender, packaged in 0.5 ml French plastic straws and suspended in static liquid nitrogen vapor at -100 degree C. For this purpose, a mathematical heat transfer model previously developed to predict freezing times (phase change was considered) of semen/extender packaged in straw was extended to predict internal temperature gradients during the cooling/freezing process. Results showed maximum temperature differences between the centre and the periphery of semen/extender "liquid" column was 1.5 degree C for an external heat transfer coefficient, h = 15 W per (m(2) K), and only 0.5 degree C for h = 5 W per (m(2) K). It is concluded that if a thermocouple wire were inserted in a 0.5 ml plastic straw to monitor the freezing process in nitrogen vapor, its radial position would have little importance since expected internal gradients may be safely neglected. This finding facilitates the interpretation of freezing rates in 0.5 ml plastic straws immersed in nitrogen vapor over liquid nitrogen, a widely used method for cryopreservation of bovine spermatozoa.

Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA.

PubMed

Majid, Farjana; Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

2014-01-01

Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log 10 copies/ml and 6.95 ± 1.08 log 10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log 10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35.

Integrated Electrode Arrays for Neuro-Prosthetic Implants

NASA Technical Reports Server (NTRS)

Brandon, Erik; Mojarradi, Mohammede

2003-01-01

Arrays of electrodes integrated with chip-scale packages and silicon-based integrated circuits have been proposed for use as medical electronic implants, including neuro-prosthetic devices that might be implanted in brains of patients who suffer from strokes, spinal-cord injuries, or amyotrophic lateral sclerosis. The electrodes of such a device would pick up signals from neurons in the cerebral cortex, and the integrated circuit would perform acquisition and preprocessing of signal data. The output of the integrated circuit could be used to generate, for example, commands for a robotic arm. Electrode arrays capable of acquiring electrical signals from neurons already exist, but heretofore, there has been no convenient means to integrate these arrays with integrated-circuit chips. Such integration is needed in order to eliminate the need for the extensive cabling now used to pass neural signals to data-acquisition and -processing equipment outside the body. The proposed integration would enable progress toward neuro-prostheses that would be less restrictive of patients mobility. An array of electrodes would comprise a set of thin wires of suitable length and composition protruding from and supported by a fine-pitch micro-ball grid array or chip-scale package (see figure). The associated integrated circuit would be mounted on the package face opposite the probe face, using the solder bumps (the balls of the ball grid array) to make the electrical connections between the probes and the input terminals of the integrated circuit. The key innovation is the insertion of probe wires of the appropriate length and material into the solder bumps through a reflow process, thereby fixing the probes in place and electrically connecting them with the integrated circuit. The probes could be tailored to any distribution of lengths and made of any suitable metal that could be drawn into fine wires. Furthermore, the wires could be coated with an insulating layer using anodization or other processes, to achieve the correct electrical impedance. The probe wires and the packaging materials must be biocompatible using such materials as lead-free solders. For protection, the chip and package can be coated with parylene.

Patient understanding of oral contraceptive pill instructions related to missed pills: a systematic review.

PubMed

Zapata, Lauren B; Steenland, Maria W; Brahmi, Dalia; Marchbanks, Polly A; Curtis, Kathryn M

2013-05-01

Instructions on what to do after pills are missed are critical to reducing unintended pregnancies resulting from patient non-adherence to oral contraceptive (OC) regimens. Missed pill instructions have previously been criticized for being too complex, lacking a definition of what is meant by "missed pills," and for being confusing to women who may not know the estrogen content of their formulation. To help inform the development of missed pill guidance to be included in the forthcoming US Selected Practice Recommendations, the objective of this systematic review was to evaluate the evidence on patient understanding of missed pill instructions. We searched the PubMed database for peer-reviewed articles that examined patient understanding of OC pill instructions that were published in any language from inception of the database through March 2012. We included studies that examined women's knowledge and understanding of missed pill instructions after exposure to some written material (e.g., patient package insert, brochure), as well as studies that compared different types of missed pill instructions on women's comprehension. We used standard abstract forms and grading systems to summarize and assess the quality of the evidence. From 1620 articles, nine studies met our inclusion criteria. Evidence from one randomized controlled trial (RCT) and two descriptive studies found that more women knew what to do after missing 1 pill than after missing 2 or 3 pills (Level I, good, to Level II-3, poor), and two descriptive studies found that more women knew what to do after missing 2 pills than after missing 3 pills (Level II-3, fair). Data from two descriptive studies documented the difficulty women have understanding missed pill instructions contained in patient package inserts (Level II-3, poor), and evidence from two RCTs found that providing written brochures with information on missed pill instructions in addition to contraceptive counseling significantly improved knowledge of how to manage missed pills for up to three months compared to contraceptive counseling alone (Level I, fair). Evidence from one RCT found that graphic-based missed pill instructions were better than text-only instructions (Level I, good), and data from two RCTs found that less information resulted in improved comprehension (Level I, good to fair). Evidence from one descriptive study found that many women missing pills did not intend to follow recommended actions per missed pill instructions despite understanding the guidance (Level II-3, poor). There is wide variability in the percent of women having correct knowledge on what to do when pills are missed after exposure to written missed pills instructions, with more women knowing what to do after missing 1 pill than after missing 2 or 3 pills. Women have difficulty understanding missed pill instructions contained in patient package inserts. Providing written brochures with information on missed pill instructions in addition to contraceptive counseling may improve knowledge of how to manage missed pills. Graphic-based missed pill instructions and those containing less information may result in improved comprehension. Even with clear instructions, many women missing pills may choose not to follow the recommended actions. Published by Elsevier Inc.

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control.

PubMed Central

Kox, L F; Noordhoek, G T; Kunakorn, M; Mulder, S; Sterrenburg, M; Kolk, A H

1996-01-01

A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization. PMID:8862568

Rapid detection of rifampin-resistant clinical isolates of Mycobacterium tuberculosis by reverse dot blot hybridization.

PubMed

Guo, Qian; Yu, Yan; Zhu, Yan Ling; Zhao, Xiu Qin; Liu, Zhi Guang; Zhang, Yuan Yuan; Li, Gui Lian; Wei, Jian Hao; Wu, Yi Mou; Wan, Kang Lin

2015-01-01

A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis). 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.