Digital gene expression analysis with sample multiplexing and PCR duplicate detection: A straightforward protocol.

PubMed

Rozenberg, Andrey; Leese, Florian; Weiss, Linda C; Tollrian, Ralph

2016-01-01

Tag-Seq is a high-throughput approach used for discovering SNPs and characterizing gene expression. In comparison to RNA-Seq, Tag-Seq eases data processing and allows detection of rare mRNA species using only one tag per transcript molecule. However, reduced library complexity raises the issue of PCR duplicates, which distort gene expression levels. Here we present a novel Tag-Seq protocol that uses the least biased methods for RNA library preparation combined with a novel approach for joint PCR template and sample labeling. In our protocol, input RNA is fragmented by hydrolysis, and poly(A)-bearing RNAs are selected and directly ligated to mixed DNA-RNA P5 adapters. The P5 adapters contain i5 barcodes composed of sample-specific (moderately) degenerate base regions (mDBRs), which later allow detection of PCR duplicates. The P7 adapter is attached via reverse transcription with individual i7 barcodes added during the amplification step. The resulting libraries can be sequenced on an Illumina sequencer. After sample demultiplexing and PCR duplicate removal with a free software tool we designed, the data are ready for downstream analysis. Our protocol was tested on RNA samples from predator-induced and control Daphnia microcrustaceans.

Computational fragment-based screening using RosettaLigand: the SAMPL3 challenge

NASA Astrophysics Data System (ADS)

Kumar, Ashutosh; Zhang, Kam Y. J.

2012-05-01

SAMPL3 fragment based virtual screening challenge provides a valuable opportunity for researchers to test their programs, methods and screening protocols in a blind testing environment. We participated in SAMPL3 challenge and evaluated our virtual fragment screening protocol, which involves RosettaLigand as the core component by screening a 500 fragments Maybridge library against bovine pancreatic trypsin. Our study reaffirmed that the real test for any virtual screening approach would be in a blind testing environment. The analyses presented in this paper also showed that virtual screening performance can be improved, if a set of known active compounds is available and parameters and methods that yield better enrichment are selected. Our study also highlighted that to achieve accurate orientation and conformation of ligands within a binding site, selecting an appropriate method to calculate partial charges is important. Another finding is that using multiple receptor ensembles in docking does not always yield better enrichment than individual receptors. On the basis of our results and retrospective analyses from SAMPL3 fragment screening challenge we anticipate that chances of success in a fragment screening process could be increased significantly with careful selection of receptor structures, protein flexibility, sufficient conformational sampling within binding pocket and accurate assignment of ligand and protein partial charges.

A COMPARISON OF BENTHIC MACROINVERTEBRATE SAMPLING METHODS ON SELECTED LARGE RIVER TRIBUTARIES TO THE MISSISSIPPI

EPA Science Inventory

We compared three benthic macroinvertebrate sampling methods on the St. Croix, Wisconsin and Scioto Rivers in summer 2004 and 2005. EPA's newly developed, multi-habitat Large River Bioassessment Protocol (LR-BP) was compared to the multi-habitat method of the Minnesota Pollution...

INFORMATION MANAGEMENT AND RELATED QUALITY ASSURANCE FOR A LARGE SCALE, MULTI-SITE RESEARCH PROJECT

EPA Science Inventory

During the summer of 2000, as part of a U.S. Environmental Protection Agency study designed to improve microbial water quality monitoring protocols at public beaches, over 11,000 water samples were collected at five selected beaches across the country. At each beach, samples wer...

ISOLATION AND GENOTYPING OF CLOSTRIDIUM PERFRINGENS FROM FREE-LIVING SOUTH AMERICAN COATI (NASUA NASUA).

PubMed

Silva, Rodrigo O S; Almeida, Lara R; Oliveira Junior, Carlos A; Lima, Paula C S; Soares, Danielle F M; Pereira, Pedro L L; Silva, Israel J; Lobato, Francisco C F

2016-03-01

The importance of Clostridium perfringens for most wild animal species remains unclear. This study aimed to isolate and genotype C. perfringens in stool samples from free-living South American coati (Nasua nasua) in Brazil. Forty-six free-living N. nasua were trapped and stool samples were collected. Two different protocols for C. perfringens isolation were tested: direct plating onto selective agar and pre-enrichment in broth followed by plating in selective agar. Clostridium perfringens type A was isolated from 15 (32.6%) animals by direct plating and 36 (78.3%) animals by broth PE, and the rate of isolation was significantly different between these two methods (P < 0.01). Twelve of the 36 (33.3%) isolated strains by the PE protocol were positive for the β-2 toxin-encoding gene (cpb2) whereas the enterotoxin-encoding gene (cpe) and necrotic enteritis like-B toxin gene (netb) were not found. These results suggest that C. perfringens is commonly part of the microbiota of free-living coatis. Additionally, the use of a PE protocol appears to be essential for studies on C. perfringens in this species.

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.

PubMed

Treweek, Jennifer B; Chan, Ken Y; Flytzanis, Nicholas C; Yang, Bin; Deverman, Benjamin E; Greenbaum, Alon; Lignell, Antti; Xiao, Cheng; Cai, Long; Ladinsky, Mark S; Bjorkman, Pamela J; Fowlkes, Charless C; Gradinaru, Viviana

2015-11-01

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1-2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.

A semi-nested real-time PCR method to detect low chimerism percentage in small quantity of hematopoietic stem cell transplant DNA samples.

PubMed

Aloisio, Michelangelo; Bortot, Barbara; Gandin, Ilaria; Severini, Giovanni Maria; Athanasakis, Emmanouil

2017-02-01

Chimerism status evaluation of post-allogeneic hematopoietic stem cell transplantation samples is essential to predict post-transplant relapse. The most commonly used technique capable of detecting small increments of chimerism is quantitative real-time PCR. Although this method is already used in several laboratories, previously described protocols often lack sensitivity and the amount of the DNA required for each chimerism analysis is too high. In the present study, we compared a novel semi-nested allele-specific real-time PCR (sNAS-qPCR) protocol with our in-house standard allele-specific real-time PCR (gAS-qPCR) protocol. We selected two genetic markers and analyzed technical parameters (slope, y-intercept, R2, and standard deviation) useful to determine the performances of the two protocols. The sNAS-qPCR protocol showed better sensitivity and precision. Moreover, the sNAS-qPCR protocol requires, as input, only 10 ng of DNA, which is at least 10-fold less than the gAS-qPCR protocols described in the literature. Finally, the proposed sNAS-qPCR protocol could prove very useful for performing chimerism analysis with a small amount of DNA, as in the case of blood cell subsets.

Quantification of the overall measurement uncertainty associated with the passive moss biomonitoring technique: Sample collection and processing.

PubMed

Aboal, J R; Boquete, M T; Carballeira, A; Casanova, A; Debén, S; Fernández, J A

2017-05-01

In this study we examined 6080 data gathered by our research group during more than 20 years of research on the moss biomonitoring technique, in order to quantify the variability generated by different aspects of the protocol and to calculate the overall measurement uncertainty associated with the technique. The median variance of the concentrations of different pollutants measured in moss tissues attributed to the different methodological aspects was high, reaching values of 2851 (ng·g -1 ) 2 for Cd (sample treatment), 35.1 (μg·g -1 ) 2 for Cu (sample treatment), 861.7 (ng·g -1 ) 2 and for Hg (material selection). These variances correspond to standard deviations that constitute 67, 126 and 59% the regional background levels of these elements in the study region. The overall measurement uncertainty associated with the worst experimental protocol (5 subsamples, refrigerated, washed, 5 × 5 m size of the sampling area and once a year sampling) was between 2 and 6 times higher than that associated with the optimal protocol (30 subsamples, dried, unwashed, 20 × 20 m size of the sampling area and once a week sampling), and between 1.5 and 7 times higher than that associated with the standardized protocol (30 subsamples and once a year sampling). The overall measurement uncertainty associated with the standardized protocol could generate variations of between 14 and 47% in the regional background levels of Cd, Cu, Hg, Pb and Zn in the study area and much higher levels of variation in polluted sampling sites. We demonstrated that although the overall measurement uncertainty of the technique is still high, it can be reduced by using already well defined aspects of the protocol. Further standardization of the protocol together with application of the information on the overall measurement uncertainty would improve the reliability and comparability of the results of different biomonitoring studies, thus extending use of the technique beyond the context of scientific research. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparison of the effect of a CIDR-Select Synch versus a long-term CIDR based AI protocol on reproductive performance in multiparous dairy cows in Swiss dairy farms.

PubMed

Rudolph, Jürn; Bruckmaier, Rupert M; Kasimanickam, Ramanathan; Steiner, Adrian; Kirchhofer, Marc; Hüsler, Jürg; Hirsbrunner, Gaby

2011-11-25

Synchronization programs have become standard in the dairy industry in many countries. In Switzerland, these programs are not routinely used for groups of cows, but predominantly as a therapy for individual problem cows. The objective of this study was to compare the effect of a CIDR-Select Synch and a 12-d CIDR protocol on the pregnancy rate in healthy, multiparous dairy cows in Swiss dairy farms. Cows (N = 508) were randomly assigned to CIDR-Select Synch (N = 262) or 12-d CIDR (N = 246) protocols. Cows in the CIDR-Select Synch group received a CIDR and 2.5 ml of buserelin i.m. on d 0. On d 7, the CIDR insert was removed and 5 ml of dinoprost was administered i.m.. Cows in the 12-d CIDR group received the CIDR on d 0 and it was removed on d 12 (the routine CIDR protocol in Swiss dairies). On d 0 a milk sample for progesterone analysis was taken. Cows were inseminated upon observed estrus. Pregnancy was determined at or more than 35 days after artificial insemination. As a first step, the two groups were compared as to indication for treatment, breed, stud book, stall, pasture, and farmer's business using chi square tests or Fisher's exact test. Furthermore, groups were compared as to age, DIM, number of AI's, number of cows per farm, and yearly milk yield per cow using nonparametric ANOVA. A multiple logistic model was used to relate the success of the protocols to all of the available factors; in particular treatment (CIDR-Select Synch/12-d CIDR), milk progesterone value, age, DIM, previous treatment of the uterus, previous gynecological treatment, and number of preceding inseminations. The pregnancy rate was higher in cows following the CIDR-Select Synch compared to the 12-d CIDR protocol (50.4% vs. 22.4%; P < 0.0001). The CIDR-Select Synch protocol may be highly recommended for multiparous dairy cows. The reduced time span of the progesterone insert decreased the number of days open, improved the pregnancy rate compared to the 12-d CIDR protocol and the cows did not to have to be handled more often.

Comparison of the effect of a CIDR-Select Synch versus a long-term CIDR based AI protocol on reproductive performance in multiparous dairy cows in Swiss dairy farms

PubMed Central

2011-01-01

Background Synchronization programs have become standard in the dairy industry in many countries. In Switzerland, these programs are not routinely used for groups of cows, but predominantly as a therapy for individual problem cows. The objective of this study was to compare the effect of a CIDR-Select Synch and a 12-d CIDR protocol on the pregnancy rate in healthy, multiparous dairy cows in Swiss dairy farms. Methods Cows (N = 508) were randomly assigned to CIDR-Select Synch (N = 262) or 12-d CIDR (N = 246) protocols. Cows in the CIDR-Select Synch group received a CIDR and 2.5 ml of buserelin i.m. on d 0. On d 7, the CIDR insert was removed and 5 ml of dinoprost was administered i.m.. Cows in the 12-d CIDR group received the CIDR on d 0 and it was removed on d 12 (the routine CIDR protocol in Swiss dairies). On d 0 a milk sample for progesterone analysis was taken. Cows were inseminated upon observed estrus. Pregnancy was determined at or more than 35 days after artificial insemination. As a first step, the two groups were compared as to indication for treatment, breed, stud book, stall, pasture, and farmer's business using chi square tests or Fisher's exact test. Furthermore, groups were compared as to age, DIM, number of AI's, number of cows per farm, and yearly milk yield per cow using nonparametric ANOVA. A multiple logistic model was used to relate the success of the protocols to all of the available factors; in particular treatment (CIDR-Select Synch/12-d CIDR), milk progesterone value, age, DIM, previous treatment of the uterus, previous gynecological treatment, and number of preceding inseminations. Results The pregnancy rate was higher in cows following the CIDR-Select Synch compared to the 12-d CIDR protocol (50.4% vs. 22.4%; P < 0.0001). Conclusion The CIDR-Select Synch protocol may be highly recommended for multiparous dairy cows. The reduced time span of the progesterone insert decreased the number of days open, improved the pregnancy rate compared to the 12-d CIDR protocol and the cows did not to have to be handled more often. PMID:22117599

Optimizing larval assessment to support sea lamprey control in the Great Lakes

USGS Publications Warehouse

Hansen, Michael J.; Adams, Jean V.; Cuddy, Douglas W.; Richards, Jessica M.; Fodale, Michael F.; Larson, Geraldine L.; Ollila, Dale J.; Slade, Jeffrey W.; Steeves, Todd B.; Young, Robert J.; Zerrenner, Adam

2003-01-01

Elements of the larval sea lamprey (Petromyzon marinus) assessment program that most strongly influence the chemical treatment program were analyzed, including selection of streams for larval surveys, allocation of sampling effort among stream reaches, allocation of sampling effort among habitat types, estimation of daily growth rates, and estimation of metamorphosis rates, to determine how uncertainty in each element influenced the stream selection program. First, the stream selection model based on current larval assessment sampling protocol significantly underestimated transforming sea lam-prey abundance, transforming sea lampreys killed, and marginal costs per sea lamprey killed, compared to a protocol that included more years of data (especially for large streams). Second, larval density in streams varied significantly with Type-I habitat area, but not with total area or reach length. Third, the ratio of larval density between Type-I and Type-II habitat varied significantly among streams, and that the optimal allocation of sampling effort varied with the proportion of habitat types and variability of larval density within each habitat. Fourth, mean length varied significantly among streams and years. Last, size at metamorphosis varied more among years than within or among regions and that metamorphosis varied significantly among streams within regions. Study results indicate that: (1) the stream selection model should be used to identify streams with potentially high residual populations of larval sea lampreys; (2) larval sampling in Type-II habitat should be initiated in all streams by increasing sampling in Type-II habitat to 50% of the sampling effort in Type-I habitat; and (3) methods should be investigated to reduce uncertainty in estimates of sea lamprey production, with emphasis on those that reduce the uncertainty associated with larval length at the end of the growing season and those used to predict metamorphosis.

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens.

PubMed

Saeidi, Saman; McKain, Michael R; Kellogg, Elizabeth A

2018-03-08

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

An optimised protocol for molecular identification of Eimeria from chickens☆

PubMed Central

Kumar, Saroj; Garg, Rajat; Moftah, Abdalgader; Clark, Emily L.; Macdonald, Sarah E.; Chaudhry, Abdul S.; Sparagano, Olivier; Banerjee, Partha S.; Kundu, Krishnendu; Tomley, Fiona M.; Blake, Damer P.

2014-01-01

Molecular approaches supporting identification of Eimeria parasites infecting chickens have been available for more than 20 years, although they have largely failed to replace traditional measures such as microscopy and pathology. Limitations of microscopy-led diagnostics, including a requirement for specialist parasitological expertise and low sample throughput, are yet to be outweighed by the difficulties associated with accessing genomic DNA from environmental Eimeria samples. A key step towards the use of Eimeria species-specific PCR as a sensitive and reproducible discriminatory tool for use in the field is the production of a standardised protocol that includes sample collection and DNA template preparation, as well as primer selection from the numerous PCR assays now published. Such a protocol will facilitate development of valuable epidemiological datasets which may be easily compared between studies and laboratories. The outcome of an optimisation process undertaken in laboratories in India and the UK is described here, identifying four steps. First, samples were collected into a 2% (w/v) potassium dichromate solution. Second, oocysts were enriched by flotation in saturated saline. Third, genomic DNA was extracted using a QIAamp DNA Stool mini kit protocol including a mechanical homogenisation step. Finally, nested PCR was carried out using previously published primers targeting the internal transcribed spacer region 1 (ITS-1). Alternative methods tested included sample processing in the presence of faecal material, DNA extraction using a traditional phenol/chloroform protocol, the use of SCAR multiplex PCR (one tube and two tube versions) and speciation using the morphometric tool COCCIMORPH for the first time with field samples. PMID:24138724

Discovery of Novel Gene Elements Associated with Prostate Cancer Progression

DTIC Science & Technology

2014-12-01

consent under an Institutional Review Board (IRB) approved protocol at the University of Michigan [SPORE in Prostate Cancer (Tissue/Serum/Urine) Bank IRB...1994-0481]. For the Weill Cornell Medical College patient samples, prostate tissues were collected as part of an IRB- approved protocol at Weill...PCAT-1 or nontargeting short hairpin RNA (shRNA) lentiviral constructs for 48 hours. GFPþ cells were drug -selected using 1 mg/mL puromycin. PCAT-1

Well installation and documentation, and ground-water sampling protocols for the pilot National Water-Quality Assessment Program

USGS Publications Warehouse

Hardy, M.A.; Leahy, P.P.; Alley, W.M.

1989-01-01

Several pilot projects are being conducted as part of the National Water Quality Assessment (NAWQA) Program. The purpose of the pilot program is to test and refine concepts for a proposed full-scale program. Three of the pilot projects are specifically designed to assess groundwater. The purpose of this report is to describe the criteria that are being used in the NAWQA pilot projects for selecting and documenting wells, installing new wells, and sampling wells for different water quality constituents. Guidelines are presented for the selection of wells for sampling. Information needed to accurately document each well includes site characteristics related to the location of the well, land use near the well, and important well construction features. These guidelines ensure the consistency of the information collected and will provide comparable data for interpretive purposes. Guidelines for the installation of wells are presented and include procedures that need to be followed for preparations prior to drilling, the selection of the drilling technique and casing type, the grouting procedure, and the well-development technique. A major component of the protocols is related to water quality sampling. Tasks are identified that need to be completed prior to visiting the site for sampling. Guidelines are presented for purging the well prior t sampling, both in terms of the volume of water pumped and the chemical stability of field parameters. Guidelines are presented concerning sampler selection as related to both inorganic and organic constituents. Documentation needed to describe the measurements and observations related to sampling each well and treating and preserving the samples are also presented. Procedures are presented for the storage and shipping of water samples, equipment cleaning, and quality assurance. Quality assurance guidelines include the description of the general distribution of the various quality assurance samples (blanks, spikes, duplicates, and reference samples) that will be used in the pilot program. (Lantz-PTT)

Monitoring fish distributions along electrofishing segments

USGS Publications Warehouse

Miranda, Leandro E.

2014-01-01

Electrofishing is widely used to monitor fish species composition and relative abundance in streams and lakes. According to standard protocols, multiple segments are selected in a body of water to monitor population relative abundance as the ratio of total catch to total sampling effort. The standard protocol provides an assessment of fish distribution at a macrohabitat scale among segments, but not within segments. An ancillary protocol was developed for assessing fish distribution at a finer scale within electrofishing segments. The ancillary protocol was used to estimate spacing, dispersion, and association of two species along shore segments in two local reservoirs. The added information provided by the ancillary protocol may be useful for assessing fish distribution relative to fish of the same species, to fish of different species, and to environmental or habitat characteristics.

Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments

PubMed Central

Goode, Angela E.; Skepper, Jeremy N.; Thorley, Andrew J.; Seiffert, Joanna M.; Chung, K. Fan; Tetley, Teresa D.; Shaffer, Milo S. P.; Ryan, Mary P.

2015-01-01

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinised. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data. PMID:25606708

A practical guideline for intracranial volume estimation in patients with Alzheimer's disease

PubMed Central

2015-01-01

Background Intracranial volume (ICV) is an important normalization measure used in morphometric analyses to correct for head size in studies of Alzheimer Disease (AD). Inaccurate ICV estimation could introduce bias in the outcome. The current study provides a decision aid in defining protocols for ICV estimation in patients with Alzheimer disease in terms of sampling frequencies that can be optimally used on the volumetric MRI data, and the type of software most suitable for use in estimating the ICV measure. Methods Two groups of 22 subjects are considered, including adult controls (AC) and patients with Alzheimer Disease (AD). Reference measurements were calculated for each subject by manually tracing intracranial cavity by the means of visual inspection. The reliability of reference measurements were assured through intra- and inter- variation analyses. Three publicly well-known software packages (Freesurfer, FSL, and SPM) were examined in their ability to automatically estimate ICV across the groups. Results Analysis of the results supported the significant effect of estimation method, gender, cognitive condition of the subject and the interaction among method and cognitive condition factors in the measured ICV. Results on sub-sampling studies with a 95% confidence showed that in order to keep the accuracy of the interleaved slice sampling protocol above 99%, the sampling period cannot exceed 20 millimeters for AC and 15 millimeters for AD. Freesurfer showed promising estimates for both adult groups. However SPM showed more consistency in its ICV estimation over the different phases of the study. Conclusions This study emphasized the importance in selecting the appropriate protocol, the choice of the sampling period in the manual estimation of ICV and selection of suitable software for the automated estimation of ICV. The current study serves as an initial framework for establishing an appropriate protocol in both manual and automatic ICV estimations with different subject populations. PMID:25953026

Robowell: An automated process for monitoring ground water quality using established sampling protocols

USGS Publications Warehouse

Granato, G.E.; Smith, K.P.

1999-01-01

Robowell is an automated process for monitoring selected ground water quality properties and constituents by pumping a well or multilevel sampler. Robowell was developed and tested to provide a cost-effective monitoring system that meets protocols expected for manual sampling. The process uses commercially available electronics, instrumentation, and hardware, so it can be configured to monitor ground water quality using the equipment, purge protocol, and monitoring well design most appropriate for the monitoring site and the contaminants of interest. A Robowell prototype was installed on a sewage treatment plant infiltration bed that overlies a well-studied unconfined sand and gravel aquifer at the Massachusetts Military Reservation, Cape Cod, Massachusetts, during a time when two distinct plumes of constituents were released. The prototype was operated from May 10 to November 13, 1996, and quality-assurance/quality-control measurements demonstrated that the data obtained by the automated method was equivalent to data obtained by manual sampling methods using the same sampling protocols. Water level, specific conductance, pH, water temperature, dissolved oxygen, and dissolved ammonium were monitored by the prototype as the wells were purged according to U.S Geological Survey (USGS) ground water sampling protocols. Remote access to the data record, via phone modem communications, indicated the arrival of each plume over a few days and the subsequent geochemical reactions over the following weeks. Real-time availability of the monitoring record provided the information needed to initiate manual sampling efforts in response to changes in measured ground water quality, which proved the method and characterized the screened portion of the plume in detail through time. The methods and the case study described are presented to document the process for future use.

An optimised protocol for molecular identification of Eimeria from chickens.

PubMed

Kumar, Saroj; Garg, Rajat; Moftah, Abdalgader; Clark, Emily L; Macdonald, Sarah E; Chaudhry, Abdul S; Sparagano, Olivier; Banerjee, Partha S; Kundu, Krishnendu; Tomley, Fiona M; Blake, Damer P

2014-01-17

Molecular approaches supporting identification of Eimeria parasites infecting chickens have been available for more than 20 years, although they have largely failed to replace traditional measures such as microscopy and pathology. Limitations of microscopy-led diagnostics, including a requirement for specialist parasitological expertise and low sample throughput, are yet to be outweighed by the difficulties associated with accessing genomic DNA from environmental Eimeria samples. A key step towards the use of Eimeria species-specific PCR as a sensitive and reproducible discriminatory tool for use in the field is the production of a standardised protocol that includes sample collection and DNA template preparation, as well as primer selection from the numerous PCR assays now published. Such a protocol will facilitate development of valuable epidemiological datasets which may be easily compared between studies and laboratories. The outcome of an optimisation process undertaken in laboratories in India and the UK is described here, identifying four steps. First, samples were collected into a 2% (w/v) potassium dichromate solution. Second, oocysts were enriched by flotation in saturated saline. Third, genomic DNA was extracted using a QIAamp DNA Stool mini kit protocol including a mechanical homogenisation step. Finally, nested PCR was carried out using previously published primers targeting the internal transcribed spacer region 1 (ITS-1). Alternative methods tested included sample processing in the presence of faecal material, DNA extraction using a traditional phenol/chloroform protocol, the use of SCAR multiplex PCR (one tube and two tube versions) and speciation using the morphometric tool COCCIMORPH for the first time with field samples. Copyright © 2013 Dirk Vulpius The Authors. Published by Elsevier B.V. All rights reserved.

An evaluation of the properties of the variance estimator used by FIA

Treesearch

John P. Brown; James A. Westfall

2012-01-01

The Forest Inventory and Analysis (FIA) program of the U.S. Forest Service currently conducts inventories utilizing the protocols of the national enhanced FIA Program. Due to the permanent locations of the sample plots, the stratification of the population occurs after the selection of sample units, i.e., post-stratification. In situations where the population is of...

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

PubMed Central

Treweek, Jennifer B; Deverman, Benjamin E; Greenbaum, Alon; Lignell, Antti; Xiao, Cheng; Cai, Long; Ladinsky, Mark S; Bjorkman, Pamela J; Fowlkes, Charless C; Gradinaru, Viviana

2016-01-01

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks. PMID:26492141

Optimized probability sampling of study sites to improve generalizability in a multisite intervention trial.

PubMed

Kraschnewski, Jennifer L; Keyserling, Thomas C; Bangdiwala, Shrikant I; Gizlice, Ziya; Garcia, Beverly A; Johnston, Larry F; Gustafson, Alison; Petrovic, Lindsay; Glasgow, Russell E; Samuel-Hodge, Carmen D

2010-01-01

Studies of type 2 translation, the adaption of evidence-based interventions to real-world settings, should include representative study sites and staff to improve external validity. Sites for such studies are, however, often selected by convenience sampling, which limits generalizability. We used an optimized probability sampling protocol to select an unbiased, representative sample of study sites to prepare for a randomized trial of a weight loss intervention. We invited North Carolina health departments within 200 miles of the research center to participate (N = 81). Of the 43 health departments that were eligible, 30 were interested in participating. To select a representative and feasible sample of 6 health departments that met inclusion criteria, we generated all combinations of 6 from the 30 health departments that were eligible and interested. From the subset of combinations that met inclusion criteria, we selected 1 at random. Of 593,775 possible combinations of 6 counties, 15,177 (3%) met inclusion criteria. Sites in the selected subset were similar to all eligible sites in terms of health department characteristics and county demographics. Optimized probability sampling improved generalizability by ensuring an unbiased and representative sample of study sites.

Evaluating Protocols for Porcine Faecal Microbiome Recollection, Storage and DNA Extraction: from the Farm to the Lab.

PubMed

Muiños-Bühl, Anixa; González-Recio, Oscar; Muñoz, María; Óvilo, Cristina; García-Casco, Juan; Fernández, Ana I

2018-06-01

There is a growing interest in understanding the role of the gut microbiome on productive and meat quality-related traits in livestock species in order to develop new useful tools for improving pig production systems and industry. Faecal samples are analysed as a proxy of gut microbiota and here the selection of suitable protocols for faecal sampling and DNA isolation is a critical first step in order to obtain reliable results, even more to compare results obtained from different studies. The aim of the current study was to establish in a cost-effective way, using automated ribosomal intergenic spacer analysis technique, a protocol for porcine faecal sampling and storage at farm and slaughterhouse and to determine the most efficient microbiota DNA isolation kit among those most widely used. Operational Taxonomic Unit profiles were compared from Iberian pig faecal samples collected from rectum or ground, stored with liquid N 2 , room temperature or RNAlater, and processed with QIAamp DNA Stool (Qiagen), PowerFecal DNA Isolation (Mobio) or SpeedTools Tissue DNA extraction (Biotools) commercial kits. The results, focused on prokaryote sampling, based on DNA yield and quality, OTU number and Sørensen similarity Indexes, indicate that the recommended protocol for porcine faecal microbiome sampling at farm should include: the collection from porcine rectum to avoid contamination; the storage in liquid N 2 or even at room temperature, but not in RNAlater; and the isolation of microbiota DNA using PowerFecal DNA Isolation kit. These conditions provide more reliable DNA samples for further microbiome analysis.

Characterisation of a reference site for quantifying uncertainties related to soil sampling.

PubMed

Barbizzi, Sabrina; de Zorzi, Paolo; Belli, Maria; Pati, Alessandra; Sansone, Umberto; Stellato, Luisa; Barbina, Maria; Deluisa, Andrea; Menegon, Sandro; Coletti, Valter

2004-01-01

The paper reports a methodology adopted to face problems related to quality assurance in soil sampling. The SOILSAMP project, funded by the Environmental Protection Agency of Italy (APAT), is aimed at (i) establishing protocols for soil sampling in different environments; (ii) assessing uncertainties associated with different soil sampling methods in order to select the "fit-for-purpose" method; (iii) qualifying, in term of trace elements spatial variability, a reference site for national and international inter-comparison exercises. Preliminary results and considerations are illustrated.

Water quality monitoring protocol for wadeable streams and rivers in the Northern Great Plains Network

USGS Publications Warehouse

Wilson, Marcia H.; Rowe, Barbara L.; Gitzen, Robert A.; Wilson, Stephen K.; Paintner-Green, Kara J.

2014-01-01

As recommended by Oakley et al. (2003), this protocol provides a narrative and the rationale for selection of streams and rivers within the NGPN that will be measured for water quality, including dissolved oxygen, pH, specific conductivity, and temperature. Standard operating procedures (SOPs) that detail the steps to collect, manage, and disseminate the NGPN water quality data are in an accompanying document. The sampling design documented in this protocol may be updated as monitoring information is collected and interpreted, and as refinement of methodologies develop through time. In addition, evaluation of data and refinement of the program may necessitate potential changes of program objectives. Changes to the NGPN water quality protocols and SOPs will be carefully documented in a revision history log.

Environmental DNA as a Tool for Inventory and Monitoring of Aquatic Vertebrates

DTIC Science & Technology

2017-07-01

geomorphic calculations and description of each reach. Methods Channel Surveys We initially selected reaches based on access and visual indicators...WA 99164 I-2 Environmental DNA lab protocol: designing species-specific qPCR assays Species-specific surveys should use quantitative polymerase...to traditional field sampling with respect to sensitivity, detection probabilities, and cost efficiency. Compared to field surveys , eDNA sampling

Integration of GC-MSD and ER-Calux® assay into a single protocol for determining steroid estrogens in environmental samples.

PubMed

Avberšek, Miha; Žegura, Bojana; Filipič, Metka; Heath, Ester

2011-11-01

There are many published studies that use either chemical or biological methods to investigate steroid estrogens in the aquatic environment, but rarer are those that combine both. In this study, gas chromatography with mass selective detection (GC-MSD) and the ER-Calux(®) estrogenicity assay were integrated into a single protocol for simultaneous determination of natural (estrone--E1, 17β-estradiol--E2, estriol--E3) and synthetic (17α-ethinylestradiol--EE2) steroid estrogens concentrations and the total estrogenic potential of environmental samples. For integration purposes, several solvents were investigated and the commonly used dimethyl sulphoxide (DMSO) in the ER-Calux(®) assay was replaced by ethyl acetate, which is more compatible with gas chromatography and enables the same sample to be analysed by both GC-MSD and the ER-Calux(®) assay. The integrated protocol was initially tested using a standard mixture of estrogens. The results for pure standards showed that the estrogenicity calculated on the basis of GC-MSD and the ER-Calux(®) assay exhibited good correlation (r(2)=0.96; α=0.94). The result remained the same when spiked waste water extracts were tested (r(2)=0.92, α=1.02). When applied to real waste water influent and effluent samples the results proved (r(2)=0.93; α=0.99) the applicability of the protocol. The main advantages of this newly developed protocol are simple sample handling for both methods, and reduced material consumption and labour. In addition, it can be applied as either a complete or sequential analysis where the ER-Calux(®) assay is used as a pre-screening method prior to the chemical analysis. Copyright © 2011 Elsevier B.V. All rights reserved.

Determination of hydrazine in drinking water: Development and multivariate optimization of a rapid and simple solid phase microextraction-gas chromatography-triple quadrupole mass spectrometry protocol.

PubMed

Gionfriddo, Emanuela; Naccarato, Attilio; Sindona, Giovanni; Tagarelli, Antonio

2014-07-04

In this work, the capabilities of solid phase microextraction were exploited in a fully optimized SPME-GC-QqQ-MS analytical approach for hydrazine assay. A rapid and easy method was obtained by a simple derivatization reaction with propyl chloroformate and pyridine carried out directly in water samples, followed by automated SPME analysis in the same vial without further sample handling. The affinity of the different derivatized compounds obtained towards five commercially available SPME coatings was evaluated, in order to achieve the best extraction efficiency. GC analyses were carried out using a GC-QqQ-MS instrument in selected reaction monitoring (SRM) acquisition mode which has allowed the achievement of high specificity by selecting appropriate precursor-product ion couples improving the capability in analyte identification. The multivariate approach of experimental design was crucial in order to optimize derivatization reaction, SPME process and tandem mass spectrometry parameters. Accuracy of the proposed protocol, tested at 60, 200 and 800 ng L(-1), provided satisfactory values (114.2%, 83.6% and 98.6%, respectively), whereas precision (RSD%) at the same concentration levels were of 10.9%, 7.9% and 7.7% respectively. Limit of detection and quantification of 4.4 and 8.3 ng L(-1) were obtained. The reliable application of the proposed protocol to real drinking water samples confirmed its capability to be used as analytical tool for routine analyses. Copyright © 2014 Elsevier B.V. All rights reserved.

A HIGHLY SELECTIVE PCR PROTOCOL FOR DETECTING 16S RRNA GENES OF THE GENUS PSEUDOMONAS (SENSU STRICTO) IN ENVIRONMENTAL SAMPLES

EPA Science Inventory

Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation. Specific detection of Pseudomonas species in the environment may ...

Optimization of an extraction protocol for organic matter from soils and sediments using high resolution mass spectrometry: selectivity and biases

NASA Astrophysics Data System (ADS)

Chu, R. K.; Tfaily, M. M.; Tolic, N.; Kyle, J. E.; Robinson, E. R.; Hess, N. J.; Paša-Tolić, L.

2015-12-01

Soil organic matter (SOM) is a complex mixture of above and belowground plant litter and microbial residues, and is a key reservoir for carbon (C) and nutrient biogeochemical cycling in different ecosystems. A limited understanding of the molecular composition of SOM prohibits the ability to routinely decipher chemical processes within soil and predict how terrestrial C fluxes will response to changing climatic conditions. Here, we present that the choice of solvent can be used to selectively extract different compositional fractions from SOM to either target a specific class of compounds or gain a better understanding of the entire composition of the soil sample using 12T Fourier transform ion cyclotron resonance mass spectrometry. Specifically, we found that hexane and chloroform were selective for lipid-like compounds with very low O:C ratios; water was selective for carbohydrates with high O:C ratios; acetonitrile preferentially extracts lignin, condensed structures, and tannin polyphenolic compounds with O:C > 0.5; methanol has higher selectivity towards lignin and lipid compounds characterized with relatively low O:C < 0.5. Hexane, chloroform, methanol, acetonitrile and water increase the number and types of organic molecules extracted from soil for a broader range of chemically diverse soil types. Since each solvent extracts a selective group of compounds, using a suite of solvents with varying polarity for analysis results in more comprehensive representation of the diversity of organic molecules present in soil and a better representation of the whole spectrum of available substrates for microorganisms. Moreover, we have developed a sequential extraction protocol that permits sampling diverse classes of organic compounds while minimizing ionization competition during ESI while increasing sample throughput and decreasing sample volume. This allowed us to hypothesize about possible chemical reactions relating classes of organic molecules that reflect abiotic and biotic processes impacting SOM composition.

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects.

PubMed

Schlipalius, D I; Waldron, J; Carroll, B J; Collins, P J; Ebert, P R

2001-12-01

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability in three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced approximately 50 scoreable polymorphic DNA markers, between individuals of three independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from individual DNA samples that had been combined to create the bulked samples.

Measuring stream temperature with digital data loggers: a user's guide

Treesearch

Jason Dunham; Gwynne Chandler; Bruce Rieman; Don Martin

2005-01-01

Digital data loggers (thermographs) are among the most widespread instruments in use for monitoring physical conditions in aquatic ecosystems. The intent of this protocol is to provide guidelines for selecting and programming data loggers, sampling water temperatures in the field, data screening and analysis, and data archiving.

Detection of Legionella pneumophila on clinical samples and susceptibility assessment by flow cytometry.

PubMed

Faria-Ramos, I; Costa-de-Oliveira, S; Barbosa, J; Cardoso, A; Santos-Antunes, J; Rodrigues, A G; Pina-Vaz, C

2012-12-01

Culture in selective media represents the standard diagnostic method to confirm Legionella pneumophila infection, despite requiring a prolonged incubation period; antigen detection by immunofluorescence (IFS) and molecular techniques are also available, but they do not allow antimicrobial susceptibility evaluation. Our objective was to optimise flow cytometry (FC) protocols for the detection of L. pneumophila in respiratory samples and for susceptibility evaluation to first-line drugs. In order to optimise the FC protocol, a specific monoclonal antibody, conjugated with fluorescein isothiocyanate (FITC), was incubated with type strain L. pneumophila ATCC 33152. The limit of detection was established by analysing serial dilutions of bacterial suspension; specificity was assayed using mixtures of prokaryotic and eukaryotic microorganisms. The optimised FC protocol was used to assess 50 respiratory samples and compared with IFS evaluation. The susceptibility profile to erythromycin, ciprofloxacin and levofloxacin was evaluated by FC using propidium iodide and SYBR Green fluorescent dyes; the results were compared with the Etest afterwards. The optimal specific antibody concentration was 20 μg/ml; 10(2)/ml Legionella organisms were detected by this protocol and no cross-reactions with other microorganisms were detected. The five positive respiratory samples (10 %) determined by IFS were also detected by FC, showing 100 % correlation. After 1 h of incubation at 37 °C with different antimicrobials, SYBR Green staining could discriminate between treated and non-treated cells. A novel flow cytometric approach for the detection of L. pneumophila from clinical samples and susceptibility evaluation is now available, representing an important step forward for the diagnosis of this very relevant agent.

Recommendations for the use of mist nets for inventory and monitoring of bird populations

USGS Publications Warehouse

Ralph, C. John; Dunn, Erica H.; Peach, Will J.; Handel, Colleen M.; Ralph, C. John; Dunn, Erica H.

2004-01-01

We provide recommendations on the best practices for mist netting for the purposes of monitoring population parameters such as abundance and demography. Studies should be carefully thought out before nets are set up, to ensure that sampling design and estimated sample size will allow study objectives to be met. Station location, number of nets, type of nets, net placement, and schedule of operation should be determined by the goals of the particular project, and we provide guidelines for typical mist-net studies. In the absence of study-specific requirements for novel protocols, commonly used protocols should be used to enable comparison of results among studies. Regardless of the equipment, net layout, or netting schedule selected, it is important for all studies that operations be strictly standardized, and a well-written operation protocol will help in attaining this goal. We provide recommendations for data to be collected on captured birds, and emphasize the need for good training of project personnel

Protocol for monitoring metals in Ozark National Scenic Riverways, Missouri: Version 1.0

USGS Publications Warehouse

Schmitt, Christopher J.; Brumbaugh, William G.; Besser, John M.; Hinck, Jo Ellen; Bowles, David E.; Morrison, Lloyd W.; Williams, Michael H.

2008-01-01

The National Park Service is developing a monitoring plan for the Ozark National Scenic Riverways in southeastern Missouri. Because of concerns about the release of lead, zinc, and other metals from lead-zinc mining to streams, the monitoring plan will include mining-related metals. After considering a variety of alternatives, the plan will consist of measuring the concentrations of cadmium, cobalt, lead, nickel, and zinc in composite samples of crayfish (Orconectes luteus or alternate species) and Asian clam (Corbicula fluminea) collected periodically from selected sites. This document, which comprises a protocol narrative and supporting standard operating procedures, describes the methods to be employed prior to, during, and after collection of the organisms, along with procedures for their chemical analysis and quality assurance; statistical analysis, interpretation, and reporting of the data; and for modifying the protocol narrative and supporting standard operating procedures. A list of supplies and equipment, data forms, and sample labels are also included. An example based on data from a pilot study is presented.

Establishment of a protocol for the gene expression analysis of laser microdissected rat kidney samples with affymetrix genechips

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

Stemmer, Kerstin; Ellinger-Ziegelbauer, Heidrun; Lotz, Kerstin

2006-11-15

Laser microdissection in conjunction with microarray technology allows selective isolation and analysis of specific cell populations, e.g., preneoplastic renal lesions. To date, only limited information is available on sample preparation and preservation techniques that result in both optimal histomorphological preservation of sections and high-quality RNA for microarray analysis. Furthermore, amplification of minute amounts of RNA from microdissected renal samples allowing analysis with genechips has only scantily been addressed to date. The objective of this study was therefore to establish a reliable and reproducible protocol for laser microdissection in conjunction with microarray technology using kidney tissue from Eker rats p.o. treatedmore » for 7 days and 6 months with 10 and 1 mg Aristolochic acid/kg bw, respectively. Kidney tissues were preserved in RNAlater or snap frozen. Cryosections were cut and stained with either H and E or cresyl violet for subsequent morphological and RNA quality assessment and laser microdissection. RNA quality was comparable in snap frozen and RNAlater-preserved samples, however, the histomorphological preservation of renal sections was much better following cryopreservation. Moreover, the different staining techniques in combination with sample processing time at room temperature can have an influence on RNA quality. Different RNA amplification protocols were shown to have an impact on gene expression profiles as demonstrated with Affymetrix Rat Genome 230{sub 2}.0 arrays. Considering all the parameters analyzed in this study, a protocol for RNA isolation from laser microdissected samples with subsequent Affymetrix chip hybridization was established that was also successfully applied to preneoplastic lesions laser microdissected from Aristolochic acid-treated rats.« less

Automated selected reaction monitoring data analysis workflow for large-scale targeted proteomic studies.

PubMed

Surinova, Silvia; Hüttenhain, Ruth; Chang, Ching-Yun; Espona, Lucia; Vitek, Olga; Aebersold, Ruedi

2013-08-01

Targeted proteomics based on selected reaction monitoring (SRM) mass spectrometry is commonly used for accurate and reproducible quantification of protein analytes in complex biological mixtures. Strictly hypothesis-driven, SRM assays quantify each targeted protein by collecting measurements on its peptide fragment ions, called transitions. To achieve sensitive and accurate quantitative results, experimental design and data analysis must consistently account for the variability of the quantified transitions. This consistency is especially important in large experiments, which increasingly require profiling up to hundreds of proteins over hundreds of samples. Here we describe a robust and automated workflow for the analysis of large quantitative SRM data sets that integrates data processing, statistical protein identification and quantification, and dissemination of the results. The integrated workflow combines three software tools: mProphet for peptide identification via probabilistic scoring; SRMstats for protein significance analysis with linear mixed-effect models; and PASSEL, a public repository for storage, retrieval and query of SRM data. The input requirements for the protocol are files with SRM traces in mzXML format, and a file with a list of transitions in a text tab-separated format. The protocol is especially suited for data with heavy isotope-labeled peptide internal standards. We demonstrate the protocol on a clinical data set in which the abundances of 35 biomarker candidates were profiled in 83 blood plasma samples of subjects with ovarian cancer or benign ovarian tumors. The time frame to realize the protocol is 1-2 weeks, depending on the number of replicates used in the experiment.

Measurement of DNA damage in rat urinary bladder transitional cells: improved selective harvest of transitional cells and detailed Comet assay protocols.

PubMed

Wang, Amy; Robertson, John L; Holladay, Steven D; Tennant, Alan H; Lengi, Andrea J; Ahmed, S Ansar; Huckle, William R; Kligerman, Andrew D

2007-12-01

Urinary bladder transitional epithelium is the main site of bladder cancer, and the use of transitional cells to study carcinogenesis/genotoxicity is recommended over the use of whole bladders. Because the transitional epithelium is only a small fraction of the whole bladder, the alkaline single cell gel electrophoresis assay (Comet assay), which requires only a small number of cells per sample, is especially suitable for measuring DNA damage in transitional cells. However, existed procedures of cell collection did not yield transitional cells with a high purity, and pooling of samples was needed for Comet assay. The goal of this study was to develop an optimized protocol to evaluate DNA damage in the urinary bladder transitional epithelium. This was achieved by an enzymatic stripping method (trypsin-EDTA incubation plus gentle scraping) to selectively harvest transitional cells from rat bladders, and the use of the alkaline Comet assay to detect DNA strand breaks, alkaline labile sites, and DNA-protein crosslinks. Step by step procedures are reported here. Cells collected from a single rat bladder were sufficient for multiple Comet assays. With this new protocol, increases in DNA damage were detected in transitional cells after in vitro exposure to the positive control agents, hydrogen peroxide or formaldehyde. Repair of the induced DNA damage occurred within 4h. This indicated the capacity for DNA repair was maintained in the harvested cells. The new protocol provides a simple and inexpensive method to detect various types of DNA damage and to measure DNA damage repair in urinary bladder transitional cells.

Technical standards and guidelines: prenatal screening for Down syndrome that includes first-trimester biochemistry and/or ultrasound measurements.

PubMed

Palomaki, Glenn E; Lee, Jo Ellen S; Canick, Jacob A; McDowell, Geraldine A; Donnenfeld, Alan E

2009-09-01

This statement is intended to augment the current general ACMG Standards and Guidelines for Clinical Genetics Laboratories and to address guidelines specific to first-trimester screening for Down syndrome. The aim is to provide the laboratory the necessary information to ensure accurate and reliable Down syndrome screening results given a screening protocol (e.g., combined first trimester and integrated testing). Information about various test combinations and their expected performance are provided, but other issues such as availability of reagents, patient interest in early test results, access to open neural tube defect screening, and availability of chorionic villus sampling are all contextual factors in deciding which screening protocol(s) will be selected by individual health care providers. Individual laboratories are responsible for meeting the quality assurance standards described by the Clinical Laboratory Improvement Act, the College of American Pathologists, and other regulatory agencies, with respect to appropriate sample documentation, assay validation, general proficiency, and quality control measures. These guidelines address first-trimester screening that includes ultrasound measurement and interpretation of nuchal translucency thickness and protocols that combine markers from both the first and second trimesters. Laboratories can use their professional judgment to make modification or additions.

Fully Automated Sample Preparation for Ultrafast N-Glycosylation Analysis of Antibody Therapeutics.

PubMed

Szigeti, Marton; Lew, Clarence; Roby, Keith; Guttman, Andras

2016-04-01

There is a growing demand in the biopharmaceutical industry for high-throughput, large-scale N-glycosylation profiling of therapeutic antibodies in all phases of product development, but especially during clone selection when hundreds of samples should be analyzed in a short period of time to assure their glycosylation-based biological activity. Our group has recently developed a magnetic bead-based protocol for N-glycosylation analysis of glycoproteins to alleviate the hard-to-automate centrifugation and vacuum-centrifugation steps of the currently used protocols. Glycan release, fluorophore labeling, and cleanup were all optimized, resulting in a <4 h magnetic bead-based process with excellent yield and good repeatability. This article demonstrates the next level of this work by automating all steps of the optimized magnetic bead-based protocol from endoglycosidase digestion, through fluorophore labeling and cleanup with high-throughput sample processing in 96-well plate format, using an automated laboratory workstation. Capillary electrophoresis analysis of the fluorophore-labeled glycans was also optimized for rapid (<3 min) separation to accommodate the high-throughput processing of the automated sample preparation workflow. Ultrafast N-glycosylation analyses of several commercially relevant antibody therapeutics are also shown and compared to their biosimilar counterparts, addressing the biological significance of the differences. © 2015 Society for Laboratory Automation and Screening.

Efficiency in nonequilibrium molecular dynamics Monte Carlo simulations

DOE PAGES

Radak, Brian K.; Roux, Benoît

2016-10-07

Hybrid algorithms combining nonequilibrium molecular dynamics and Monte Carlo (neMD/MC) offer a powerful avenue for improving the sampling efficiency of computer simulations of complex systems. These neMD/MC algorithms are also increasingly finding use in applications where conventional approaches are impractical, such as constant-pH simulations with explicit solvent. However, selecting an optimal nonequilibrium protocol for maximum efficiency often represents a non-trivial challenge. This work evaluates the efficiency of a broad class of neMD/MC algorithms and protocols within the theoretical framework of linear response theory. The approximations are validated against constant pH-MD simulations and shown to provide accurate predictions of neMD/MC performance.more » An assessment of a large set of protocols confirms (both theoretically and empirically) that a linear work protocol gives the best neMD/MC performance. Lastly, a well-defined criterion for optimizing the time parameters of the protocol is proposed and demonstrated with an adaptive algorithm that improves the performance on-the-fly with minimal cost.« less

Detection of Salmonella sp in chicken cuts using immunomagnetic separation

PubMed Central

de Cássia dos Santos da Conceição, Rita; Moreira, Ângela Nunes; Ramos, Roberta Juliano; Goularte, Fabiana Lemos; Carvalhal, José Beiro; Aleixo, José Antonio Guimarães

2008-01-01

The immunomagnetic separation (IMS) is a technique that has been used to increase sensitivity and specificity and to decrease the time required for detection of Salmonella in foods through different methodologies. In this work we report on the development of a method for detection of Salmonella in chicken cuts using in house antibody-sensitized microspheres associated to conventional plating in selective agar (IMS-plating). First, protein A-coated microspheres were sensitized with polyclonal antibodies against lipopolysacharide and flagella from salmonellae and used to standardize a procedure for capturing Salmonella Enteritidis from pure cultures and detection in selective agar. Subsequently, samples of chicken meat experimentally contaminated with S. Enteritidis were analyzed immediately after contamination and after 24h of refrigeration using three enrichment protocols. The detection limit of the IMS-plating procedure after standardization with pure culture was about 2x10 CFU/mL. The protocol using non-selective enrichment for 6-8h, selective enrichment for 16-18h and a post-enrichment for 4h gave the best results of S. Enteritidis detection by IMS-plating in experimentally contaminated meat. IMS-plating using this protocol was compared to the standard culture method for salmonellae detection in naturally contaminated chicken cuts and yielded 100% sensitivity and 94% specificity. The method developed using in house prepared magnetic microespheres for IMS and plating in selective agar was able to diminish by at least one day the time required for detection of Salmonella in chicken products by the conventional culture method. PMID:24031199

What are the keys to successful adrenal venous sampling (AVS) in patients with primary aldosteronism?

PubMed

Young, William F; Stanson, Anthony W

2009-01-01

Adrenal venous sampling (AVS) is the criterion standard to distinguish between unilateral and bilateral adrenal disease in patients with primary aldosteronism. The keys to successful AVS include appropriate patient selection, careful patient preparation, focused technical expertise, defined protocol, and accurate data interpretation. The use of AVS should be based on patient preferences, patient age, clinical comorbidities, and the clinical probability of finding an aldosterone-producing adenoma. AVS is optimally performed in the fasting state in the morning. AVS is an intricate procedure because the right adrenal vein is small and may be difficult to locate - the success rate depends on the proficiency of the angiographer. The key factors that determine the successful catheterization of both adrenal veins are experience, dedication and repetition. With experience, and focusing the expertise to 1 or 2 radiologists at a referral centre, the AVS success rate can be as high as 96%. A centre-specific, written protocol is mandatory. The protocol should be developed by an interested group of endocrinologists, radiologists and laboratory personnel. Safeguards should be in place to prevent mislabelling of the blood tubes in the radiology suite and to prevent sample mix-up in the laboratory.

An Assessment of Intervention Fidelity in Published Social Work Intervention Research Studies

ERIC Educational Resources Information Center

Corley, Nicole A.; Kim, Irang

2016-01-01

Objectives: Intervention fidelity is a critical strategy to help advance the usefulness and integrity of social work research. This study assessed the extent to which a selected sample of published social work intervention researchers reported its intervention protocols. Methods: Six core social work journals were reviewed in this analysis. The…

Modeling and Analysis of Asynchronous Systems Using SAL and Hybrid SAL

NASA Technical Reports Server (NTRS)

Tiwari, Ashish; Dutertre, Bruno

2013-01-01

We present formal models and results of formal analysis of two different asynchronous systems. We first examine a mid-value select module that merges the signals coming from three different sensors that are each asynchronously sampling the same input signal. We then consider the phase locking protocol proposed by Daly, Hopkins, and McKenna. This protocol is designed to keep a set of non-faulty (asynchronous) clocks phase locked even in the presence of Byzantine-faulty clocks on the network. All models and verifications have been developed using the SAL model checking tools and the Hybrid SAL abstractor.

Highlighting the complexities of a groundwater pilot study during an avian influenza outbreak: Methods, lessons learned, and select contaminant results.

PubMed

Hubbard, Laura E; Kolpin, Dana W; Fields, Chad L; Hladik, Michelle L; Iwanowicz, Luke R

2017-10-01

The highly pathogenic avian influenza (H5N2) outbreak in the Midwestern United States (US) in 2015 was historic due to the number of birds and poultry operations impacted and the corresponding economic loss to the poultry industry and was the largest animal health emergency in US history. The U.S. Geological Survey (USGS), with the assistance of several state and federal agencies, aided the response to the outbreak by developing a study to determine the extent of virus transport in the environment. The study goals were to: develop the appropriate sampling methods and protocols for measuring avian influenza virus (AIV) in groundwater, provide the first baseline data on AIV and outbreak- and poultry-related contaminant occurrence and movement into groundwater, and document climatological factors that may have affected both survival and transport of AIV to groundwater during the months of the 2015 outbreak. While site selection was expedient, there were often delays in sample response times due to both relationship building between agencies, groups, and producers and logistical time constraints. This study's design and sampling process highlights the unpredictable nature of disease outbreaks and the corresponding difficulty in environmental sampling of such events. The lessons learned, including field protocols and approaches, can be used to improve future research on AIV in the environment. Published by Elsevier Inc.

Highlighting the complexities of a groundwater pilot study during an avian influenza outbreak: Methods, lessons learned, and select contaminant results

USGS Publications Warehouse

Hubbard, Laura E.; Kolpin, Dana W.; Fields, Chad L.; Hladik, Michelle L.; Iwanowicz, Luke R.

2017-01-01

The highly pathogenic avian influenza (H5N2) outbreak in the Midwestern United States (US) in 2015 was historic due to the number of birds and poultry operations impacted and the corresponding economic loss to the poultry industry and was the largest animal health emergency in US history. The U.S. Geological Survey (USGS), with the assistance of several state and federal agencies, aided the response to the outbreak by developing a study to determine the extent of virus transport in the environment. The study goals were to: develop the appropriate sampling methods and protocols for measuring avian influenza virus (AIV) in groundwater, provide the first baseline data on AIV and outbreak- and poultry-related contaminant occurrence and movement into groundwater, and document climatological factors that may have affected both survival and transport of AIV to groundwater during the months of the 2015 outbreak. While site selection was expedient, there were often delays in sample response times due to both relationship building between agencies, groups, and producers and logistical time constraints. This study's design and sampling process highlights the unpredictable nature of disease outbreaks and the corresponding difficulty in environmental sampling of such events. The lessons learned, including field protocols and approaches, can be used to improve future research on AIV in the environment.

Neighborhood sampling: how many streets must an auditor walk?

PubMed

McMillan, Tracy E; Cubbin, Catherine; Parmenter, Barbara; Medina, Ashley V; Lee, Rebecca E

2010-03-12

This study tested the representativeness of four street segment sampling protocols using the Pedestrian Environment Data Scan (PEDS) in eleven neighborhoods surrounding public housing developments in Houston, TX. The following four street segment sampling protocols were used (1) all segments, both residential and arterial, contained within the 400 meter radius buffer from the center point of the housing development (the core) were compared with all segments contained between the 400 meter radius buffer and the 800 meter radius buffer (the ring); all residential segments in the core were compared with (2) 75% (3) 50% and (4) 25% samples of randomly selected residential street segments in the core. Analyses were conducted on five key variables: sidewalk presence; ratings of attractiveness and safety for walking; connectivity; and number of traffic lanes. Some differences were found when comparing all street segments, both residential and arterial, in the core to the ring. Findings suggested that sampling 25% of residential street segments within the 400 m radius of a residence sufficiently represents the pedestrian built environment. Conclusions support more cost effective environmental data collection for physical activity research.

Neighborhood sampling: how many streets must an auditor walk?

PubMed Central

2010-01-01

This study tested the representativeness of four street segment sampling protocols using the Pedestrian Environment Data Scan (PEDS) in eleven neighborhoods surrounding public housing developments in Houston, TX. The following four street segment sampling protocols were used (1) all segments, both residential and arterial, contained within the 400 meter radius buffer from the center point of the housing development (the core) were compared with all segments contained between the 400 meter radius buffer and the 800 meter radius buffer (the ring); all residential segments in the core were compared with (2) 75% (3) 50% and (4) 25% samples of randomly selected residential street segments in the core. Analyses were conducted on five key variables: sidewalk presence; ratings of attractiveness and safety for walking; connectivity; and number of traffic lanes. Some differences were found when comparing all street segments, both residential and arterial, in the core to the ring. Findings suggested that sampling 25% of residential street segments within the 400 m radius of a residence sufficiently represents the pedestrian built environment. Conclusions support more cost effective environmental data collection for physical activity research. PMID:20226052

Nationwide cross-sectional survey of schistosomiasis and soil-transmitted helminthiasis in Sudan: study protocol.

PubMed

Cha, Seungman; Hong, Sung-Tae; Lee, Young-Ha; Lee, Keon Hoon; Cho, Dae Seong; Lee, Jinmoo; Chai, Jong-Yil; Elhag, Mousab Siddig; Khaled, Soheir Gabralla Ahmad; Elnimeiri, Mustafa Khidir Mustafa; Siddig, Nahid Abdelgadeir Ali; Abdelrazig, Hana; Awadelkareem, Sarah; Elshafie, Azza Tag Eldin; Ismail, Hassan Ahmed Hassan Ahmed; Amin, Mutamad

2017-09-12

Schistosomiasis and soil-transmitted helminthiasis (STHs) are target neglected tropical diseases (NTDs) of preventive chemotherapy, but the control and elimination of these diseases have been impeded due to resource constraints. Few reports have described study protocol to draw on when conducting a nationwide survey. We present a detailed methodological description of the integrated mapping of schistosomiasis and STHs on the basis of our experiences, hoping that this protocol can be applied to future surveys in similar settings. In addition to determining the ecological zones requiring mass drug administration interventions, we aim to provide precise estimates of the prevalence of these diseases. A school-based cross-sectional design will be applied for the nationwide survey across Sudan. The survey is designed to cover all districts in every state. We have divided each district into 3 different ecological zones depending on proximity to bodies of water. We will employ a probability-proportional-to-size sampling method for schools and systematic sampling for student selection to provide adequate data regarding the prevalence for schistosomiasis and STHs in Sudan at the state level. A total of 108,660 students will be selected from 1811 schools across Sudan. After the survey is completed, 391 ecological zones will be mapped out. To carry out the survey, 655 staff members were recruited. The feces and urine samples are microscopically examined by the Kato-Katz method and the sediment smears for helminth eggs respectively. For quality control, a minimum of 10% of the slides will be rechecked by the federal supervisors in each state and also 5% of the smears are validated again within one day by independent supervisors. This nationwide mapping is expected to generate important epidemiological information and indicators about schistosomiasis and STHs that will be useful for monitoring and evaluating the control program. The mapping data will also be used for overviewing the status and policy formulation and updates to the control strategies. This paper, which describes a feasible and practical study protocol, is to be shared with the global health community, especially those who are planning to perform nationwide mapping of NTDs by feces or urine sampling.

Development of a new protocol for rapid bacterial identification and susceptibility testing directly from urine samples.

PubMed

Zboromyrska, Y; Rubio, E; Alejo, I; Vergara, A; Mons, A; Campo, I; Bosch, J; Marco, F; Vila, J

2016-06-01

The current gold standard method for the diagnosis of urinary tract infections (UTI) is urine culture that requires 18-48 h for the identification of the causative microorganisms and an additional 24 h until the results of antimicrobial susceptibility testing (AST) are available. The aim of this study was to shorten the time of urine sample processing by a combination of flow cytometry for screening and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for bacterial identification followed by AST directly from urine. The study was divided into two parts. During the first part, 675 urine samples were processed by a flow cytometry device and a cut-off value of bacterial count was determined to select samples for direct identification by MALDI-TOF-MS at ≥5 × 10(6) bacteria/mL. During the second part, 163 of 1029 processed samples reached the cut-off value. The sample preparation protocol for direct identification included two centrifugation and two washing steps. Direct AST was performed by the disc diffusion method if a reliable direct identification was obtained. Direct MALDI-TOF-MS identification was performed in 140 urine samples; 125 of the samples were positive by urine culture, 12 were contaminated and 3 were negative. Reliable direct identification was obtained in 108 (86.4%) of the 125 positive samples. AST was performed in 102 identified samples, and the results were fully concordant with the routine method among 83 monomicrobial infections. In conclusion, the turnaround time of the protocol described to diagnose UTI was about 1 h for microbial identification and 18-24 h for AST. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Academic Performance Enhancement: A Qualitative Study of the Perceptions and Habits of Prescription Stimulant-Using College Students

ERIC Educational Resources Information Center

Aikins, Ross D.

2011-01-01

This study explores the developmental implications of cognitive enhancement among college students. Data comes from interviews with a purposive sample of licit and illicit users of stimulant medications at a highly selective West Coast University. A semistructured protocol revolved around students' habits and perceptions relating to stimulant…

Cross-Validation of a PACER Prediction Equation for Assessing Aerobic Capacity in Hungarian Youth

ERIC Educational Resources Information Center

Saint-Maurice, Pedro F.; Welk, Gregory J.; Finn, Kevin J.; Kaj, Mónika

2015-01-01

Purpose: The purpose of this article was to evaluate the validity of the Progressive Aerobic Cardiovascular and Endurance Run (PACER) test in a sample of Hungarian youth. Method: Approximately 500 participants (aged 10-18 years old) were randomly selected across Hungary to complete both laboratory (maximal treadmill protocol) and field assessments…

Rapid microfluidic analysis of a Y-STR multiplex for screening of forensic samples.

PubMed

Gibson-Daw, Georgiana; Albani, Patricia; Gassmann, Marcus; McCord, Bruce

2017-02-01

In this paper, we demonstrate a rapid analysis procedure for use with a small set of rapidly mutating Y chromosomal short tandem repeat (Y-STR) loci that combines both rapid polymerase chain reaction (PCR) and microfluidic separation elements. The procedure involves a high-speed polymerase and a rapid cycling protocol to permit PCR amplification in 16 min. The resultant amplified sample is next analysed using a short 1.8-cm microfluidic electrophoresis system that permits a four-locus Y-STR genotype to be produced in 80 s. The entire procedure takes less than 25 min from sample collection to result. This paper describes the rapid amplification protocol as well as studies of the reproducibility and sensitivity of the procedure and its optimisation. The amplification process utilises a small high-speed thermocycler, microfluidic device and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The four loci used for the multiplex were selected due to their rapid mutation rates and should proved useful in preliminary screening of samples and suspects. Overall, this technique provides a method for rapid sample screening of suspect and crime scene samples in forensic casework. Graphical abstract ᅟ.

Assessment of Sample Preparation Bias in Mass Spectrometry-Based Proteomics.

PubMed

Klont, Frank; Bras, Linda; Wolters, Justina C; Ongay, Sara; Bischoff, Rainer; Halmos, Gyorgy B; Horvatovich, Péter

2018-04-17

For mass spectrometry-based proteomics, the selected sample preparation strategy is a key determinant for information that will be obtained. However, the corresponding selection is often not based on a fit-for-purpose evaluation. Here we report a comparison of in-gel (IGD), in-solution (ISD), on-filter (OFD), and on-pellet digestion (OPD) workflows on the basis of targeted (QconCAT-multiple reaction monitoring (MRM) method for mitochondrial proteins) and discovery proteomics (data-dependent acquisition, DDA) analyses using three different human head and neck tissues (i.e., nasal polyps, parotid gland, and palatine tonsils). Our study reveals differences between the sample preparation methods, for example, with respect to protein and peptide losses, quantification variability, protocol-induced methionine oxidation, and asparagine/glutamine deamidation as well as identification of cysteine-containing peptides. However, none of the methods performed best for all types of tissues, which argues against the existence of a universal sample preparation method for proteome analysis.

Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples.

PubMed

Kresse, Stine H; Namløs, Heidi M; Lorenz, Susanne; Berner, Jeanne-Marie; Myklebost, Ola; Bjerkehagen, Bodil; Meza-Zepeda, Leonardo A

2018-01-01

Nucleic acid material of adequate quality is crucial for successful high-throughput sequencing (HTS) analysis. DNA and RNA isolated from archival FFPE material are frequently degraded and not readily amplifiable due to chemical damage introduced during fixation. To identify optimal nucleic acid extraction kits, DNA and RNA quantity, quality and performance in HTS applications were evaluated. DNA and RNA were isolated from five sarcoma archival FFPE blocks, using eight extraction protocols from seven kits from three different commercial vendors. For DNA extraction, the truXTRAC FFPE DNA kit from Covaris gave higher yields and better amplifiable DNA, but all protocols gave comparable HTS library yields using Agilent SureSelect XT and performed well in downstream variant calling. For RNA extraction, all protocols gave comparable yields and amplifiable RNA. However, for fusion gene detection using the Archer FusionPlex Sarcoma Assay, the truXTRAC FFPE RNA kit from Covaris and Agencourt FormaPure kit from Beckman Coulter showed the highest percentage of unique read-pairs, providing higher complexity of HTS data and more frequent detection of recurrent fusion genes. truXTRAC simultaneous DNA and RNA extraction gave similar outputs as individual protocols. These findings show that although successful HTS libraries could be generated in most cases, the different protocols gave variable quantity and quality for FFPE nucleic acid extraction. Selecting the optimal procedure is highly valuable and may generate results in borderline quality specimens.

Molecular discrimination of Echinococcus granulosus and Echinococcus multilocularis by sequencing and a new PCR-RFLP method with the potential use for other Echinococcus species.

PubMed

Şakalar, Çağrı; Kuk, Salih; Erensoy, Ahmet; Dağli, Adile Ferda; Özercan, İbrahim Hanifi; Çetınkaya, Ülfet; Yazar, Süleyman

2014-01-01

To develop a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial genomic marker region was amplified and sequenced using a novel primer pair and a new PCR-RFLP protocol was developed for the detection and discrimination of E. granulosus and E. multilocularis using a set of restriction enzymes including AccI, MboI, MboII, and TsoI. The selected marker region was amplified using DNA isolated from FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis and the discrimination of E. granulosus and E. multilocularis was accomplished by use of the novel PCR-RFLP method. In this PCR-RFLP protocol, use of any single restriction enzyme is enough for the discrimination of E. granulosus and E. multilocularis. The PCR-RFLP protocol can be potentially used for the discrimination of 5 other Echinococcus species: E. oligarthus, E. shiquicus, E. ortleppi, E. canadensis, and E. vogeli.

Development of a spatial sampling protocol using GIS to measure health disparities in Bobo-Dioulasso, Burkina Faso, a medium-sized African city.

PubMed

Kassié, Daouda; Roudot, Anna; Dessay, Nadine; Piermay, Jean-Luc; Salem, Gérard; Fournet, Florence

2017-04-18

Many cities in developing countries experience an unplanned and rapid growth. Several studies have shown that the irregular urbanization and equipment of cities produce different health risks and uneven exposure to specific diseases. Consequently, health surveys within cities should be carried out at the micro-local scale and sampling methods should try to capture this urban diversity. This article describes the methodology used to develop a multi-stage sampling protocol to select a population for a demographic survey that investigates health disparities in the medium-sized city of Bobo-Dioulasso, Burkina Faso. It is based on the characterization of Bobo-Dioulasso city typology by taking into account the city heterogeneity, as determined by analysis of the built environment and of the distribution of urban infrastructures, such as healthcare structures or even water fountains, by photo-interpretation of aerial photographs and satellite images. Principal component analysis and hierarchical ascendant classification were then used to generate the city typology. Five groups of spaces with specific profiles were identified according to a set of variables which could be considered as proxy indicators of health status. Within these five groups, four sub-spaces were randomly selected for the study. We were then able to survey 1045 households in all the selected sub-spaces. The pertinence of this approach is discussed regarding to classical sampling as random walk method for example. This urban space typology allowed to select a population living in areas representative of the uneven urbanization process, and to characterize its health status in regards to several indicators (nutritional status, communicable and non-communicable diseases, and anaemia). Although this method should be validated and compared with more established methods, it appears as an alternative in developing countries where geographic and population data are scarce.

Insights into biodiversity sampling strategies for freshwater microinvertebrate faunas through bioblitz campaigns and DNA barcoding.

PubMed

Laforest, Brandon J; Winegardner, Amanda K; Zaheer, Omar A; Jeffery, Nicholas W; Boyle, Elizabeth E; Adamowicz, Sarah J

2013-04-04

Biodiversity surveys have long depended on traditional methods of taxonomy to inform sampling protocols and to determine when a representative sample of a given species pool of interest has been obtained. Questions remain as to how to design appropriate sampling efforts to accurately estimate total biodiversity. Here we consider the biodiversity of freshwater ostracods (crustacean class Ostracoda) from the region of Churchill, Manitoba, Canada. Through an analysis of observed species richness and complementarity, accumulation curves, and richness estimators, we conduct an a posteriori analysis of five bioblitz-style collection strategies that differed in terms of total duration, number of sites, protocol flexibility to heterogeneous habitats, sorting of specimens for analysis, and primary purpose of collection. We used DNA barcoding to group specimens into molecular operational taxonomic units for comparison. Forty-eight provisional species were identified through genetic divergences, up from the 30 species previously known and documented in literature from the Churchill region. We found differential sampling efficiency among the five strategies, with liberal sorting of specimens for molecular analysis, protocol flexibility (and particularly a focus on covering diverse microhabitats), and a taxon-specific focus to collection having strong influences on garnering more accurate species richness estimates. Our findings have implications for the successful design of future biodiversity surveys and citizen-science collection projects, which are becoming increasingly popular and have been shown to produce reliable results for a variety of taxa despite relying on largely untrained collectors. We propose that efficiency of biodiversity surveys can be increased by non-experts deliberately selecting diverse microhabitats; by conducting two rounds of molecular analysis, with the numbers of samples processed during round two informed by the singleton prevalence during round one; and by having sub-teams (even if all non-experts) focus on select taxa. Our study also provides new insights into subarctic diversity of freshwater Ostracoda and contributes to the broader "Barcoding Biotas" campaign at Churchill. Finally, we comment on the associated implications and future research directions for community ecology analyses and biodiversity surveys through DNA barcoding, which we show here to be an efficient technique enabling rapid biodiversity quantification in understudied taxa.

Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

PubMed

Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

2017-01-01

Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

The resident microflora of voided midstream urine of healthy controls: standard versus expanded urine culture protocols.

PubMed

Coorevits, L; Heytens, S; Boelens, J; Claeys, G

2017-04-01

The workup and interpretation of urine cultures is not always clear-cut, especially for midstream samples contaminated with commensals. Standard urine culture (SUC) protocols are designed in favor of growth of uropathogens at the expense of commensals. In selected clinical situations, however, it is essential to trace fastidious or new uropathogens by expanding the urine culture conditions (EUC). The aim of our study was to map the microflora in midstream urine specimens from healthy controls by means of EUC, in view of the interpretation of bacterial culture results in symptomatic patients. Midstream urine specimens from 101 healthy controls (86 females and 15 males) were examined using both SUC and EUC. Whilst 73 % of samples examined by SUC showed no growth at 10 3  colony-forming units (CFU)/mL, 91 % of samples examined by EUC grew bacterial species in large numbers (≥10 4  CFU/mL). Asymptomatic bacteriuria, as defined by the European guidelines for urinalysis, was detected in six samples with both protocols. EUC revealed 98 different species, mostly Lactobacillus, Staphylococcus, Streptococcus, and Corynebacterium. None of the samples grew Staphylococcus saprophyticus, Corynebacterium urealyticum, or Aerococcus urinae. Samples from females contained higher bacterial loads and showed higher bacterial diversity compared to males. Midstream urine of healthy controls contains large communities of living bacteria that comprise a resident microflora, only revealed by EUC. Hence, the use of EUC instead of SUC in a routine setting would result in more sensitive but less specific results, requiring critical interpretation. In our view, EUC should be reserved for limited indications.

Returning Individual Research Results: Development of a Cancer Genetics Education and Risk Communication Protocol

PubMed Central

Roberts, J. Scott; Shalowitz, David I.; Christensen, Kurt D.; Everett, Jessica N.; Kim, Scott Y. H.; Raskin, Leon; Gruber, Stephen B.

2011-01-01

The obligations of researchers to disclose clinically and/or personally significant individual research results are highly debated, but few empirical studies have addressed this topic. We describe the development of a protocol for returning research results to participants at one site of a multicenter study of the genetic epidemiology of melanoma. Protocol development involved numerous challenges: (1) deciding whether genotype results merited disclosure; (2) achieving an appropriate format for communicating results; (3) developing education materials; (4) deciding whether to retest samples for additional laboratory validation; (5) identifying and notifying selected participants; and (6) assessing the impact of disclosure. Our experience suggests potential obstacles depending on researcher resources and the design of the parent study, but offers a process by which researchers can responsibly return individual study results and evaluate the impact of disclosure. PMID:20831418

Simple and Sensitive Paper-Based Device Coupling Electrochemical Sample Pretreatment and Colorimetric Detection.

PubMed

Silva, Thalita G; de Araujo, William R; Muñoz, Rodrigo A A; Richter, Eduardo M; Santana, Mário H P; Coltro, Wendell K T; Paixão, Thiago R L C

2016-05-17

We report the development of a simple, portable, low-cost, high-throughput visual colorimetric paper-based analytical device for the detection of procaine in seized cocaine samples. The interference of most common cutting agents found in cocaine samples was verified, and a novel electrochemical approach was used for sample pretreatment in order to increase the selectivity. Under the optimized experimental conditions, a linear analytical curve was obtained for procaine concentrations ranging from 5 to 60 μmol L(-1), with a detection limit of 0.9 μmol L(-1). The accuracy of the proposed method was evaluated using seized cocaine samples and an addition and recovery protocol.

Selective isolation of gonyautoxins 1,4 from the dinoflagellate Alexandrium minutum based on molecularly imprinted solid-phase extraction.

PubMed

Lian, Ziru; Wang, Jiangtao

2017-09-15

Gonyautoxins 1,4 (GTX1,4) from Alexandrium minutum samples were isolated selectively and recognized specifically by an innovative and effective extraction procedure based on molecular imprinting technology. Novel molecularly imprinted polymer microspheres (MIPMs) were prepared by double-templated imprinting strategy using caffeine and pentoxifylline as dummy templates. The synthesized polymers displayed good affinity to GTX1,4 and were applied as sorbents. Further, an off-line molecularly imprinted solid-phase extraction (MISPE) protocol was optimized and an effective approach based on the MISPE coupled with HPLC-FLD was developed for selective isolation of GTX1,4 from the cultured A. minutum samples. The separation method showed good extraction efficiency (73.2-81.5%) for GTX1,4 and efficient removal of interferences matrices was also achieved after the MISPE process for the microalgal samples. The outcome demonstrated the superiority and great potential of the MISPE procedure for direct separation of GTX1,4 from marine microalgal extracts. Copyright © 2017. Published by Elsevier Ltd.

Data Collection Protocols for Adhesive Testing Results Using the Materials Selection and Analysis Tool

DTIC Science & Technology

2012-06-01

20090110) was 12.0 MPa. The C100 sample failed at 3.92 mm of crosshead displacement. The total area under the load versus displacement curve is 12600 ... 12600 (12200  300 This paper is declared a work of the U.S. Government and is not subject to copyright protection in the United States

Evaluating adequacy of the representative stream reach used in invertebrate monitoring programs

USGS Publications Warehouse

Rabeni, C.F.; Wang, N.; Sarver, R.J.

1999-01-01

Selection of a representative stream reach is implicitly or explicitly recommended in many biomonitoring protocols using benthic invertebrates. We evaluated the adequacy of sampling a single stream reach selected on the basis of its appearance. We 1st demonstrated the precision of our within-reach sampling. Then we sampled 3 or 4 reaches (each ~20x mean width) within an 8-16 km segment on each of 8 streams in 3 ecoregions and calculated 4 common metrics: 1) total taxa; 2) Ephemeroptera, Plecoptera, and Trichoptera taxa; 3) biotic index; and 4) Sharmon's diversity index. In only 6% of possible cases was the coefficient of variation for any of the metrics reduced >10% by sampling additional reaches. Sampling a 2nd reach on a stream improved the ability to detect impairment by an average of only 9.3%. Sampling a 3rd reach on a stream additionally improved ability to detect impairment by only 4.5%. We concluded that a single well-chosen reach, if adequately sampled, can be representative of an entire stream segment, and sampling additional reaches within a segment may not be cost effective.

Laboratory evaluation of a protocol for personal sampling of airborne particles in welding and allied processes.

PubMed

Chung, K Y; Carter, G J; Stancliffe, J D

1999-02-01

A new European/International Standard (ISOprEN 10882-1) on the sampling of airborne particulates generated during welding and allied processes has been proposed. The use of a number of samplers and sampling procedures is allowable within the defined protocol. The influence of these variables on welding fume exposures measured during welding and grinding of stainless and mild steel using the gas metal arc (GMA) and flux-cored arc (FCA) and GMA welding of aluminium has been examined. Results show that use of any of the samplers will not give significantly different measured exposures. The effect on exposure measurement of placing the samplers on either side of the head was variable; consequently, sampling position cannot be meaningfully defined. All samplers collected significant amounts of grinding dust. Therefore, gravimetric determination of welding fume exposure in atmospheres containing grinding dust will be inaccurate. The use of a new size selective sampler can, to some extent, be used to give a more accurate estimate of exposure. The reliability of fume analysis data of welding consumables has caused concern; and the reason for differences that existed between the material safety data sheet and the analysis of fume samples collected requires further investigation.

Use of probability analysis to establish routine bioassay screening levels

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

Carbaugh, E.H.; Sula, M.J.; McFadden, K.M.

1990-09-01

Probability analysis was used by the Hanford Internal Dosimetry Program to establish bioassay screening levels for tritium and uranium in urine. Background environmental levels of these two radionuclides are generally detectable by the highly sensitive urine analysis procedures routinely used at Hanford. Establishing screening levels requires balancing the impact of false detection with the consequence of potentially undetectable occupation dose. To establish the screening levels, tritium and uranium analyses were performed on urine samples collected from workers exposed only to environmental sources. All samples were collected at home using a simulated 12-hour protocol for tritium and a simulated 24-hour collectionmore » protocol for uranium. Results of the analyses of these samples were ranked according to tritium concentration or total sample uranium. The cumulative percentile was calculated and plotted using log-probability coordinates. Geometric means and screening levels corresponding to various percentiles were estimated by graphical interpolation and standard calculations. The potentially annual internal dose associated with a screening level was calculated. Screening levels were selected corresponding to the 99.9 percentile, implying that, on the average, 1 out of 1000 samples collected from an unexposed worker population would be expected to exceed the screening level. 4 refs., 2 figs.« less

Preliminary neutron crystallographic analysis of selectively CH3-protonated, deuterated rubredoxin from Pyrococcus furiosus

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

Weiss, Kevin L; Meilleur, Flora; Blakeley, Matthew

2008-01-01

Neutron crystallography is used to locate hydrogen atoms in biological materials and can distinguish between negatively scattering hydrogen and positively scattering deuterium substituted positions in isomorphous neutron structures. Recently, Hauptman and Langs (2003) have shown that neutron diffraction data can be used to solve macromolecular structures by direct methods and that solution is aided by the presence of negatively scattering hydrogen atoms in the structure. Selective labeling protocols allow the design and production of H/D-labeled macromolecular structures in which the ratio of hydrogen to deuterium atoms can be precisely controlled. We have applied methyl-selective labeling protocols to introduce (1H-delta methyl)-leucinemore » and (1H-gamma methyl)-valine into deuterated rubredoxin from Pyrococcus furiosus (PfRd). Here we report on the production, crystallization, and preliminary neutron analysis of the selectively CH3-protonated, deuterated PfRd sample, which provided a high quality neutron data set extending to 1.75 resolution at the new LADI-III instrument at the Insititut Laue-Langevin. Preliminary analysis of neutron density maps allows unambiguous assignment of the positions of hydrogen atoms at the methyl groups of the valine and leucine residues in the otherwise deuterated rubredoxin structure.« less

Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines.

PubMed

Santos, E M; Paula, J F R; Motta, P M C; Heinemann, M B; Leite, R C; Haddad, J P A; Del Puerto, H L; Reis, J K P

2010-08-17

We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from lung fragments, producing high DNA concentrations, with high sensitivity in PCR amplification (100%), followed by Protocols C and B. On the other hand, for PBMC samples, Protocol B gave the highest sensitivity in PCR amplification (100%), followed by Protocols C and A. We conclude that Protocol A should be used for PCR diagnosis from lung fragment samples, while Protocol B should be used for PBMC.

Validation of a standardized extraction method for formalin-fixed paraffin-embedded tissue samples.

PubMed

Lagheden, Camilla; Eklund, Carina; Kleppe, Sara Nordqvist; Unger, Elizabeth R; Dillner, Joakim; Sundström, Karin

2016-07-01

Formalin-fixed paraffin-embedded (FFPE) samples can be DNA-extracted and used for human papillomavirus (HPV) genotyping. The xylene-based gold standard for extracting FFPE samples is laborious, suboptimal and involves health hazards for the personnel involved. To compare extraction with the standard xylene method to a xylene-free method used in an HPV LabNet Global Reference Laboratory at the Centers for Disease Control (CDC); based on a commercial method with an extra heating step. Fifty FFPE samples were randomly selected from a national audit of all cervical cancer cases diagnosed in Sweden during 10 years. For each case-block, a blank-block was sectioned, as a control for contamination. For xylene extraction, the standard WHO Laboratory Manual protocol was used. For the CDC method, the manufacturers' protocol was followed except for an extra heating step, 120°C for 20min. Samples were extracted and tested in parallel with β-globin real-time PCR, HPV16 real-time PCR and HPV typing using modified general primers (MGP)-PCR and Luminex assays. For a valid result the blank-block had to be betaglobin-negative in all tests and the case-block positive for beta-globin. Overall, detection was improved with the heating method and the amount of HPV-positive samples increased from 70% to 86% (p=0.039). For all samples where HPV type concordance could be evaluated, there was 100% type concordance. A xylene-free and robust extraction method for HPV-DNA typing in FFPE material is currently in great demand. Our proposed standardized protocol appears to be generally useful. Copyright © 2016. Published by Elsevier B.V.

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two-dimensional gel electrophoresis.

PubMed

Rodrigues, Silas Pessini; Ventura, José Aires; Zingali, R B; Fernandes, P M B

2009-01-01

A variety of sample preparation protocols for plant proteomic analysis using two-dimensional gel electrophoresis (2-DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2-DE. Four sample preparation methods were tested: (1) phenol extraction and methanol-ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid-acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS-PAGE (1-DE) and 2-DE. Fifteen selected protein spots were trypsinised and analysed by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Methods number 3 and 4 resulted in large quantities of protein with good 1-DE separation and were chosen for 2-DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. The described sample preparation method allows the proteomic analysis of papaya leaves by 2-DE and mass spectrometry (MALDI-TOF-MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species.

FuGeF: A Resource Bound Secure Forwarding Protocol for Wireless Sensor Networks.

PubMed

Umar, Idris Abubakar; Mohd Hanapi, Zurina; Sali, A; Zulkarnain, Zuriati A

2016-06-22

Resource bound security solutions have facilitated the mitigation of spatio-temporal attacks by altering protocol semantics to provide minimal security while maintaining an acceptable level of performance. The Dynamic Window Secured Implicit Geographic Forwarding (DWSIGF) routing protocol for Wireless Sensor Network (WSN) has been proposed to achieve a minimal selection of malicious nodes by introducing a dynamic collection window period to the protocol's semantics. However, its selection scheme suffers substantial packet losses due to the utilization of a single distance based parameter for node selection. In this paper, we propose a Fuzzy-based Geographic Forwarding protocol (FuGeF) to minimize packet loss, while maintaining performance. The FuGeF utilizes a new form of dynamism and introduces three selection parameters: remaining energy, connectivity cost, and progressive distance, as well as a Fuzzy Logic System (FLS) for node selection. These introduced mechanisms ensure the appropriate selection of a non-malicious node. Extensive simulation experiments have been conducted to evaluate the performance of the proposed FuGeF protocol as compared to DWSIGF variants. The simulation results show that the proposed FuGeF outperforms the two DWSIGF variants (DWSIGF-P and DWSIGF-R) in terms of packet delivery.

Working towards accreditation by the International Standards Organization 15189 Standard: how to validate an in-house developed method an example of lead determination in whole blood by electrothermal atomic absorption spectrometry.

PubMed

Garcia Hejl, Carine; Ramirez, Jose Manuel; Vest, Philippe; Chianea, Denis; Renard, Christophe

2014-09-01

Laboratories working towards accreditation by the International Standards Organization (ISO) 15189 standard are required to demonstrate the validity of their analytical methods. The different guidelines set by various accreditation organizations make it difficult to provide objective evidence that an in-house method is fit for the intended purpose. Besides, the required performance characteristics tests and acceptance criteria are not always detailed. The laboratory must choose the most suitable validation protocol and set the acceptance criteria. Therefore, we propose a validation protocol to evaluate the performance of an in-house method. As an example, we validated the process for the detection and quantification of lead in whole blood by electrothermal absorption spectrometry. The fundamental parameters tested were, selectivity, calibration model, precision, accuracy (and uncertainty of measurement), contamination, stability of the sample, reference interval, and analytical interference. We have developed a protocol that has been applied successfully to quantify lead in whole blood by electrothermal atomic absorption spectrometry (ETAAS). In particular, our method is selective, linear, accurate, and precise, making it suitable for use in routine diagnostics.

Guidelines and sample protocol for sampling forest gaps.

Treesearch

J.R. Runkle

1992-01-01

A protocol for sampling forest canopy gaps is presented. Methods used in published gap studies are reviewed. The sample protocol will be useful in developing a broader understanding of forest structure and dynamics through comparative studies across different forest ecosystems.

It's Time to Develop a New "Draft Test Protocol" for a Mars Sample Return Mission (or Two…).

PubMed

Rummel, John D; Kminek, Gerhard

2018-04-01

The last time NASA envisioned a sample return mission from Mars, the development of a protocol to support the analysis of the samples in a containment facility resulted in a "Draft Test Protocol" that outlined required preparations "for the safe receiving, handling, testing, distributing, and archiving of martian materials here on Earth" (Rummel et al., 2002 ). This document comprised a specific protocol to be used to conduct a biohazard test for a returned martian sample, following the recommendations of the Space Studies Board of the US National Academy of Sciences. Given the planned launch of a sample-collecting and sample-caching rover (Mars 2020) in 2 years' time, and with a sample return planned for the end of the next decade, it is time to revisit the Draft Test Protocol to develop a sample analysis and biohazard test plan to meet the needs of these future missions. Key Words: Biohazard detection-Mars sample analysis-Sample receiving facility-Protocol-New analytical techniques-Robotic sample handling. Astrobiology 18, 377-380.

Comparison of sampling procedures and microbiological and non-microbiological parameters to evaluate cleaning and disinfection in broiler houses.

PubMed

Luyckx, K; Dewulf, J; Van Weyenberg, S; Herman, L; Zoons, J; Vervaet, E; Heyndrickx, M; De Reu, K

2015-04-01

Cleaning and disinfection of the broiler stable environment is an essential part of farm hygiene management. Adequate cleaning and disinfection is essential for prevention and control of animal diseases and zoonoses. The goal of this study was to shed light on the dynamics of microbiological and non-microbiological parameters during the successive steps of cleaning and disinfection and to select the most suitable sampling methods and parameters to evaluate cleaning and disinfection in broiler houses. The effectiveness of cleaning and disinfection protocols was measured in six broiler houses on two farms through visual inspection, adenosine triphosphate hygiene monitoring and microbiological analyses. Samples were taken at three time points: 1) before cleaning, 2) after cleaning, and 3) after disinfection. Before cleaning and after disinfection, air samples were taken in addition to agar contact plates and swab samples taken from various sampling points for enumeration of total aerobic flora, Enterococcus spp., and Escherichia coli and the detection of E. coli and Salmonella. After cleaning, air samples, swab samples, and adenosine triphosphate swabs were taken and a visual score was also assigned for each sampling point. The mean total aerobic flora determined by swab samples decreased from 7.7±1.4 to 5.7±1.2 log CFU/625 cm2 after cleaning and to 4.2±1.6 log CFU/625 cm2 after disinfection. Agar contact plates were used as the standard for evaluating cleaning and disinfection, but in this study they were found to be less suitable than swabs for enumeration. In addition to measuring total aerobic flora, Enterococcus spp. seemed to be a better hygiene indicator to evaluate cleaning and disinfection protocols than E. coli. All stables were Salmonella negative, but the detection of its indicator organism E. coli provided additional information for evaluating cleaning and disinfection protocols. Adenosine triphosphate analyses gave additional information about the hygiene level of the different sampling points. © 2015 Poultry Science Association Inc.

Laboratory and quality assurance protocols for the analysis of herbicides in ground water from the Management Systems Evaluation Area, Princeton, Minnesota

USGS Publications Warehouse

Larson, S.J.; Capel, P.D.; VanderLoop, A.G.

1996-01-01

Laboratory and quality assurance procedures for the analysis of ground-water samples for herbicides at the Management Systems Evaluation Area near Princeton, Minnesota are described. The target herbicides include atrazine, de-ethylatrazine, de-isopropylatrazine, metribuzin, alachlor, 2,6-diethylaniline, and metolachlor. The analytical techniques used are solid-phase extraction, and analysis by gas chromatography with mass-selective detection. Descriptions of cleaning procedures, preparation of standard solutions, isolation of analytes from water, sample transfer methods, instrumental analysis, and data analysis are included.

Evaluation of a cleanser for petroleum-contaminated skin.

PubMed

Phieffer, Laura S; Banks, David M; Bosse, Michael J; Meyer, Martha H; Meyer, Ralph A; Smith, Kevin

2003-12-01

Extremity injuries contaminated with petroleum products pose clinical dilemmas. This project was designed to evaluate the efficacy of a dioctyl sulfosuccinate (DS) solution for cleansing petroleum-contaminated skin. One hundred Sprague-Dawley rats were subjected to a contamination protocol followed by a cleansing procedure. Four petroleum contaminants and five cleansing solutions were selected. The protocol consisted of shaving, initial punch biopsy, contamination, precleansing punch biopsy, standardized scrub protocol, and postcleansing punch biopsy. Biopsy samples were analyzed for petroleum residue using fluorometry. The 10% DS solution had the highest reduction of crude oil, grease, and tar: 99.6 +/- 0.4% (mean +/- SD) contaminant reduction for crude oil, 99.8 +/- 0.2% for grease, and 99.8 +/- 0.2% for tar. The other cleansers showed less efficacy (p < 0.05). Concentrated DS appears to be significantly more effective at cleaning petroleum products from skin than the commonly chosen surgical and commercial cleansers.

Isolation of bacteria-containing phagosomes by magnetic selection

PubMed Central

Lönnbro, Per; Nordenfelt, Pontus; Tapper, Hans

2008-01-01

Background There is a growing awareness of the importance of intracellular events in determining the outcome of infectious disease. To improve the understanding of such events, like phagosome maturation, we set out to develop a versatile technique for phagosome isolation that is rapid and widely applicable to different pathogens. Results We developed two different protocols to isolate phagosomes containing dead or live bacteria modified with small magnetic particles, in conjunction with a synchronized phagocytosis protocol and nitrogen cavitation. For dead bacteria, we performed analysis of the phagosome samples by microscopy and immunoblot, and demonstrated the appearance of maturation markers on isolated phagosomes. Conclusion We have presented detailed protocols for phagosome isolation, which can be adapted for use with different cell types and prey. The versatility and simplicity of the approach allow better control of phagosome isolation, the parameters of which are critical in studies of host-bacteria interaction and phagosome maturation. PMID:18588680

The United States Department Of Agriculture Northeast Area-wide Tick Control Project: history and protocol.

PubMed

Pound, Joe Mathews; Miller, John Allen; George, John E; Fish, Durland

2009-08-01

The Northeast Area-wide Tick Control Project (NEATCP) was funded by the United States Department of Agriculture (USDA) as a large-scale cooperative demonstration project of the USDA-Agricultural Research Service (ARS)-patented 4-Poster tick control technology (Pound et al. 1994) involving the USDA-ARS and a consortium of universities, state agencies, and a consulting firm at research locations in the five states of Connecticut (CT), Maryland (MD), New Jersey (NJ), New York (NY), and Rhode Island (RI). The stated objective of the project was "A community-based field trial of ARS-patented tick control technology designed to reduce the risk of Lyme disease in northeastern states." Here we relate the rationale and history of the technology, a chronological listing of events leading to implementation of the project, the original protocol for selecting treatment, and control sites, and protocols for deployment of treatments, sampling, assays, data analyses, and estimates of efficacy.

Development of Analytical Protocols For Organics and Isotopes Analysis on the 2009 MARS Science Laboratory.

NASA Technical Reports Server (NTRS)

Mahaffy, P. R.

2006-01-01

The Mars Science Laboratory, under development for launch in 2009, is designed explore and quantitatively asses a local region on Mars as a potential habitat for present or past life. Its ambitious goals are to (1) assess the past or present biological potential of the target environment, (2) to characterize the geology and geochemistry at the MSL landing site, and (3) to investigate planetary processes that influence habitability. The planned capabilities of the rover payload will enable a comprehensive search for organic molecules, a determination of definitive mineralogy of sampled rocks and fines, chemical and isotopic analysis of both atmospheric and solid samples, and precision isotope measurements of several volatile elements. A range of contact and remote surface and subsurface survey tools will establish context for these measurements and will facilitate sample identification and selection. The Sample Analysis at Mars (SAM) suite of MSL addresses several of the mission's core measurement goals. It includes a gas chromatograph, a mass spectrometer, and a tunable laser spectrometer. These instruments will be designed to analyze either atmospheric samples or gases extracted from solid phase samples such as rocks and fines. We will describe the range of measurement protocols under development and study by the SAM engineering and science teams for use on the surface of Mars.

Profile and birthing practices of Maranao traditional birth attendants.

PubMed

Maghuyop-Butalid, Roselyn; Mayo, Norhanifa A; Polangi, Hania T

2015-01-01

This study determined the profile and birthing practices in both modern and traditional ways among Maranao traditional birth attendants (TBAs) in Lanao del Norte, Philippines. It employed a descriptive research design. The respondents were 50 Maranao TBAs selected through the snowball sampling technique. A questionnaire was developed by the researchers to identify the respondents' modern birthing practices utilizing the Essential Intrapartum and Newborn Care (EINC) Protocol. To determine their profile and traditional birthing practices, items from a previous study and the respondents' personal claims were adapted. This study shows that Maranao TBAs have less compliance to the EINC Protocol and they often practice the traditional birthing interventions, thus increasing the risk of complications to both mother and newborn.

Sampling protocol for post-landfall Deepwater Horizon oil release, Gulf of Mexico, 2010

USGS Publications Warehouse

Wilde, F.D.; Skrobialowski, S.C.; Hart, J.S.

2010-01-01

The protocols and procedures described in this report are designed to be used by U.S. Geological Survey (USGS) field teams for the collection of environmental data and samples in coastal areas affected by the 2010 Deepwater Horizon oil spill in the Gulf of Mexico. This sampling protocol focuses specifically on sampling for water, sediments, benthic invertebrates, and microorganisms (ambient bacterial populations) after shoreline arrival of petroleum-associated product on beach, barrier island, and wetland environments of the Gulf of Mexico coastal states. Deployment to sampling sites, site setup, and sample collection in these environments necessitates modifications to standard USGS sampling procedures in order to address the regulatory, logistical, and legal requirements associated with samples collected in oil-impacted coastal areas. This document, therefore, has been written as an addendum to the USGS National Field Manual for the Collection of Water-Quality Data (NFM) (http://pubs.water.usgs.gov/twri9A/), which provides the basis for training personnel in the use of standard USGS sampling protocols. The topics covered in this Gulf of Mexico oil-spill sampling protocol augment NFM protocols for field-deployment preparations, health and safety precautions, sampling and quality-assurance procedures, and decontamination requirements under potentially hazardous environmental conditions. Documentation procedures and maintenance of sample integrity by use of chain-of-custody procedures also are described in this protocol.

Preparation and guest-uptake protocol for a porous complex useful for 'crystal-free' crystallography.

PubMed

Inokuma, Yasuhide; Yoshioka, Shota; Ariyoshi, Junko; Arai, Tatsuhiko; Fujita, Makoto

2014-02-01

We recently reported a new method for single-crystal X-ray diffraction (SCD) analysis that does not require the crystallization of the target compound. In this 'crystal-free' crystallography, a tiny crystal of a porous complex is soaked in the solution of the target guest. The guest molecules are absorbed and oriented in the crystal pores and can be analyzed by X-ray diffraction. We describe here a detailed synthetic protocol for the preparation of uniform single crystals of the porous host complex and for the subsequent guest uptake. The protocol describes our most versatile porous complex, which is prepared from commercially available ZnI2 and 2,4,6-tri(4-pyridyl)-1,3,5-triazine. The host complex has large pores with a cross-section of 8 × 5 Å(2). Single crystals of the complex are grown from layered solutions of the two components. The pores of the as-synthesized complex are filled with nitrobenzene, which is replaced with the inert solvent cyclohexane. This solvent exchange is essential for the rapid and effective inclusion of target compounds. The most crucial and delicate step is the selection of high-quality single crystals from the mixture of crystals of various shapes and sizes. We suggest using the facial indices of the single crystals as a criterion for crystal selection. Single-crystal samples for X-ray analysis can be prepared by immersing the selected crystals in a cyclohexane/dichloromethane solution of target compound. After a very slow evaporation of the solvent, typically over 2 d, the final crystal can be picked and directly subjected to SCD analysis. The protocol can be completed within ∼16 d.

Chronology of desert margin in western India using improved luminescence dating protocols

NASA Astrophysics Data System (ADS)

Chauhan, Naveen; Morthekai, P.

2017-12-01

The present study provides improved chronology for the desert margin fluvial sediments of semi-arid region located in the Mahi river basin, western India. The sequence has preserved a near-continuous record of climate change since the Last Interglacial. An earlier attempt of dating based on feldspar IRSL chronology shows a combined effect of anomalous fading and unbleached components resulting in age inversions. The present work tries to explore the possibility of using blue light stimulated luminescence (BLSL) of quartz, infra-red stimulated luminescence (IRSL) of feldspar and the newly developed methodologies, like natural correction factor based single aliquot regeneration (NCF-SAR) protocol and decision making schemes based on distribution of doses and beta heterogeneity concept for luminescence dating of sediments. Observations suggest that quartz suffered from significant sensitivity changes during natural signal measurement and partial bleaching. A combination of NCF-SAR protocol and sample specific equivalent dose computation helped in arriving at better age estimate for present samples. The study also compares the criteria for the selection of different age models that are used at present. The age of the alluvial sequence is now bracketed between 10 ka (upper aeolian unit) and 75 ka (lowermost fluvial unit).

Exploiting automatic on-line renewable molecularly imprinted solid-phase extraction in lab-on-valve format as front end to liquid chromatography: application to the determination of riboflavin in foodstuffs.

PubMed

Oliveira, Hugo M; Segundo, Marcela A; Lima, José L F C; Miró, Manuel; Cerdà, Victor

2010-05-01

In the present work, it is proposed, for the first time, an on-line automatic renewable molecularly imprinted solid-phase extraction (MISPE) protocol for sample preparation prior to liquid chromatographic analysis. The automatic microscale procedure was based on the bead injection (BI) concept under the lab-on-valve (LOV) format, using a multisyringe burette as propulsion unit for handling solutions and suspensions. A high precision on handling the suspensions containing irregularly shaped molecularly imprinted polymer (MIP) particles was attained, enabling the use of commercial MIP as renewable sorbent. The features of the proposed BI-LOV manifold also allowed a strict control of the different steps within the extraction protocol, which are essential for promoting selective interactions in the cavities of the MIP. By using this on-line method, it was possible to extract and quantify riboflavin from different foodstuff samples in the range between 0.450 and 5.00 mg L(-1) after processing 1,000 microL of sample (infant milk, pig liver extract, and energy drink) without any prior treatment. For milk samples, LOD and LOQ values were 0.05 and 0.17 mg L(-1), respectively. The method was successfully applied to the analysis of two certified reference materials (NIST 1846 and BCR 487) with high precision (RSD < 5.5%). Considering the downscale and simplification of the sample preparation protocol and the simultaneous performance of extraction and chromatographic assays, a cost-effective and enhanced throughput (six determinations per hour) methodology for determination of riboflavin in foodstuff samples is deployed here.

Determination of doping peptides via solid-phase microelution and accurate-mass quadrupole time-of-flight LC-MS.

PubMed

Cuervo, Darío; Loli, Cynthia; Fernández-Álvarez, María; Muñoz, Gloria; Carreras, Daniel

2017-10-15

A complete analytical protocol for the determination of 25 doping-related peptidic drugs and 3 metabolites in urine was developed by means of accurate-mass quadrupole time-of-flight (Q-TOF) LC-MS analysis following solid-phase extraction (SPE) on microplates and conventional SPE pre-treatment for initial testing and confirmation, respectively. These substances included growth hormone releasing factors, gonadotropin releasing factors and anti-diuretic hormones, with molecular weights ranging from 540 to 1320Da. Optimal experimental conditions were stablished after investigation of different parameters concerning sample preparation and instrumental analysis. Weak cation exchange SPE followed by C18 HPLC chromatography and accurate mass detection provided the required sensitivity and selectivity for all the target peptides under study. 2mg SPE on 96-well microplates can be used in combination with full scan MS detection for the initial testing, thus providing a fast, cost-effective and high-throughput protocol for the processing of a large batch of samples simultaneously. On the other hand, extraction on 30mg SPE cartridges and subsequent target MS/MS determination was the protocol of choice for confirmatory purposes. The methodology was validated in terms of selectivity, recovery, matrix effect, precision, sensitivity (limit of detection, LOD), cross contamination, carryover, robustness and stability. Recoveries ranged from 6 to 70% (microplates) and 17-95% (cartridges), with LODs from 0.1 to 1ng/mL. The suitability of the method was assessed by analyzing different spiked or excreted urines containing some of the target substances. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of storage and filtration protocols for alpine/subalpine lake water quality samples

Treesearch

John L. Korfmacher; Robert C. Musselman

2007-01-01

Many government agencies and other organizations sample natural alpine and subalpine surface waters using varying protocols for sample storage and filtration. Simplification of protocols would be beneficial if it could be shown that sample quality is unaffected. In this study, samples collected from low ionic strength waters in alpine and subalpine lake inlets...

7 CFR 301.92-11 - Inspection and sampling protocols.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 5 2010-01-01 2010-01-01 false Inspection and sampling protocols. 301.92-11 Section... Inspection and sampling protocols. Type(s) of plants in the nursery Type(s) of plants shipped interstate... interstate. (1) Annual inspection, sampling, and testing—(i) Inspection. The nursery must be inspected...

7 CFR 301.92-11 - Inspection and sampling protocols.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 5 2011-01-01 2011-01-01 false Inspection and sampling protocols. 301.92-11 Section... Inspection and sampling protocols. Type(s) of plants in the nursery Type(s) of plants shipped interstate... interstate. (1) Annual inspection, sampling, and testing—(i) Inspection. The nursery must be inspected...

A comparison of single and multiple stressor protocols to assess acute stress in a coastal shark species, Rhizoprionodon terraenovae.

PubMed

Hoffmayer, Eric R; Hendon, Jill M; Parsons, Glenn R; Driggers, William B; Campbell, Matthew D

2015-10-01

Elasmobranch stress responses are traditionally measured in the field by either singly or serially sampling an animal after a physiologically stressful event. Although capture and handling techniques are effective at inducing a stress response, differences in protocols could affect the degree of stress experienced by an individual, making meaningful comparisons between the protocols difficult, if not impossible. This study acutely stressed Atlantic sharpnose sharks, Rhizoprionodon terraenovae, by standardized capture (rod and reel) and handling methods and implemented either a single or serial blood sampling protocol to monitor four indicators of the secondary stress response. Single-sampled sharks were hooked and allowed to swim around the boat until retrieved for a blood sample at either 0, 15, 30, 45, or 60 min post-hooking. Serially sampled sharks were retrieved, phlebotomized, released while still hooked, and subsequently resampled at 15, 30, 45, and 60 min intervals post-hooking. Blood was analyzed for hematocrit, and plasma glucose, lactate, and osmolality levels. Although both single and serial sampling protocols resulted in an increase in glucose, no significant difference in glucose level was found between protocols. Serially sampled sharks exhibited cumulatively heightened levels for lactate and osmolality at all time intervals when compared to single-sampled animals at the same time. Maximal concentration differences of 217.5, 9.8, and 41.6 % were reported for lactate, osmolality, and glucose levels, respectively. Hematocrit increased significantly over time for the single sampling protocol but did not change significantly during the serial sampling protocol. The differences in resultant blood chemistry levels between implemented stress protocols and durations are significant and need to be considered when assessing stress in elasmobranchs.

A Draft Test Protocol for Detecting Possible Biohazards in Martian Samples Returned to Earth

NASA Technical Reports Server (NTRS)

Rummel, John D. (Editor); Race, Margaret S.; DeVincenzi, Donald L.; Schad, P. Jackson; Stabekis, Pericles D.; Viso, Michel; Acevedo, Sara E.

2002-01-01

This document presents the first complete draft of a protocol for detecting possible biohazards in Mars samples returned to Earth: it is the final product of the Mars Sample Handling Protocol Workshop Series. convened in 2000-2001 by NASA's Planetary Protection Officer. The goal of the five-workshop Series vas to develop a comprehensive protocol by which returned martian sample materials could be assessed k r the presence of any biological hazard(s) while safeguarding the purity of the samples from possible terrestrial contamination.

Development of a simple fluorescence-based microplate method for the high-throughput analysis of proline in wine samples.

PubMed

Robert-Peillard, Fabien; Boudenne, Jean-Luc; Coulomb, Bruno

2014-05-01

This paper presents a simple, accurate and multi-sample method for the determination of proline in wines thanks to a 96-well microplate technique. Proline is the most abundant amino acid in wine and is an important parameter related to wine characteristics or maturation processes of grape. In the current study, an improved application of the general method based on sodium hypochlorite oxidation and o-phthaldialdehyde (OPA)-thiol spectrofluorometric detection is described. The main interfering compounds for specific proline detection in wines are strongly reduced by selective reaction with OPA in a preliminary step under well-defined pH conditions. Application of the protocol after a 500-fold dilution of wine samples provides a working range between 0.02 and 2.90gL(-1), with a limit of detection of 7.50mgL(-1). Comparison and validation on real wine samples by ion-exchange chromatography prove that this procedure yields accurate results. Simplicity of the protocol used, with no need for centrifugation or filtration, organic solvents or high temperature enables its full implementation in plastic microplates and efficient application for routine analysis of proline in wines. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of freeze/thaw cycles on several biomarkers in urine from patients with kidney disease.

PubMed

Zhang, Yinan; Luo, Yi; Lu, Huijuan; Wang, Niansong; Shen, Yixie; Chen, Ruihua; Fang, Pingyan; Yu, Hong; Wang, Congrong; Jia, Weiping

2015-04-01

Urine samples were collected from eleven randomly selected patients with kidney disease, including diabetic nephropathy, chronic nephritis, and nephritic syndrome. Urine samples were treated with one of four protocols for freezing and thawing: freeze directly and thaw directly; freeze directly and thaw by temperature gradient; freeze by temperature gradient and thaw directly; and freeze by temperature gradient and thaw by temperature gradient. After one to six freeze/thaw cycles at -20°C or -80°C, different biomarkers showed differential stabilities. The concentrations of total protein, calcium, and potassium did not change significantly after five freeze/thaw cycles at either -20°C or -80°C. Albumin could only sustain three freeze/thaw cycles at -20°C before it started to degrade. We recommend that urine be stored at -80°C as albumin and the organic ions could sustain five and six freeze/thaw cycles, respectively, using the simple "direct freeze and direct thaw" protocol. Furthermore, in most cases, gradient freeze/thaw cycles are not necessary for urine sample storage.

Bounce Back Now! Protocol of a population-based randomized controlled trial to examine the efficacy of a Web-based intervention with disaster-affected families.

PubMed

Ruggiero, Kenneth J; Davidson, Tatiana M; McCauley, Jenna; Gros, Kirstin Stauffacher; Welsh, Kyleen; Price, Matthew; Resnick, Heidi S; Danielson, Carla Kmett; Soltis, Kathryn; Galea, Sandro; Kilpatrick, Dean G; Saunders, Benjamin E; Nissenboim, Josh; Muzzy, Wendy; Fleeman, Anna; Amstadter, Ananda B

2015-01-01

Disasters have far-reaching and potentially long-lasting effects on youth and families. Research has consistently shown a clear increase in the prevalence of several mental health disorders after disasters, including depression and posttraumatic stress disorder. Widely accessible evidence-based interventions are needed to address this unmet need for youth and families, who are underrepresented in disaster research. Rapid growth in Internet and Smartphone access, as well as several Web based evaluation studies with various adult populations has shown that Web-based interventions are likely to be feasible in this context and can improve clinical outcomes. Such interventions also are generally cost-effective, can be targeted or personalized, and can easily be integrated in a stepped care approach to screening and intervention delivery. This is a protocol paper that describes an innovative study design in which we evaluate a self-help Web-based resource, Bounce Back Now, with a population-based sample of disaster affected adolescents and families. The paper includes description and justification for sampling selection and procedures, selection of assessment measures and methods, design of the intervention, and statistical evaluation of critical outcomes. Unique features of this study design include the use of address-based sampling to recruit a population-based sample of disaster-affected adolescents and parents, telephone and Web-based assessments, and development and evaluation of a highly individualized Web intervention for adolescents. Challenges related to large-scale evaluation of technology-delivered interventions with high-risk samples in time-sensitive research are discussed, as well as implications for future research and practice. Published by Elsevier Inc.

Bounce Back Now! Protocol of a Population-Based Randomized Controlled Trial to Examine the Efficacy of a Web-based Intervention with Disaster-Affected Families

PubMed Central

Ruggiero, Kenneth J.; Davidson, Tatiana M.; McCauley, Jenna; Gros, Kirstin Stauffacher; Welsh, Kyleen; Price, Matthew; Resnick, Heidi S.; Danielson, Carla Kmett; Soltis, Kathryn; Galea, Sandro; Kilpatrick, Dean G.; Saunders, Benjamin E.; Nissenboim, Josh; Muzzy, Wendy; Fleeman, Anna; Amstadter, Ananda B.

2014-01-01

Disasters have far-reaching and potentially long-lasting effects on youth and families. Research has consistently shown a clear increase in the prevalence of several mental health disorders after disasters, including depression and posttraumatic stress disorder. Widely accessible evidence-based interventions are needed to address this unmet need for youth and families, who are underrepresented in disaster research. Rapid growth in Internet and Smartphone access, as well as several web based evaluation studies with various adult populations has shown that web-based interventions are likely to be feasible in this context and can improve clinical outcomes. Such interventions also are generally cost-effective, can be targeted or personalized, and can easily be integrated in a stepped care approach to screening and intervention delivery. This is a protocol paper that describes an innovative study design in which we evaluate a self-help web-based resource, Bounce Back Now, with a population-based sample of disaster affected adolescents and families. The paper includes description and justification for sampling selection and procedures, selection of assessment measures and methods, design of the intervention, and statistical evaluation of critical outcomes. Unique features of this study design include the use of address-based sampling to recruit a population-based sample of disaster-affected adolescents and parents, telephone and web-based assessments, and development and evaluation of a highly individualized web intervention for adolescents. Challenges related to large-scale evaluation of technology-delivered interventions with high-risk samples in time-sensitive research are discussed, as well as implications for future research and practice. PMID:25478956

Non-targeted workflow for identification of antimicrobial compounds in animal feed using bioassay-directed screening in combination with liquid chromatography-high resolution mass spectrometry.

PubMed

Wegh, Robin S; Berendsen, Bjorn J A; Driessen-Van Lankveld, Wilma D M; Pikkemaat, Mariël G; Zuidema, Tina; Van Ginkel, Leen A

2017-11-01

A non-targeted workflow is reported for the isolation and identification of antimicrobial active compounds using bioassay-directed screening and LC coupled to high-resolution MS. Suspect samples are extracted using a generic protocol and fractionated using two different LC conditions (A and B). The behaviour of the bioactive compound under these different conditions yields information about the physicochemical properties of the compound and introduces variations in co-eluting compounds in the fractions, which is essential for peak picking and identification. The fractions containing the active compound(s) obtained with conditions A and B are selected using a microbiological effect-based bioassay. The selected bioactive fractions from A and B are analysed using LC combined with high-resolution MS. Selection of relevant signals is automatically carried out by selecting all signals present in both bioactive fractions A and B, yielding tremendous data reduction. The method was assessed using two spiked feed samples and subsequently applied to two feed samples containing an unidentified compound showing microbial growth inhibition. In all cases, the identity of the compound causing microbiological inhibition was successfully confirmed.

Adaptive transmission based on multi-relay selection and rate-compatible LDPC codes

NASA Astrophysics Data System (ADS)

Su, Hualing; He, Yucheng; Zhou, Lin

2017-08-01

In order to adapt to the dynamical changeable channel condition and improve the transmissive reliability of the system, a cooperation system of rate-compatible low density parity check (RC-LDPC) codes combining with multi-relay selection protocol is proposed. In traditional relay selection protocol, only the channel state information (CSI) of source-relay and the CSI of relay-destination has been considered. The multi-relay selection protocol proposed by this paper takes the CSI between relays into extra account in order to obtain more chances of collabration. Additionally, the idea of hybrid automatic request retransmission (HARQ) and rate-compatible are introduced. Simulation results show that the transmissive reliability of the system can be significantly improved by the proposed protocol.

77 FR 5492 - Magnuson-Stevens Act Provisions; General Provisions for Domestic Fisheries; Application for...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-03

... assess the performance of an approved sampling protocol and to allow for continued sample collection and... developmental sampling protocol. While this application was being reviewed and was available for public comment, the sampling protocol being tested was adopted into the National Shellfish Sanitation Program by the...

Recent select Sample Analysis at Mars (SAM) Testbed analog results

NASA Astrophysics Data System (ADS)

Malespin, C.; McAdam, A.; Teinturier, S.; Eigenbrode, J. L.; Freissinet, C.; Knudson, C. A.; Lewis, J. M.; Millan, M.; Steele, A.; Stern, J. C.; Williams, A. J.

2017-12-01

The Sample Analysis at Mars (SAM) testbed (TB) is a high fidelity replica of the flight instrument currently onboard the Curiosity rover in Gale Crater, Mars1. The SAM testbed is housed in a Mars environment chamber at NASA Goddard Space Flight Center (GSFC), which can replicate both thermal and environmental conditions. The testbed is used to validate and test new experimental procedures before they are implemented on Mars, but it is also used to analyze analog samples which assists in the interpretation of results from the surface. Samples are heated using the same experimental protocol as on Mars to allow for direct comparison with Martian sampling conditions. Here we report preliminary results from select samples that were loaded into the SAM TB, including meteorites, an organically rich iron oxide, and a synthetic analog to the Martian Cumberland sample drilled by the rover at Yellowknife Bay. Each of these samples have been analyzed under SAM-like conditions using breadboard and lab instrument systems. By comparing the data from the lab systems and SAM TB, further insight on results from Mars can be gained. References: [1] Mahaffy, P. R., et al. (2013), Science, 341(6143), 263-266, doi:10.1126/science.1237966.

Recommended protocols for sampling macrofungi

Treesearch

Gregory M. Mueller; John Paul Schmit; Sabine M. Hubndorf Leif Ryvarden; Thomas E. O' Dell; D. Jean Lodge; Patrick R. Leacock; Milagro Mata; Loengrin Umania; Qiuxin (Florence) Wu; Daniel L. Czederpiltz

2004-01-01

This chapter discusses several issues regarding reommended protocols for sampling macrofungi: Opportunistic sampling of macrofungi, sampling conspicuous macrofungi using fixed-size, sampling small Ascomycetes using microplots, and sampling a fixed number of downed logs.

National Sample Assessment Protocols

ERIC Educational Resources Information Center

Ministerial Council on Education, Employment, Training and Youth Affairs (NJ1), 2012

2012-01-01

These protocols represent a working guide for planning and implementing national sample assessments in connection with the national Key Performance Measures (KPMs). The protocols are intended for agencies involved in planning or conducting national sample assessments and personnel responsible for administering associated tenders or contracts,…

The natural history of conducting and reporting clinical trials: interviews with trialists.

PubMed

Smyth, Rebecca M D; Jacoby, Ann; Altman, Douglas G; Gamble, Carrol; Williamson, Paula R

2015-01-26

To investigate the nature of the research process as a whole, factors that might influence the way in which research is carried out, and how researchers ultimately report their findings. Semi-structured qualitative telephone interviews with authors of trials, identified from two sources: trials published since 2002 included in Cochrane systematic reviews selected for the ORBIT project; and trial reports randomly sampled from 14,758 indexed on PubMed over the 12-month period from August 2007 to July 2008. A total of 268 trials were identified for inclusion, 183 published since 2002 and included in the Cochrane systematic reviews selected for the ORBIT project and 85 randomly selected published trials indexed on PubMed. The response rate from researchers in the former group was 21% (38/183) and in the latter group was 25% (21/85). Overall, 59 trialists were interviewed from the two different sources. A number of major but related themes emerged regarding the conduct and reporting of trials: establishment of the research question; identification of outcome variables; use of and adherence to the study protocol; conduct of the research; reporting and publishing of findings. Our results reveal that, although a substantial proportion of trialists identify outcome variables based on their clinical experience and knowing experts in the field, there can be insufficient reference to previous research in the planning of a new trial. We have revealed problems with trial recruitment: not reaching the target sample size, over-estimation of recruitment potential and recruiting clinicians not being in equipoise. We found a wide variation in the completeness of protocols, in terms of detailing study rationale, outlining the proposed methods, trial organisation and ethical considerations. Our results confirm that the conduct and reporting of some trials can be inadequate. Interviews with researchers identified aspects of clinical research that can be especially challenging: establishing appropriate and relevant outcome variables to measure, use of and adherence to the study protocol, recruiting of study participants and reporting and publishing the study findings. Our trialists considered the prestige and impact factors of academic journals to be the most important criteria for selecting those to which they would submit manuscripts.

The Resin-Embedded Cornea Prepared Via Rapid Processing Protocol : A Good Histomorphometric Target for Clinical Investigation in Ophthalmology and Optometry

PubMed Central

Cheah, Pike See; Mohidin, Norhani; Mohd Ali, Bariah; Maung, Myint; Latif, Azian Abdul

2008-01-01

This study illustrates and quantifies the changes on corneal tissue between the paraffin-embedded and resin-embedded blocks and thus, selects a better target in investigational ophthalmology and optometry via light microscopy. Corneas of two cynomolgus monkeys (Macaca fascicularis) were used in this study. The formalin-fixed cornea was prepared in paraffin block via the conventional tissue processing protocol (4-day protocol) and stained with haematoxylin and eosin. The glutaraldehyde-fixed cornea was prepared in resin block via the rapid and modified tissue processing procedure (1.2-day protocol) and stained with toluidine blue. The paraffin-embedded sample exhibits various undesired tissue damage and artifact such as thinner epithelium (due to the substantial volumic extraction from the tissue), thicker stroma layer (due to the separation of lamellae and the presence of voids) and the distorted endothelium. In contrast, the resin-embedded corneal tissue has demonstrated satisfactory corneal ultrastructural preservation. The rapid and modified tissue processing method for preparing the resin-embedded is particularly beneficial to accelerate the microscopic evaluation in ophthalmology and optometry. PMID:22570589

Testing the efficiency of rover science protocols for robotic sample selection: A GeoHeuristic Operational Strategies Test

NASA Astrophysics Data System (ADS)

Yingst, R. A.; Bartley, J. K.; Chidsey, T. C.; Cohen, B. A.; Gilleaudeau, G. J.; Hynek, B. M.; Kah, L. C.; Minitti, M. E.; Williams, R. M. E.; Black, S.; Gemperline, J.; Schaufler, R.; Thomas, R. J.

2018-05-01

The GHOST field tests are designed to isolate and test science-driven rover operations protocols, to determine best practices. During a recent field test at a potential Mars 2020 landing site analog, we tested two Mars Science Laboratory data-acquisition and decision-making methods to assess resulting science return and sample quality: a linear method, where sites of interest are studied in the order encountered, and a "walkabout-first" method, where sites of interest are examined remotely before down-selecting to a subset of sites that are interrogated with more resource-intensive instruments. The walkabout method cost less time and fewer resources, while increasing confidence in interpretations. Contextual data critical to evaluating site geology was acquired earlier than for the linear method, and given a higher priority, which resulted in development of more mature hypotheses earlier in the analysis process. Combined, this saved time and energy in the collection of data with more limited spatial coverage. Based on these results, we suggest that the walkabout method be used where doing so would provide early context and time for the science team to develop hypotheses-critical tests; and that in gathering context, coverage may be more important than higher resolution.

Conduct of a personal radiofrequency electromagnetic field measurement study: proposed study protocol.

PubMed

Röösli, Martin; Frei, Patrizia; Bolte, John; Neubauer, Georg; Cardis, Elisabeth; Feychting, Maria; Gajsek, Peter; Heinrich, Sabine; Joseph, Wout; Mann, Simon; Martens, Luc; Mohler, Evelyn; Parslow, Roger C; Poulsen, Aslak Harbo; Radon, Katja; Schüz, Joachim; Thuroczy, György; Viel, Jean-François; Vrijheid, Martine

2010-05-20

The development of new wireless communication technologies that emit radio frequency electromagnetic fields (RF-EMF) is ongoing, but little is known about the RF-EMF exposure distribution in the general population. Previous attempts to measure personal exposure to RF-EMF have used different measurement protocols and analysis methods making comparisons between exposure situations across different study populations very difficult. As a result, observed differences in exposure levels between study populations may not reflect real exposure differences but may be in part, or wholly due to methodological differences. The aim of this paper is to develop a study protocol for future personal RF-EMF exposure studies based on experience drawn from previous research. Using the current knowledge base, we propose procedures for the measurement of personal exposure to RF-EMF, data collection, data management and analysis, and methods for the selection and instruction of study participants. We have identified two basic types of personal RF-EMF measurement studies: population surveys and microenvironmental measurements. In the case of a population survey, the unit of observation is the individual and a randomly selected representative sample of the population is needed to obtain reliable results. For microenvironmental measurements, study participants are selected in order to represent typical behaviours in different microenvironments. These two study types require different methods and procedures. Applying our proposed common core procedures in future personal measurement studies will allow direct comparisons of personal RF-EMF exposures in different populations and study areas.

Conduct of a personal radiofrequency electromagnetic field measurement study: proposed study protocol

PubMed Central

2010-01-01

Background The development of new wireless communication technologies that emit radio frequency electromagnetic fields (RF-EMF) is ongoing, but little is known about the RF-EMF exposure distribution in the general population. Previous attempts to measure personal exposure to RF-EMF have used different measurement protocols and analysis methods making comparisons between exposure situations across different study populations very difficult. As a result, observed differences in exposure levels between study populations may not reflect real exposure differences but may be in part, or wholly due to methodological differences. Methods The aim of this paper is to develop a study protocol for future personal RF-EMF exposure studies based on experience drawn from previous research. Using the current knowledge base, we propose procedures for the measurement of personal exposure to RF-EMF, data collection, data management and analysis, and methods for the selection and instruction of study participants. Results We have identified two basic types of personal RF-EMF measurement studies: population surveys and microenvironmental measurements. In the case of a population survey, the unit of observation is the individual and a randomly selected representative sample of the population is needed to obtain reliable results. For microenvironmental measurements, study participants are selected in order to represent typical behaviours in different microenvironments. These two study types require different methods and procedures. Conclusion Applying our proposed common core procedures in future personal measurement studies will allow direct comparisons of personal RF-EMF exposures in different populations and study areas. PMID:20487532

FuGeF: A Resource Bound Secure Forwarding Protocol for Wireless Sensor Networks

PubMed Central

Umar, Idris Abubakar; Mohd Hanapi, Zurina; Sali, A.; Zulkarnain, Zuriati A.

2016-01-01

Resource bound security solutions have facilitated the mitigation of spatio-temporal attacks by altering protocol semantics to provide minimal security while maintaining an acceptable level of performance. The Dynamic Window Secured Implicit Geographic Forwarding (DWSIGF) routing protocol for Wireless Sensor Network (WSN) has been proposed to achieve a minimal selection of malicious nodes by introducing a dynamic collection window period to the protocol’s semantics. However, its selection scheme suffers substantial packet losses due to the utilization of a single distance based parameter for node selection. In this paper, we propose a Fuzzy-based Geographic Forwarding protocol (FuGeF) to minimize packet loss, while maintaining performance. The FuGeF utilizes a new form of dynamism and introduces three selection parameters: remaining energy, connectivity cost, and progressive distance, as well as a Fuzzy Logic System (FLS) for node selection. These introduced mechanisms ensure the appropriate selection of a non-malicious node. Extensive simulation experiments have been conducted to evaluate the performance of the proposed FuGeF protocol as compared to DWSIGF variants. The simulation results show that the proposed FuGeF outperforms the two DWSIGF variants (DWSIGF-P and DWSIGF-R) in terms of packet delivery. PMID:27338411

A Draft Test Protocol for Detecting Possible Biohazards in Martian Samples Returned to Earth

NASA Technical Reports Server (NTRS)

Rummel, John D.; Race, Margaret S.; DeVinenzi, Donald L.; Schad, P. Jackson; Stabekis, Pericles D.; Viso, Michel; Acevedo, Sara E.

2002-01-01

This document presents the first complete draft of a protocol for detecting possible biohazards in Mars samples returned to Earth; it is the final product of the Mars Sample Handling Protocol Workshop Series, convened in 2000-2001 by NASA's Planetary Protection Officer. The goal of the five-workshop Series vas to develop a comprehensive protocol by which returned martian sample materials could be assessed for the presence of any biological hazard(s) while safeguarding the purity of the samples from possible terrestrial contamination The reference numbers for the proceedings from the five individual Workshops.

Sequential extraction protocol for organic matter from soils and sediments using high resolution mass spectrometry

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

Tfaily, Malak M.; Chu, Rosalie K.; Toyoda, Jason

A vast number of organic compounds are present in soil organic matter (SOM) and play an important role in the terrestrial carbon cycle, facilitate interactions between organisms, and represent a sink for atmospheric CO2. The diversity of different SOM compounds and their molecular characteristics is a function of the organic source material and biogeochemical history. By understanding how SOM composition changes with sources and the processes by which it is biogeochemically altered in different terrestrial ecosystems, it may be possible to predict nutrient and carbon cycling, response to system perturbations, and impact of climate change will have on SOM composition.more » In this study, a sequential chemical extraction procedure was developed to reveal the diversity of organic matter (OM) in different ecosystems and was compared to the previously published protocol using parallel solvent extraction (PSE). We compared six extraction methods using three sample types, peat soil, spruce forest soil and river sediment, so as to select the best method for extracting a representative fraction of organic matter from soils and sediments from a wide range of ecosystems. We estimated the extraction yield of dissolved organic carbon (DOC) by total organic carbon analysis, and measured the composition of extracted OM using high resolution mass spectrometry. This study showed that OM composition depends primarily on soil and sediment characteristics. Two sequential extraction protocols, progressing from polar to non-polar solvents, were found to provide the highest number and diversity of organic compounds extracted from the soil and sediments. Water (H2O) is the first solvent used for both protocols followed by either co-extraction with methanol-chloroform (MeOH-CHCl3) mixture, or acetonitrile (ACN) and CHCl3 sequentially. The sequential extraction protocol developed in this study offers improved sensitivity, and requires less sample compared to the PSE workflow where a new sample is used for each solvent type. Furthermore, a comparison of SOM composition from the different sample types revealed that our sequential protocol allows for ecosystem comparisons based on the diversity of compounds present, which in turn could provide new insights about source and processing of organic compounds in different soil and sediment types.« less

Sequential extraction protocol for organic matter from soils and sediments using high resolution mass spectrometry.

PubMed

Tfaily, Malak M; Chu, Rosalie K; Toyoda, Jason; Tolić, Nikola; Robinson, Errol W; Paša-Tolić, Ljiljana; Hess, Nancy J

2017-06-15

A vast number of organic compounds are present in soil organic matter (SOM) and play an important role in the terrestrial carbon cycle, facilitate interactions between organisms, and represent a sink for atmospheric CO 2 . The diversity of different SOM compounds and their molecular characteristics is a function of the organic source material and biogeochemical history. By understanding how SOM composition changes with sources and the processes by which it is biogeochemically altered in different terrestrial ecosystems, it may be possible to predict nutrient and carbon cycling, response to system perturbations, and impact of climate change will have on SOM composition. In this study, a sequential chemical extraction procedure was developed to reveal the diversity of organic matter (OM) in different ecosystems and was compared to the previously published protocol using parallel solvent extraction (PSE). We compared six extraction methods using three sample types, peat soil, spruce forest soil and river sediment, so as to select the best method for extracting a representative fraction of organic matter from soils and sediments from a wide range of ecosystems. We estimated the extraction yield of dissolved organic carbon (DOC) by total organic carbon analysis, and measured the composition of extracted OM using high resolution mass spectrometry. This study showed that OM composition depends primarily on soil and sediment characteristics. Two sequential extraction protocols, progressing from polar to non-polar solvents, were found to provide the highest number and diversity of organic compounds extracted from the soil and sediments. Water (H 2 O) is the first solvent used for both protocols followed by either co-extraction with methanol-chloroform (MeOH-CHCl 3 ) mixture, or acetonitrile (ACN) and CHCl 3 sequentially. The sequential extraction protocol developed in this study offers improved sensitivity, and requires less sample compared to the PSE workflow where a new sample is used for each solvent type. Furthermore, a comparison of SOM composition from the different sample types revealed that our sequential protocol allows for ecosystem comparisons based on the diversity of compounds present, which in turn could provide new insights about source and processing of organic compounds in different soil and sediment types. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of cerebrospinal fluid leakage by specific measurement of transferrin glycoforms.

PubMed

Kwon, Seok-Joon; Zhang, Fuming; Dordick, Jonathan S; Sonstein, William J; Linhardt, Robert J

2015-10-01

A simple and rapid detection of cerebrospinal fluid (CSF) leakage would benefit spine surgeons making critical postoperative decisions on patient care. We have assessed novel approaches to selectively determine CSF β2-transferrin (β2TF), an asialo-transferrin (aTF) biomarker, without interference from serum sialo-transferrin (sTF) in test samples. First, we performed mild periodate oxidation to selectively generate aldehyde groups in sTF for capture with magnetic hydrazide microparticles, and selective removal with a magnetic separator. Using this protocol sTF was selectively removed from mixtures of CSF and serum containing CSF aTF (β2TF) and serum sTF, respectively. Second, a two-step enzymatic method was developed with neuraminidase and galactose oxidase for generating aldehyde groups in sTF present in CSF and serum mixtures for magnetic hydrazide microparticle capture. After selectively removing sTF from mixtures of CSF and serum, ELISA could detect significant TF signal only in CSF, while the TF signal in serum was negligible. The new approach for selective removal of only sTF in test samples will be promising for the required intervention by a spine surgeon. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antimicrobial usage and risk of retreatment for mild to moderate clinical mastitis cases on dairy farms following on-farm bacterial culture and selective therapy.

PubMed

McDougall, S; Niethammer, J; Graham, E M

2018-03-01

To assess antimicrobial usage for treatment of mild to moderate clinical mastitis, and risk of retreatment, following implementation of an on-farm bacterial culture system and selective therapy based on culture results, and to assess compliance with treatment decision tree protocols and the level of agreement between results from on-farm culture and laboratory-based microbiology methods. Herdowners from seven dairy herds were asked to collect milk samples from cases of mild to moderate clinical mastitis between July 2015 and May 2016. All samples were cultured on-farm using a commercially available selective media and were also submitted for laboratory-based culture. Within sequential pairs of cows with mastitis, half were assigned to be treated without regard to culture results (Blanket group), and half were treated based on the on-farm culture results (Selective group) according to decision tree diagrams provided to the farmers. Culture results, treatments, and retreatments for clinical mastitis were recorded. The sum of the daily doses of antimicrobials used per cow, the number of retreatments and interval to first retreatment were compared between treatment groups. The geometric mean sum of daily doses for quarters assigned to the Selective (1.72 (95% CI=1.55-1.90)) group was lower than for the Blanket (2.38 (95% CI=2.17-2.60)) group (p=0.005). The percentage of cows retreated for clinical mastitis did not differ between the Selective (21.7 (95% CI=10.5-25.9)%) and Blanket (26.1 (95% CI=20.9-31.3)%) groups (p=0.13), and there was no difference between groups in the hazard that cows would be retreated within 60 days of enrolment (hazard ratio=0.82 (95% CI=0.39-1.69); p=0.59). Compliance with the treatment protocols was higher amongst quarters assigned to the Selective (199/233; 85.4%) compared with the Blanket (171/249; 68.7%) group (p<0.001), and varied between farms from 64-94%. The overall agreement between results from on-farm and laboratory culture was 188/331 (56.9%; kappa=0.31; p<0.001), but varied between farms from 44.7-88.2% (p<0.001). Use of on-farm culture with selective antimicrobial therapy resulted in approximately 25% lower antimicrobial usage, but was not associated with an increase in the proportion of cows retreated for clinical mastitis. This study has demonstrated that on-farm culture and selective therapy based on culture results can be implemented on-farm. However, farms varied in their implementation of both the treatment protocols and microbiology procedures. Where such systems are to be used on-farm, specific training and on-going monitoring is required.

A Constrained and Versioned Data Model for TEAM Data

NASA Astrophysics Data System (ADS)

Andelman, S.; Baru, C.; Chandra, S.; Fegraus, E.; Lin, K.

2009-04-01

The objective of the Tropical Ecology Assessment and Monitoring Network (www.teamnetwork.org) is "To generate real time data for monitoring long-term trends in tropical biodiversity through a global network of TEAM sites (i.e. field stations in tropical forests), providing an early warning system on the status of biodiversity to effectively guide conservation action". To achieve this, the TEAM Network operates by collecting data via standardized protocols at TEAM Sites. The standardized TEAM protocols include the Climate, Vegetation and Terrestrial Vertebrate Protocols. Some sites also implement additional protocols. There are currently 7 TEAM Sites with plans to grow the network to 15 by June 30, 2009 and 50 TEAM Sites by the end of 2010. At each TEAM Site, data is gathered as defined by the protocols and according to a predefined sampling schedule. The TEAM data is organized and stored in a database based on the TEAM spatio-temporal data model. This data model is at the core of the TEAM Information System - it consumes and executes spatio-temporal queries, and analytical functions that are performed on TEAM data, and defines the object data types, relationships and operations that maintain database integrity. The TEAM data model contains object types including types for observation objects (e.g. bird, butterfly and trees), sampling unit, person, role, protocol, site and the relationship of these object types. Each observation data record is a set of attribute values of an observation object and is always associated with a sampling unit, an observation timestamp or time interval, a versioned protocol and data collectors. The operations on the TEAM data model can be classified as read operations, insert operations and update operations. Following are some typical operations: The operation get(site, protocol, [sampling unit block, sampling unit,] start time, end time) returns all data records using the specified protocol and collected at the specified site, block, sampling unit and time range. The operation insertSamplingUnit(sampling unit, site, protocol) saves a new sampling unit into the data model and links it with the site and protocol. The operation updateSampligUnit(sampling_unit_id, attribute, value) changes the attribute (e.g. latitude or longitude) of the sampling unit to the specified value. The operation insertData(observation record, site, protocol, sampling unit, timestamps, data collectors) saves a new observation record into the database and associates it with specified objects. The operation updateData(protocol, data_id, attribute, value) modifies the attribute of an existing observation record to the specified value. All the insert or update operations require: 1) authorization to ensure the user has necessary privileges to perform the operation; 2) timestamp validation to ensure the observation timestamps are in the designated time range specified in the sampling schedule; 3) data validation to check that the data records use correct taxonomy terms and data values. No authorization is performed for get operations, but under some specific condition, a username may be required for the purpose of authentication. Along with the validations above, the TEAM data model also supports human based data validation on observed data through the Data Review subsystem to ensure data quality. The data review is implemented by adding two attributes review_tag and review_comment to each observation data record. The attribute review_tag is used by a reviewer to specify the quality of data, and the attribute review_comment is for reviewers to give more information when a problem is identified. The review_tag attribute can be populated by either the system conducting QA/QC tests or by pre-specified scientific experts. The following is the review operation, which is actually a special case of the operation updateData: The operation updateReview(protocol, data_id, judgment, comment) sets the attribute review_tag and review_comment to the specified values. By systematically tracking every step, The TEAM data model can roll back to any previous state. This is achieved by introducing a historical data container for each editable object type. When the operation updateData is applied to an object to modify its attribute, the object will be tagged with the current timestamp and the name of the user who conducts the operation, the tagged object will then be moved into the historical data container, and finally a new object will be created with the new value for the specified attribute. The diagram illustrates the architecture of the TEAM data management system. A data collector can use the Data Ingestion subsystem to load new data records into the TEAM data model. The system establishes a first level of review (i.e. meets minimum data standards via QA/QC tests). Further review is done via experts and they can verify and provide their comments on data records through the Data Review subsystem. The data editor can then address data records based on the reviewer's comments. Users can use the Data Query and Download application to find data by sites, protocols and time ranges. The Data Query and Download system packages selected data with the data license and important metadata information into a single package and delivers it to the user.

In vitro selection and amplification protocols for isolation of aptameric sensors for small molecules

PubMed Central

Yang, Kyung-Ae; Pei, Renjun; Stojanovic, Milan N.

2016-01-01

We recently optimized a procedure that directly yields aptameric sensors for small molecules in so-called structure-switching format. The protocol has a high success rate, short time, and is sufficiently simple to be readily implemented in a non-specialist laboratory. We provide a stepwise guide to this selection protocol. PMID:27155227

Structure refinement of membrane proteins via molecular dynamics simulations.

PubMed

Dutagaci, Bercem; Heo, Lim; Feig, Michael

2018-07-01

A refinement protocol based on physics-based techniques established for water soluble proteins is tested for membrane protein structures. Initial structures were generated by homology modeling and sampled via molecular dynamics simulations in explicit lipid bilayer and aqueous solvent systems. Snapshots from the simulations were selected based on scoring with either knowledge-based or implicit membrane-based scoring functions and averaged to obtain refined models. The protocol resulted in consistent and significant refinement of the membrane protein structures similar to the performance of refinement methods for soluble proteins. Refinement success was similar between sampling in the presence of lipid bilayers and aqueous solvent but the presence of lipid bilayers may benefit the improvement of lipid-facing residues. Scoring with knowledge-based functions (DFIRE and RWplus) was found to be as good as scoring using implicit membrane-based scoring functions suggesting that differences in internal packing is more important than orientations relative to the membrane during the refinement of membrane protein homology models. © 2018 Wiley Periodicals, Inc.

Recent advances in hopanoids analysis: Quantification protocols overview, main research targets and selected problems of complex data exploration.

PubMed

Zarzycki, Paweł K; Portka, Joanna K

2015-09-01

Pentacyclic triterpenoids, particularly hopanoids, are organism-specific compounds and are generally considered as useful biomarkers that allow fingerprinting and classification of biological, environmental and geological samples. Simultaneous quantification of various hopanoids together with battery of related non-polar and low-molecular mass compounds may provide principal information for geochemical and environmental research focusing on both modern and ancient investigations. Target compounds can be derived from microbial biomass, water columns, sediments, coals, crude fossils or rocks. This create number of analytical problems due to different composition of the analytical matrix and interfering compounds and therefore, proper optimization of quantification protocols for such biomarkers is still the challenge. In this work we summarizing typical analytical protocols that were recently applied for quantification of hopanoids like compounds from different samples. Main steps including components of interest extraction, pre-purification, fractionation, derivatization and quantification involving gas (1D and 2D) as well as liquid separation techniques (liquid-liquid extraction, solid-phase extraction, planar and low resolution column chromatography, high-performance liquid chromatography) are described and discussed from practical point of view, mainly based on the experimental papers that were published within last two years, where significant increase in hopanoids research was noticed. The second aim of this review is to describe the latest research trends concerning determination of hopanoids and related low-molecular mass lipids analyzed in various samples including sediments, rocks, coals, crude oils and plant fossils as well as stromatolites and microbial biomass cultivated under different conditions. It has been found that majority of the most recent papers are based on uni- or bivariate approach for complex data analysis. Data interpretation involves number of physicochemical parameters and hopanoids quantities or given biomarkers mass ratios derived from high-throughput separation and detection systems, typically GC-MS and HPLC-MS. Based on quantitative data reported in recently published experimental works it has been demonstrated that multivariate data analysis using e.g. principal components computations may significantly extend our knowledge concerning proper biomarkers selection and samples classification by means of hopanoids and related non-polar compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

A prospective gating method to acquire a diverse set of free-breathing CT images for model-based 4DCT

NASA Astrophysics Data System (ADS)

O'Connell, D.; Ruan, D.; Thomas, D. H.; Dou, T. H.; Lewis, J. H.; Santhanam, A.; Lee, P.; Low, D. A.

2018-02-01

Breathing motion modeling requires observation of tissues at sufficiently distinct respiratory states for proper 4D characterization. This work proposes a method to improve sampling of the breathing cycle with limited imaging dose. We designed and tested a prospective free-breathing acquisition protocol with a simulation using datasets from five patients imaged with a model-based 4DCT technique. Each dataset contained 25 free-breathing fast helical CT scans with simultaneous breathing surrogate measurements. Tissue displacements were measured using deformable image registration. A correspondence model related tissue displacement to the surrogate. Model residual was computed by comparing predicted displacements to image registration results. To determine a stopping criteria for the prospective protocol, i.e. when the breathing cycle had been sufficiently sampled, subsets of N scans where 5  ⩽  N  ⩽  9 were used to fit reduced models for each patient. A previously published metric was employed to describe the phase coverage, or ‘spread’, of the respiratory trajectories of each subset. Minimum phase coverage necessary to achieve mean model residual within 0.5 mm of the full 25-scan model was determined and used as the stopping criteria. Using the patient breathing traces, a prospective acquisition protocol was simulated. In all patients, phase coverage greater than the threshold necessary for model accuracy within 0.5 mm of the 25 scan model was achieved in six or fewer scans. The prospectively selected respiratory trajectories ranked in the (97.5  ±  4.2)th percentile among subsets of the originally sampled scans on average. Simulation results suggest that the proposed prospective method provides an effective means to sample the breathing cycle with limited free-breathing scans. One application of the method is to reduce the imaging dose of a previously published model-based 4DCT protocol to 25% of its original value while achieving mean model residual within 0.5 mm.

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites.

PubMed

Xie, Li; Chen, Liqin; Gu, Pan; Wei, Lanlan; Kang, Xuejun

2018-03-01

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

23 CFR 1340.5 - Selection of observation sites.

Code of Federal Regulations, 2013 CFR

2013-04-01

... STATE OBSERVATIONAL SURVEYS OF SEAT BELT USE Survey Design Requirements § 1340.5 Selection of... observation sites. The survey design shall include at a minimum the following protocols: (1) Protocol when...

23 CFR 1340.5 - Selection of observation sites.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STATE OBSERVATIONAL SURVEYS OF SEAT BELT USE Survey Design Requirements § 1340.5 Selection of... observation sites. The survey design shall include at a minimum the following protocols: (1) Protocol when...

23 CFR 1340.5 - Selection of observation sites.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STATE OBSERVATIONAL SURVEYS OF SEAT BELT USE Survey Design Requirements § 1340.5 Selection of... observation sites. The survey design shall include at a minimum the following protocols: (1) Protocol when...

Characterizing biological variability in livestock blood cholinesterase activity for biomonitoring organophosphate nerve agent exposure

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

Halbrook, R.S.; Shugart, L.R.; Watson, A.P.

1992-09-01

A biomonitoring protocol, using blood cholinesterase (ChE) activity in livestock as a monitor of potential organophosphate nerve agent exposure during the planned destruction of US unitary chemical warfare agent stockpiles, is described. The experimental design included analysis of blood ChE activity in individual healthy sheep, horses, and dairy and beef cattle during a 10- to 12-month period. Castrated and sexually intact males, pregnant and lactating females, and adult and immature animals were examined through at least one reproductive cycle. The same animals were used throughout the period of observation and were not exposed to ChE-inhibiting organophosphate or carbamate compounds. Amore » framework for an effective biomonitoring protocol within a monitoring area includes establishing individual baseline blood ChE activity for a sentinel group of 6 animals on the bases of blood samples collected over a 6-month period, monthly collection of blood samples for ChE-activity determination during monitoring, and selection of adult animals as sentinels. Exposure to ChE-inhibiting compounds would be suspected when all blood ChE activity of all animals within the sentinel group are decreased greater than 20% from their own baseline value. Sentinel species selection is primarily a logistical and operational concern; however, sheep appear to be the species of choice because within-individual baseline ChE activity and among age and gender group ChE activity in sheep had the least variability, compared with data from other species. This protocol provides an effective and efficient means for detecting abnormal depressions in blood ChE activity in livestock and can serve as a valuable indicator of the extent of actual plume movement and/or deposition in the event of organophosphate nerve agent release.« less

Isolation of 1E4 IgM Anti-Fasciola hepatica Rediae Monoclonal Antibody from Ascites: Comparison of Two Purification Protocols.

PubMed

Alba, Annia; Marcet, Ricardo; Otero, Oscar; Hernández, Hilda M; Figueredo, Mabel; Sarracent, Jorge

2016-02-01

Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were compared to purify, from ascites, the 1E4 IgM monoclonal antibody (mAb) previously raised against the stage of redia of the trematode Fasciola hepatica. This immunoglobulin is used as capture antibody in an immunoenzymatic assay to detect parasite ongoing infection in its intermediate hosts. The first purification protocol of the 1E4 mAb involved two chromatographic steps: an affinity chromatography on a Concanavalin A matrix followed by size exclusion chromatography. An immunoaffinity chromatography was selected as the second protocol for one-step purification of the antibody using the crude extract of adult parasites coupled to a commercial matrix. Immunoreactivity of the fractions during purification schemes was assessed by indirect immunoenzymatic assays against the crude extract of F. hepatica rediae, while purity was estimated by protein electrophoresis. Losses on the recovery of the antibody isolated by the first purification protocol occurred due to protein precipitation during the concentration of the sample and to low resolution of the size exclusion molecular chromatography step regarding this particular immunoglobulin. The immunoaffinity chromatography using F. hepatica antigens as ligands proved to be the most suitable protocol yielding a pure and immunoreactive antibody. The purification protocols used are discussed regarding efficiency and difficulties.

A direct morphometric comparison of five labeling protocols for multi-atlas driven automatic segmentation of the hippocampus in Alzheimer's disease.

PubMed

Nestor, Sean M; Gibson, Erin; Gao, Fu-Qiang; Kiss, Alex; Black, Sandra E

2013-02-01

Hippocampal volumetry derived from structural MRI is increasingly used to delineate regions of interest for functional measurements, assess efficacy in therapeutic trials of Alzheimer's disease (AD) and has been endorsed by the new AD diagnostic guidelines as a radiological marker of disease progression. Unfortunately, morphological heterogeneity in AD can prevent accurate demarcation of the hippocampus. Recent developments in automated volumetry commonly use multi-template fusion driven by expert manual labels, enabling highly accurate and reproducible segmentation in disease and healthy subjects. However, there are several protocols to define the hippocampus anatomically in vivo, and the method used to generate atlases may impact automatic accuracy and sensitivity - particularly in pathologically heterogeneous samples. Here we report a fully automated segmentation technique that provides a robust platform to directly evaluate both technical and biomarker performance in AD among anatomically unique labeling protocols. For the first time we test head-to-head the performance of five common hippocampal labeling protocols for multi-atlas based segmentation, using both the Sunnybrook Longitudinal Dementia Study and the entire Alzheimer's Disease Neuroimaging Initiative 1 (ADNI-1) baseline and 24-month dataset. We based these atlas libraries on the protocols of (Haller et al., 1997; Killiany et al., 1993; Malykhin et al., 2007; Pantel et al., 2000; Pruessner et al., 2000), and a single operator performed all manual tracings to generate de facto "ground truth" labels. All methods distinguished between normal elders, mild cognitive impairment (MCI), and AD in the expected directions, and showed comparable correlations with measures of episodic memory performance. Only more inclusive protocols distinguished between stable MCI and MCI-to-AD converters, and had slightly better associations with episodic memory. Moreover, we demonstrate that protocols including more posterior anatomy and dorsal white matter compartments furnish the best voxel-overlap accuracies (Dice Similarity Coefficient=0.87-0.89), compared to expert manual tracings, and achieve the smallest sample sizes required to power clinical trials in MCI and AD. The greatest distribution of errors was localized to the caudal hippocampus and the alveus-fimbria compartment when these regions were excluded. The definition of the medial body did not significantly alter accuracy among more comprehensive protocols. Voxel-overlap accuracies between automatic and manual labels were lower for the more pathologically heterogeneous Sunnybrook study in comparison to the ADNI-1 sample. Finally, accuracy among protocols appears to significantly differ the most in AD subjects compared to MCI and normal elders. Together, these results suggest that selection of a candidate protocol for fully automatic multi-template based segmentation in AD can influence both segmentation accuracy when compared to expert manual labels and performance as a biomarker in MCI and AD. Copyright © 2012 Elsevier Inc. All rights reserved.

A Direct Morphometric Comparison of Five Labeling Protocols for Multi-Atlas Driven Automatic Segmentation of the Hippocampus in Alzheimer’s Disease

PubMed Central

Nestor, Sean M.; Gibson, Erin; Gao, Fu-Qiang; Kiss, Alex; Black, Sandra E.

2012-01-01

Hippocampal volumetry derived from structural MRI is increasingly used to delineate regions of interest for functional measurements, assess efficacy in therapeutic trials of Alzheimer’s disease (AD) and has been endorsed by the new AD diagnostic guidelines as a radiological marker of disease progression. Unfortunately, morphological heterogeneity in AD can prevent accurate demarcation of the hippocampus. Recent developments in automated volumetry commonly use multitemplate fusion driven by expert manual labels, enabling highly accurate and reproducible segmentation in disease and healthy subjects. However, there are several protocols to define the hippocampus anatomically in vivo, and the method used to generate atlases may impact automatic accuracy and sensitivity – particularly in pathologically heterogeneous samples. Here we report a fully automated segmentation technique that provides a robust platform to directly evaluate both technical and biomarker performance in AD among anatomically unique labeling protocols. For the first time we test head-to-head the performance of five common hippocampal labeling protocols for multi-atlas based segmentation, using both the Sunnybrook Longitudinal Dementia Study and the entire Alzheimer’s Disease Neuroimaging Initiative 1 (ADNI-1) baseline and 24-month dataset. We based these atlas libraries on the protocols of (Haller et al., 1997; Killiany et al., 1993; Malykhin et al., 2007; Pantel et al., 2000; Pruessner et al., 2000), and a single operator performed all manual tracings to generate de facto “ground truth” labels. All methods distinguished between normal elders, mild cognitive impairment (MCI), and AD in the expected directions, and showed comparable correlations with measures of episodic memory performance. Only more inclusive protocols distinguished between stable MCI and MCI-to-AD converters, and had slightly better associations with episodic memory. Moreover, we demonstrate that protocols including more posterior anatomy and dorsal white matter compartments furnish the best voxel-overlap accuracies (Dice Similarity Coefficient = 0.87–0.89), compared to expert manual tracings, and achieve the smallest sample sizes required to power clinical trials in MCI and AD. The greatest distribution of errors was localized to the caudal hippocampus and alveus-fimbria compartment when these regions were excluded. The definition of the medial body did not significantly alter accuracy among more comprehensive protocols. Voxel-overlap accuracies between automatic and manual labels were lower for the more pathologically heterogeneous Sunnybrook study in comparison to the ADNI-1 sample. Finally, accuracy among protocols appears to significantly differ the most in AD subjects compared to MCI and normal elders. Together, these results suggest that selection of a candidate protocol for fully automatic multi-template based segmentation in AD can influence both segmentation accuracy when compared to expert manual labels and performance as a biomarker in MCI and AD. PMID:23142652

A multigear protocol for sampling crayfish assemblages in Gulf of Mexico coastal streams

Treesearch

William R. Budnick; William E. Kelso; Susan B. Adams; Michael D. Kaller

2018-01-01

Identifying an effective protocol for sampling crayfish in streams that vary in habitat and physical/chemical characteristics has proven problematic. We evaluated an active, combined-gear (backpack electrofishing and dipnetting) sampling protocol in 20 Coastal Plain streams in Louisiana. Using generalized linear models and rarefaction curves, we evaluated environmental...

How to Improve the Peer Review Method: Free-Selection vs Assigned-Pair Protocol Evaluated in a Computer Networking Course

ERIC Educational Resources Information Center

Papadopoulos, Pantelis M.; Lagkas, Thomas D.; Demetriadis, Stavros N.

2012-01-01

This study provides field research evidence on the efficiency of a "free-selection" peer review assignment protocol as compared to the typically implemented "assigned-pair" protocol. The study employed 54 sophomore students who were randomly assigned into three groups: Assigned-Pair (AP) (the teacher assigns student works for review to student…

A Randomized Trial Comparing Mail versus In-Office Distribution of the CAHPS Clinician and Group Survey

PubMed Central

Anastario, Michael P; Rodriguez, Hector P; Gallagher, Patricia M; Cleary, Paul D; Shaller, Dale; Rogers, William H; Bogen, Karen; Safran, Dana Gelb

2010-01-01

Objective To assess the effect of survey distribution protocol (mail versus handout) on data quality and measurement of patient care experiences. Data Sources/Study Setting Multisite randomized trial of survey distribution protocols. Analytic sample included 2,477 patients of 15 clinicians at three practice sites in New York State. Data Collection/Extraction Methods Mail and handout distribution modes were alternated weekly at each site for 6 weeks. Principal Findings Handout protocols yielded an incomplete distribution rate (74 percent) and lower overall response rates (40 percent versus 58 percent) compared with mail. Handout distribution rates decreased over time and resulted in more favorable survey scores compared with mailed surveys. There were significant mode–physician interaction effects, indicating that data cannot simply be pooled and adjusted for mode. Conclusions In-office survey distribution has the potential to bias measurement and comparison of physicians and sites on patient care experiences. Incomplete distribution rates observed in-office, together with between-office differences in distribution rates and declining rates over time suggest staff may be burdened by the process and selective in their choice of patients. Further testing with a larger physician and site sample is important to definitively establish the potential role for in-office distribution in obtaining reliable, valid assessment of patient care experiences. PMID:20579126

An accessible protocol for solid-phase extraction of N-linked glycopeptides through reductive amination by amine-functionalized magnetic nanoparticles.

PubMed

Zhang, Ying; Kuang, Min; Zhang, Lijuan; Yang, Pengyuan; Lu, Haojie

2013-06-04

In light of the significance of glycosylation for wealthy biological events, it is important to prefractionate glycoproteins/glycopeptides from complex biological samples. Herein, we reported a novel protocol of solid-phase extraction of glycopeptides through a reductive amination reaction by employing the easily accessible 3-aminopropyltriethoxysilane (APTES)-functionalized magnetic nanoparticles. The amino groups from APTES, which were assembled onto the surface of the nanoparticles through a one-step silanization reaction, could conjugate with the aldehydes from oxidized glycopeptides and, therefore, completed the extraction. To the best of our knowledge, this is the first example of applying the reductive amination reaction into the isolation of glycopeptides. Due to the elimination of the desalting step, the detection limit of glycopeptides was improved by 2 orders of magnitude, compared to the traditional hydrazide chemistry-based solid phase extraction, while the extraction time was shortened to 4 h, suggesting the high sensitivity, specificity, and efficiency for the extraction of N-linked glycopeptides by this method. In the meantime, high selectivity toward glycoproteins was also observed in the separation of Ribonuclease B from the mixtures contaminated with bovine serum albumin. What's more, this technique required significantly less sample volume, as demonstrated in the successful mapping of glycosylation of human colorectal cancer serum with the sample volume as little as 5 μL. Because of all these attractive features, we believe that the innovative protocol proposed here will shed new light on the research of glycosylation profiling.

Screening protocol for dysphagia in adults: comparison with videofluoroscopic findings.

PubMed

Sassi, Fernanda C; Medeiros, Gisele C; Zilberstein, Bruno; Jayanthi, Shri Krishna; de Andrade, Claudia R F

2017-12-01

To compare the videofluoroscopic findings of patients with suspected oropharyngeal dysphagia with the results of a clinical screening protocol. A retrospective observational cohort study was conducted on all consecutive patients with suspected oropharyngeal dysphagia between March 2015 and February 2016 who were assigned to receive a videofluoroscopic assessment of swallowing. All patients were first submitted to videofluoroscopy and then to the clinical assessment of swallowing. The clinical assessment was performed within the first 24 hours after videofluoroscopy. The videofluoroscopy results were analyzed regarding penetration/aspiration using an 8-point multidimensional perceptual scale. The accuracy of the clinical protocol was analyzed using the sensitivity, specificity, likelihood ratios and predictive values. The selected sample consisted of 50 patients. The clinical protocol presented a sensitivity of 50% and specificity of 95%, with an accuracy of 88%. "Cough" and "wet-hoarse" vocal quality after/during swallowing were clinical indicators that appeared to correctly identify the presence of penetration/aspiration risk. The clinical protocol used in the present study is a simple, rapid and reliable clinical assessment. Despite the absence of a completely satisfactory result, especially in terms of the sensitivity and positive predictive values, we suggest that lower rates of pneumonia can be achieved using a formal dysphagia screening method.

Consumer use of health-related endorsements on food labels in the United Kingdom and Australia.

PubMed

Rayner, M; Boaz, A; Higginson, C

2001-01-01

The objective of this research was to examine how consumers use health-related food endorsements on food labels. Three endorsement programs were examined: those of the two major retailers in the United Kingdom, Tesco and Sainsbury's, and the "Pick the Tick" program of the National Heart Foundation of Australia. The main methodology used was protocol analysis. This involves the subject "thinking aloud" while performing a task--in this case, (a) shopping normally and (b) shopping "healthily" for foods on a predetermined list--to generate a protocol. Each subject was also interviewed to investigate reported use of endorsements. Subjects were a quota sample (N = 44) of shoppers representative of the U.K. and Australian populations. Information about the subjects, the protocols, and interview data were analyzed quantitatively; the protocols were also analyzed qualitatively. Sainsbury's and Australian shoppers never used the endorsements when shopping but Tesco shoppers did, albeit rarely. Tesco shoppers used the endorsement in complex ways and not just as a trigger to food selection. They sometimes used the endorsement to reject endorsed foods. Subjects claimed to use the endorsements even though the protocol analysis revealed no actual use. There are features of the Tesco endorsement program that make it more helpful to consumers than the other programs.

A new standard of visual data representation for imaging mass spectrometry.

PubMed

O'Rourke, Matthew B; Padula, Matthew P

2017-03-01

MALDI imaging MS (IMS) is principally used for cancer diagnostics. In our own experience with publishing IMS data, we have been requested to modify our protocols with respect to the areas of the tissue that are imaged in order to comply with the wider literature. In light of this, we have determined that current methodologies lack effective controls and can potentially introduce bias by only imaging specific areas of the targeted tissue EXPERIMENTAL DESIGN: A previously imaged sample was selected and then cropped in different ways to show the potential effect of only imaging targeted areas. By using a model sample, we were able to effectively show how selective imaging of samples can misinterpret tissue features and by changing the areas that are acquired, according to our new standard, an effective internal control can be introduced. Current IMS sampling convention relies on the assumption that sample preparation has been performed correctly. This prevents users from checking whether molecules have moved beyond borders of the tissue due to delocalization and consequentially products of improper sample preparation could be interpreted as biological features that are of critical importance when encountered in a visual diagnostic. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel qPCR protocol for the specific detection and quantification of the fuel-deteriorating fungus Hormoconis resinae.

PubMed

Martin-Sanchez, Pedro M; Gorbushina, Anna A; Kunte, Hans-Jörg; Toepel, Jörg

2016-07-01

A wide variety of fungi and bacteria are known to contaminate fuels and fuel systems. These microbial contaminants have been linked to fuel system fouling and corrosion. The fungus Hormoconis resinae, a common jet fuel contaminant, is used in this study as a model for developing innovative risk assessment methods. A novel qPCR protocol to detect and quantify H. resinae in, and together with, total fungal contamination of fuel systems is reported. Two primer sets, targeting the markers RPB2 and ITS, were selected for their remarkable specificity and sensitivity. These primers were successfully applied on fungal cultures and diesel samples demonstrating the validity and reliability of the established qPCR protocol. This novel tool allows clarification of the current role of H. resinae in fuel contamination cases, as well as providing a technique to detect fungal outbreaks in fuel systems. This tool can be expanded to other well-known fuel-deteriorating microorganisms.

Sample processing, protocol, and statistical analysis of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) of protein, cell, and tissue samples.

PubMed

Barreto, Goncalo; Soininen, Antti; Sillat, Tarvo; Konttinen, Yrjö T; Kaivosoja, Emilia

2014-01-01

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is increasingly being used in analysis of biological samples. For example, it has been applied to distinguish healthy and osteoarthritic human cartilage. This chapter discusses ToF-SIMS principle and instrumentation including the three modes of analysis in ToF-SIMS. ToF-SIMS sets certain requirements for the samples to be analyzed; for example, the samples have to be vacuum compatible. Accordingly, sample processing steps for different biological samples, i.e., proteins, cells, frozen and paraffin-embedded tissues and extracellular matrix for the ToF-SIMS are presented. Multivariate analysis of the ToF-SIMS data and the necessary data preprocessing steps (peak selection, data normalization, mean-centering, and scaling and transformation) are discussed in this chapter.

An investigation into current protocols and radiographer opinions on contrast extravasation in Irish CT departments.

PubMed

Cleary, N; McNulty, J P; Foley, S J; Kelly, E

2017-11-01

Iodinated contrast extravasation is a serious complication associated with intravenous administration in radiology. Departmental protocols and the radiographer's approach on both prevention techniques and treatment will affect the prevalence of extravasation, and the eventual outcome for the patient when it does occur. To examine contrast extravasation protocols in place in Irish CT departments for alignment with European Society of Urogenital Radiology (ESUR) Guidelines (2014); to establish radiographer's opinions on contrast extravasation; and to examine radiographer adherence to protocols. Contrast extravasation protocols from a purposively selected sample of CT departments across Ireland (n = 6) were compared to ESUR guidelines, followed by an online survey of CT radiographers practicing in the participating centres. All participating CT departments (n = 5) had written protocols in place. High risk patients, such as elderly or unconscious, were identified in most protocols, however, children were mentioned in just one protocol and obese patients were not specified in any. The response rate of CT radiographers was 23% (n = 24). 58% (n = 14) of respondents indicated that contrast extravasation was more likely during CTA examinations. While high levels of confidence in managing extravasation were reported, suggested treatment approaches, and confidence in same, was more variable. Clinical workload in CT departments was also identified as a factor impacting on patient care and management. While contrast extravasation protocols were generally in line with ESUR Guidelines, high risk patients may not be getting sufficient attention. More radiographer awareness of patient monitoring needs, particularly in busy departments with a heavy workload may also reduce extravasation risk, and improve management of same. Copyright © 2017 The College of Radiographers. Published by Elsevier Ltd. All rights reserved.

A distance limited method for sampling downed coarse woody debris

Treesearch

Jeffrey H. Gove; Mark J. Ducey; Harry T. Valentine; Michael S. Williams

2012-01-01

A new sampling method for down coarse woody debris is proposed based on limiting the perpendicular distance from individual pieces to a randomly chosen sample point. Two approaches are presented that allow different protocols to be used to determine field measurements; estimators for each protocol are also developed. Both protocols are compared via simulation against...

An Assessment Protocol for Selective Mutism: Analogue Assessment Using Parents as Facilitators.

ERIC Educational Resources Information Center

Schill, Melissa T.; And Others

1996-01-01

Assesses protocol for conducting a functional analysis of maintaining variables for children with selective mutism. A parent was trained in and later applied various behavior strategies designed to increase speech in an eight-year-old girl with selective mutism. Parent and child ratings of treatment were positive. Presents implications for future…

Measurement of stable isotopic enrichment and concentration of long-chain fatty acyl-carnitines in tissue by HPLC-MS.

PubMed

Sun, Dayong; Cree, Melanie G; Zhang, Xiao-Jun; Bøersheim, Elisabet; Wolfe, Robert R

2006-02-01

We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.

A new, ultra-low latency data transmission protocol for Earthquake Early Warning Systems

NASA Astrophysics Data System (ADS)

Hill, P.; Hicks, S. P.; McGowan, M.

2016-12-01

One measure used to assess the performance of Earthquake Early Warning Systems (EEWS) is the delay time between earthquake origin and issued alert. EEWS latency is dependent on a number of sources (e.g. P-wave propagation, digitisation, transmission, receiver processing, triggering, event declaration). Many regional seismic networks use the SEEDlink protocol; however, packet size is fixed to 512-byte miniSEED records, resulting in transmission latencies of >0.5 s. Data packetisation is seen as one of the main sources of delays in EEWS (Brown et al., 2011). Optimising data-logger and telemetry configurations is a cost-effective strategy to improve EEWS alert times (Behr et al., 2015). Digitisers with smaller, selectable packets can result in faster alerts (Sokos et al., 2016). We propose a new seismic protocol for regional seismic networks benefiting low-latency applications such as EEWS. The protocol, based on Güralp's existing GDI-link format is an efficient and flexible method to exchange data between seismic stations and data centers for a range of network configurations. The main principle is to stream data sample-by-sample instead of fixed-length packets to minimise transmission latency. Self-adaptive packetisation with compression maximises available telemetry bandwidth. Highly flexible metadata fields within GDI-link are compatible with existing miniSEED definitions. Data is sent as integers or floats, supporting a wide range of data formats, including discrete parameters such as Pd & τC for on-site earthquake early warning. Other advantages include: streaming station state-of-health information, instrument control, support of backfilling and fail-over strategies during telemetry outages. Based on tests carried out on the Güralp Minimus data-logger, we show our new protocol can reduce transmission latency to as low as 1 ms. The low-latency protocol is currently being implemented with common processing packages. The results of these tests will help to highlight latency levels that can be achieved with next-generation EEWS.

Effects of strongman training on salivary testosterone levels in a sample of trained men.

PubMed

Ghigiarelli, Jamie J; Sell, Katie M; Raddock, Jessica M; Taveras, Kurt

2013-03-01

Strongman exercises consist of multi-joint movements that incorporate large muscle mass groups and impose a substantial amount of neuromuscular stress. The purpose of this study was to examine salivary testosterone responses from 2 novel strongman training (ST) protocols in comparison with an established hypertrophic (H) protocol reported to acutely elevate testosterone levels. Sixteen men (24 ± 4.4 years, 181.2 ± 6.8 cm, and 95.3 ± 20.3 kg) volunteered to participate in this study. Subjects completed 3 protocols designed to ensure equal total volume (sets and repetitions), rest period, and intensity between the groups. Exercise sets were performed to failure. Exercise selection and intensity (3 sets × 10 repetitions at 75% 1 repetition maximum) were chosen as they reflected commonly prescribed resistance exercise protocols recognized to elicit a large acute hormonal response. In each of the protocols, subjects were required to perform 3 sets to muscle failure of 5 different exercises (tire flip, chain drag, farmers walk, keg carry, and atlas stone lift) with a 2-minute rest interval between sets and a 3-minute rest interval between exercises. Saliva samples were collected pre-exercise (PRE), immediate postexercise (PST), and 30 minutes postexercise (30PST). Delta scores indicated a significant difference between PRE and PST testosterone level within each group (p ≤ 0.05), with no significant difference between the groups. Testosterone levels spiked 136% (225.23 ± 148.01 pg·ml(-1)) for the H group, 74% (132.04 ± 98.09 pg·ml(-1)) for the ST group, and 54% (122.10 ± 140.67 pg·ml) for the mixed strongman/hypertrophy (XST) group. A significant difference for testosterone level occurred over time (PST to 30PST) for the H group p ≤ 0.05. In conclusion, ST elicits an acute endocrine response similar to a recognized H protocol when equated for duration and exercise intensity.

Two alternative DNA extraction methods to improve the detection of Mycobacterium-tuberculosis-complex members in cattle and red deer tissue samples.

PubMed

Fell, Shari; Bröckl, Stephanie; Büttner, Mathias; Rettinger, Anna; Zimmermann, Pia; Straubinger, Reinhard K

2016-09-15

Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis and M. caprae, is a notifiable animal disease in Germany. Diagnostic procedure is based on a prescribed protocol that is published in the framework of German bTB legislation. In this protocol small sample volumes are used for DNA extraction followed by real-time PCR analyses. As mycobacteria tend to concentrate in granuloma and the infected tissue in early stages of infection does not necessarily show any visible lesions, it is likely that DNA extraction from only small tissue samples (20-40 mg) of a randomly chosen spot from the organ and following PCR testing may result in false negative results. In this study two DNA extraction methods were developed to process larger sample volumes to increase the detection sensitivity of mycobacterial DNA in animal tissue. The first extraction method is based on magnetic capture, in which specific capture oligonucleotides were utilized. These nucleotides are linked to magnetic particles and capture Mycobacterium-tuberculosis-complex (MTC) DNA released from 10 to 15 g of tissue material. In a second approach remaining sediments from the magnetic capture protocol were further processed with a less complex extraction protocol that can be used in daily routine diagnostics. A total number of 100 tissue samples from 34 cattle (n = 74) and 18 red deer (n = 26) were analyzed with the developed protocols and results were compared to the prescribed protocol. All three extraction methods yield reliable results by the real-time PCR analysis. The use of larger sample volume led to a sensitivity increase of DNA detection which was shown by the decrease of Ct-values. Furthermore five samples which were tested negative or questionable by the official extraction protocol were detected positive by real time PCR when the alternative extraction methods were used. By calculating the kappa index, the three extraction protocols resulted in a moderate (0.52; protocol 1 vs 3) to almost perfect agreement (1.00; red deer sample testing with all protocols). Both new methods yielded increased detection rates for MTC DNA detection in large sample volumes and consequently improve the official diagnostic protocol.

IJS: An Intelligent Junction Selection Based Routing Protocol for VANET to Support ITS Services.

PubMed

Bhoi, Sourav Kumar; Khilar, Pabitra Mohan

2014-01-01

Selecting junctions intelligently for data transmission provides better intelligent transportation system (ITS) services. The main problem in vehicular communication is high disturbances of link connectivity due to mobility and less density of vehicles. If link conditions are predicted earlier, then there is a less chance of performance degradation. In this paper, an intelligent junction selection based routing protocol (IJS) is proposed to transmit the data in a quickest path, in which the vehicles are mostly connected and have less link connectivity problem. In this protocol, a helping vehicle is set at every junction to control the communication by predicting link failures or network gaps in a route. Helping vehicle at the junction produces a score for every neighboring junction to forward the data to the destination by considering the current traffic information and selects that junction which has minimum score. IJS protocol is implemented and compared with GyTAR, A-STAR, and GSR routing protocols. Simulation results show that IJS performs better in terms of average end-to-end delay, network gap encounter, and number of hops.

IJS: An Intelligent Junction Selection Based Routing Protocol for VANET to Support ITS Services

PubMed Central

Khilar, Pabitra Mohan

2014-01-01

Selecting junctions intelligently for data transmission provides better intelligent transportation system (ITS) services. The main problem in vehicular communication is high disturbances of link connectivity due to mobility and less density of vehicles. If link conditions are predicted earlier, then there is a less chance of performance degradation. In this paper, an intelligent junction selection based routing protocol (IJS) is proposed to transmit the data in a quickest path, in which the vehicles are mostly connected and have less link connectivity problem. In this protocol, a helping vehicle is set at every junction to control the communication by predicting link failures or network gaps in a route. Helping vehicle at the junction produces a score for every neighboring junction to forward the data to the destination by considering the current traffic information and selects that junction which has minimum score. IJS protocol is implemented and compared with GyTAR, A-STAR, and GSR routing protocols. Simulation results show that IJS performs better in terms of average end-to-end delay, network gap encounter, and number of hops. PMID:27433485

A highly efficient protocol for micropropagation of Begonia tuberous.

PubMed

Duong, Tan Nhut; Nguyen, Thanh Hai; Mai, Xuan Phan

2010-01-01

A protocol for micropropagation of begonia was established utilizing a thin cell layer (TCL) system. This system has been employed to produce several thousand shoots per sample. Explant size and position, and plant growth regulators (PGRs) contribute to the tissue morphogenesis. By optimizing the size of the tissue and applying an improved selection procedure, shoots were elongated in 8 weeks of culture, with an average number of 210 +/- 9.7 shoots per segment. This system has facilitated a number of studies using TCL as a model for micropropagation and will enable the large-scale production of begonia. On an average, the best treatment would allow production of about 10,000 plantlets by the micropropagation of the axillary buds of one plant with five petioles, within a period of 8 months.

Dinucleotide repeat polymorphisms in waterfowl (family Anatidae): Characterization of a sex-linked (Z-specific) and 14 autosomal loci

USGS Publications Warehouse

Buchholz, W.G.; Pearce, J.M.; Pierson, B.J.; Scribner, K.T.

1998-01-01

Canada goose (Branta Canadensis) and harlequin duck (Histrionicus histrionicus) DNAs were digested with Sau3AI, and size selected (300-700 bp) fragments were ligated into BamHI-digested pBluscriptII KS+. The enrichment protocol of Ostrander et al.1 was followed. The resulting libraries were screened using a [ƴ-32P]ATP end-labelled (CA)20 oligonucleotides as a hybridization probe. Positive clones were sequenced using cycle-sequencing protocols (Epicentre Technologies, Madison, WI) and primers flanking the inserts. PCR primers were designed to amplify the repeat and yield amplification products of ≈100-200 bp. DNA  samples were screened for variation at these loci using [ƴ-32P]ATP end-labelled primers. The products were resolved using 6% denaturing polyacrylamide gels and autoradiography.

Differentiating primary progressive aphasias in a brief sample of connected speech

PubMed Central

Evans, Emily; O'Shea, Jessica; Powers, John; Boller, Ashley; Weinberg, Danielle; Haley, Jenna; McMillan, Corey; Irwin, David J.; Rascovsky, Katya; Grossman, Murray

2013-01-01

Objective: A brief speech expression protocol that can be administered and scored without special training would aid in the differential diagnosis of the 3 principal forms of primary progressive aphasia (PPA): nonfluent/agrammatic PPA, logopenic variant PPA, and semantic variant PPA. Methods: We used a picture-description task to elicit a short speech sample, and we evaluated impairments in speech-sound production, speech rate, lexical retrieval, and grammaticality. We compared the results with those obtained by a longer, previously validated protocol and further validated performance with multimodal imaging to assess the neuroanatomical basis of the deficits. Results: We found different patterns of impaired grammar in each PPA variant, and additional language production features were impaired in each: nonfluent/agrammatic PPA was characterized by speech-sound errors; logopenic variant PPA by dysfluencies (false starts and hesitations); and semantic variant PPA by poor retrieval of nouns. Strong correlations were found between this brief speech sample and a lengthier narrative speech sample. A composite measure of grammaticality and other measures of speech production were correlated with distinct regions of gray matter atrophy and reduced white matter fractional anisotropy in each PPA variant. Conclusions: These findings provide evidence that large-scale networks are required for fluent, grammatical expression; that these networks can be selectively disrupted in PPA syndromes; and that quantitative analysis of a brief speech sample can reveal the corresponding distinct speech characteristics. PMID:23794681

FAST: Size-Selective, Clog-Free Isolation of Rare Cancer Cells from Whole Blood at a Liquid-Liquid Interface.

PubMed

Kim, Tae-Hyeong; Lim, Minji; Park, Juhee; Oh, Jung Min; Kim, Hyeongeun; Jeong, Hyunjin; Lee, Sun Ju; Park, Hee Chul; Jung, Sungmok; Kim, Byung Chul; Lee, Kyusang; Kim, Mi-Hyun; Park, Do Youn; Kim, Gwang Ha; Cho, Yoon-Kyoung

2017-01-17

Circulating tumor cells (CTCs) have great potential to provide minimally invasive ways for the early detection of cancer metastasis and for the response monitoring of various cancer treatments. Despite the clinical importance and progress of CTC-based cancer diagnostics, most of the current methods of enriching CTCs are difficult to implement in general hospital settings due to complex and time-consuming protocols. Among existing technologies, size-based isolation methods provide antibody-independent, relatively simple, and high throughput protocols. However, the clogging issues and lower than desired recovery rates and purity are the key challenges. In this work, inspired by antifouling membranes with liquid-filled pores in nature, clog-free, highly sensitive (95.9 ± 3.1% recovery rate), selective (>2.5 log depletion of white blood cells), rapid (>3 mL/min), and label-free isolation of viable CTCs from whole blood without prior sample treatment is achieved using a stand-alone lab-on-a-disc system equipped with fluid-assisted separation technology (FAST). Numerical simulation and experiments show that this method provides uniform, clog-free, ultrafast cell enrichment with pressure drops much less than in conventional size-based filtration, at 1 kPa. We demonstrate the clinical utility of the point-of-care detection of CTCs with samples taken from 142 patients suffering from breast, stomach, or lung cancer.

Epidemiology of Psychiatric Disorders in Iranian Children and Adolescents (IRCAP) and Its Relationship with Social Capital, Life Style and Parents' Personality Disorders: Study Protocol.

PubMed

Mohammadi, Mohammad Reza; Ahmadi, Nastaran; Kamali, Koorosh; Khaleghi, Ali; Ahmadi, Ameneh

2017-01-01

Objective: We aimed at designing a cross sectional study to investigate the prevalence of psychiatric disorders in Iranian children and adolescents (IRCAP) and to determine its relationship with social capital, life style, and parents' personality disorders. Method: This cross sectional study was a national project implemented in all provinces of Iran. In this community-based study, using ‎multistage cluster sampling method, we selected 1000 children and adolescents aged 6 to 18 years in each province. The total sample size reached to 31 000. ‎We randomly collected 170 blocks. Then, of each cluster head, we selected 6 cases including 3 cases of each gender in ‎different age groups (6- 9 years, 10- 14 years, and 15- 18 years). The clinical psychologists instructed the participants to complete the Persian version of Kiddie-Sads-‎Present and Lifetime Version (K-SADS-PL). In addition, demographic data (gender, age, education, parent education, and economic situation) and information on lifestyle, social capital, and parents' personality disorders were obtained from the participants. Discussion: IRCAP study presents a protocol for an epidemiological survey on the first estimates for the prevalence of psychiatric disorders in children and adolescents across the country. This large body of data, on a range of individual behavioural and emotional items and scores, allows us to compare the rates and patterns of deviance between urban and rural places of residence in 31 provinces of Iran with non Iranian samples surveyed with the same measures.

[A study of global ecological adaptability and field selection practices of Panax ginseng].

PubMed

Shen, Liang; Wu, Jie; Li, Xi-Wen; Xu, Jiang; Dong, Lin-Lin; Sang, Ming-Chun; Sun, Xi-Wen; Naoki, Fujihara; Chen, Shi-Lin

2016-09-01

Through the development of ecological suitability analysis of producing area and the selection criteria of farmland cultivation in the global range of ginseng, we aim to provide scientific basis for rational planning, production layout and standardized planting of farmland. We analyze the data based on the ecological factors from 271 sample plots of Panax ginseng, including both the traditional producing regions recorded in past dynasties medicinal works and the popular production regions in the world, using global geographic information system for medicinal plant(GMPGIS) developed by ICMM (Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences). We concluded that the suitable producing areas in global for P. ginseng mainly included America, Canada, China, Russia, Japan, North Korea, France, Italy, Ukraine, and South Korea. In addition, the suitable producing areas in China mainly included Heilongjiang, Jilin, Liaoning, Shanxi, Gansu, Hubei, Sichuan, Inner Mongolia, Shandong, and Shanxi. Besides, based on the references and the experience of ginseng-producing and our many years' work on the 1,000-hectare plantation of P. ginseng, we established a standard land selection protocol for cultivation of P. ginseng. The use of GMPGIS to select the most optimum ginseng production regions provides a new scientific basis for introduction, cultivation, tending, protection, cultivation normalization for P. ginseng and the standard land selection protocol would lay a solid foundation for the high quality P. ginseng production. Copyright© by the Chinese Pharmaceutical Association.

A comparison of microscopic and spectroscopic identification methods for analysis of microplastics in environmental samples.

PubMed

Song, Young Kyoung; Hong, Sang Hee; Jang, Mi; Han, Gi Myung; Rani, Manviri; Lee, Jongmyoung; Shim, Won Joon

2015-04-15

The analysis of microplastics in various environmental samples requires the identification of microplastics from natural materials. The identification technique lacks a standardized protocol. Herein, stereomicroscope and Fourier transform infrared spectroscope (FT-IR) identification methods for microplastics (<1mm) were compared using the same samples from the sea surface microlayer (SML) and beach sand. Fragmented microplastics were significantly (p<0.05) underestimated and fiber was significantly overestimated using the stereomicroscope both in the SML and beach samples. The total abundance by FT-IR was higher than by microscope both in the SML and beach samples, but they were not significantly (p>0.05) different. Depending on the number of samples and the microplastic size range of interest, the appropriate identification method should be determined; selecting a suitable identification method for microplastics is crucial for evaluating microplastic pollution. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enhancement of the NMSU Channel Error Simulator to Provide User-Selectable Link Delays

NASA Technical Reports Server (NTRS)

Horan, Stephen; Wang, Ru-Hai

2000-01-01

This is the third in a continuing series of reports describing the development of the Space-to-Ground Link Simulator (SGLS) to be used for testing data transfers under simulated space channel conditions. The SGLS is based upon Virtual Instrument (VI) software techniques for managing the error generation, link data rate configuration, and, now, selection of the link delay value. In this report we detail the changes that needed to be made to the SGLS VI configuration to permit link delays to be added to the basic error generation and link data rate control capabilities. This was accomplished by modifying the rate-splitting VIs to include a buffer the hold the incoming data for the duration selected by the user to emulate the channel link delay. In sample tests of this configuration, the TCP/IP(sub ftp) service and the SCPS(sub fp) service were used to transmit 10-KB data files using both symmetric (both forward and return links set to 115200 bps) and unsymmetric (forward link set at 2400 bps and a return link set at 115200 bps) link configurations. Transmission times were recorded at bit error rates of 0 through 10(exp -5) to give an indication of the link performance. In these tests. we noted separate timings for the protocol setup time to initiate the file transfer and the variation in the actual file transfer time caused by channel errors. Both protocols showed similar performance to that seen earlier for the symmetric and unsymmetric channels. This time, the delays in establishing the file protocol also showed that these delays could double the transmission time and need to be accounted for in mission planning. Both protocols also showed a difficulty in transmitting large data files over large link delays. In these tests, there was no clear favorite between the TCP/IP(sub ftp) and the SCPS(sub fp). Based upon these tests, further testing is recommended to extend the results to different file transfer configurations.

Laser capture microdissection of embryonic cells and preparation of RNA for microarray assays.

PubMed

Redmond, Latasha C; Pang, Christopher J; Dumur, Catherine; Haar, Jack L; Lloyd, Joyce A

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice-isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure(®) LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM.

Laser Capture Microdissection of Embryonic Cells and Preparation of RNA for Microarray Assays

PubMed Central

Redmond, Latasha C.; Pang, Christopher J.; Dumur, Catherine; Haar, Jack L.; Lloyd, Joyce A.

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice–isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure® LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM. PMID:24318813

Energy Efficient Medium Access Control Protocol for Clustered Wireless Sensor Networks with Adaptive Cross-Layer Scheduling.

PubMed

Sefuba, Maria; Walingo, Tom; Takawira, Fambirai

2015-09-18

This paper presents an Energy Efficient Medium Access Control (MAC) protocol for clustered wireless sensor networks that aims to improve energy efficiency and delay performance. The proposed protocol employs an adaptive cross-layer intra-cluster scheduling and an inter-cluster relay selection diversity. The scheduling is based on available data packets and remaining energy level of the source node (SN). This helps to minimize idle listening on nodes without data to transmit as well as reducing control packet overhead. The relay selection diversity is carried out between clusters, by the cluster head (CH), and the base station (BS). The diversity helps to improve network reliability and prolong the network lifetime. Relay selection is determined based on the communication distance, the remaining energy and the channel quality indicator (CQI) for the relay cluster head (RCH). An analytical framework for energy consumption and transmission delay for the proposed MAC protocol is presented in this work. The performance of the proposed MAC protocol is evaluated based on transmission delay, energy consumption, and network lifetime. The results obtained indicate that the proposed MAC protocol provides improved performance than traditional cluster based MAC protocols.

Energy Efficient Medium Access Control Protocol for Clustered Wireless Sensor Networks with Adaptive Cross-Layer Scheduling

PubMed Central

Sefuba, Maria; Walingo, Tom; Takawira, Fambirai

2015-01-01

This paper presents an Energy Efficient Medium Access Control (MAC) protocol for clustered wireless sensor networks that aims to improve energy efficiency and delay performance. The proposed protocol employs an adaptive cross-layer intra-cluster scheduling and an inter-cluster relay selection diversity. The scheduling is based on available data packets and remaining energy level of the source node (SN). This helps to minimize idle listening on nodes without data to transmit as well as reducing control packet overhead. The relay selection diversity is carried out between clusters, by the cluster head (CH), and the base station (BS). The diversity helps to improve network reliability and prolong the network lifetime. Relay selection is determined based on the communication distance, the remaining energy and the channel quality indicator (CQI) for the relay cluster head (RCH). An analytical framework for energy consumption and transmission delay for the proposed MAC protocol is presented in this work. The performance of the proposed MAC protocol is evaluated based on transmission delay, energy consumption, and network lifetime. The results obtained indicate that the proposed MAC protocol provides improved performance than traditional cluster based MAC protocols. PMID:26393608

Chapter A6. Section 6.6. Alkalinity and Acid Neutralizing Capacity

USGS Publications Warehouse

Rounds, Stewart A.; Wilde, Franceska D.

2002-01-01

Alkalinity (determined on a filtered sample) and Acid Neutralizing Capacity (ANC) (determined on a whole-water sample) are measures of the ability of a water sample to neutralize strong acid. Alkalinity and ANC provide information on the suitability of water for uses such as irrigation, determining the efficiency of wastewater processes, determining the presence of contamination by anthropogenic wastes, and maintaining ecosystem health. In addition, alkalinity is used to gain insights on the chemical evolution of an aqueous system. This section of the National Field Manual (NFM) describes the USGS field protocols for alkalinity/ANC determination using either the inflection-point or Gran function plot methods, including calculation of carbonate species, and provides guidance on equipment selection.

A Protocol-Based Decision for Choosing a Proper Surgical Treatment Option for Carotid Artery Stenosis.

PubMed

Jang, E-Wook; Chung, Joonho; Seo, Kwon-Duk; Suh, Sang Hyun; Kim, Yong Bae; Lee, Kyung-Yul

2015-06-01

There are two established surgical treatment options for carotid artery stenosis. Carotid endarterectomy (CEA) has been accepted as a gold standard for surgical treatment while carotid artery stenting (CAS) has recently become an alternative option. Each treatment option has advantages and disadvantages for the treatment outcomes. We propose a protocol for selection of a proper surgical treatment option for carotid artery stenosis. A total of 192 published articles on management of carotid artery stenosis were reviewed. Preoperatively considerable factors which had been repeatedly noted in those articles for the risk/benefits of CEA or CAS were selected. According to those factors, a protocol with four categories was established. CEA or CAS is indicated when the patient has a symptomatic stenosis ≥ 50%, or when the patient has an asymptomatic stenosis ≥ 80%. Each treatment option has absolute indications and favorable indications. Each absolute indication is scored with three points, and each favorable indication, one point. Based on the highest scores, a proper treatment option (CEA or CAS) is selected. We have been treating patients according to this protocol and evaluating the outcomes of our protocol-based decision because this protocol might be helpful in assessment of risk/benefit for selection of a proper surgical treatment option in patients with carotid artery stenosis.

Evaluation protocol for amusia: Portuguese sample.

PubMed

Peixoto, Maria Conceição; Martins, Jorge; Teixeira, Pedro; Alves, Marisa; Bastos, José; Ribeiro, Carlos

2012-12-01

Amusia is a disorder that affects the processing of music. Part of this processing happens in the primary auditory cortex. The study of this condition allows us to evaluate the central auditory pathways. To explore the diagnostic evaluation tests of amusia. The authors propose an evaluation protocol for patients with suspected amusia (after brain injury or complaints of poor musical perception), in parallel with the assessment of central auditory processing, already implemented in the department. The Montreal Evaluation of Battery of amusia was the basis for the selection of the tests. From this comprehensive battery of tests we selected some of the musical examples to evaluate different musical aspects, including memory and perception of music, ability concerning musical recognition and discrimination. In terms of memory there is a test for assessing delayed memory, adapted to the Portuguese culture. Prospective study. Although still experimental, with the possibility of adjustments in the assessment, we believe that this assessment, combined with the study of central auditory processing, will allow us to understand some central lesions, congenital or acquired hearing perception limitations.

Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA.

PubMed

Liu, Yu; Holmstrom, Erik; Yu, Ping; Tan, Kemin; Zuo, Xiaobing; Nesbitt, David J; Sousa, Rui; Stagno, Jason R; Wang, Yun-Xing

2018-05-01

Site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid-liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a 13 C 15 N-labeled segment; (ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues. The flexible procedure enables a wide range of downstream biophysical analyses using precisely localized functionalized nucleotides. All three RNAs were obtained in <2 d, excluding time for preparing reagents and optimizing experimental conditions. With optimization, the protocol can be applied to other RNAs with various labeling schemes, such as ligation of segmentally labeled fragments.

Gossip-Based Dissemination

NASA Astrophysics Data System (ADS)

Friedman, Roy; Kermarrec, Anne-Marie; Miranda, Hugo; Rodrigues, Luís

Gossip-based networking has emerged as a viable approach to disseminate information reliably and efficiently in large-scale systems. Initially introduced for database replication [222], the applicability of the approach extends much further now. For example, it has been applied for data aggregation [415], peer sampling [416] and publish/subscribe systems [845]. Gossip-based protocols rely on a periodic peer-wise exchange of information in wired systems. By changing the way each peer is selected for the gossip communication, and which data are exchanged and processed [451], gossip systems can be used to perform different distributed tasks, such as, among others: overlay maintenance, distributed computation, and information dissemination (a collection of papers on gossip can be found in [451]). In a wired setting, the peer sampling service, allowing for a random or specific peer selection, is often provided as an independent service, able to operate independently from other gossip-based services [416].

Correlation Between Iron and alpha and pi Glutathione-S-Transferase Levels in Humans

DTIC Science & Technology

2012-09-01

assays were performed as described in the Biotrin High Sensitivity Alpha GST EIA kit protocol. First, serum samples were diluted 1:10 with wash solution...immunosorbent assays were performed as described in the Biotrin Pi GST EIA kit protocol. First, plasma samples were diluted 1:5 with sample diluent...immunosorbent assays were performed as described in the AssayMax Human Transferrin ELISA kit protocol. First, serum samples were diluted 1:2000 with MIX

Concordance and Reliability of Photogrammetric Protocols for Measuring the Cervical Lordosis Angle: A Systematic Review of the Literature.

PubMed

de Albuquerque, Priscila Maria Nascimento Martins; de Alencar, Geisa Guimarães; de Oliveira, Daniela Araújo; de Siqueira, Gisela Rocha

2018-01-01

The aim of this study was to examine and interpret the concordance, accuracy, and reliability of photogrammetric protocols available in the literature for evaluating cervical lordosis in an adult population aged 18 to 59 years. A systematic search of 6 electronic databases (MEDLINE via PubMed, LILACS, CINAHL, Scopus, ScienceDirect, and Web of Science) located studies that assessed the reliability and/or concordance and/or accuracy of photogrammetric protocols for evaluating cervical lordosis, compared with radiography. Articles published through April 2016 were selected. Two independent reviewers used a critical appraisal tool (QUADAS and QAREL) to assess the quality of the selected studies. Two studies were included in the review and had high levels of reliability (intraclass correlation coefficient: 0.974-0.98). Only 1 study assessed the concordance between the methods, which was calculated using Pearson's correlation coefficient. To date, the accuracy of photogrammetry has not been investigated thoroughly. We encountered no study in the literature that investigated the accuracy of photogrammetry in diagnosing hyperlordosis of cervical spine. However, both current studies report high levels of intra- and interrater reliability. To increase the level of evidence of photogrammetry in the evaluation of cervical lordosis, it is necessary to conduct further studies using a larger sample to increase the external validity of the findings. Copyright © 2018. Published by Elsevier Inc.

Tissue Sampling Guides for Porcine Biomedical Models.

PubMed

Albl, Barbara; Haesner, Serena; Braun-Reichhart, Christina; Streckel, Elisabeth; Renner, Simone; Seeliger, Frank; Wolf, Eckhard; Wanke, Rüdiger; Blutke, Andreas

2016-04-01

This article provides guidelines for organ and tissue sampling adapted to porcine animal models in translational medical research. Detailed protocols for the determination of sampling locations and numbers as well as recommendations on the orientation, size, and trimming direction of samples from ∼50 different porcine organs and tissues are provided in the Supplementary Material. The proposed sampling protocols include the generation of samples suitable for subsequent qualitative and quantitative analyses, including cryohistology, paraffin, and plastic histology; immunohistochemistry;in situhybridization; electron microscopy; and quantitative stereology as well as molecular analyses of DNA, RNA, proteins, metabolites, and electrolytes. With regard to the planned extent of sampling efforts, time, and personnel expenses, and dependent upon the scheduled analyses, different protocols are provided. These protocols are adjusted for (I) routine screenings, as used in general toxicity studies or in analyses of gene expression patterns or histopathological organ alterations, (II) advanced analyses of single organs/tissues, and (III) large-scale sampling procedures to be applied in biobank projects. Providing a robust reference for studies of porcine models, the described protocols will ensure the efficiency of sampling, the systematic recovery of high-quality samples representing the entire organ or tissue as well as the intra-/interstudy comparability and reproducibility of results. © The Author(s) 2016.

Comparison between publicly accessible publications, registries, and protocols of phase III trials indicated persistence of selective outcome reporting.

PubMed

Zhang, Sheng; Liang, Fei; Li, Wenfeng

2017-11-01

The decision to make protocols of phase III randomized controlled trials (RCTs) publicly accessible by leading journals was a landmark event in clinical trial reporting. Here, we compared primary outcomes defined in protocols with those in publications describing the trials and in trial registration. We identified phase III RCTs published between January 1, 2012, and June 30, 2015, in The New England Journal of Medicine, The Lancet, The Journal of the American Medical Association, and The BMJ with available protocols. Consistency in primary outcomes between protocols and registries (articles) was evaluated. We identified 299 phase III RCTs with available protocols in this analysis. Out of them, 25 trials (8.4%) had some discrepancy for primary outcomes between publications and protocols. Types of discrepancies included protocol-defined primary outcome reported as nonprimary outcome in publication (11 trials, 3.7%), protocol-defined primary outcome omitted in publication (10 trials, 3.3%), new primary outcome introduced in publication (8 trials, 2.7%), protocol-defined nonprimary outcome reported as primary outcome in publication (4 trials, 1.3%), and different timing of assessment of primary outcome (4 trials, 1.3%). Out of trials with discrepancies in primary outcome, 15 trials (60.0%) had discrepancies that favored statistically significant results. Registration could be seen as a valid surrogate of protocol in 237 of 299 trials (79.3%) with regard to primary outcome. Despite unrestricted public access to protocols, selective outcome reporting persists in a small fraction of phase III RCTs. Only studies from four leading journals were included, which may cause selection bias and limit the generalizability of this finding. Copyright © 2017 Elsevier Inc. All rights reserved.

Lead Sampling Protocols: Why So Many and What Do They Tell You?

EPA Science Inventory

Sampling protocols can be broadly categorized based on their intended purpose of 1) Pb regulatory compliance/corrosion control efficacy, 2) Pb plumbing source determination or Pb type identification, and 3) Pb exposure assessment. Choosing the appropriate protocol is crucial to p...

NHEXAS PHASE I MARYLAND STUDY--LIST OF AVAILABLE DOCUMENTS: PROTOCOLS AND SOPS

EPA Science Inventory

This document lists available protocols and SOPs for the NHEXAS Phase I Maryland study. It identifies protocols and SOPs for the following study components: (1) Sample collection and field operations, (2) Sample analysis and general laboratory procedures, (3) Data Analysis Proced...

Defining standardized protocols for determining the efficacy of a postmilking teat disinfectant following experimental exposure of teats to mastitis pathogens.

PubMed

Schukken, Y H; Rauch, B J; Morelli, J

2013-04-01

The objective of this paper was to define standardized protocols for determining the efficacy of a postmilking teat disinfectant following experimental exposure of teats to both Staphylococcus aureus and Streptococcus agalactiae. The standardized protocols describe the selection of cows and herds and define the critical points in performing experimental exposure, performing bacterial culture, evaluating the culture results, and finally performing statistical analyses and reporting of the results. The protocols define both negative control and positive control trials. For negative control trials, the protocol states that an efficacy of reducing new intramammary infections (IMI) of at least 40% is required for a teat disinfectant to be considered effective. For positive control trials, noninferiority to a control disinfectant with a published efficacy of reducing new IMI of at least 70% is required. Sample sizes for both negative and positive control trials are calculated. Positive control trials are expected to require a large trial size. Statistical analysis methods are defined and, in the proposed methods, the rate of IMI may be analyzed using generalized linear mixed models. The efficacy of the test product can be evaluated while controlling for important covariates and confounders in the trial. Finally, standards for reporting are defined and reporting considerations are discussed. The use of the defined protocol is shown through presentation of the results of a recent trial of a test product against a negative control. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection

NASA Astrophysics Data System (ADS)

Tsao, Shih-Ming; Lai, Ji-Ching; Horng, Horng-Er; Liu, Tu-Chen; Hong, Chin-Yih

2017-04-01

Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5‧ and 3‧ termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.

Representativeness of laboratory sampling procedures for the analysis of trace metals in soil.

PubMed

Dubé, Jean-Sébastien; Boudreault, Jean-Philippe; Bost, Régis; Sona, Mirela; Duhaime, François; Éthier, Yannic

2015-08-01

This study was conducted to assess the representativeness of laboratory sampling protocols for purposes of trace metal analysis in soil. Five laboratory protocols were compared, including conventional grab sampling, to assess the influence of sectorial splitting, sieving, and grinding on measured trace metal concentrations and their variability. It was concluded that grinding was the most important factor in controlling the variability of trace metal concentrations. Grinding increased the reproducibility of sample mass reduction by rotary sectorial splitting by up to two orders of magnitude. Combined with rotary sectorial splitting, grinding increased the reproducibility of trace metal concentrations by almost three orders of magnitude compared to grab sampling. Moreover, results showed that if grinding is used as part of a mass reduction protocol by sectorial splitting, the effect of sieving on reproducibility became insignificant. Gy's sampling theory and practice was also used to analyze the aforementioned sampling protocols. While the theoretical relative variances calculated for each sampling protocol qualitatively agreed with the experimental variances, their quantitative agreement was very poor. It was assumed that the parameters used in the calculation of theoretical sampling variances may not correctly estimate the constitutional heterogeneity of soils or soil-like materials. Finally, the results have highlighted the pitfalls of grab sampling, namely, the fact that it does not exert control over incorrect sampling errors and that it is strongly affected by distribution heterogeneity.

In Search of a Dipole Field during the Plio-Pleistocene

NASA Astrophysics Data System (ADS)

Asefaw, H. F.; Tauxe, L.; Staudigel, H.; Shaar, R.; Cai, S.; Cromwell, G.; Behar, N.; Koppers, A. A. P.

2017-12-01

A geocentric axial dipole (GAD) field accounts for the majority of the modern field and is assumed to be a good first order approximation for the time averaged ancient field. A GAD field predicts a latitudinal dependence of intensity. Given this relationship, the intensity of the field measured at the North and South poles should be twice as strong as the intensity recorded at the equator. The current paleointensity database- archived at both http://earth.liv.ac.uk/pint/ and http://earthref.org/MagIC - shows no such dependency over the last 5 Myr (e.g. Lawrence et al., 2009, doi: 10.1029/2008GC002072; Cromwell et al., 2015; doi: 10.1002/2014JB011828). In order to investigate whether better experimental protocol or data selection approaches could resolve the problem, we: 1) applied a new data selection protocol (CCRIT) which has recovered historical field values with high precision and accuracy (Cromwell et al., 2015), 2) re-sampled the fine grained tops of lava flows in Antarctica (77.9° S) that were previously studied for paleodirections but failed to meet our strict selection criteria, 3) sampled cinder cones in the Golan Heights (33.08° N), and 4) acquired data from lava flows from the HSDP2 drill core in Hawaii (19.71° N ). New and published Ar-Ar dates demonstrate that all the samples formed in the last 5 Myr. We conducted IZZI modified Thellier-Thellier experiments and then calculated paleointensities from the samples that passed a set of strict selection criteria. After applying the CCRIT criteria to our data, we find a time averaged paleointensity of 35.7 ±6.86 μT in the Golan Heights, 34.5 μT in Hawaii, and 34.22 ±3.4 μT in Antarctica. New results from Iceland (64° N), published by Cromwell et al. (2015, doi: 10.1002/2014JB011828), also pass the CCRIT criteria and record an average intensity of 33.1 ± 8.3 μT. The average paleointensities from the Golan Heights, Antarctica, Iceland and Hawaii, that span the last 5 Myr and pass the CCRIT criteria, fail to show the variation of intensity with latitude that is expected of an ideal GAD field. The question remains as to why.

Two New Fluorogenic Aptasensors Based on Capped Mesoporous Silica Nanoparticles to Detect Ochratoxin A

PubMed Central

Ribes, Àngela; Santiago‐Felipe, Sara; Bernardos, Andrea; Marcos, M. Dolores; Pardo, Teresa; Sancenón, Félix; Aznar, Elena

2017-01-01

Abstract Aptamers have been used as recognition elements for several molecules due to their great affinity and selectivity. Additionally, mesoporous nanomaterials have demonstrated great potential in sensing applications. Based on these concepts, we report herein the use of two aptamer‐capped mesoporous silica materials for the selective detection of ochratoxin A (OTA). A specific aptamer for OTA was used to block the pores of rhodamine B‐loaded mesoporous silica nanoparticles. Two solids were prepared in which the aptamer capped the porous scaffolds by using a covalent or electrostatic approach. Whereas the prepared materials remained capped in water, dye delivery was selectively observed in the presence of OTA. The protocol showed excellent analytical performance in terms of sensitivity (limit of detection: 0.5–0.05 nm), reproducibility, and selectivity. Moreover, the aptasensors were tested for OTA detection in commercial foodstuff matrices, which demonstrated their potential applicability in real samples. PMID:29046860

Sticky trap and stem-tap sampling protocols for the Asian citrus psyllid (Hemiptera: Psyllidae)

USDA-ARS?s Scientific Manuscript database

Sampling statistics were obtained to develop a sampling protocol for estimating numbers of adult Diaphorina citri in citrus using two different sampling methods: yellow sticky traps and stem–tap samples. A 4.0 ha block of mature orange trees was stratified into ten 0.4 ha strata and sampled using...

An Energy Balanced and Lifetime Extended Routing Protocol for Underwater Sensor Networks.

PubMed

Wang, Hao; Wang, Shilian; Zhang, Eryang; Lu, Luxi

2018-05-17

Energy limitation is an adverse problem in designing routing protocols for underwater sensor networks (UWSNs). To prolong the network lifetime with limited battery power, an energy balanced and efficient routing protocol, called energy balanced and lifetime extended routing protocol (EBLE), is proposed in this paper. The proposed EBLE not only balances traffic loads according to the residual energy, but also optimizes data transmissions by selecting low-cost paths. Two phases are operated in the EBLE data transmission process: (1) candidate forwarding set selection phase and (2) data transmission phase. In candidate forwarding set selection phase, nodes update candidate forwarding nodes by broadcasting the position and residual energy level information. The cost value of available nodes is calculated and stored in each sensor node. Then in data transmission phase, high residual energy and relatively low-cost paths are selected based on the cost function and residual energy level information. We also introduce detailed analysis of optimal energy consumption in UWSNs. Numerical simulation results on a variety of node distributions and data load distributions prove that EBLE outperforms other routing protocols (BTM, BEAR and direct transmission) in terms of network lifetime and energy efficiency.

Preliminary report for analysis of genome wide mutations from four ciprofloxacin resistant B. anthracis Sterne isolates generated by Illumina, 454 sequencing and microarrays for DHS

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

Jaing, Crystal; Vergez, Lisa; Hinckley, Aubree

2011-06-21

The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, Taqman PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. As the result of a different DHS project, we have selected for and isolated a large number of ciprofloxacin resistant B. anthracis Sterne isolates. These isolates vary in the concentrations of ciprofloxacin that they can tolerate, suggesting multiple mutations in the samples. In collaboration with University of Houston, Eureka Genomics and Oak Ridge National Laboratory, we analyzedmore » the ciprofloxacin resistant B. anthracis Sterne isolates by microarray hybridization, Illumina and Roche 454 sequencing to understand the error rates and sensitivity of the different methods. The report provides an assessment of the results and a complete set of all protocols used and all data generated along with information to interpret the protocols and data sets.« less

Study of a scanning HIFU therapy protocol, Part II: Experiment and results

NASA Astrophysics Data System (ADS)

Andrew, Marilee A.; Kaczkowski, Peter; Cunitz, Bryan W.; Brayman, Andrew A.; Kargl, Steven G.

2003-04-01

Instrumentation and protocols for creating scanned HIFU lesions in freshly excised bovine liver were developed in order to study the in vitro HIFU dose response and validate models. Computer-control of the HIFU transducer and 3-axis positioning system provided precise spatial placement of the thermal lesions. Scan speeds were selected in the range of 1 to 8 mm/s, and the applied electrical power was varied from 20 to 60 W. These parameters were chosen to hold the thermal dose constant. A total of six valid scans of 15 mm length were created in each sample; a 3.5 MHz single-element, spherically focused transducer was used. Treated samples were frozen, then sliced in 1.27 mm increments. Digital photographs of slices were downloaded to computer for image processing and analysis. Lesion characteristics, including the depth within the tissue, axial length, and radial width, were computed. Results were compared with those generated from modified KZK and BHTE models, and include a comparison of the statistical variation in the across-scan lesion radial width. [Work supported by USAMRMC.

Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples.

PubMed

Coêlho, Diego F; Saturnino, Thais Peron; Fernandes, Fernanda Freitas; Mazzola, Priscila Gava; Silveira, Edgar; Tambourgi, Elias Basile

2016-01-01

Given the importance of protease's worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM). We also quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0.072 and 0.494 mg·mL(-1) of azocasein, resp.) and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%), which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise.

Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples

PubMed Central

Mazzola, Priscila Gava

2016-01-01

Given the importance of protease's worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM). We also quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0.072 and 0.494 mg·mL−1 of azocasein, resp.) and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%), which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise. PMID:26925415

Standard operating procedures for collection of soil and sediment samples for the Sediment-bound Contaminant Resiliency and Response (SCoRR) strategy pilot study

USGS Publications Warehouse

Fisher, Shawn C.; Reilly, Timothy J.; Jones, Daniel K.; Benzel, William M.; Griffin, Dale W.; Loftin, Keith A.; Iwanowicz, Luke R.; Cohl, Jonathan A.

2015-12-17

An understanding of the effects on human and ecological health brought by major coastal storms or flooding events is typically limited because of a lack of regionally consistent baseline and trends data in locations proximal to potential contaminant sources and mitigation activities, sensitive ecosystems, and recreational facilities where exposures are probable. In an attempt to close this gap, the U.S. Geological Survey (USGS) has implemented the Sediment-bound Contaminant Resiliency and Response (SCoRR) strategy pilot study to collect regional sediment-quality data prior to and in response to future coastal storms. The standard operating procedure (SOP) detailed in this document serves as the sample-collection protocol for the SCoRR strategy by providing step-by-step instructions for site preparation, sample collection and processing, and shipping of soil and surficial sediment (for example, bed sediment, marsh sediment, or beach material). The objectives of the SCoRR strategy pilot study are (1) to create a baseline of soil-, sand-, marsh sediment-, and bed-sediment-quality data from sites located in the coastal counties from Maine to Virginia based on their potential risk of being contaminated in the event of a major coastal storm or flooding (defined as Resiliency mode); and (2) respond to major coastal storms and flooding by reoccupying select baseline sites and sampling within days of the event (defined as Response mode). For both modes, samples are collected in a consistent manner to minimize bias and maximize quality control by ensuring that all sampling personnel across the region collect, document, and process soil and sediment samples following the procedures outlined in this SOP. Samples are analyzed using four USGS-developed screening methods—inorganic geochemistry, organic geochemistry, pathogens, and biological assays—which are also outlined in this SOP. Because the SCoRR strategy employs a multi-metric approach for sample analyses, this protocol expands upon and reconciles differences in the sample collection protocols outlined in the USGS “National Field Manual for the Collection of Water-Quality Data,” which should be used in conjunction with this SOP. A new data entry and sample tracking system also is presented to ensure all relevant data and metadata are gathered at the sample locations and in the laboratories.

Virtual standardized patients: an interactive method to examine variation in depression care among primary care physicians

PubMed Central

Hooper, Lisa M.; Weinfurt, Kevin P.; Cooper, Lisa A.; Mensh, Julie; Harless, William; Kuhajda, Melissa C.; Epstein, Steven A.

2009-01-01

Background Some primary care physicians provide less than optimal care for depression (Kessler et al., Journal of the American Medical Association 291, 2581–90, 2004). However, the literature is not unanimous on the best method to use in order to investigate this variation in care. To capture variations in physician behaviour and decision making in primary care settings, 32 interactive CD-ROM vignettes were constructed and tested. Aim and method The primary aim of this methods-focused paper was to review the extent to which our study method – an interactive CD-ROM patient vignette methodology – was effective in capturing variation in physician behaviour. Specifically, we examined the following questions: (a) Did the interactive CD-ROM technology work? (b) Did we create believable virtual patients? (c) Did the research protocol enable interviews (data collection) to be completed as planned? (d) To what extent was the targeted study sample size achieved? and (e) Did the study interview protocol generate valid and reliable quantitative data and rich, credible qualitative data? Findings Among a sample of 404 randomly selected primary care physicians, our voice-activated interactive methodology appeared to be effective. Specifically, our methodology – combining interactive virtual patient vignette technology, experimental design, and expansive open-ended interview protocol – generated valid explanations for variations in primary care physician practice patterns related to depression care. PMID:20463864

Evaluation of a Progressive Mobility Protocol in Postoperative Cardiothoracic Surgical Patients.

PubMed

Floyd, Shawn; Craig, Sarah W; Topley, Darla; Tullmann, Dorothy

2016-01-01

Cardiothoracic surgical patients are at high risk for complications related to immobility, such as increased intensive care and hospital length of stay, intensive care unit readmission, pressure ulcer development, and deep vein thrombosis/pulmonary embolus. A progressive mobility protocol was started in the thoracic cardiovascular intensive care unit in a rural academic medical center. The purpose of the progressive mobility protocol was to increase mobilization of postoperative patients and decrease complications related to immobility in this unique patient population. A matched-pairs design was used to compare a randomly selected sample of the preintervention group (n = 30) to a matched postintervention group (n = 30). The analysis compared outcomes including intensive care unit and hospital length of stay, intensive care unit readmission occurrence, pressure ulcer prevalence, and deep vein thrombosis/pulmonary embolism prevalence between the 2 groups. Although this comparison does not achieve statistical significance (P < .05) for any of the outcomes measured, it does show clinical significance in a reduction in hospital length of stay, intensive care unit days, in intensive care unit readmission rate, and a decline in pressure ulcer prevalence, which is the overall goal of progressive mobility. This study has implications for nursing, hospital administration, and therapy services with regard to staffing and cost savings related to fewer complications of immobility. Future studies with a larger sample size and other populations are warranted.

SELECTING PLANT SPECIES FOR PESTICIDE REGISTRATION TESTS

EPA Science Inventory

Current test protocols used by the US EPA for the registration of pesticides examines plant responses of 10 crop species but may not examine regionally important native plants or crops. In order to test the efficiency of current test protocols we selected six native plant species...

Using machine learning for sequence-level automated MRI protocol selection in neuroradiology.

PubMed

Brown, Andrew D; Marotta, Thomas R

2018-05-01

Incorrect imaging protocol selection can lead to important clinical findings being missed, contributing to both wasted health care resources and patient harm. We present a machine learning method for analyzing the unstructured text of clinical indications and patient demographics from magnetic resonance imaging (MRI) orders to automatically protocol MRI procedures at the sequence level. We compared 3 machine learning models - support vector machine, gradient boosting machine, and random forest - to a baseline model that predicted the most common protocol for all observations in our test set. The gradient boosting machine model significantly outperformed the baseline and demonstrated the best performance of the 3 models in terms of accuracy (95%), precision (86%), recall (80%), and Hamming loss (0.0487). This demonstrates the feasibility of automating sequence selection by applying machine learning to MRI orders. Automated sequence selection has important safety, quality, and financial implications and may facilitate improvements in the quality and safety of medical imaging service delivery.

Protocol for determining bull trout presence

USGS Publications Warehouse

Peterson, James; Dunham, Jason B.; Howell, Philip; Thurow, Russell; Bonar, Scott

2002-01-01

The Western Division of the American Fisheries Society was requested to develop protocols for determining presence/absence and potential habitat suitability for bull trout. The general approach adopted is similar to the process for the marbled murrelet, whereby interim guidelines are initially used, and the protocols are subsequently refined as data are collected. Current data were considered inadequate to precisely identify suitable habitat but could be useful in stratifying sampling units for presence/absence surveys. The presence/absence protocol builds on previous approaches (Hillman and Platts 1993; Bonar et al. 1997), except it uses the variation in observed bull trout densities instead of a minimum threshold density and adjusts for measured differences in sampling efficiency due to gear types and habitat characteristics. The protocol consists of: 1. recommended sample sizes with 80% and 95% detection probabilities for juvenile and resident adult bull trout for day and night snorkeling and electrofishing adjusted for varying habitat characteristics for 50m and 100m sampling units, 2. sampling design considerations, including possible habitat characteristics for stratification, 3. habitat variables to be measured in the sampling units, and 3. guidelines for training sampling crews. Criteria for habitat strata consist of coarse, watershed-scale characteristics (e.g., mean annual air temperature) and fine-scale, reach and habitat-specific features (e.g., water temperature, channel width). The protocols will be revised in the future using data from ongoing presence/absence surveys, additional research on sampling efficiencies, and development of models of habitat/species occurrence.

21 CFR 660.6 - Samples; protocols; official release.

Code of Federal Regulations, 2013 CFR

2013-04-01

... Hepatitis B Surface Antigen § 660.6 Samples; protocols; official release. (a) Samples. (1) For the purposes... of official release is no longer required under paragraph (c)(2) of this section. (ii) One sample at... required under paragraph (c)(2) of this section. The sample submitted at the 90-day interval shall be from...

21 CFR 660.6 - Samples; protocols; official release.

Code of Federal Regulations, 2014 CFR

2014-04-01

... Hepatitis B Surface Antigen § 660.6 Samples; protocols; official release. (a) Samples. (1) For the purposes... of official release is no longer required under paragraph (c)(2) of this section. (ii) One sample at... required under paragraph (c)(2) of this section. The sample submitted at the 90-day interval shall be from...

21 CFR 660.6 - Samples; protocols; official release.

Code of Federal Regulations, 2012 CFR

2012-04-01

... Hepatitis B Surface Antigen § 660.6 Samples; protocols; official release. (a) Samples. (1) For the purposes... of official release is no longer required under paragraph (c)(2) of this section. (ii) One sample at... required under paragraph (c)(2) of this section. The sample submitted at the 90-day interval shall be from...

Simultaneous determination of four plant hormones in bananas by molecularly imprinted solid-phase extraction coupled with high performance liquid chromatography.

PubMed

Yan, Hongyuan; Wang, Fang; Han, Dandan; Yang, Gengliang

2012-06-21

A highly selective molecularly imprinted solid-phase extraction (MISPE) combined with liquid chromatography-ultraviolet detection was developed for the simultaneous isolation and determination of four plant hormones including indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) in banana samples. The new molecularly imprinted microspheres (MIMs) prepared by aqueous suspension polymerization using 3-hydroxy-2-naphthoic acid and 1-methylpiperazine as mimic templates performed with high selectivity and affinity for the four plant hormones, and applied as selective sorbents of solid-phase extraction could effectively eliminate the interferences of the banana matrix. Good linearity was obtained in a range of 0.04-4.00 μg g(-1) and the recoveries of the four plant hormones at three spiked levels ranged from 78.5 to 107.7% with the relative standard deviations (RSD) of less than 4.6%. The developed MISPE-HPLC protocol obviously improved the selectivity and eliminated the effect of template leakage on quantitative analysis, and could be applied for the determination of plant hormones in complicated biological samples.

Tuning of automatic exposure control strength in lumbar spine CT.

PubMed

D'Hondt, A; Cornil, A; Bohy, P; De Maertelaer, V; Gevenois, P A; Tack, D

2014-05-01

To investigate the impact of tuning the automatic exposure control (AEC) strength curve (specific to Care Dose 4D®; Siemens Healthcare, Forchheim, Germany) from "average" to "strong" on image quality, radiation dose and operator dependency during lumbar spine CT examinations. Two hospitals (H1, H2), both using the same scanners, were considered for two time periods (P1 and P2). During P1, the AEC curve was "average" and radiographers had to select one of two protocols according to the body mass index (BMI): "standard" if BMI <30.0 kg m(-2) (120 kV-330 mAs) or "large" if BMI >30.0 kg m(-2) (140 kV-280 mAs). During P2, the AEC curve was changed to "strong", and all acquisitions were obtained with one protocol (120 kV and 270 mAs). Image quality was scored and patients' diameters calculated for both periods. 497 examinations were analysed. There was no significant difference in mean diameters according to hospitals and periods (p > 0.801) and in quality scores between periods (p > 0.172). There was a significant difference between hospitals regarding how often the "large" protocol was assigned [13 (10%)/132 patients in H1 vs 37 (28%)/133 in H2] (p < 0.001). During P1, volume CT dose index (CTDIvol) was higher in H2 (+13%; p = 0.050). In both hospitals, CTDIvol was reduced between periods (-19.2% in H1 and -29.4% in H2; p < 0.001). An operator dependency in protocol selection, unexplained by patient diameters or highlighted by image quality scores, has been observed. Tuning the AEC curve from average to strong enables suppression of the operator dependency in protocol selection and related dose increase, while preserving image quality. CT acquisition protocols based on weight are responsible for biases in protocol selection. Using an appropriate AEC strength curve reduces the number of protocols to one. Operator dependency of protocol selection is thereby eliminated.

A new real-time PCR protocol for detection of avian haemosporidians.

PubMed

Bell, Jeffrey A; Weckstein, Jason D; Fecchio, Alan; Tkach, Vasyl V

2015-07-19

Birds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great genetic diversity and insufficient sampling in highly diverse regions. Studies aimed at surveying haemosporidian diversity involve collecting and screening samples from hundreds to thousands of individuals. Currently, screening relies on microscopy and/or single or nested standard PCR. Although effective, these methods are time and resource consuming, and in the case of microscopy require substantial expertise. Here we report a newly developed real-time PCR protocol designed to quickly and reliably detect all three genera of avian haemosporidians in a single biochemical reaction. Using available DNA sequences from avian haemosporidians we designed primers R330F and R480RL, which flank a 182 base pair fragment of mitochondrial conserved rDNA. These primers were initially tested using real-time PCR on samples from Malawi, Africa, previously screened for avian haemosporidians using traditional nested PCR. Our real time protocol was further tested on 94 samples from the Cerrado biome of Brazil, previously screened using a single PCR assay for haemosporidian parasites. These samples were also amplified using modified nested PCR protocols, allowing for comparisons between the three different screening methods (single PCR, nested PCR, real-time PCR). The real-time PCR protocol successfully identified all three genera of avian haemosporidians from both single and mixed infections previously detected from Malawi. There was no significant difference between the three different screening protocols used for the 94 samples from the Brazilian Cerrado (χ(2) = 0.3429, df = 2, P = 0.842). After proving effective, the real-time protocol was used to screen 2113 Brazilian samples, identifying 693 positive samples. Our real-time PCR assay proved as effective as two widely used molecular screening techniques, single PCR and nested PCR. However, the real-time protocol has the distinct advantage of detecting all three genera in a single reaction, which significantly increases efficiency by greatly decreasing screening time and cost. Our real-time PCR protocol is therefore a valuable tool in the quickly expanding field of avian haemosporidian research.

Updated archaeointensity dataset from the SW Pacific

NASA Astrophysics Data System (ADS)

Hill, Mimi; Nilsson, Andreas; Holme, Richard; Hurst, Elliot; Turner, Gillian; Herries, Andy; Sheppard, Peter

2016-04-01

It is well known that there are far more archaeomagnetic data from the Northern Hemisphere than from the Southern. Here we present a compilation of archaeointensity data from the SW Pacific region covering the past 3000 years. The results have primarily been obtained from a collection of ceramics from the SW Pacific Islands including Fiji, Tonga, Papua New Guinea, New Caledonia and Vanuatu. In addition we present results obtained from heated clay balls from Australia. The microwave method has predominantly been used with a variety of experimental protocols including IZZI and Coe variants. Standard Thellier archaeointensity experiments using the IZZI protocol have also been carried out on selected samples. The dataset is compared to regional predictions from current global geomagnetic field models, and the influence of the new data on constraining the pfm9k family of global geomagnetic field models is explored.

Ion-Exchange Chromatography: Basic Principles and Application.

PubMed

Cummins, Philip M; Rochfort, Keith D; O'Connor, Brendan F

2017-01-01

Ion-Exchange Chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline basic laboratory protocols to partially purify a soluble serine peptidase from bovine whole brain tissue, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described.

Sequence polymorphism in an insect RNA virus field population: A snapshot from a single point in space and time reveals stochastic differences among and within individual hosts

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

Stenger, Drake C., E-mail: drake.stenger@ars.usda.

Population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected insects sampled from a single point in space and time was examined. Polymorphism in complete consensus sequences among single-insect isolates was dominated by synonymous substitutions. The mutant spectrum of the C2 helicase region within each single-insect isolate was unique and dominated by nonsynonymous singletons. Bootstrapping was used to correct the within-isolate nonsynonymous:synonymous arithmetic ratio (N:S) for RT-PCR error, yielding an N:S value ~one log-unit greater than that of consensus sequences. Probability of all possible single-base substitutions for the C2 region predicted N:S values within 95% confidence limits of themore » corrected within-isolate N:S when the only constraint imposed was viral polymerase error bias for transitions over transversions. These results indicate that bottlenecks coupled with strong negative/purifying selection drive consensus sequences toward neutral sequence space, and that most polymorphism within single-insect isolates is composed of newly-minted mutations sampled prior to selection. -- Highlights: •Sampling protocol minimized differential selection/history among isolates. •Polymorphism among consensus sequences dominated by negative/purifying selection. •Within-isolate N:S ratio corrected for RT-PCR error by bootstrapping. •Within-isolate mutant spectrum dominated by new mutations yet to undergo selection.« less

Quarantine and protocol

NASA Technical Reports Server (NTRS)

1981-01-01

The purpose of the Orbiting Quarantine Facility is to provide maximum protection of the terrestrial biosphere by ensuring that the returned Martian samples are safe to bring to Earth. The protocol designed to detect the presence of biologically active agents in the Martian soil is described. The protocol determines one of two things about the sample: (1) that it is free from nonterrestrial life forms and can be sent to a terrestrial containment facility where extensive chemical, biochemical, geological, and physical investigations can be conducted; or (2) that it exhibits "biological effects" of the type that dictate second order testing. The quarantine protocol is designed to be conducted on a small portion of the returned sample, leaving the bulk of the sample undisturbed for study on Earth.

COMPARISON OF USEPA FIELD SAMPLING METHODS FOR BENTHIC MACROINVERTEBRATE STUDIES

EPA Science Inventory

Two U.S. Environmental Protection Agency (USEPA) macroinvertebrate sampling protocols were compared in the Mid-Atlantic Highlands region. The Environmental Monitoring and Assessment Program (EMAP) wadeable streams protocol results in a single composite sample from nine transects...

A Field Comparison of Sampling Protocols for Measuring Lead in Drinking Water

EPA Science Inventory

US EPA Region 5 conducted a sampling study that demonstrates existing sampling protocols used for the Lead and Copper Rule (LCR) underestimate peak and probable mass of lead released in a system with lead service lines (LSLs). This comparative stagnation sampling was conducted i...

Judges' Agreement and Disagreement Patterns When Encoding Verbal Protocols.

ERIC Educational Resources Information Center

Schael, Jocelyne; Dionne, Jean-Paul

The basis of agreement or disagreement among judges/evaluators when applying a coding scheme to concurrent verbal protocols was studied. The sample included 20 university graduates, from varied backgrounds; 10 subjects had and 10 subjects did not have experience in protocol analysis. The total sample was divided into four balanced groups according…

A Natural Language Processing-based Model to Automate MRI Brain Protocol Selection and Prioritization.

PubMed

Brown, Andrew D; Marotta, Thomas R

2017-02-01

Incorrect imaging protocol selection can contribute to increased healthcare cost and waste. To help healthcare providers improve the quality and safety of medical imaging services, we developed and evaluated three natural language processing (NLP) models to determine whether NLP techniques could be employed to aid in clinical decision support for protocoling and prioritization of magnetic resonance imaging (MRI) brain examinations. To test the feasibility of using an NLP model to support clinical decision making for MRI brain examinations, we designed three different medical imaging prediction tasks, each with a unique outcome: selecting an examination protocol, evaluating the need for contrast administration, and determining priority. We created three models for each prediction task, each using a different classification algorithm-random forest, support vector machine, or k-nearest neighbor-to predict outcomes based on the narrative clinical indications and demographic data associated with 13,982 MRI brain examinations performed from January 1, 2013 to June 30, 2015. Test datasets were used to calculate the accuracy, sensitivity and specificity, predictive values, and the area under the curve. Our optimal results show an accuracy of 82.9%, 83.0%, and 88.2% for the protocol selection, contrast administration, and prioritization tasks, respectively, demonstrating that predictive algorithms can be used to aid in clinical decision support for examination protocoling. NLP models developed from the narrative clinical information provided by referring clinicians and demographic data are feasible methods to predict the protocol and priority of MRI brain examinations. Copyright © 2017 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.

Preparation of Low-Input and Ligation-Free ChIP-seq Libraries Using Template-Switching Technology.

PubMed

Bolduc, Nathalie; Lehman, Alisa P; Farmer, Andrew

2016-10-10

Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low-abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP-seq workflow that can be applied to small cell numbers, including a robust single-tube and ligation-free method for preparation of sequencing libraries from sub-nanogram amounts of ChIP DNA. An example ChIP protocol is first presented, resulting in selective enrichment of DNA-binding proteins and cross-linked DNA fragments immobilized on beads via an antibody bridge. This is followed by a protocol for fast and easy cross-linking reversal and DNA recovery. Finally, we describe a fast, ligation-free library preparation protocol, featuring DNA SMART technology, resulting in samples ready for Illumina sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Pepper, chili (Capsicum annuum).

PubMed

Min, Jung; Shin, Sun Hee; Jeon, En Mi; Park, Jung Mi; Hyun, Ji Young; Harn, Chee Hark

2015-01-01

Pepper is a recalcitrant plant for Agrobacterium-mediated genetic transformation. Several obstacles to genetic transformation remain such as extremely low transformation rates; the choice of correct genotype is critical; and there is a high frequency of false positives due to direct shoot formation. Here, we report a useful protocol with a suitable selection method. The most important aspect of the pepper transformation protocol is selecting shoots growing from the callus, which is referred to as callus-mediated shoot formation. This protocol is a reproducible and reliable system for pepper transformation.

GENERIC VERIFICATION PROTOCOL FOR DETERMINATION OF EMISSIONS REDUCTIONS FROM SELECTIVE CATALYTIC REDUCTIONS CONTROL TECHNOLOGIES FOR HIGHWAY, NONROAD, AND STATIONARY USE DIESEL ENGINES

EPA Science Inventory

The protocol describes the Environmental Technology Verification (ETV) Program's considerations and requirements for verification of emissions reduction provided by selective catalytic reduction (SCR) technologies. The basis of the ETV will be comparison of the emissions and perf...

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

PubMed

Monsanto, Megan M; White, Kevin S; Kim, Taeyong; Wang, Bingyan J; Fisher, Kristina; Ilves, Kelli; Khalafalla, Farid G; Casillas, Alexandria; Broughton, Kathleen; Mohsin, Sadia; Dembitsky, Walter P; Sussman, Mark A

2017-07-07

The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm 3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit + cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit - mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit + population is further enriched by selection for a CD133 + endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients. © 2017 American Heart Association, Inc.

Evaluation and optimization of microbial DNA extraction from fecal samples of wild Antarctic bird species

PubMed Central

Eriksson, Per; Mourkas, Evangelos; González-Acuna, Daniel; Olsen, Björn; Ellström, Patrik

2017-01-01

ABSTRACT Introduction: Advances in the development of nucleic acid-based methods have dramatically facilitated studies of host–microbial interactions. Fecal DNA analysis can provide information about the host’s microbiota and gastrointestinal pathogen burden. Numerous studies have been conducted in mammals, yet birds are less well studied. Avian fecal DNA extraction has proved challenging, partly due to the mixture of fecal and urinary excretions and the deficiency of optimized protocols. This study presents an evaluation of the performance in avian fecal DNA extraction of six commercial kits from different bird species, focusing on penguins. Material and methods: Six DNA extraction kits were first tested according to the manufacturers’ instructions using mallard feces. The kit giving the highest DNA yield was selected for further optimization and evaluation using Antarctic bird feces. Results: Penguin feces constitute a challenging sample type: most of the DNA extraction kits failed to yield acceptable amounts of DNA. The QIAamp cador Pathogen kit (Qiagen) performed the best in the initial investigation. Further optimization of the protocol resulted in good yields of high-quality DNA from seven bird species of different avian orders. Conclusion: This study presents an optimized approach to DNA extraction from challenging avian fecal samples. PMID:29152162

Reliable paleointensity determinations from Late Cretaceous volcanic rocks in Korea with constraint of thermochemical alteration

NASA Astrophysics Data System (ADS)

Kim, Wonnyon; Doh, Seong-Jae; Yu, Yongjae

2018-06-01

Paleointensity determinations were carried out from Late Cretaceous (∼77 Ma) volcanic rocks in Korea using a Thellier-type IZZI experimental protocol with systematic partial thermal remanent magnetization (pTRM) checks. Various data selection criteria were used to estimate reliable paleointensities. We set stringent threshold values for each parameter to ensure that there was: (1) a linear relationship between natural remanent magnetization (NRM) lost and TRM gained; (2) negligible thermal alteration of magnetic minerals; and (3) uni-vectorial decay of NRM towards the origin. From the 336 samples, ∼88% were rejected because of an insufficient extrapolated NRM fraction in the best-fit line (fvds < 0.6), highlighting that fvds is the most stringent selection criterion in this study. For the 31 accepted samples, paleointensities range from 6.4 to 30.4 μT. Among the 31 samples, eight samples yielded extremely low paleointensities. Although single-domain (titano)magnetite was identified as the stable paleointensity recorder, oxidation of superparamagnetic fractions upon repeated heating probably caused enhancement of pTRM acquisition and yielded a low paleointensity estimate. Apart from these low paleointensities (n = 8) as screened by a newly proposed data selection criterion of Δk < 0.2, the remaining 23 samples have a mean paleointensity of 23.1 ± 4.8 μT, corresponding to a virtual axial dipole moment (VADM) of 40.8 ± 8.5 ZAm2, which is ∼50% of the present-day VADM.

Thermogravimetric Analysis of Single-Wall Carbon Nanotubes

NASA Technical Reports Server (NTRS)

Arepalli, Sivram; Nikolaev, Pavel; Gorelik, Olga

2010-01-01

An improved protocol for thermogravimetric analysis (TGA) of samples of single-wall carbon nanotube (SWCNT) material has been developed to increase the degree of consistency among results so that meaningful comparisons can be made among different samples. This improved TGA protocol is suitable for incorporation into the protocol for characterization of carbon nanotube material. In most cases, TGA of carbon nanotube materials is performed in gas mixtures that contain oxygen at various concentrations. The improved protocol is summarized.

Automatic spectral imaging protocol selection and iterative reconstruction in abdominal CT with reduced contrast agent dose: initial experience.

PubMed

Lv, Peijie; Liu, Jie; Chai, Yaru; Yan, Xiaopeng; Gao, Jianbo; Dong, Junqiang

2017-01-01

To evaluate the feasibility, image quality, and radiation dose of automatic spectral imaging protocol selection (ASIS) and adaptive statistical iterative reconstruction (ASIR) with reduced contrast agent dose in abdominal multiphase CT. One hundred and sixty patients were randomly divided into two scan protocols (n = 80 each; protocol A, 120 kVp/450 mgI/kg, filtered back projection algorithm (FBP); protocol B, spectral CT imaging with ASIS and 40 to 70 keV monochromatic images generated per 300 mgI/kg, ASIR algorithm. Quantitative parameters (image noise and contrast-to-noise ratios [CNRs]) and qualitative visual parameters (image noise, small structures, organ enhancement, and overall image quality) were compared. Monochromatic images at 50 keV and 60 keV provided similar or lower image noise, but higher contrast and overall image quality as compared with 120-kVp images. Despite the higher image noise, 40-keV images showed similar overall image quality compared to 120-kVp images. Radiation dose did not differ between the two protocols, while contrast agent dose in protocol B was reduced by 33 %. Application of ASIR and ASIS to monochromatic imaging from 40 to 60 keV allowed contrast agent dose reduction with adequate image quality and without increasing radiation dose compared to 120 kVp with FBP. • Automatic spectral imaging protocol selection provides appropriate scan protocols. • Abdominal CT is feasible using spectral imaging and 300 mgI/kg contrast agent. • 50-keV monochromatic images with 50 % ASIR provide optimal image quality.

Comparative assessment of the physico-chemical and bacteriological qualities of selected streams in Louisiana.

PubMed

Hill, Dagne D; Owens, William E; Tchounwou, Paul B

2005-04-01

The objective of this research was to compare the chemical/physical parameters and bacterial qualities of selected surface water streams in Louisiana, including a natural stream (control) and an animal waste related stream. Samples were collected and analyzed for fecal coliforms. Fecal coliforms isolated from these samples were identified to the species level. Chemical analysis was performed following standard test protocols (LaMotte 2002). An analysis of biological oxygen demand (BOD), chemical oxygen demand (COD), total organic carbon (TOC), total dissolved solids (TDS), conductivity, pH, temperature, ammonia nitrogen, nitrate nitrogen, iron, copper, phosphate, potassium, sulfate, turbidity, zinc and bacterial levels was performed following standard test protocols as presented in Standard Methods for the Examination of Water and Wastewater [9]. Results of the comparisons of the various surface water streams showed that phosphate levels, according to Mitchell and Stapp, were considered good for Lake Claiborne (control) and Bayou Dorcheat. The levels were found to be .001 mg/L and .007 mg/L respectively. Other streams associated with animal waste, had higher phosphate levels of 2.07 mg/L and 2.78 mg/L, respectively. Conductivity and total dissolved solids (TDS) levels were the lowest in Lake Claiborne and highest in the Hill Farm Research Station stream. It can be concluded from the data that some bacterial levels and various nutrient levels can be affected in water resources due to non-point source pollution. Many of these levels will remain unaffected.

Sodium fluoroacetate toxicity: a case report of malicious poisoning in dogs across a Phoenix, Arizona neighborhood.

PubMed

Brower, Alexandra; Struthers, Jason; Schmidt, Jemima

2017-12-01

In May 2016, thirteen dogs housed in backyards within a single neighborhood were reported to have developed convulsions and died within a 24 h period. An investigation of the scene by law enforcement resulted in submission of eight dogs for postmortem examination. It was suspected that a rapid acting toxin was the cause of death. A gas chromatography-mass spectrophotometry (GC-MS) protocol combined with thin-layer chromatography that allows screening for common convulsants failed to identify a toxin in either pooled gastric content or liver samples from select cases. After consultation with a veterinary toxicologist, sodium fluoroacetate poisoning was investigated. Sodium fluoroacetate, also known as 1080, is a pesticide that was available in the United States from the 1940's to the 1970's, but since 1972 has been banned or under EPA restricted use. When gastric content was re-tested using a GC-MS protocol with selective fluoroacetate ion monitoring and carbon 14 radiolabeling to facilitate quantification, 379 ppb sodium fluoroacetate was detected in a pooled gastric content sample. In spite of its banned status, sodium fluoroacetate remains a rarely reported cause of malicious poisoning in domestic dogs in the United Sates. This compound is highly toxic and is capable of causing death in dogs, humans, other mammals, and insects in ingested quantities as small as a few droplets. Even when geographic or historical proximity to a source is not evident, this intoxication should be considered in dogs exhibiting compatible clinical signs.

Simple and economic colloidal centrifugation protocols may be incorporated into the clinical equine sperm processing procedure.

PubMed

Gutiérrez-Cepeda, L; Fernández, A; Crespo, F; Gosálvez, J; Serres, C

2011-03-01

For many years in human assisted-reproduction procedures there have been special protocols to prepare and improve sperm quality. Colloidal centrifugation (CC) is a useful technique that has been proved to enhance semen quality by selection of the best spermatozoa for different species. Its use is recommended to improve fertility of subfertile stallions but current CC protocols are clinically complicated in the equine sperm processing technique due to economic and technical difficulties. The aim of this study was to determine the optimal processing procedures to adapt the use of a CC product (EquiPure™) in the equine reproduction industry. A total of nineteen ejaculates were collected from 10 Purebred Spanish Horses (P.R.E horses) using a Missouri artificial vagina. Gel-free semen aliquots were analyzed prior to treatment (control). Semen was subjected to one of six CC protocols with EquiPure™ and centrifuged samples were statistically evaluated by ANOVA and Duncan tests (p<0.05) for sperm quality and recovery rate. We obtained higher values by colloidal centrifugation in LIN, STR and BCF variables and DNA fragmentation index trended to be lower in most of the CC protocols. The studied protocols were shown to be as efficient in improving equine sperm quality as the current commercial EquiPure™, with the added advantage of being much more economical and simple to use. According to these results it seems to be possible to incorporate single layer and or high colloidal centrifugation volume protocols what would make them simple, economic and clinically viable for the equine sperm processing procedure. Copyright © 2011 Elsevier B.V. All rights reserved.

Homogenization of sample absorption for the imaging of large and dense fossils with synchrotron microtomography.

PubMed

Sanchez, Sophie; Fernandez, Vincent; Pierce, Stephanie E; Tafforeau, Paul

2013-09-01

Propagation phase-contrast synchrotron radiation microtomography (PPC-SRμCT) has proved to be very successful for examining fossils. Because fossils range widely in taphonomic preservation, size, shape and density, X-ray computed tomography protocols are constantly being developed and refined. Here we present a 1-h procedure that combines a filtered high-energy polychromatic beam with long-distance PPC-SRμCT (sample to detector: 4-16 m) and an attenuation protocol normalizing the absorption profile (tested on 13-cm-thick and 5.242 g cm(-3) locally dense samples but applicable to 20-cm-thick samples). This approach provides high-quality imaging results, which show marked improvement relative to results from images obtained without the attenuation protocol in apparent transmission, contrast and signal-to-noise ratio. The attenuation protocol involves immersing samples in a tube filled with aluminum or glass balls in association with a U-shaped aluminum profiler. This technique therefore provides access to a larger dynamic range of the detector used for tomographic reconstruction. This protocol homogenizes beam-hardening artifacts, thereby rendering it effective for use with conventional μCT scanners.

FIELD SAMPLING PROTOCOLS AND ANALYSIS

EPA Science Inventory

I have been asked to speak again to the environmental science class regarding actual research scenarios related to my work at Kerr Lab. I plan to discuss sampling protocols along with various field analyses performed during sampling activities. Many of the students have never see...

Communications protocol

NASA Technical Reports Server (NTRS)

Zhou, Xiaoming (Inventor); Baras, John S. (Inventor)

2010-01-01

The present invention relates to an improved communications protocol which increases the efficiency of transmission in return channels on a multi-channel slotted Alohas system by incorporating advanced error correction algorithms, selective retransmission protocols and the use of reserved channels to satisfy the retransmission requests.

21 CFR 660.46 - Samples; protocols; official release.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Samples; protocols; official release. 660.46 Section 660.46 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES..., a sample of product not iodinated with 125I means a sample from each filling of each lot packaged as...

Performance Improvement in Geographic Routing for Vehicular Ad Hoc Networks

PubMed Central

Kaiwartya, Omprakash; Kumar, Sushil; Lobiyal, D. K.; Abdullah, Abdul Hanan; Hassan, Ahmed Nazar

2014-01-01

Geographic routing is one of the most investigated themes by researchers for reliable and efficient dissemination of information in Vehicular Ad Hoc Networks (VANETs). Recently, different Geographic Distance Routing (GEDIR) protocols have been suggested in the literature. These protocols focus on reducing the forwarding region towards destination to select the Next Hop Vehicles (NHV). Most of these protocols suffer from the problem of elevated one-hop link disconnection, high end-to-end delay and low throughput even at normal vehicle speed in high vehicle density environment. This paper proposes a Geographic Distance Routing protocol based on Segment vehicle, Link quality and Degree of connectivity (SLD-GEDIR). The protocol selects a reliable NHV using the criteria segment vehicles, one-hop link quality and degree of connectivity. The proposed protocol has been simulated in NS-2 and its performance has been compared with the state-of-the-art protocols: P-GEDIR, J-GEDIR and V-GEDIR. The empirical results clearly reveal that SLD-GEDIR has lower link disconnection and end-to-end delay, and higher throughput as compared to the state-of-the-art protocols. It should be noted that the performance of the proposed protocol is preserved irrespective of vehicle density and speed. PMID:25429415

Performance improvement in geographic routing for Vehicular Ad Hoc Networks.

PubMed

Kaiwartya, Omprakash; Kumar, Sushil; Lobiyal, D K; Abdullah, Abdul Hanan; Hassan, Ahmed Nazar

2014-11-25

Geographic routing is one of the most investigated themes by researchers for reliable and efficient dissemination of information in Vehicular Ad Hoc Networks (VANETs). Recently, different Geographic Distance Routing (GEDIR) protocols have been suggested in the literature. These protocols focus on reducing the forwarding region towards destination to select the Next Hop Vehicles (NHV). Most of these protocols suffer from the problem of elevated one-hop link disconnection, high end-to-end delay and low throughput even at normal vehicle speed in high vehicle density environment. This paper proposes a Geographic Distance Routing protocol based on Segment vehicle, Link quality and Degree of connectivity (SLD-GEDIR). The protocol selects a reliable NHV using the criteria segment vehicles, one-hop link quality and degree of connectivity. The proposed protocol has been simulated in NS-2 and its performance has been compared with the state-of-the-art protocols: P-GEDIR, J-GEDIR and V-GEDIR. The empirical results clearly reveal that SLD-GEDIR has lower link disconnection and end-to-end delay, and higher throughput as compared to the state-of-the-art protocols. It should be noted that the performance of the proposed protocol is preserved irrespective of vehicle density and speed.

Active SAmpling Protocol (ASAP) to Optimize Individual Neurocognitive Hypothesis Testing: A BCI-Inspired Dynamic Experimental Design.

PubMed

Sanchez, Gaëtan; Lecaignard, Françoise; Otman, Anatole; Maby, Emmanuel; Mattout, Jérémie

2016-01-01

The relatively young field of Brain-Computer Interfaces has promoted the use of electrophysiology and neuroimaging in real-time. In the meantime, cognitive neuroscience studies, which make extensive use of functional exploration techniques, have evolved toward model-based experiments and fine hypothesis testing protocols. Although these two developments are mostly unrelated, we argue that, brought together, they may trigger an important shift in the way experimental paradigms are being designed, which should prove fruitful to both endeavors. This change simply consists in using real-time neuroimaging in order to optimize advanced neurocognitive hypothesis testing. We refer to this new approach as the instantiation of an Active SAmpling Protocol (ASAP). As opposed to classical (static) experimental protocols, ASAP implements online model comparison, enabling the optimization of design parameters (e.g., stimuli) during the course of data acquisition. This follows the well-known principle of sequential hypothesis testing. What is radically new, however, is our ability to perform online processing of the huge amount of complex data that brain imaging techniques provide. This is all the more relevant at a time when physiological and psychological processes are beginning to be approached using more realistic, generative models which may be difficult to tease apart empirically. Based upon Bayesian inference, ASAP proposes a generic and principled way to optimize experimental design adaptively. In this perspective paper, we summarize the main steps in ASAP. Using synthetic data we illustrate its superiority in selecting the right perceptual model compared to a classical design. Finally, we briefly discuss its future potential for basic and clinical neuroscience as well as some remaining challenges.

Criteria for the Collection of Useful Respirator Performance Data in the Workplace

PubMed Central

Janssen, Larry; Zhuang, Ziqing; Shaffer, Ronald

2016-01-01

Workplace protection factors (WPFs) are intended to measure the ability of a respiratory protective device (RPD) to reduce contaminant exposure when used in the context of an effective respiratory protection program. In 1992, members of the American Industrial Hygiene Association Respiratory Protection Committee (RPC) published a review of important issues and considerations for measuring respirator performance in the workplace. The RPC recognized that respirator testing in workplaces can have a variety of objectives and endpoints, and that not all workplace measurements are WPFs. That paper addressed concerns in the general categories of 1) study objectives; 2) site selection; 3) subject selection and preparation; 4) sampling and analytical methods; and 5) data analysis. No specific protocol for measuring WPFs was recommended by the RPC, and attempts to reach a U.S. consensus on a WPF protocol since 1992 have not succeeded. Numerous studies have implemented the principles for WPF measurement described in the RPC paper. Modifications to the original recommendations have been made to reflect the current state of the art. This article describes what has been learned in recent years in each of the five categories identified in the 1992 discussion. Because of the wide variety of workplaces and work activities, contaminants and respiratory protective devices, a strict protocol is not appropriate for collecting WPF data. Rather, the minimum requirements for the collection and presentation of meaningful respirator performance data in the workplace are described. Understanding of these principles will permit useful RPD performance data to be generated. PMID:24579751

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

PubMed Central

Kovač Viršek, Manca; Palatinus, Andreja; Koren, Špela; Peterlin, Monika; Horvat, Petra; Kržan, Andrej

2016-01-01

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an »A frame« attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world. PMID:28060297

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis.

PubMed

Kovač Viršek, Manca; Palatinus, Andreja; Koren, Špela; Peterlin, Monika; Horvat, Petra; Kržan, Andrej

2016-12-16

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an »A frame« attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

Jack Healy Remembers - Anecdotal Evidence for the Origin of the Approximate 24-hour Urine Sampling Protocol Used for Worker Bioassay Monitoring

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

Carbaugh, Eugene H.

2008-10-01

The origin of the approximate 24-hour urine sampling protocol used at Hanford for routine bioassay is attributed to an informal study done in the mid-1940s. While the actual data were never published and have been lost, anecdotal recollections by staff involved in the initial bioassay program design and administration suggest that the sampling protocol had a solid scientific basis. Numerous alternate methods for normalizing partial day samples to represent a total 24-hour collection have since been proposed and used, but no one method is obviously preferred.

Comparison of methods for the extraction of DNA from formalin-fixed, paraffin-embedded archival tissues.

PubMed

Sengüven, Burcu; Baris, Emre; Oygur, Tulin; Berktas, Mehmet

2014-01-01

Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues. DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability. The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification. Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE.

Demonstration and Certification of Amphibian Ecological Risk Assessment Protocol. Cost and Performance Report (Version 2)

DTIC Science & Technology

2009-07-01

sediment protocol, respectively). The soil protocol evaluates impacts to adult salamanders and the sediment protocol evaluates impacts to larval tadpoles ...the sediment protocol evaluates impacts to larval tadpoles . When selecting appropriate receptors to derive ERA-based remedial goals, amphibians...Sediment tests are conducted with recently hatched tadpoles (i.e., Rana pipiens; Gosner Stages 17-20). Young tadpoles are placed in beakers

40 CFR Appendix A - Protocol for Using an Electrochemical Analyzer to Determine Oxygen and Carbon Monoxide...

Code of Federal Regulations, 2014 CFR

2014-07-01

..., and Process Heaters Using Portable Analyzers”, EMC Conditional Test Protocol 30 (CTM-30), Gas Research... cell design(s) conforming to this protocol will determine the analytical range for each gas component..., selective gas scrubbers, etc.) to meet the design specifications of this protocol. Do not make changes to...

40 CFR Appendix A - Protocol for Using an Electrochemical Analyzer to Determine Oxygen and Carbon Monoxide...

Code of Federal Regulations, 2013 CFR

2013-07-01

..., and Process Heaters Using Portable Analyzers”, EMC Conditional Test Protocol 30 (CTM-30), Gas Research... cell design(s) conforming to this protocol will determine the analytical range for each gas component..., selective gas scrubbers, etc.) to meet the design specifications of this protocol. Do not make changes to...

Four studies on effects of environmental factors on the quality of National Atmospheric Deposition Program measurements

USGS Publications Warehouse

Wetherbee, Gregory A.; Latysh, Natalie E.; Lehmann, Christopher M.B.; Rhodes, Mark F.

2011-01-01

Selected aspects of National Atmospheric Deposition Program / National Trends Network (NADP/NTN) protocols are evaluated in four studies. Meteorological conditions have minor impacts on the error in NADP/NTN sampling. Efficiency of frozen precipitation sample collection is lower than for liquid precipitation samples. Variability of NTN measurements is higher for relatively low-intensity deposition of frozen precipitation than for higher-intensity deposition of liquid precipitation. Urbanization of the landscape surrounding NADP/NTN sites is not affecting trends in wet-deposition chemistry data to a measureable degree. Five NADP siting criteria intended to preserve wet-deposition sample integrity have varying degrees of effectiveness. NADP siting criteria for objects within the 90 degrees cones and trees within the 120 degrees cones projected from the collector bucket to sky are important for protecting sample integrity. Tall vegetation, fences, and other objects located within 5 meters of the collectors are related to the frequency of visible sample contamination, indicating the importance of these factors in NADP siting criteria.

Sampling design for an integrated socioeconomic and ecological survey by using satellite remote sensing and ordination

PubMed Central

Binford, Michael W.; Lee, Tae Jeong; Townsend, Robert M.

2004-01-01

Environmental variability is an important risk factor in rural agricultural communities. Testing models requires empirical sampling that generates data that are representative in both economic and ecological domains. Detrended correspondence analysis of satellite remote sensing data were used to design an effective low-cost sampling protocol for a field study to create an integrated socioeconomic and ecological database when no prior information on ecology of the survey area existed. We stratified the sample for the selection of tambons from various preselected provinces in Thailand based on factor analysis of spectral land-cover classes derived from satellite data. We conducted the survey for the sampled villages in the chosen tambons. The resulting data capture interesting variations in soil productivity and in the timing of good and bad years, which a purely random sample would likely have missed. Thus, this database will allow tests of hypotheses concerning the effect of credit on productivity, the sharing of idiosyncratic risks, and the economic influence of environmental variability. PMID:15254298

Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols.

PubMed

Campoy, Irene; Lanau, Lucia; Altadill, Tatiana; Sequeiros, Tamara; Cabrera, Silvia; Cubo-Abert, Montserrat; Pérez-Benavente, Assumpción; Garcia, Angel; Borrós, Salvador; Santamaria, Anna; Ponce, Jordi; Matias-Guiu, Xavier; Reventós, Jaume; Gil-Moreno, Antonio; Rigau, Marina; Colas, Eva

2016-06-18

Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 ± 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology.

Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications

PubMed Central

Peña-Llopis, Samuel; Brugarolas, James

2014-01-01

Genomic technologies have revolutionized our understanding of complex Mendelian diseases and cancer. Solid tumors present several challenges for genomic analyses, such as tumor heterogeneity and tumor contamination with surrounding stroma and infiltrating lymphocytes. We developed a protocol to (i) select tissues of high cellular purity on the basis of histological analyses of immediately flanking sections and (ii) simultaneously extract genomic DNA (gDNA), messenger RNA (mRNA), noncoding RNA (ncRNA; enriched in microRNA (miRNA)) and protein from the same tissues. After tissue selection, about 12–16 extractions of DNA/RNA/protein can be obtained per day. Compared with other similar approaches, this fast and reliable methodology allowed us to identify mutations in tumors with remarkable sensitivity and to perform integrative analyses of whole-genome and exome data sets, DNA copy numbers (by single-nucleotide polymorphism (SNP) arrays), gene expression data (by transcriptome profiling and quantitative PCR (qPCR)) and protein levels (by western blotting and immunohistochemical analysis) from the same samples. Although we focused on renal cell carcinoma, this protocol may be adapted with minor changes to any human or animal tissue to obtain high-quality and high-yield nucleic acids and proteins. PMID:24136348

Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols

PubMed Central

GARBIERI, Thais Francini; BROZOSKI, Daniel Thomas; DIONÍSIO, Thiago José; SANTOS, Carlos Ferreira; NEVES, Lucimara Teixeira das

2017-01-01

Abstract Saliva when compared to blood collection has the following advantages: it requires no specialized personnel for collection, allows for remote collection by the patient, is painless, well accepted by participants, has decreased risks of disease transmission, does not clot, can be frozen before DNA extraction and possibly has a longer storage time. Objective and Material and Methods This study aimed to compare the quantity and quality of human DNA extracted from saliva that was fresh or frozen for three, six and twelve months using five different DNA extraction protocols: protocol 1 – Oragene™ commercial kit, protocol 2 – QIAamp DNA mini kit, protocol 3 – DNA extraction using ammonium acetate, protocol 4 – Instagene™ Matrix and protocol 5 – Instagene™ Matrix diluted 1:1 using proteinase K and 1% SDS. Briefly, DNA was analyzed using spectrophotometry, electrophoresis and PCR. Results Results indicated that time spent in storage typically decreased the DNA quantity with the exception of protocol 1. The purity of DNA was generally not affected by storage times for the commercial based protocols, while the purity of the DNA samples extracted by the noncommercial protocols typically decreased when the saliva was stored longer. Only protocol 1 consistently extracted unfragmented DNA samples. In general, DNA samples extracted through protocols 1, 2, 3 and 4, regardless of storage time, were amplified by human specific primers whereas protocol 5 produced almost no samples that were able to be amplified by human specific primers. Depending on the protocol used, it was possible to extract DNA in high quantities and of good quality using whole saliva, and furthermore, for the purposes of DNA extraction, saliva can be reliably stored for relatively long time periods. Conclusions In summary, a complicated picture emerges when taking into account the extracted DNA’s quantity, purity and quality; depending on a given researchers needs, one protocol’s particular strengths and costs might be the deciding factor for its employment. PMID:28403355

NHEXAS PHASE I REGION 5 STUDY--STANDARD OPERATING PROCEDURE--HANDLING QUALITY CONTROL SAMPLES IN THE FIELD (RTI/ACS-AP-209-090)

EPA Science Inventory

This protocol describes how quality control samples should be handled in the field, and was designed as a quick reference source for the field staff. The protocol describes quality control samples for air-VOCs, air-particles, water samples, house dust, soil, urine, blood, hair, a...

Protocol for Detection of Yersinia pestis in Environmental ...

EPA Pesticide Factsheets

Methods Report This is the first ever open-access and detailed protocol available to all government departments and agencies, and their contractors to detect Yersinia pestis, the pathogen that causes plague, from multiple environmental sample types including water. Each analytical method includes sample processing procedure for each sample type in a step-by-step manner. It includes real-time PCR, traditional microbiological culture, and the Rapid Viability PCR (RV-PCR) analytical methods. For large volume water samples it also includes an ultra-filtration-based sample concentration procedure. Because of such a non-restrictive availability of this protocol to all government departments and agencies, and their contractors, the nation will now have increased laboratory capacity to analyze large number of samples during a wide-area plague incident.

Processing Protocol for Soil Samples Potentially ...

EPA Pesticide Factsheets

Method Operating Procedures This protocol describes the processing steps for 45 g and 9 g soil samples potentially contaminated with Bacillus anthracis spores. The protocol is designed to separate and concentrate the spores from bulk soil down to a pellet that can be used for further analysis. Soil extraction solution and mechanical shaking are used to disrupt soil particle aggregates and to aid in the separation of spores from soil particles. Soil samples are washed twice with soil extraction solution to maximize recovery. Differential centrifugation is used to separate spores from the majority of the soil material. The 45 g protocol has been demonstrated by two laboratories using both loamy and sandy soil types. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol would be robust enough to use at multiple laboratories while achieving comparable recoveries. The 45 g protocol has demonstrated a matrix limit of detection at 14 spores/gram of soil for loamy and sandy soils.

Processing protocol for soil samples potentially contaminated with Bacillus anthracis spores [HS7.52.02 - 514

USGS Publications Warehouse

Silvestri, Erin E.; Griffin, Dale W.

2017-01-01

This protocol describes the processing steps for 45 g and 9 g soil samples potentially contaminated with Bacillus anthracis spores. The protocol is designed to separate and concentrate the spores from bulk soil down to a pellet that can be used for further analysis. Soil extraction solution and mechanical shaking are used to disrupt soil particle aggregates and to aid in the separation of spores from soil particles. Soil samples are washed twice with soil extraction solution to maximize recovery. Differential centrifugation is used to separate spores from the majority of the soil material. The 45 g protocol has been demonstrated by two laboratories using both loamy and sandy soil types. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol would be robust enough to use at multiple laboratories while achieving comparable recoveries. The 45 g protocol has demonstrated a matrix limit of detection at 14 spores/gram of soil for loamy and sandy soils.

Surface morphology and dislocation characteristics near the surface of 4H-SiC wafer using multi-directional scanning transmission electron microscopy.

PubMed

Sato, Takahiro; Orai, Yoshihisa; Suzuki, Yuya; Ito, Hiroyuki; Isshiki, Toshiyuki; Fukui, Munetoshi; Nakamura, Kuniyasu; Schamp, C T

2017-10-01

To improve the reliability of silicon carbide (SiC) electronic power devices, the characteristics of various kinds of crystal defects should be precisely understood. Of particular importance is understanding the correlation between the surface morphology and the near surface dislocations. In order to analyze the dislocations near the surface of 4H-SiC wafers, a dislocation analysis protocol has been developed. This protocol consists of the following process: (1) inspection of surface defects using low energy scanning electron microscopy (LESEM), (2) identification of small and shallow etch pits using KOH low temperature etching, (3) classification of etch pits using LESEM, (4) specimen preparation of several hundred nanometer thick sample using the in-situ focused ion beam micro-sampling® technique, (5) crystallographic analysis using the selected diffraction mode of the scanning transmission electron microscope (STEM), and (6) determination of the Burgers vector using multi-directional STEM (MD-STEM). The results show a correlation between the triangular terrace shaped surface defects and an hexagonal etch pit arising from threading dislocations, linear shaped surface defects and elliptical shaped etch pits arising from basal plane dislocations. Through the observation of the sample from two orthogonal directions via the MD-STEM technique, a basal plane dislocation is found to dissociate into an extended dislocation bound by two partial dislocations. A protocol developed and presented in this paper enables one to correlate near surface defects of a 4H-SiC wafer with the root cause dislocations giving rise to those surface defects. © The Author 2017. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

World Health Organization Generic Protocol to Assess Drug-Resistant HIV Among Children <18 Months of Age and Newly Diagnosed With HIV in Resource-Limited Countries

PubMed Central

Penazzato, Martina; Jordan, Michael R.; Persaud, Deborah; Mofenson, Lynne M.; Bennett, Diane E.

2012-01-01

Increased use of nonnucleoside reverse transcriptase inhibitors (NNRTIs) in pregnant and breastfeeding women will result in fewer children infected with human immunodeficiency virus (HIV). However, among children infected despite prevention of mother-to-child transmission (PMTCT), a substantial proportion will acquire NNRTI-resistant HIV, potentially compromising response to NNRTI-based antiretroviral therapy (ART). In countries scaling up PMTCT and pediatric ART programs, it is crucial to assess the proportion of young children with drug-resistant HIV to improve health outcomes and support national and global decision making on optimal selection of pediatric first-line ART. This article summarizes a new World Health Organization surveillance protocol to assess resistance using remnant dried blood spot specimens from a representative sample of children aged <18 months being tested for early infant diagnosis. PMID:22544184

A simplified protocol for molecular identification of Eimeria species in field samples.

PubMed

Haug, Anita; Thebo, Per; Mattsson, Jens G

2007-05-15

This study aimed to find a fast, sensitive and efficient protocol for molecular identification of chicken Eimeria spp. in field samples. Various methods for each of the three steps of the protocol were evaluated: oocyst wall rupturing methods, DNA extraction methods, and identification of species-specific DNA sequences by PCR. We then compared and evaluated five complete protocols. Three series of oocyst suspensions of known number of oocysts from Eimeria mitis, Eimeria praecox, Eimeria maxima and Eimeria tenella were prepared and ground using glass beads or mini-pestle. DNA was extracted from ruptured oocysts using commercial systems (GeneReleaser, Qiagen Stoolkit and Prepman) or phenol-chloroform DNA extraction, followed by identification of species-specific ITS-1 sequences by optimised single species PCR assays. The Stoolkit and Prepman protocols showed insufficient repeatability, and the former was also expensive and relatively time-consuming. In contrast, both the GeneReleaser protocol and phenol-chloroform protocols were robust and sensitive, detecting less than 0.4 oocysts of each species per PCR. Finally, we evaluated our new protocol on 68 coccidia positive field samples. Our data suggests that rupturing the oocysts by mini-pestle grinding, preparing the DNA with GeneReleaser, followed by optimised single species PCR assays, makes a robust and sensitive procedure for identifying chicken Eimeria species in field samples. Importantly, it also provides minimal hands-on-time in the pre-PCR process, lower contamination risk and no handling of toxic chemicals.

Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples.

PubMed

Lewandowska, Dagmara W; Zagordi, Osvaldo; Geissberger, Fabienne-Desirée; Kufner, Verena; Schmutz, Stefan; Böni, Jürg; Metzner, Karin J; Trkola, Alexandra; Huber, Michael

2017-08-08

Sequence-specific PCR is the most common approach for virus identification in diagnostic laboratories. However, as specific PCR only detects pre-defined targets, novel virus strains or viruses not included in routine test panels will be missed. Recently, advances in high-throughput sequencing allow for virus-sequence-independent identification of entire virus populations in clinical samples, yet standardized protocols are needed to allow broad application in clinical diagnostics. Here, we describe a comprehensive sample preparation protocol for high-throughput metagenomic virus sequencing using random amplification of total nucleic acids from clinical samples. In order to optimize metagenomic sequencing for application in virus diagnostics, we tested different enrichment and amplification procedures on plasma samples spiked with RNA and DNA viruses. A protocol including filtration, nuclease digestion, and random amplification of RNA and DNA in separate reactions provided the best results, allowing reliable recovery of viral genomes and a good correlation of the relative number of sequencing reads with the virus input. We further validated our method by sequencing a multiplexed viral pathogen reagent containing a range of human viruses from different virus families. Our method proved successful in detecting the majority of the included viruses with high read numbers and compared well to other protocols in the field validated against the same reference reagent. Our sequencing protocol does work not only with plasma but also with other clinical samples such as urine and throat swabs. The workflow for virus metagenomic sequencing that we established proved successful in detecting a variety of viruses in different clinical samples. Our protocol supplements existing virus-specific detection strategies providing opportunities to identify atypical and novel viruses commonly not accounted for in routine diagnostic panels.

Open PHACTS computational protocols for in silico target validation of cellular phenotypic screens: knowing the knowns† †The authors declare no competing interests. ‡ ‡Electronic supplementary information (ESI) available: Pipeline Pilot protocols, xls file with the output of the Pipeline Pilot protocols, KNIME workflows, and supplementary figures showing the Pipeline Pilot protocols. See DOI: 10.1039/c6md00065g Click here for additional data file.

PubMed Central

Zdrazil, B.; Neefs, J.-M.; Van Vlijmen, H.; Herhaus, C.; Caracoti, A.; Brea, J.; Roibás, B.; Loza, M. I.; Queralt-Rosinach, N.; Furlong, L. I.; Gaulton, A.; Bartek, L.; Senger, S.; Chichester, C.; Engkvist, O.; Evelo, C. T.; Franklin, N. I.; Marren, D.; Ecker, G. F.

2016-01-01

Phenotypic screening is in a renaissance phase and is expected by many academic and industry leaders to accelerate the discovery of new drugs for new biology. Given that phenotypic screening is per definition target agnostic, the emphasis of in silico and in vitro follow-up work is on the exploration of possible molecular mechanisms and efficacy targets underlying the biological processes interrogated by the phenotypic screening experiments. Herein, we present six exemplar computational protocols for the interpretation of cellular phenotypic screens based on the integration of compound, target, pathway, and disease data established by the IMI Open PHACTS project. The protocols annotate phenotypic hit lists and allow follow-up experiments and mechanistic conclusions. The annotations included are from ChEMBL, ChEBI, GO, WikiPathways and DisGeNET. Also provided are protocols which select from the IUPHAR/BPS Guide to PHARMACOLOGY interaction file selective compounds to probe potential targets and a correlation robot which systematically aims to identify an overlap of active compounds in both the phenotypic as well as any kinase assay. The protocols are applied to a phenotypic pre-lamin A/C splicing assay selected from the ChEMBL database to illustrate the process. The computational protocols make use of the Open PHACTS API and data and are built within the Pipeline Pilot and KNIME workflow tools. PMID:27774140

Investigation of Rhodopsin Dynamics in its Signaling State by Solid-State Deuterium NMR Spectroscopy

PubMed Central

Struts, Andrey V.; Chawla, Udeep; Perera, Suchithranga M.D.C.; Brown, Michael F.

2017-01-01

Site-directed deuterium NMR spectroscopy is a valuable tool to study the structural dynamics of biomolecules in cases where solution NMR is inapplicable. Solid-state 2H NMR spectral studies of aligned membrane samples of rhodopsin with selectively labeled retinal provide information on structural changes of the chromophore in different protein states. In addition, solid-state 2H NMR relaxation time measurements allow one to study the dynamics of the ligand during the transition from the inactive to the active state. Here we describe the methodological aspects of solid-state 2H NMR spectroscopy for functional studies of rhodopsin, with an emphasis on the dynamics of the retinal cofactor. We provide complete protocols for the preparation of NMR samples of rhodopsin with 11-cis-retinal selectively deuterated at the methyl groups in aligned membranes. In addition, we review optimized conditions for trapping the rhodopsin photointermediates; and lastly we address the challenging problem of trapping the signaling state of rhodopsin in aligned membrane films. PMID:25697522

A MORE COST-EFFECTIVE EMAP-ESTUARIES BENTHIC MACROFAUNAL SAMPLING PROTOCOL

EPA Science Inventory

The standard benthic macrofaunal sampling protocol in the U.S. Environmental Protection Agency's Pacific Coast Environmental Monitoring and Assessment Program (EMAP) is to collect a minimum of 30 random benthic samples per reporting unit (e.g., estuary) using a 0.1 m2 grab and to...

It's Time to Develop a New "Draft Test Protocol" for a Mars Sample Return Mission (or Two....)

NASA Astrophysics Data System (ADS)

Rummel, J. D.

2018-04-01

A Mars Sample Return (MSR) will involve analysis of those samples in containment, including their safe receiving, handling, testing, and archiving. With an MSR planned for the end of the next decade, it is time to update the existing MSR protocol.

Method selection for sustainability assessments: The case of recovery of resources from waste water.

PubMed

Zijp, M C; Waaijers-van der Loop, S L; Heijungs, R; Broeren, M L M; Peeters, R; Van Nieuwenhuijzen, A; Shen, L; Heugens, E H W; Posthuma, L

2017-07-15

Sustainability assessments provide scientific support in decision procedures towards sustainable solutions. However, in order to contribute in identifying and choosing sustainable solutions, the sustainability assessment has to fit the decision context. Two complicating factors exist. First, different stakeholders tend to have different views on what a sustainability assessment should encompass. Second, a plethora of sustainability assessment methods exist, due to the multi-dimensional characteristic of the concept. Different methods provide other representations of sustainability. Based on a literature review, we present a protocol to facilitate method selection together with stakeholders. The protocol guides the exploration of i) the decision context, ii) the different views of stakeholders and iii) the selection of pertinent assessment methods. In addition, we present an online tool for method selection. This tool identifies assessment methods that meet the specifications obtained with the protocol, and currently contains characteristics of 30 sustainability assessment methods. The utility of the protocol and the tool are tested in a case study on the recovery of resources from domestic waste water. In several iterations, a combination of methods was selected, followed by execution of the selected sustainability assessment methods. The assessment results can be used in the first phase of the decision procedure that leads to a strategic choice for sustainable resource recovery from waste water in the Netherlands. Copyright © 2017 Elsevier Ltd. All rights reserved.

System for Configuring Modular Telemetry Transponders

NASA Technical Reports Server (NTRS)

Varnavas, Kosta A. (Inventor); Sims, William Herbert, III (Inventor)

2014-01-01

A system for configuring telemetry transponder cards uses a database of error checking protocol data structures, each containing data to implement at least one CCSDS protocol algorithm. Using a user interface, a user selects at least one telemetry specific error checking protocol from the database. A compiler configures an FPGA with the data from the data structures to implement the error checking protocol.

Effect of colloid (Androcoll-Bear, Percoll, and PureSperm) selection on the freezability of brown bear (Ursus arctos) sperm.

PubMed

Álvarez-Rodríguez, M; Álvarez, M; Anel-López, L; López-Urueña, E; Manrique, P; Borragán, S; Morrell, J M; de Paz, P; Anel, L

2016-04-01

The development of a species-specific conservation protocol that involves artificial insemination with frozen semen needs to validate an effective methodology for freezing semen. Colloid centrifugation has been suggested and widely applied as an effective tool for selecting animal spermatozoa for artificial breeding. The objective of the present study was to compare different methods of centrifugation, single layer using Androcoll-Bear and Percoll and double layer using PureSperm 100 (in two different discontinuous gradients 40%-80% and 45%-90%), for the selection of fresh brown bear sperm samples. In the before freezing group, all selected samples showed a higher progressive motility and viability (except Percoll for motility 43.0 ± 5.3 [P < 0.05]); all colloids except PureSperm 45/90% rendered samples with fewer damaged acrosomes. In the after thawing group, all tested centrifugation colloids showed a good capacity to decrease the number of damaged acrosomes. Furthermore, PureSperm treatment (45/90%) resulted in an increase in apoptotic-like changes not only immediately after thawing but also after the incubation test, leading us to suggest that this gradient could induce some kind of deleterious effects on the sperm samples. On the other hand, PureSperm treatment (40/80%) yielded a quality preservation capacity similar to Androcoll-Bear in number of damaged acrosomes, different relative to the control (control, 5.3 ± 0.6; PureSperm 80, 2.0 ± 0.3; Androcoll, 2.1 ± 0.9 [P < 0.05]) but a decrease in the number of viable spermatozoa recovered after thawing relative to the control (control, 21.2 ± 3.1; PureSperm 80, 13.7 ± 2.7 [P < 0.05]). In conclusion, Androcoll-Bear constitutes a useful tool for handling of brown bear ejaculates owing to its simple handling and procedure with a reliable sperm selection and freezability. This colloid yielded an improvement in several sperm parameters in brown bear frozen-thawed semen; the selected spermatozoa of fresh samples with this colloid showed a better resistance to freezing compared with the control sample not only for motility but also for viability. Copyright © 2016 Elsevier Inc. All rights reserved.

Refinement of NMR structures using implicit solvent and advanced sampling techniques.

PubMed

Chen, Jianhan; Im, Wonpil; Brooks, Charles L

2004-12-15

NMR biomolecular structure calculations exploit simulated annealing methods for conformational sampling and require a relatively high level of redundancy in the experimental restraints to determine quality three-dimensional structures. Recent advances in generalized Born (GB) implicit solvent models should make it possible to combine information from both experimental measurements and accurate empirical force fields to improve the quality of NMR-derived structures. In this paper, we study the influence of implicit solvent on the refinement of protein NMR structures and identify an optimal protocol of utilizing these improved force fields. To do so, we carry out structure refinement experiments for model proteins with published NMR structures using full NMR restraints and subsets of them. We also investigate the application of advanced sampling techniques to NMR structure refinement. Similar to the observations of Xia et al. (J.Biomol. NMR 2002, 22, 317-331), we find that the impact of implicit solvent is rather small when there is a sufficient number of experimental restraints (such as in the final stage of NMR structure determination), whether implicit solvent is used throughout the calculation or only in the final refinement step. The application of advanced sampling techniques also seems to have minimal impact in this case. However, when the experimental data are limited, we demonstrate that refinement with implicit solvent can substantially improve the quality of the structures. In particular, when combined with an advanced sampling technique, the replica exchange (REX) method, near-native structures can be rapidly moved toward the native basin. The REX method provides both enhanced sampling and automatic selection of the most native-like (lowest energy) structures. An optimal protocol based on our studies first generates an ensemble of initial structures that maximally satisfy the available experimental data with conventional NMR software using a simplified force field and then refines these structures with implicit solvent using the REX method. We systematically examine the reliability and efficacy of this protocol using four proteins of various sizes ranging from the 56-residue B1 domain of Streptococcal protein G to the 370-residue Maltose-binding protein. Significant improvement in the structures was observed in all cases when refinement was based on low-redundancy restraint data. The proposed protocol is anticipated to be particularly useful in early stages of NMR structure determination where a reliable estimate of the native fold from limited data can significantly expedite the overall process. This refinement procedure is also expected to be useful when redundant experimental data are not readily available, such as for large multidomain biomolecules and in solid-state NMR structure determination.

An automated baseline correction protocol for infrared spectra of atmospheric aerosols collected on polytetrafluoroethylene (Teflon) filters

NASA Astrophysics Data System (ADS)

Kuzmiakova, Adele; Dillner, Ann M.; Takahama, Satoshi

2016-06-01

A growing body of research on statistical applications for characterization of atmospheric aerosol Fourier transform infrared (FT-IR) samples collected on polytetrafluoroethylene (PTFE) filters (e.g., Russell et al., 2011; Ruthenburg et al., 2014) and a rising interest in analyzing FT-IR samples collected by air quality monitoring networks call for an automated PTFE baseline correction solution. The existing polynomial technique (Takahama et al., 2013) is not scalable to a project with a large number of aerosol samples because it contains many parameters and requires expert intervention. Therefore, the question of how to develop an automated method for baseline correcting hundreds to thousands of ambient aerosol spectra given the variability in both environmental mixture composition and PTFE baselines remains. This study approaches the question by detailing the statistical protocol, which allows for the precise definition of analyte and background subregions, applies nonparametric smoothing splines to reproduce sample-specific PTFE variations, and integrates performance metrics from atmospheric aerosol and blank samples alike in the smoothing parameter selection. Referencing 794 atmospheric aerosol samples from seven Interagency Monitoring of PROtected Visual Environment (IMPROVE) sites collected during 2011, we start by identifying key FT-IR signal characteristics, such as non-negative absorbance or analyte segment transformation, to capture sample-specific transitions between background and analyte. While referring to qualitative properties of PTFE background, the goal of smoothing splines interpolation is to learn the baseline structure in the background region to predict the baseline structure in the analyte region. We then validate the model by comparing smoothing splines baseline-corrected spectra with uncorrected and polynomial baseline (PB)-corrected equivalents via three statistical applications: (1) clustering analysis, (2) functional group quantification, and (3) thermal optical reflectance (TOR) organic carbon (OC) and elemental carbon (EC) predictions. The discrepancy rate for a four-cluster solution is 10 %. For all functional groups but carboxylic COH the discrepancy is ≤ 10 %. Performance metrics obtained from TOR OC and EC predictions (R2 ≥ 0.94 %, bias ≤ 0.01 µg m-3, and error ≤ 0.04 µg m-3) are on a par with those obtained from uncorrected and PB-corrected spectra. The proposed protocol leads to visually and analytically similar estimates as those generated by the polynomial method. More importantly, the automated solution allows us and future users to evaluate its analytical reproducibility while minimizing reducible user bias. We anticipate the protocol will enable FT-IR researchers and data analysts to quickly and reliably analyze a large amount of data and connect them to a variety of available statistical learning methods to be applied to analyte absorbances isolated in atmospheric aerosol samples.

Cost Analysis of the Addition of Hyperacute Magnetic Resonance Imaging for Selection of Patients for Endovascular Stroke Therapy.

PubMed

John, Seby; Thompson, Nicolas R; Lesko, Terry; Papesh, Nancy; Obuchowski, Nancy; Tomic, Dan; Wisco, Dolora; Khawaja, Zeshaun; Uchino, Ken; Man, Shumei; Cheng-Ching, Esteban; Toth, Gabor; Masaryk, Thomas; Ruggieri, Paul; Modic, Michael; Hussain, Muhammad Shazam

2017-10-01

Patient selection is important to determine the best candidates for endovascular stroke therapy. In application of a hyperacute magnetic resonance imaging (MRI) protocol for patient selection, we have shown decreased utilization with improved outcomes. A cost analysis comparing the pre- and post-MRI protocol time periods was performed to determine if the previous findings translated into cost opportunities. We retrospectively identified individuals considered for endovascular stroke therapy from January 2008 to August 2012 who were ≤8 h from stroke symptoms onset. Patients prior to April 30, 2010 were selected based on results of the computed tomography/computed tomography angiography alone (pre-hyperacute), whereas patients after April 30, 2010 were selected based on results of MRI (post-hyperacute MRI). Demographic, outcome, and financial information was collected. Log-transformed average daily direct costs were regressed on time period. The regression model included demographic and clinical covariates as potential confounders. Multiple imputation was used to account for missing data. We identified 267 patients in our database (88 patients in pre-hyperacute MRI period, 179 in hyperacute MRI protocol period). Patient length of stay was not significantly different in the hyperacute MRI protocol period as compared to the pre-hyperacute MRI period (10.6 vs. 9.9 days, p < 0.42). The median of average daily direct costs was reduced by 24.5% (95% confidence interval 14.1-33.7%, p < 0.001). Use of the hyperacute MRI protocol translated into reduced costs, in addition to reduced utilization and better outcomes. MRI selection of patients is an effective strategy, both for patients and hospital systems.

Improving Leishmania Species Identification in Different Types of Samples from Cutaneous Lesions

PubMed Central

Cruz-Barrera, Mónica L.; Ovalle-Bracho, Clemencia; Ortegon-Vergara, Viviana; Pérez-Franco, Jairo E.

2015-01-01

The discrimination of Leishmania species from patient samples has epidemiological and clinical relevance. In this study, different gene target PCR-restriction fragment length polymorphism (RFLP) protocols were evaluated for their robustness as Leishmania species discriminators in 61 patients with cutaneous leishmaniasis. We modified the hsp70-PCR-RFLP protocol and found it to be the most reliable protocol for species identification. PMID:25609727

Is the Prosthetic Homologue Necessary for Embodiment?

PubMed Central

Dornfeld, Chelsea; Swanston, Michelle; Cassella, Joseph; Beasley, Casey; Green, Jacob; Moshayev, Yonatan; Wininger, Michael

2016-01-01

Embodiment is the process by which patients with limb loss come to accept their peripheral device as a natural extension of self. However, there is little guidance as to how exacting the prosthesis must be in order for embodiment to take place: is it necessary for the prosthetic hand to look just like the absent hand? Here, we describe a protocol for testing whether an individual would select a hand that looks like their own from among a selection of five hands, and whether the hand selection (regardless of homology) is consistent across multiple exposures to the same (but reordered) set of candidate hands. Pilot results using healthy volunteers reveals that hand selection is only modestly consistent, and that selection of the prosthetic homologue is atypical (61 of 192 total exposures). Our protocol can be executed in minutes, and makes use of readily available equipment and softwares. We present both a face-to-face and a virtual protocol, for maximum flexibility of implementation. PMID:28066228

Inhibitory effects of lupene-derived pentacyclic triterpenoids from Bursera simaruba on HSV-1 and HSV-2 in vitro replication.

PubMed

Álvarez, Ángel L; Habtemariam, Solomon; Parra, Francisco

2015-01-01

The cytotoxicity and antiviral properties of Bursera simaruba against herpes simplex viruses (HSV-1 and HSV-2) were investigated through a bioactivity-guided isolation protocol. The plant material was fractionated using solvent-solvent partitioning, size-exclusion and thin-layer chromatography. The antiviral compounds present in the most active fractions were identified by means of LC-MS and NMR. Three different methods were compared during the evaluation of antiviral activity of samples. Four lupene-related pentacyclic triterpenes were found to be responsible for the anti-herpesvirus effects of B. simaruba and were isolated from this species for the first time. The selective indexes (SI) of B. simaruba-derived samples ranged from 7.7 to 201.9.

EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

PubMed Central

Kalina, T; Flores-Montero, J; van der Velden, V H J; Martin-Ayuso, M; Böttcher, S; Ritgen, M; Almeida, J; Lhermitte, L; Asnafi, V; Mendonça, A; de Tute, R; Cullen, M; Sedek, L; Vidriales, M B; Pérez, J J; te Marvelde, J G; Mejstrikova, E; Hrusak, O; Szczepański, T; van Dongen, J J M; Orfao, A

2012-01-01

The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database. PMID:22948490

Protocol for Biomarker Ratio Imaging Microscopy with Specific Application to Ductal Carcinoma In situ of the Breast

PubMed Central

Clark, Andrea J.; Petty, Howard R.

2016-01-01

This protocol describes the methods and steps involved in performing biomarker ratio imaging microscopy (BRIM) using formalin fixed paraffin-embedded (FFPE) samples of human breast tissue. The technique is based on the acquisition of two fluorescence images of the same microscopic field using two biomarkers and immunohistochemical tools. The biomarkers are selected such that one biomarker correlates with breast cancer aggressiveness while the second biomarker anti-correlates with aggressiveness. When the former image is divided by the latter image, a computed ratio image is formed that reflects the aggressiveness of tumor cells while increasing contrast and eliminating path-length and other artifacts from the image. For example, the aggressiveness of epithelial cells may be assessed by computing ratio images of N-cadherin and E-cadherin images or CD44 and CD24 images, which specifically reflect the mesenchymal or stem cell nature of the constituent cells, respectively. This methodology is illustrated for tissue samples of ductal carcinoma in situ (DCIS) and invasive breast cancer. This tool should be useful in tissue studies of experimental cancer as well as the management of cancer patients. PMID:27857940

The predictive value of skin prick testing for challenge-proven food allergy: a systematic review.

PubMed

Peters, Rachel L; Gurrin, Lyle C; Allen, Katrina J

2012-06-01

Immunoglobulin E-mediated (IgE) food allergy affects 6-8% of children, and the prevalence is believed to be increasing. The gold standard of food allergy diagnosis is oral food challenges (OFCs); however, they are resource-consuming and potentially dangerous. Skin prick tests (SPTs) are able to detect the presence of allergen-specific IgE antibodies (sensitization), but they have low specificity for clinically significant food allergy. To reduce the need for OFCs, it has been suggested that children forgo an OFC if their SPT wheal size exceeds a cutoff that has a high predictability for food allergy. Although data for these studies are almost always gathered from high-risk populations, the 95% positive predictive values (PPVs) vary substantially between studies. SPT thresholds with a high probability of food allergy generated from these studies may not be generalizable to other populations, because of highly selective samples and variability in participant's age, test allergens, and food challenge protocol. Standardization of SPT devices and allergens, OFC protocols including standardized cessation criteria, and population-based samples would all help to improve generalizability of PPVs of SPTs. © 2011 John Wiley & Sons A/S.

Process and Outcome of Fecal Microbiota Transplants in Patients With Recurrent Clostridium difficile Infection: A Prospective Study.

PubMed

Walton, Janice; Burns, Denise; Gaehle, Kay E

The incidence of Clostridium difficile infection is on the rise worldwide, causing high mortality rates and costing patients, hospitals, and insurance companies millions of dollars annually. Fecal microbiota transplants successfully treat recurrent C. difficile infections unresponsive to standard pharmacologic treatment such as flagyl, vancomycin, or rifaximin. Evidence in the literature provided the foundation for the development and refinement of this fecal microbiota transplant protocol. During the initial phase of the project, the protocol included patient selection criteria, donor screening/selection, infection control, fecal processing and delivery, and patient pre and postprocedure education. This article highlights the second phase of prospective testing of a nurse-driven protocol to implement fecal microbiota transplantation in patients with recurrent C. difficile infection. All stages of the protocol are explained as well as rationale for component parts to achieve successful patient outcomes when the protocol is carefully followed.

High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants.

PubMed

Rech, Elibio L; Vianna, Giovanni R; Aragão, Francisco J L

2008-01-01

This protocol describes a method for high-frequency recovery of transgenic soybean, bean and cotton plants, by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques. This protocol involves the following stages: plasmid design, preparation of soybean, common bean and cotton apical meristems for bombardment, microparticle-coated DNA bombardment of apical meristems and in vitro culture and selection of transgenic plants. The average frequencies (the total number of fertile transgenic plants divided by the total number of bombarded embryonic axes) of producing germline transgenic soybean and bean and cotton plants using this protocol are 9, 2.7 and 0.55%, respectively. This protocol is suitable for studies of gene function as well as the production of transgenic cultivars carrying different traits for breeding programs. This protocol can be completed in 7-10 months.

Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry

PubMed Central

Abelin, Jennifer G; Trantham, Paisley D; Penny, Sarah A; Patterson, Andrea M; Ward, Stephen T; Hildebrand, William H; Cobbold, Mark; Bai, Dina L; Shabanowitz, Jeffrey; Hunt, Donald F

2015-01-01

Phosphorylation events within cancer cells often become dysregulated, leading to oncogenic signaling and abnormal cell growth. Phosphopeptides derived from aberrantly phosphorylated proteins that are presented on tumors and not on normal tissues by human leukocyte antigen (HLA) class I molecules are promising candidates for future cancer immunotherapies, because they are tumor specific and have been shown to elicit cytotoxic T cell responses. Robust phosphopeptide enrichments that are suitable for low input amounts must be developed to characterize HLA-associated phosphopeptides from clinical samples that are limited by material availability. We present two complementary mass spectrometry–compatible, iron(III)-immobilized metal affinity chromatography (IMAC) methods that use either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) in-house-fabricated columns. We developed these protocols to enrich for subfemtomole-level phosphopeptides from cell line and human tissue samples containing picograms of starting material, which is an order of magnitude less material than what is commonly used. In addition, we added a peptide esterification step to increase phosphopeptide specificity from these low-input samples. To date, hundreds of phosphopeptides displayed on melanoma, ovarian cancer, leukemia and colorectal cancer have been identified using these highly selective phosphopeptide enrichment protocols in combination with a program called ‘CAD Neutral Loss Finder’ that identifies all spectra containing the characteristic neutral loss of phosphoric acid from phosphorylated serine and threonine residues. This methodology enables the identification of HLA-associated phosphopeptides presented by human tissue samples containing as little as nanograms of peptide material in 2 d. PMID:26247297

Comparison of Health Examination Survey Methods in Brazil, Chile, Colombia, Mexico, England, Scotland, and the United States.

PubMed

Mindell, Jennifer S; Moody, Alison; Vecino-Ortiz, Andres I; Alfaro, Tania; Frenz, Patricia; Scholes, Shaun; Gonzalez, Silvia A; Margozzini, Paula; de Oliveira, Cesar; Sanchez Romero, Luz Maria; Alvarado, Andres; Cabrera, Sebastián; Sarmiento, Olga L; Triana, Camilo A; Barquera, Simón

2017-09-15

Comparability of population surveys across countries is key to appraising trends in population health. Achieving this requires deep understanding of the methods used in these surveys to examine the extent to which the measurements are comparable. In this study, we obtained detailed protocols of 8 nationally representative surveys from 2007-2013 from Brazil, Chile, Colombia, Mexico, the United Kingdom (England and Scotland), and the United States-countries that that differ in economic and inequity indicators. Data were collected on sampling frame, sample selection procedures, recruitment, data collection methods, content of interview and examination modules, and measurement protocols. We also assessed their adherence to the World Health Organization's "STEPwise Approach to Surveillance" framework for population health surveys. The surveys, which included half a million participants, were highly comparable on sampling methodology, survey questions, and anthropometric measurements. Heterogeneity was found for physical activity questionnaires and biological samples collection. The common age range included by the surveys was adults aged 18-64 years. The methods used in these surveys were similar enough to enable comparative analyses of the data across the 7 countries. This comparability is crucial in assessing and comparing national and subgroup population health, and to assisting the transfer of research and policy knowledge across countries. © The Author(s) 2017. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A paleointensity technique for multidomain igneous rocks

NASA Astrophysics Data System (ADS)

Wang, Huapei; Kent, Dennis V.

2013-10-01

We developed a paleointensity technique to account for concave-up Arai diagrams due to multidomain (MD) contributions to determine unbiased paleointensities for 24 trial samples from site GA-X in Pleistocene lavas from Floreana Island, Galapagos Archipelago. The main magnetization carrier is fine-grained low-titanium magnetite of variable grain size. We used a comprehensive back-zero-forth (BZF) heating technique by adding an additional zero-field heating between the Thellier two opposite in-field heating steps in order to estimate paleointensities in various standard protocols and provide internal self-consistency checks. After the first BZF experiment, we gave each sample a total thermal remanent magnetization (tTRM) by cooling from the Curie point in the presence of a low (15 µT) laboratory-applied field. Then we repeated the BZF protocol, with the laboratory-applied tTRM as a synthetic natural remanent magnetization (NRM), using the same laboratory-applied field and temperature steps to obtain the synthetic Arai signatures, which should only represent the domain-state dependent properties of the samples. We corrected the original Arai diagrams from the first BZF experiment by using the Arai signatures from the repeated BZF experiment, which neutralizes the typical MD concave-up effect. Eleven samples meet the Arai diagram post-selection criteria and provide qualified paleointensity estimates with a mean value for site GA-X of 4.23 ± 1.29 µT, consistent with an excursional geomagnetic field direction reported for this site.

Multiplex Real-Time PCR for Detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) Genes from Selective Enrichments from Animals and Retail Meat

PubMed Central

Velasco, Valeria; Sherwood, Julie S.; Rojas-García, Pedro P.; Logue, Catherine M.

2014-01-01

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method. PMID:24849624

Multiplex real-time PCR for detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) genes from selective enrichments from animals and retail meat.

PubMed

Velasco, Valeria; Sherwood, Julie S; Rojas-García, Pedro P; Logue, Catherine M

2014-01-01

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68-0.88 (from substantial to almost perfect agreement) and 0.29-0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0-0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method.

Calibrated work function mapping by Kelvin probe force microscopy

NASA Astrophysics Data System (ADS)

Fernández Garrillo, Pablo A.; Grévin, Benjamin; Chevalier, Nicolas; Borowik, Łukasz

2018-04-01

We propose and demonstrate the implementation of an alternative work function tip calibration procedure for Kelvin probe force microscopy under ultrahigh vacuum, using monocrystalline metallic materials with known crystallographic orientation as reference samples, instead of the often used highly oriented pyrolytic graphite calibration sample. The implementation of this protocol allows the acquisition of absolute and reproducible work function values, with an improved uncertainty with respect to unprepared highly oriented pyrolytic graphite-based protocols. The developed protocol allows the local investigation of absolute work function values over nanostructured samples and can be implemented in electronic structures and devices characterization as demonstrated over a nanostructured semiconductor sample presenting Al0.7Ga0.3As and GaAs layers with variable thickness. Additionally, using our protocol we find that the work function of annealed highly oriented pyrolytic graphite is equal to 4.6 ± 0.03 eV.

Effectiveness of the Preservation Protocol within EPA Method 200.8 for Soluble and Particulate Lead Recovery in Drinking Water

EPA Science Inventory

The purpose of this project was to investigate the effectiveness of the sample preservation protocol outlined in Method 200.8 in recovering lead from water samples. Lead recoveries were studied in various water samples spiked with lead by evaluating lead sorption and desorption f...

21 CFR 660.36 - Samples and protocols.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Samples and protocols. 660.36 Section 660.36 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS... Research Sample Custodian (ATTN: HFM-672) (see mailing addresses in § 600.2 of this chapter), within 30...

BIASES IN CASTNET FILTER PACK RESULTS ASSOCIATED WITH SAMPLING PROTOCOL

EPA Science Inventory

In the current study, single filter weekly (w) results are compared with weekly results aggregated from day and night (dn) weekly samples. Comparisons of the two sampling protocols for all major constituents (SO42-, NO3-, NH4+, HNO3, and SO2) show median bias (MB) of < 5 nmol m-3...

A standardized sampling protocol for channel catfish in prairie streams

USGS Publications Warehouse

Vokoun, Jason C.; Rabeni, Charles F.

2001-01-01

Three alternative gears—an AC electrofishing raft, bankpoles, and a 15-hoop-net set—were used in a standardized manner to sample channel catfish Ictalurus punctatus in three prairie streams of varying size in three seasons. We compared these gears as to time required per sample, size selectivity, mean catch per unit effort (CPUE) among months, mean CPUE within months, effect of fluctuating stream stage, and sensitivity to population size. According to these comparisons, the 15-hoop-net set used during stable water levels in October had the most desirable characteristics. Using our catch data, we estimated the precision of CPUE and size structure by varying sample sizes for the 15-hoop-net set. We recommend that 11–15 repetitions of the 15-hoop-net set be used for most management activities. This standardized basic unit of effort will increase the precision of estimates and allow better comparisons among samples as well as increased confidence in management decisions.

Spatial cluster analysis of nanoscopically mapped serotonin receptors for classification of fixed brain tissue

NASA Astrophysics Data System (ADS)

Sams, Michael; Silye, Rene; Göhring, Janett; Muresan, Leila; Schilcher, Kurt; Jacak, Jaroslaw

2014-01-01

We present a cluster spatial analysis method using nanoscopic dSTORM images to determine changes in protein cluster distributions within brain tissue. Such methods are suitable to investigate human brain tissue and will help to achieve a deeper understanding of brain disease along with aiding drug development. Human brain tissue samples are usually treated postmortem via standard fixation protocols, which are established in clinical laboratories. Therefore, our localization microscopy-based method was adapted to characterize protein density and protein cluster localization in samples fixed using different protocols followed by common fluorescent immunohistochemistry techniques. The localization microscopy allows nanoscopic mapping of serotonin 5-HT1A receptor groups within a two-dimensional image of a brain tissue slice. These nanoscopically mapped proteins can be confined to clusters by applying the proposed statistical spatial analysis. Selected features of such clusters were subsequently used to characterize and classify the tissue. Samples were obtained from different types of patients, fixed with different preparation methods, and finally stored in a human tissue bank. To verify the proposed method, samples of a cryopreserved healthy brain have been compared with epitope-retrieved and paraffin-fixed tissues. Furthermore, samples of healthy brain tissues were compared with data obtained from patients suffering from mental illnesses (e.g., major depressive disorder). Our work demonstrates the applicability of localization microscopy and image analysis methods for comparison and classification of human brain tissues at a nanoscopic level. Furthermore, the presented workflow marks a unique technological advance in the characterization of protein distributions in brain tissue sections.

Integrated Cryptosporidium Assay To Determine Oocyst Density, Infectivity, and Genotype for Risk Assessment of Source and Reuse Water

PubMed Central

King, Brendon; Fanok, Stella; Phillips, Renae; Swaffer, Brooke

2015-01-01

Cryptosporidium continues to be problematic for the water industry, with risk assessments often indicating that treatment barriers may fail under extreme conditions. However, risk analyses have historically used oocyst densities and not considered either oocyst infectivity or species/genotype, which can result in an overestimation of risk if the oocysts are not human infective. We describe an integrated assay for determining oocyst density, infectivity, and genotype from a single-sample concentrate, an important advance that overcomes the need for processing multiple-grab samples or splitting sample concentrates for separate analyses. The assay incorporates an oocyst recovery control and is compatible with standard primary concentration techniques. Oocysts were purified from primary concentrates using immunomagnetic separation prior to processing by an infectivity assay. Plate-based cell culture was used to detect infectious foci, with a monolayer washing protocol developed to allow recovery and enumeration of oocysts. A simple DNA extraction protocol was developed to allow typing of any wells containing infectious Cryptosporidium. Water samples from a variety of source water and wastewater matrices, including a semirural catchment, wastewater, an aquifer recharge site, and storm water, were analyzed using the assay. Results demonstrate that the assay can reliably determine oocyst densities, infectivity, and genotype from single-grab samples for a variety of water matrices and emphasize the varying nature of Cryptosporidium risk extant throughout source waters and wastewaters. This assay should therefore enable a more comprehensive understanding of Cryptosporidium risk for different water sources, assisting in the selection of appropriate risk mitigation measures. PMID:25769833

Integrated cryptosporidium assay to determine oocyst density, infectivity, and genotype for risk assessment of source and reuse water.

PubMed

King, Brendon; Fanok, Stella; Phillips, Renae; Swaffer, Brooke; Monis, Paul

2015-05-15

Cryptosporidium continues to be problematic for the water industry, with risk assessments often indicating that treatment barriers may fail under extreme conditions. However, risk analyses have historically used oocyst densities and not considered either oocyst infectivity or species/genotype, which can result in an overestimation of risk if the oocysts are not human infective. We describe an integrated assay for determining oocyst density, infectivity, and genotype from a single-sample concentrate, an important advance that overcomes the need for processing multiple-grab samples or splitting sample concentrates for separate analyses. The assay incorporates an oocyst recovery control and is compatible with standard primary concentration techniques. Oocysts were purified from primary concentrates using immunomagnetic separation prior to processing by an infectivity assay. Plate-based cell culture was used to detect infectious foci, with a monolayer washing protocol developed to allow recovery and enumeration of oocysts. A simple DNA extraction protocol was developed to allow typing of any wells containing infectious Cryptosporidium. Water samples from a variety of source water and wastewater matrices, including a semirural catchment, wastewater, an aquifer recharge site, and storm water, were analyzed using the assay. Results demonstrate that the assay can reliably determine oocyst densities, infectivity, and genotype from single-grab samples for a variety of water matrices and emphasize the varying nature of Cryptosporidium risk extant throughout source waters and wastewaters. This assay should therefore enable a more comprehensive understanding of Cryptosporidium risk for different water sources, assisting in the selection of appropriate risk mitigation measures. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Secure and Fair Cluster Head Selection Protocol for Enhancing Security in Mobile Ad Hoc Networks

PubMed Central

Paramasivan, B.; Kaliappan, M.

2014-01-01

Mobile ad hoc networks (MANETs) are wireless networks consisting of number of autonomous mobile devices temporarily interconnected into a network by wireless media. MANETs become one of the most prevalent areas of research in the recent years. Resource limitations, energy efficiency, scalability, and security are the great challenging issues in MANETs. Due to its deployment nature, MANETs are more vulnerable to malicious attack. The secure routing protocols perform very basic security related functions which are not sufficient to protect the network. In this paper, a secure and fair cluster head selection protocol (SFCP) is proposed which integrates security factors into the clustering approach for achieving attacker identification and classification. Byzantine agreement based cooperative technique is used for attacker identification and classification to make the network more attack resistant. SFCP used to solve this issue by making the nodes that are totally surrounded by malicious neighbors adjust dynamically their belief and disbelief thresholds. The proposed protocol selects the secure and energy efficient cluster head which acts as a local detector without imposing overhead to the clustering performance. SFCP is simulated in network simulator 2 and compared with two protocols including AODV and CBRP. PMID:25143986

Secure and fair cluster head selection protocol for enhancing security in mobile ad hoc networks.

PubMed

Paramasivan, B; Kaliappan, M

2014-01-01

Mobile ad hoc networks (MANETs) are wireless networks consisting of number of autonomous mobile devices temporarily interconnected into a network by wireless media. MANETs become one of the most prevalent areas of research in the recent years. Resource limitations, energy efficiency, scalability, and security are the great challenging issues in MANETs. Due to its deployment nature, MANETs are more vulnerable to malicious attack. The secure routing protocols perform very basic security related functions which are not sufficient to protect the network. In this paper, a secure and fair cluster head selection protocol (SFCP) is proposed which integrates security factors into the clustering approach for achieving attacker identification and classification. Byzantine agreement based cooperative technique is used for attacker identification and classification to make the network more attack resistant. SFCP used to solve this issue by making the nodes that are totally surrounded by malicious neighbors adjust dynamically their belief and disbelief thresholds. The proposed protocol selects the secure and energy efficient cluster head which acts as a local detector without imposing overhead to the clustering performance. SFCP is simulated in network simulator 2 and compared with two protocols including AODV and CBRP.

Biofeedback treatment of constipation: a critical review.

PubMed

Heymen, Steve; Jones, Kenneth R; Scarlett, Yolanda; Whitehead, William E

2003-09-01

This review was designed to 1) critically examine the research design used in investigations of biofeedback for pelvic floor dyssynergia, 2) compare the various biofeedback treatment protocols for pelvic floor dyssynergia-type constipation used in this research, 3) identify factors that influence treatment outcome, and 4) identify goals for future biofeedback research for pelvic floor dyssynergia. A comprehensive review of both the pediatric and adult research from 1970 to 2002 on "biofeedback for constipation" was conducted using a Medline search in all languages. Only prospective studies including five or more subjects that described the treatment protocol were included. In addition, a meta-analysis of these studies was performed to compare the outcome of different biofeedback protocols for treating constipation. Thirty-eight studies were reviewed, and sample size, treatment protocol, outcome rates, number of sessions, and etiology are shown in a table. Ten studies using a parallel treatment design were reviewed in detail, including seven that randomized subjects to treatment groups. A meta-analysis (weighted by subjects) was performed to compare the results of two treatment protocols prevalent in the literature. The mean success rate of studies using pressure biofeedback (78 percent) was superior (P = 0.018) to the mean success rate for studies using electromyography biofeedback (70 percent). However, the mean success rates comparing studies using intra-anal electromyography sensors to studies using perianal electromyography sensors were 69 and 72 percent, respectively, indicating no advantages for one type of electromyography protocol over the other (P = 0.428). In addition to the varied protocols and instrumentation used, there also are inconsistencies in the literature regarding the severity and etiology of symptoms, patient selection criteria, and the definition of a successful outcome. Finally, no anatomic, physiologic, or demographic variables were identified that would assist in predicting successful outcome. Having significant psychological symptoms was identified as a factor that may influence treatment outcome, but this requires further study. Although most studies report positive results using biofeedback to treat constipation, quality research is lacking. Specific recommendations are made for future investigations to 1) improve experimental design, 2) clearly define outcome measures, 3) identify the etiology and severity of symptoms, 4) determine which treatment protocol and which component of treatment is most effective for different types of subjects, 5) systematically explore the role of psychopathology in this population, 6) use an adequate sample size that allows for meaningful analysis, and 7) include long-term follow-up data.

Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA.

PubMed

Weiberg, Arne; Pöhler, Dirk; Morgenstern, Burkhard; Karlovsky, Petr

2008-10-13

cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively. With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation) was written in Perl. cDNA-AFLP protocols described in the literature and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and Arabidopsis thaliana. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set. Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each of the marking enzymes is recommended.

The National Cohort of Dairy Farms--a data collection platform for mastitis research in Canada.

PubMed

Reyher, K K; Dufour, S; Barkema, H W; Des Côteaux, L; Devries, T J; Dohoo, I R; Keefe, G P; Roy, J-P; Scholl, D T

2011-03-01

Costs and feasibility of extensive sample collection and processing are major obstacles to mastitis epidemiology research. Studies are often consequentially limited, and fundamental mastitis researchers rarely have the opportunity to conduct their work in epidemiologically valid populations. To mitigate these limitations, the Canadian Bovine Mastitis Research Network has optimized research funds by creating a data collection platform to provide epidemiologically meaningful data for several simultaneous research endeavors. This platform consists of a National Cohort of Dairy Farms (NCDF), Mastitis Laboratory Network, and Mastitis Pathogen Culture Collection. This paper describes the implementation and operation of the NCDF, explains its sampling protocols and data collection, and documents characteristics, strengths and limitations of these data for current and potential users. The NCDF comprises 91 commercial dairy farms in 6 provinces sampled over a 2-yr period. Primarily Holstein-Friesian herds participating in Dairy Herd Improvement milk recording were selected in order to achieve a uniform distribution among 3 strata of bulk tank somatic cell counts and to reflect regional proportions of freestall housing systems. Standardized protocols were implemented for repeated milk samplings on clinical mastitis cases, fresh and randomly selected lactating cows, and cows at dry-off and after calving. Just fewer than 133,000 milk samples were collected. Demographic and production data were recorded at individual cow and farm levels. Health management data are documented and extensive questionnaire data detailing farm management and cleanliness information are also captured. The Laboratory Network represents coordinated regional mastitis bacteriology laboratories using standardized procedures. The Culture Collection archives isolates recovered from intramammary infections of cows in the NCDF and contains over 16,500 isolates, all epidemiologically cross-referenced between linked databases. The NCDF is similar to Canadian dairies in relation to mean herd size, average production, and freestall percentages. Pathogen recovery was greater than anticipated, particularly for coagulase-negative staphylococci and Corynebacterium spp. International scientists are encouraged to use this extensive archive of data and material to enhance their own mastitis research. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

[Identification of difficulties at the beginning of breastfeeding by means of protocol application].

PubMed

Carvalhaes, Maria Antonieta de Barros Leite; Corrêa, Cláudia Regina Hostin

2003-01-01

To assess a group of mothers/newborns with the necessity of special support at the beginning of breastfeeding by means of protocol application recommended by UNICEF and to verify assisting practices associated with difficulties of breastfeeding.\\par In this descriptive study, the sample comprised 50 mother/newborn pairs randomly selected in a maternity where low risk deliveries are cared by SUS (Brazilian Unified Health System). The breastfeeding observation protocol was used to record the behavior of each pair, including the frequency of negative behavior regarding breastfeeding. Next, each aspect was scored as good, regular or poor. The association between negative scores and particular assisting practices was also investigated. A critical level of p < 0.05 was used.\\par The frequency of pairs presenting evidence of severe problems (poor score) at the beginning of breastfeeding ranged from 2% to 22% according to the aspects assessed. The most frequently observed difficulties were mother and infant's bad positioning during breastfeeding and inappropriate mother/newborn interaction. These problems were significantly more frequent after surgical deliveries (p < 0.05). Milk formula and/or glucose solution was also associated with the worst scores in some breastfeeding aspects. \\par The protocol application to observe and evaluate breastfeeding identified a high prevalence of mothers/newborn pairs with difficulties to begin breastfeeding, especially when the delivery was surgically performed and when the newborn was offered supplementary liquids.

Phenytoin speciation with potentiometric and chronopotentiometric ion-selective membrane electrodes.

PubMed

Jansod, Sutida; Afshar, Majid Ghahraman; Crespo, Gastón A; Bakker, Eric

2016-05-15

We report on an electrochemical protocol based on perm-selective membranes to provide valuable information about the speciation of ionizable drugs, with phenytoin as a model example. Membranes containing varying amounts of tetradodecylammonium chloride (TDDA) were read out at zero current (potentiometry) and with applied current techniques (chronopotentiometry). Potentiometry allows one to assess the ionized form of phenytoin (pKa~8.2) that corresponds to a negatively monocharged ion. A careful optimization of the membrane components resulted in a lower limit of detection (~1.6 µM) than previous reports. Once the pH (from 9 to 10) or the concentration of albumin is varied in the sample (from 0 to 30 g L(-1)), the potentiometric signal changes abruptly as a result of reducing/increasing the ionized concentration of phenytoin. Therefore, potentiometry as a single technique is by itself not sufficient to obtain information about the concentration and speciation of the drug in the system. For this reason, a tandem configuration with chronopotentiometry as additional readout principle was used to determine the total and ionized concentration of phenytoin. In samples containing excess albumin the rate-limiting step for the chronopotentiometry readout appears to be the diffusion of ionized phenytoin preceded by comparatively rapid deprotonation and decomplexation reactions. This protocol was applied to measure phenytoin in pharmaceutical tables (100mg per tablet). This tandem approach can likely be extended to more ionizable drugs and may eventually be utilized in view of pharmacological monitoring of drugs during the delivery process. Copyright © 2015 Elsevier B.V. All rights reserved.

Manipulation and selective exercises decrease pelvic anteversion and low-back pain: a pilot study.

PubMed

Barbosa, Alexandre Carvalho; Martins, Fábio Luiz Mendonça; Barbosa, Michelle Cristina Sales Almeida; Dos Santos, Rúbia Tenile

2013-01-01

To study the effect of a protocol involving joint manipulation and specific exercises for pelvic stability to influence proprioceptive input to the spinal tissues and to observe the effects on sensorimotor function. Seven patients with pelvic anteversion and low back pain participated in an eight-week protocol (three sessions per week/nonconsecutive days). At each session, a high-velocity, low-amplitude manipulative thrust was applied to the sacroiliac joint, followed by quadriceps eccentric and hamstring concentric contractions. The perceived pain symptoms, pelvic anteversion as determined by photogrammetry analysis, and the electromyographic activity of the rectus femoris and lateral and medial hamstrings during flexion and extension exercises were assessed before and after treatment. Non-parametric tests were used to compare the groups before and after treatment with α=0.05. Perceived pain symptoms decreased after treatment (p=0.0007). The differences in the pelvis angles (p=0.0130) suggested significant differences between the assessments, and the electromyographic activities of all the muscles during isometric voluntary contraction increased. The eight-week manipulation/exercise protocol was effective for these subjects' needs. Further research should include a greater sample size to confirm the results and to determine the lead factors of pelvic stability.

gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples

PubMed Central

Vezzulli, Luigi; Stauder, Monica; Grande, Chiara; Pezzati, Elisabetta; Verheye, Hans M.; Owens, Nicholas J. P.; Pruzzo, Carla

2015-01-01

The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 102 genomic units/l for drinking and seawater samples, 101 genomic units/g for sediment and 102 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies. PMID:25915771

gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples.

PubMed

Vezzulli, Luigi; Stauder, Monica; Grande, Chiara; Pezzati, Elisabetta; Verheye, Hans M; Owens, Nicholas J P; Pruzzo, Carla

2015-01-01

The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 102 genomic units/l for drinking and seawater samples, 101 genomic units/g for sediment and 102 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.

Identification and Quantification of Microplastics in Wastewater Using Focal Plane Array-Based Reflectance Micro-FT-IR Imaging.

PubMed

Tagg, Alexander S; Sapp, Melanie; Harrison, Jesse P; Ojeda, Jesús J

2015-06-16

Microplastics (<5 mm) have been documented in environmental samples on a global scale. While these pollutants may enter aquatic environments via wastewater treatment facilities, the abundance of microplastics in these matrices has not been investigated. Although efficient methods for the analysis of microplastics in sediment samples and marine organisms have been published, no methods have been developed for detecting these pollutants within organic-rich wastewater samples. In addition, there is no standardized method for analyzing microplastics isolated from environmental samples. In many cases, part of the identification protocol relies on visual selection before analysis, which is open to bias. In order to address this, a new method for the analysis of microplastics in wastewater was developed. A pretreatment step using 30% hydrogen peroxide (H2O2) was employed to remove biogenic material, and focal plane array (FPA)-based reflectance micro-Fourier-transform (FT-IR) imaging was shown to successfully image and identify different microplastic types (polyethylene, polypropylene, nylon-6, polyvinyl chloride, polystyrene). Microplastic-spiked wastewater samples were used to validate the methodology, resulting in a robust protocol which was nonselective and reproducible (the overall success identification rate was 98.33%). The use of FPA-based micro-FT-IR spectroscopy also provides a considerable reduction in analysis time compared with previous methods, since samples that could take several days to be mapped using a single-element detector can now be imaged in less than 9 h (circular filter with a diameter of 47 mm). This method for identifying and quantifying microplastics in wastewater is likely to provide an essential tool for further research into the pathways by which microplastics enter the environment.

A set of plastid loci for use in multiplex fragment length genotyping for intraspecific variation in Pinus (Pinaceae)1

PubMed Central

Wofford, Austin M.; Finch, Kristen; Bigott, Adam; Willyard, Ann

2014-01-01

• Premise of the study: Recently released Pinus plastome sequences support characterization of 15 plastid simple sequence repeat (cpSSR) loci originally published for P. contorta and P. thunbergii. This allows selection of loci for single-tube PCR multiplexed genotyping in any subsection of the genus. • Methods: Unique placement of primers and primer conservation across the genus were investigated, and a set of six loci were selected for single-tube multiplexing. We compared interspecific variation between cpSSRs and nucleotide sequences of ycf1 and tested intraspecific variation for cpSSRs using 911 samples in the P. ponderosa species complex. • Results: The cpSSR loci contain mononucleotide and complex repeats with additional length variation in flanking regions. They are not located in hypervariable regions, and most primers are conserved across the genus. A single PCR per sample multiplexed for six loci yielded 45 alleles in 911 samples. • Discussion: The protocol allows efficient genotyping of many samples. The cpSSR loci are too variable for Pinus phylogenies but are useful for the study of genetic structure within and among populations. The multiplex method could easily be extended to other plant groups by choosing primers for cpSSR loci in a plastome alignment for the target group. PMID:25202625

Recommended features of protocols for long-term ecological monitoring

USGS Publications Warehouse

Oakley, Karen L.; Boudreau, Susan L.; Humphrey, Sioux-Z

2001-01-01

In 1991, the National Park Service (NPS) selected seven parks to serve as prototypes for development of a long-term ecological monitoring program. Denali National Park and Preserve was one of the prototype parks selected. The principal focus of this national program was to detect and document resource changes and to understand the forces driving those changes. One of the major tasks of each prototype park was to develop monitoring protocols. In this paper, we discuss some lessons learned and what we believe to be the most important features of protocols.One of the many lessons we have learned is that monitoring protocols vary greatly in content and format. This variation leads to confusion about what information protocols should contain and how they should be formatted. Problems we have observed in existing protocols include (1) not providing enough detail, (2) omitting critical topics (such as data management), and (3) mixing explanation with instructions. Once written, protocols often sit on the shelf to collect dust, allowing methods changes to occur without being adequately considered, tested, or documented. Because a lengthy and costly research effort is often needed to develop protocols, a vision of what the final product should look like is helpful. Based on our involvement with the prototype monitoring program for Denali (Oakley and Boudreau 2000), we recommend key features of protocols, including a scheme for linking protocols to data in the data management system and for tracking protocol revisions. A protocol system is crucial for producing long-term data sets of known quality that meet program objectives.

Strategies for Achieving High Sequencing Accuracy for Low Diversity Samples and Avoiding Sample Bleeding Using Illumina Platform

PubMed Central

Mitra, Abhishek; Skrzypczak, Magdalena; Ginalski, Krzysztof; Rowicka, Maga

2015-01-01

Sequencing microRNA, reduced representation sequencing, Hi-C technology and any method requiring the use of in-house barcodes result in sequencing libraries with low initial sequence diversity. Sequencing such data on the Illumina platform typically produces low quality data due to the limitations of the Illumina cluster calling algorithm. Moreover, even in the case of diverse samples, these limitations are causing substantial inaccuracies in multiplexed sample assignment (sample bleeding). Such inaccuracies are unacceptable in clinical applications, and in some other fields (e.g. detection of rare variants). Here, we discuss how both problems with quality of low-diversity samples and sample bleeding are caused by incorrect detection of clusters on the flowcell during initial sequencing cycles. We propose simple software modifications (Long Template Protocol) that overcome this problem. We present experimental results showing that our Long Template Protocol remarkably increases data quality for low diversity samples, as compared with the standard analysis protocol; it also substantially reduces sample bleeding for all samples. For comprehensiveness, we also discuss and compare experimental results from alternative approaches to sequencing low diversity samples. First, we discuss how the low diversity problem, if caused by barcodes, can be avoided altogether at the barcode design stage. Second and third, we present modified guidelines, which are more stringent than the manufacturer’s, for mixing low diversity samples with diverse samples and lowering cluster density, which in our experience consistently produces high quality data from low diversity samples. Fourth and fifth, we present rescue strategies that can be applied when sequencing results in low quality data and when there is no more biological material available. In such cases, we propose that the flowcell be re-hybridized and sequenced again using our Long Template Protocol. Alternatively, we discuss how analysis can be repeated from saved sequencing images using the Long Template Protocol to increase accuracy. PMID:25860802

An Overview and Empirical Comparison of Distance Metric Learning Methods.

PubMed

Moutafis, Panagiotis; Leng, Mengjun; Kakadiaris, Ioannis A

2016-02-16

In this paper, we first offer an overview of advances in the field of distance metric learning. Then, we empirically compare selected methods using a common experimental protocol. The number of distance metric learning algorithms proposed keeps growing due to their effectiveness and wide application. However, existing surveys are either outdated or they focus only on a few methods. As a result, there is an increasing need to summarize the obtained knowledge in a concise, yet informative manner. Moreover, existing surveys do not conduct comprehensive experimental comparisons. On the other hand, individual distance metric learning papers compare the performance of the proposed approach with only a few related methods and under different settings. This highlights the need for an experimental evaluation using a common and challenging protocol. To this end, we conduct face verification experiments, as this task poses significant challenges due to varying conditions during data acquisition. In addition, face verification is a natural application for distance metric learning because the encountered challenge is to define a distance function that: 1) accurately expresses the notion of similarity for verification; 2) is robust to noisy data; 3) generalizes well to unseen subjects; and 4) scales well with the dimensionality and number of training samples. In particular, we utilize well-tested features to assess the performance of selected methods following the experimental protocol of the state-of-the-art database labeled faces in the wild. A summary of the results is presented along with a discussion of the insights obtained and lessons learned by employing the corresponding algorithms.

Heart rate variability indexes as a marker of chronic adaptation in athletes: a systematic review.

PubMed

da Silva, Vanessa Pereira; de Oliveira, Natacha Alves; Silveira, Heitor; Mello, Roger Gomes Tavares; Deslandes, Andrea Camaz

2015-03-01

Regular exercise promotes functional and structural changes in the central and peripheral mechanisms of the cardiovascular system. Heart rate variability (HRV) measurement provides a sensitive indicator of the autonomic balance. However, because of the diversity of methods and variables used, the results are difficult to compare in the sports sciences. Since the protocol (supine, sitting, or standing position) and measure (time or frequency domain) are not well defined, the aim of this study is to investigate the HRV measures that better indicates the chronic adaptations of physical exercise in athletes. PubMed (MEDLINE), Web of Science, SciELO (Scientific Electronic Library), and Scopus databases were consulted. Original complete articles in English with short-term signals evaluating young and adult athletes, between 17 and 40 years old, with a control group, published up to 2013 were included. Selected 19 of 1369 studies, for a total sample pool of 333 male and female athletes who practice different sports. The main protocols observed were the supine or standing positions in free or controlled breathing conditions. The main statistical results found in this study were the higher mean RR, standard deviation of RR intervals, and high frequency in athletes group. In addition, the analyses of Cohen's effect size showed that factors as modality of sport, protocol used and unit of measure selected could influence this expected results. Our findings indicate that time domain measures are more consistent than frequency domain to describe the chronic cardiovascular autonomic adaptations in athletes. © 2014 Wiley Periodicals, Inc.

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma.

PubMed

Li, Congfen; Takahashi, Chikara; Zhang, Liangxuan; Huseni, Mahrukh; Stankovich, Basha; Mashhedi, Haider; Lee, Joanna; French, Dorothy; Anderson, Jeff Eastham; Kim, Doris; Howell, Kathy; Brauer, Matthew J; Kowanetz, Marcin; Yan, Yibing; Humke, Eric; Ebens, Allen; Hampton, Garret; Lackner, Mark R; Hegde, Priti; Jia, Shidong

2013-03-23

The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

PubMed Central

2013-01-01

Background The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. Methods We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. Results The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. Conclusions We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials. PMID:23522020

Clauser-Horne-Shimony-Holt versus three-party pseudo-telepathy: on the optimal number of samples in device-independent quantum private query

NASA Astrophysics Data System (ADS)

Basak, Jyotirmoy; Maitra, Subhamoy

2018-04-01

In device-independent (DI) paradigm, the trustful assumptions over the devices are removed and CHSH test is performed to check the functionality of the devices toward certifying the security of the protocol. The existing DI protocols consider infinite number of samples from theoretical point of view, though this is not practically implementable. For finite sample analysis of the existing DI protocols, we may also consider strategies for checking device independence other than the CHSH test. In this direction, here we present a comparative analysis between CHSH and three-party Pseudo-telepathy game for the quantum private query protocol in DI paradigm that appeared in Maitra et al. (Phys Rev A 95:042344, 2017) very recently.

Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS

NASA Astrophysics Data System (ADS)

Sitnikov, Dmitri G.; Monnin, Cian S.; Vuckovic, Dajana

2016-12-01

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34-80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics.

Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS

PubMed Central

Sitnikov, Dmitri G.; Monnin, Cian S.; Vuckovic, Dajana

2016-01-01

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34–80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics. PMID:28000704

Structure-based prediction of subtype selectivity of histamine H3 receptor selective antagonists in clinical trials.

PubMed

Kim, Soo-Kyung; Fristrup, Peter; Abrol, Ravinder; Goddard, William A

2011-12-27

Histamine receptors (HRs) are excellent drug targets for the treatment of diseases, such as schizophrenia, psychosis, depression, migraine, allergies, asthma, ulcers, and hypertension. Among them, the human H(3) histamine receptor (hH(3)HR) antagonists have been proposed for specific therapeutic applications, including treatment of Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), epilepsy, and obesity. However, many of these drug candidates cause undesired side effects through the cross-reactivity with other histamine receptor subtypes. In order to develop improved selectivity and activity for such treatments, it would be useful to have the three-dimensional structures for all four HRs. We report here the predicted structures of four HR subtypes (H(1), H(2), H(3), and H(4)) using the GEnSeMBLE (GPCR ensemble of structures in membrane bilayer environment) Monte Carlo protocol, sampling ∼35 million combinations of helix packings to predict the 10 most stable packings for each of the four subtypes. Then we used these 10 best protein structures with the DarwinDock Monte Carlo protocol to sample ∼50 000 × 10(20) poses to predict the optimum ligand-protein structures for various agonists and antagonists. We find that E206(5.46) contributes most in binding H(3) selective agonists (5, 6, 7) in agreement with experimental mutation studies. We also find that conserved E5.46/S5.43 in both of hH(3)HR and hH(4)HR are involved in H(3)/ H(4) subtype selectivity. In addition, we find that M378(6.55) in hH(3)HR provides additional hydrophobic interactions different from hH(4)HR (the corresponding amino acid of T323(6.55) in hH(4)HR) to provide additional subtype bias. From these studies, we developed a pharmacophore model based on our predictions for known hH(3)HR selective antagonists in clinical study [ABT-239 1, GSK-189,254 2, PF-3654746 3, and BF2.649 (tiprolisant) 4] that suggests critical selectivity directing elements are: the basic proton interacting with D114(3.32), the spacer, the aromatic ring substituted with the hydrophilic or lipophilic groups interacting with lipophilic pockets in transmembranes (TMs) 3-5-6 and the aliphatic ring located in TMs 2-3-7. These 3D structures for all four HRs should help guide the rational design of novel drugs for the subtype selective antagonists and agonists with reduced side effects.

Assay based on electrical impedance spectroscopy to discriminate between normal and cancerous mammalian cells

NASA Astrophysics Data System (ADS)

Giana, Fabián Eduardo; Bonetto, Fabián José; Bellotti, Mariela Inés

2018-03-01

In this work we present an assay to discriminate between normal and cancerous cells. The method is based on the measurement of electrical impedance spectra of in vitro cell cultures. We developed a protocol consisting on four consecutive measurement phases, each of them designed to obtain different information about the cell cultures. Through the analysis of the measured data, 26 characteristic features were obtained for both cell types. From the complete set of features, we selected the most relevant in terms of their discriminant capacity by means of conventional statistical tests. A linear discriminant analysis was then carried out on the selected features, allowing the classification of the samples in normal or cancerous with 4.5% of false positives and no false negatives.

Assay based on electrical impedance spectroscopy to discriminate between normal and cancerous mammalian cells.

PubMed

Giana, Fabián Eduardo; Bonetto, Fabián José; Bellotti, Mariela Inés

2018-03-01

In this work we present an assay to discriminate between normal and cancerous cells. The method is based on the measurement of electrical impedance spectra of in vitro cell cultures. We developed a protocol consisting on four consecutive measurement phases, each of them designed to obtain different information about the cell cultures. Through the analysis of the measured data, 26 characteristic features were obtained for both cell types. From the complete set of features, we selected the most relevant in terms of their discriminant capacity by means of conventional statistical tests. A linear discriminant analysis was then carried out on the selected features, allowing the classification of the samples in normal or cancerous with 4.5% of false positives and no false negatives.

Comparison of two DNA extraction protocols from leave samples of Cotinus coggygria, Citrus sinensis and Genus juglans.

PubMed

Fallah, F; Minaei Chenar, H; Amiri, H; Omodipour, S; Shirbande Ghods, F; Kahrizi, D; Sohrabi, M; Ghorbani, T; Kazemi, E

2017-02-28

High quality DNA is essential for molecular research. Secondary metabolites can affect the quantity and quality DNA. In current research two DNA isolation methods including CTAB and Delaporta (protocols 1 & 2 respectively) were applied in three leave samples from Cotinus coggygria, Citrus sinensis and Genus juglans that their leaves are rich of secondary metabolites. We successfully isolated DNA from C. coggygria, C. sinensis and Genus Juglans using the two protocols described above. Good quality DNA was isolated from C. coggygria, C. sinensis and Genus Juglans using protocol 1, while protocol 2 failed to produce usable DNA from these sources. The highest amount of DNA (1.3-1.6) was obtained from them using protocol 1. As we discovered, procedure 1 may work better for plants with secondary metabolites.

Distributional assumptions in food and feed commodities- development of fit-for-purpose sampling protocols.

PubMed

Paoletti, Claudia; Esbensen, Kim H

2015-01-01

Material heterogeneity influences the effectiveness of sampling procedures. Most sampling guidelines used for assessment of food and/or feed commodities are based on classical statistical distribution requirements, the normal, binomial, and Poisson distributions-and almost universally rely on the assumption of randomness. However, this is unrealistic. The scientific food and feed community recognizes a strong preponderance of non random distribution within commodity lots, which should be a more realistic prerequisite for definition of effective sampling protocols. Nevertheless, these heterogeneity issues are overlooked as the prime focus is often placed only on financial, time, equipment, and personnel constraints instead of mandating acquisition of documented representative samples under realistic heterogeneity conditions. This study shows how the principles promulgated in the Theory of Sampling (TOS) and practically tested over 60 years provide an effective framework for dealing with the complete set of adverse aspects of both compositional and distributional heterogeneity (material sampling errors), as well as with the errors incurred by the sampling process itself. The results of an empirical European Union study on genetically modified soybean heterogeneity, Kernel Lot Distribution Assessment are summarized, as they have a strong bearing on the issue of proper sampling protocol development. TOS principles apply universally in the food and feed realm and must therefore be considered the only basis for development of valid sampling protocols free from distributional constraints.

High sensitivity Troponin T: an audit of implementation of its protocol in a district general hospital.

PubMed

Kalim, Shahid; Nazir, Shaista; Khan, Zia Ullah

2013-01-01

Protocols based on newer high sensitivity Troponin T (hsTropT) assays can rule in a suspected Acute Myocardial Infarction (AMI) as early as 3 hours. We conducted this study to audit adherence to our Trust's newly introduced AMI diagnostic protocol based on paired hsTropT testing at 0 and 3 hours. We retrospectively reviewed data of all patients who had hsTropT test done between 1st and 7th May 2012. Patient's demographics, utility of single or paired samples, time interval between paired samples, patient's presenting symptoms and ECG findings were noted and their means, medians, Standard deviations and proportions were calculated. A total of 66 patients had hsTropT test done during this period. Mean age was 63.30 +/- 17.46 years and 38 (57.57%) were males. Twenty-four (36.36%) patients had only single, rather than protocol recommended paired hsTropT samples, taken. Among the 42 (63.63%) patients with paired samples, the mean time interval was found to be 4.41 +/- 5.7 hours. Contrary to the recommendations, 15 (22.73%) had a very long whereas 2 (3.03%) had a very short time interval between two samples. A subgroup analysis of patients with single samples, found only 2 (3.03%) patient with ST-segment elevation, appropriate for single testing. Our study confirmed that in a large number of patients the protocol for paired sampling or a recommended time interval of 3 hours between 2 samples was not being followed.

Performance of Identifiler Direct and PowerPlex 16 HS on the Applied Biosystems 3730 DNA Analyzer for processing biological samples archived on FTA cards.

PubMed

Laurin, Nancy; DeMoors, Anick; Frégeau, Chantal

2012-09-01

Direct amplification of STR loci from biological samples collected on FTA cards without prior DNA purification was evaluated using Identifiler Direct and PowerPlex 16 HS in conjunction with the use of a high throughput Applied Biosystems 3730 DNA Analyzer. In order to reduce the overall sample processing cost, reduced PCR volumes combined with various FTA disk sizes were tested. Optimized STR profiles were obtained using a 0.53 mm disk size in 10 μL PCR volume for both STR systems. These protocols proved effective in generating high quality profiles on the 3730 DNA Analyzer from both blood and buccal FTA samples. Reproducibility, concordance, robustness, sample stability and profile quality were assessed using a collection of blood and buccal samples on FTA cards from volunteer donors as well as from convicted offenders. The new developed protocols offer enhanced throughput capability and cost effectiveness without compromising the robustness and quality of the STR profiles obtained. These results support the use of these protocols for processing convicted offender samples submitted to the National DNA Data Bank of Canada. Similar protocols could be applied to the processing of casework reference samples or in paternity or family relationship testing. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Multisite tumor sampling enhances the detection of intratumor heterogeneity at all different temporal stages of tumor evolution.

PubMed

Erramuzpe, Asier; Cortés, Jesús M; López, José I

2018-02-01

Intratumor heterogeneity (ITH) is an inherent process of tumor development that has received much attention in previous years, as it has become a major obstacle for the success of targeted therapies. ITH is also temporally unpredictable across tumor evolution, which makes its precise characterization even more problematic since detection success depends on the precise temporal snapshot at which ITH is analyzed. New and more efficient strategies for tumor sampling are needed to overcome these difficulties which currently rely entirely on the pathologist's interpretation. Recently, we showed that a new strategy, the multisite tumor sampling, works better than the routine sampling protocol for the ITH detection when the tumor time evolution was not taken into consideration. Here, we extend this work and compare the ITH detections of multisite tumor sampling and routine sampling protocols across tumor time evolution, and in particular, we provide in silico analyses of both strategies at early and late temporal stages for four different models of tumor evolution (linear, branched, neutral, and punctuated). Our results indicate that multisite tumor sampling outperforms routine protocols in detecting ITH at all different temporal stages of tumor evolution. We conclude that multisite tumor sampling is more advantageous than routine protocols in detecting intratumor heterogeneity.

Environmental DNA sampling protocol - filtering water to capture DNA from aquatic organisms

USGS Publications Warehouse

Laramie, Matthew B.; Pilliod, David S.; Goldberg, Caren S.; Strickler, Katherine M.

2015-09-29

Environmental DNA (eDNA) analysis is an effective method of determining the presence of aquatic organisms such as fish, amphibians, and other taxa. This publication is meant to guide researchers and managers in the collection, concentration, and preservation of eDNA samples from lentic and lotic systems. A sampling workflow diagram and three sampling protocols are included as well as a list of suggested supplies. Protocols include filter and pump assembly using: (1) a hand-driven vacuum pump, ideal for sample collection in remote sampling locations where no electricity is available and when equipment weight is a primary concern; (2) a peristaltic pump powered by a rechargeable battery-operated driver/drill, suitable for remote sampling locations when weight consideration is less of a concern; (3) a 120-volt alternating current (AC) powered peristaltic pump suitable for any location where 120-volt AC power is accessible, or for roadside sampling locations. Images and detailed descriptions are provided for each step in the sampling and preservation process.

Evaluating the impact of environmental interventions across 2 countries: the International Bikeshare Impacts on Cycling and Collisions Study (IBICCS) Study protocol.

PubMed

Fuller, Daniel; Gauvin, Lise; Dubé, Anne-Sophie; Winters, Meghan; Teschke, Kay; Russo, Elizabeth T; Camden, Andi; Mee, Carol; Friedman, Steven Marc

2014-10-25

Few international studies examine public bicycle share programs (PBSP) health impacts. We describe the protocol for the International Bikeshare Impacts on Cycling and Collisions Study (IBICCS). A quasi-experimental non-equivalent groups design was used. Intervention cities (Montreal, Toronto, Boston, New York and Vancouver) were matched to control cities (Chicago, Detroit, and Philadelphia) on total population, population density, cycling rates, and average yearly temperature. The study used three repeated, cross-sectional surveys in intervention and control cities in Fall 2012 (baseline), 2013 (year 1), and 2014 (year 2). A non-probabilistic online panel survey with a sampling frame of individuals residing in and around areas where PBSP are/would be implemented was used. A total of 12,000 respondents will be sampled. In each of the 8 cities 1000 respondents will be sampled with an additional 4000 respondents sampled based on the total population of the city. Survey questions include measures of self-rated health, and self-reported height and weight, knowledge and experience using PBSP, physical activity, bicycle helmet use and history of collisions and injuries while cycling, socio-demographic questions, and home/workplace locations. Respondents could complete questionnaires in English, French, and Spanish. Two weights will be applied to the data: inverse probability of selection and post-stratification on age and sex.A triple difference analysis will be used. This approach includes in the models, time, exposure, and treatment group, and interaction terms between these variables to estimate changes across time, between exposure groups and between cities. There are scientific and practical challenges in evaluating PBSP. Methodological challenges included: appropriate sample recruitment, exchangeability of treatment and control groups, controlling unmeasured confounding, and specifying exposure. Practical challenges arise in the evaluation of environmental interventions such as a PBSP: one of the companies involved filed for bankruptcy, a Hurricane devastated New York City, and one PBSP was not implemented. Overall, this protocol provides methodological and practical guidance for researchers wanting to study PBSP impacts on health.

Performance assessment of two lysis methods for direct identification of yeasts from clinical blood cultures using MALDI-TOF mass spectrometry.

PubMed

Jeddi, Fakhri; Yapo-Kouadio, Gisèle Cha; Normand, Anne-Cécile; Cassagne, Carole; Marty, Pierre; Piarroux, Renaud

2017-02-01

In cases of fungal infection of the bloodstream, rapid species identification is crucial to provide adapted therapy and thereby ameliorate patient outcome. Currently, the commercial Sepsityper kit and the sodium-dodecyl sulfate (SDS) method coupled with MALDI-TOF mass spectrometry are the most commonly reported lysis protocols for direct identification of fungi from positive blood culture vials. However, the performance of these two protocols has never been compared on clinical samples. Accordingly, we performed a two-step survey on two distinct panels of clinical positive blood culture vials to identify the most efficient protocol, establish an appropriate log score (LS) cut-off, and validate the best method. We first compared the performance of the Sepsityper and the SDS protocols on 71 clinical samples. For 69 monomicrobial samples, mass spectrometry LS values were significantly higher with the SDS protocol than with the Sepsityper method (P < .0001), especially when the best score of four deposited spots was considered. Next, we established the LS cut-off for accurate identification at 1.7, based on specimen DNA sequence data. Using this LS cut-off, 66 (95.6%) and 46 (66.6%) isolates were correctly identified at the species level with the SDS and the Sepsityper protocols, respectively. In the second arm of the survey, we validated the SDS protocol on an additional panel of 94 clinical samples. Ninety-two (98.9%) of 93 monomicrobial samples were correctly identified at the species level (median LS = 2.061). Overall, our data suggest that the SDS method yields more accurate species identification of yeasts, than the Sepsityper protocol. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A feeding protocol for delivery of agents to assess development in Varroa mites

PubMed Central

2017-01-01

A novel feeding protocol for delivery of bio-active agents to Varroa mites was developed by providing mites with honey bee larva hemolymph supplemented with cultured insect cells and selected materials delivered on a fibrous cotton substrate. Mites were starved, fed on treated hemolymph to deliver selected agents and then returned to bee larvae. Transcript levels of two reference genes, actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as well as for nine selected genes involved in reproductive processes showed that the starvation and feeding protocol periods did not pose a high level of stress to the mites as transcript levels remained comparable between phoretic mites and those completing the protocol. The feeding protocol was used to deliver molecules such as hormone analogs or plasmids. Mites fed with Tebufenozide, an ecdysone analog, had higher transcript levels of shade than untreated or solvent treated mites. In order to extend this feeding protocol, cultured insect cells were incorporated to a final ratio of 1 part cells and 2 parts hemolymph. Although supplementation with Bombyx mori Bm5 cells increased the amount of hemolymph consumed per mite, there was a significant decrease in the percentage of mites that fed and survived. On the other hand, Drosophila melanogaster S2 cells reduced significantly the percentage of mites that fed and survived as well as the amount of hemolymph consumed. The feeding protocol provides a dynamic platform with which to challenge the Varroa mite to establish efficacy of control agents for this devastating honey bee pest. PMID:28448606

To nearly come full circle: Nonoperative management of high-grade IV-V blunt splenic trauma is safe using a protocol with routine angioembolization.

PubMed

Bhullar, Indermeet S; Tepas, Joseph J; Siragusa, Daniel; Loper, Todd; Kerwin, Andrew; Frykberg, Eric R

2017-04-01

Nonoperative management (NOM) of hemodynamically stable high-grade (IV-V) blunt splenic trauma remains controversial given the high failure rates (19%) that persist despite angioembolization (AE) protocols. The NOM protocol was modified in 2011 to include mandatory AE of all grade (IV-V) injuries without contrast blush (CB) along with selective AE of grade (I-V) with CB. The purpose of this study was to determine if this new AE (NAE) protocol significantly lowered the failure rates for grade (IV-V) injuries allowing for safe observation without surgery and if the exclusion of grade III injuries allowed for the prevention of unnecessary angiograms without affecting the overall failure rates. The records of patients with blunt splenic trauma from January 2000 to October 2014 at a Level I trauma center were retrospectively reviewed. Patients were divided into two groups and failure of NOM (FNOM) rates compared: NAE protocol (2011-2014) with mandatory AE for all grade (IV-V) injuries without CB and selective AE for grade (I-V) with CB versus old AE (OAE) protocol (2000-2010) with selective AE for grade (I-V) with CB. Seven hundred twelve patients underwent NOM with 522 (73%) in the OAE group and 190 (27%) in the NAE group. Evolving from the OAE to the NAE strategy resulted in a significantly lower FNOM rate for the overall group (grade I-V) (OAE vs. NAE, 4% to 1%, p = 0.04) and the grade (IV-V) group (OAE vs. NAE, 19% vs. 3%, p = 0.01). Angiograms were avoided in 113 grade (I-III) injuries with no CB; these patients had NOM with observation alone and none failed. A protocol using mandatory AE of all high-grade (IV-V) injuries without CB and selective AE of grade (I-V) with CB may provide for optimum salvage with safe NOM of the high-grade injuries (IV-V) and limited unnecessary angiograms. Therapeutic study, level IV.

Isolation of Campylobacter from Brazilian broiler flocks using different culturing procedures.

PubMed

Vaz, C S L; Voss-Rech, D; Pozza, J S; Coldebella, A; Silva, V S

2014-11-01

Conventional culturing methods enable the detection of Campylobacter in broiler flocks. However, laboratory culture of Campylobacter is laborious because of its fastidious behavior and the presence of competing nontarget bacteria. This study evaluated different protocols to isolate Campylobacter from broiler litter, feces, and cloacal and drag swabs. Samples taken from commercial Brazilian broiler flocks were directly streaked onto Preston agar (PA), Campy-Line agar (CLA), and modified charcoal cefoperazone deoxycholate agar (mCCDA) and also enriched in blood-free Bolton broth (bfBB) for 24 and 48 h followed by plating onto the different selective media. Higher numbers of Campylobacter-positive cloacal and drag swab samples were observed using either direct plating or enrichment for 24 h before plating onto PA, compared with enrichment for 48 h (P < 0.05). Furthermore, direct plating was a more sensitive method to detect Campylobacter in broiler litter and feces samples. Analysis of directly plated samples revealed that higher Campylobacter levels were detected in feces streaked onto PA (88.8%), cloacal swabs plated onto mCCDA (72.2%), drag swabs streaked onto CLA or mCCDA (69.4%), and litter samples inoculated onto PA (63.8%). Preston agar was the best agar to isolate Campylobacter from directly plated litter samples (P < 0.05), but there was no difference in the efficacies of PA, mCCDA, and CLA in detecting Campylobacter in other samples. The isolated Campylobacter strains were phenotypically identified as Campylobacter jejuni or Campylobacter coli. The predominant contaminant observed in the Campylobacter cultures was Proteus mirabilis, which was resistant to the majority of antimicrobial agents in selective media. Together, these data showed that direct plating onto PA and onto either CLA or mCCDA as the second selective agar enabled the reliable isolation of thermophilic Campylobacter species from broiler samples. Finally, Campylobacter was detected in all broiler flocks sampled. ©2014 Poultry Science Association Inc.

Protocols | Office of Cancer Clinical Proteomics Research

Cancer.gov

Each reagent on the Antibody Portal has been characterized by a combination of methods specific for that antibody. To view the customized antibody methods and protocols (Standard Operating Procedures) used to generate and characterize each reagent, select an antibody of interest and open the protocols associated with their respective characterization methods along with characterization data.

Development of Research-Based Protocol Aligned to Predict High Levels of Teaching Quality

ERIC Educational Resources Information Center

Schumacher, Gary; Grigsby, Bettye; Vesey, Winona

2011-01-01

This study proposes a research-based teacher selection protocol. The protocol is intended to offer school district hiring authorities a tool to identify teacher candidates with the behaviors expected to predict effective teaching. It is hypothesized that a particular series of research-based interview questions focusing on teaching behaviors in…

Optimization of Native and Formaldehyde iPOND Techniques for Use in Suspension Cells.

PubMed

Wiest, Nathaniel E; Tomkinson, Alan E

2017-01-01

The isolation of proteins on nascent DNA (iPOND) technique developed by the Cortez laboratory allows a previously unparalleled ability to examine proteins associated with replicating and newly synthesized DNA in mammalian cells. Both the original, formaldehyde-based iPOND technique and a more recent derivative, accelerated native iPOND (aniPOND), have mostly been performed in adherent cell lines. Here, we describe modifications to both protocols for use with suspension cell lines. These include cell culture, pulse, and chase conditions that optimize sample recovery in both protocols using suspension cells and several key improvements to the published aniPOND technique that reduce sample loss, increase signal to noise, and maximize sample recovery. Additionally, we directly and quantitatively compare the iPOND and aniPOND protocols to test the strengths and limitations of both. Finally, we present a detailed protocol to perform the optimized aniPOND protocol in suspension cell lines. © 2017 Elsevier Inc. All rights reserved.

Soil and leaf litter metaproteomics—a brief guideline from sampling to understanding

PubMed Central

Keiblinger, Katharina M.; Fuchs, Stephan; Zechmeister-Boltenstern, Sophie; Riedel, Katharina

2016-01-01

The increasing application of soil metaproteomics is providing unprecedented, in-depth characterization of the composition and functionality of in situ microbial communities. Despite recent advances in high-resolution mass spectrometry, soil metaproteomics still suffers from a lack of effective and reproducible protein extraction protocols and standardized data analyses. This review discusses the opportunities and limitations of selected techniques in soil-, and leaf litter metaproteomics, and presents a step-by-step guideline on their application, covering sampling, sample preparation, extraction and data evaluation strategies. In addition, we present recent applications of soil metaproteomics and discuss how such approaches, linking phylogenetics and functionality, can help gain deeper insights into terrestrial microbial ecology. Finally, we strongly recommend that to maximize the insights environmental metaproteomics may provide, such methods should be employed within a holistic experimental approach considering relevant aboveground and belowground ecosystem parameters. PMID:27549116

Investigation of therapeutic potentials of some selected medicinal plants using neutron activation analysis

NASA Astrophysics Data System (ADS)

Abubakar, Sani; Usman, Ahmed Rufa'i.; Isa, Nasiru Fage; Khandaker, Mayeen Uddin; Abubakar, Nuraddeen

2015-04-01

Series of attempts were made to investigate concentrations of trace elements and their therapeutic properties in various medicinal plants. In this study, samples of some commonly used plants were collected from Bauchi State, Nigeria. They includes leaves of azadirachta indica (neem), Moringa Oleifera (moringa), jatropha curcas (purgin Nut), guiera senegalensis (custard apple) and anogeissus leiocarpus (African birch). These samples were analyzed for their trace elements contents with both short and long irradiation protocols of Instrumental Neutron Activation Analysis (INAA) at Nigerian Research Reactor-1 (NIRR-1) of Ahmadu Bello University, Zaria, Nigeria. The level of trace elements found varies from one sample to another, with some reported at hundreds of mg/Kg dry weight. The results have been compared with the available literature data. The presence of these trace elements indicates promising potentials of these plants for relief of certain ailments.

Experimental and computational studies on molecularly imprinted solid-phase extraction for gonyautoxins 2,3 from dinoflagellate Alexandrium minutum.

PubMed

Lian, Ziru; Li, Hai-Bei; Wang, Jiangtao

2016-08-01

An innovative and effective extraction procedure based on molecularly imprinted solid-phase extraction (MISPE) was developed for the isolation of gonyautoxins 2,3 (GTX2,3) from Alexandrium minutum sample. Molecularly imprinted polymer microspheres were prepared by suspension polymerization and and were employed as sorbents for the solid-phase extraction of GTX2,3. An off-line MISPE protocol was optimized. Subsequently, the extract samples from A. minutum were analyzed. The results showed that the interference matrices in the extract were obviously cleaned up by MISPE procedures. This outcome enabled the direct extraction of GTX2,3 in A. minutum samples with extraction efficiency as high as 83 %, rather significantly, without any need for a cleanup step prior to the extraction. Furthermore, computational approach also provided direct evidences of the high selective isolation of GTX2,3 from the microalgal extracts.

Corrosion and mechanical performance of AZ91 exposed to simulated inflammatory conditions.

PubMed

Brooks, Emily K; Der, Stephanie; Ehrensberger, Mark T

2016-03-01

Magnesium (Mg) and its alloys, including Mg-9%Al-1%Zn (AZ91), are biodegradable metals with potential use as temporary orthopedic implants. Invasive orthopedic procedures can provoke an inflammatory response that produces hydrogen peroxide (H2O2) and an acidic environment near the implant. This study assessed the influence of inflammation on both the corrosion and mechanical properties of AZ91. The AZ91 samples in the inflammatory protocol were immersed for three days in a complex biologically relevant electrolyte (AMEM culture media) that contained serum proteins (FBS), 150 mM of H2O2, and was titrated to a pH of 5. The control protocol immersed AZ91 samples in the same biologically relevant electrolyte (AMEM & FBS) but without H2O2 and the acid titration. After 3 days all samples were switched into fresh AMEM & FBS for an additional 3-day immersion. During the initial immersion, inflammatory protocol samples showed increased corrosion rate determined by mass loss testing, increased Mg and Al ion released to solution, and a completely corroded surface morphology as compared to the control protocol. Although corrosion in both protocols slowed once the test electrolyte solution was replaced at 3 days, the samples originally exposed to the simulated inflammatory conditions continued to display enhanced corrosion rates as compared to the control protocol. These lingering effects may indicate the initial inflammatory corrosion processes modified components of the surface oxide and corrosion film or initiated aggressive localized processes that subsequently left the interface more vulnerable to continued enhanced corrosion. The electrochemical properties of the interfaces were also evaluated by EIS, which found that the corrosion characteristics of the AZ91 samples were potentially influenced by the role of intermediate adsorption layer processes. The increased corrosion observed for the inflammatory protocol did not affect the flexural mechanical properties of the AZ91 at any time point assessed. Copyright © 2015 Elsevier B.V. All rights reserved.

Vascular Blood Collection protocol samples into MELFI

NASA Image and Video Library

2011-10-18

iss029e028495 (10/18/2011) --- Japan Aerospace Exploration Agency astronaut Satoshi Furukawa,Expedition 29 flight engineer,prepares to put samples from the CSA (Canadian Space Agency) Vascular Blood Collection protocol into the MELFI-1 (Minus Eighty Laboratory Freezer for ISS 1) unit.

NHEXAS PHASE I ARIZONA STUDY--LIST OF STANDARD OPERATING PROCEDURES

EPA Science Inventory

This document lists available protocols and SOPs for the NHEXAS Phase I Arizona study. It identifies protocols and SOPs for the following study components: (1) Sample collection and field operations, (2) Sample analysis, (3) General laboratory procedures, (4) Quality Assurance, (...

Development of pig welfare assessment protocol integrating animal-, environment-, and management-based measures.

PubMed

Renggaman, Anriansyah; Choi, Hong L; Sudiarto, Sartika Ia; Alasaarela, Laura; Nam, Ok S

2015-01-01

Due to increased interest in animal welfare, there is now a need for a comprehensive assessment protocol to be used in intensive pig farming systems. There are two current welfare assessment protocols for pigs: Welfare Quality® Assessment Protocols (applicable in the Europe Union), that mostly focuses on animal-based measures, and the Swine Welfare Assurance Program (applicable in the United States), that mostly focuses on management- and environment-based measures. In certain cases, however, animal-based measures might not be adequate for properly assessing pig welfare status. Similarly, welfare assessment that relies only on environment- and management-based measures might not represent the actual welfare status of pigs. Therefore, the objective of this paper was to develop a new welfare protocol by integrating animal-, environment-, and management-based measures. The background for selection of certain welfare criteria and modification of the scoring systems from existing welfare assessment protocols are described. The developed pig welfare assessment protocol consists of 17 criteria that are related to four main principles of welfare (good feeding, good housing, good health, and appropriate behavior). Good feeding, good housing, and good health were assessed using a 3-point scale: 0 (good welfare), 1 (moderate welfare), and 2 (poor welfare). In certain cases, only a 2-point scale was used: 0 (certain condition is present) or 2 (certain condition is absent). Appropriate behavior was assessed by scan sampling of positive and negative social behaviors based on qualitative behavior assessment and human-animal relationship tests. Modification of the body condition score into a 3-point scale revealed pigs with a moderate body condition (score 1). Moreover, additional criteria such as feed quality confirmed that farms had moderate (score 1) or poor feed quality (score 2), especially those farms located in a high relative humidity region. The developed protocol can be utilized to assess welfare status in an intensive pig farming system. Although further improvements are still needed, this study is a first step in developing a pig welfare assessment protocol that combines animal-, environment-, and management-based measures.

Key exchange using biometric identity based encryption for sharing encrypted data in cloud environment

NASA Astrophysics Data System (ADS)

Hassan, Waleed K.; Al-Assam, Hisham

2017-05-01

The main problem associated with using symmetric/ asymmetric keys is how to securely store and exchange the keys between the parties over open networks particularly in the open environment such as cloud computing. Public Key Infrastructure (PKI) have been providing a practical solution for session key exchange for loads of web services. The key limitation of PKI solution is not only the need for a trusted third partly (e.g. certificate authority) but also the absent link between data owner and the encryption keys. The latter is arguably more important where accessing data needs to be linked with identify of the owner. Currently available key exchange protocols depend on using trusted couriers or secure channels, which can be subject to man-in-the-middle attack and various other attacks. This paper proposes a new protocol for Key Exchange using Biometric Identity Based Encryption (KE-BIBE) that enables parties to securely exchange cryptographic keys even an adversary is monitoring the communication channel between the parties. The proposed protocol combines biometrics with IBE in order to provide a secure way to access symmetric keys based on the identity of the users in unsecure environment. In the KE-BIOBE protocol, the message is first encrypted by the data owner using a traditional symmetric key before migrating it to a cloud storage. The symmetric key is then encrypted using public biometrics of the users selected by data owner to decrypt the message based on Fuzzy Identity-Based Encryption. Only the selected users will be able to decrypt the message by providing a fresh sample of their biometric data. The paper argues that the proposed solution eliminates the needs for a key distribution centre in traditional cryptography. It will also give data owner the power of finegrained sharing of encrypted data by control who can access their data.

Sampled-data-based consensus and containment control of multiple harmonic oscillators: A motion-planning approach

NASA Astrophysics Data System (ADS)

Liu, Yongfang; Zhao, Yu; Chen, Guanrong

2016-11-01

This paper studies the distributed consensus and containment problems for a group of harmonic oscillators with a directed communication topology. First, for consensus without a leader, a class of distributed consensus protocols is designed by using motion planning and Pontryagin's principle. The proposed protocol only requires relative information measurements at the sampling instants, without requiring information exchange over the sampled interval. By using stability theory and the properties of stochastic matrices, it is proved that the distributed consensus problem can be solved in the motion planning framework. Second, for the case with multiple leaders, a class of distributed containment protocols is developed for followers such that their positions and velocities can ultimately converge to the convex hull formed by those of the leaders. Compared with the existing consensus algorithms, a remarkable advantage of the proposed sampled-data-based protocols is that the sampling periods, communication topologies and control gains are all decoupled and can be separately designed, which relaxes many restrictions in controllers design. Finally, some numerical examples are given to illustrate the effectiveness of the analytical results.

Selection for production-related traits in Pelargonium zonale: improved design and analysis make all the difference

PubMed Central

Molenaar, Heike; Glawe, Martin; Boehm, Robert; Piepho, Hans-Peter

2017-01-01

Ornamental plant variety improvement is limited by current phenotyping approaches and neglected use of experimental designs. The present study was conducted to show the benefits of using an experimental design and corresponding analysis in ornamental breeding regarding simulated response to selection in Pelargonium zonale for production-related traits. This required establishment of phenotyping protocols for root formation and stem cutting counts, with which 974 genotypes were assessed in a two-phase experimental design. The present paper evaluates this protocol. The possibility of varietal improvement through indirect selection on secondary traits such as branch count and flower count was assessed by genetic correlations. Simulated response to selection varied greatly, depending on the genotypic variances of the breeding population and traits. A varietal improvement of over 20% is possible for stem cutting count, root formation, branch count and flower count. In contrast, indirect selection of stem cutting count by branch count or flower count was found to be ineffective. The established phenotypic protocols and two-phase experimental designs are valuable tools for breeding of P. zonale. PMID:28243453

Selection for production-related traits in Pelargonium zonale: improved design and analysis make all the difference.

PubMed

Molenaar, Heike; Glawe, Martin; Boehm, Robert; Piepho, Hans-Peter

2017-01-01

Ornamental plant variety improvement is limited by current phenotyping approaches and neglected use of experimental designs. The present study was conducted to show the benefits of using an experimental design and corresponding analysis in ornamental breeding regarding simulated response to selection in Pelargonium zonale for production-related traits. This required establishment of phenotyping protocols for root formation and stem cutting counts, with which 974 genotypes were assessed in a two-phase experimental design. The present paper evaluates this protocol. The possibility of varietal improvement through indirect selection on secondary traits such as branch count and flower count was assessed by genetic correlations. Simulated response to selection varied greatly, depending on the genotypic variances of the breeding population and traits. A varietal improvement of over 20% is possible for stem cutting count, root formation, branch count and flower count. In contrast, indirect selection of stem cutting count by branch count or flower count was found to be ineffective. The established phenotypic protocols and two-phase experimental designs are valuable tools for breeding of P. zonale .

Quality assurance and quality control of geochemical data—A primer for the research scientist

USGS Publications Warehouse

Geboy, Nicholas J.; Engle, Mark A.

2011-01-01

Geochemistry is a constantly expanding science. More and more, scientists are employing geochemical tools to help answer questions about the Earth and earth system processes. Scientists may assume that the responsibility of examining and assessing the quality of the geochemical data they generate is not theirs but rather that of the analytical laboratories to which their samples have been submitted. This assumption may be partially based on knowledge about internal and external quality assurance and quality control (QA/QC) programs in which analytical laboratories typically participate. Or there may be a perceived lack of time or resources to adequately examine data quality. Regardless of the reason, the lack of QA/QC protocols can lead to the generation and publication of erroneous data. Because the interpretations drawn from the data are primary products to U.S. Geological Survey (USGS) stakeholders, the consequences of publishing erroneous results can be significant. The principal investigator of a scientific study ultimately is responsible for the quality and interpretation of the project's findings, and thus must also play a role in the understanding, implementation, and presentation of QA/QC information about the data. Although occasionally ignored, QA/QC protocols apply not only to procedures in the laboratory but also in the initial planning of a research study and throughout the life of the project. Many of the tenets of developing a sound QA/QC program or protocols also parallel the core concepts of developing a good study: What is the main objective of the study? Will the methods selected provide data of enough resolution to answer the hypothesis? How should samples be collected? Are there known or unknown artifacts or contamination sources in the sampling and analysis methods? Assessing data quality requires communication between the scientists responsible for designing the study and those collecting samples, analyzing samples, treating data, and interpreting results. This primer has been developed to provide basic information and guidance about developing QA/QC protocols for geochemical studies. It is not intended to be a comprehensive guide but rather an introduction to key concepts tied to a list of relevant references for further reading. The guidelines are presented in stepwise order beginning with presampling considerations and continuing through final data interpretation. The goal of this primer is to outline basic QA/QC practices that scientists can use before, during, and after chemical analysis to ensure the validity of the data they collect with the goal of providing defendable results and conclusions.

Optimized Enrichment of Phosphoproteomes by Fe-IMAC Column Chromatography.

PubMed

Ruprecht, Benjamin; Koch, Heiner; Domasinska, Petra; Frejno, Martin; Kuster, Bernhard; Lemeer, Simone

2017-01-01

Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. In contrast to other formats such as StageTips or batch incubations using TiO 2 or Ti-IMAC beads, Fe-IMAC HPLC columns do not suffer from issues regarding incomplete phosphopeptide binding or elution and enrichment efficiency scales linearly with the amount of starting material. Here, we provide a step-by-step protocol for the entire phosphopeptide enrichment procedure including sample preparation (lysis, digestion, desalting), Fe-IMAC column chromatography (column setup, operation, charging), measurement by LC-MS/MS (nHPLC gradient, MS parameters) and data analysis (MaxQuant). To increase throughput, we have optimized several key steps such as the gradient time of the Fe-IMAC separation (15 min per enrichment), the number of consecutive enrichments possible between two chargings (>20) and the column recharging itself (<1 h). We show that the application of this protocol enables the selective (>90 %) identification of more than 10,000 unique phosphopeptides from 1 mg of HeLa digest within 2 h of measurement time (Q Exactive Plus).

A randomized controlled trial of a community health worker intervention in a population of patients with multiple chronic diseases: Study design and protocol

PubMed Central

Kangovi, Shreya; Mitra, Nandita; Turr, Lindsey; Huo, Hairong; Grande, David; Long, Judith A.

2017-01-01

Upstream interventions – e.g. housing programs and community health worker interventions-address socioeconomic and behavioral factors that influence health outcomes across diseases. Studying these types of interventions in clinical trials raises a methodological challenge: how should researchers measure the effect of an upstream intervention in a sample of patients with different diseases? This paper addresses this question using an illustrative protocol of a randomized controlled trial of collaborative-goal setting versus goal-setting plus community health worker support among patients multiple chronic diseases: diabetes, obesity, hypertension and tobacco dependence. At study enrollment, patients met with their primary care providers to select one of their chronic diseases to focus on during the study, and to collaboratively set a goal for that disease. Patients randomly assigned to a community health worker also received six months of support to address socioeconomic and behavioral barriers to chronic disease control. The primary hypothesis was that there would be differences in patients’ selected chronic disease control as measured by HbA1c, body mass index, systolic blood pressure and cigarettes per day, between the goal-setting alone and community health worker support arms. To test this hypothesis, we will conduct a stratum specific multivariate analysis of variance which allows all patients (regardless of their selected chronic disease) to be included in a single model for the primary outcome. Population health researchers can use this approach to measure clinical outcomes across diseases. PMID:27965180

Sampling protocol, estimation, and analysis procedures for the down woody materials indicator of the FIA program

Treesearch

Christopher W. Woodall; Vicente J. Monleon

2008-01-01

The USDA Forest Service's Forest Inventory and Analysis program conducts an inventory of forests of the United States including down woody materials (DWM). In this report we provide the rationale and context for a national inventory of DWM, describe the components sampled, discuss the sampling protocol used and corresponding estimation procedures, and provide...

Simple Sodium Dodecyl Sulfate-Assisted Sample Preparation Method for LC-MS-based Proteomic Applications

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

Zhou, Jianying; Dann, Geoffrey P.; Shi, Tujin

2012-03-10

Sodium dodecyl sulfate (SDS) is one of the most popular laboratory reagents used for highly efficient biological sample extraction; however, SDS presents a significant challenge to LC-MS-based proteomic analyses due to its severe interference with reversed-phase LC separations and electrospray ionization interfaces. This study reports a simple SDS-assisted proteomic sample preparation method facilitated by a novel peptide-level SDS removal protocol. After SDS-assisted protein extraction and digestion, SDS was effectively (>99.9%) removed from peptides through ion substitution-mediated DS- precipitation with potassium chloride (KCl) followed by {approx}10 min centrifugation. Excellent peptide recovery (>95%) was observed for less than 20 {mu}g of peptides.more » Further experiments demonstrated the compatibility of this protocol with LC-MS/MS analyses. The resulting proteome coverage from this SDS-assisted protocol was comparable to or better than those obtained from other standard proteomic preparation methods in both mammalian tissues and bacterial samples. These results suggest that this SDS-assisted protocol is a practical, simple, and broadly applicable proteomic sample processing method, which can be particularly useful when dealing with samples difficult to solubilize by other methods.« less

DNA elution from buccal cells stored on Whatman FTA Classic Cards using a modified methanol fixation method.

PubMed

Johanson, Helene C; Hyland, Valentine; Wicking, Carol; Sturm, Richard A

2009-04-01

We describe here a method for DNA elution from buccal cells and whole blood both collected onto Whatman FTA technology, using methanol fixation followed by an elution PCR program. Extracted DNA is comparable in quality to published Whatman FTA protocols, as judged by PCR-based genotyping. Elution of DNA from the dried sample is a known rate-limiting step in the published Whatman FTA protocol; this method enables the use of each 3-mm punch of sample for several PCR reactions instead of the standard, one PCR reaction per sample punch. This optimized protocol therefore extends the usefulness and cost effectiveness of each buccal swab sample collected, when used for nucleic acid PCR and genotyping.

Monitoring nekton as a bioindicator in shallow estuarine habitats

USGS Publications Warehouse

Raposa, K.B.; Roman, C.T.; Heltshe, J.F.

2003-01-01

Long-term monitoring of estuarine nekton has many practical and ecological benefits but efforts are hampered by a lack of standardized sampling procedures. This study provides a rationale for monitoring nekton in shallow (< 1 m), temperate, estuarine habitats and addresses some important issues that arise when developing monitoring protocols. Sampling in seagrass and salt marsh habitats is emphasized due to the susceptibility of each habitat to anthropogenic stress and to the abundant and rich nekton assemblages that each habitat supports. Extensive sampling with quantitative enclosure traps that estimate nekton density is suggested. These gears have a high capture efficiency in most habitats and are small enough (e.g., 1 m(2)) to permit sampling in specific microhabitats. Other aspects of nekton monitoring are discussed, including spatial and temporal sampling considerations, station selection, sample size estimation, and data collection and analysis. Developing and initiating long-term nekton monitoring programs will help evaluate natural and human-induced changes in estuarine nekton over time and advance our understanding of the interactions between nekton and the dynamic estuarine environment.

Protein crystal screening and characterization for serial femtosecond nanocrystallography

PubMed Central

Darmanin, Connie; Strachan, Jamie; Adda, Christopher G.; Ve, Thomas; Kobe, Bostjan; Abbey, Brian

2016-01-01

The recent development of X-ray free electron lasers (XFELs) has spurred the development of serial femtosecond nanocrystallography (SFX) which, for the first time, is enabling structure retrieval from sub-micron protein crystals. Although there are already a growing number of structures published using SFX, the technology is still very new and presents a number of unique challenges as well as opportunities for structural biologists. One of the biggest barriers to the success of SFX experiments is the preparation and selection of suitable protein crystal samples. Here we outline a protocol for preparing and screening for suitable XFEL targets. PMID:27139248

Solution Hybrid Selection Capture for the Recovery of Functional Full-Length Eukaryotic cDNAs From Complex Environmental Samples

PubMed Central

Bragalini, Claudia; Ribière, Céline; Parisot, Nicolas; Vallon, Laurent; Prudent, Elsa; Peyretaillade, Eric; Girlanda, Mariangela; Peyret, Pierre; Marmeisse, Roland; Luis, Patricia

2014-01-01

Eukaryotic microbial communities play key functional roles in soil biology and potentially represent a rich source of natural products including biocatalysts. Culture-independent molecular methods are powerful tools to isolate functional genes from uncultured microorganisms. However, none of the methods used in environmental genomics allow for a rapid isolation of numerous functional genes from eukaryotic microbial communities. We developed an original adaptation of the solution hybrid selection (SHS) for an efficient recovery of functional complementary DNAs (cDNAs) synthesized from soil-extracted polyadenylated mRNAs. This protocol was tested on the Glycoside Hydrolase 11 gene family encoding endo-xylanases for which we designed 35 explorative 31-mers capture probes. SHS was implemented on four soil eukaryotic cDNA pools. After two successive rounds of capture, >90% of the resulting cDNAs were GH11 sequences, of which 70% (38 among 53 sequenced genes) were full length. Between 1.5 and 25% of the cloned captured sequences were expressed in Saccharomyces cerevisiae. Sequencing of polymerase chain reaction-amplified GH11 gene fragments from the captured sequences highlighted hundreds of phylogenetically diverse sequences that were not yet described, in public databases. This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families within microbial communities thriving in any type of environment. PMID:25281543

Avoidance of harvesting and sampling artefacts in hydraulic analyses: a protocol tested on Malus domestica

PubMed Central

Beikircher, Barbara; Mayr, Stefan

2016-01-01

A prerequisite for reliable hydraulic measurements is an accurate collection of the plant material. Thereby, the native hydraulic state of the sample has to be preserved during harvesting (i.e., cutting the plant or plant parts) and preparation (i.e., excising the target section). This is particularly difficult when harvesting has to be done under transpiring conditions. In this article, we present a harvesting and sampling protocol designed for hydraulic measurements on Malus domestica Borkh. and checked for possible sampling artefacts. To test for artefacts, we analysed the percentage loss of hydraulic conductivity, maximum specific conductivity and water contents of bark and wood of branches, taking into account conduit length, time of day of harvesting, different shoot ages and seasonal effects. Our results prove that use of appropriate protocols can avoid artefactual embolization or refilling even when the xylem is under tension at harvest. The presented protocol was developed for Malus but may also be applied for other angiosperms with similar anatomy and refilling characteristics. PMID:26705311

Performance Evaluation of Relay Selection Schemes in Beacon-Assisted Dual-Hop Cognitive Radio Wireless Sensor Networks under Impact of Hardware Noises.

PubMed

Hieu, Tran Dinh; Duy, Tran Trung; Dung, Le The; Choi, Seong Gon

2018-06-05

To solve the problem of energy constraints and spectrum scarcity for cognitive radio wireless sensor networks (CR-WSNs), an underlay decode-and-forward relaying scheme is considered, where the energy constrained secondary source and relay nodes are capable of harvesting energy from a multi-antenna power beacon (PB) and using that harvested energy to forward the source information to the destination. Based on the time switching receiver architecture, three relaying protocols, namely, hybrid partial relay selection (H-PRS), conventional opportunistic relay selection (C-ORS), and best opportunistic relay selection (B-ORS) protocols are considered to enhance the end-to-end performance under the joint impact of maximal interference constraint and transceiver hardware impairments. For performance evaluation and comparison, we derive the exact and asymptotic closed-form expressions of outage probability (OP) and throughput (TP) to provide significant insights into the impact of our proposed protocols on the system performance over Rayleigh fading channel. Finally, simulation results validate the theoretical results.

Rapid Waterborne Pathogen Detection with Mobile Electronics.

PubMed

Wu, Tsung-Feng; Chen, Yu-Chen; Wang, Wei-Chung; Kucknoor, Ashwini S; Lin, Che-Jen; Lo, Yu-Hwa; Yao, Chun-Wei; Lian, Ian

2017-06-09

Pathogen detection in water samples, without complex and time consuming procedures such as fluorescent-labeling or culture-based incubation, is essential to public safety. We propose an immunoagglutination-based protocol together with the microfluidic device to quantify pathogen levels directly from water samples. Utilizing ubiquitous complementary metal-oxide-semiconductor (CMOS) imagers from mobile electronics, a low-cost and one-step reaction detection protocol is developed to enable field detection for waterborne pathogens. 10 mL of pathogen-containing water samples was processed using the developed protocol including filtration enrichment, immune-reaction detection and imaging processing. The limit of detection of 10 E. coli O157:H7 cells/10 mL has been demonstrated within 10 min of turnaround time. The protocol can readily be integrated into a mobile electronics such as smartphones for rapid and reproducible field detection of waterborne pathogens.

FISH-in-CHIPS: A Microfluidic Platform for Molecular Typing of Cancer Cells.

PubMed

Perez-Toralla, Karla; Mottet, Guillaume; Tulukcuoglu-Guneri, Ezgi; Champ, Jérôme; Bidard, François-Clément; Pierga, Jean-Yves; Klijanienko, Jerzy; Draskovic, Irena; Malaquin, Laurent; Viovy, Jean-Louis; Descroix, Stéphanie

2017-01-01

Microfluidics offer powerful tools for the control, manipulation, and analysis of cells, in particular for the assessment of cell malignancy or the study of cell subpopulations. However, implementing complex biological protocols on chip remains a challenge. Sample preparation is often performed off chip using multiple manually performed steps, and protocols usually include different dehydration and drying steps that are not always compatible with a microfluidic format.Here, we report the implementation of a Fluorescence in situ Hybridization (FISH) protocol for the molecular typing of cancer cells in a simple and low-cost device. The geometry of the chip allows integrating the sample preparation steps to efficiently assess the genomic content of individual cells using a minute amount of sample. The FISH protocol can be fully automated, thus enabling its use in routine clinical practice.

Near-optimal protocols in complex nonequilibrium transformations

DOE PAGES

Gingrich, Todd R.; Rotskoff, Grant M.; Crooks, Gavin E.; ...

2016-08-29

The development of sophisticated experimental means to control nanoscale systems has motivated efforts to design driving protocols that minimize the energy dissipated to the environment. Computational models are a crucial tool in this practical challenge. In this paper, we describe a general method for sampling an ensemble of finite-time, nonequilibrium protocols biased toward a low average dissipation. In addition, we show that this scheme can be carried out very efficiently in several limiting cases. As an application, we sample the ensemble of low-dissipation protocols that invert the magnetization of a 2D Ising model and explore how the diversity of themore » protocols varies in response to constraints on the average dissipation. In this example, we find that there is a large set of protocols with average dissipation close to the optimal value, which we argue is a general phenomenon.« less

Study on Quality of IUD Services Provided by Trained Professionals at Teaching Institutes.

PubMed

Prasad, Noopur; Jain, M L; Meena, B S

2018-06-01

Access the completeness in IUD services provided by trained professionals and find out the weak links. Study was conducted on 100 IUD trained professionals of tertiary care hospital and nursing teaching institute. All were given questionnaire that was duly filled by them. Data obtained were analysed. Protocols of case selection, pre-insertion counselling, insertion process and follow-up were assessed. All the four criteria were assessed on score of ten. Study group could not get ten points under any of the set criteria. Average of 53% case selection, 31.4% pre-insertion counselling, 42.5% insertion protocols and 46.1% follow-up counselling criteria were observed by study group. Highest compliance of protocols was seen among postgraduate students. Although IUD training is given to all medical professionals and IUD facility is available up to subcentres but the study shows that completeness in services is still lacking. Ensuring ideal place for IUD insertion, proper case selection, use of specific instruments for insertion and observance of insertion protocols are very vital for the success of IUD.

Exploring the Implementation of Steganography Protocols on Quantum Audio Signals

NASA Astrophysics Data System (ADS)

Chen, Kehan; Yan, Fei; Iliyasu, Abdullah M.; Zhao, Jianping

2018-02-01

Two quantum audio steganography (QAS) protocols are proposed, each of which manipulates or modifies the least significant qubit (LSQb) of the host quantum audio signal that is encoded as an FRQA (flexible representation of quantum audio) audio content. The first protocol (i.e. the conventional LSQb QAS protocol or simply the cLSQ stego protocol) is built on the exchanges between qubits encoding the quantum audio message and the LSQb of the amplitude information in the host quantum audio samples. In the second protocol, the embedding procedure to realize it implants information from a quantum audio message deep into the constraint-imposed most significant qubit (MSQb) of the host quantum audio samples, we refer to it as the pseudo MSQb QAS protocol or simply the pMSQ stego protocol. The cLSQ stego protocol is designed to guarantee high imperceptibility between the host quantum audio and its stego version, whereas the pMSQ stego protocol ensures that the resulting stego quantum audio signal is better immune to illicit tampering and copyright violations (a.k.a. robustness). Built on the circuit model of quantum computation, the circuit networks to execute the embedding and extraction algorithms of both QAS protocols are determined and simulation-based experiments are conducted to demonstrate their implementation. Outcomes attest that both protocols offer promising trade-offs in terms of imperceptibility and robustness.

Surface-enhanced Raman spectroscopy studies of yellow organic dyestuffs and lake pigments in oil paint.

PubMed

Mayhew, Hannah E; Fabian, David M; Svoboda, Shelley A; Wustholz, Kristin L

2013-08-21

Identifying natural, organic dyes and pigments is important for the conservation, preservation, and historical interpretation of works of art. Although previous SERS studies have demonstrated high sensitivity and selectivity for red lake pigments using various pretreatment conditions, corresponding investigations of yellow lake pigments and paints are relatively sparse. Here, surface-enhanced Raman scattering (SERS) spectroscopy is used to identify a variety of yellow organic dyestuffs and lake pigments in oil paint. High-quality SERS spectra of yellow dyestuffs (i.e., turmeric, old fustic, Buckthorn berries) and corresponding paints could be obtained with or without sample pretreatment using microliter quantities of HCl and methanol at room temperature. However, the SERS spectra of yellow lake pigments (i.e., Stil de Grain, Reseda lake) and their corresponding oil paints were only observed upon sample pretreatment. Ultimately, we demonstrate a reliable sample treatment protocol for SERS-based identification of turmeric, old fustic, Buckthorn berries, Stil de Grain, and Reseda lake as well as for microscopic samples of the corresponding oil paints.

Comparison of Methods for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded Archival Tissues

PubMed Central

Sengüven, Burcu; Baris, Emre; Oygur, Tulin; Berktas, Mehmet

2014-01-01

Aim: Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues. Methods: DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability. Results: The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification. Conclusion: Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE. PMID:24688314

USGS/EPA collection protocol for bacterial pathogens in soil

USGS Publications Warehouse

Griffin, Dale W.; Shaefer, F.L.; Charlena Bowling,; Dino Mattorano,; Tonya Nichols,; Erin Silvestri,

2014-01-01

This Sample Collection Procedure (SCP) describes the activities and considerations for the collection of bacterial pathogens from representative surface soil samples (0-5 cm). This sampling depth can be reached without the use of a drill rig, direct-push technology, or other mechanized equipment. This procedure can be used in most soil types but is limited to sampling at or near the ground surface. This protocol has components for two different types of sampling applications: (1) typical sampling, when there is no suspicion of contamination (e.g., surveillance or background studies); and (2) in response to known or suspected accidental contamination (e.g., the presence of animal carcasses). This protocol does not cover sampling in response to a suspected bioterrorist or intentional release event. Surface material is removed to the required depth (0-5 cm) and clean trowel or 50 ml sample tube is used to collect the sample. Sample containers are sealed, bagged, and shipped to the laboratory for analysis. Associated documentation, including a Field Data Log and Chain-of-Custody are also included in this document.

Forensic genetic SNP typing of low-template DNA and highly degraded DNA from crime case samples.

PubMed

Børsting, Claus; Mogensen, Helle Smidt; Morling, Niels

2013-05-01

Heterozygote imbalances leading to allele drop-outs and disproportionally large stutters leading to allele drop-ins are known stochastic phenomena related to STR typing of low-template DNA (LtDNA). The large stutters and the many drop-ins in typical STR stutter positions are artifacts from the PCR amplification of tandem repeats. These artifacts may be avoided by typing bi-allelic markers instead of STRs. In this work, the SNPforID multiplex assay was used to type LtDNA. A sensitized SNP typing protocol was introduced, that increased signal strengths without increasing noise and without affecting the heterozygote balance. Allele drop-ins were only observed in experiments with 25 pg of DNA and not in experiments with 50 and 100 pg of DNA. The allele drop-in rate in the 25 pg experiments was 0.06% or 100 times lower than what was previously reported for STR typing of LtDNA. A composite model and two different consensus models were used to interpret the SNP data. Correct profiles with 42-49 SNPs were generated from the 50 and 100 pg experiments, whereas a few incorrect genotypes were included in the generated profiles from the 25 pg experiments. With the strict consensus model, between 35 and 48 SNPs were correctly typed in the 25 pg experiments and only one allele drop-out (error rate: 0.07%) was observed in the consensus profiles. A total of 28 crime case samples were selected for typing with the sensitized SNPforID protocol. The samples were previously typed with old STR kits during the crime case investigation and only partial profiles (0-6 STRs) were obtained. Eleven of the samples could not be quantified with the Quantifiler™ Human DNA Quantification kit because of partial or complete inhibition of the PCR. For eight of these samples, SNP typing was only possible when the buffer and DNA polymerase used in the original protocol was replaced with the AmpFℓSTR(®) SEfiler Plus™ Master Mix, which was developed specifically for challenging forensic samples. All the crime case samples were successfully typed with the SNPforID multiplex assay and the match probabilities ranged from 1.1×10(-15) to 7.9×10(-23). In comparison, four of the samples could not be typed with the AmpFℓSTR(®) SEfiler Plus™ kit and the match probabilities were higher than 10(-7) for another six samples. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Optimization of scat detection methods for a social ungulate, the wild pig, and experimental evaluation of factors affecting detection of scat

USGS Publications Warehouse

Keiter, David A.; Cunningham, Fred L.; Rhodes, Olin E.; Irwin, Brian J.; Beasley, James

2016-01-01

Collection of scat samples is common in wildlife research, particularly for genetic capture-mark-recapture applications. Due to high degradation rates of genetic material in scat, large numbers of samples must be collected to generate robust estimates. Optimization of sampling approaches to account for taxa-specific patterns of scat deposition is, therefore, necessary to ensure sufficient sample collection. While scat collection methods have been widely studied in carnivores, research to maximize scat collection and noninvasive sampling efficiency for social ungulates is lacking. Further, environmental factors or scat morphology may influence detection of scat by observers. We contrasted performance of novel radial search protocols with existing adaptive cluster sampling protocols to quantify differences in observed amounts of wild pig (Sus scrofa) scat. We also evaluated the effects of environmental (percentage of vegetative ground cover and occurrence of rain immediately prior to sampling) and scat characteristics (fecal pellet size and number) on the detectability of scat by observers. We found that 15- and 20-m radial search protocols resulted in greater numbers of scats encountered than the previously used adaptive cluster sampling approach across habitat types, and that fecal pellet size, number of fecal pellets, percent vegetative ground cover, and recent rain events were significant predictors of scat detection. Our results suggest that use of a fixed-width radial search protocol may increase the number of scats detected for wild pigs, or other social ungulates, allowing more robust estimation of population metrics using noninvasive genetic sampling methods. Further, as fecal pellet size affected scat detection, juvenile or smaller-sized animals may be less detectable than adult or large animals, which could introduce bias into abundance estimates. Knowledge of relationships between environmental variables and scat detection may allow researchers to optimize sampling protocols to maximize utility of noninvasive sampling for wild pigs and other social ungulates.

Optimization of Scat Detection Methods for a Social Ungulate, the Wild Pig, and Experimental Evaluation of Factors Affecting Detection of Scat.

PubMed

Keiter, David A; Cunningham, Fred L; Rhodes, Olin E; Irwin, Brian J; Beasley, James C

2016-01-01

Collection of scat samples is common in wildlife research, particularly for genetic capture-mark-recapture applications. Due to high degradation rates of genetic material in scat, large numbers of samples must be collected to generate robust estimates. Optimization of sampling approaches to account for taxa-specific patterns of scat deposition is, therefore, necessary to ensure sufficient sample collection. While scat collection methods have been widely studied in carnivores, research to maximize scat collection and noninvasive sampling efficiency for social ungulates is lacking. Further, environmental factors or scat morphology may influence detection of scat by observers. We contrasted performance of novel radial search protocols with existing adaptive cluster sampling protocols to quantify differences in observed amounts of wild pig (Sus scrofa) scat. We also evaluated the effects of environmental (percentage of vegetative ground cover and occurrence of rain immediately prior to sampling) and scat characteristics (fecal pellet size and number) on the detectability of scat by observers. We found that 15- and 20-m radial search protocols resulted in greater numbers of scats encountered than the previously used adaptive cluster sampling approach across habitat types, and that fecal pellet size, number of fecal pellets, percent vegetative ground cover, and recent rain events were significant predictors of scat detection. Our results suggest that use of a fixed-width radial search protocol may increase the number of scats detected for wild pigs, or other social ungulates, allowing more robust estimation of population metrics using noninvasive genetic sampling methods. Further, as fecal pellet size affected scat detection, juvenile or smaller-sized animals may be less detectable than adult or large animals, which could introduce bias into abundance estimates. Knowledge of relationships between environmental variables and scat detection may allow researchers to optimize sampling protocols to maximize utility of noninvasive sampling for wild pigs and other social ungulates.

An Organic Decontamination Method for Sampling Devices used in Life-detection Studies

NASA Technical Reports Server (NTRS)

Eigenbrode, Jennifer; Maule, Jake; Wainwright, Norm; Steele, Andrew; Amundsen, Hans E.F.

2008-01-01

Organic decontamination of sampling and storage devices are crucial steps for life-detection, habitability, and ecological investigations of extremophiles living in the most inhospitable niches of Earth, Mars and elsewhere. However, one of the main stumbling blocks for Mars-analogue life-detection studies in terrestrial remote field-sites is the capability to clean instruments and sampling devices to organic levels consistent with null values. Here we present a new seven-step, multi-reagent cleaning and decontamination protocol that was adapted and tested on a glacial ice-coring device and on a rover-guided scoop used for sediment sampling both deployed multiple times during two field seasons of the Arctic Mars Analog Svalbard Expedition AMASE). The effectiveness of the protocols for both devices was tested by (1)in situ metabolic measurements via APT, (2)in situ lipopolysacchride (LPS) quantifications via low-level endotoxin assays, and(3) laboratory-based molecular detection via gas chromatography-mass spectrometry. Our results show that the combination and step-wise application of disinfectants with oxidative and solvation properties for sterilization are effective at removing cellular remnants and other organic traces to levels necessary for molecular organic- and life-detection studies. The validation of this seven-step protocol - specifically for ice sampling - allows us to proceed with confidence in kmskia4 analogue investigations of icy environments. However, results from a rover scoop test showed that this protocol is also suitable for null-level decontamination of sample acquisition devices. Thus, this protocol may be applicable to a variety of sampling devices and analytical instrumentation used for future astrobiology missions to Enceladus, and Europa, as well as for sample-return missions.

DEVELOPMENT OF LARGE RIVER BIOASSESSMENT PROTOCOLS (LR-BPS) FOR BENTHIC MACROINVERTEBRATES IN EPA REGION 5

EPA Science Inventory

Non-wadeable rivers have been largely overlooked by bioassessment programs because of sampling difficulties and a lack of appropriate methods and biological indicators. We are in the process of developing a Large River Bioassessment Protocol (LR-BP) for sampling macroinvertebrat...

U.S.-MEXICO BORDER PROGRAM ARIZONA BORDER STUDY--LIST OF STANDARD OPERATING PROCEDURES

EPA Science Inventory

This document lists available protocols and SOPs for the U.S.-Mexico Border Program study. It identifies protocols and SOPs for the following study components: (1) Sample collection and field operations, (2) Sample analysis, (3) General laboratory procedures, (4) Quality Assuranc...

21 CFR 660.46 - Samples; protocols; official release.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Samples; protocols; official release. 660.46 Section 660.46 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface...

A method for real-time visual stimulus selection in the study of cortical object perception.

PubMed

Leeds, Daniel D; Tarr, Michael J

2016-06-01

The properties utilized by visual object perception in the mid- and high-level ventral visual pathway are poorly understood. To better establish and explore possible models of these properties, we adopt a data-driven approach in which we repeatedly interrogate neural units using functional Magnetic Resonance Imaging (fMRI) to establish each unit's image selectivity. This approach to imaging necessitates a search through a broad space of stimulus properties using a limited number of samples. To more quickly identify the complex visual features underlying human cortical object perception, we implemented a new functional magnetic resonance imaging protocol in which visual stimuli are selected in real-time based on BOLD responses to recently shown images. Two variations of this protocol were developed, one relying on natural object stimuli and a second based on synthetic object stimuli, both embedded in feature spaces based on the complex visual properties of the objects. During fMRI scanning, we continuously controlled stimulus selection in the context of a real-time search through these image spaces in order to maximize neural responses across pre-determined 1cm(3) rain regions. Elsewhere we have reported the patterns of cortical selectivity revealed by this approach (Leeds et al., 2014). In contrast, here our objective is to present more detailed methods and explore the technical and biological factors influencing the behavior of our real-time stimulus search. We observe that: 1) Searches converged more reliably when exploring a more precisely parameterized space of synthetic objects; 2) real-time estimation of cortical responses to stimuli is reasonably consistent; 3) search behavior was acceptably robust to delays in stimulus displays and subject motion effects. Overall, our results indicate that real-time fMRI methods may provide a valuable platform for continuing study of localized neural selectivity, both for visual object representation and beyond. Copyright © 2016 Elsevier Inc. All rights reserved.

A method for real-time visual stimulus selection in the study of cortical object perception

PubMed Central

Leeds, Daniel D.; Tarr, Michael J.

2016-01-01

The properties utilized by visual object perception in the mid- and high-level ventral visual pathway are poorly understood. To better establish and explore possible models of these properties, we adopt a data-driven approach in which we repeatedly interrogate neural units using functional Magnetic Resonance Imaging (fMRI) to establish each unit’s image selectivity. This approach to imaging necessitates a search through a broad space of stimulus properties using a limited number of samples. To more quickly identify the complex visual features underlying human cortical object perception, we implemented a new functional magnetic resonance imaging protocol in which visual stimuli are selected in real-time based on BOLD responses to recently shown images. Two variations of this protocol were developed, one relying on natural object stimuli and a second based on synthetic object stimuli, both embedded in feature spaces based on the complex visual properties of the objects. During fMRI scanning, we continuously controlled stimulus selection in the context of a real-time search through these image spaces in order to maximize neural responses across predetermined 1 cm3 brain regions. Elsewhere we have reported the patterns of cortical selectivity revealed by this approach (Leeds 2014). In contrast, here our objective is to present more detailed methods and explore the technical and biological factors influencing the behavior of our real-time stimulus search. We observe that: 1) Searches converged more reliably when exploring a more precisely parameterized space of synthetic objects; 2) Real-time estimation of cortical responses to stimuli are reasonably consistent; 3) Search behavior was acceptably robust to delays in stimulus displays and subject motion effects. Overall, our results indicate that real-time fMRI methods may provide a valuable platform for continuing study of localized neural selectivity, both for visual object representation and beyond. PMID:26973168

Optimization of a Sample Processing Protocol for Recovery of ...

EPA Pesticide Factsheets

Journal Article Following a release of Bacillus anthracis spores into the environment, there is a potential for lasting environmental contamination in soils. There is a need for detection protocols for B. anthracis in environmental matrices. However, identification of B. anthracis within a soil is a difficult task. Processing soil samples helps to remove debris, chemical components, and biological impurities that can interfere with microbiological detection. This study aimed to optimize a previously used indirect processing protocol, which included a series of washing and centrifugation steps.

Screening apatites for (U-Th)/He thermochronometry via continuous ramped heating: He age components and implications for age dispersion

NASA Astrophysics Data System (ADS)

McDannell, Kalin T.; Zeitler, Peter K.; Janes, Darwin G.; Idleman, Bruce D.; Fayon, Annia K.

2018-02-01

Old slowly-cooled apatites often yield dispersed (U-Th)/He ages for a variety of reasons, some well understood and some not. Analytical protocols like careful grain selection can reduce the impact of this dispersion but add costs in time and resources and too often have proven insufficient. We assess a new analytical protocol that utilizes static-gas measurement during continuous ramped heating (CRH) as a means to rapidly screen apatite samples. In about the time required for a conventional total-gas analysis, this method can discriminate between samples showing expected volume-diffusion behavior and those showing anomalous release patterns inconsistent with their direct use in thermochronologic applications. This method also appears able to discriminate between the radiogenic and extraneous 4He fractions released by a sample, potentially allowing ages to be corrected. Well-behaved examples such as the Durango standard and other apatites with good age reproducibility show the expected smooth, sigmoidal gas-release curves predicted for volume diffusion using typical apatite kinetics, with complete exhaustion by ∼900 °C for linear heating at 20 °C/min. Secondary factors such as U and Th zoning and alpha-loss distribution have a relatively minor impact on such profiles. In contrast, samples having greater age dispersion show significant He release in the form of outgassing spikes and He release deferred to higher temperatures. Screening results for a range of samples permit us to assess the degree to which CRH screening can identify misbehaving grains, give insight into the source of extraneous He, and suggest that in some cases it may be possible to correct ages for the presence of such components.

Comparison of the Sensitivity of Laboratory Diagnostic Methods from a Well-Characterized Outbreak of Mumps in New York City in 2009

PubMed Central

Rosen, Jennifer B.; Doll, Margaret K.; McNall, Rebecca J.; McGrew, Marcia; Williams, Nobia; Lopareva, Elena N.; Barskey, Albert E.; Punsalang, Amado; Rota, Paul A.; Oleszko, William R.; Hickman, Carole J.; Zimmerman, Christopher M.; Bellini, William J.

2013-01-01

A mumps outbreak in upstate New York in 2009 at a summer camp for Orthodox Jewish boys spread into Orthodox Jewish communities in the Northeast, including New York City. The availability of epidemiologic information, including vaccination records and parotitis onset dates, allowed an enhanced analysis of laboratory methods for mumps testing. Serum and buccal swab samples were collected from 296 confirmed cases with onsets from September through December 2009. All samples were tested using the Centers for Disease Control and Prevention (CDC) capture IgM enzyme immunoassay (EIA) and a real-time reverse transcription-PCR (rRT-PCR) that targets the short hydrophobic gene. A subset of the samples (n = 205) was used to evaluate 3 commercial mumps IgM assays and to assess the sensitivity of using an alternative target gene (nucleoprotein) in the rRT-PCR protocol. Among 115 cases of mumps with 2 documented doses of measles, mumps, and rubella (MMR) vaccine, the CDC capture IgM EIA detected IgM in 51% of serum samples compared to 9% to 24% using three commercial IgM assays. The rRT-PCR that targeted the nucleoprotein gene increased RNA detection by 14% compared to that obtained with the original protocol. The ability to detect IgM improved when serum was collected 3 days or more after symptom onset, whereas sensitivity of RNA detection by rRT-PCR declined when buccal swabs were collected later than 2 days after onset. Selection of testing methods and timing of sample collection are important factors in the ability to confirm infection among vaccinated persons. These results reinforce the need to use virus detection assays in addition to serologic tests. PMID:23324519

A novel contact assay for testing aryl hydrocarbon receptor (AhR)-mediated toxicity of chemicals and whole sediments in zebrafish (Danio rerio) embryos.

PubMed

Schiwy, Sabrina; Bräunig, Jennifer; Alert, Henriette; Hollert, Henner; Keiter, Steffen H

2015-11-01

The European Water Framework Directive aims to achieve a good ecological and chemical status in surface waters until 2015. Sediment toxicology plays a major role in this intention as sediments can act as a secondary source of pollution. In order to fulfill this legal obligation, there is an urgent need to develop whole-sediment exposure protocols, since sediment contact assays represent the most realistic scenario to simulate in situ exposure conditions. Therefore, in the present study, a vertebrate sediment contact assay to determine aryl hydrocarbon receptor (AhR)-mediated activity of particle-bound pollutants was developed. Furthermore, the activity and the expression of the CYP1 family in early life stages of zebrafish after exposure to freeze-dried sediment samples were investigated. In order to validate the developed protocol, effects of β-naphthoflavone and three selected sediment on zebrafish embryos were investigated. Results documented clearly AhR-mediated toxicity after exposure to β-naphthoflavone (β-NF) and to the sediment from the Vering canal. Upregulation of mRNA levels was observed for all investigated sediment samples. The highest levels of all investigated cyp genes (cyp1a, cyp1b1, cyp1c1, and cyp1c2) were recorded after exposure to the sediment sample of the Vering canal. In conclusion, the newly developed sediment contact assay can be recommended for the investigation of dioxin-like activities of single substances and the bioavailable fraction of complex environmental samples. Moreover, the exposure of whole zebrafish embryos to native (freeze-dried) sediment samples represents a highly realistic and ecologically relevant exposure scenario.

Omics for Precious Rare Biosamples: Characterization of Ancient Human Hair by a Proteomic Approach.

PubMed

Fresnais, Margaux; Richardin, Pascale; Sepúlveda, Marcela; Leize-Wagner, Emmanuelle; Charrié-Duhaut, Armelle

2017-07-01

Omics technologies have far-reaching applications beyond clinical medicine. A case in point is the analysis of ancient hair samples. Indeed, hair is an important biological indicator that has become a material of choice in archeometry to study the ancient civilizations and their environment. Current characterization of ancient hair is based on elemental and structural analyses, but only few studies have focused on the molecular aspects of ancient hair proteins-keratins-and their conservation state. In such cases, applied extraction protocols require large amounts of raw hair, from 30 to 100 mg. In the present study, we report an optimized new proteomic approach to accurately identify archeological hair proteins, and assess their preservation state, while using a minimum of raw material. Testing and adaptation of three protocols and of nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) parameters were performed on modern hair. On the basis of mass spectrometry data quality, and of the required initial sample amount, the most promising workflow was selected and applied to an ancient archeological sample, dated to about 3880 years before present. Finally, and importantly, we were able to identify 11 ancient hair proteins and to visualize the preservation state of mummy's hair from only 500 μg of raw material. The results presented here pave the way for new insights into the understanding of hair protein alteration processes such as those due to aging and ecological exposures. This work could enable omics scientists to apply a proteomic approach to precious and rare samples, not only in the context of archeometrical studies but also for future applications that would require the use of very small amounts of sample.

Organic and Isotope Measurement Protocols under Development for the 2009 Mars Science Laboratory

NASA Technical Reports Server (NTRS)

Mahaffy, Paul R.; Atreya, Sushil K.

2006-01-01

The Mars Science Laboratory (MSL) is under development by NASA with several international partners for launch in 2009. MSL is designed to quantitatively explore a local region on Mars as a potential habitat for present or past life (http://mars.jpl.nasa.gov/msl). The goals of MSL are to (1) assess the past or present biological potential of the target environment, (2) to characterize its geology and geochemistry, (3) to study planetary processes that influence habitability, and (4) to characterize the surface radiation. The last substantial search for organic molecules on the surface of Mars was with the Viking Landers in 1976 [Biemann, et al., 19771. In that mission, no organics were detected in near surface fines and presently a more comprehensive search is required to understand the potential of that planet to support life. While the Mars Exploration Rovers are able to identify mineralogical signatures of aqueous alteration, they are not equipped to search for organics. The planned capabilities of the MSL rover payload will enable a search for a wide range of organic molecules in both solid samples of rocks and fines and atmospheric samples. MSL will also provide a determination of definitive mineralogy of the solid samples and precision isotope measurements of several volatile elements. Contact and remote surface and subsurface survey tools will establish context for Analytical Laboratory measurements and will facilitate sample selection. The Sample Analysis at Mars (SAM) suite of MSL addresses several of the mission's core measurement goals. SAM includes a gas chromatograph, a mass spectrometer, and a tunable laser spectrometer. We will describe the range of measurement protocols under development and test for SAM and the relationship of our planned measurements to outstanding issues of martian habitability.

Non-invasive biomonitoring for PFRs and PBDEs: new insights in analysis of human hair externally exposed to selected flame retardants.

PubMed

Kucharska, Agnieszka; Covaci, Adrian; Vanermen, Guido; Voorspoels, Stefan

2015-02-01

In this study, we investigated the hypothesis whether externally adsorbed and internally deposited flame retardants (FRs) in hair could be distinguished. To this extent, hair samples collected from one volunteer were exposed under controlled conditions to phosphate FR (PFR) and polybrominated diphenyl ether (PBDE) standards to mimic external contamination. Afterwards, suitable washing procedures to selectively remove contaminants from the hair surface were investigated. The samples were measured by GC-(ECNI)-MS for PBDEs and LC-(ESI+)-MS/MS for PFRs. All investigated compounds were transferred onto the hair surface. One of the most important finding was that dust particles are not mandatory to transfer compounds on the hair surface and to be able to measure high levels of compounds in human hair. To assess different protocols to selectively remove external contamination, the exposed hair samples were washed in different media before analysis: water, methanol, hexane:dichloromethane (1:1, v/v), acetone and shampoo. Results indicated that there is no washing medium able to entirely and exclusively remove external contamination. Among investigated media, methanol removed a meaningful part of the external contamination (42-105%), but the removal efficiencies differed among compounds. We therefore concluded that hair should not be washed prior to analysis and in case of visible contamination (e.g. with cosmetic products), water would be the recommended agent. Organic solvents should not be used for the washing step. Although it is impossible to distinguish external from internal exposure, hair samples may be used as valuable biomarker of human exposure, providing a measure of integral exposure. To the best of our knowledge, this is the first study which has used externally exposed hair samples to PBDEs and PFRs. Copyright © 2014 Elsevier B.V. All rights reserved.

Flow cytometric sorting of fecal bacteria after in situ hybridization with polynucleotide probes.

PubMed

Bruder, Lena M; Dörkes, Marcel; Fuchs, Bernhard M; Ludwig, Wolfgang; Liebl, Wolfgang

2016-10-01

The gut microbiome represents a key contributor to human physiology, metabolism, immune function, and nutrition. Elucidating the composition and genetics of the gut microbiota under various conditions is essential to understand how microbes function individually and as a community. Metagenomic analyses are increasingly used to study intestinal microbiota. However, for certain scientific questions it is sufficient to examine taxon-specific submetagenomes, covering selected bacterial genera in a targeted manner. Here we established a new variant of fluorescence in situ hybridization (FISH) combined with fluorescence-activated cell sorting (FACS), providing access to the genomes of specific taxa belonging to the complex community of the intestinal microbiota. In contrast to standard oligonucleotide probes, the RNA polynucleotide probe used here, which targets domain III of the 23S rRNA gene, extends the resolution power in environmental samples by increasing signal intensity. Furthermore, cells hybridized with the polynucleotide probe are not subjected to harsh pretreatments, and their genetic information remains intact. The protocol described here was tested on genus-specifically labeled cells in various samples, including complex fecal samples from different laboratory mouse types that harbor diverse intestinal microbiota. Specifically, as an example for the protocol described here, RNA polynucleotide probes could be used to label Enterococcus cells for subsequent sorting by flow cytometry. To detect and quantify enterococci in fecal samples prior to enrichment, taxon-specific PCR and qPCR detection systems have been developed. The accessibility of the genomes from taxon-specifically sorted cells for subsequent molecular analyses was demonstrated by amplification of functional genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

Integrating occupancy modeling and interview data for corridor identification: A case study for jaguars in Nicaragua

USGS Publications Warehouse

Zeller, K.A.; Nijhawan, S.; Salom-Perez, R.; Potosme, S.H.; Hines, J.E.

2011-01-01

Corridors are critical elements in the long-term conservation of wide-ranging species like the jaguar (Panthera onca). Jaguar corridors across the range of the species were initially identified using a GIS-based least-cost corridor model. However, due to inherent errors in remotely sensed data and model uncertainties, these corridors warrant field verification before conservation efforts can begin. We developed a novel corridor assessment protocol based on interview data and site occupancy modeling. We divided our pilot study area, in southeastern Nicaragua, into 71, 6. ??. 6 km sampling units and conducted 160 structured interviews with local residents. Interviews were designed to collect data on jaguar and seven prey species so that detection/non-detection matrices could be constructed for each sampling unit. Jaguars were reportedly detected in 57% of the sampling units and had a detection probability of 28%. With the exception of white-lipped peccary, prey species were reportedly detected in 82-100% of the sampling units. Though the use of interview data may violate some assumptions of the occupancy modeling approach for determining 'proportion of area occupied', we countered these shortcomings through study design and interpreting the occupancy parameter, psi, as 'probability of habitat used'. Probability of habitat use was modeled for each target species using single state or multistate models. A combination of the estimated probabilities of habitat use for jaguar and prey was selected to identify the final jaguar corridor. This protocol provides an efficient field methodology for identifying corridors for easily-identifiable species, across large study areas comprised of unprotected, private lands. ?? 2010 Elsevier Ltd.

Protocols for dry DNA storage and shipment at room temperature

PubMed Central

Ivanova, Natalia V; Kuzmina, Masha L

2013-01-01

The globalization of DNA barcoding will require core analytical facilities to develop cost-effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry-state DNA stabilization systems: commercial Biomatrica® DNAstable® plates, home-made trehalose and polyvinyl alcohol (PVA) plates on 96-well panels of insect DNA stored at 56 °C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56 °C, and diluted samples held at 4 °C and at −20 °C. PCR and selective sequencing were performed over a 4-year interval to test the condition of DNA extracts. Biomatrica® provided better protection of DNA at 56 °C and at room temperature than trehalose and PVA, especially for diluted samples. PVA was the second best protectant after Biomatrica® at room temperature, whereas trehalose was the second best protectant at 56 °C. In spite of lower PCR success, the DNA stored at −20 °C yielded longer sequence reads and stronger signal, indicating that temperature is a crucial factor for DNA quality which has to be considered especially for long-term storage. Although it is premature to advocate a transition to DNA storage at room temperature, dry storage provides an additional layer of security for frozen samples, protecting them from degradation in the event of freezer failure. All three forms of DNA preservation enable shipment of dry DNA and PCR products between barcoding facilities. PMID:23789643

Modeling and Simulation of a Novel Relay Node Based Secure Routing Protocol Using Multiple Mobile Sink for Wireless Sensor Networks.

PubMed

Perumal, Madhumathy; Dhandapani, Sivakumar

2015-01-01

Data gathering and optimal path selection for wireless sensor networks (WSN) using existing protocols result in collision. Increase in collision further increases the possibility of packet drop. Thus there is a necessity to eliminate collision during data aggregation. Increasing the efficiency is the need of the hour with maximum security. This paper is an effort to come up with a reliable and energy efficient WSN routing and secure protocol with minimum delay. This technique is named as relay node based secure routing protocol for multiple mobile sink (RSRPMS). This protocol finds the rendezvous point for optimal transmission of data using a "splitting tree" technique in tree-shaped network topology and then to determine all the subsequent positions of a sink the "Biased Random Walk" model is used. In case of an event, the sink gathers the data from all sources, when they are in the sensing range of rendezvous point. Otherwise relay node is selected from its neighbor to transfer packets from rendezvous point to sink. A symmetric key cryptography is used for secure transmission. The proposed relay node based secure routing protocol for multiple mobile sink (RSRPMS) is experimented and simulation results are compared with Intelligent Agent-Based Routing (IAR) protocol to prove that there is increase in the network lifetime compared with other routing protocols.

What is in your cup of tea? DNA Verity Test to characterize black and green commercial teas

PubMed Central

Comparone, Maria; Di Maio, Antonietta; Del Guacchio, Emanuele; Menale, Bruno; Troisi, Jacopo; Aliberti, Francesco

2017-01-01

In this study, we used several molecular techniques to develop a fast and reliable protocol (DNA Verity Test, DVT) for the characterization and confirmation of the species or taxa present in herbal infusions. As a model plant for this protocol, Camellia sinensis, a traditional tea plant, was selected due to the following reasons: its historical popularity as a (healthy) beverage, its high selling value, the importation of barely recognizable raw product (i.e., crushed), and the scarcity of studies concerning adulterants or contamination. The DNA Verity Test includes both the sequencing of DNA barcoding markers and genotyping of labeled-PCR DNA barcoding fragments for each sample analyzed. This protocol (DVT) was successively applied to verify the authenticity of 32 commercial teas (simple or admixture), and the main results can be summarized as follows: (1) the DVT protocol is suitable to detect adulteration in tea matrices (contaminations or absence of certified ingredients), and the method can be exported for the study of other similar systems; (2) based on the BLAST analysis of the sequences of rbcL+matK±rps7-trnV(GAC) chloroplast markers, C. sinensis can be taxonomically characterized; (3) rps7-trnV(GAC) can be employed to discriminate C. sinensis from C. pubicosta; (4) ITS2 is not an ideal DNA barcode for tea samples, reflecting potential incomplete lineage sorting and hybridization/introgression phenomena in C. sinensis taxa; (5) the genotyping approach is an easy, inexpensive and rapid pre-screening method to detect anomalies in the tea templates using the trnH(GUG)-psbA barcoding marker; (6) two herbal companies provided no authentic products with a contaminant or without some of the listed ingredients; and (7) the leaf matrices present in some teabags could be constituted using an admixture of different C. sinensis haplotypes and/or allied species (C. pubicosta). PMID:28542606

A Secure Region-Based Geographic Routing Protocol (SRBGR) for Wireless Sensor Networks

PubMed Central

Adnan, Ali Idarous; Hanapi, Zurina Mohd; Othman, Mohamed; Zukarnain, Zuriati Ahmad

2017-01-01

Due to the lack of dependency for routing initiation and an inadequate allocated sextant on responding messages, the secure geographic routing protocols for Wireless Sensor Networks (WSNs) have attracted considerable attention. However, the existing protocols are more likely to drop packets when legitimate nodes fail to respond to the routing initiation messages while attackers in the allocated sextant manage to respond. Furthermore, these protocols are designed with inefficient collection window and inadequate verification criteria which may lead to a high number of attacker selections. To prevent the failure to find an appropriate relay node and undesirable packet retransmission, this paper presents Secure Region-Based Geographic Routing Protocol (SRBGR) to increase the probability of selecting the appropriate relay node. By extending the allocated sextant and applying different message contention priorities more legitimate nodes can be admitted in the routing process. Moreover, the paper also proposed the bound collection window for a sufficient collection time and verification cost for both attacker identification and isolation. Extensive simulation experiments have been performed to evaluate the performance of the proposed protocol in comparison with other existing protocols. The results demonstrate that SRBGR increases network performance in terms of the packet delivery ratio and isolates attacks such as Sybil and Black hole. PMID:28121992

A Secure Region-Based Geographic Routing Protocol (SRBGR) for Wireless Sensor Networks.

PubMed

Adnan, Ali Idarous; Hanapi, Zurina Mohd; Othman, Mohamed; Zukarnain, Zuriati Ahmad

2017-01-01

Due to the lack of dependency for routing initiation and an inadequate allocated sextant on responding messages, the secure geographic routing protocols for Wireless Sensor Networks (WSNs) have attracted considerable attention. However, the existing protocols are more likely to drop packets when legitimate nodes fail to respond to the routing initiation messages while attackers in the allocated sextant manage to respond. Furthermore, these protocols are designed with inefficient collection window and inadequate verification criteria which may lead to a high number of attacker selections. To prevent the failure to find an appropriate relay node and undesirable packet retransmission, this paper presents Secure Region-Based Geographic Routing Protocol (SRBGR) to increase the probability of selecting the appropriate relay node. By extending the allocated sextant and applying different message contention priorities more legitimate nodes can be admitted in the routing process. Moreover, the paper also proposed the bound collection window for a sufficient collection time and verification cost for both attacker identification and isolation. Extensive simulation experiments have been performed to evaluate the performance of the proposed protocol in comparison with other existing protocols. The results demonstrate that SRBGR increases network performance in terms of the packet delivery ratio and isolates attacks such as Sybil and Black hole.

Direct Sensing of Total Acidity by Chronopotentiometric Flash Titrations at Polymer Membrane Ion-Selective Electrodes

PubMed Central

Gemene, Kebede L.; Bakker, Eric

2008-01-01

Polymer membrane ion-selective electrodes containing lipophilic ionophores are traditionally interrogated by zero current potentiometry, which, ideally, gives information on the sample activity of ionic species. It is shown here that a discrete cathodic current pulse across an H+-selective polymeric membrane doped with the ionophore ETH 5294 may be used for the chronopotentiometric detection of pH in well buffered samples. However, a reduction in the buffer capacity leads to large deviations from the expected Nernstian response slope. This is explained by the local depletion of hydrogen ions at the sample-membrane interface as a result of the galvanostatically imposed ion flux in direction of the membrane. This depletion is found to be a function of the total acidity of the sample and can be directly monitored chronopotentiometrically in a flash titration experiment. The subsequent application of a baseline potential pulse reverses the extraction process of the current pulse, allowing one to interrogate the sample with minimal perturbation. In one protocol, total acidity is found to be proportional to the magnitude of applied current at the flash titration endpoint. More conveniently, the square root of the flash titration endpoint time observed at a fixed applied current is a linear function of the total acid concentration. This suggests that it is possible to perform rapid localized pH titrations at ion-selective electrodes without the need for volumetric titrimetry. The technique is explored here for acetic acid, MES and citric acid with promising results. Polymeric membrane electrodes on the basis of poly(vinyl chloride) plasticized with o-nitrophenyloctylether in a 1:2 mass ratio may be used for the detection of acids of up to ca. 1 mM concentration, with flash titration times on the order of a few seconds. Possible limitations of the technique are discussed, including variations of the acid diffusion coefficients and influence of electrical migration. PMID:18370399

Metabolic profiling of body fluids and multivariate data analysis.

PubMed

Trezzi, Jean-Pierre; Jäger, Christian; Galozzi, Sara; Barkovits, Katalin; Marcus, Katrin; Mollenhauer, Brit; Hiller, Karsten

2017-01-01

Metabolome analyses of body fluids are challenging due pre-analytical variations, such as pre-processing delay and temperature, and constant dynamical changes of biochemical processes within the samples. Therefore, proper sample handling starting from the time of collection up to the analysis is crucial to obtain high quality samples and reproducible results. A metabolomics analysis is divided into 4 main steps: 1) Sample collection, 2) Metabolite extraction, 3) Data acquisition and 4) Data analysis. Here, we describe a protocol for gas chromatography coupled to mass spectrometry (GC-MS) based metabolic analysis for biological matrices, especially body fluids. This protocol can be applied on blood serum/plasma, saliva and cerebrospinal fluid (CSF) samples of humans and other vertebrates. It covers sample collection, sample pre-processing, metabolite extraction, GC-MS measurement and guidelines for the subsequent data analysis. Advantages of this protocol include: •Robust and reproducible metabolomics results, taking into account pre-analytical variations that may occur during the sampling process•Small sample volume required•Rapid and cost-effective processing of biological samples•Logistic regression based determination of biomarker signatures for in-depth data analysis.

Information Theory for Gabor Feature Selection for Face Recognition

NASA Astrophysics Data System (ADS)

Shen, Linlin; Bai, Li

2006-12-01

A discriminative and robust feature—kernel enhanced informative Gabor feature—is proposed in this paper for face recognition. Mutual information is applied to select a set of informative and nonredundant Gabor features, which are then further enhanced by kernel methods for recognition. Compared with one of the top performing methods in the 2004 Face Verification Competition (FVC2004), our methods demonstrate a clear advantage over existing methods in accuracy, computation efficiency, and memory cost. The proposed method has been fully tested on the FERET database using the FERET evaluation protocol. Significant improvements on three of the test data sets are observed. Compared with the classical Gabor wavelet-based approaches using a huge number of features, our method requires less than 4 milliseconds to retrieve a few hundreds of features. Due to the substantially reduced feature dimension, only 4 seconds are required to recognize 200 face images. The paper also unified different Gabor filter definitions and proposed a training sample generation algorithm to reduce the effects caused by unbalanced number of samples available in different classes.

Rationale, design and methodology for the Navajo Health and Nutrition Survey.

PubMed

White, L L; Goldberg, H I; Gilbert, T J; Ballew, C; Mendlein, J M; Peter, D G; Percy, C A; Mokdad, A H

1997-10-01

As recently as 1990, there was no reservation-wide, population-based health status information about Navajo Indians. To remedy this shortcoming, the Navajo Health and Nutrition Survey was conducted from 1991 to 1992 to assess the health and nutritional status of Navajo Reservation residents using a population-based sample. Using a three-stage design, a representative sample of reservation households was selected for inclusion. All members of selected households 12 y of age and older were invited to participate. A total of 985 people in 459 households participated in the study. Survey protocols were modeled on those of previous national surveys and included a standard blood chemistry profile, complete blood count, oral glucose tolerance test, blood pressure, anthropometric measurements, a single 24-h dietary recall and a questionnaire on health behaviors. The findings from this survey, reported in the accompanying papers, inform efforts to prevent and control chronic disease among the Navajo. Lessons learned from this survey may be of interest to those conducting similar surveys in other American Indian and Alaska Native populations.

76 FR 24862 - Proposed Information Collection; Comment Request; Protocol for Access to Tissue Specimen Samples...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-03

... Collection; Comment Request; Protocol for Access to Tissue Specimen Samples From the National Marine Mammal Tissue Bank AGENCY: National Oceanic and Atmospheric Administration (NOAA), Commerce. ACTION: Notice... National Marine Mammal Tissue Bank (NMMTB) was established by the National Marine Fisheries Service (NMFS...

Development of reagents for immunoassay of Phytophthora ramorum in nursery water samples

Treesearch

Douglas G. Luster; Timothy Widmer; Michael McMahon; C. André Lévesque

2017-01-01

Current regulations under the August 6, 2014 USDA APHIS Official Regulatory Protocol (Confirmed Nursery Protocol: Version 8.2) for Nurseries Containing Plants Infected with Phytophthora ramorum mandates the sampling of water in affected nurseries to demonstrate they are free of P. ramorum. Currently, detection of

A MORE COST-EFFECTIVE EMAP BENTHIC MACROFAUNAL SAMPLING PROTOCOL

EPA Science Inventory

Benthic macrofaunal sampling protocols in the U.S. Environmental Protection Agency's Environmental Monitoring and Assessment Program (EMAP) are to collect 30 to 50 random benthic macrofauna [defined as animals retained on a 0.5 mm (East and Gulf Coasts, USA) or a 1.0 mm mesh siev...

21 CFR 660.6 - Samples; protocols; official release.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Research, determines that the reliability and consistency of the finished product can be assured with a... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Samples; protocols; official release. 660.6 Section 660.6 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES...

21 CFR 660.6 - Samples; protocols; official release.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Research, determines that the reliability and consistency of the finished product can be assured with a... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Samples; protocols; official release. 660.6 Section 660.6 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

STANDARD MEASUREMENT PROTOCOLS - FLORIDA RADON RESEARCH PROGRAM

EPA Science Inventory

The manual, in support of the Florida Radon Research Program, contains standard protocols for key measurements where data quality is vital to the program. t contains two sections. he first section, soil measurements, contains field sampling protocols for soil gas permeability and...

Method and Apparatus for Processing UDP Data Packets

NASA Technical Reports Server (NTRS)

Murphy, Brandon M. (Inventor)

2017-01-01

A method and apparatus for processing a plurality of data packets. A data packet is received. A determination is made as to whether a portion of the data packet follows a selected digital recorder standard protocol based on a header of the data packet. Raw data in the data packet is converted into human-readable information in response to a determination that the portion of the data packet follows the selected digital recorder standard protocol.

Exponential Sensitivity and its Cost in Quantum Physics

PubMed Central

Gilyén, András; Kiss, Tamás; Jex, Igor

2016-01-01

State selective protocols, like entanglement purification, lead to an essentially non-linear quantum evolution, unusual in naturally occurring quantum processes. Sensitivity to initial states in quantum systems, stemming from such non-linear dynamics, is a promising perspective for applications. Here we demonstrate that chaotic behaviour is a rather generic feature in state selective protocols: exponential sensitivity can exist for all initial states in an experimentally realisable optical scheme. Moreover, any complex rational polynomial map, including the example of the Mandelbrot set, can be directly realised. In state selective protocols, one needs an ensemble of initial states, the size of which decreases with each iteration. We prove that exponential sensitivity to initial states in any quantum system has to be related to downsizing the initial ensemble also exponentially. Our results show that magnifying initial differences of quantum states (a Schrödinger microscope) is possible; however, there is a strict bound on the number of copies needed. PMID:26861076

Exponential Sensitivity and its Cost in Quantum Physics.

PubMed

Gilyén, András; Kiss, Tamás; Jex, Igor

2016-02-10

State selective protocols, like entanglement purification, lead to an essentially non-linear quantum evolution, unusual in naturally occurring quantum processes. Sensitivity to initial states in quantum systems, stemming from such non-linear dynamics, is a promising perspective for applications. Here we demonstrate that chaotic behaviour is a rather generic feature in state selective protocols: exponential sensitivity can exist for all initial states in an experimentally realisable optical scheme. Moreover, any complex rational polynomial map, including the example of the Mandelbrot set, can be directly realised. In state selective protocols, one needs an ensemble of initial states, the size of which decreases with each iteration. We prove that exponential sensitivity to initial states in any quantum system has to be related to downsizing the initial ensemble also exponentially. Our results show that magnifying initial differences of quantum states (a Schrödinger microscope) is possible; however, there is a strict bound on the number of copies needed.

Molecular Typing of Clostridium perfringens from a Food-Borne Disease Outbreak in a Nursing Home: Ribotyping versus Pulsed-Field Gel Electrophoresis

PubMed Central

Schalch, Barbara; Bader, Lutz; Schau, Hans-Peter; Bergmann, Rolf; Rometsch, Andrea; Maydl, Gertraud; Keßler, Silvia

2003-01-01

In 1998, 21 inhabitants of a German nursing home fell ill with acute gastroenteritis after consumption of minced beef heart (P. Graf and L. Bader, Epidemiol. Bull. 41:327-329, 2000). Two residents died during hospital treatment. Seventeen Clostridium perfringens strains were collected from two different dishes and from patients' stool samples and autopsy materials. A majority of these isolates was not typeable by restriction fragment length polymorphism-pulsed-field gel electrophoresis (PFGE). Subsequent ribotyping of C. perfringens distinguished four different groups. The same ribopattern was detected in a minced beef heart dish, in autopsy material from the two deceased patients, and additionally in stool samples from six further residents who had fallen ill with diarrhea. Three further ribopatterns from food and autopsy materials could be differentiated. As chromosomal macrorestriction with subsequent PFGE is generally regarded more useful than ribotyping for molecular strain analysis, four selected isolates were lysed in parallel with a standard protocol and two nucleases inhibiting modifications. Neither of these methods could differentiate all of the isolates. These results suggest that PFGE with the current standard protocols is not able to characterize all C. perfringens isolates from food-borne disease investigations and that ribotyping is still a helpful method for molecular identification of clonal relationships. PMID:12574310

Laser confocal microscope for analysis of 3013 inner container closure weld region

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

Martinez-Rodriguez, M. J.

As part of the protocol to investigate the corrosion in the inner container closure weld region (ICCWR) a laser confocal microscope (LCM) was used to perform close visual examination of the surface and measurements of corrosion features on the surface. However, initial analysis of selected destructively evaluated (DE) containers using the LCM revealed several challenges for acquiring, processing and interpreting the data. These challenges include topography of the ICCWR sample, surface features, and the amount of surface area for collecting data at high magnification conditions. In FY17, the LCM parameters were investigated to identify the appropriate parameter values for datamore » acquisition and identification of regions of interest. Using these parameter values, selected DE containers were analyzed to determine the extent of the ICCWR to be examined.« less

Conducting field studies for testing pesticide leaching models

USGS Publications Warehouse

Smith, Charles N.; Parrish, Rudolph S.; Brown, David S.

1990-01-01

A variety of predictive models are being applied to evaluate the transport and transformation of pesticides in the environment. These include well known models such as the Pesticide Root Zone Model (PRZM), the Risk of Unsaturated-Saturated Transport and Transformation Interactions for Chemical Concentrations Model (RUSTIC) and the Groundwater Loading Effects of Agricultural Management Systems Model (GLEAMS). The potentially large impacts of using these models as tools for developing pesticide management strategies and regulatory decisions necessitates development of sound model validation protocols. This paper offers guidance on many of the theoretical and practical problems encountered in the design and implementation of field-scale model validation studies. Recommendations are provided for site selection and characterization, test compound selection, data needs, measurement techniques, statistical design considerations and sampling techniques. A strategy is provided for quantitatively testing models using field measurements.

Microbial Groundwater Sampling Protocol for Fecal-Rich Environments

PubMed Central

Harter, Thomas; Watanabe, Naoko; Li, Xunde; Atwill, Edward R; Samuels, William

2014-01-01

Inherently, confined animal farming operations (CAFOs) and other intense fecal-rich environments are potential sources of groundwater contamination by enteric pathogens. The ubiquity of microbial matter poses unique technical challenges in addition to economic constraints when sampling wells in such environments. In this paper, we evaluate a groundwater sampling protocol that relies on extended purging with a portable submersible stainless steel pump and Teflon® tubing as an alternative to equipment sterilization. The protocol allows for collecting a large number of samples quickly, relatively inexpensively, and under field conditions with limited access to capacity for sterilizing equipment. The protocol is tested on CAFO monitoring wells and considers three cross-contamination sources: equipment, wellbore, and ambient air. For the assessment, we use Enterococcus, a ubiquitous fecal indicator bacterium (FIB), in laboratory and field tests with spiked and blank samples, and in an extensive, multi-year field sampling campaign on 17 wells within 2 CAFOs. The assessment shows that extended purging can successfully control for equipment cross-contamination, but also controls for significant contamination of the well-head, within the well casing and within the immediate aquifer vicinity of the well-screen. Importantly, our tests further indicate that Enterococcus is frequently entrained in water samples when exposed to ambient air at a CAFO during sample collection. Wellbore and air contamination pose separate challenges in the design of groundwater monitoring strategies on CAFOs that are not addressed by equipment sterilization, but require adequate QA/QC procedures and can be addressed by the proposed sampling strategy. PMID:24903186

Development of Protocol Materials in Teacher Education: A Case Study in Relating Theory and Practice.

ERIC Educational Resources Information Center

University of South Florida, Tampa.

A protocol materials program was developed to (1) train personnel who produce, use, and make budgetary decisions about protocol materials and (2) produce and disseminate effective materials and knowledge acquired as a result of the program. Two groups were selected to meet the goals of the program--one group consisting of project directors who…

A General Self-Organized Tree-Based Energy-Balance Routing Protocol for Wireless Sensor Network

NASA Astrophysics Data System (ADS)

Han, Zhao; Wu, Jie; Zhang, Jie; Liu, Liefeng; Tian, Kaiyun

2014-04-01

Wireless sensor network (WSN) is a system composed of a large number of low-cost micro-sensors. This network is used to collect and send various kinds of messages to a base station (BS). WSN consists of low-cost nodes with limited battery power, and the battery replacement is not easy for WSN with thousands of physically embedded nodes, which means energy efficient routing protocol should be employed to offer a long-life work time. To achieve the aim, we need not only to minimize total energy consumption but also to balance WSN load. Researchers have proposed many protocols such as LEACH, HEED, PEGASIS, TBC and PEDAP. In this paper, we propose a General Self-Organized Tree-Based Energy-Balance routing protocol (GSTEB) which builds a routing tree using a process where, for each round, BS assigns a root node and broadcasts this selection to all sensor nodes. Subsequently, each node selects its parent by considering only itself and its neighbors' information, thus making GSTEB a dynamic protocol. Simulation results show that GSTEB has a better performance than other protocols in balancing energy consumption, thus prolonging the lifetime of WSN.

Continuous-variable measurement-device-independent quantum key distribution with virtual photon subtraction

NASA Astrophysics Data System (ADS)

Zhao, Yijia; Zhang, Yichen; Xu, Bingjie; Yu, Song; Guo, Hong

2018-04-01

The method of improving the performance of continuous-variable quantum key distribution protocols by postselection has been recently proposed and verified. In continuous-variable measurement-device-independent quantum key distribution (CV-MDI QKD) protocols, the measurement results are obtained from untrusted third party Charlie. There is still not an effective method of improving CV-MDI QKD by the postselection with untrusted measurement. We propose a method to improve the performance of coherent-state CV-MDI QKD protocol by virtual photon subtraction via non-Gaussian postselection. The non-Gaussian postselection of transmitted data is equivalent to an ideal photon subtraction on the two-mode squeezed vacuum state, which is favorable to enhance the performance of CV-MDI QKD. In CV-MDI QKD protocol with non-Gaussian postselection, two users select their own data independently. We demonstrate that the optimal performance of the renovated CV-MDI QKD protocol is obtained with the transmitted data only selected by Alice. By setting appropriate parameters of the virtual photon subtraction, the secret key rate and tolerable excess noise are both improved at long transmission distance. The method provides an effective optimization scheme for the application of CV-MDI QKD protocols.

Dual phylogenetic staining protocol for simultaneous analysis of yeast and bacteria in artworks

NASA Astrophysics Data System (ADS)

González-Pérez, Marina; Brinco, Catarina; Vieira, Ricardo; Rosado, Tânia; Mauran, Guilhem; Pereira, António; Candeias, António; Caldeira, Ana Teresa

2017-02-01

The detection and analysis of metabolically active microorganisms are useful to determine those directly involved in the biodeterioration of cultural heritage (CH). Fluorescence in situ hybridization with oligonucleotide probes targeted at rRNA (RNA-FISH) has demonstrated to be a powerful tool for signaling them. However, more efforts are required for the technique to become a vital tool for the analysis of CH's microbiological communities. Simultaneous analysis of microorganisms belonging to different kingdoms, by RNA-FISH in-suspension approach, could represent an important progress: it could open the door for the future use of the technique to analyze the microbial communities by flow cytometry, which has shown to be a potent tool in environmental microbiology. Thus, in this work, various already implemented in-suspension RNA-FISH protocols for ex situ analysis of yeast and bacteria were investigated and adapted for allowing the simultaneous detection of these types of microorganisms. A deep investigation of the factors that can affect the results was carried out, focusing particular attention on the selection of the fluorochromes used for labelling the probe set. The resultant protocol, involving the use of EUK516-6-FAM/EUB338-Cy3 probes combination, was validated using artificial consortia and gave positive preliminary results when applied in samples from a real case study: the Paleolithic archaeological site of Escoural Cave (Alentejo, Portugal). This approach represents the first dual-staining RNA-FISH in-suspension protocol developed and applied for the simultaneous investigation of CH biodeteriogenic agents belonging to different kingdoms.

A novel method of genomic DNA extraction for Cactaceae1

PubMed Central

Fehlberg, Shannon D.; Allen, Jessica M.; Church, Kathleen

2013-01-01

• Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. • Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. • Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies. PMID:25202521

The Tehran Eye Study: research design and eye examination protocol

PubMed Central

Hashemi, Hassan; Fotouhi, Akbar; Mohammad, Kazem

2003-01-01

Background Visual impairment has a profound impact on society. The majority of visually impaired people live in developing countries, and since most disorders leading to visual impairment are preventable or curable, their control is a priority in these countries. Considering the complicated epidemiology of visual impairment and the wide variety of factors involved, region specific intervention strategies are required for every community. Therefore, providing appropriate data is one of the first steps in these communities, as it is in Iran. The objectives of this study are to describe the prevalence and causes of visual impairment in the population of Tehran city; the prevalence of refractive errors, lens opacity, ocular hypertension, and color blindness in this population, and also the familial aggregation of refractive errors, lens opacity, ocular hypertension, and color blindness within the study sample. Methods Design Through a population-based, cross-sectional study, a total of 5300 Tehran citizens will be selected from 160 clusters using a stratified cluster random sampling strategy. The eligible people will be enumerated through a door-to-door household survey in the selected clusters and will be invited. All participants will be transferred to a clinic for measurements of uncorrected, best corrected and presenting visual acuity; manifest, subjective and cycloplegic refraction; color vision test; Goldmann applanation tonometry; examination of the external eye, anterior segment, media, and fundus; and an interview about demographic characteristics and history of eye diseases, eye trauma, diabetes mellitus, high blood pressure, and ophthalmologic cares. The study design and eye examination protocol are described. Conclusion We expect that findings from the TES will show the status of visual problems and their causes in the community. This study can highlight the people who should be targeted by visual impairment prevention programs. PMID:12859794

The Tehran Eye Study: research design and eye examination protocol.

PubMed

Hashemi, Hassan; Fotouhi, Akbar; Mohammad, Kazem

2003-07-15

Visual impairment has a profound impact on society. The majority of visually impaired people live in developing countries, and since most disorders leading to visual impairment are preventable or curable, their control is a priority in these countries. Considering the complicated epidemiology of visual impairment and the wide variety of factors involved, region specific intervention strategies are required for every community. Therefore, providing appropriate data is one of the first steps in these communities, as it is in Iran. The objectives of this study are to describe the prevalence and causes of visual impairment in the population of Tehran city; the prevalence of refractive errors, lens opacity, ocular hypertension, and color blindness in this population, and also the familial aggregation of refractive errors, lens opacity, ocular hypertension, and color blindness within the study sample. Through a population-based, cross-sectional study, a total of 5300 Tehran citizens will be selected from 160 clusters using a stratified cluster random sampling strategy. The eligible people will be enumerated through a door-to-door household survey in the selected clusters and will be invited. All participants will be transferred to a clinic for measurements of uncorrected, best corrected and presenting visual acuity; manifest, subjective and cycloplegic refraction; color vision test; Goldmann applanation tonometry; examination of the external eye, anterior segment, media, and fundus; and an interview about demographic characteristics and history of eye diseases, eye trauma, diabetes mellitus, high blood pressure, and ophthalmologic cares. The study design and eye examination protocol are described. We expect that findings from the TES will show the status of visual problems and their causes in the community. This study can highlight the people who should be targeted by visual impairment prevention programs.

Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples

PubMed Central

Kopek, Benjamin G.; Paez-Segala, Maria G.; Shtengel, Gleb; Sochacki, Kem A.; Sun, Mei G.; Wang, Yalin; Xu, C. Shan; van Engelenburg, Schuyler B.; Taraska, Justin W.; Looger, Loren L.; Hess, Harald F.

2017-01-01

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM datasets on aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. Choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replica creates high-contrast, 3-dimensional images of the cytoplasmic surface of the plasma membrane, but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples, but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (~10–50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2–7 days, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology. PMID:28384138

A rapid and efficient DNA extraction protocol from fresh and frozen human blood samples.

PubMed

Guha, Pokhraj; Das, Avishek; Dutta, Somit; Chaudhuri, Tapas Kumar

2018-01-01

Different methods available for extraction of human genomic DNA suffer from one or more drawbacks including low yield, compromised quality, cost, time consumption, use of toxic organic solvents, and many more. Herein, we aimed to develop a method to extract DNA from 500 μL of fresh or frozen human blood. Five hundred microliters of fresh and frozen human blood samples were used for standardization of the extraction procedure. Absorbance at 260 and 280 nm, respectively, (A 260 /A 280 ) were estimated to check the quality and quantity of the extracted DNA sample. Qualitative assessment of the extracted DNA was checked by Polymerase Chain reaction and double digestion of the DNA sample. Our protocol resulted in average yield of 22±2.97 μg and 20.5±3.97 μg from 500 μL of fresh and frozen blood, respectively, which were comparable to many reference protocols and kits. Besides yielding bulk amount of DNA, our protocol is rapid, economical, and avoids toxic organic solvents such as Phenol. Due to unaffected quality, the DNA is suitable for downstream applications. The protocol may also be useful for pursuing basic molecular researches in laboratories having limited funds. © 2017 Wiley Periodicals, Inc.

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA.

PubMed

Miya, Masaki; Minamoto, Toshifumi; Yamanaka, Hiroki; Oka, Shin-Ichiro; Sato, Keiichi; Yamamoto, Satoshi; Sado, Tetsuya; Doi, Hideyuki

2016-11-25

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes ≤4 L or >4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m 3 ) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products.

PubMed

Ferrarini, Alberto; Forcato, Claudio; Buson, Genny; Tononi, Paola; Del Monaco, Valentina; Terracciano, Mario; Bolognesi, Chiara; Fontana, Francesca; Medoro, Gianni; Neves, Rui; Möhlendick, Birte; Rihawi, Karim; Ardizzoni, Andrea; Sumanasuriya, Semini; Flohr, Penny; Lambros, Maryou; de Bono, Johann; Stoecklein, Nikolas H; Manaresi, Nicolò

2018-01-01

Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the Ampli1™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.

Assessment protocols of maximum oxygen consumption in young people with Down syndrome--a review.

PubMed

Seron, Bruna Barboza; Greguol, Márcia

2014-03-01

Maximum oxygen consumption is considered the gold standard measure of cardiorespiratory fitness. Young people with Down syndrome (DS) present low values of this indicator compared to their peers without disabilities and to young people with an intellectual disability but without DS. The use of reliable and valid assessment methods provides more reliable results for the diagnosis of cardiorespiratory fitness and the response of this variable to exercise. The aim of the present study was to review the literature on the assessment protocols used to measure maximum oxygen consumption in children and adolescents with Down syndrome giving emphasis to the protocols used, the validation process and their feasibility. The search was carried out in eight electronic databases--Scopus, Medline-Pubmed, Web of science, SportDiscus, Cinhal, Academic Search Premier, Scielo, and Lilacs. The inclusion criteria were: (a) articles which assessed VO2peak and/or VO2max (independent of the validation method), (b) samples composed of children and/or adolescents with Down syndrome, (c) participants of up to 20 years old, and (d) studies performed after 1990. Fifteen studies were selected and, of these, 11 measured the VO2peak using tests performed in a laboratory, 2 used field tests and the remaining 2 used both laboratory and field tests. The majority of the selected studies used maximal tests and conducted familiarization sessions. All the studies took into account the clinical conditions that could hamper testing or endanger the individuals. However, a large number of studies used tests which had not been specifically validated for the evaluated population. Finally, the search emphasized the small number of studies which use field tests to evaluate oxygen consumption. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Unified Fault-Tolerance Protocol

NASA Technical Reports Server (NTRS)

Miner, Paul; Gedser, Alfons; Pike, Lee; Maddalon, Jeffrey

2004-01-01

Davies and Wakerly show that Byzantine fault tolerance can be achieved by a cascade of broadcasts and middle value select functions. We present an extension of the Davies and Wakerly protocol, the unified protocol, and its proof of correctness. We prove that it satisfies validity and agreement properties for communication of exact values. We then introduce bounded communication error into the model. Inexact communication is inherent for clock synchronization protocols. We prove that validity and agreement properties hold for inexact communication, and that exact communication is a special case. As a running example, we illustrate the unified protocol using the SPIDER family of fault-tolerant architectures. In particular we demonstrate that the SPIDER interactive consistency, distributed diagnosis, and clock synchronization protocols are instances of the unified protocol.

The DNA isolation method has effect on allele drop-out and on the results of fluorescent PCR and DNA fragment analysis.

PubMed

Nagy, Bálint; Bán, Zoltán; Papp, Zoltán

2005-10-01

The quality and the quantity of isolated DNA have an effect on PCR amplifications. The authors studied three DNA isolation protocols (resin binding method using fresh and frozen amniotic fluid samples, and silica adsorption method using fresh samples) on the quantity and on the quality of the isolated DNA. Amniotic fluid samples were obtained from 20 pregnant women. The isolated DNA concentrations were determined by real-time fluorimeter using SYBRGreen I method. Each sample was studied for the presence of 8 STR markers. The authors compared the number of the detected alleles, electrophoretograms and peak areas. There was a significant difference between the concentration of the obtained DNA and in the peak areas between the three isolation protocols. The numbers of detected alleles were different, we observed the most allele drop outs in the resin type DNA isolation protocol from the fresh sample (detected allele numbers 182), followed by resin binding protocol from the frozen samples (detected allele number 243) and by the silica adsorption method (detected allele number 264). The authors demonstrated that the DNA isolation method has an effect on the quantity and quality of the isolated DNA, and on further PCR amplifications.

A communication protocol for mobile satellite systems affected by rain attenuation

NASA Technical Reports Server (NTRS)

Lay, Norman; Dessouky, Khaled

1992-01-01

A communication protocol is described that has been developed as part of a K/Ka-band mobile terminal breadboard system to be demonstrated through NASA's Advanced Communications Technology Satellite (ACTS) in 1993. The protocol is aimed at providing the means for enhancing link availability and continuity by supporting real-time data rate selection and changes during rain events. Particular attention is given to the system architecture; types of links, connections, and packets; the protocol procedures; and design rationales.

Microextraction by packed sorbent: an emerging, selective and high-throughput extraction technique in bioanalysis.

PubMed

Pereira, Jorge; Câmara, José S; Colmsjö, Anders; Abdel-Rehim, Mohamed

2014-06-01

Sample preparation is an important analytical step regarding the isolation and concentration of desired components from complex matrices and greatly influences their reliable and accurate analysis and data quality. It is the most labor-intensive and error-prone process in analytical methodology and, therefore, may influence the analytical performance of the target analytes quantification. Many conventional sample preparation methods are relatively complicated, involving time-consuming procedures and requiring large volumes of organic solvents. Recent trends in sample preparation include miniaturization, automation, high-throughput performance, on-line coupling with analytical instruments and low-cost operation through extremely low volume or no solvent consumption. Micro-extraction techniques, such as micro-extraction by packed sorbent (MEPS), have these advantages over the traditional techniques. This paper gives an overview of MEPS technique, including the role of sample preparation in bioanalysis, the MEPS description namely MEPS formats (on- and off-line), sorbents, experimental and protocols, factors that affect the MEPS performance, and the major advantages and limitations of MEPS compared with other sample preparation techniques. We also summarize MEPS recent applications in bioanalysis. Copyright © 2014 John Wiley & Sons, Ltd.

Crystallization of Macromolecules

PubMed Central

Friedmann, David; Messick, Troy; Marmorstein, Ronen

2014-01-01

X-ray crystallography has evolved into a very powerful tool to determine the three-dimensional structure of macromolecules and macromolecular complexes. The major bottleneck in structure determination by X-ray crystallography is the preparation of suitable crystalline samples. This unit outlines steps for the crystallization of a macromolecule, starting with a purified, homogeneous sample. The first protocols describe preparation of the macromolecular sample (i.e., proteins, nucleic acids, and macromolecular complexes). The preparation and assessment of crystallization trials is then described, along with a protocol for confirming whether the crystals obtained are composed of macromolecule as opposed to a crystallization reagent . Next, the optimization of crystallization conditions is presented. Finally, protocols that facilitate the growth of larger crystals through seeding are described. PMID:22045560

New potentiometric sensor based on molecularly imprinted nanoparticles for cocaine detection.

PubMed

Smolinska-Kempisty, K; Ahmad, O Sheej; Guerreiro, A; Karim, K; Piletska, E; Piletsky, S

2017-10-15

Here we present a potentiometric sensor for cocaine detection based on molecularly imprinted polymer nanoparticles (nanoMIPs) produced by the solid-phase imprinting method. The composition of polymers with high affinity for cocaine was optimised using molecular modelling. Four compositions were selected and polymers prepared using two protocols: chemical polymerisation in water and UV-initiated polymerisation in organic solvent. All synthesised nanoparticles had very good affinity to cocaine with dissociation constants between 0.6nM and 5.3nM. Imprinted polymers produced in organic solvent using acrylamide as a functional monomer demonstrated the highest yield and affinity, and so were selected for further sensor development. For this, nanoparticles were incorporated within a PVC matrix which was then used to prepare an ion-selective membrane integrated with a potentiometric transducer. It was demonstrated that the sensor was able to quantify cocaine in blood serum samples in the range of concentrations between 1nM and 1mM. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of Standardized Material Testing Protocols for Prosthetic Liners

PubMed Central

Cagle, John C.; Reinhall, Per G.; Hafner, Brian J.; Sanders, Joan E.

2017-01-01

A set of protocols was created to characterize prosthetic liners across six clinically relevant material properties. Properties included compressive elasticity, shear elasticity, tensile elasticity, volumetric elasticity, coefficient of friction (CoF), and thermal conductivity. Eighteen prosthetic liners representing the diverse range of commercial products were evaluated to create test procedures that maximized repeatability, minimized error, and provided clinically meaningful results. Shear and tensile elasticity test designs were augmented with finite element analysis (FEA) to optimize specimen geometries. Results showed that because of the wide range of available liner products, the compressive elasticity and tensile elasticity tests required two test maxima; samples were tested until they met either a strain-based or a stress-based maximum, whichever was reached first. The shear and tensile elasticity tests required that no cyclic conditioning be conducted because of limited endurance of the mounting adhesive with some liner materials. The coefficient of friction test was based on dynamic coefficient of friction, as it proved to be a more reliable measurement than static coefficient of friction. The volumetric elasticity test required that air be released beneath samples in the test chamber before testing. The thermal conductivity test best reflected the clinical environment when thermal grease was omitted and when liner samples were placed under pressure consistent with load bearing conditions. The developed procedures provide a standardized approach for evaluating liner products in the prosthetics industry. Test results can be used to improve clinical selection of liners for individual patients and guide development of new liner products. PMID:28233885

Development of an efficient real-time quantitative PCR protocol for detection of Xanthomonas arboricola pv. pruni in Prunus species.

PubMed

Palacio-Bielsa, Ana; Cubero, Jaime; Cambra, Miguel A; Collados, Raquel; Berruete, Isabel M; López, María M

2011-01-01

Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as being X. arboricola pv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system in X. arboricola pv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 10(2) CFU ml(-1), thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed for X. arboricola pv. pruni strains from different origins as well as for closely related Xanthomonas species, non-Xanthomonas species, saprophytic bacteria, and healthy Prunus samples. The efficiency of the developed protocol was evaluated with field samples of 14 Prunus species and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, and X. arboricola pv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test for X. arboricola pv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.

Consensus for second-order multi-agent systems with position sampled data

NASA Astrophysics Data System (ADS)

Wang, Rusheng; Gao, Lixin; Chen, Wenhai; Dai, Dameng

2016-10-01

In this paper, the consensus problem with position sampled data for second-order multi-agent systems is investigated. The interaction topology among the agents is depicted by a directed graph. The full-order and reduced-order observers with position sampled data are proposed, by which two kinds of sampled data-based consensus protocols are constructed. With the provided sampled protocols, the consensus convergence analysis of a continuous-time multi-agent system is equivalently transformed into that of a discrete-time system. Then, by using matrix theory and a sampled control analysis method, some sufficient and necessary consensus conditions based on the coupling parameters, spectrum of the Laplacian matrix and sampling period are obtained. While the sampling period tends to zero, our established necessary and sufficient conditions are degenerated to the continuous-time protocol case, which are consistent with the existing result for the continuous-time case. Finally, the effectiveness of our established results is illustrated by a simple simulation example. Project supported by the Natural Science Foundation of Zhejiang Province, China (Grant No. LY13F030005) and the National Natural Science Foundation of China (Grant No. 61501331).

Development of an HPV Educational Protocol for Adolescents

PubMed Central

Wetzel, Caitlin; Tissot, Abbigail; Kollar, Linda M.; Hillard, Paula A.; Stone, Rachel; Kahn, Jessica A.

2007-01-01

Study Objectives To develop an educational protocol about HPV and Pap tests for adolescents, to evaluate the protocol for understandability and clarity, and to evaluate the protocol for its effectiveness in increasing knowledge about HPV. Design In phase 1, investigators and adolescents developed the protocol. In phase 2, adolescents evaluated the protocol qualitatively, investigators evaluated its effectiveness in increasing HPV knowledge in a sample of adolescents, and the protocol was revised. In phase 3, investigators evaluated the effectiveness of the revised protocol in an additional adolescent sample. Setting Urban, hospital-based teen health center. Participants A total of 252 adolescent girls and boys in the three study phases. Main Outcome Measures Pre- and post-protocol knowledge about HPV, measured using a 10- or 11-item scale. Results Scores on the HPV knowledge scale increased significantly (p<.0001) among adolescents who participated in phases 2 and 3 after they received the protocol. Initial differences in scores based on race, insurance type and condom use were not noted post-protocol. Conclusion The protocol significantly increased knowledge scores about HPV in this population, regardless of sociodemographic characteristics and risk behaviors. Effective, developmentally appropriate educational protocols about HPV and Pap tests are particularly important in clinical settings as cervical cancer screening guidelines evolve, HPV DNA testing is integrated into screening protocols, and HPV vaccines become available. In-depth, one-on-one education about HPV may also prevent adverse psychosocial responses and promote healthy sexual and Pap screening behaviors in adolescents with abnormal HPV or Pap test results. Synopsis The investigators developed an educational protocol about HPV and Pap tests and evaluated its effectiveness in increasing knowledge about HPV among adolescents. PMID:17868894

Rapid determination of anti-estrogens by gas chromatography/mass spectrometry in urine: Method validation and application to real samples.

PubMed

Gerace, E; Salomone, A; Abbadessa, G; Racca, S; Vincenti, M

2012-02-01

A fast screening protocol was developed for the simultaneous determination of nine anti-estrogenic agents (aminoglutethimide, anastrozole, clomiphene, drostanolone, formestane, letrozole, mesterolone, tamoxifen, testolactone) plus five of their metabolites in human urine. After an enzymatic hydrolysis, these compounds can be extracted simultaneously from urine with a simple liquid-liquid extraction at alkaline conditions. The analytes were subsequently analyzed by fast-gas chromatography/mass spectrometry (fast-GC/MS) after derivatization. The use of a short column, high-flow carrier gas velocity and fast temperature ramping produced an efficient separation of all analytes in about 4 min, allowing a processing rate of 10 samples/h. The present analytical method was validated according to UNI EN ISO/IEC 17025 guidelines for qualitative methods. The range of investigated parameters included the limit of detection, selectivity, linearity, repeatability, robustness and extraction efficiency. High MS-sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. Therefore, the performances of the method are comparable to the ones obtainable from traditional GC/MS analysis. The method was successfully tested on real samples arising from clinical treatments of hospitalized patients and could profitably be used for clinical studies on anti-estrogenic drug administration.

Rapid determination of anti-estrogens by gas chromatography/mass spectrometry in urine: Method validation and application to real samples

PubMed Central

Gerace, E.; Salomone, A.; Abbadessa, G.; Racca, S.; Vincenti, M.

2011-01-01

A fast screening protocol was developed for the simultaneous determination of nine anti-estrogenic agents (aminoglutethimide, anastrozole, clomiphene, drostanolone, formestane, letrozole, mesterolone, tamoxifen, testolactone) plus five of their metabolites in human urine. After an enzymatic hydrolysis, these compounds can be extracted simultaneously from urine with a simple liquid–liquid extraction at alkaline conditions. The analytes were subsequently analyzed by fast-gas chromatography/mass spectrometry (fast-GC/MS) after derivatization. The use of a short column, high-flow carrier gas velocity and fast temperature ramping produced an efficient separation of all analytes in about 4 min, allowing a processing rate of 10 samples/h. The present analytical method was validated according to UNI EN ISO/IEC 17025 guidelines for qualitative methods. The range of investigated parameters included the limit of detection, selectivity, linearity, repeatability, robustness and extraction efficiency. High MS-sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. Therefore, the performances of the method are comparable to the ones obtainable from traditional GC/MS analysis. The method was successfully tested on real samples arising from clinical treatments of hospitalized patients and could profitably be used for clinical studies on anti-estrogenic drug administration. PMID:29403714

Optimization of a sample processing protocol for recovery of Bacillus anthracis spores from soil

USGS Publications Warehouse

Silvestri, Erin E.; Feldhake, David; Griffin, Dale; Lisle, John T.; Nichols, Tonya L.; Shah, Sanjiv; Pemberton, A; Schaefer III, Frank W

2016-01-01

Following a release of Bacillus anthracis spores into the environment, there is a potential for lasting environmental contamination in soils. There is a need for detection protocols for B. anthracis in environmental matrices. However, identification of B. anthracis within a soil is a difficult task. Processing soil samples helps to remove debris, chemical components, and biological impurities that can interfere with microbiological detection. This study aimed to optimize a previously used indirect processing protocol, which included a series of washing and centrifugation steps. Optimization of the protocol included: identifying an ideal extraction diluent, variation in the number of wash steps, variation in the initial centrifugation speed, sonication and shaking mechanisms. The optimized protocol was demonstrated at two laboratories in order to evaluate the recovery of spores from loamy and sandy soils. The new protocol demonstrated an improved limit of detection for loamy and sandy soils over the non-optimized protocol with an approximate matrix limit of detection at 14 spores/g of soil. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol will be robust enough to use at multiple laboratories while achieving comparable recoveries.

Update of Ireland's national average indoor radon concentration - Application of a new survey protocol.

PubMed

Dowdall, A; Murphy, P; Pollard, D; Fenton, D

2017-04-01

In 2002, a National Radon Survey (NRS) in Ireland established that the geographically weighted national average indoor radon concentration was 89 Bq m -3 . Since then a number of developments have taken place which are likely to have impacted on the national average radon level. Key among these was the introduction of amending Building Regulations in 1998 requiring radon preventive measures in new buildings in High Radon Areas (HRAs). In 2014, the Irish Government adopted the National Radon Control Strategy (NRCS) for Ireland. A knowledge gap identified in the NRCS was to update the national average for Ireland given the developments since 2002. The updated national average would also be used as a baseline metric to assess the effectiveness of the NRCS over time. A new national survey protocol was required that would measure radon in a sample of homes representative of radon risk and geographical location. The design of the survey protocol took into account that it is not feasible to repeat the 11,319 measurements carried out for the 2002 NRS due to time and resource constraints. However, the existence of that comprehensive survey allowed for a new protocol to be developed, involving measurements carried out in unbiased randomly selected volunteer homes. This paper sets out the development and application of that survey protocol. The results of the 2015 survey showed that the current national average indoor radon concentration for homes in Ireland is 77 Bq m -3 , a decrease from the 89 Bq m -3 reported in the 2002 NRS. Analysis of the results by build date demonstrate that the introduction of the amending Building Regulations in 1998 have led to a reduction in the average indoor radon level in Ireland. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel quantitative real-time LCR for the sensitive detection of SNP frequencies in pooled DNA: method development, evaluation and application.

PubMed

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-19

Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food.

Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

PubMed Central

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-01

Background Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. Methods The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. Conclusions The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. Significance The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food. PMID:21283808

Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa.

PubMed

Nicolas, M; Alvarez, M; Borragán, S; Martinez-Pastor, F; Chamorro, C A; Alvarez-Rodriguez, M; de Paz, P; Anel, L

2012-04-01

Centrifugation is a crucial procedure in sperm cryopreservation protocols of brown bear (Ursus arctos), because the semen must be processed to increase sperm concentration and/or clean urine-contaminated samples. The efficacy of three media for centrifugation (Maxifreeze [IMV technologies, L'Aigle, France], Cushion Fluid Equine (Minitübe, Tiefenbach, Germany), and PureSperm [Nidacon, Gothenburg, Sweden]) on the quality of bear spermatozoa was evaluated. In experiment one, two cushioned media used for protecting against mechanical stress during centrifugation were analyzed. In experiment two, a density gradient based on PureSperm was assessed in relation to the maximum retrieval and the quality of fresh spermatozoa, and the freezability of the spermatozoa selected in this density gradient was studied in experiment three. Finally, the selection of frozen-thawed sperm using PureSperm was analyzed in experiment four. Our results indicate that the use of dense isotonic cushion solutions (Maxifreeze, Cushion Fluid Equine) in centrifugation did not improve the quality of recovered spermatozoa compared with standard centrifugation. However, a density gradient prepared with PureSperm improved the quality of spermatozoa in fresh semen and frozen-thawed semen, but the spermatozoa selected from the fresh sample with this density gradient did not show a better resistance to freezing with this density gradient in comparison with the control sample. Copyright © 2012 Elsevier Inc. All rights reserved.

The expanding cognitive-behavioural therapy treatment umbrella for the anxiety disorders: disorder-specific and transdiagnostic approaches.

PubMed

Rector, Neil A; Man, Vincent; Lerman, Bethany

2014-06-01

Cognitive-behavioural therapy (CBT) is an empirically supported treatment for anxiety disorders. CBT treatments are based on disorder-specific protocols that have been developed to target individual anxiety disorders, despite that anxiety disorders frequently co-occur and are comorbid with depression. Given the high rates of diagnostic comorbidity, substantial overlap in dimensional symptom ratings, and extensive evidence that the mood and anxiety disorders share a common set of psychological and biological vulnerabilities, transdiagnostic CBT protocols have recently been developed to treat the commonalities among the mood and anxiety disorders. We conducted a selective review of empirical developments in the transdiagnostic CBT treatment of anxiety and depression (2008-2013). Preliminary evidence suggests that theoretically based transdiagnostic CBT approaches lead to large treatment effects on the primary anxiety disorder, considerable reduction of diagnostic comorbidity, and some preliminary effects regarding the impact on the putative, shared psychological mechanisms. However, the empirical literature remains tentative owing to relatively small samples, limited direct comparisons with disorder-specific CBT protocols, and the relative absence of the study of disorder-specific compared with shared mechanisms of action in treatment. We conclude with a treatment conceptualization of the new transdiagnostic interventions as complementary, rather than contradictory, to disorder-specific CBT.

Slow histidine H/D exchange protocol for thermodynamic analysis of protein folding and stability using mass spectrometry.

PubMed

Tran, Duc T; Banerjee, Sambuddha; Alayash, Abdu I; Crumbliss, Alvin L; Fitzgerald, Michael C

2012-02-07

Described here is a mass spectrometry-based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the chemical denaturant dependence of the slow H/D exchange reaction of the imidazole C(2) proton in histidine side chains. The protocol is developed using several model protein systems including: ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA) II, hemoglobin (Hb), and the hemoglobin-haptoglobin (Hb-Hp) protein complex. Folding free energies consistent with those previously determined by other more conventional techniques were obtained for the two-state folding proteins, Rnase A and myoglobin. The protocol successfully detected a previously observed partially unfolded intermediate stabilized in the BCA II folding/unfolding reaction, and it could be used to generate a K(d) value of 0.24 nM for the Hb-Hp complex. The compatibility of the protocol with conventional mass spectrometry-based proteomic sample preparation and analysis methods was also demonstrated in an experiment in which the protocol was used to detect the binding of zinc to superoxide dismutase in the yeast cell lysate sample. The yeast cell sample analyses also helped define the scope of the technique, which requires the presence of globally protected histidine residues in a protein's three-dimensional structure for successful application. © 2011 American Chemical Society

Comparison of two cooling protocols for llama semen: with and without collagenase and seminal plasma in the medium.

PubMed

Carretero, M I; Giuliano, S M; Arraztoa, C C; Santa Cruz, R C; Fumuso, F G; Neild, D M

2017-08-01

Seminal plasma (SP) of South American Camelids could interfere with the interaction of spermatozoa with the extenders; therefore it becomes necessary to improve semen management using enzymatic treatment. Our objective was to compare two cooling protocols for llama semen. Twelve ejaculates were incubated in 0.1% collagenase and then were divided into two aliquots. One was extended in lactose and egg yolk (LEY) (Protocol A: collagenase and SP present). The other aliquot was centrifuged, and the pellet was resuspended in LEY (Protocol B: collagenase and SP absent). Both samples were maintained at 5°C during 24 hr. Routine and DNA evaluations were carried out in raw and cooled semen. Both cooling protocols maintained sperm viability, membrane function and DNA fragmentation, with Protocol A showing a significantly lowered total and progressive motility (p < .05) and Protocol B showing a significant increase in chromatin decondensation (p < .05). Protocol A avoids centrifugation, reducing processing times and making application in the field simpler. However, as neither protocol showed a significant superiority over the other, studies should be carried out in vivo to evaluate the effect on pregnancy rates of the presence of collagenase and SP in semen samples prior to either cooling or freeze-thawing. © 2016 Blackwell Verlag GmbH.

Comparison of Cryopreservation Protocols (Single and Two-steps) and Thawing (Fast and Slow) for Canine Sperm.

PubMed

Brito, Maíra M; Lúcio, Cristina F; Angrimani, Daniel S R; Losano, João Diego A; Dalmazzo, Andressa; Nichi, Marcílio; Vannucchi, Camila I

2017-01-02

In addition to the existence of several cryopreservation protocols, no systematic research has been carried out in order to confirm the suitable protocol for canine sperm. This study aims to assess the effect of adding 5% glycerol during cryopreservation at 37°C (one-step) and 5°C (two-steps), in addition of testing two thawing protocols (37°C for 30 seconds, and 70°C for 8 seconds). We used 12 sperm samples divided into four experimental groups: Single-Step - Slow Thawing Group; Two-Step - Slow Thawing Group; Single-Step - Fast Thawing Group; and Two-Step - Fast Thawing Group. Frozen-thawed samples were submitted to automated analysis of sperm motility, evaluation of plasmatic membrane integrity, acrosomal integrity, mitochondrial activity, sperm morphology, sperm susceptibility to oxidative stress, and sperm binding assay to perivitellinic membrane of chicken egg yolk. Considering the comparison between freezing protocols, no statistical differences were verified for any of the response variables. When comparison between thawing protocols was performed, slow thawing protocol presented higher sperm count bound to perivitelline membrane of chicken egg yolk, compared to fast thawing protocol. Regardless of the freezing process, the slow thawing protocol can be recommended for the large scale cryopreservation of canine semen, since it shows a consistent better functional result.

Development of bull trout sampling protocols

Treesearch

R. F. Thurow; J. T. Peterson; J. W. Guzevich

2001-01-01

This report describes results of research conducted in Washington in 2000 through Interagency Agreement #134100H002 between the U.S. Fish and Wildlife Service (USFWS) and the U.S. Forest Service Rocky Mountain Research Station (RMRS). The purpose of this agreement is to develop a bull trout (Salvelinus confluentus) sampling protocol by integrating...

Comparison of PIXE and XRF analysis of airborne particulate matter samples collected on Teflon and quartz fibre filters

NASA Astrophysics Data System (ADS)

Chiari, M.; Yubero, E.; Calzolai, G.; Lucarelli, F.; Crespo, J.; Galindo, N.; Nicolás, J. F.; Giannoni, M.; Nava, S.

2018-02-01

Within the framework of research projects focusing on the sampling and analysis of airborne particulate matter, Particle Induced X-ray Emission (PIXE) and Energy Dispersive X-ray Fluorescence (ED-XRF) techniques are routinely used in many laboratories throughout the world to determine the elemental concentration of the particulate matter samples. In this work an inter-laboratory comparison of the results obtained from analysing several samples (collected on both Teflon and quartz fibre filters) using both techniques is presented. The samples were analysed by PIXE (in Florence, at the 3 MV Tandetron accelerator of INFN-LABEC laboratory) and by XRF (in Elche, using the ARL Quant'X EDXRF spectrometer with specific conditions optimized for specific groups of elements). The results from the two sets of measurements are in good agreement for all the analysed samples, thus validating the use of the ARL Quant'X EDXRF spectrometer and the selected measurement protocol for the analysis of aerosol samples. Moreover, thanks to the comparison of PIXE and XRF results on Teflon and quartz fibre filters, possible self-absorption effects due to the penetration of the aerosol particles inside the quartz fibre-filters were quantified.

Development of Gold Standard Ion-Selective Electrode-Based Methods for Fluoride Analysis

PubMed Central

Martínez-Mier, E.A.; Cury, J.A.; Heilman, J.R.; Katz, B.P.; Levy, S.M.; Li, Y.; Maguire, A.; Margineda, J.; O’Mullane, D.; Phantumvanit, P.; Soto-Rojas, A.E.; Stookey, G.K.; Villa, A.; Wefel, J.S.; Whelton, H.; Whitford, G.M.; Zero, D.T.; Zhang, W.; Zohouri, V.

2011-01-01

Background/Aims: Currently available techniques for fluoride analysis are not standardized. Therefore, this study was designed to develop standardized methods for analyzing fluoride in biological and nonbiological samples used for dental research. Methods A group of nine laboratories analyzed a set of standardized samples for fluoride concentration using their own methods. The group then reviewed existing analytical techniques for fluoride analysis, identified inconsistencies in the use of these techniques and conducted testing to resolve differences. Based on the results of the testing undertaken to define the best approaches for the analysis, the group developed recommendations for direct and microdiffusion methods using the fluoride ion-selective electrode. Results Initial results demonstrated that there was no consensus regarding the choice of analytical techniques for different types of samples. Although for several types of samples, the results of the fluoride analyses were similar among some laboratories, greater differences were observed for saliva, food and beverage samples. In spite of these initial differences, precise and true values of fluoride concentration, as well as smaller differences between laboratories, were obtained once the standardized methodologies were used. Intraclass correlation coefficients ranged from 0.90 to 0.93, for the analysis of a certified reference material, using the standardized methodologies. Conclusion The results of this study demonstrate that the development and use of standardized protocols for F analysis significantly decreased differences among laboratories and resulted in more precise and true values. PMID:21160184

Protocol for selecting ASR-affected structures for lithium treatment

DOT National Transportation Integrated Search

2006-02-01

This TechBrief describes a protocol for evaluating damaged concrete structures to determine whether they are suitable candidates for lithium treatment to address alkali-silica reactivity (ASR). A major part of the TechBrief's source document, Protoco...

Assessment of levels of bacterial contamination of large wild game meat in Europe.

PubMed

Membré, Jeanne-Marie; Laroche, Michel; Magras, Catherine

2011-08-01

The variations in prevalence and levels of pathogens and fecal contamination indicators in large wild game meat were studied to assess their potential impact on consumers. This analysis was based on hazard analysis, data generation and statistical analysis. A total of 2919 meat samples from three species (red deer, roe deer, wild boar) were collected at French game meat traders' facilities using two sampling protocols. Information was gathered on the types of meat cuts (forequarter or haunch; first sampling protocol) or type of retail-ready meat (stewing meat or roasting meat; second protocol), and also on the meat storage conditions (frozen or chilled), country of origin (eight countries) and shooting season (autumn, winter, spring). The samples were analyzed in both protocols for detection and enumeration of Escherichia coli, coagulase+staphylococci and Clostridium perfringens. In addition, detection and enumeration of thermotolerant coliforms and Listeria monocytogenes were performed for samples collected in the first and second protocols, respectively. The levels of bacterial contamination of the raw meat were determined by performing statistical analysis involving probabilistic techniques and Bayesian inference. C. perfringens was found in the highest numbers for the three indicators of microbial quality, hygiene and good handling, and L. monocytogenes in the lowest. Differences in contamination levels between game species and between meats distributed as chilled or frozen products were not significant. These results might be included in quantitative exposure assessments. Copyright © 2011 Elsevier Ltd. All rights reserved.

Demonstrating Patterns in the Views Of Stakeholders Regarding Ethically-Salient Issues in Clinical Research: A Novel Use of Graphical Models in Empirical Ethics Inquiry.

PubMed

Kim, Jane Paik; Roberts, Laura Weiss

Empirical ethics inquiry works from the notion that stakeholder perspectives are necessary for gauging the ethical acceptability of human studies and assuring that research aligns with societal expectations. Although common, studies involving different populations often entail comparisons of trends that problematize the interpretation of results. Using graphical model selection - a technique aimed at transcending limitations of conventional methods - this report presents data on the ethics of clinical research with two objectives: (1) to display the patterns of views held by ill and healthy individuals in clinical research as a test of the study's original hypothesis and (2) to introduce graphical model selection as a key analytic tool for ethics research. In this IRB-approved, NIH-funded project, data were collected from 60 mentally ill and 43 physically ill clinical research protocol volunteers, 47 healthy protocol-consented participants, and 29 healthy individuals without research protocol experience. Respondents were queried on the ethical acceptability of research involving people with mental and physical illness (i.e., cancer, HIV, depression, schizophrenia, and post-traumatic stress disorder) and non-illness related sources of vulnerability (e.g., age, class, gender, ethnicity). Using a statistical algorithm, we selected graphical models to display interrelationships among responses to questions. Both mentally and physically ill protocol volunteers revealed a high degree of connectivity among ethically-salient perspectives. Healthy participants, irrespective of research protocol experience, revealed patterns of views that were not highly connected. Between ill and healthy protocol participants, the pattern of views is vastly different. Experience with illness was tied to dense connectivity, whereas healthy individuals expressed views with sparse connections. In offering a nuanced perspective on the interrelation of ethically relevant responses, graphical model selection has the potential to bring new insights to the field of ethics.

An efficient and high-throughput protocol for Agrobacterium-mediated transformation based on phosphomannose isomerase positive selection in Japonica rice (Oryza sativa L.).

PubMed

Duan, Yongbo; Zhai, Chenguang; Li, Hao; Li, Juan; Mei, Wenqian; Gui, Huaping; Ni, Dahu; Song, Fengshun; Li, Li; Zhang, Wanggen; Yang, Jianbo

2012-09-01

A number of Agrobacterium-mediated rice transformation systems have been developed and widely used in numerous laboratories and research institutes. However, those systems generally employ antibiotics like kanamycin and hygromycin, or herbicide as selectable agents, and are used for the small-scale experiments. To address high-throughput production of transgenic rice plants via Agrobacterium-mediated transformation, and to eliminate public concern on antibiotic markers, we developed a comprehensive efficient protocol, covering from explant preparation to the acquisition of low copy events by real-time PCR analysis before transplant to field, for high-throughput production of transgenic plants of Japonica rice varieties Wanjing97 and Nipponbare using Escherichia coli phosphomannose isomerase gene (pmi) as a selectable marker. The transformation frequencies (TF) of Wanjing97 and Nipponbare were achieved as high as 54.8 and 47.5%, respectively, in one round of selection of 7.5 or 12.5 g/L mannose appended with 5 g/L sucrose. High-throughput transformation from inoculation to transplant of low copy events was accomplished within 55-60 days. Moreover, the Taqman assay data from a large number of transformants showed 45.2% in Wanjing97 and 31.5% in Nipponbare as a low copy rate, and the transformants are fertile and follow the Mendelian segregation ratio. This protocol facilitates us to perform genome-wide functional annotation of the open reading frames and utilization of the agronomically important genes in rice under a reduced public concern on selectable markers. We describe a comprehensive protocol for large scale production of transgenic Japonica rice plants using non-antibiotic selectable agent, at simplified, cost- and labor-saving manners.

[Primary care screening of problems in the elderly and a proposal for a screening protocol with a multidimensional approach].

PubMed

Lino, Valéria Teresa Saraiva; Portela, Margareth Crisóstomo; Camacho, Luiz Antonio Bastos; Rodrigues, Nadia Cristina Pinheiro; Andrade, Monica Kramer de Noronha; O'Dwyer, Gisele

2016-07-21

The objectives were to examine psychometric properties of a screening test for the elderly and to propose a protocol for use in primary care. The method consisted of four stages: (1) inter-evaluator reliability for performance tests and self-assessment questions for eight functions; (2) sensitivity and specificity of questions on depression and social support; (3) meeting of experts to select instrumental activities of daily living (IADL); and (4) elaboration of the protocol. Screening lasted 16 minutes. Inter-evaluator reliability was excellent for performance tests but poor for questions. Depression and social support showed satisfactory sensitivity and specificity (0.74/0.77 and 0.77/0.96). Four IADL were selected by more than 55% of the experts. Following the results, a screening protocol was elaborated that prioritized the use of performance tests, maintaining questions on mood, social support, and IADL. The study suggests better reproducibility of performance tests when compared to questions. For mood and social support, the questions may provide a first screening stage. The proposed protocol allows rapid screening of problems.

Determination of total procyanidins in selected chocolate and confectionery products using DMAC.

PubMed

Payne, Mark J; Hurst, William Jeffrey; Stuart, David A; Ou, Boxin; Fan, Ellen; Ji, Hongping; Kou, Yan

2010-01-01

A simple, specific, high-throughput colorimetric method based on the reaction of 4-dimethylaminocinnamaldehyde (DMAC) with flavan-3-ols was developed to determine total procyanidins in selected cacao-based products. Extracts of defatted samples were dispensed into a 96-well plate and reacted with DMAC. The absorbance of the reaction products was measured at 640 nm and compared to commercially available procyanidin B2 as a standard. The use of the 96-well plates and a plate reader dramatically improved sample throughput. A standard protocol was established and used for further studies. The calibration was found to be linear from 1-100 ppm. The DMAC reagent reacted relatively specifically to (-)-epicatechin, (+)-catechin, epigallocatechin, gallocatechin, the gallates of catechin, epicatechin, gallocatechin, and epigallocatechin, oligomeric procyanidins of cocoa up to n=4, and A-type procyanidins. Little or no reaction occurred with cyanidins and representative compounds of phenolic acids, flavones, flavanones, flavonols, anthocyanidins, isoflavones, and stilbenes. Sample precision studies were carried out on 10 different test materials over several weeks, and yielded RSD values of 4.0 to 9.5%. The method was ring-tested in three laboratories using blinded test materials including cocoa beans, cocoa powder, chocolate liquor, dark chocolate, and milk chocolate. There was excellent agreement of the results between laboratories.

Wastewater GHG Accounting Protocols as Compared to the State of GHG Science.

PubMed

Willis, John L; Yuan, Zhiguo; Murthy, Sudhir

2016-08-01

Greenhouse gas (GHG) accounting protocols have addressed emissions from wastewater conveyance and treatment using a variety of simplifying methodologies. While these methodologies vary to some degree by protocol, within each protocol they provide consistent tools for organizational entities of varying size and scope to report and verify GHG emissions. Much of the science supporting these methodologies is either limited or the protocols have failed to keep abreast of developing GHG research. This state-of-the-art review summarizes the sources of direct GHG emissions (both those covered and not covered in current protocols) from wastewater handling; provides a review of the wastewater-related methodologies in a select group of popular protocols; and discusses where research has out-paced protocol methodologies and other areas where the supporting science is relatively weak and warrants further exploration.

Water quality data for selected wells in the Coastal Plain of New Jersey, 1996-98

USGS Publications Warehouse

Hibbs, Kathleen L.; Stackelberg, Paul E.; Kauffman, Leon J.; Ayers, Mark A.

2001-01-01

Water-quality data were collected during 1996-98 for 217 wells in New Jersey and 3 wells in New York as part of the U. S. Geological Survey's National Water Quality Assessment Program. Samples were collected for five ground-water surveys that were designed to assess water quality in major aquifer systems, with an emphasis on recently recharged (shallow) ground water associated with present and recent human activities. This report (1) summarizes the hydrogeologic framework in the areas of data collection; (2) describes the objectives and procedures for designing each ground-water survey; (3) summarizes the procedures and protocols for data collec-tion, analysis, and quality control; and (4) lists the concentrations of inorganic constituents, volatile organic compounds, pesticides, nutrients, and trace elements present in the ground-water samples.

Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae.

PubMed

Klauser, Benedikt; Atanasov, Janina; Siewert, Lena K; Hartig, Jörg S

2015-05-15

Systems for conditional gene expression are powerful tools in basic research as well as in biotechnology. For future applications, it is of great importance to engineer orthogonal genetic switches that function reliably in diverse contexts. RNA-based switches have the advantage that effector molecules interact immediately with regulatory modules inserted into the target RNAs, getting rid of the need of transcription factors usually mediating genetic control. Artificial riboswitches are characterized by their simplicity and small size accompanied by a high degree of modularity. We have recently reported a series of hammerhead ribozyme-based artificial riboswitches that allow for post-transcriptional regulation of gene expression via switching mRNA, tRNA, or rRNA functions. A more widespread application was so far hampered by moderate switching performances and a limited set of effector molecules available. Here, we report the re-engineering of hammerhead ribozymes in order to respond efficiently to aminoglycoside antibiotics. We first established an in vivo selection protocol in Saccharomyces cerevisiae that enabled us to search large sequence spaces for optimized switches. We then envisioned and characterized a novel strategy of attaching the aptamer to the ribozyme catalytic core, increasing the design options for rendering the ribozyme ligand-dependent. These innovations enabled the development of neomycin-dependent RNA modules that switch gene expression up to 25-fold. The presented aminoglycoside-responsive riboswitches belong to the best-performing RNA-based genetic regulators reported so far. The developed in vivo selection protocol should allow for sampling of large sequence spaces for engineering of further optimized riboswitches.

Comparison of Sensorimotor Rhythm (SMR) and Beta Training on Selective Attention and Symptoms in Children with Attention Deficit/Hyperactivity Disorder (ADHD): A Trend Report.

PubMed

Mohammadi, Mohammad Reza; Malmir, Nastaran; Khaleghi, Ali; Aminiorani, Majd

2015-06-01

The aim of this study was to assess and compare the effect of two neurofeedback protocols (SMR/theta and beta/theta) on ADHD symptoms, selective attention and EEG (electroencephalogram) parameters in children with ADHD. The sample consisted of 16 children (9-15 year old: 13 boys; 3 girls) with ADHD-combined type (ADHD-C). All of children used methylphenidate (MPH) during the study. The neurofeedback training consisted of two phases of 15 sessions, each lasting 45 minutes. In the first phase, participants were trained to enhance sensorimotor rhythm (12-15 Hz) and reduce theta activity (4-8 Hz) at C4 and in the second phase; they had to increase beta (15-18 Hz) and reduce theta activity at C3. Assessments consisted of d2 attention endurance test, ADHD rating scale (parent form) at three time periods: before, middle and the end of the training. EEG signals were recorded just before and after the training. Based on parents' reports, inattention after beta/theta training, and hyperactivity/impulsivity were improved after the end of the training. All subscales of d2 test were improved except for the difference between maximum and minimum responses. However, EEG analysis showed no significant differences. Neurofeedback in conjunction with Methylphenidate may cause further improvement in ADHD symptoms reported by parents and selective attention without long-term impact on EEG patterns. However, determining the exact relationship between EEG parameters, neurofeedback protocols and ADHD symptoms remain unclear.

A Protocol for Collecting Human Cardiac Tissue for Research.

PubMed

Blair, Cheavar A; Haynes, Premi; Campbell, Stuart G; Chung, Charles; Mitov, Mihail I; Dennis, Donna; Bonnell, Mark R; Hoopes, Charles W; Guglin, Maya; Campbell, Kenneth S

2016-01-01

This manuscript describes a protocol at the University of Kentucky that allows a translational research team to collect human myocardium that can be used for biological research. We have gained a great deal of practical experience since we started this protocol in 2008, and we hope that other groups might be able to learn from our endeavors. To date, we have procured ~4000 samples from ~230 patients. The tissue that we collect comes from organ donors and from patients who are receiving a heart transplant or a ventricular assist device because they have heart failure. We begin our manuscript by describing the importance of human samples in cardiac research. Subsequently, we describe the process for obtaining consent from patients, the cost of running the protocol, and some of the issues and practical difficulties that we have encountered. We conclude with some suggestions for other researchers who may be considering starting a similar protocol.

A Protocol for Collecting Human Cardiac Tissue for Research

PubMed Central

Blair, Cheavar A.; Haynes, Premi; Campbell, Stuart G.; Chung, Charles; Mitov, Mihail I.; Dennis, Donna; Bonnell, Mark R.; Hoopes, Charles W.; Guglin, Maya; Campbell, Kenneth S.

2016-01-01

This manuscript describes a protocol at the University of Kentucky that allows a translational research team to collect human myocardium that can be used for biological research. We have gained a great deal of practical experience since we started this protocol in 2008, and we hope that other groups might be able to learn from our endeavors. To date, we have procured ~4000 samples from ~230 patients. The tissue that we collect comes from organ donors and from patients who are receiving a heart transplant or a ventricular assist device because they have heart failure. We begin our manuscript by describing the importance of human samples in cardiac research. Subsequently, we describe the process for obtaining consent from patients, the cost of running the protocol, and some of the issues and practical difficulties that we have encountered. We conclude with some suggestions for other researchers who may be considering starting a similar protocol. PMID:28042604

A Mobile Satellite Experiment (MSAT-X) network definition

NASA Technical Reports Server (NTRS)

Wang, Charles C.; Yan, Tsun-Yee

1990-01-01

The network architecture development of the Mobile Satellite Experiment (MSAT-X) project for the past few years is described. The results and findings of the network research activities carried out under the MSAT-X project are summarized. A framework is presented upon which the Mobile Satellite Systems (MSSs) operator can design a commercial network. A sample network configuration and its capability are also included under the projected scenario. The Communication Interconnection aspect of the MSAT-X network is discussed. In the MSAT-X network structure two basic protocols are presented: the channel access protocol, and the link connection protocol. The error-control techniques used in the MSAT-X project and the packet structure are also discussed. A description of two testbeds developed for experimentally simulating the channel access protocol and link control protocol, respectively, is presented. A sample network configuration and some future network activities of the MSAT-X project are also presented.

New archaeomagnetic data recovered from the study of celtiberic remains from central Spain (Numantia and Ciadueña, 3rd-1st centuries BC). Implications on the fidelity of the Iberian paleointensity database

NASA Astrophysics Data System (ADS)

Osete, M. L.; Chauvin, A.; Catanzariti, G.; Jimeno, A.; Campuzano, S. A.; Benito-Batanero, J. P.; Tabernero-Galán, C.; Roperch, P.

2016-11-01

Variations of geomagnetic field in the Iberian Peninsula prior to roman times are poorly constrained. Here we report new archaeomagnetic results from four ceramic collections and two combustion structures recovered in two pre-roman (celtiberic) archaeological sites in central Spain. The studied materials have been dated by archaeological evidences and supported by five radiocarbon dates. Rock magnetic experiments indicate that the characteristic remanent manetization (ChRM) is carried by a low coercivity magnetic phase with Curie temperatures of 530-575 °C, most likely Ti-poor titanomagnetite/titanomaghemite. Archaeointensity determinations were carried out by using the classical Thellier-Thellier protocol including tests and corrections for magnetic anisotropy and cooling rate dependency. Two magnetic behaviours were depicted during the laboratory treatment. Black potsherds and poor heated samples from the kilns, presented two magnetization components, alterations or curved Arai plots and were therefore rejected. In contrast, well heated specimens (red ceramic fragments and well heated samples from the kilns) show one single well defined component of magnetization going through the origin and linear Arai plots providing successful archaeointensity determinations. The effect of anisotropy of the thermoremanent magnetization (ATRM) on paleointensity analysis was systematically investigated obtaining very high ATRM corrections on fine pottery specimens. In some cases, differences between the uncorrected and ATRM corrected paleointensity values reached up to 86 %. The mean intensity values obtained from three selected set of samples were 64.3 ± 5.8 μT; 56.8 ± 3.8 and 56.7 ± 4.6 μT (NUS2, CI2 and CIA, respectively), which contribute to better understand the evolution of the palaeofield intensity in central Iberia during the 3rd-1st centuries BC. The direction of the field at first century BC has also been determined from oriented samples from CIA kilns (D = 357.2°; I = 62.2°; N = 10, α95 = 2.7°). The new archaeointensity data disagrees with previous results from Iberian ceramics which were not corrected for the ATRM effect. On the contrary, they are in agreement with the most recent French paleointensity curve and the latest European intensity model; both based on a selection of high quality paleointensity data. This result reinforces the idea that the puzzling scatter often observed in the global paleointensity database is likely due to differences in the laboratory protocols. Further data from well-established laboratory protocols are still necessary to delineate confidently the evolution of the geomagnetic palaeofield during the first millennium BC.

Whole-Genome Sequencing and Assembly with High-Throughput, Short-Read Technologies

PubMed Central

Sundquist, Andreas; Ronaghi, Mostafa; Tang, Haixu; Pevzner, Pavel; Batzoglou, Serafim

2007-01-01

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology. PMID:17534434

Assessing competence: the European Survey on Aging Protocol (ESAP).

PubMed

Fernández-Ballesteros, Rocío; Zamarrón, María Dolores; Rudinger, Georg; Schroots, Johannes J F; Hekkinnen, Eino; Drusini, Andrea; Paul, Constanza; Charzewska, Jadwiga; Rosenmayr, Leopold

2004-01-01

The main goal of this research project was to translate and adapt the European Survey on Ageing Protocol (ESAP) to 7 European countries/cultures. This article presents preliminary results from the ESAP, the basic assessment instrument of EXCELSA (European Longitudinal Study of Aging). 672 individuals aged 30-85, selected through quota sampling (by age, gender, education and living conditions), participated in this study, with 96 subjects from each of the 7 European countries. The basic research protocol for assessing competence and its determinants was designed to be administered in a 90-min in-home face-to-face interview. It contains a series of questions, instruments, scales and physical tests assessing social relationships and caregiving, mental abilities, well-being, personality, mastery and perceived control, self-reported health, lifestyles, anthropometry, biobehavioral measures and sociodemographic variables. 84% of ESAP measures are age-dependent and 75% of them discriminate between education levels. Minor differences were found due to gender, and between people living in rural and urban areas. Exploratory factor analysis yielded 10 factors accounting for 67.85% of total variance, one of which was identified as cognitive and physical 'competence'. This factorial structure was tested across countries through concordance coefficients. Finally, using structural equation modeling, our data were fitted into a model of competence. When the sample was split into younger groups (aged 30-49 years) and older ones (50 and more years), the same model was appropriate for our data. The results are discussed in accordance with other findings on psychosocial, biophysical and sociodemographic components of competence, and also in accordance with theories on competence and successful aging. Copyright 2004 S. Karger AG, Basel

Mars Sample Quarantine Protocol Workshop

NASA Technical Reports Server (NTRS)

DeVincenzi, Donald L. (Editor); Bagby, John (Editor); Race, Margaret (Editor); Rummel, John (Editor)

1999-01-01

The Mars Sample Quarantine Protocol (QP) Workshop was convened to deal with three specific aspects of the initial handling of a returned Mars sample: 1) biocontainment, to prevent uncontrolled release of sample material into the terrestrial environment; 2) life detection, to examine the sample for evidence of live organisms; and 3) biohazard testing, to determine if the sample poses any threat to terrestrial life forms and the Earth's biosphere. During the first part of the Workshop, several tutorials were presented on topics related to the workshop in order to give all participants a common basis in the technical areas necessary to achieve the objectives of the Workshop.

Adaptive Peer Sampling with Newscast

NASA Astrophysics Data System (ADS)

Tölgyesi, Norbert; Jelasity, Márk

The peer sampling service is a middleware service that provides random samples from a large decentralized network to support gossip-based applications such as multicast, data aggregation and overlay topology management. Lightweight gossip-based implementations of the peer sampling service have been shown to provide good quality random sampling while also being extremely robust to many failure scenarios, including node churn and catastrophic failure. We identify two problems with these approaches. The first problem is related to message drop failures: if a node experiences a higher-than-average message drop rate then the probability of sampling this node in the network will decrease. The second problem is that the application layer at different nodes might request random samples at very different rates which can result in very poor random sampling especially at nodes with high request rates. We propose solutions for both problems. We focus on Newscast, a robust implementation of the peer sampling service. Our solution is based on simple extensions of the protocol and an adaptive self-control mechanism for its parameters, namely—without involving failure detectors—nodes passively monitor local protocol events using them as feedback for a local control loop for self-tuning the protocol parameters. The proposed solution is evaluated by simulation experiments.

Adaptive consensus of scale-free multi-agent system by randomly selecting links

NASA Astrophysics Data System (ADS)

Mou, Jinping; Ge, Huafeng

2016-06-01

This paper investigates an adaptive consensus problem for distributed scale-free multi-agent systems (SFMASs) by randomly selecting links, where the degree of each node follows a power-law distribution. The randomly selecting links are based on the assumption that every agent decides to select links among its neighbours according to the received data with a certain probability. Accordingly, a novel consensus protocol with the range of the received data is developed, and each node updates its state according to the protocol. By the iterative method and Cauchy inequality, the theoretical analysis shows that all errors among agents converge to zero, and in the meanwhile, several criteria of consensus are obtained. One numerical example shows the reliability of the proposed methods.

Point of a space experiment proposal.

PubMed

Fukui, Keiji; Shimazu, Toru; Higashibata, Akira; Fujimoto, Nobuyoshi; Ishioka, Noriaki

2003-10-01

JAXA will solicit research proposals for space flight experiments that would be conducted for less than three years after the selection. In principle, available samples will be limited to Arabidopsis and C. elegans and flight hardware and protocol of space flight experiment will be pre-fixed. Proposals using different combinations of species and flight hardware will not be acceptable. Besides scientific issues, it is very important for proposer to write an impressive proposal. Hypothesis basis research proposal is the accepted standard. Reviewers will dislike a descriptive and unfocused research proposal without hypothesis. Ground preparation experiments, which are not related directly to space experiments, should not be included in the solicitation.

Hybridization of Environmental Microbial Community Nucleic Acids by GeoChip.

PubMed

Van Nostrand, Joy D; Yin, Huaqin; Wu, Liyou; Yuan, Tong; Zhou, Jizhong

2016-01-01

Functional gene arrays, like the GeoChip, allow for the study of tens of thousands of genes in a single assay. The GeoChip array (5.0) contains probes for genes involved in geochemical cycling (N, C, S, and P), metal homeostasis, stress response, organic contaminant degradation, antibiotic resistance, secondary metabolism, and virulence factors as well as genes specific for fungi, protists, and viruses. Here, we briefly describe GeoChip design strategies (gene selection and probe design) and discuss minimum quantity and quality requirements for nucleic acids. We then provide detailed protocols for amplification, labeling, and hybridization of samples to the GeoChip.

Exposure assessment for endocrine disruptors: some considerations in the design of studies.

PubMed Central

Rice, Carol; Birnbaum, Linda S; Cogliano, James; Mahaffey, Kathryn; Needham, Larry; Rogan, Walter J; vom Saal, Frederick S

2003-01-01

In studies designed to evaluate exposure-response relationships in children's development from conception through puberty, multiple factors that affect the generation of meaningful exposure metrics must be considered. These factors include multiple routes of exposure; the timing, frequency, and duration of exposure; need for qualitative and quantitative data; sample collection and storage protocols; and the selection and documentation of analytic methods. The methods for exposure data collection and analysis must be sufficiently robust to accommodate the a priori hypotheses to be tested, as well as hypotheses generated from the data. A number of issues that must be considered in study design are summarized here. PMID:14527851

Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices: Sample preparation methods and liquid chromatography tandem mass spectrometric separations.

PubMed

Tölgyesi, Ádám; Barta, Enikő; Simon, Andrea; McDonald, Thomas J; Sharma, Virender K

2017-10-25

Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed for their applications in livestock of the European Union (EU). This paper presents analyses of twelve selected steroids and six nitroimidazole antibiotics at low levels (1.56μg/L-4.95μg/L and 0.17μg/kg-2.14μg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up procedures, high performance liquid chromatography (HPLC) separation, and tandem mass spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples were cleaned by two independent supported liquid extraction and solid phase extraction procedures. Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost effective clean-up. The confirmatory methods were improved by extending the number of matrices and compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new methods were validated according to the recommendation of the European Union Reference Laboratories and the performance characteristics evaluated met fully the criteria. The methods were applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the applicability of developed protocols of the methods to real samples. The confirmatory methods were applied to the national monitoring program and natural contamination of prednisolone could be detected in urine at low concentration in few samples. Copyright © 2017 Elsevier B.V. All rights reserved.

An efficacious oral health care protocol for immunocompromised patients.

PubMed

Solomon, C S; Shaikh, A B; Arendorf, T M

1995-01-01

A twice-weekly oral and perioral examination was provided to 120 patients receiving antineoplastic therapy. Sixty patients were monitored while following the traditional hospital oral care protocol (chlorhexidine, hydrogen peroxide, sodium bicarbonate, thymol glycol, benzocaine mouthrinse, and nystatin). The mouth care protocol was then changed (experimental protocol = chlorhexidine, benzocaine lozenges, amphotericin B lozenges), and patients were monitored until the sample size matched that of the hospital mouth care regime. There was a statistically significant reduction in oral complications upon introduction and maintenance of the experimental protocol.

PROTOCOL FOR EXAMINATION OF THE INNER CAN CLOSURE WELD REGION FOR 3013 DE CONTAINERS

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

Mickalonis, J.

2014-09-16

The protocol for the examination of the inner can closure weld region (ICCWR) for 3013 DE containers is presented within this report. The protocol includes sectioning of the inner can lid section, documenting the surface condition, measuring corrosion parameters, and storing of samples. This protocol may change as the investigation develops since findings may necessitate additional steps be taken. Details of the previous analyses, which formed the basis for this protocol, are also presented.

On fixed-area plot sampling for downed coarse woody debris

Treesearch

Jeffrey H. Gove; Paul C. Van Deusen

2011-01-01

The use of fixed-area plots for sampling down coarse woody debris is reviewed. A set of clearly defined protocols for two previously described methods is established and a new method, which we call the 'sausage' method, is developed. All methods (protocols) are shown to be unbiased for volume estimation, but not necessarily for estimation of population...

U.S.-MEXICO BORDER PROGRAM ARIZONA BORDER STUDY--STANDARD OPERATING PROCEDURE FOR LABORATORY ANALYSIS OF HAIR SAMPLES FOR MERCURY (RTI-L-1.0)

EPA Science Inventory

The purpose of this protocol is to provide guidelines for the analysis of hair samples for total mercury by cold vapor atomic fluorescence (CVAFS) spectrometry. This protocol describes the methodology and all other analytical aspects involved in the analysis. Keywords: hair; s...

21 CFR 610.2 - Requests for samples and protocols; official release.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Biologics Evaluation and Research, a manufacturer shall not distribute a lot of a product until the lot is... Evaluation and Research, a manufacturer shall not distribute a lot of a biological product until the lot is... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Requests for samples and protocols; official...

21 CFR 610.2 - Requests for samples and protocols; official release.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Biologics Evaluation and Research, a manufacturer shall not distribute a lot of a product until the lot is... Evaluation and Research, a manufacturer shall not distribute a lot of a biological product until the lot is... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Requests for samples and protocols; official...

Sampling and measurement protocols for long-term silvicultural studies on the Penobscot Experimental Forest

Treesearch

Justin D. Waskiewicz; Laura S. Kenefic; Nicole S. Rogers; Joshua J. Puhlick; John C. Brissette; Richard J. Dionne

2015-01-01

The U.S. Forest Service, Northern Research Station has been conducting research on the silviculture of northern conifers on the Penobscot Experimental Forest (PEF) in Maine since 1950. Formal study plans provide guidance and specifications for the experimental treatments, but documentation is also needed to ensure consistency in data collection and sampling protocols....

Educational Module Intervention for Radiographers to Reduce Repetition Rate of Routine Digital Chest Radiography in Makkah Region of Saudi Arabia Tertiary Hospitals: Protocol of a Quasi-Experimental Study.

PubMed

Almalki, Abdullah A; Abdul Manaf, Rosliza; Hanafiah Juni, Muhamad; Kadir Shahar, Hayati; Noor, Noramaliza Mohd; Gabbad, Abdelsafi

2017-09-26

Repetition of an image is a critical event in any radiology department. When the repetition rate of routine digital chest radiographs is high, radiation exposure of staff and patients is increased. In addition, repetition consumes the equipment's life span, thus affecting the annual budget of the department. The aim of this study is to determine the impact of a printed educational module on reducing the repetition rate of routine digital chest radiography among radiographers in Makkah Region tertiary hospitals. A quasi-experimental time series with a control group will be conducted in Makkah Region tertiary hospitals for 8 months starting in the second quarter of 2017. Four hospitals out of 5 in the region will be selected; 2 of them will be selected as the control group and the other 2 as the intervention group. Stratification and a simple random sampling technique will be used to sample 56 radiographers in each group. Pre- and postintervention assessments will be conducted to determine the radiographer knowledge, motivation, and skills and repetition rate of chest radiographs. Radiographs of the chest performed by sampled radiographers in the selected hospitals will be collected for 2 weeks before and after the intervention. A piloted questionnaire will be distributed and collected by a researcher in both groups. One-way multivariate analysis of variance and 2-way repeated multivariate analysis of variance will be used to analyze the data. It is expected that the repetition rate in the intervention group will decline after implementing the intervention and the change will be statistically significant (P<.05). Furthermore, it is expected that the knowledge, motivation, and skill levels in the intervention group will increase significantly among radiographers after implementation of the intervention (P<.05). Meanwhile, knowledge, motivation, and skills in the control group will not change. A quasi-experimental time series with a control will be conducted to investigate the effect of printed educational material in reducing the repetition rate of routine digital chest radiographs among radiographers in tertiary hospitals in the Makkah Region of Saudi Arabia. ©Abdullah A. Almalki, Rosliza Abdul Manaf, Muhamad Hanafiah Juni, Hayati Kadir Shahar, Noramaliza Mohd Noor, Abdelsafi Gabbad. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 26.09.2017.

Effect of promoter driving selectable marker on corn transformation.

PubMed

Prakash, N Shiva; Prasad, V; Chidambram, Thillai P; Cherian, Shoba; Jayaprakash, T L; Dasgupta, Santanu; Wang, Qi; Mann, Michael T; Spencer, T Michael; Boddupalli, Raghava S

2008-08-01

Identification of an appropriate selection agent and its corresponding selectable marker gene is one of the first steps in establishing a transformation protocol for a given plant species. As the promoter controls expression level of the genes, the promoter driving the selectable marker gene can affect transformation. However, investigations into the direct effect of promoters driving selectable marker on transformation are lacking in the literature though many reports of relative strengths of promoters driving reporter genes like GUS or CAT or GFP are available. In the present study, we have compared rice Actin1 and CaMV.35S (commonly used promoters in monocotyledonous plant transformation) promoters driving nptII for their effectiveness in paromomycin selection of transgenic corn events. To enable statistically meaningful analysis of the results, a large sample size of nearly 5,000 immature embryos (explants) was employed producing approximately 1,250 independent events from each of the two constructs in four independent experiments. The rate of appearance of resistant calli and percentage of resistant calli recovered was higher with P-Os.Actin1/nptII/nos3' as compared to P-CaMV.35S/nptII/nos3' in all four experiments. There was no appreciable difference either in the frequency of plant regeneration or in the morphological characteristics of plants recovered from the two constructs. Although the escape rate trended lower with P-Os.Actin1 as compared to P-CaMV.35S, the recovery of low copy events was significantly higher with P-CaMV.35S. The higher transformation frequency with P-Os.Actin1 could be related to the strength of this promoter as compared to P-CaMV.35S in the explants and/or calli. Based on these results, we infer that the promoter driving the selectable marker is an important factor to be considered while establishing a high throughput transformation protocol as it could not only influence the transformation frequency but also the copy number of the transgene in the recovered transgenics.

Toxoplasma Gondii and Pre-treatment Protocols for Polymerase Chain Reaction Analysis of Milk Samples: A Field Trial in Sheep from Southern Italy.

PubMed

Vismarra, Alice; Barilli, Elena; Miceli, Maura; Mangia, Carlo; Bacci, Cristina; Brindani, Franco; Kramer, Laura

2017-01-24

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii. Ingestion of raw milk has been suggested as a risk for transmission to humans. Here the authors evaluated pre-treatment protocols for DNA extraction on T. gondii tachyzoite-spiked sheep milk with the aim of identifying the method that resulted in the most rapid and reliable polymerase chain reaction (PCR) positivity. This protocol was then used to analyse milk samples from sheep of three different farms in Southern Italy, including real time PCR for DNA quantification and PCR-restriction fragment length polymorphism for genotyping. The pre-treatment protocol using ethylenediaminetetraacetic acid and Tris-HCl to remove casein gave the best results in the least amount of time compared to the others on spiked milk samples. One sample of 21 collected from sheep farms was positive on one-step PCR, real time PCR and resulted in a Type I genotype at one locus (SAG3). Milk usually contains a low number of tachyzoites and this could be a limiting factor for molecular identification. Our preliminary data has evaluated a rapid, cost-effective and sensitive protocol to treat milk before DNA extraction. The results of the present study also confirm the possibility of T. gondii transmission through consumption of raw milk and its unpasteurised derivatives.

A simplified field protocol for genetic sampling of birds using buccal swabs

USGS Publications Warehouse

Vilstrup, Julia T.; Mullins, Thomas D.; Miller, Mark P.; McDearman, Will; Walters, Jeffrey R.; Haig, Susan M.

2018-01-01

DNA sampling is an essential prerequisite for conducting population genetic studies. For many years, blood sampling has been the preferred method for obtaining DNA in birds because of their nucleated red blood cells. Nonetheless, use of buccal swabs has been gaining favor because they are less invasive yet still yield adequate amounts of DNA for amplifying mitochondrial and nuclear markers; however, buccal swab protocols often include steps (e.g., extended air-drying and storage under frozen conditions) not easily adapted to field settings. Furthermore, commercial extraction kits and swabs for buccal sampling can be expensive for large population studies. We therefore developed an efficient, cost-effective, and field-friendly protocol for sampling wild birds after comparing DNA yield among 3 inexpensive buccal swab types (2 with foam tips and 1 with a cotton tip). Extraction and amplification success was high (100% and 97.2% respectively) using inexpensive generic swabs. We found foam-tipped swabs provided higher DNA yields than cotton-tipped swabs. We further determined that omitting a drying step and storing swabs in Longmire buffer increased efficiency in the field while still yielding sufficient amounts of DNA for detailed population genetic studies using mitochondrial and nuclear markers. This new field protocol allows time- and cost-effective DNA sampling of juveniles or small-bodied birds for which drawing blood may cause excessive stress to birds and technicians alike.

Intercomparison of thermal-optical method with different temperature protocols: Implications from source samples and solvent extraction

NASA Astrophysics Data System (ADS)

Cheng, Yuan; Duan, Feng-kui; He, Ke-bin; Du, Zhen-yu; Zheng, Mei; Ma, Yong-liang

2012-12-01

Three temperature protocols with different peak inert mode temperature (Tpeak-inert) were compared based on source and ambient samples (both untreated and extracted using a mixture of hexane, methylene chloride, and acetone) collected in Beijing, China. The ratio of EC580 (elemental carbon measured by the protocol with a Tpeak-inert of 580 °C; similar hereinafter) to EC850 could be as high as 4.8 for biomass smoke samples whereas the ratio was about 1.0 for diesel and gasoline exhaust samples. The EC580 to EC850 ratio averaged 1.95 ± 0.89 and 1.13 ± 0.20 for the untreated and extracted ambient samples, whereas the EC580 to EC650 ratio of ambient samples was 1.22 ± 0.10 and 1.20 ± 0.12 before and after extraction. It was suggested that there are two competing mechanisms for the effects of Tpeak-inert on the EC results such that when Tpeak-inert is increased, one mechanism tends to decrease EC by increasing the amount of charring whereas the other tends to increase EC through promoting more charring to evolve before native EC. Results from this study showed that EC does not always decrease when increasing the peak inert mode temperature. Moreover, reducing the charring amount could improve the protocols agreement on EC measurements, whereas temperature protocol would not influence the EC results if no charring is formed. This study also demonstrated the benefits of allowing for the OC and EC split occurring in the inert mode when a high Tpeak-inert is used (e.g., 850 °C).

Thermal/optical methods for elemental carbon quantification in soils and urban dusts: equivalence of different analysis protocols.

PubMed

Han, Yongming; Chen, Antony; Cao, Junji; Fung, Kochy; Ho, Fai; Yan, Beizhan; Zhan, Changlin; Liu, Suixin; Wei, Chong; An, Zhisheng

2013-01-01

Quantifying elemental carbon (EC) content in geological samples is challenging due to interferences of crustal, salt, and organic material. Thermal/optical analysis, combined with acid pretreatment, represents a feasible approach. However, the consistency of various thermal/optical analysis protocols for this type of samples has never been examined. In this study, urban street dust and soil samples from Baoji, China were pretreated with acids and analyzed with four thermal/optical protocols to investigate how analytical conditions and optical correction affect EC measurement. The EC values measured with reflectance correction (ECR) were found always higher and less sensitive to temperature program than the EC values measured with transmittance correction (ECT). A high-temperature method with extended heating times (STN120) showed the highest ECT/ECR ratio (0.86) while a low-temperature protocol (IMPROVE-550), with heating time adjusted for sample loading, showed the lowest (0.53). STN ECT was higher than IMPROVE ECT, in contrast to results from aerosol samples. A higher peak inert-mode temperature and extended heating times can elevate ECT/ECR ratios for pretreated geological samples by promoting pyrolyzed organic carbon (PyOC) removal over EC under trace levels of oxygen. Considering that PyOC within filter increases ECR while decreases ECT from the actual EC levels, simultaneous ECR and ECT measurements would constrain the range of EC loading and provide information on method performance. Further testing with standard reference materials of common environmental matrices supports the findings. Char and soot fractions of EC can be further separated using the IMPROVE protocol. The char/soot ratio was lower in street dusts (2.2 on average) than in soils (5.2 on average), most likely reflecting motor vehicle emissions. The soot concentrations agreed with EC from CTO-375, a pure thermal method.

Soil sampling and analytical strategies for mapping fallout in nuclear emergencies based on the Fukushima Dai-ichi Nuclear Power Plant accident.

PubMed

Onda, Yuichi; Kato, Hiroaki; Hoshi, Masaharu; Takahashi, Yoshio; Nguyen, Minh-Long

2015-01-01

The Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident resulted in extensive radioactive contamination of the environment via deposited radionuclides such as radiocesium and (131)I. Evaluating the extent and level of environmental contamination is critical to protecting citizens in affected areas and to planning decontamination efforts. However, a standardized soil sampling protocol is needed in such emergencies to facilitate the collection of large, tractable samples for measuring gamma-emitting radionuclides. In this study, we developed an emergency soil sampling protocol based on preliminary sampling from the FDNPP accident-affected area. We also present the results of a preliminary experiment aimed to evaluate the influence of various procedures (e.g., mixing, number of samples) on measured radioactivity. Results show that sample mixing strongly affects measured radioactivity in soil samples. Furthermore, for homogenization, shaking the plastic sample container at least 150 times or disaggregating soil by hand-rolling in a disposable plastic bag is required. Finally, we determined that five soil samples within a 3 m × 3-m area are the minimum number required for reducing measurement uncertainty in the emergency soil sampling protocol proposed here. Copyright © 2014 Elsevier Ltd. All rights reserved.

Clones identification of Sequoia sempervirens (D. Don) Endl. in Chile by using PCR-RAPDs technique.

PubMed

Toral Ibañez, Manuel; Caru, Margarita; Herrera, Miguel A; Gonzalez, Luis; Martin, Luis M; Miranda, Jorge; Navarro-Cerrillo, Rafael M

2009-02-01

A protocol of polymerase chain reaction-random amplified polymorphic DNAs (PCR-RAPDs) was established to analyse the gene diversity and genotype identification for clones of Sequoia sempervirens (D. Don) Endl. in Chile. Ten (out of 34) clones from introduction trial located in Voipir-Villarrica, Chile, were studied. The PCR-RAPDs technique and a modified hexadecyltrimethylammonium bromide (CTAB) protocol were used for genomic DNA extraction. The PCR tests were carried out employing 10-mer random primers. The amplification products were detected by electrophoresis in agarose gels. Forty nine polymorphic bands were obtained with the selected primers (BG04, BF07, BF12, BF13, and BF14) and were ordered according to their molecular size. The genetic similarity between samples was calculated by the Jaccard index and a dendrogram was constructed using a cluster analysis of unweighted pair group method using arithmetic averages (UPGMA). Of the primers tested, 5 (out of 60) RAPD primers were selected for their reproducibility and high polymorphism. A total of 49 polymorphic RAPD bands were detected out of 252 bands. The genetic similarity analysis demonstrates an extensive genetic variability between the tested clones and the dendrogram depicts the genetic relationships among the clones, suggesting a geographic relationship. The results indicate that the RAPD markers permitted the identification of the assayed clones, although they are derived from the same geographic origin.

Clones identification of Sequoia sempervirens (D. Don) Endl. in Chile by using PCR-RAPDs technique*

PubMed Central

Toral Ibañez, Manuel; Caru, Margarita; Herrera, Miguel A.; Gonzalez, Luis; Martin, Luis M.; Miranda, Jorge; Navarro-Cerrillo, Rafael M.

2009-01-01

A protocol of polymerase chain reaction-random amplified polymorphic DNAs (PCR-RAPDs) was established to analyse the gene diversity and genotype identification for clones of Sequoia sempervirens (D. Don) Endl. in Chile. Ten (out of 34) clones from introduction trial located in Voipir-Villarrica, Chile, were studied. The PCR-RAPDs technique and a modified hexadecyltrimethylammonium bromide (CTAB) protocol were used for genomic DNA extraction. The PCR tests were carried out employing 10-mer random primers. The amplification products were detected by electrophoresis in agarose gels. Forty nine polymorphic bands were obtained with the selected primers (BG04, BF07, BF12, BF13, and BF14) and were ordered according to their molecular size. The genetic similarity between samples was calculated by the Jaccard index and a dendrogram was constructed using a cluster analysis of unweighted pair group method using arithmetic averages (UPGMA). Of the primers tested, 5 (out of 60) RAPD primers were selected for their reproducibility and high polymorphism. A total of 49 polymorphic RAPD bands were detected out of 252 bands. The genetic similarity analysis demonstrates an extensive genetic variability between the tested clones and the dendrogram depicts the genetic relationships among the clones, suggesting a geographic relationship. The results indicate that the RAPD markers permitted the identification of the assayed clones, although they are derived from the same geographic origin. PMID:19235269

Flow cytometry for enrichment and titration in massively parallel DNA sequencing

PubMed Central

Sandberg, Julia; Ståhl, Patrik L.; Ahmadian, Afshin; Bjursell, Magnus K.; Lundeberg, Joakim

2009-01-01

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols. PMID:19304748

Feasibility of Providing Safe Mouth Care and Collecting Oral and Fecal Microbiome Samples from Nursing Home Residents with Dysphagia: Proof of Concept Study.

PubMed

Jablonski, Rita A; Winstead, Vicki; Azuero, Andres; Ptacek, Travis; Jones-Townsend, Corteza; Byrd, Elizabeth; Geisinger, Maria L; Morrow, Casey

2017-09-01

Individuals with dysphagia who reside in nursing homes often receive inadequate mouth care and experience poor oral health. From a policy perspective, the combination of absent evidence-based mouth care protocols coupled with insufficient dental coverage create a pool of individuals at great risk for preventable infectious illnesses that contribute to high health care costs. The purpose of the current study was to determine (a) the safety of a mouth care protocol tailored for individuals with dysphagia residing in nursing homes without access to suction equipment, and (b) the feasibility of collecting oral and fecal samples for microbiota analyses. The mouth care protocol resulted in improved oral hygiene without aspiration, and oral and fecal samples were safely collected from participants. Policies supporting ongoing testing of evidence-based mouth care protocols for individuals with dysphagia are important to improve quality, demonstrate efficacy, and save health care costs. [Journal of Gerontological Nursing, 43(9), 9-15.]. Copyright 2017, SLACK Incorporated.

METHOD FOR MICRORNA ISOLATION FROM CLINICAL SERUM SAMPLES

PubMed Central

Li, Yu; Kowdley, Kris V.

2012-01-01

MicroRNAs are a group of intracellular non-coding RNA molecules that have been implicated in a variety of human diseases. Due to their high stability in blood, microRNAs released into circulation could be potentially utilized as non-invasive biomarkers for diagnosis or prognosis. Current microRNA isolation protocols are specifically designed for solid tissues and are impractical for biomarker development utilizing small-volume serum samples on a large scale. Thus, a protocol for microRNA isolation from serum is needed to accommodate these conditions in biomarker development. To establish such a protocol, we developed a simplified approach to normalize sample input by using single synthetic spike-in microRNA. We evaluated three commonly used commercial microRNA isolation kits for the best performance by comparing RNA quality and yield. The manufacturer’s protocol was further modified to improve the microRNA yield from 200 μL of human serum. MicroRNAs isolated from a large set of clinical serum samples were tested on the miRCURY LNA real-time PCR panel and confirmed to be suitable for high-throughput microRNA profiling. In conclusion, we have established a proven method for microRNA isolation from clinical serum samples suitable for microRNA biomarker development. PMID:22982505

Finite-key analysis for quantum key distribution with weak coherent pulses based on Bernoulli sampling

NASA Astrophysics Data System (ADS)

Kawakami, Shun; Sasaki, Toshihiko; Koashi, Masato

2017-07-01

An essential step in quantum key distribution is the estimation of parameters related to the leaked amount of information, which is usually done by sampling of the communication data. When the data size is finite, the final key rate depends on how the estimation process handles statistical fluctuations. Many of the present security analyses are based on the method with simple random sampling, where hypergeometric distribution or its known bounds are used for the estimation. Here we propose a concise method based on Bernoulli sampling, which is related to binomial distribution. Our method is suitable for the Bennett-Brassard 1984 (BB84) protocol with weak coherent pulses [C. H. Bennett and G. Brassard, Proceedings of the IEEE Conference on Computers, Systems and Signal Processing (IEEE, New York, 1984), Vol. 175], reducing the number of estimated parameters to achieve a higher key generation rate compared to the method with simple random sampling. We also apply the method to prove the security of the differential-quadrature-phase-shift (DQPS) protocol in the finite-key regime. The result indicates that the advantage of the DQPS protocol over the phase-encoding BB84 protocol in terms of the key rate, which was previously confirmed in the asymptotic regime, persists in the finite-key regime.

[Legionella spp. contamination in indoor air: preliminary results of an Italian multicenter study].

PubMed

Montagna, Maria Teresa; De Giglio, Osvalda; Napoli, Christian; Cannova, Lucia; Cristina, Maria Luisa; Deriu, Maria Grazia; Delia, Santi Antonino; Giuliano, Ada; Guida, Marco; Laganà, Pasqualina; Liguori, Giorgio; Mura, Ida; Pennino, Francesca; Rossini, Angelo; Tardivo, Stefano; Torre, Ida; Torregrossa, Maria Valeria; Villafrate, Maria Rosaria; Albertini, Roberto; Pasquarella, Cesira

2014-01-01

To propose a standardized protocol for the evaluation of Legionella contamination in air. A bathroom having a Legionella contamination in water >1,000 cfu/l was selected in 10 different healthcare facilities. Air contamination was assessed by active (Surface Air System, SAS) and passive (Index of Microbial Air, IMA) sampling for 8 hours, about 1 m away from the floor and 50 cm from the tap water. Two hundred liters of air were sampled by SAS every 12 min, after flushing water for 2 min. The IMA value was calculated as the mean value of colony forming units/16 plates exposed during sampling (2 plates/hour). Water contamination was evaluated at T0, after 4 and 8 hours, according to the standard methods. Air contamination by Legionella was found in three healthcare facilities (one with active and two with passive sampling), showing a concomitant tap water contamination (median=40,000; range 1,100-43,000 cfu/l). The remaining seven hospitals isolated Legionella spp. exclusively from water samples (median=8,000; range 1,200-70,000 cfu/l). Our data suggest that environmental Legionella contamination cannot be assessed only through the air sampling, even in the presence of an important water contamination.

Protocols for dry DNA storage and shipment at room temperature.

PubMed

Ivanova, Natalia V; Kuzmina, Masha L

2013-09-01

The globalization of DNA barcoding will require core analytical facilities to develop cost-effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry-state DNA stabilization systems: commercial Biomatrica(®) DNAstable(®) plates, home-made trehalose and polyvinyl alcohol (PVA) plates on 96-well panels of insect DNA stored at 56 °C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56 °C, and diluted samples held at 4 °C and at -20 °C. PCR and selective sequencing were performed over a 4-year interval to test the condition of DNA extracts. Biomatrica(®) provided better protection of DNA at 56 °C and at room temperature than trehalose and PVA, especially for diluted samples. PVA was the second best protectant after Biomatrica(®) at room temperature, whereas trehalose was the second best protectant at 56 °C. In spite of lower PCR success, the DNA stored at -20 °C yielded longer sequence reads and stronger signal, indicating that temperature is a crucial factor for DNA quality which has to be considered especially for long-term storage. Although it is premature to advocate a transition to DNA storage at room temperature, dry storage provides an additional layer of security for frozen samples, protecting them from degradation in the event of freezer failure. All three forms of DNA preservation enable shipment of dry DNA and PCR products between barcoding facilities. © 2013 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.

Standardization and optimization of fluorescence in situ hybridization (FISH) for HER-2 assessment in breast cancer: A single center experience.

PubMed

Bogdanovska-Todorovska, Magdalena; Petrushevska, Gordana; Janevska, Vesna; Spasevska, Liljana; Kostadinova-Kunovska, Slavica

2018-05-20

Accurate assessment of human epidermal growth factor receptor 2 (HER-2) is crucial in selecting patients for targeted therapy. Commonly used methods for HER-2 testing are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Here we presented the implementation, optimization and standardization of two FISH protocols using breast cancer samples and assessed the impact of pre-analytical and analytical factors on HER-2 testing. Formalin fixed paraffin embedded (FFPE) tissue samples from 70 breast cancer patients were tested for HER-2 using PathVysion™ HER-2 DNA Probe Kit and two different paraffin pretreatment kits, Vysis/Abbott Paraffin Pretreatment Reagent Kit (40 samples) and DAKO Histology FISH Accessory Kit (30 samples). The concordance between FISH and IHC results was determined. Pre-analytical and analytical factors (i.e., fixation, baking, digestion, and post-hybridization washing) affected the efficiency and quality of hybridization. The overall hybridization success in our study was 98.6% (69/70); the failure rate was 1.4%. The DAKO pretreatment kit was more time-efficient and resulted in more uniform signals that were easier to interpret, compared to the Vysis/Abbott kit. The overall concordance between IHC and FISH was 84.06%, kappa coefficient 0.5976 (p < 0.0001). The greatest discordance (82%) between IHC and FISH was observed in IHC 2+ group. A standardized FISH protocol for HER-2 assessment, with high hybridization efficiency, is necessary due to variability in tissue processing and individual tissue characteristics. Differences in the pre-analytical and analytical steps can affect the hybridization quality and efficiency. The use of DAKO pretreatment kit is time-saving and cost-effective.

Aptamer-based polyhedral oligomeric silsesquioxane (POSS)-containing hybrid affinity monolith prepared via a "one-pot" process for selective extraction of ochratoxin A.

PubMed

Chen, Yiqiong; Chen, Maolin; Chi, Jinxin; Yu, Xia; Chen, Yongxuan; Lin, Xucong; Xie, Zenghong

2018-08-17

A novel aptamer-based polyhedral oligomeric silsesquioxane (POSS)-containing hybrid affinity monolith has been prepared with a facile "one-pot" process simultaneously via "free radical polymerization" and "thiol-ene" click reaction, and used for on-line selective extraction and practical analysis to trace ochratoxin A (OTA). By using the ternary porogenic mixture composed of water/DMF/PEG, a homogeneous polymerization mixture with POSS chemicals, acrylate-based monomers and aptamer aqueous solution was obtained, and the copolymerization of POSS chemicals, polymer monomers and aptamer aqueous solution was systematically studied. Characterizations such as the morphology, FT-IR and fluorescence spectra, mechanical stability, dynamic binding capacity, cross-reactivity and selectivity of the resultant affinity monolith were also evaluated. Attributed to the porous monolithic structure and aptamer-based affinity interaction, acceptable selective recognition and recovery yields towards trace OTA were obtained. With a 5-fold volume enrichment, the limit of detection (LOD) and limit of quantitation (LOQ) of OTA in fortified beer samples were gained at 0.025 ng/mL (S/N = 3) and 0.045 ng/mL (S/N = 10), respectively. It could be competent for the sensitive measure of actual OTA residues in real beer samples. In comparison with the previously reported strategies containing common "sol-gel" chemistry, the proposed protocol to fabricating aptamer-modified POSS-containing hybrid affinity monolith showed a simpler preparation with acceptable selectivity and higher recovery to trace OTA. Copyright © 2018 Elsevier B.V. All rights reserved.

Optical Trap Loading of Dielectric Microparticles In Air.

PubMed

Park, Haesung; LeBrun, Thomas W

2017-02-05

We demonstrate a method to trap a selected dielectric microparticle in air using radiation pressure from a single-beam gradient optical trap. Randomly scattered dielectric microparticles adhered to a glass substrate are momentarily detached using ultrasonic vibrations generated by a piezoelectric transducer (PZT). Then, the optical beam focused on a selected particle lifts it up to the optical trap while the vibrationally excited microparticles fall back to the substrate. A particle may be trapped at the nominal focus of the trapping beam or at a position above the focus (referred to here as the levitation position) where gravity provides the restoring force. After the measurement, the trapped particle can be placed at a desired position on the substrate in a controlled manner. In this protocol, an experimental procedure for selective optical trap loading in air is outlined. First, the experimental setup is briefly introduced. Second, the design and fabrication of a PZT holder and a sample enclosure are illustrated in detail. The optical trap loading of a selected microparticle is then demonstrated with step-by-step instructions including sample preparation, launching into the trap, and use of electrostatic force to excite particle motion in the trap and measure charge. Finally, we present recorded particle trajectories of Brownian and ballistic motions of a trapped microparticle in air. These trajectories can be used to measure stiffness or to verify optical alignment through time domain and frequency domain analysis. Selective trap loading enables optical tweezers to track a particle and its changes over repeated trap loadings in a reversible manner, thereby enabling studies of particle-surface interaction.

Quality testing of an innovative cascade separation system for multiple cell separation

NASA Astrophysics Data System (ADS)

Pierzchalski, Arkadiusz; Moszczynska, Aleksandra; Albrecht, Bernd; Heinrich, Jan-Michael; Tarnok, Attila

2012-03-01

Isolation of different cell types from mixed samples in one separation step by FACS is feasible but expensive and slow. It is cheaper and faster but still challenging by magnetic separation. An innovative bead-based cascade-system (pluriSelect GmbH, Leipzig, Germany) relies on simultaneous physical separation of different cell types. It is based on antibody-mediated binding of cells to beads of different size and isolation with sieves of different mesh-size. We validated pluriSelect system for single parameter (CD3) and simultaneous separation of CD3 and CD15 cells from EDTA blood-samples. Results were compared with those obtained by MACS (Miltenyi-Biotech) magnetic separation (CD3 separation). pluriSelect separation was done in whole blood, MACS on Ficoll gradient isolated leukocytes, according to the manufacturer's protocols. Isolated and residual cells were immunophenotyped (7-color 8-antibody panel (CD3; CD16/56; CD4; CD8; CD14; CD19; CD45; HLADR) on a CyFlowML flow cytometer (Partec GmbH). Cell count (Coulter), purity, yield and viability (7-AAD exclusion) were determined. There were no significant differences between both systems regarding purity (92-98%), yield (50-60%) and viability (92-98%) of isolated cells. PluriSelect separation was slightly faster than MACS (1.15 h versus 1.5h). Moreover, no preenrichment steps were necessary. In conclusion, pluriSelect is a fast, simple and gentle system for efficient simultaneous separation of two cell subpopulation directly from whole blood and can provide a simple alternative to FACS. The isolated cells can be used for further research applications.

Study on the Flexibility in Cross-Border Water Resources Cooperation Governance

NASA Astrophysics Data System (ADS)

Liu, Zongrui; Wang, Teng; Zhou, Li

2018-02-01

Flexible strategy is very important to cross-border cooperation in international rivers water resources, which may be employed to reconcile contradictions and ease conflicts. Flexible characters of cross-border cooperation in international rivers water resources could be analyzed and revealed, using flexible strategic management framework, by taking international cooperation protocols related to water from Transboundary Freshwater Disputes Database (TFDD) as samples from the number of cooperation issues, the amount of management layers and regulator agencies in cooperation organization and the categories of income (cost) distribution (allocation) mode. The research demonstrates that there are some flexible features of cross-border cooperation in international rivers water resources: Riparian countries would select relative diversification strategies related to water, tend to construct a flexible cooperation organization featured with moderate hierarchies from vertical perspective and simplified administrations from horizontal perspective, and adopt selective inducement modes to respect ‘joint and several liability’.

Searching for life in extreme environments relevant to Jovian's Europa: Lessons from subglacial ice studies at Lake Vostok (East Antarctica)

NASA Astrophysics Data System (ADS)

Bulat, Sergey A.; Alekhina, Irina A.; Marie, Dominique; Martins, Jean; Petit, Jean Robert

2011-08-01

The objective was to estimate the genuine microbial content of ice samples from refrozen water (accretion ice) from the subglacial Lake Vostok (Antarctica) buried beneath the 4-km thick East Antarctic ice sheet. The samples were extracted by heavy deep ice drilling from 3659 m below the surface. High pressure, a low carbon and chemical content, isolation, complete darkness and the probable excess of oxygen in water for millions of years characterize this extreme environment. A decontamination protocol was first applied to samples selected for the absence of cracks to remove the outer part contaminated by handling and drilling fluid. Preliminary indications showed the accretion ice samples to be almost gas free with a low impurity content. Flow cytometry showed the very low unevenly distributed biomass while repeated microscopic observations were unsuccessful.We used strategies of Ancient DNA research that include establishing contaminant databases and criteria to validate the amplification results. To date, positive results that passed the artifacts and contaminant databases have been obtained for a pair of bacterial phylotypes only in accretion ice samples featured by some bedrock sediments. The phylotypes included the chemolithoautotrophic thermophile Hydrogenophilus thermoluteolus and one unclassified phylotype. Combined with geochemical and geophysical considerations, our results suggest the presence of a deep biosphere, possibly thriving within some active faults of the bedrock encircling the subglacial lake, where the temperature is as high as 50 °C and in situ hydrogen is probably present.Our approach indicates that the search for life in the subglacial Lake Vostok is constrained by a high probability of forward-contamination. Our strategy includes strict decontamination procedures, thorough tracking of contaminants at each step of the analysis and validation of the results along with geophysical and ecological considerations for the lake setting. This may serve to establish a guideline protocol for studying extraterrestrial ice samples.

Miniaturized Temperature-Controlled Planar Chromatography (Micro-TLC) as a Versatile Technique for Fast Screening of Micropollutants and Biomarkers Derived from Surface Water Ecosystems and During Technological Processes of Wastewater Treatment.

PubMed

Ślączka-Wilk, Magdalena M; Włodarczyk, Elżbieta; Kaleniecka, Aleksandra; Zarzycki, Paweł K

2017-07-01

There is increasing interest in the development of simple analytical systems enabling the fast screening of target components in complex samples. A number of newly invented protocols are based on quasi separation techniques involving microfluidic paper-based analytical devices and/or micro total analysis systems. Under such conditions, the quantification of target components can be performed mainly due to selective detection. The main goal of this paper is to demonstrate that miniaturized planar chromatography has the capability to work as an efficient separation and quantification tool for the analysis of multiple targets within complex environmental samples isolated and concentrated using an optimized SPE method. In particular, we analyzed various samples collected from surface water ecosystems (lakes, rivers, and the Baltic Sea of Middle Pomerania in the northern part of Poland) in different seasons, as well as samples collected during key wastewater technological processes (originating from the "Jamno" wastewater treatment plant in Koszalin, Poland). We documented that the multiple detection of chromatographic spots on RP-18W microplates-under visible light, fluorescence, and fluorescence quenching conditions, and using the visualization reagent phosphomolybdic acid-enables fast and robust sample classification. The presented data reveal that the proposed micro-TLC system is useful, inexpensive, and can be considered as a complementary method for the fast control of treated sewage water discharged by a municipal wastewater treatment plant, particularly for the detection of low-molecular mass micropollutants with polarity ranging from estetrol to progesterone, as well as chlorophyll-related dyes. Due to the low consumption of mobile phases composed of water-alcohol binary mixtures (less than 1 mL/run for the simultaneous separation of up to nine samples), this method can be considered an environmentally friendly and green chemistry analytical tool. The described analytical protocol can be complementary to those involving classical column chromatography (HPLC) or various planar microfluidic devices.

Sample Preparation and Mounting of Drosophila Embryos for Multiview Light Sheet Microscopy.

PubMed

Schmied, Christopher; Tomancak, Pavel

2016-01-01

Light sheet fluorescent microscopy (LSFM), and in particular its most widespread flavor Selective Plane Illumination Microscopy (SPIM), promises to provide unprecedented insights into developmental dynamics of entire living systems. By combining minimal photo-damage with high imaging speed and sample mounting tailored toward the needs of the specimen, it enables in toto imaging of embryogenesis with high spatial and temporal resolution. Drosophila embryos are particularly well suited for SPIM imaging because the volume of the embryo does not change from the single cell embryo to the hatching larva. SPIM microscopes can therefore image Drosophila embryos embedded in rigid media, such as agarose, from multiple angles every few minutes from the blastoderm stage until hatching. Here, we describe sample mounting strategies to achieve such a recording. We also provide detailed protocols to realize multiview, long-term, time-lapse recording of Drosophila embryos expressing fluorescent markers on the commercially available Zeiss Lightsheet Z.1 microscope and the OpenSPIM.

'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples.

PubMed

Eichmann, Cordula; Parson, Walther

2008-09-01

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.

USDA's National Food and Nutrient Analysis Program (NFNAP) produces high-quality data for USDA food composition databases: Two decades of collaboration.

PubMed

Haytowitz, David B; Pehrsson, Pamela R

2018-01-01

For nearly 20years, the National Food and Nutrient Analysis Program (NFNAP) has expanded and improved the quantity and quality of data in US Department of Agriculture's (USDA) food composition databases (FCDB) through the collection and analysis of nationally representative food samples. NFNAP employs statistically valid sampling plans, the Key Foods approach to identify and prioritize foods and nutrients, comprehensive quality control protocols, and analytical oversight to generate new and updated analytical data for food components. NFNAP has allowed the Nutrient Data Laboratory to keep up with the dynamic US food supply and emerging scientific research. Recently generated results for nationally representative food samples show marked changes compared to previous database values for selected nutrients. Monitoring changes in the composition of foods is critical in keeping FCDB up-to-date, so that they remain a vital tool in assessing the nutrient intake of national populations, as well as for providing dietary advice. Published by Elsevier Ltd.

The UK Biobank sample handling and storage protocol for the collection, processing and archiving of human blood and urine.

PubMed

Elliott, Paul; Peakman, Tim C

2008-04-01

UK Biobank is a large prospective study in the UK to investigate the role of genetic factors, environmental exposures and lifestyle in the causes of major diseases of late and middle age. Extensive data and biological samples are being collected from 500,000 participants aged between 40 and 69 years. The biological samples that are collected and how they are processed and stored will have a major impact on the future scientific usefulness of the UK Biobank resource. The aim of the UK Biobank sample handling and storage protocol is to specify methods for the collection and storage of participant samples that give maximum scientific return within the available budget. Processing or storage methods that, as far as can be predicted, will preclude current or future assays have been avoided. The protocol was developed through a review of the literature on sample handling and processing, wide consultation within the academic community and peer review. Protocol development addressed which samples should be collected, how and when they should be processed and how the processed samples should be stored to ensure their long-term integrity. The recommended protocol was extensively tested in a series of validation studies. UK Biobank collects about 45 ml blood and 9 ml of urine with minimal local processing from each participant using the vacutainer system. A variety of preservatives, anti-coagulants and clot accelerators is used appropriate to the expected end use of the samples. Collection of other material (hair, nails, saliva and faeces) was also considered but rejected for the full cohort. Blood and urine samples from participants are transported overnight by commercial courier to a central laboratory where they are processed and aliquots of urine, plasma, serum, white cells and red cells stored in ultra-low temperature archives. Aliquots of whole blood are also stored for potential future production of immortalized cell lines. A standard panel of haematology assays is completed on whole blood from all participants, since such assays need to be conducted on fresh samples (whereas other assays can be done on stored samples). By the end of the recruitment phase, 15 million sample aliquots will be stored in two geographically separate archives: 9.5 million in a -80 degrees C automated archive and 5.5 million in a manual liquid nitrogen archive at -180 degrees C. Because of the size of the study and the numbers of samples obtained from participants, the protocol stipulates a highly automated approach for the processing and storage of samples. Implementation of the processes, technology, systems and facilities has followed best practices used in manufacturing industry to reduce project risk and to build in quality and robustness. The data produced from sample collection, processing and storage are highly complex and are managed by a commercially available LIMS system fully integrated with the entire process. The sample handling and storage protocol adopted by UK Biobank provides quality assured and validated methods that are feasible within the available funding and reflect the size and aims of the project. Experience from recruiting and processing the first 40,000 participants to the study demonstrates that the adopted methods and technologies are fit-for-purpose and robust.

A quarantine protocol for analysis of returned extraterrestrial samples

NASA Technical Reports Server (NTRS)

Bagby, J. R.; Sweet, H. C.; Devincenzi, D. L.

1983-01-01

A protocol is presented for the analysis at an earth-orbiting quarantine facility of return samples of extraterrestrial material that might contain (nonterrestrial) life forms. The protocol consists of a series of tests designed to determine whether the sample, conceptualized as a 1-kg sample of Martian soil, is free from nonterrestrial biologically active agents and so may safely be sent to a terrestrial containment facility, or it exhibits biological activity requiring further (second-order) testing outside the biosphere. The first-order testing procedure seeks to detect the presence of any replicating organisms or toxic substances through a series of experiments including gas sampling, analysis of radioactivity, stereomicroscopic inspection, chemical analysis, microscopic examination, the search for metabolic products under growth conditions, microbiologicl assays, and the challenge of cultured cells with any agents found or with the extraterrestrial material as is. Detailed plans for the second-order testing would be developed in response to the actual data received from primary testing.

A Field-Based Cleaning Protocol for Sampling Devices Used in Life-Detection Studies

NASA Astrophysics Data System (ADS)

Eigenbrode, Jennifer; Benning, Liane G.; Maule, Jake; Wainwright, Norm; Steele, Andrew; Amundsen, Hans E. F.

2009-06-01

Analytical approaches to extant and extinct life detection involve molecular detection often at trace levels. Thus, removal of biological materials and other organic molecules from the surfaces of devices used for sampling is essential for ascertaining meaningful results. Organic decontamination to levels consistent with null values on life-detection instruments is particularly challenging at remote field locations where Mars analog field investigations are carried out. Here, we present a seven-step, multi-reagent decontamination method that can be applied to sampling devices while in the field. In situ lipopolysaccharide detection via low-level endotoxin assays and molecular detection via gas chromatography-mass spectrometry were used to test the effectiveness of the decontamination protocol for sampling of glacial ice with a coring device and for sampling of sediments with a rover scoop during deployment at Arctic Mars-analog sites in Svalbard, Norway. Our results indicate that the protocols and detection technique sufficiently remove and detect low levels of molecular constituents necessary for life-detection tests.

A field-based cleaning protocol for sampling devices used in life-detection studies.

PubMed

Eigenbrode, Jennifer; Benning, Liane G; Maule, Jake; Wainwright, Norm; Steele, Andrew; Amundsen, Hans E F

2009-06-01

Analytical approaches to extant and extinct life detection involve molecular detection often at trace levels. Thus, removal of biological materials and other organic molecules from the surfaces of devices used for sampling is essential for ascertaining meaningful results. Organic decontamination to levels consistent with null values on life-detection instruments is particularly challenging at remote field locations where Mars analog field investigations are carried out. Here, we present a seven-step, multi-reagent decontamination method that can be applied to sampling devices while in the field. In situ lipopolysaccharide detection via low-level endotoxin assays and molecular detection via gas chromatography-mass spectrometry were used to test the effectiveness of the decontamination protocol for sampling of glacial ice with a coring device and for sampling of sediments with a rover scoop during deployment at Arctic Mars-analog sites in Svalbard, Norway. Our results indicate that the protocols and detection technique sufficiently remove and detect low levels of molecular constituents necessary for life-detection tests.

Counting at low concentrations: the statistical challenges of verifying ballast water discharge standards

USGS Publications Warehouse

Frazier, Melanie; Miller, A. Whitman; Lee, Henry; Reusser, Deborah A.

2013-01-01

Discharge from the ballast tanks of ships is one of the primary vectors of nonindigenous species in marine environments. To mitigate this environmental and economic threat, international, national, and state entities are establishing regulations to limit the concentration of living organisms that may be discharged from the ballast tanks of ships. The proposed discharge standards have ranged from zero detectable organisms to 3. If standard sampling methods are used, verifying whether ballast discharge complies with these stringent standards will be challenging due to the inherent stochasticity of sampling. Furthermore, at low concentrations, very large volumes of water must be sampled to find enough organisms to accurately estimate concentration. Despite these challenges, adequate sampling protocols comprise a critical aspect of establishing standards because they help define the actual risk level associated with a standard. A standard that appears very stringent may be effectively lax if it is paired with an inadequate sampling protocol. We describe some of the statistical issues associated with sampling at low concentrations to help regulators understand the uncertainties of sampling as well as to inform the development of sampling protocols that ensure discharge standards are adequately implemented.

Wound-healing outcomes using standardized assessment and care in clinical practice.

PubMed

Bolton, Laura; McNees, Patrick; van Rijswijk, Lia; de Leon, Jean; Lyder, Courtney; Kobza, Laura; Edman, Kelly; Scheurich, Anne; Shannon, Ron; Toth, Michelle

2004-01-01

Wound-healing outcomes applying standardized protocols have typically been measured within controlled clinical trials, not natural settings. Standardized protocols of wound care have been validated for clinical use, creating an opportunity to measure the resulting outcomes. Wound-healing outcomes were explored during clinical use of standardized validated protocols of care based on patient and wound assessments. This was a prospective multicenter study of wound-healing outcomes management in real-world clinical practice. Healing outcomes from March 26 to October 31, 2001, were recorded on patients in 3 long-term care facilities, 1 long-term acute care hospital, and 12 home care agencies for wounds selected by staff to receive care based on computer-generated validated wound care algorithms. After diagnosis, wound dimensions and status were assessed using a tool adapted from the Pressure Sore Status Toolfor use on all wounds. Wound, ostomy, and continence nursing professionals accessed consistent protocols of care, via telemedicine in home care or paper forms in long-term care. A physician entered assessments into a desktop computer in the wound clinic. Based on evidence that healing proceeds faster with fewer infections in environments without gauze, the protocols generally avoided gauze dressings. Most of the 767 wounds selected to receive the standardized-protocols of care were stage III-IV pressure ulcers (n = 373; mean healing time 62 days) or full-thickness venous ulcers (n = 124; mean healing time 57 days). Partial-thickness wounds healed faster than same-etiology full-thickness wounds. These results provide benchmarks for natural-setting healing outcomes and help to define and address wound care challenges. Outcomes primarily using nongauze protocols of care matched or surpassed best previously published results on similar wounds using gauze-based protocols of care, including protocols applying gauze impregnated with growth factors or other agents.

Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation.

PubMed

Shamseer, Larissa; Moher, David; Clarke, Mike; Ghersi, Davina; Liberati, Alessandro; Petticrew, Mark; Shekelle, Paul; Stewart, Lesley A

2015-01-02

Protocols of systematic reviews and meta-analyses allow for planning and documentation of review methods, act as a guard against arbitrary decision making during review conduct, enable readers to assess for the presence of selective reporting against completed reviews, and, when made publicly available, reduce duplication of efforts and potentially prompt collaboration. Evidence documenting the existence of selective reporting and excessive duplication of reviews on the same or similar topics is accumulating and many calls have been made in support of the documentation and public availability of review protocols. Several efforts have emerged in recent years to rectify these problems, including development of an international register for prospective reviews (PROSPERO) and launch of the first open access journal dedicated to the exclusive publication of systematic review products, including protocols (BioMed Central's Systematic Reviews). Furthering these efforts and building on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines, an international group of experts has created a guideline to improve the transparency, accuracy, completeness, and frequency of documented systematic review and meta-analysis protocols--PRISMA-P (for protocols) 2015. The PRISMA-P checklist contains 17 items considered to be essential and minimum components of a systematic review or meta-analysis protocol.This PRISMA-P 2015 Explanation and Elaboration paper provides readers with a full understanding of and evidence about the necessity of each item as well as a model example from an existing published protocol. This paper should be read together with the PRISMA-P 2015 statement. Systematic review authors and assessors are strongly encouraged to make use of PRISMA-P when drafting and appraising review protocols. © BMJ Publishing Group Ltd 2014.

Enhancing QKD security with weak measurements

NASA Astrophysics Data System (ADS)

Farinholt, Jacob M.; Troupe, James E.

2016-10-01

Publisher's Note: This paper, originally published on 10/24/2016, was replaced with a corrected/revised version on 11/8/2016. If you downloaded the original PDF but are unable to access the revision, please contact SPIE Digital Library Customer Service for assistance. In the late 1980s, Aharonov and colleagues developed the notion of a weak measurement of a quantum observable that does not appreciably disturb the system.1, 2 The measurement results are conditioned on both the pre-selected and post-selected state of the quantum system. While any one measurement reveals very little information, by making the same measurement on a large ensemble of identically prepared pre- and post-selected (PPS) states and averaging the results, one may obtain what is known as the weak value of the observable with respect to that PPS ensemble. Recently, weak measurements have been proposed as a method of assessing the security of QKD in the well-known BB84 protocol.3 This weak value augmented QKD protocol (WV-QKD) works by additionally requiring the receiver, Bob, to make a weak measurement of a particular observable prior to his strong measurement. For the subset of measurement results in which Alice and Bob's measurement bases do not agree, the weak measurement results can be used to detect any attempt by an eavesdropper, Eve, to correlate her measurement results with Bob's. Furthermore, the well-known detector blinding attacks, which are known to perfectly correlate Eve's results with Bob's without being caught by conventional BB84 implementations, actually make the eavesdropper more visible in the new WV-QKD protocol. In this paper, we will introduce the WV-QKD protocol and discuss its generalization to the 6-state single qubit protocol. We will discuss the types of weak measurements that are optimal for this protocol, and compare the predicted performance of the 6- and 4-state WV-QKD protocols.

Efficacy of a polyvalent mastitis vaccine against Staphylococcus aureus on a dairy Mediterranean buffalo farm: results of two clinical field trials.

PubMed

Guccione, Jacopo; Pesce, Antonella; Pascale, Massimo; Salzano, Caterina; Tedeschi, Gianni; D'Andrea, Luigi; De Rosa, Angela; Ciaramella, Paolo

2017-01-19

In the last years the knowledges on Mediterranean Buffalo (MB) mastitis is remarkably improving, nevertheless the attention has been never focused on vaccination as preventive strategy for the control of mastitis in these ruminates. The aim of the current study was to assess clinical efficacy over time of two different preventive vaccination protocols against S. aureus mastitis, in primiparous MB.Vaccinated (VG) and not-vaccinated (N-VG) groups, of 30 MB each one, were selected from two different herds (herd A: VG1 and N-VG1; herd B: VG2 and N-VG2) of the same farm. Herd A received a double vaccination (Startvac®, 45 and 10 days before calving, protocol A), while in herd B an additional administration was performed (52 days after calving, protocol B). Bacteriological milk culture and assessment of somatic cell count (SCC) were performed at 10, 30, 60 and 90 days in milk (DIM) from composite milk samples. After 90 DIM, daily milk yields and SCC values were monthly detected until dry-off. The overall incidence of positive MB for S. aureus was 40.8% (49/120) in VG1 and 43.3% (52/120) in N-VG1 (Protocol A), while 45.8% (55/120) and 50.8% (61/120) in VG2 and N-VG2 (Protocol B). The latter was associated with a significant decreased in prevalence (at 90 DIM) and incidence of mastitis (animals positive for S. aureus, SCC > 200^10 3 , with or without clinical signs) in the vaccinated MB, while no difference occurred in protocol A. Moreover, herd B showed a significant reduction in prevalence of intramammary infection (animals positive for S. aureus, SCC < 200^10 3 , no clinical signs) in the vaccinated MB at 60 DIM while no differences were detected in herd A, at any sampling time; N-VG2 had significantly higher overall SCC values than VG2 (4.97 ± 4.75 and 4.84 ± 4.60 Log10 cells/mL ± standard deviation, respectively), while no differences were recorded in herd A. The current investigation explores for the first time the clinical efficacy of vaccinations against S. aureus infections in MB, showing encouraging results regarding reduction in mastitis and somatic cell count; the polyvalent mastitis vaccine may be considered an additional tool for in-herd S aureus infection and should be associated to other control procedures to maximize its properties.

Molecular detection of Rift Valley fever virus in serum samples from selected areas of Tanzania.

PubMed

Chengula, Augustino Alfred; Kasanga, Christopher Jacob; Mdegela, Robinson Hammerthon; Sallu, Raphael; Yongolo, Mmeta

2014-04-01

Rift Valley fever (RVF) is an acute mosquito-borne viral zoonotic disease affecting domestic animals and humans caused by the Rift Valley fever virus (RVFV). The virus belongs to the genus Phlebovirus of the family Bunyaviridae. The main aim of this study was to detect the presence of antibodies to RVFV as well as the virus in the serum samples that were collected from livestock during the 2006/2007 RVF outbreaks in different locations in Tanzania. Analysis of selected samples was done using a RVF-specific inhibition enzyme-linked immunosorbent assay (I-ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Genomic viral RNA was extracted directly from serum samples using a QIAamp Viral RNA Mini Kit (QIAGEN), and a one-step RT-PCR protocol was used to amplify the S segment of RVFV. Positive results were obtained in 39.5% (n = 200) samples using the RVF I-ELISA, and 17.6% (n = 108) of samples were positive by RT-PCR. I-ELISA detected 41 (38.7%), 32 (39.0%), and 6 (50.0%) positive results in cattle, goats, and sheep sera, respectively, whereas the RT-PCR detected 11 (0.2%), 7 (0.2%), and 1 (0.1%) positive results in cattle, goats, and sheep sera, respectively. These findings have demonstrated the presence of RVFV in Tanzania during the 2006/2007 RVF outbreaks. To our knowledge, this is the first report to detect RVFV in serum samples from domestic animals in Tanzania using PCR technique. Therefore, a detailed molecular study to characterize the virus from different geographical locations in order to establish the profile of strains circulating in the country and develop more effective and efficient control strategies should be done.

Development of an analytical microbial consortia method for enhancing performance monitoring at aerobic wastewater treatment plants.

PubMed

Razban, Behrooz; Nelson, Kristina Y; McMartin, Dena W; Cullimore, D Roy; Wall, Michelle; Wang, Dunling

2012-01-01

An analytical method to produce profiles of bacterial biomass fatty acid methyl esters (FAME) was developed employing rapid agitation followed by static incubation (RASI) using selective media of wastewater microbial communities. The results were compiled to produce a unique library for comparison and performance analysis at a Wastewater Treatment Plant (WWTP). A total of 146 samples from the aerated WWTP, comprising 73 samples of each secondary and tertiary effluent, were included analyzed. For comparison purposes, all samples were evaluated via a similarity index (SI) with secondary effluents producing an SI of 0.88 with 2.7% variation and tertiary samples producing an SI 0.86 with 5.0% variation. The results also highlighted significant differences between the fatty acid profiles of the tertiary and secondary effluents indicating considerable shifts in the bacterial community profile between these treatment phases. The WWTP performance results using this method were highly replicable and reproducible indicating that the protocol has potential as a performance-monitoring tool for aerated WWTPs. The results quickly and accurately reflect shifts in dominant bacterial communities that result when processes operations and performance change.