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.

Biolistic Transformation of Wheat.

PubMed

Tassy, Caroline; Barret, Pierre

2017-01-01

The wheat genome encodes some 100,000 genes. To understand how the expression of these genes is regulated it will be necessary to carry out many genetic transformation experiments. Robust protocols that allow scientists to transform a wide range of wheat genotypes are therefore required. In this chapter, we describe a protocol for biolistic transformation of wheat that uses immature embryos and small quantities of DNA cassettes. An original method for DNA cassette purification is also described. This protocol can be used to transform a wide range of wheat genotypes and other related species.

Imaging Tumor Cell Movement In Vivo

PubMed Central

Entenberg, David; Kedrin, Dmitriy; Wyckoff, Jeffrey; Sahai, Erik; Condeelis, John; Segall, Jeffrey E.

2013-01-01

This unit describes the methods that we have been developing for analyzing tumor cell motility in mouse and rat models of breast cancer metastasis. Rodents are commonly used both to provide a mammalian system for studying human tumor cells (as xenografts in immunocompromised mice) as well as for following the development of tumors from a specific tissue type in transgenic lines. The Basic Protocol in this unit describes the standard methods used for generation of mammary tumors and imaging them. Additional protocols for labeling macrophages, blood vessel imaging, and image analysis are also included. PMID:23456602

Cell synchrony and chromosomal protocols for somatic cells of cotton gossypium hirsutum

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

Van`t Hof, J.; Lamm, S.S.

1994-10-10

The intent of this pamphlet is to share information with others who are interested in useful protocols for cotton cytogenetics and cytology. The protocols described are a beginning effort and they are expected to be improved. All methods, results and data given pertain to cultivar MD 51ne. We are not satisfied with our results of in situ hybridization.

Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads.

PubMed

Baskaran, Padmamalini; Thyagarajan, Baskaran

2017-07-21

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes the method for the measurement of basal and forskolin (FSK)-stimulated lipolysis in the inguinal fat pads isolated from wild type mice fed either normal chow diet (NCD), high fat diet (HFD) or a high fat diet containing 0.01% of capsaicin (CAP; transient receptor potential vanilloid subfamily 1 (TRPV1) agonist) for 32 weeks. The method described here for performing ex vivo lipolysis is adopted from Schweiger et al. 1 We present a detailed protocol for measuring glycerol levels by UV-Visible (UV/VIS) spectrophotometry. The method described here can be used to successfully isolate inguinal fat pads for lipolysis measurements to obtain consistent results. The protocol described for inguinal fat pads can readily be extended to measure lipolysis in other tissues.

Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins.

PubMed

Liang, Zhen; Chen, Kunling; Zhang, Yi; Liu, Jinxing; Yin, Kangquan; Qiu, Jin-Long; Gao, Caixia

2018-03-01

This protocol is an extension to: Nat. Protoc. 9, 2395-2410 (2014); doi:10.1038/nprot.2014.157; published online 18 September 2014In recent years, CRISPR/Cas9 has emerged as a powerful tool for improving crop traits. Conventional plant genome editing mainly relies on plasmid-carrying cassettes delivered by Agrobacterium or particle bombardment. Here, we describe DNA-free editing of bread wheat by delivering in vitro transcripts (IVTs) or ribonucleoprotein complexes (RNPs) of CRISPR/Cas9 by particle bombardment. This protocol serves as an extension of our previously published protocol on genome editing in bread wheat using CRISPR/Cas9 plasmids delivered by particle bombardment. The methods we describe not only eliminate random integration of CRISPR/Cas9 into genomic DNA, but also reduce off-target effects. In this protocol extension article, we present detailed protocols for preparation of IVTs and RNPs; validation by PCR/restriction enzyme (RE) and next-generation sequencing; delivery by biolistics; and recovery of mutants and identification of mutants by pooling methods and Sanger sequencing. To use these protocols, researchers should have basic skills and experience in molecular biology and biolistic transformation. By using these protocols, plants edited without the use of any foreign DNA can be generated and identified within 9-11 weeks.

Optimized protocols for Mycobacterium leprae strain management: frozen stock preservation and maintenance in athymic nude mice.

PubMed

Trombone, Ana Paula Fávaro; Pedrini, Sílvia Cristina Barbosa; Diório, Suzana Madeira; Belone, Andréa de Faria Fernandes; Fachin, Luciana Raquel Vicenzi; do Nascimento, Dejair Caitano; Rosa, Patricia Sammarco

2014-03-23

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies.

PubMed

Nguyen, Hung X; Kirkton, Robert D; Bursac, Nenad

2018-05-01

We describe a two-stage protocol to generate electrically excitable and actively conducting cell networks with stable and customizable electrophysiological phenotypes. Using this method, we have engineered monoclonally derived excitable tissues as a robust and reproducible platform to investigate how specific ion channels and mutations affect action potential (AP) shape and conduction. In the first stage of the protocol, we combine computational modeling, site-directed mutagenesis, and electrophysiological techniques to derive optimal sets of mammalian and/or prokaryotic ion channels that produce specific AP shape and conduction characteristics. In the second stage of the protocol, selected ion channels are stably expressed in unexcitable human cells by means of viral or nonviral delivery, followed by flow cytometry or antibiotic selection to purify the desired phenotype. This protocol can be used with traditional heterologous expression systems or primary excitable cells, and application of this method to primary fibroblasts may enable an alternative approach to cardiac cell therapy. Compared with existing methods, this protocol generates a well-defined, relatively homogeneous electrophysiological phenotype of excitable cells that facilitates experimental and computational studies of AP conduction and can decrease arrhythmogenic risk upon cell transplantation. Although basic cell culture and molecular biology techniques are sufficient to generate excitable tissues using the described protocol, experience with patch-clamp techniques is required to characterize and optimize derived cell populations.

Protocols for the delivery of small molecules to the two-spotted spider mite, Tetranychus urticae

PubMed Central

Nunes, Maria Andreia; Zhurov, Vladimir; Dermauw, Wannes; Osakabe, Masahiro; Van Leeuwen, Thomas; Grbic, Miodrag

2017-01-01

The two-spotted spider mite, Tetranychus urticae, is a chelicerate herbivore with an extremely wide host range and an extraordinary ability to develop pesticide resistance. Due to its responsiveness to natural and synthetic xenobiotics, the spider mite is becoming a prime pest herbivore model for studies of the evolution of host range, plant-herbivore interactions and mechanisms of xenobiotic resistance. The spider mite genome has been sequenced and its transcriptional responses to developmental and various biotic and abiotic cues have been documented. However, to identify biological and evolutionary roles of T. urticae genes and proteins, it is necessary to develop methods for the efficient manipulation of mite gene function or protein activity. Here, we describe protocols developed for the delivery of small molecules into spider mites. Starting with mite maintenance and the preparation of the experimental mite populations of developmentally synchronized larvae and adults, we describe 3 methods for delivery of small molecules including artificial diet, leaf coating, and soaking. The presented results define critical steps in these methods and demonstrate that they can successfully deliver tracer dyes into mites. Described protocols provide guidelines for high-throughput setups for delivery of experimental compounds that could be used in reverse genetics platforms to modulate gene expression or protein activity, or for screens focused on discovery of new molecules for mite control. In addition, described protocols could be adapted for other Tetranychidae and related species of economic importance such as Varroa, dust and poultry mites. PMID:28686745

Polyethylene glycol 3350 based colon cleaning protocol: 2 d vs 4 d head to head comparison

PubMed Central

Elitsur, Rotem; Butcher, Lisa; Vicki, Lund; Elitsur, Yoram

2013-01-01

AIM: To compare between 2 and 4 d colon cleansing protocols. METHODS: Children who were scheduled for colonoscopy procedure (2010-2012) for various medical reasons, were recruited from the pediatric gastroenterology clinic at Marshall University School of Medicine, Huntington, WV. Exclusion criteria were patients who were allergic to the medication used in the protocols [polyethylene glycol (PEG) 3350, Bisacodyl], or children with metabolic or renal diseases. Two PEG 3350 protocols for 4 d (A) and 2 d (B) were prescribed as previously described. A questionnaire describing the volume of PEG consumed, clinical data, and side effects were recorded. Colon preparation was graded by two observers according to previously described method. Main outcome measurements: Rate of adequate colon preparation. RESULTS: A total of 78 patients were considered for final calculation (group A: 40, group B: 38). Age and stool consistency at the last day was comparable in both groups, but the number of stools/day was significantly higher in group B (P = 0.001). Adequate colon preparation was reached in 57.5% (A) and 73.6% (B), respectively (P = 0.206). Side effects were minimal and comparable in both groups. There was no difference in children’s age, stool characteristics, or side effects between the children with adequate or inadequate colon preparation. Correlation and agreement between observers was excellent (Pearson correlation = 0.972, kappa = 1.0). CONCLUSION: No difference between protocols was observed, but the 2 d protocol was superior for its shorter time. Direct comparison between different colon cleansing protocols is crucial in order to establish the “gold standard” protocol for children. PMID:23596539

Evaluation of telomere length in human cardiac tissues using cardiac quantitative FISH.

PubMed

Sharifi-Sanjani, Maryam; Meeker, Alan K; Mourkioti, Foteini

2017-09-01

Telomere length has been correlated with various diseases, including cardiovascular disease and cancer. The use of currently available telomere-length measurement techniques is often restricted by the requirement of a large amount of cells (Southern-based techniques) or the lack of information on individual cells or telomeres (PCR-based methods). Although several methods have been used to measure telomere length in tissues as a whole, the assessment of cell-type-specific telomere length provides valuable information on individual cell types. The development of fluorescence in situ hybridization (FISH) technologies enables the quantification of telomeres in individual chromosomes, but the use of these methods is dependent on the availability of isolated cells, which prevents their use with fixed archival samples. Here we describe an optimized quantitative FISH (Q-FISH) protocol for measuring telomere length that bypasses the previous limitations by avoiding contributions from undesired cell types. We have used this protocol on small paraffin-embedded cardiac-tissue samples. This protocol describes step-by-step procedures for tissue preparation, permeabilization, cardiac-tissue pretreatment and hybridization with a Cy3-labeled telomeric repeat complementing (CCCTAA) 3 peptide nucleic acid (PNA) probe coupled with cardiac-specific antibody staining. We also describe how to quantify telomere length by means of the fluorescence intensity and area of each telomere within individual nuclei. This protocol provides comparative cell-type-specific telomere-length measurements in relatively small human cardiac samples and offers an attractive technique to test hypotheses implicating telomere length in various cardiac pathologies. The current protocol (from tissue collection to image procurement) takes ∼28 h along with three overnight incubations. We anticipate that the protocol could be easily adapted for use on different tissue types.

Metal- and additive-free photoinduced borylation of haloarenes.

PubMed

Mfuh, Adelphe M; Schneider, Brett D; Cruces, Westley; Larionov, Oleg V

2017-03-01

Boronic acids and esters have critical roles in the areas of synthetic organic chemistry, molecular sensors, materials science, drug discovery, and catalysis. Many of the current applications of boronic acids and esters require materials with very low levels of transition metal contamination. Most of the current methods for the synthesis of boronic acids, however, require transition metal catalysts and ligands that must be removed via additional purification procedures. This protocol describes a simple, metal- and additive-free method of conversion of haloarenes directly to boronic acids and esters. This photoinduced borylation protocol does not require expensive and toxic metal catalysts or ligands, and it produces innocuous and easy-to-remove by-products. Furthermore, the reaction can be carried out on multigram scales in common-grade solvents without the need for reaction mixtures to be deoxygenated. The setup and purification steps are typically accomplished within 1-3 h. The reactions can be run overnight, and the protocol can be completed within 13-16 h. Two representative procedures that are described in this protocol provide details for preparation of a boronic acid (3-cyanopheylboronic acid) and a boronic ester (1,4-benzenediboronic acid bis(pinacol)ester). We also discuss additional details of the method that will be helpful in the application of the protocol to other haloarene substrates.

A protocol using coho salmon to monitor Tongass National Forest Land and Resource Management Plan standards and guidelines for fish habitat.

Treesearch

M.D. Bryant; Trent McDonald; R. Aho; B.E. Wright; Michelle Bourassa Stahl

2008-01-01

We describe a protocol to monitor the effectiveness of the Tongass Land Management Plan (TLMP) management standards for maintaining fish habitat. The protocol uses juvenile coho salmon (Oncorhynchus kisutch) in small tributary streams in forested watersheds. We used a 3-year pilot study to develop detailed methods to estimate juvenile salmonid...

Single-step affinity purification for fungal proteomics.

PubMed

Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

2010-05-01

A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

A Method to Identify Nucleolus-Associated Chromatin Domains (NADs).

PubMed

Carpentier, Marie-Christine; Picart-Picolo, Ariadna; Pontvianne, Frédéric

2018-01-01

The nuclear context needs to be taken into consideration to better understand the mechanisms shaping the epigenome and its organization, and therefore its impact on gene expression. For example, in Arabidopsis, heterochromatin is preferentially localized at the nuclear and the nucleolar periphery. Although chromatin domains associating with the nuclear periphery remain to be identified in plant cells, Nucleolus Associated chromatin Domains (NADs) can be identified thanks to a protocol allowing the isolation of pure nucleoli. We describe here the protocol enabling the identification of NADs in Arabidopsis. Providing the transfer of a nucleolus marker as described here in other crop species, this protocol is broadly applicable.

An Analysis of Transmission and Storage Gains from Sliding Checksum Methods

DTIC Science & Technology

1998-11-01

analysed a protocol "rsync" for synchronising related files at different ends of a communications channel with a minimum of transmitted data. This report...Sliding Checksum Methods Executive Summary In a previous report we described, modelled and analysed a protocol "rsync" for synchronising related...collaborative writing of documentation and synchronisation of distributed databases in the situation where no one location is aware of the differences

FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry

PubMed Central

Arrigucci, Riccardo; Bushkin, Yuri; Radford, Felix; Lakehal, Karim; Vir, Pooja; Pine, Richard; Martin, December; Sugarman, Jeffrey; Zhao, Yanlin; Yap, George S; Lardizabal, Alfred A; Tyagi, Sanjay; Gennaro, Maria Laura

2017-01-01

We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ~30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described. PMID:28518171

Protocols for efficient simulations of long-time protein dynamics using coarse-grained CABS model.

PubMed

Jamroz, Michal; Kolinski, Andrzej; Kmiecik, Sebastian

2014-01-01

Coarse-grained (CG) modeling is a well-acknowledged simulation approach for getting insight into long-time scale protein folding events at reasonable computational cost. Depending on the design of a CG model, the simulation protocols vary from highly case-specific-requiring user-defined assumptions about the folding scenario-to more sophisticated blind prediction methods for which only a protein sequence is required. Here we describe the framework protocol for the simulations of long-term dynamics of globular proteins, with the use of the CABS CG protein model and sequence data. The simulations can start from a random or a selected (e.g., native) structure. The described protocol has been validated using experimental data for protein folding model systems-the prediction results agreed well with the experimental results.

Clinical protocol with digital cad/cam chairside workflow for the rehabilitation of severely worn dentition patients

PubMed Central

GERMANO, FED.; GERMANO, FRA.; PIRO, M.; ARCURI, C.; OTTRIA, L.

2017-01-01

SUMMARY Objectives The purpose of this paper is to investigate the effectiveness and to describe a clinical protocol with digital CAD CAM chairside workflow for the rehabilitation of severely compromised and worn dentitions. Methods This article reports 4 consecutive cases, where a clinical digital chairside workflow is used for the rehabilitation of severely compromised and worn dentitions. Advantages and limitations of this method compared with the traditional prosthetic protocol are also described and discussed. Results With all four patients treated with this protocol, we obtained a good aesthetic and functional result, improvement in chewing function, loss of cold sensitivity, better preservation of most of the left hard tissue and a good level of satisfaction. In a two-year follow-up, all patients also maintained the condition obtained with prosthetic chairside rehabilitation, resulting in almost 100% cumulative survival rate. Conclusion Within the limitations of this study, we can assert that the aforementioned restorative treatment with digital CAD/CAM chairside workflow represents a valid alternative to rehabilitate this kind of patients, because it is a safe, predictable and personalized procedure but also it seems easier, faster and cheaper than traditional protocols. PMID:29285327

Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors.

PubMed

Kutner, Robert H; Zhang, Xian-Yang; Reiser, Jakob

2009-01-01

Over the past decade, lentiviral vectors have emerged as powerful tools for transgene delivery. The use of lentiviral vectors has become commonplace and applications in the fields of neuroscience, hematology, developmental biology, stem cell biology and transgenesis are rapidly emerging. Also, lentiviral vectors are at present being explored in the context of human clinical trials. Here we describe improved protocols to generate highly concentrated lentiviral vector pseudotypes involving different envelope glycoproteins. In this protocol, vector stocks are prepared by transient transfection using standard cell culture media or serum-free media. Such stocks are then concentrated by ultracentrifugation and/or ion exchange chromatography, or by precipitation using polyethylene glycol 6000, resulting in vector titers of up to 10(10) transducing units per milliliter and above. We also provide reliable real-time PCR protocols to titrate lentiviral vectors based on proviral DNA copies present in genomic DNA extracted from transduced cells or on vector RNA. These production/concentration methods result in high-titer vector preparations that show reduced toxicity compared with lentiviral vectors produced using standard protocols involving ultracentrifugation-based methods. The vector production and titration protocol described here can be completed within 8 d.

Bacterial molecular networks: bridging the gap between functional genomics and dynamical modelling.

PubMed

van Helden, Jacques; Toussaint, Ariane; Thieffry, Denis

2012-01-01

This introductory review synthesizes the contents of the volume Bacterial Molecular Networks of the series Methods in Molecular Biology. This volume gathers 9 reviews and 16 method chapters describing computational protocols for the analysis of metabolic pathways, protein interaction networks, and regulatory networks. Each protocol is documented by concrete case studies dedicated to model bacteria or interacting populations. Altogether, the chapters provide a representative overview of state-of-the-art methods for data integration and retrieval, network visualization, graph analysis, and dynamical modelling.

Where Words Fail, Music Speaks: A Mixed Method Study of an Evidence-Based Music Protocol.

PubMed

Daniels, Ruby A; Torres, David; Reeser, Cathy

2016-01-01

Despite numerous studies documenting the benefits of music, hospice social workers are often unfamiliar with evidence-based music practices that may improve end of life care. This mixed method study tested an intervention to teach hospice social workers and chaplains (N = 10) an evidence-based music protocol. Participants used the evidence-based practice (EBP) for 30 days, recording 226 journal entries that described observations of 84 patients and their families. There was a significant increase in EBP knowledge (35%). Prompting behavioral and emotional responses, music was described frequently as a catalyst that facilitated deeper dialogue between patients, families, social workers, and chaplains.

Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

PubMed

Leonard, Annemarie K; Loughran, Elizabeth A; Klymenko, Yuliya; Liu, Yueying; Kim, Oleg; Asem, Marwa; McAbee, Kevin; Ravosa, Matthew J; Stack, M Sharon

2018-01-01

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders. © 2018 Elsevier Inc. All rights reserved.

Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering.

PubMed

Shin, Jongoh; Lee, Namil; Cho, Suhyung; Cho, Byung-Kwan

2018-01-01

Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects. Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.

A COMPARISON OF SINGLE AND MULTIPLE HABITAT RAPID BIOASSESSMENT SAMPLING METHODS FOR MACROINVERTEBRATES IN PIEDMONT AND NORTHERN PIEDMONT STREAMS

EPA Science Inventory

Stream macroinvertebrate collection methods described in the Rapid Bioassessment Protocols (RBPs) have been used widely throughout the U.S. The first edition of the RBP manual in 1989 described a single habitat approach that focused on riffles and runs, where macroinvertebrate d...

Measuring the Levels of Ribonucleotides Embedded in Genomic DNA.

PubMed

Meroni, Alice; Nava, Giulia M; Sertic, Sarah; Plevani, Paolo; Muzi-Falconi, Marco; Lazzaro, Federico

2018-01-01

Ribonucleotides (rNTPs) are incorporated into genomic DNA at a relatively high frequency during replication. They have beneficial effects but, if not removed from the chromosomes, increase genomic instability. Here, we describe a fast method to easily estimate the amounts of embedded ribonucleotides into the genome. The protocol described is performed in Saccharomyces cerevisiae and allows us to quantify altered levels of rNMPs due to different mutations in the replicative polymerase ε. However, this protocol can be easily applied to cells derived from any organism.

Measuring Feedforward Inhibition and Its Impact on Local Circuit Function.

PubMed

Hull, Court

2017-05-01

This protocol describes a series of approaches to measure feedforward inhibition in acute brain slices from the cerebellar cortex. Using whole-cell voltage and current clamp recordings from Purkinje cells in conjunction with electrical stimulation of the parallel fibers, these methods demonstrate how to measure the relationship between excitation and inhibition in a feedforward circuit. This protocol also describes how to measure the impact of feedforward inhibition on Purkinje cell excitability, with an emphasis on spike timing. © 2017 Cold Spring Harbor Laboratory Press.

Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred.

PubMed

Raji, Jennifer A; Frame, Bronwyn; Little, Daniel; Santoso, Tri Joko; Wang, Kan

2018-01-01

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell's ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14 days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T 0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16-21 weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

A field protocol to monitor cavity-nesting birds

Treesearch

J. Dudley; V. Saab

2003-01-01

We developed a field protocol to monitor populations of cavity-nesting birds in burned and unburned coniferous forests of western North America. Standardized field methods are described for implementing long-term monitoring strategies and for conducting field research to evaluate the effects of habitat change on cavity-nesting birds. Key references (but not...

Monitoring wilderness stream ecosystems

Treesearch

Jeffrey C. Davis; G. Wayne Minshall; Christopher T. Robinson; Peter Landres

2001-01-01

A protocol and methods for monitoring the major physical, chemical, and biological components of stream ecosystems are presented. The monitoring protocol is organized into four stages. At stage 1 information is obtained on a basic set of parameters that describe stream ecosystems. Each following stage builds upon stage 1 by increasing the number of parameters and the...

Antimicrobial Testing Methods & Procedures: MB-03-07

EPA Pesticide Factsheets

This protocol describes the procedures for the preparation of carriers for the following methods: the AOAC UDM, AOAC Tuberculocidal Activity of Disinfectants Test, AOAC GSPT, the Disinfectant Towelette Test, and the AOAC SAT.

Chapter 1: Introduction. The Uniform Methods Project: Methods for Determining Energy-Efficiency Savings for Specific Measures

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

Li, Michael; Haeri, Hossein; Reynolds, Arlis

This chapter provides a set of model protocols for determining energy and demand savings that result from specific energy efficiency measures implemented through state and utility efficiency programs. The methods described here are approaches that are or are among the most commonly used and accepted in the energy efficiency industry for certain measures or programs. As such, they draw from the existing body of research and best practices for energy efficiency program evaluation, measurement, and verification (EM&V). These protocols were developed as part of the Uniform Methods Project (UMP), funded by the U.S. Department of Energy (DOE). The principal objectivemore » for the project was to establish easy-to-follow protocols based on commonly accepted methods for a core set of widely deployed energy efficiency measures.« less

High-throughput real-time quantitative reverse transcription PCR.

PubMed

Bookout, Angie L; Cummins, Carolyn L; Mangelsdorf, David J; Pesola, Jean M; Kramer, Martha F

2006-02-01

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Novel methodology to isolate microplastics from vegetal-rich samples.

PubMed

Herrera, Alicia; Garrido-Amador, Paloma; Martínez, Ico; Samper, María Dolores; López-Martínez, Juan; Gómez, May; Packard, Theodore T

2018-04-01

Microplastics are small plastic particles, globally distributed throughout the oceans. To properly study them, all the methodologies for their sampling, extraction, and measurement should be standardized. For heterogeneous samples containing sediments, animal tissues and zooplankton, several procedures have been described. However, definitive methodologies for samples, rich in algae and plant material, have not yet been developed. The aim of this study was to find the best extraction protocol for vegetal-rich samples by comparing the efficacies of five previously described digestion methods, and a novel density separation method. A protocol using 96% ethanol for density separation was better than the five digestion methods tested, even better than using H 2 O 2 digestion. As it was the most efficient, simple, safe and inexpensive method for isolating microplastics from vegetal rich samples, we recommend it as a standard separation method. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

Copper(I)/TEMPO Catalyzed Aerobic Oxidation of Primary Alcohols to Aldehydes with Ambient Air

PubMed Central

Hoover, Jessica M.; Steves, Janelle E.; Stahl, Shannon S.

2012-01-01

This protocol describes a practical laboratory-scale method for aerobic oxidation of primary alcohols to aldehydes, using a chemoselective CuI/TEMPO catalyst system. The catalyst is prepared in situ from commercially available reagents, and the reactions are performed in a common organic solvent (acetonitrile) with ambient air as the oxidant. Three different reaction conditions and three procedures for the isolation and purification of the aldehyde product are presented. The oxidations of eight different alcohols, described here, include representative examples of each reaction condition and purification method. Reaction times vary from 20 min to 24 h, depending on the alcohol, while the purification methods each take about 2 h. The total time necessary for the complete protocol ranges from 3 – 26 h. PMID:22635108

Extraction of High Quality DNA from Seized Moroccan Cannabis Resin (Hashish)

PubMed Central

El Alaoui, Moulay Abdelaziz; Melloul, Marouane; Alaoui Amine, Sanaâ; Stambouli, Hamid; El Bouri, Aziz; Soulaymani, Abdelmajid; El Fahime, Elmostafa

2013-01-01

The extraction and purification of nucleic acids is the first step in most molecular biology analysis techniques. The objective of this work is to obtain highly purified nucleic acids derived from Cannabis sativa resin seizure in order to conduct a DNA typing method for the individualization of cannabis resin samples. To obtain highly purified nucleic acids from cannabis resin (Hashish) free from contaminants that cause inhibition of PCR reaction, we have tested two protocols: the CTAB protocol of Wagner and a CTAB protocol described by Somma (2004) adapted for difficult matrix. We obtained high quality genomic DNA from 8 cannabis resin seizures using the adapted protocol. DNA extracted by the Wagner CTAB protocol failed to give polymerase chain reaction (PCR) amplification of tetrahydrocannabinolic acid (THCA) synthase coding gene. However, the extracted DNA by the second protocol permits amplification of THCA synthase coding gene using different sets of primers as assessed by PCR. We describe here for the first time the possibility of DNA extraction from (Hashish) resin derived from Cannabis sativa. This allows the use of DNA molecular tests under special forensic circumstances. PMID:24124454

Revised Methods for Characterizing Stream Habitat in the National Water-Quality Assessment Program

USGS Publications Warehouse

Fitzpatrick, Faith A.; Waite, Ian R.; D'Arconte, Patricia J.; Meador, Michael R.; Maupin, Molly A.; Gurtz, Martin E.

1998-01-01

Stream habitat is characterized in the U.S. Geological Survey's National Water-Quality Assessment (NAWQA) Program as part of an integrated physical, chemical, and biological assessment of the Nation's water quality. The goal of stream habitat characterization is to relate habitat to other physical, chemical, and biological factors that describe water-quality conditions. To accomplish this goal, environmental settings are described at sites selected for water-quality assessment. In addition, spatial and temporal patterns in habitat are examined at local, regional, and national scales. This habitat protocol contains updated methods for evaluating habitat in NAWQA Study Units. Revisions are based on lessons learned after 6 years of applying the original NAWQA habitat protocol to NAWQA Study Unit ecological surveys. Similar to the original protocol, these revised methods for evaluating stream habitat are based on a spatially hierarchical framework that incorporates habitat data at basin, segment, reach, and microhabitat scales. This framework provides a basis for national consistency in collection techniques while allowing flexibility in habitat assessment within individual Study Units. Procedures are described for collecting habitat data at basin and segment scales; these procedures include use of geographic information system data bases, topographic maps, and aerial photographs. Data collected at the reach scale include channel, bank, and riparian characteristics.

A method for acetylcholinesterase staining of brain sections previously processed for receptor autoradiography.

PubMed

Lim, M M; Hammock, E A D; Young, L J

2004-02-01

Receptor autoradiography using selective radiolabeled ligands allows visualization of brain receptor distribution and density on film. The resolution of specific brain regions on the film often can be difficult to discern owing to the general spread of the radioactive label and the lack of neuroanatomical landmarks on film. Receptor binding is a chemically harsh protocol that can render the tissue virtually unstainable by Nissl and other conventional stains used to delineate neuroanatomical boundaries of brain regions. We describe a method for acetylcholinesterase (AChE) staining of slides previously processed for receptor binding. AChE staining is a useful tool for delineating major brain nuclei and tracts. AChE staining on sections that have been processed for receptor autoradiography provides a direct comparison of brain regions for more precise neuroanatomical description. We report a detailed thiocholine protocol that is a modification of the Koelle-Friedenwald method to amplify the AChE signal in brain sections previously processed for autoradiography. We also describe several temporal and experimental factors that can affect the density and clarity of the AChE signal when using this protocol.

Direct Measurement of Intracellular Pressure

PubMed Central

Petrie, Ryan J.; Koo, Hyun

2014-01-01

A method to directly measure the intracellular pressure of adherent, migrating cells is described in the Basic Protocol. This approach is based on the servo-null method where a microelectrode is introduced into the cell to directly measure the physical pressure of the cytoplasm. We also describe the initial calibration of the microelectrode as well as the application of the method to cells migrating inside three-dimensional (3D) extracellular matrix (ECM). PMID:24894836

Detection of isotype switch rearrangement in bulk culture by PCR.

PubMed

Max, E E; Mills, F C; Chu, C

2001-05-01

When a B lymphocyte changes from synthesizing IgM to synthesizing IgG, IgA, or IgE, this isotype switch is generally accompanied by a unique DNA rearrangement. The protocols in this unit describe two polymerase chain reaction (PCR)-based strategies for detecting switch rearrangements in bulk culture. The first involves direct PCR across the switch junctions, providing the opportunity for characterizing the recombination products by nucleotide sequence analysis; however, because of characteristics inherent to the PCR methodology this strategy cannot easily be used as a quantitative assay for recombination. A support protocol details the preparation of the 5' Su PCR probe for this protocol. The second basic protocol describes a method known as digestion-circularization PCR (DCPCR) that is more amenable to quantitation but yields no information on structure of the recombination products. Both techniques should be capable of detecting reciprocal deletion circles as well as functional recombination products remaining on the expressed chromosome.

Animal Models of Mycobacteria Infection

PubMed Central

Ordway, Diane J.; Orme, Ian M.

2011-01-01

This unit describes the infection of mice and guinea pigs with mycobacteria via various routes, as well as necropsy methods for the determination of mycobacterial loads within target organs. Additionally, methods for cultivating mycobacteria and preparing stocks are described. The protocols outlined are primarily used for M. tuberculosis, but can also be used for the study of other non-tuberculosis mycobacterial species. PMID:18432756

Polyethylene glycol 3350 based colon cleaning protocol: 2 d vs 4 d head to head comparison.

PubMed

Elitsur, Rotem; Butcher, Lisa; Vicki, Lund; Elitsur, Yoram

2013-04-16

To compare between 2 and 4 d colon cleansing protocols. Children who were scheduled for colonoscopy procedure (2010-2012) for various medical reasons, were recruited from the pediatric gastroenterology clinic at Marshall University School of Medicine, Huntington, WV. Exclusion criteria were patients who were allergic to the medication used in the protocols [polyethylene glycol (PEG) 3350, Bisacodyl], or children with metabolic or renal diseases. Two PEG 3350 protocols for 4 d (A) and 2 d (B) were prescribed as previously described. A questionnaire describing the volume of PEG consumed, clinical data, and side effects were recorded. Colon preparation was graded by two observers according to previously described method. Rate of adequate colon preparation. A total of 78 patients were considered for final calculation (group A: 40, group B: 38). Age and stool consistency at the last day was comparable in both groups, but the number of stools/day was significantly higher in group B (P = 0.001). Adequate colon preparation was reached in 57.5% (A) and 73.6% (B), respectively (P = 0.206). Side effects were minimal and comparable in both groups. There was no difference in children's age, stool characteristics, or side effects between the children with adequate or inadequate colon preparation. Correlation and agreement between observers was excellent (Pearson correlation = 0.972, kappa = 1.0). No difference between protocols was observed, but the 2 d protocol was superior for its shorter time. Direct comparison between different colon cleansing protocols is crucial in order to establish the "gold standard" protocol for children.

Hysterectomy for placenta accreta; methods for gross and microscopic pathology examination.

PubMed

Dannheim, Katelyn; Shainker, Scott A; Hecht, Jonathan L

2016-05-01

Placenta accreta is an abnormal adherence of the placenta to the uterine wall. As the incidence of placenta accreta continues to rise, it has been useful to develop standard protocols for the diagnosis and management of affected patients. Pathologists have the opportunity to take an active role in evaluating these resource intensive protocols. We describe methods of gross dissection, microscopic examination and reporting of hysterectomy specimens containing placenta accreta. This protocol facilitates retrospective correlation with surgical and radiographic findings as well as standardized tissue sampling for potential research. Through regular review of such quality measures pathologists can give feedback on the quality of surgical planning and use of imaging.

Method for training honeybees to respond to olfactory stimuli and enhancement of memory retention therein

DOEpatents

McCade, Kirsten J.; Wingo, Robert M.; Haarmann, Timothy K.; Sutherland, Andrew; Gubler, Walter D.

2015-12-15

A specialized conditioning protocol for honeybees that is designed for use within a complex agricultural ecosystem. This method ensures that the conditioned bees will be less likely to exhibit a conditioned response to uninfected plants, a false positive response that would render such a biological sensor unreliable for agricultural decision support. Also described is a superboosting training regime that allows training without the aid of expensive equipment and protocols for training in out in the field. Also described is a memory enhancing cocktail that aids in long term memory retention of a vapor signature. This allows the bees to be used in the field for longer durations and with fewer bees trained overall.

Reconstitution of mouse oogenesis in a dish from pluripotent stem cells.

PubMed

Hayashi, Katsuhiko; Hikabe, Orie; Obata, Yayoi; Hirao, Yuji

2017-09-01

This protocol is an extension to: Nat. Protoc. 8, 1513-1524 (2013); doi: 10.1038/nprot.2013.090; published online 11 July 2013Generation of functional oocytes in culture from pluripotent stem cells should provide a useful model system for improving our understanding of the basic mechanisms underlying oogenesis. In addition, it has potential applications as an alternative source of oocytes for reproduction. Using the most advanced mouse model in regard to reproductive engineering and stem cell biology, we previously developed a culture method that produces functional primorial germ cells starting from pluripotent cells in culture and described it in a previous protocol. This Protocol Extension describes an adaptation of this existing Protocol in which oogenesis also occurs in vitro, thus substantially modifying the technique. Oocytes generated from embryonic stem cells (ESCs) or induced pluripotent stem cells give rise to healthy pups. Here, we describe the protocol for oocyte generation in culture. The protocol is mainly composed of three different culture stages: in vitro differentiation (IVDi), in vitro growth (IVG), and in vitro maturation (IVM), which in total take ∼5 weeks. In each culture period, there are several checkpoints that enable the number of oocytes being produced in the culture to be monitored. The basic structure of the culture system should provide a useful tool for clarifying the complicated sequence of oogenesis in mammals.

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines.

PubMed

Aspinall-O'Dea, Mark; Pierce, Andrew; Pellicano, Francesca; Williamson, Andrew J; Scott, Mary T; Walker, Michael J; Holyoake, Tessa L; Whetton, Anthony D

2015-01-01

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein CrkL, a major substrate of the oncogenic tyrosine kinase BCR-ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <10(4) cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-CrkL and the protein tyrosine phosphatase PTPRC/CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents.

Chapter 10: Peak Demand and Time-Differentiated Energy Savings Cross-Cutting Protocol. The Uniform Methods Project: Methods for Determining Energy Efficiency Savings for Specific Measures

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

Kurnik, Charles W; Stern, Frank; Spencer, Justin

Savings from electric energy efficiency measures and programs are often expressed in terms of annual energy and presented as kilowatt-hours per year (kWh/year). However, for a full assessment of the value of these savings, it is usually necessary to consider the measure or program's impact on peak demand as well as time-differentiated energy savings. This cross-cutting protocol describes methods for estimating the peak demand and time-differentiated energy impacts of measures implemented through energy efficiency programs.

A protocol for lifetime energy and environmental impact assessment of building insulation materials

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

Shrestha, Som S., E-mail: shresthass@ornl.gov; Biswas, Kaushik; Desjarlais, Andre O.

This article describes a proposed protocol that is intended to provide a comprehensive list of factors to be considered in evaluating the direct and indirect environmental impacts of building insulation materials, as well as detailed descriptions of standardized calculation methodologies to determine those impacts. The energy and environmental impacts of insulation materials can generally be divided into two categories: (1) direct impact due to the embodied energy of the insulation materials and other factors and (2) indirect or environmental impacts avoided as a result of reduced building energy use due to addition of insulation. Standards and product category rules exist,more » which provide guidelines about the life cycle assessment (LCA) of materials, including building insulation products. However, critical reviews have suggested that these standards fail to provide complete guidance to LCA studies and suffer from ambiguities regarding the determination of the environmental impacts of building insulation and other products. The focus of the assessment protocol described here is to identify all factors that contribute to the total energy and environmental impacts of different building insulation products and, more importantly, provide standardized determination methods that will allow comparison of different insulation material types. Further, the intent is not to replace current LCA standards but to provide a well-defined, easy-to-use comparison method for insulation materials using existing LCA guidelines. - Highlights: • We proposed a protocol to evaluate the environmental impacts of insulation materials. • The protocol considers all life cycle stages of an insulation material. • Both the direct environmental impacts and the indirect impacts are defined. • Standardized calculation methods for the ‘avoided operational energy’ is defined. • Standardized calculation methods for the ‘avoided environmental impact’ is defined.« less

Gene Delivery to the Airway

PubMed Central

Keiser, Nicholas W.; Engelhardt, John F.

2013-01-01

This unit describes generation of and gene transfer to several commonly used airway models. Isolation and transduction of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined. Transduction of these polarized cells is also described. Methods are presented for generation of tracheal xenografts as well as both ex vivo and in vivo gene transfer to these xenografts. Finally, a method for in vivo gene delivery to the lungs of rodents is included. Methods for evaluating transgene expression are given in the support protocols. PMID:23853081

Micro-chromatin Immunoprecipation (μChIP) Protocol for Real-time PCR Analysis of a Limited Amount of Cells.

PubMed

Gillotin, Sébastien; Guillemot, François

2016-06-20

Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is an important strategy to study gene regulation. When availability of cells is limited, however, it can be useful to focus on specific genes to investigate in depth the role of transcription factors or histone marks. Unfortunately, performing ChIP experiments to study transcription factors' binding to DNA can be difficult when biological material is restricted. This protocol describes a robust method to perform μChIP for over-expressed or endogenous transcription factors using ~100,000 cells per ChIP experiment (Masserdotti et al ., 2015). We also describe optimization steps, which we think are critical for this protocol to work and which can be used to further reduce the number of cells.

Verification and validation of a reliable multicast protocol

NASA Technical Reports Server (NTRS)

Callahan, John R.; Montgomery, Todd L.

1995-01-01

This paper describes the methods used to specify and implement a complex communications protocol that provides reliable delivery of data in multicast-capable, packet-switching telecommunication networks. The protocol, called the Reliable Multicasting Protocol (RMP), was developed incrementally by two complementary teams using a combination of formal and informal techniques in an attempt to ensure the correctness of the protocol implementation. The first team, called the Design team, initially specified protocol requirements using a variant of SCR requirements tables and implemented a prototype solution. The second team, called the V&V team, developed a state model based on the requirements tables and derived test cases from these tables to exercise the implementation. In a series of iterative steps, the Design team added new functionality to the implementation while the V&V team kept the state model in fidelity with the implementation through testing. Test cases derived from state transition paths in the formal model formed the dialogue between teams during development and served as the vehicles for keeping the model and implementation in fidelity with each other. This paper describes our experiences in developing our process model, details of our approach, and some example problems found during the development of RMP.

Writing standard operating procedures (SOPs) for cryostorage protocols: using shoot meristem cryopreservation as an example.

PubMed

Harding, Keith; Benson, Erica E

2015-01-01

Standard operating procedures are a systematic way of making sure that biopreservation processes, tasks, protocols, and operations are correctly and consistently performed. They are the basic documents of biorepository quality management systems and are used in quality assurance, control, and improvement. Methodologies for constructing workflows and writing standard operating procedures and work instructions are described using a plant cryopreservation protocol as an example. This chapter is pertinent to other biopreservation sectors because how methods are written, interpreted, and implemented can affect the quality of storage outcomes.

Handling and restraint.

PubMed

Donovan, John; Brown, Patricia

2006-07-01

For the safety of the handler and the animal, proper methods for handling and restraining laboratory animals should be followed. Improper handling can result in increased stress and injury to the animal. In addition, the handler risks injury from bite wounds or scratches inflicted when the animal becomes fearful or anxious. By using sure, direct movements with a determined attitude, the animal can be easily handled and restrained. Animals can be restrained either manually or in a plastic restrainer. The protocols in this unit describe handling and manual restraint of mice, rats, hamsters, and rabbits. Alternate protocols describe restraint using the plastic restrainer.

Handling and restraint.

PubMed

Donovan, John; Brown, Patricia

2004-09-01

For the safety of the handler and the animal, proper methods for handling and restraining laboratory animals should be followed. Improper handling can result in increased stress and injury to the animal. In addition, the handler risks injury from bite wounds or scratches inflicted when the animal becomes fearful or anxious. By using sure, direct movements with a determined attitude, the animal can be easily handled and restrained. Animals can be restrained either manually or in a plastic restrainer. The protocols in this unit describe handling and manual restraint of mice, rats, hamsters, and rabbits. Alternate protocols describe restraint using the plastic restrainer.

A Web Resource for Standardized Benchmark Datasets, Metrics, and Rosetta Protocols for Macromolecular Modeling and Design.

PubMed

Ó Conchúir, Shane; Barlow, Kyle A; Pache, Roland A; Ollikainen, Noah; Kundert, Kale; O'Meara, Matthew J; Smith, Colin A; Kortemme, Tanja

2015-01-01

The development and validation of computational macromolecular modeling and design methods depend on suitable benchmark datasets and informative metrics for comparing protocols. In addition, if a method is intended to be adopted broadly in diverse biological applications, there needs to be information on appropriate parameters for each protocol, as well as metrics describing the expected accuracy compared to experimental data. In certain disciplines, there exist established benchmarks and public resources where experts in a particular methodology are encouraged to supply their most efficient implementation of each particular benchmark. We aim to provide such a resource for protocols in macromolecular modeling and design. We present a freely accessible web resource (https://kortemmelab.ucsf.edu/benchmarks) to guide the development of protocols for protein modeling and design. The site provides benchmark datasets and metrics to compare the performance of a variety of modeling protocols using different computational sampling methods and energy functions, providing a "best practice" set of parameters for each method. Each benchmark has an associated downloadable benchmark capture archive containing the input files, analysis scripts, and tutorials for running the benchmark. The captures may be run with any suitable modeling method; we supply command lines for running the benchmarks using the Rosetta software suite. We have compiled initial benchmarks for the resource spanning three key areas: prediction of energetic effects of mutations, protein design, and protein structure prediction, each with associated state-of-the-art modeling protocols. With the help of the wider macromolecular modeling community, we hope to expand the variety of benchmarks included on the website and continue to evaluate new iterations of current methods as they become available.

Protocols for the Initial Treatment of Moderately Severe Juvenile Dermatomyositis: Results of a Children's Arthritis and Rheumatology Research Alliance Consensus Conference

PubMed Central

Huber, Adam M.; Giannini, Edward H.; Bowyer, Suzanne L.; Kim, Susan; Lang, Bianca; Lindsley, Carol B.; Pachman, Lauren M.; Pilkington, Clarissa; Reed, Ann M.; Rennebohm, Robert M.; Rider, Lisa G.; Wallace, Carol A.; Feldman, Brian M.

2010-01-01

Objective To use juvenile dermatomyositis (JDM) survey data and expert opinion to develop a small number of consensus treatment protocols which reflect current initial treatment of moderately severe JDM. Methods A consensus meeting was held in Toronto, Ontario, Canada on December 1-2, 2007. Nominal group technique was used to achieve consensus on treatment protocols which represented typical management of moderately severe JDM. Consensus was also reached on which patients these protocols would be applicable to (inclusion and exclusion criteria), initial investigations which should be done prior to initiating one of these protocols, data which should be collected to evaluate these protocols, concomitant interventions that would be required or recommended. Results Three protocols were developed which described the first 2 months of treatment. All protocols included corticosteroids and methotrexate. One protocol also included intravenous gammaglobulin. Consensus was achieved for all issues that were addressed by conference participants, although there were some areas of controversy Conclusions This study shows that it is possible to achieve consensus on the initial treatment of JDM, despite considerable variation in clinical practice. Once these protocols are extended beyond 2 months, these protocols will be available for clinical use. By using methods which account for differences between patients (confounding by indication), the comparative effectiveness of the protocols will be evaluated. In the future, the goal will be to identify the optimal treatment of moderately severe JDM. PMID:20191521

Peer Review and Publication of Research Protocols and Proposals: A Role for Open Access Journals

PubMed Central

2004-01-01

Peer-review and publication of research protocols offer several advantages to all parties involved. Among these are the following opportunities for authors: external expert opinion on the methods, demonstration to funding agencies of prior expert review of the protocol, proof of priority of ideas and methods, and solicitation of potential collaborators. We think that review and publication of protocols is an important role for Open Access journals. Because of their electronic form, openness for readers, and author-pays business model, they are better suited than traditional journals to ensure the sustainability and quality of protocol reviews and publications. In this editorial, we describe the workflow for investigators in eHealth research, from protocol submission to a funding agency, to protocol review and (optionally) publication at JMIR, to registration of trials at the International eHealth Study Registry (IESR), and to publication of the report. One innovation at JMIR is that protocol peer reviewers will be paid a honorarium, which will be drawn partly from a new submission fee for protocol reviews. Separating the article processing fee into a submission and a publishing fee will allow authors to opt for “peer-review only” (without subsequent publication) at reduced costs, if they wish to await a funding decision or for other reasons decide not to make the protocol public. PMID:15471763

Peer-review and publication of research protocols and proposals: a role for open access journals.

PubMed

Eysenbach, Gunther

2004-09-30

Peer-review and publication of research protocols offer several advantages to all parties involved. Among these are the following opportunities for authors: external expert opinion on the methods, demonstration to funding agencies of prior expert review of the protocol, proof of priority of ideas and methods, and solicitation of potential collaborators. We think that review and publication of protocols is an important role for Open Access journals. Because of their electronic form, openness for readers, and author-pays business model, they are better suited than traditional journals to ensure the sustainability and quality of protocol reviews and publications. In this editorial, we describe the workflow for investigators in eHealth research, from protocol submission to a funding agency, to protocol review and (optionally) publication at JMIR, to registration of trials at the International eHealth Study Registry (IESR), and to publication of the report. One innovation at JMIR is that protocol peer reviewers will be paid a honorarium, which will be drawn partly from a new submission fee for protocol reviews. Separating the article processing fee into a submission and a publishing fee will allow authors to opt for "peer-review only" (without subsequent publication) at reduced costs, if they wish to await a funding decision or for other reasons decide not to make the protocol public.

Validation of quantitative and qualitative methods for detecting allergenic ingredients in processed foods in Japan.

PubMed

Sakai, Shinobu; Adachi, Reiko; Akiyama, Hiroshi; Teshima, Reiko

2013-06-19

A labeling system for food allergenic ingredients was established in Japan in April 2002. To monitor the labeling, the Japanese government announced official methods for detecting allergens in processed foods in November 2002. The official methods consist of quantitative screening tests using enzyme-linked immunosorbent assays (ELISAs) and qualitative confirmation tests using Western blotting or polymerase chain reactions (PCR). In addition, the Japanese government designated 10 μg protein/g food (the corresponding allergenic ingredient soluble protein weight/food weight), determined by ELISA, as the labeling threshold. To standardize the official methods, the criteria for the validation protocol were described in the official guidelines. This paper, which was presented at the Advances in Food Allergen Detection Symposium, ACS National Meeting and Expo, San Diego, CA, Spring 2012, describes the validation protocol outlined in the official Japanese guidelines, the results of interlaboratory studies for the quantitative detection method (ELISA for crustacean proteins) and the qualitative detection method (PCR for shrimp and crab DNAs), and the reliability of the detection methods.

Hi-C 2.0: An optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation.

PubMed

Belaghzal, Houda; Dekker, Job; Gibcus, Johan H

2017-07-01

Chromosome conformation capture-based methods such as Hi-C have become mainstream techniques for the study of the 3D organization of genomes. These methods convert chromatin interactions reflecting topological chromatin structures into digital information (counts of pair-wise interactions). Here, we describe an updated protocol for Hi-C (Hi-C 2.0) that integrates recent improvements into a single protocol for efficient and high-resolution capture of chromatin interactions. This protocol combines chromatin digestion and frequently cutting enzymes to obtain kilobase (kb) resolution. It also includes steps to reduce random ligation and the generation of uninformative molecules, such as unligated ends, to improve the amount of valid intra-chromosomal read pairs. This protocol allows for obtaining information on conformational structures such as compartment and topologically associating domains, as well as high-resolution conformational features such as DNA loops. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Standard methods for fungal brood disease research

USDA-ARS?s Scientific Manuscript database

In this paper we describe the disease symptoms and protocols commonly used in research of honey bee fungal diseases chalkbrood and stonebrood, caused by Ascosphaera apis and Aspergillus spp. respectively....

ChIP-seq and RNA-seq methods to study circadian control of transcription in mammals

PubMed Central

Takahashi, Joseph S.; Kumar, Vivek; Nakashe, Prachi; Koike, Nobuya; Huang, Hung-Chung; Green, Carla B.; Kim, Tae-Kyung

2015-01-01

Genome-wide analyses have revolutionized our ability to study the transcriptional regulation of circadian rhythms. The advent of next-generation sequencing methods has facilitated the use of two such technologies, ChIP-seq and RNA-seq. In this chapter, we describe detailed methods and protocols for these two techniques, with emphasis on their usage in circadian rhythm experiments in the mouse liver, a major target organ of the circadian clock system. Critical factors for these methods are highlighted and issues arising with time series samples for ChIP-seq and RNA-seq are discussed. Finally detailed protocols for library preparation suitable for Illumina sequencing platforms are presented. PMID:25662462

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult.

PubMed

Herrlinger, Stephanie A; Shao, Qiang; Ma, Li; Brindley, Melinda; Chen, Jian-Fu

2018-04-26

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Hydroxylapatite chromatography.

PubMed

Broadhurst, A V

2001-05-01

Hydroxylapatite (also called hydroxyapatite), a form of calcium phosphate, can be used as a matrix for the chromatography of both proteins and nucleic acids. Protocols are provided for both standard low-pressure chromatography of a protein mixture using a hydroxylapatite column prepared in the laboratory, and an HPLC method, applicable to proteins and nucleic acids, that uses a commercially available column. Alternate protocols describe column chromatography using a step gradient or batch binding and step-gradient elution.

Similarity-based modeling in large-scale prediction of drug-drug interactions.

PubMed

Vilar, Santiago; Uriarte, Eugenio; Santana, Lourdes; Lorberbaum, Tal; Hripcsak, George; Friedman, Carol; Tatonetti, Nicholas P

2014-09-01

Drug-drug interactions (DDIs) are a major cause of adverse drug effects and a public health concern, as they increase hospital care expenses and reduce patients' quality of life. DDI detection is, therefore, an important objective in patient safety, one whose pursuit affects drug development and pharmacovigilance. In this article, we describe a protocol applicable on a large scale to predict novel DDIs based on similarity of drug interaction candidates to drugs involved in established DDIs. The method integrates a reference standard database of known DDIs with drug similarity information extracted from different sources, such as 2D and 3D molecular structure, interaction profile, target and side-effect similarities. The method is interpretable in that it generates drug interaction candidates that are traceable to pharmacological or clinical effects. We describe a protocol with applications in patient safety and preclinical toxicity screening. The time frame to implement this protocol is 5-7 h, with additional time potentially necessary, depending on the complexity of the reference standard DDI database and the similarity measures implemented.

Overcoming pain thresholds with multilevel models-an example using quantitative sensory testing (QST) data.

PubMed

Hirschfeld, Gerrit; Blankenburg, Markus R; Süß, Moritz; Zernikow, Boris

2015-01-01

The assessment of somatosensory function is a cornerstone of research and clinical practice in neurology. Recent initiatives have developed novel protocols for quantitative sensory testing (QST). Application of these methods led to intriguing findings, such as the presence lower pain-thresholds in healthy children compared to healthy adolescents. In this article, we (re-) introduce the basic concepts of signal detection theory (SDT) as a method to investigate such differences in somatosensory function in detail. SDT describes participants' responses according to two parameters, sensitivity and response-bias. Sensitivity refers to individuals' ability to discriminate between painful and non-painful stimulations. Response-bias refers to individuals' criterion for giving a "painful" response. We describe how multilevel models can be used to estimate these parameters and to overcome central critiques of these methods. To provide an example we apply these methods to data from the mechanical pain sensitivity test of the QST protocol. The results show that adolescents are more sensitive to mechanical pain and contradict the idea that younger children simply use more lenient criteria to report pain. Overall, we hope that the wider use of multilevel modeling to describe somatosensory functioning may advance neurology research and practice.

Solid-State NMR Studies of Amyloid Materials: A Protocol to Define an Atomic Model of Aβ(1-42) in Amyloid Fibrils.

PubMed

Xiao, Yiling; McElheny, Dan; Hoshi, Minako; Ishii, Yoshitaka

2018-01-01

Intense efforts have been made to understand the molecular structures of misfolded amyloid β (Aβ) in order to gain insight into the pathological mechanism of Alzheimer's disease. Solid-state NMR spectroscopy (SSNMR) is considered a primary tool for elucidating the structures of insoluble and noncrystalline amyloid fibrils and other amyloid assemblies. In this chapter, we describe a detailed protocol to obtain the first atomic model of the 42-residue human Aβ peptide Aβ(1-42) in structurally homogeneous amyloid fibrils from our recent SSNMR study (Nat Struct Mol Biol 22:499-505, 2015). Despite great biological and clinical interest in Aβ(1-42) fibrils, their structural details have been long-elusive until this study. The protocol is divided into four sections. First, the solid-phase peptide synthesis (SPPS) and purification of monomeric Aβ(1-42) is described. We illustrate a controlled incubation method to prompt misfolding of Aβ(1-42) into homogeneous amyloid fibrils in an aqueous solution with fragmented Aβ(1-42) fibrils as seeds. Next, we detail analysis of Aβ(1-42) fibrils by SSNMR to obtain structural restraints. Finally, we describe methods to construct atomic models of Aβ(1-42) fibrils based on SSNMR results through two-stage molecular dynamics calculations.

Aptamers and methods for their in vitro selection and uses thereof

DOEpatents

Doyle, Sharon A [Walnut Creek, CA; Murphy, Michael B [Severna Park, MD

2008-02-12

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

Aptamers and methods for their in vitro selection and uses thereof

DOEpatents

Doyle, Sharon A [Walnut Creek, CA; Murphy, Michael B [Severna Park, MD

2012-01-31

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

Protocol for Short- and Longer-term Spatial Learning and Memory in Mice

PubMed Central

Willis, Emily F.; Bartlett, Perry F.; Vukovic, Jana

2017-01-01

Studies on the role of the hippocampus in higher cognitive functions such as spatial learning and memory in rodents are reliant upon robust and objective behavioral tests. This protocol describes one such test—the active place avoidance (APA) task. This behavioral task involves the mouse continuously integrating visual cues to orientate itself within a rotating arena in order to actively avoid a shock zone, the location of which remains constant relative to the room. This protocol details the step-by-step procedures for a novel paradigm of the hippocampal-dependent APA task, measuring acquisition of spatial learning during a single 20-min trial (i.e., short-term memory), with spatial memory encoding and retrieval (i.e., long-term memory) assessed by trials conducted over consecutive days. Using the APA task, cognitive flexibility can be assessed using the reversal learning paradigm, as this increases the cognitive load required for efficient performance in the task. In addition to a detailed experimental protocol, this paper also describes the range of its possible applications, the expected key results, as well as the analytical methods to assess the data, and the pitfalls/troubleshooting measures. The protocol described herein is highly robust and produces replicable results, thus presenting an important paradigm that enables the assessment of subtle short-term changes in spatial learning and memory, such as those observed for many experimental interventions. PMID:29089878

Calcium phosphate transfection of primary hippocampal neurons.

PubMed

Sun, Miao; Bernard, Laura P; Dibona, Victoria L; Wu, Qian; Zhang, Huaye

2013-11-12

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

A Protocol Specification-Based Intrusion Detection System for VoIP and Its Evaluation

NASA Astrophysics Data System (ADS)

Phit, Thyda; Abe, Kôki

We propose an architecture of Intrusion Detection System (IDS) for VoIP using a protocol specification-based detection method to monitor the network traffics and alert administrator for further analysis of and response to suspicious activities. The protocol behaviors and their interactions are described by state machines. Traffic that behaves differently from the standard specifications are considered to be suspicious. The IDS has been implemented and simulated using OPNET Modeler, and verified to detect attacks. It was found that our system can detect typical attacks within a reasonable amount of delay time.

Using the Internet in Rural Special Education: Accessing Resources.

ERIC Educational Resources Information Center

Bull, Kay S.; Kimball, Sarah L.

This paper provides basic information on searching the Internet and describes sites of interest in the area of rural special education. The first section traces the evolution of the Internet through various phases--ARPANET, NSFNET, CERNET, and the beginnings of the World Wide Web--and describes various protocols (methods and tools) developed to…

Extended memory management under RTOS

NASA Technical Reports Server (NTRS)

Plummer, M.

1981-01-01

A technique for extended memory management in ROLM 1666 computers using FORTRAN is presented. A general software system is described for which the technique can be ideally applied. The memory manager interface with the system is described. The protocols by which the manager is invoked are presented, as well as the methods used by the manager.

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

Proposal for founding mistrustful quantum cryptography on coin tossing

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

Kent, Adrian; Hewlett-Packard Laboratories, Filton Road, Stoke Gifford, Bristol BS34 8QZ,

2003-07-01

A significant branch of classical cryptography deals with the problems which arise when mistrustful parties need to generate, process, or exchange information. As Kilian showed a while ago, mistrustful classical cryptography can be founded on a single protocol, oblivious transfer, from which general secure multiparty computations can be built. The scope of mistrustful quantum cryptography is limited by no-go theorems, which rule out, inter alia, unconditionally secure quantum protocols for oblivious transfer or general secure two-party computations. These theorems apply even to protocols which take relativistic signaling constraints into account. The best that can be hoped for, in general, aremore » quantum protocols which are computationally secure against quantum attack. Here a method is described for building a classically certified bit commitment, and hence every other mistrustful cryptographic task, from a secure coin-tossing protocol. No security proof is attempted, but reasons are sketched why these protocols might resist quantum computational attack.« less

Optimization of methods for the genetic modification of human T cells.

PubMed

Bilal, Mahmood Y; Vacaflores, Aldo; Houtman, Jon Cd

2015-11-01

CD4(+) T cells are not only critical in the fight against parasitic, bacterial and viral infections, but are also involved in many autoimmune and pathological disorders. Studies of protein function in human T cells are confined to techniques such as RNA interference (RNAi) owing to ethical reasons and relative simplicity of these methods. However, introduction of RNAi or genes into primary human T cells is often hampered by toxic effects from transfection or transduction methods that yield cell numbers inadequate for downstream assays. Additionally, the efficiency of recombinant DNA expression is frequently low because of multiple factors including efficacy of the method and strength of the targeting RNAs. Here, we describe detailed protocols that will aid in the study of primary human CD4(+) T cells. First, we describe a method for development of effective microRNA/shRNAs using available online algorithms. Second, we illustrate an optimized protocol for high efficacy retroviral or lentiviral transduction of human T-cell lines. Importantly, we demonstrate that activated primary human CD4(+) T cells can be transduced efficiently with lentiviruses, with a highly activated population of T cells receiving the largest number of copies of integrated DNA. We also illustrate a method for efficient lentiviral transduction of hard-to-transduce un-activated primary human CD4(+) T cells. These protocols will significantly assist in understanding the activation and function of human T cells and will ultimately aid in the development or improvement of current drugs that target human CD4(+) T cells.

Proteomic Analysis of Cytoskeleton Proteins in Fish.

PubMed

Gotesman, Michael; Menanteau-Ledouble, Simon; El-Matbouli, Mansour

2016-01-01

In this chapter, we describe laboratory protocols for rearing fish and a simple and efficient method of extracting and identifying pathogen and host proteins that may be involved in entry and replication of commercially important fish viruses. We have used the common carp (Cyprinus carpio L.) and goldfish (Cyprinus auratus) as a model system for studies of proteins involved in viral entry and replication. The chapter describes detailed protocols for maintenance of carp, cell culture, antibody purification of proteins, and use of electrospray-ionization mass spectrometry analysis to screen and identify cytoskeleton and other proteins that may be involved in viral infection and propagation in fish.

Standardized terminology for clinical trial protocols based on top-level ontological categories.

PubMed

Heller, B; Herre, H; Lippoldt, K; Loeffler, M

2004-01-01

This paper describes a new method for the ontologically based standardization of concepts with regard to the quality assurance of clinical trial protocols. We developed a data dictionary for medical and trial-specific terms in which concepts and relations are defined context-dependently. The data dictionary is provided to different medical research networks by means of the software tool Onto-Builder via the internet. The data dictionary is based on domain-specific ontologies and the top-level ontology of GOL. The concepts and relations described in the data dictionary are represented in natural language, semi-formally or formally according to their use.

Protocol and standard operating procedures for common use in a worldwide multicenter study on reference values.

PubMed

Ozarda, Yesim; Ichihara, Kiyoshi; Barth, Julian H; Klee, George

2013-05-01

The reference intervals (RIs) given in laboratory reports have an important role in aiding clinicians in interpreting test results in reference to values of healthy populations. In this report, we present a proposed protocol and standard operating procedures (SOPs) for common use in conducting multicenter RI studies on a national or international scale. The protocols and consensus on their contents were refined through discussions in recent C-RIDL meetings. The protocol describes in detail (1) the scheme and organization of the study, (2) the target population, inclusion/exclusion criteria, ethnicity, and sample size, (3) health status questionnaire, (4) target analytes, (5) blood collection, (6) sample processing and storage, (7) assays, (8) cross-check testing, (9) ethics, (10) data analyses, and (11) reporting of results. In addition, the protocol proposes the common measurement of a panel of sera when no standard materials exist for harmonization of test results. It also describes the requirements of the central laboratory, including the method of cross-check testing between the central laboratory of each country and local laboratories. This protocol and the SOPs remain largely exploratory and may require a reevaluation from the practical point of view after their implementation in the ongoing worldwide study. The paper is mainly intended to be a basis for discussion in the scientific community.

Detection of proteins on blot transfer membranes.

PubMed

Sasse, Joachim; Gallagher, Sean R

2003-11-01

In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.

Copper-catalyzed oxidative C-O bond formation of 2-acyl phenols and 1,3-dicarbonyl compounds with ethers: direct access to phenol esters and enol esters.

PubMed

Park, Jihye; Han, Sang Hoon; Sharma, Satyasheel; Han, Sangil; Shin, Youngmi; Mishra, Neeraj Kumar; Kwak, Jong Hwan; Lee, Cheong Hoon; Lee, Jeongmi; Kim, In Su

2014-05-16

A copper-catalyzed oxidative coupling of 2-carbonyl-substituted phenols and 1,3-dicarbonyl compounds with a wide range of dibenzyl or dialkyl ethers is described. This protocol provides an efficient preparation of phenol esters and enol esters in good yields with high chemoselectivity. This method represents an alternative protocol for classical esterification reactions.

Toward fidelity between specification and implementation

NASA Technical Reports Server (NTRS)

Callahan, John R.; Montgomery, Todd L.; Morrison, Jeff; Wu, Yunqing

1994-01-01

This paper describes the methods used to specify and implement a complex communications protocol that provides reliable delivery of data in multicast-capable, packet-switching telecommunication networks. The protocol, called the Reliable Multicasting Protocol (RMP), was developed incrementally by two complementary teams using a combination of formal and informal techniques in an attempt to ensure the correctness of the protocol implementation. The first team, called the Design team, initially specified protocol requirements using a variant of SCR requirements tables and implemented a prototype solution. The second team, called the V&V team, developed a state model based on the requirements tables and derived test cases from these tables to exercise the implementation. In a series of iterative steps, the Design team added new functionality to the implementation while the V&V team kept the state model in fidelity with the implementation through testing. Test cases derived from state transition paths in the formal model formed the dialogue between teams during development and served as the vehicles for keeping the model and implementation in fidelity with each other. This paper describes our experiences in developing our process model, details of our approach, and some example problems found during the development of RMP.

CT protocol management: simplifying the process by using a master protocol concept.

PubMed

Szczykutowicz, Timothy P; Bour, Robert K; Rubert, Nicholas; Wendt, Gary; Pozniak, Myron; Ranallo, Frank N

2015-07-08

This article explains a method for creating CT protocols for a wide range of patient body sizes and clinical indications, using detailed tube current information from a small set of commonly used protocols. Analytical expressions were created relating CT technical acquisition parameters which can be used to create new CT protocols on a given scanner or customize protocols from one scanner to another. Plots of mA as a function of patient size for specific anatomical regions were generated and used to identify the tube output needs for patients as a function of size for a single master protocol. Tube output data were obtained from the DICOM header of clinical images from our PACS and patient size was measured from CT localizer radiographs under IRB approval. This master protocol was then used to create 11 additional master protocols. The 12 master protocols were further combined to create 39 single and multiphase clinical protocols. Radiologist acceptance rate of exams scanned using the clinical protocols was monitored for 12,857 patients to analyze the effectiveness of the presented protocol management methods using a two-tailed Fisher's exact test. A single routine adult abdominal protocol was used as the master protocol to create 11 additional master abdominal protocols of varying dose and beam energy. Situations in which the maximum tube current would have been exceeded are presented, and the trade-offs between increasing the effective tube output via 1) decreasing pitch, 2) increasing the scan time, or 3) increasing the kV are discussed. Out of 12 master protocols customized across three different scanners, only one had a statistically significant acceptance rate that differed from the scanner it was customized from. The difference, however, was only 1% and was judged to be negligible. All other master protocols differed in acceptance rate insignificantly between scanners. The methodology described in this paper allows a small set of master protocols to be adapted among different clinical indications on a single scanner and among different CT scanners.

Potato transformation and potato cyst nematode infection on potato plantlets in tissue culture

USDA-ARS?s Scientific Manuscript database

These two protocols describe the methods for generating transgenic potato plants and for evaluating potato cyst nematode (Globodera rostochiensis and G. pallida) infection on potato plantlets in tissue culture. These methods are useful tools that can be used in the study of the interactions between ...

Techtalk: Telecommunications for Improving Developmental Education.

ERIC Educational Resources Information Center

Caverly, David C.; Broderick, Bill

1993-01-01

Explains how to access the Internet, discussing hardware and software considerations, connectivity, and types of access available to users. Describes the uses of electronic mail; TELNET, a method for remotely logging onto another computer; and anonymous File Transfer Protocol (FTP), a method for downloading files from a remote computer. (MAB)

Lipid Extraction and Cholesterol Quantification: A Simple Protocol

ERIC Educational Resources Information Center

Barreto, M. Carmo

2005-01-01

Enzymatic methods are used to measure cholesterol levels but a simple and inexpensive method is described, which is particularly important when one has repeated lab sessions during the week. It can be carried out on the organic phase containing the lipids, without evaporating the solvent, yielding quick results.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-Fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidum and Giardia spp.

EPA Science Inventory

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

Calcium Phosphate Transfection of Primary Hippocampal Neurons

PubMed Central

DiBona, Victoria L.; Wu, Qian; Zhang, Huaye

2013-01-01

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells. PMID:24300106

Comparison of Four Protocols to Generate Chondrocyte-Like Cells from Human Induced Pluripotent Stem Cells (hiPSCs).

PubMed

Suchorska, Wiktoria Maria; Augustyniak, Ewelina; Richter, Magdalena; Trzeciak, Tomasz

2017-04-01

Stem cells (SCs) are a promising approach to regenerative medicine, with the potential to treat numerous orthopedic disorders, including osteo-degenerative diseases. The development of human-induced pluripotent stem cells (hiPSCs) has increased the potential of SCs for new treatments. However, current methods of differentiating hiPSCs into chondrocyte-like cells are suboptimal and better methods are needed. The aim of the present study was to assess four different chondrogenic differentiation protocols to identify the most efficient method of generating hiPSC-derived chondrocytes. For this study, hiPSCs were obtained from primary human dermal fibroblasts (PHDFs) and differentiated into chondrocyte-like cells using four different protocols: 1) monolayer culture with defined growth factors (GF); 2) embryoid bodies (EBs) in a chondrogenic medium with TGF-β3 cells; 3) EBs in chondrogenic medium conditioned with human chondrocytes (HC-402-05a cell line) and 4) EBs in chondrogenic medium conditioned with human chondrocytes and supplemented with TGF-β3. The cells obtained through these four protocols were evaluated and compared at the mRNA and protein levels. Although chondrogenic differentiation of hiPSCs was successfully achieved with all of these protocols, the two fastest and most cost-effective methods were the monolayer culture with GFs and the medium conditioned with human chondrocytes. Both of these methods are superior to other available techniques. The main advantage of the conditioned medium is that the technique is relatively simple and inexpensive while the directed method (i.e., monolayer culture with GFs) is faster than any protocol described to date because it is does not require additional steps such as EB formation.

Data transmission protocol for Pi-of-the-Sky cameras

NASA Astrophysics Data System (ADS)

Uzycki, J.; Kasprowicz, G.; Mankiewicz, M.; Nawrocki, K.; Sitek, P.; Sokolowski, M.; Sulej, R.; Tlaczala, W.

2006-10-01

The large amount of data collected by the automatic astronomical cameras has to be transferred to the fast computers in a reliable way. The method chosen should ensure data streaming in both directions but in nonsymmetrical way. The Ethernet interface is very good choice because of its popularity and proven performance. However it requires TCP/IP stack implementation in devices like cameras for full compliance with existing network and operating systems. This paper describes NUDP protocol, which was made as supplement to standard UDP protocol and can be used as a simple-network protocol. The NUDP does not need TCP protocol implementation and makes it possible to run the Ethernet network with simple devices based on microcontroller and/or FPGA chips. The data transmission idea was created especially for the "Pi of the Sky" project.

Systematic Evaluation of the Patient-Reported Outcome (PRO) Content of Clinical Trial Protocols

PubMed Central

Kyte, Derek; Duffy, Helen; Fletcher, Benjamin; Gheorghe, Adrian; Mercieca-Bebber, Rebecca; King, Madeleine; Draper, Heather; Ives, Jonathan; Brundage, Michael; Blazeby, Jane; Calvert, Melanie

2014-01-01

Background Qualitative evidence suggests patient-reported outcome (PRO) information is frequently absent from clinical trial protocols, potentially leading to inconsistent PRO data collection and risking bias. Direct evidence regarding PRO trial protocol content is lacking. The aim of this study was to systematically evaluate the PRO-specific content of UK National Institute for Health Research (NIHR) Health Technology Assessment (HTA) programme trial protocols. Methods and Findings We conducted an electronic search of the NIHR HTA programme database (inception to August 2013) for protocols describing a randomised controlled trial including a primary/secondary PRO. Two investigators independently reviewed the content of each protocol, using a specially constructed PRO-specific protocol checklist, alongside the ‘Standard Protocol Items: Recommendations for Interventional Trials’ (SPIRIT) checklist. Disagreements were resolved through discussion with a third investigator. 75 trial protocols were included in the analysis. Protocols included a mean of 32/51 (63%) SPIRIT recommendations (range 16–41, SD 5.62) and 11/33 (33%) PRO-specific items (range 4–18, SD 3.56). Over half (61%) of the PRO items were incomplete. Protocols containing a primary PRO included slightly more PRO checklist items (mean 14/33 (43%)). PRO protocol content was not associated with general protocol completeness; thus, protocols judged as relatively ‘complete’ using SPIRIT were still likely to have omitted a large proportion of PRO checklist items. Conclusions The PRO components of HTA clinical trial protocols require improvement. Information on the PRO rationale/hypothesis, data collection methods, training and management was often absent. This low compliance is unsurprising; evidence shows existing PRO guidance for protocol developers remains difficult to access and lacks consistency. Study findings suggest there are a number of PRO protocol checklist items that are not fully addressed by the current SPIRIT statement. We therefore advocate the development of consensus-based supplementary guidelines, aimed at improving the completeness and quality of PRO content in clinical trial protocols. PMID:25333349

Purification of FLAG-tagged Secreted Proteins from Mammalian Cells

PubMed Central

Itakura, Eisuke; Chen, Changchun; de Bono, Mario

2017-01-01

This protocol describes a method for purifying glycosylated FLAG-tagged secreted proteins with disulfide bonds from mammalian cells. The purified products can be used for various applications, such as ligand binding assays. PMID:29075655

A Simple XML Producer-Consumer Protocol

NASA Technical Reports Server (NTRS)

Smith, Warren; Gunter, Dan; Quesnel, Darcy

2000-01-01

This document describes a simple XML-based protocol that can be used for producers of events to communicate with consumers of events. The protocol described here is not meant to be the most efficient protocol, the most logical protocol, or the best protocol in any way. This protocol was defined quickly and it's intent is to give us a reasonable protocol that we can implement relatively easily and then use to gain experience in distributed event services. This experience will help us evaluate proposals for event representations, XML-based encoding of information, and communication protocols. The next section of this document describes how we represent events in this protocol and then defines the two events that we choose to use for our initial experiments. These definitions are made by example so that they are informal and easy to understand. The following section then proceeds to define the producer-consumer protocol we have agreed upon for our initial experiments.

Isolation of Primary Human Skeletal Muscle Cells

PubMed Central

Spinazzola, Janelle M.; Gussoni, Emanuela

2017-01-01

Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study. PMID:29152538

Long-term seafloor monitoring at an open ocean aquaculture site in the western Gulf of Maine, USA: development of an adaptive protocol.

PubMed

Grizzle, R E; Ward, L G; Fredriksson, D W; Irish, J D; Langan, R; Heinig, C S; Greene, J K; Abeels, H A; Peter, C R; Eberhardt, A L

2014-11-15

The seafloor at an open ocean finfish aquaculture facility in the western Gulf of Maine, USA was monitored from 1999 to 2008 by sampling sites inside a predicted impact area modeled by oceanographic conditions and fecal and food settling characteristics, and nearby reference sites. Univariate and multivariate analyses of benthic community measures from box core samples indicated minimal or no significant differences between impact and reference areas. These findings resulted in development of an adaptive monitoring protocol involving initial low-cost methods that required more intensive and costly efforts only when negative impacts were initially indicated. The continued growth of marine aquaculture is dependent on further development of farming methods that minimize negative environmental impacts, as well as effective monitoring protocols. Adaptive monitoring protocols, such as the one described herein, coupled with mathematical modeling approaches, have the potential to provide effective protection of the environment while minimize monitoring effort and costs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

PubMed Central

Assadian, Farzaneh; Sandström, Karl; Laurell, Göran; Svensson, Catharina; Akusjärvi, Göran; Punga, Tanel

2015-01-01

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term “tonsils” refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses. PMID:26650582

Using the emergency department as a screening site for high blood pressure. A method for improving hypertension detection and appropriate referral.

PubMed

Mamon, J; Green, L; Levine, D M; Gibson, G; Gurley, H T

1987-08-01

This study describes the development and testing of a high blood pressure protocol for use in emergency departments (ED) to enhance detection of those patients appropriate for subsequent referral. The protocol involves two serial blood pressure measurements and a patient interview to determine: 1) previous history of high blood pressure (HBP), 2) treatment in past year for HBP, and 3) usual source of medical care. The accuracy of patient reporting was validated by comparison with the patients' hospital record (reflecting outpatient and inpatient visits). Results indicate that these self-reports have high levels of sensitivity (range 90-100%) and specificity (range 79-96%). Use of the additional patient information increased the sensitivity of the screening protocol in identifying when and where a patient should be referred. Use of this methodology indicates that the protocol is a simple and effective method for HBP screening. The findings also suggest that the ED is an ideal site for screening the "hard-to-reach" hypertensive population.

Crystallizing Membrane Proteins Using Lipidic Mesophases

PubMed Central

Caffrey, Martin; Cherezov, Vadim

2009-01-01

A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and α-helical and β-barrel proteins. Its most recent successes are the human engineered β2-adrenergic and adenosine A2A G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase, and for setting up crystallizations in manual mode. Methods for harvesting micro-crystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour. PMID:19390528

A safe and accurate method to perform esthetic mandibular contouring surgery for Far Eastern Asians.

PubMed

Hsieh, A M-C; Huon, L-K; Jiang, H-R; Liu, S Y-C

2017-05-01

A tapered mandibular contour is popular with Far Eastern Asians. This study describes a safe and accurate method of using preoperative virtual surgical planning (VSP) and an intraoperative ostectomy guide to maximize the esthetic outcomes of mandibular symmetry and tapering while mitigating injury to the inferior alveolar nerve (IAN). Twelve subjects with chief complaints of a wide and square lower face underwent this protocol from January to June 2015. VSP was used to confirm symmetry and preserve the IAN while maximizing the surgeon's ability to taper the lower face via mandibular inferior border ostectomy. The accuracy of this method was confirmed by superimposition of the perioperative computed tomography scans in all subjects. No subjects complained of prolonged paresthesia after 3 months. A safe and accurate protocol for achieving an esthetic lower face in indicated Far Eastern individuals is described. Copyright © 2016 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Guide for Regional Integrated Assessments: Handbook of Methods and Procedures, Version 5.1. Appendix 1

NASA Technical Reports Server (NTRS)

Rosenzweig, Cynthia E.; Jones, James W.; Hatfield, Jerry; Antle, John; Ruane, Alex; Boote, Ken; Thorburn, Peter; Valdivia, Roberto; Porter, Cheryl; Janssen, Sander;

An Eco-Friendly Improved Protocol for the Synthesis of Bis(3-indolyl)methanes Using Poly(4-vinylpyridinium)hydrogen Sulfate as Efficient, Heterogeneous, and Recyclable Solid Acid Catalyst

PubMed Central

Banothu, Janardhan; Gali, Rajitha; Velpula, Ravibabu; Bavantula, Rajitha; Crooks, Peter A.

2013-01-01

Highly efficient and eco-friendly protocol for the synthesis of bis(3-indolyl)methanes by the electrophilic substitution reaction of indole with aldehydes catalyzed by poly(4-vinylpyridinium)hydrogen sulfate was described. Excellent yields, shorter reaction times, simple work-up procedure, avoiding hazardous organic solvents, and reusability of the catalyst are the most obvious advantages of this method. PMID:24052864

In vivo stem cell transplantation using reduced cell numbers.

PubMed

Tsutsui, Takeo W

2015-01-01

Dental pulp stem cell (DPSC) characterization is essential for regeneration of a dentin/pulp like complex in vivo. This is especially important for identifying the potential of DPSCs to function as stem cells. Previously reported DPSC transplantation methods have used with huge numbers of cells, along with hydroxyapatite/tricalcium phosphate (HA/TCP), gelatin and fibrin, and collagen scaffolds. This protocol describe a transplantation protocol that uses fewer cells and a temperature-responsive cell culture dish.

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining1

PubMed Central

Behbehani, Gregory K.; Thom, Colin; Zunder, Eli R.; Finck, Rachel; Gaudilliere, Brice; Fragiadakis, Gabriela K.; Fantl, Wendy J.; Nolan, Garry P.

2015-01-01

Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression. PMID:25274027

A gradient-free method for the purification of infective dengue virus for protein-level investigations.

PubMed

Jensen, Stephanie M; Nguyen, Celina T; Jewett, John C

2016-09-01

Dengue virus (DENV) is a mosquito-transmitted flavivirus that infects approximately 100 million people annually. Multi-day protocols for purification of DENV reduce the infective titer due to viral sensitivity to both temperature and pH. Herein we describe a 5-h protocol for the purification of all DENV serotypes, utilizing traditional gradient-free ultracentrifugation followed by selective virion precipitation. This protocol allows for the separation of DENV from contaminating proteins - including intact C6/36 densovirus, for the production of infective virus at high concentration for protein-level analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Incompleteness of Bluetooth protocol conformance test cases

NASA Astrophysics Data System (ADS)

Wu, Peng; Gao, Qiang

2001-10-01

This paper describes a formal method to verify the completeness of conformance testing, in which not only Implementation Under Test (IUT) is formalized in SDL, but also conformance tester is described in SDL so that conformance testing can be performed in simulator provided with CASE tool. The protocol set considered is Bluetooth, an open wireless communication technology. Our research results show that Bluetooth conformance test specification is not complete in that it has only limited coverage and many important capabilities defined in Bluetooth core specification are not tested. We also give a detail report on the missing test cases against Bluetooth core specification, and provide a guide on further test case generation in the future.

Laboratory maintenance of Treponema denticola.

PubMed

Fenno, J Christopher

2005-10-01

This unit describes the methods, media, and equipment necessary for routine laboratory culture and handling of the anaerobic oral spirochete Treponema denticola. Topics discussed include nutrient requirements, recommended media formulations, and expected growth kinetics, as well as methods and equipment necessary to maintain anaerobic conditions. An additional protocol on isolation of T. denticola from clinical samples is included.

Modelling and regulating of cardio-respiratory response for the enhancement of interval training

PubMed Central

2014-01-01

Background The interval training method has been a well known exercise protocol which helps strengthen and improve one’s cardiovascular fitness. Purpose To develop an effective training protocol to improve cardiovascular fitness based on modelling and analysis of Heart Rate (HR) and Oxygen Uptake (VO2) dynamics. Methods In order to model the cardiorespiratory response to the onset and offset exercises, the (K4b2, Cosmed) gas analyzer was used to monitor and record the heart rate and oxygen uptake for ten healthy male subjects. An interval training protocol was developed for young health users and was simulated using a proposed RC switching model which was presented to accommodate the variations of the cardiorespiratory dynamics to running exercises. A hybrid system model was presented to describe the adaptation process and a multi-loop PI control scheme was designed for the tuning of interval training regime. Results By observing the original data for each subject, we can clearly identify that all subjects have similar HR and VO2 profiles. The proposed model is capable to simulate the exercise responses during onset and offset exercises; it ensures the continuity of the outputs within the interval training protocol. Under some mild assumptions, a hybrid system model can describe the adaption process and accordingly a multi-loop PI controller can be designed for the tuning of interval training protocol. The self-adaption feature of the proposed controller gives the exerciser the opportunity to reach his desired setpoints after a certain number of training sessions. Conclusions The established interval training protocol targets a range of 70-80% of HRmax which is mainly a training zone for the purpose of cardiovascular system development and improvement. Furthermore, the proposed multi-loop feedback controller has the potential to tune the interval training protocol according to the feedback from an individual exerciser. PMID:24499131

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography.

PubMed

Trépout, Sylvain; Bastin, Philippe; Marco, Sergio

2017-03-12

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

Objective and automated protocols for the evaluation of biomedical search engines using No Title Evaluation protocols.

PubMed

Campagne, Fabien

2008-02-29

The evaluation of information retrieval techniques has traditionally relied on human judges to determine which documents are relevant to a query and which are not. This protocol is used in the Text Retrieval Evaluation Conference (TREC), organized annually for the past 15 years, to support the unbiased evaluation of novel information retrieval approaches. The TREC Genomics Track has recently been introduced to measure the performance of information retrieval for biomedical applications. We describe two protocols for evaluating biomedical information retrieval techniques without human relevance judgments. We call these protocols No Title Evaluation (NT Evaluation). The first protocol measures performance for focused searches, where only one relevant document exists for each query. The second protocol measures performance for queries expected to have potentially many relevant documents per query (high-recall searches). Both protocols take advantage of the clear separation of titles and abstracts found in Medline. We compare the performance obtained with these evaluation protocols to results obtained by reusing the relevance judgments produced in the 2004 and 2005 TREC Genomics Track and observe significant correlations between performance rankings generated by our approach and TREC. Spearman's correlation coefficients in the range of 0.79-0.92 are observed comparing bpref measured with NT Evaluation or with TREC evaluations. For comparison, coefficients in the range 0.86-0.94 can be observed when evaluating the same set of methods with data from two independent TREC Genomics Track evaluations. We discuss the advantages of NT Evaluation over the TRels and the data fusion evaluation protocols introduced recently. Our results suggest that the NT Evaluation protocols described here could be used to optimize some search engine parameters before human evaluation. Further research is needed to determine if NT Evaluation or variants of these protocols can fully substitute for human evaluations.

Protocols for the analytical characterization of therapeutic monoclonal antibodies. II - Enzymatic and chemical sample preparation.

PubMed

Bobaly, Balazs; D'Atri, Valentina; Goyon, Alexandre; Colas, Olivier; Beck, Alain; Fekete, Szabolcs; Guillarme, Davy

2017-08-15

The analytical characterization of therapeutic monoclonal antibodies and related proteins usually incorporates various sample preparation methodologies. Indeed, quantitative and qualitative information can be enhanced by simplifying the sample, thanks to the removal of sources of heterogeneity (e.g. N-glycans) and/or by decreasing the molecular size of the tested protein by enzymatic or chemical fragmentation. These approaches make the sample more suitable for chromatographic and mass spectrometric analysis. Structural elucidation and quality control (QC) analysis of biopharmaceutics are usually performed at intact, subunit and peptide levels. In this paper, general sample preparation approaches used to attain peptide, subunit and glycan level analysis are overviewed. Protocols are described to perform tryptic proteolysis, IdeS and papain digestion, reduction as well as deglycosylation by PNGase F and EndoS2 enzymes. Both historical and modern sample preparation methods were compared and evaluated using rituximab and trastuzumab, two reference therapeutic mAb products approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA). The described protocols may help analysts to develop sample preparation methods in the field of therapeutic protein analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Micropatterning hydroxy-PAAm hydrogels and Sylgard 184 silicone elastomers with tunable elastic moduli.

PubMed

Versaevel, Marie; Grevesse, Thomas; Riaz, Maryam; Lantoine, Joséphine; Gabriele, Sylvain

2014-01-01

This protocol describes a simple method to deposit protein micropatterns over a wide range of culture substrate stiffness (three orders of magnitude) by using two complementary polymeric substrates. In the first part, we introduce a novel polyacrylamide hydrogel, called hydroxy-polyacrylamide (PAAm), that permits to surmount the intrinsically nonadhesive properties of polyacrylamide with minimal requirements in cost or expertize. We present a protocol for tuning easily the rigidity of "soft" hydroxy-PAAm hydrogels between ~0.5 and 50 kPa and a micropatterning method to locally deposit protein micropatterns on these hydrogels. In a second part, we describe a protocol for tuning the rigidity of "stiff" silicone elastomers between ~100 and 1000 kPa and printing efficiently proteins from the extracellular matrix. Finally, we investigate the effect of the matrix rigidity on the nucleus of primary endothelial cells by tuning the rigidity of both polymeric substrates. We envision that the complementarity of these two polymeric substrates, combined with an efficient microprinting technique, can be further developed in the future as a powerful mechanobiology platform to investigate in vitro the effect of mechanotransduction cues on cellular functions, gene expression, and stem cell differentiation. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Aurich, Maike K.; Fleming, Ronan M. T.; Thiele, Ines

Metabolomic data sets provide a direct read-out of cellular phenotypes and are increasingly generated to study biological questions. Previous work, by us and others, revealed the potential of analyzing extracellular metabolomic data in the context of the metabolic model using constraint-based modeling. With the MetaboTools, we make our methods available to the broader scientific community. The MetaboTools consist of a protocol, a toolbox, and tutorials of two use cases. The protocol describes, in a step-wise manner, the workflow of data integration, and computational analysis. The MetaboTools comprise the Matlab code required to complete the workflow described in the protocol. Tutorialsmore » explain the computational steps for integration of two different data sets and demonstrate a comprehensive set of methods for the computational analysis of metabolic models and stratification thereof into different phenotypes. The presented workflow supports integrative analysis of multiple omics data sets. Importantly, all analysis tools can be applied to metabolic models without performing the entire workflow. Taken together, the MetaboTools constitute a comprehensive guide to the intra-model analysis of extracellular metabolomic data from microbial, plant, or human cells. In conclusion, this computational modeling resource offers a broad set of computational analysis tools for a wide biomedical and non-biomedical research community.« less

Generating gene knockout rats by homologous recombination in embryonic stem cells

PubMed Central

Tong, Chang; Huang, Guanyi; Ashton, Charles; Li, Ping; Ying, Qi-Long

2013-01-01

We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell–based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ~1 year to complete, from derivation of ES cells to generation of knockout rats. PMID:21637202

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.

Genetic Interaction Score (S-Score) Calculation, Clustering, and Visualization of Genetic Interaction Profiles for Yeast.

PubMed

Roguev, Assen; Ryan, Colm J; Xu, Jiewei; Colson, Isabelle; Hartsuiker, Edgar; Krogan, Nevan

2018-02-01

This protocol describes computational analysis of genetic interaction screens, ranging from data capture (plate imaging) to downstream analyses. Plate imaging approaches using both digital camera and office flatbed scanners are included, along with a protocol for the extraction of colony size measurements from the resulting images. A commonly used genetic interaction scoring method, calculation of the S-score, is discussed. These methods require minimal computer skills, but some familiarity with MATLAB and Linux/Unix is a plus. Finally, an outline for using clustering and visualization software for analysis of resulting data sets is provided. © 2018 Cold Spring Harbor Laboratory Press.

What is the optimal way to prepare a Bell state using measurement and feedback?

NASA Astrophysics Data System (ADS)

Martin, Leigh; Sayrafi, Mahrud; Whaley, K. Birgitta

2017-12-01

Recent work has shown that the use of quantum feedback can significantly enhance both the speed and success rate of measurement-based remote entanglement generation, but it is generally unknown what feedback protocols are optimal for these tasks. Here we consider two common measurements that are capable of projecting into pairwise entangled states, namely half- and full-parity measurements of two qubits, and determine in each case a globally optimal protocol for generation of entanglement. For the half-parity measurement, we rederive a previously described protocol using more general methods and prove that it is globally optimal for several figures of merit, including maximal concurrence or fidelity and minimal time to reach a specified concurrence or fidelity. For the full-parity measurement, we derive a protocol for rapid entanglement generation related to that of (Hill, Ralph, Phys. Rev. A 77, 014305), and then map the dynamics of the concurrence of the state to the Bloch vector length of an effective qubit. This mapping allows us to prove several optimality results for feedback protocols with full-parity measurements. We further show that our full-parity protocol transfers entanglement optimally from one qubit to the other amongst all measurement-based schemes. The methods developed here will be useful for deriving feedback protocols and determining their optimality properties in many other quantum systems subject to measurement and unitary operations.

Factors affecting adoption, implementation fidelity, and sustainability of the Redesigned Community Health Fund in Tanzania: a mixed methods protocol for process evaluation in the Dodoma region

PubMed Central

Kalolo, Albino; Radermacher, Ralf; Stoermer, Manfred; Meshack, Menoris; De Allegri, Manuela

2015-01-01

Background Despite the implementation of various initiatives to address low enrollment in voluntary micro health insurance (MHI) schemes in sub-Saharan Africa, the problem of low enrollment remains unresolved. The lack of process evaluations of such interventions makes it difficult to ascertain whether their poor results are because of design failures or implementation weaknesses. Objective In this paper, we describe a process evaluation protocol aimed at opening the ‘black box’ to evaluate the implementation processes of the Redesigned Community Health Fund (CHF) program in the Dodoma region of Tanzania. Design The study employs a cross-sectional mixed methods design and is being carried out 3 years after the launch of the Redesigned CHF program. The study is grounded in a conceptual framework which rests on the Diffusion of Innovation Theory and the Implementation Fidelity Framework. The study utilizes a mixture of quantitative and qualitative data collection tools (questionnaires, focus group discussions, in-depth interviews, and document review), and aligns the evaluation to the Theory of Intervention developed by our team. Quantitative data will be used to measure program adoption, implementation fidelity, and their moderating factors. Qualitative data will be used to explore the responses of stakeholders to the intervention, contextual factors, and moderators of adoption, implementation fidelity, and sustainability. Discussion This protocol describes a systematic process evaluation in relation to the implementation of a reformed MHI. We trust that the theoretical approaches and methodologies described in our protocol may be useful to inform the design of future process evaluations focused on the assessment of complex interventions, such as MHI schemes. PMID:26679408

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.

Components of Effective Cognitive-Behavioral Therapy for Pediatric Headache: A Mixed Methods Approach

PubMed Central

Law, Emily F.; Beals-Erickson, Sarah E.; Fisher, Emma; Lang, Emily A.; Palermo, Tonya M.

2017-01-01

Internet-delivered treatment has the potential to expand access to evidence-based cognitive-behavioral therapy (CBT) for pediatric headache, and has demonstrated efficacy in small trials for some youth with headache. We used a mixed methods approach to identify effective components of CBT for this population. In Study 1, component profile analysis identified common interventions delivered in published RCTs of effective CBT protocols for pediatric headache delivered face-to-face or via the Internet. We identified a core set of three treatment components that were common across face-to-face and Internet protocols: 1) headache education, 2) relaxation training, and 3) cognitive interventions. Biofeedback was identified as an additional core treatment component delivered in face-to-face protocols only. In Study 2, we conducted qualitative interviews to describe the perspectives of youth with headache and their parents on successful components of an Internet CBT intervention. Eleven themes emerged from the qualitative data analysis, which broadly focused on patient experiences using the treatment components and suggestions for new treatment components. In the Discussion, these mixed methods findings are integrated to inform the adaptation of an Internet CBT protocol for youth with headache. PMID:29503787

Components of Effective Cognitive-Behavioral Therapy for Pediatric Headache: A Mixed Methods Approach.

PubMed

Law, Emily F; Beals-Erickson, Sarah E; Fisher, Emma; Lang, Emily A; Palermo, Tonya M

2017-01-01

Internet-delivered treatment has the potential to expand access to evidence-based cognitive-behavioral therapy (CBT) for pediatric headache, and has demonstrated efficacy in small trials for some youth with headache. We used a mixed methods approach to identify effective components of CBT for this population. In Study 1, component profile analysis identified common interventions delivered in published RCTs of effective CBT protocols for pediatric headache delivered face-to-face or via the Internet. We identified a core set of three treatment components that were common across face-to-face and Internet protocols: 1) headache education, 2) relaxation training, and 3) cognitive interventions. Biofeedback was identified as an additional core treatment component delivered in face-to-face protocols only. In Study 2, we conducted qualitative interviews to describe the perspectives of youth with headache and their parents on successful components of an Internet CBT intervention. Eleven themes emerged from the qualitative data analysis, which broadly focused on patient experiences using the treatment components and suggestions for new treatment components. In the Discussion, these mixed methods findings are integrated to inform the adaptation of an Internet CBT protocol for youth with headache.

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

Diastereoselective chain-elongation reactions using microreactors for applications in complex molecule assembly.

PubMed

Carter, Catherine F; Lange, Heiko; Sakai, Daiki; Baxendale, Ian R; Ley, Steven V

2011-03-14

Diastereoselective chain-elongation reactions are important transformations for the assembly of complex molecular structures, such as those present in polyketide natural products. Here we report new methods for performing crotylation reactions and homopropargylation reactions by using newly developed low-temperature flow-chemistry technology. In-line purification protocols are described, as well as the application of the crotylation protocol in an automated multi-step sequence. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computer-aided fabrication of a zirconia 14-unit removable dental prosthesis: a technical report.

PubMed

Grösser, Julian; Sachs, Caroline; Stadelmann, Markus; Schweiger, Josef; Güthe, Jan-Frederik; Beuer, Florian

2014-01-01

Double crown systems with primary crowns made from zirconia are used to support removable dental prostheses (RDPs). However, the fabrication of RDPs is labor-intensive and costly. Manufacturing primary and secondary crowns from zirconia with a CAD/CAM system might simplify the fabrication protocol and reduce costs. Furthermore, only ceramic materials are used in this method, providing an RDP with the highest possible biocompatibility and greatest possible esthetics. This article describes the fabrication protocol step by step.

A genotyping protocol for multiple tissue types from the polyploid tree species Sequoia sempervirens (Cupressaceae)1

PubMed Central

Narayan, Lakshmi; Dodd, Richard S.; O’Hara, Kevin L.

2015-01-01

Premise of the study: Identifying clonal lineages in asexually reproducing plants using microsatellite markers is complicated by the possibility of nonidentical genotypes from the same clonal lineage due to somatic mutations, null alleles, and scoring errors. We developed and tested a clonal identification protocol that is robust to these issues for the asexually reproducing hexaploid tree species coast redwood (Sequoia sempervirens). Methods: Microsatellite data from four previously published and two newly developed primers were scored using a modified protocol, and clones were identified using Bruvo genetic distances. The effectiveness of this clonal identification protocol was assessed using simulations and by genotyping a test set of paired samples of different tissue types from the same trees. Results: Data from simulations showed that our protocol allowed us to accurately identify clonal lineages. Multiple test samples from the same trees were identified correctly, although certain tissue type pairs had larger genetic distances on average. Discussion: The methods described in this paper will allow for the accurate identification of coast redwood clones, facilitating future studies of the reproductive ecology of this species. The techniques used in this paper can be applied to studies of other clonal organisms as well. PMID:25798341

Lipids and Fatty Acids in Algae: Extraction, Fractionation into Lipid Classes, and Analysis by Gas Chromatography Coupled with Flame Ionization Detector (GC-FID).

PubMed

Guihéneuf, Freddy; Schmid, Matthias; Stengel, Dagmar B

2015-01-01

Despite the number of biochemical studies exploring algal lipids and fatty acid biosynthesis pathways and profiles, analytical methods used by phycologists for this purpose are often diverse and incompletely described. Potential confusion and potential variability of the results between studies can therefore occur due to change of protocols for lipid extraction and fractionation, as well as fatty acid methyl esters (FAME) preparation before gas chromatography (GC) analyses. Here, we describe a step-by-step procedure for the profiling of neutral and polar lipids using techniques such as solid-liquid extraction (SLE), thin-layer chromatography (TLC), and gas chromatography coupled with flame ionization detector (GC-FID). As an example, in this protocol chapter, analyses of neutral and polar lipids from the marine microalga Pavlova lutheri (an EPA/DHA-rich haptophyte) will be outlined to describe the distribution of fatty acid residues within its major lipid classes. This method has been proven to be a reliable technique to assess changes in lipid and fatty acid profiles in several other microalgal species and seaweeds.

CT protocol management: simplifying the process by using a master protocol concept

PubMed Central

Bour, Robert K.; Rubert, Nicholas; Wendt, Gary; Pozniak, Myron; Ranallo, Frank N.

2015-01-01

This article explains a method for creating CT protocols for a wide range of patient body sizes and clinical indications, using detailed tube current information from a small set of commonly used protocols. Analytical expressions were created relating CT technical acquisition parameters which can be used to create new CT protocols on a given scanner or customize protocols from one scanner to another. Plots of mA as a function of patient size for specific anatomical regions were generated and used to identify the tube output needs for patients as a function of size for a single master protocol. Tube output data were obtained from the DICOM header of clinical images from our PACS and patient size was measured from CT localizer radiographs under IRB approval. This master protocol was then used to create 11 additional master protocols. The 12 master protocols were further combined to create 39 single and multiphase clinical protocols. Radiologist acceptance rate of exams scanned using the clinical protocols was monitored for 12,857 patients to analyze the effectiveness of the presented protocol management methods using a two‐tailed Fisher's exact test. A single routine adult abdominal protocol was used as the master protocol to create 11 additional master abdominal protocols of varying dose and beam energy. Situations in which the maximum tube current would have been exceeded are presented, and the trade‐offs between increasing the effective tube output via 1) decreasing pitch, 2) increasing the scan time, or 3) increasing the kV are discussed. Out of 12 master protocols customized across three different scanners, only one had a statistically significant acceptance rate that differed from the scanner it was customized from. The difference, however, was only 1% and was judged to be negligible. All other master protocols differed in acceptance rate insignificantly between scanners. The methodology described in this paper allows a small set of master protocols to be adapted among different clinical indications on a single scanner and among different CT scanners. PACS number: 87.57.Q PMID:26219005

Biomimetic, Mild Chemical Synthesis of CdTe-GSH Quantum Dots with Improved Biocompatibility

PubMed Central

Pérez-Donoso, José M.; Monrás, Juan P.; Bravo, Denisse; Aguirre, Adam; Quest, Andrew F.; Osorio-Román, Igor O.; Aroca, Ricardo F.; Chasteen, Thomas G.; Vásquez, Claudio C.

2012-01-01

Multiple applications of nanotechnology, especially those involving highly fluorescent nanoparticles (NPs) or quantum dots (QDs) have stimulated the research to develop simple, rapid and environmentally friendly protocols for synthesizing NPs exhibiting novel properties and increased biocompatibility. In this study, a simple protocol for the chemical synthesis of glutathione (GSH)-capped CdTe QDs (CdTe-GSH) resembling conditions found in biological systems is described. Using only CdCl2, K2TeO3 and GSH, highly fluorescent QDs were obtained under pH, temperature, buffer and oxygen conditions that allow microorganisms growth. These CdTe-GSH NPs displayed similar size, chemical composition, absorbance and fluorescence spectra and quantum yields as QDs synthesized using more complicated and expensive methods. CdTe QDs were not freely incorporated into eukaryotic cells thus favoring their biocompatibility and potential applications in biomedicine. In addition, NPs entry was facilitated by lipofectamine, resulting in intracellular fluorescence and a slight increase in cell death by necrosis. Toxicity of the as prepared CdTe QDs was lower than that observed with QDs produced by other chemical methods, probably as consequence of decreased levels of Cd+2 and higher amounts of GSH. We present here the simplest, fast and economical method for CdTe QDs synthesis described to date. Also, this biomimetic protocol favors NPs biocompatibility and helps to establish the basis for the development of new, “greener” methods to synthesize cadmium-containing QDs. PMID:22292028

Novel methods to estimate antiretroviral adherence: protocol for a longitudinal study

PubMed Central

Saberi, Parya; Ming, Kristin; Legnitto, Dominique; Neilands, Torsten B; Gandhi, Monica; Johnson, Mallory O

2018-01-01

Background There is currently no gold standard for assessing antiretroviral (ARV) adherence, so researchers often resort to the most feasible and cost-effective methods possible (eg, self-report), which may be biased or inaccurate. The goal of our study was to evaluate the feasibility and acceptability of innovative and remote methods to estimate ARV adherence, which can potentially be conducted with less time and financial resources in a wide range of clinic and research settings. Here, we describe the research protocol for studying these novel methods and some lessons learned. Methods The 6-month pilot study aimed to examine the feasibility and acceptability of a remotely conducted study to evaluate the correlation between: 1) text-messaged photographs of pharmacy refill dates for refill-based adherence; 2) text-messaged photographs of pills for pill count-based adherence; and 3) home-collected hair sample measures of ARV concentration for pharmacologic-based adherence. Participants were sent monthly automated text messages to collect refill dates and pill counts that were taken and sent via mobile telephone photographs, and hair collection kits every 2 months by mail. At the study end, feasibility was calculated by specific metrics, such as the receipt of hair samples and responses to text messages. Participants completed a quantitative survey and qualitative exit interviews to examine the acceptability of these adherence evaluation methods. The relationship between the 3 novel metrics of adherence and self-reported adherence will be assessed. Discussion Investigators conducting adherence research are often limited to using either self-reported adherence, which is subjective, biased, and often overestimated, or other more complex methods. Here, we describe the protocol for evaluating the feasibility and acceptability of 3 novel and remote methods of estimating adherence, with the aim of evaluating the relationships between them. Additionally, we note the lessons learned from the protocol implementation to date. We expect that these novel measures will be feasible and acceptable. The implications of this research will be the identification and evaluation of innovative and accurate metrics of ARV adherence for future implementation. PMID:29950816

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

A novel permeabilization protocol to obtain intracellular 3D immunolabeling for electron tomography.

PubMed

Jiménez, Nuria; Post, Jan A

2014-01-01

Electron tomography (ET) is a very important high-resolution tool for 3D imaging in cell biology. By combining the technique with immunolabeling, ET can provide essential insights into both cellular architecture and dynamics. We recently developed a protocol to achieve 3D immunolabeling of intracellular antigens without the need for uncontrolled permeabilization steps that cause random, extensive cell membrane disruption. Here we describe this novel method based on well-controlled permeabilization by targeted laser cell perforation. Mechanical permeabilization of the plasma membrane can be applied at specific sites without affecting other parts of the plasma membrane and intracellular membranes. Despite the relatively small opening created in the plasma membrane, the method allows specific 3D immunolocalization of cytoplasmic antigens in cultured cells by a pre-embedment protocol. The approach is unique and leads to a superior ultrastructural preservation for transmission electron microscopy and electron tomography.

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

PubMed Central

Baars, Destiny L.; Pelegri, Francisco

2016-01-01

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species. PMID:28060351

Methods and Management of the Healthy Brain Study: A Large Multisite Qualitative Research Project

ERIC Educational Resources Information Center

Laditka, Sarah B.; Corwin, Sara J.; Laditka, James N.; Liu, Rui; Friedman, Daniela B.; Mathews, Anna E.; Wilcox, Sara

2009-01-01

Purpose of the study: To describe processes used in the Healthy Brain project to manage data collection, coding, and data distribution in a large qualitative project, conducted by researchers at 9 universities in 9 states. Design and Methods: Project management protocols included: (a) managing audiotapes and surveys to ensure data confidentiality,…

Assessment of Murine Retinal Function by Electroretinography

PubMed Central

Benchorin, Gillie; Calton, Melissa A.; Beaulieu, Marielle O.; Vollrath, Douglas

2017-01-01

The electroretinogram (ERG) is a sensitive and noninvasive method for testing retinal function. In this protocol, we describe a method for performing ERGs in mice. Contact lenses on the mouse cornea measure the electrical response to a light stimulus of photoreceptors and downstream retinal cells, and the collected data are analyzed to evaluate retinal function. PMID:29177186

Validation protocol of analytical procedures for quantification of drugs in polymeric systems for parenteral administration: dexamethasone phosphate disodium microparticles.

PubMed

Martín-Sabroso, Cristina; Tavares-Fernandes, Daniel Filipe; Espada-García, Juan Ignacio; Torres-Suárez, Ana Isabel

2013-12-15

In this work a protocol to validate analytical procedures for the quantification of drug substances formulated in polymeric systems that comprise both drug entrapped into the polymeric matrix (assay:content test) and drug released from the systems (assay:dissolution test) is developed. This protocol is applied to the validation two isocratic HPLC analytical procedures for the analysis of dexamethasone phosphate disodium microparticles for parenteral administration. Preparation of authentic samples and artificially "spiked" and "unspiked" samples is described. Specificity (ability to quantify dexamethasone phosphate disodium in presence of constituents of the dissolution medium and other microparticle constituents), linearity, accuracy and precision are evaluated, in the range from 10 to 50 μg mL(-1) in the assay:content test procedure and from 0.25 to 10 μg mL(-1) in the assay:dissolution test procedure. The robustness of the analytical method to extract drug from microparticles is also assessed. The validation protocol developed allows us to conclude that both analytical methods are suitable for their intended purpose, but the lack of proportionality of the assay:dissolution analytical method should be taken into account. The validation protocol designed in this work could be applied to the validation of any analytical procedure for the quantification of drugs formulated in controlled release polymeric microparticles. Copyright © 2013 Elsevier B.V. All rights reserved.

The Best of Both Worlds: Building on the COPUS and RTOP Observation Protocols to Easily and Reliably Measure Various Levels of Reformed Instructional Practice

PubMed Central

Lund, Travis J.; Pilarz, Matthew; Velasco, Jonathan B.; Chakraverty, Devasmita; Rosploch, Kaitlyn; Undersander, Molly; Stains, Marilyne

2015-01-01

Researchers, university administrators, and faculty members are increasingly interested in measuring and describing instructional practices provided in science, technology, engineering, and mathematics (STEM) courses at the college level. Specifically, there is keen interest in comparing instructional practices between courses, monitoring changes over time, and mapping observed practices to research-based teaching. While increasingly common observation protocols (Reformed Teaching Observation Protocol [RTOP] and Classroom Observation Protocol in Undergraduate STEM [COPUS]) at the postsecondary level help achieve some of these goals, they also suffer from weaknesses that limit their applicability. In this study, we leverage the strengths of these protocols to provide an easy method that enables the reliable and valid characterization of instructional practices. This method was developed empirically via a cluster analysis using observations of 269 individual class periods, corresponding to 73 different faculty members, 28 different research-intensive institutions, and various STEM disciplines. Ten clusters, called COPUS profiles, emerged from this analysis; they represent the most common types of instructional practices enacted in the classrooms observed for this study. RTOP scores were used to validate the alignment of the 10 COPUS profiles with reformed teaching. Herein, we present a detailed description of the cluster analysis method, the COPUS profiles, and the distribution of the COPUS profiles across various STEM courses at research-intensive universities. PMID:25976654

Validation of a new radiographic protocol for Asian elephant feet and description of their radiographic anatomy.

PubMed

Mumby, C; Bouts, T; Sambrook, L; Danika, S; Rees, E; Parry, A; Rendle, M; Masters, N; Weller, R

2013-10-05

Foot problems are extremely common in elephants and radiography is the only imaging method available but the radiographic anatomy has not been described in detail. The aims of this study were to develop a radiographic protocol for elephant feet using digital radiography, and to describe the normal radiographic anatomy of the Asian elephant front and hind foot. A total of fifteen cadaver foot specimens from captive Asian elephants were radiographed using a range of projections and exposures to determine the best radiographic technique. This was subsequently tested in live elephants in a free-contact setting. The normal radiographic anatomy of the Asian elephant front and hind foot was described with the use of three-dimensional models based on CT reconstructions. The projection angles that were found to be most useful were 65-70° for the front limb and 55-60° in the hind limb. The beam was centred 10-15 cm proximal to the cuticle in the front and 10-15 cm dorsal to the plantar edge of the sole in the hind foot depending on the size of the foot. The protocol developed can be used for larger-scale diagnostic investigations of captive elephant foot disorders, while the normal radiographic anatomy described can improve the diagnostic reliability of elephant feet radiography.

Data exchange technology based on handshake protocol for industrial automation system

NASA Astrophysics Data System (ADS)

Astafiev, A. V.; Shardin, T. O.

2018-05-01

In the article, questions of data exchange technology based on the handshake protocol for industrial automation system are considered. The methods of organizing the technology in client-server applications are analyzed. In the process of work, the main threats of client-server applications that arise during the information interaction of users are indicated. Also, a comparative analysis of analogue systems was carried out, as a result of which the most suitable option was chosen for further use. The basic schemes for the operation of the handshake protocol are shown, as well as the general scheme of the implemented application, which describes the entire process of interaction between the client and the server.

Data Acquisition for a Patient-directed Intervention Protocol in the Dynamic Intensive Care Unit Setting

PubMed Central

Chlan, Linda; Patterson, Robert P.; Heiderscheit, Annie

2011-01-01

Methods to easily, accurately, and efficiently obtain data in an ICU-based clinical trial can be challenging in this high-tech setting. Patient medical status and the dynamic nature of this clinical setting further complicates data collection. The purpose of this paper is to describe the modifications of commercially available headphones and the application of a data logging device to capture frequency and length of protocol use (music listening or headphones only for noise cancellation) without burdening participants or busy ICU nurses. With the automatic capture of protocol use by research participants, there have been no instances of lost data for this clinical trial. PMID:21382515

Data acquisition for a patient-directed intervention protocol in the dynamic intensive care unit setting.

PubMed

Chlan, Linda; Patterson, Robert P; Heiderscheit, Annie

2011-07-01

Methods to easily, accurately, and efficiently obtain data in an ICU-based clinical trial can be challenging in this high-tech setting. Patient medical status and the dynamic nature of this clinical setting further complicate data collection. The purpose of this paper is to describe the modifications of commercially available headphones and the application of a data logging device to capture frequency and length of protocol use (music listening or headphones only for noise cancellation) without burdening participants or busy ICU nurses. With the automatic capture of protocol use by research participants, there have been no instances of lost data for this clinical trial. Copyright © 2011 Elsevier Inc. All rights reserved.

Detection of DNA Methylation by Whole-Genome Bisulfite Sequencing.

PubMed

Li, Qing; Hermanson, Peter J; Springer, Nathan M

2018-01-01

DNA methylation plays an important role in the regulation of the expression of transposons and genes. Various methods have been developed to assay DNA methylation levels. Bisulfite sequencing is considered to be the "gold standard" for single-base resolution measurement of DNA methylation levels. Coupled with next-generation sequencing, whole-genome bisulfite sequencing (WGBS) allows DNA methylation to be evaluated at a genome-wide scale. Here, we described a protocol for WGBS in plant species with large genomes. This protocol has been successfully applied to assay genome-wide DNA methylation levels in maize and barley. This protocol has also been successfully coupled with sequence capture technology to assay DNA methylation levels in a targeted set of genomic regions.

Methods to Assess Mitochondrial Morphology in Mammalian Cells Mounting Autophagic or Mitophagic Responses.

PubMed

Marchi, S; Bonora, M; Patergnani, S; Giorgi, C; Pinton, P

2017-01-01

It is widely acknowledged that mitochondria are highly active structures that rapidly respond to cellular and environmental perturbations by changing their shape, number, and distribution. Mitochondrial remodeling is a key component of diverse biological processes, ranging from cell cycle progression to autophagy. In this chapter, we describe different methodologies for the morphological study of the mitochondrial network. Instructions are given for the preparation of samples for fluorescent microscopy, based on genetically encoded strategies or the employment of synthetic fluorescent dyes. We also propose detailed protocols to analyze mitochondrial morphometric parameters from both three-dimensional and bidimensional datasets. Finally, we describe a protocol for the visualization and quantification of mitochondrial structures through electron microscopy. © 2017 Elsevier Inc. All rights reserved.

The time-adjusted gradual replacement injection method enables better visualization of the right heart.

PubMed

Nakahara, Takehiro; Jinzaki, Masahiro; Niwamae, Nogiku; Saito, Yuuichirou; Arai, Masashi; Tsushima, Yoshito; Kuribayashi, Sachio; Kurabayashi, Masahiko

2014-01-01

Despite the improvement of cardiac CT, right heart visualization remains challenging. We herein describe a new method, called the time-adjusted gradual replacement injection protocol. The aim of this study was to compare this protocol with the split-bolus injection protocol. Fifty-two patients who had undergone dual-source cardiac CT were retrospectively recruited. Twenty-six patients were injected by using the split-bolus injection protocol, and 26 patients were injected by using the time-adjusted gradual replacement injection protocol. For this method, we injected contrast medium for 10 seconds at a flow rate of 0.07 × body weight mL/s, then gradually replaced the contrast material with saline until 2 seconds before finishing the scans. The CT attenuation was measured in 4 chambers, the aorta, and the coronary arteries. The visualization of the anatomic structures and the occurrence and severity of streak artifacts were scored for the cardiac structures in the heart. For the analyses, either Welch t-test or Student t-test was performed. In the right heart, the CT values and visualization scores were significantly higher in the time-adjusted replacement injection group than in the split-bolus injection group, whereas the artifact scores were comparable between the 2 groups. The CT values, visualization scores, and artifact scores of the left heart were not significantly different between the 2 groups. In this study, the time-adjusted gradual replacement injection protocol provided excellent attenuation for visualization of the right heart. This method may help to accurately evaluate the right cardiac anatomy and thereby identify any potential diseases. Copyright © 2014 Society of Cardiovascular Computed Tomography. Published by Elsevier Inc. All rights reserved.

Effectiveness of muscle strengthening and description of protocols for preventing falls in the elderly: a systematic review

PubMed Central

Ishigaki, Erika Y.; Ramos, Lidiane G.; Carvalho, Elisa S.; Lunardi, Adriana C.

2014-01-01

Background Falls are a geriatric syndrome that is considered a significant public health problem in terms of morbidity and mortality because they lead to a decline in functional capacity and an impaired quality of life in the elderly. Lower limb muscle strengthening seems to be an effective intervention for preventing falls; however, there is no consensus regarding the best method for increasing lower limb muscle strength. Objectives To analyze the effectiveness of lower limb muscle strengthening and to investigate and describe the protocols used for preventing falls in elderly subjects. Method We performed a systematic review of randomized and controlled clinical trials published between 2002 and 2012 in the databases PubMed, EMBASE, Scopus, Web of Science, and PEDro that cited some type of lower limb muscle strengthening protocol and that evaluated the incidence of falls as the primary outcome exclusively in elderly subjects. Twelve studies met the inclusion criteria. Qualitative analysis was performed by independent reviewers applying the PEDro scale. Results The data obtained from the selected studies showed lower fall rates in the intervention groups compared to controls. Six studies described the lower limb muscle strengthening protocol in detail. High methodological quality was found in 6 studies (PEDro score ≥7/10 points). Conclusions The methodological quality of the studies in this area appears to leave little doubt regarding the effectiveness of lower limb strengthening exercises for preventing falls in elderly subjects, however the interventions in these studies were poorly reported. PMID:24760166

Electroencephalographic reactivity testing in unconscious patients: a systematic review of methods and definitions.

PubMed

Admiraal, M M; van Rootselaar, A-F; Horn, J

2017-02-01

Electroencephalographic (EEG) reactivity testing is often presented as a clear-cut element of electrophysiological testing. Absence of EEG reactivity is generally considered an indicator of poor outcome, especially in patients after cardiac arrest. However, guidelines do not clearly describe how to test for reactivity and how to evaluate the results. In a quest for clear guidelines, we performed a systematic review aimed at identifying testing methods and definitions of EEG reactivity. We systematically searched the literature between 1970 and May 2016. Methodological quality of the studies was assessed using the QUality In Prognostic Studies tool. Quality of the descriptions of stimulus protocol and reactivity definition was rated on a four-category grading scale based on reproducibility. We found that protocols for EEG reactivity testing vary greatly and descriptions of protocols are almost never replicable. Furthermore, replicable definitions of presence or absence of EEG reactivity are never provided. In order to draw firm conclusions on EEG reactivity as a prognostic factor, future studies should include a precise stimulation protocol and reactivity definition to facilitate guideline formation. © 2016 EAN.

Embedding and Chemical Reactivation of Green Fluorescent Protein in the Whole Mouse Brain for Optical Micro-Imaging

PubMed Central

Gang, Yadong; Zhou, Hongfu; Jia, Yao; Liu, Ling; Liu, Xiuli; Rao, Gong; Li, Longhui; Wang, Xiaojun; Lv, Xiaohua; Xiong, Hanqing; Yang, Zhongqin; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

2017-01-01

Resin embedding has been widely applied to fixing biological tissues for sectioning and imaging, but has long been regarded as incompatible with green fluorescent protein (GFP) labeled sample because it reduces fluorescence. Recently, it has been reported that resin-embedded GFP-labeled brain tissue can be imaged with high resolution. In this protocol, we describe an optimized protocol for resin embedding and chemical reactivation of fluorescent protein labeled mouse brain, we have used mice as experiment model, but the protocol should be applied to other species. This method involves whole brain embedding and chemical reactivation of the fluorescent signal in resin-embedded tissue. The whole brain embedding process takes a total of 7 days. The duration of chemical reactivation is ~2 min for penetrating 4 μm below the surface in the resin-embedded brain. This protocol provides an efficient way to prepare fluorescent protein labeled sample for high-resolution optical imaging. This kind of sample was demonstrated to be imaged by various optical micro-imaging methods. Fine structures labeled with GFP across a whole brain can be detected. PMID:28352214

Global Land Product Validation Protocols: An Initiative of the CEOS Working Group on Calibration and Validation to Evaluate Satellite-derived Essential Climate Variables

NASA Astrophysics Data System (ADS)

Guillevic, P. C.; Nickeson, J. E.; Roman, M. O.; camacho De Coca, F.; Wang, Z.; Schaepman-Strub, G.

2016-12-01

The Global Climate Observing System (GCOS) has specified the need to systematically produce and validate Essential Climate Variables (ECVs). The Committee on Earth Observation Satellites (CEOS) Working Group on Calibration and Validation (WGCV) and in particular its subgroup on Land Product Validation (LPV) is playing a key coordination role leveraging the international expertise required to address actions related to the validation of global land ECVs. The primary objective of the LPV subgroup is to set standards for validation methods and reporting in order to provide traceable and reliable uncertainty estimates for scientists and stakeholders. The Subgroup is comprised of 9 focus areas that encompass 10 land surface variables. The activities of each focus area are coordinated by two international co-leads and currently include leaf area index (LAI) and fraction of absorbed photosynthetically active radiation (FAPAR), vegetation phenology, surface albedo, fire disturbance, snow cover, land cover and land use change, soil moisture, land surface temperature (LST) and emissivity. Recent additions to the focus areas include vegetation indices and biomass. The development of best practice validation protocols is a core activity of CEOS LPV with the objective to standardize the evaluation of land surface products. LPV has identified four validation levels corresponding to increasing spatial and temporal representativeness of reference samples used to perform validation. Best practice validation protocols (1) provide the definition of variables, ancillary information and uncertainty metrics, (2) describe available data sources and methods to establish reference validation datasets with SI traceability, and (3) describe evaluation methods and reporting. An overview on validation best practice components will be presented based on the LAI and LST protocol efforts to date.

An introduction to planar chromatography and its application to natural products isolation.

PubMed

Gibbons, Simon

2012-01-01

Thin-layer chromatography (TLC) is an easy, inexpensive, rapid, and the most widely used method for the analysis and isolation of small organic natural and synthetic products. It also has use in the biological evaluation of organic compounds, particularly in the areas of antimicrobial and antioxidant metabolites and for the evaluation of acetylcholinesterase inhibitors which have utility in the treatment of Alzheimer's disease. The ease and inexpensiveness of use of this technique, coupled with the ability to rapidly develop separation and bioassay protocols will ensure that TLC will be used for some considerable time alongside conventional instrumental methods. This chapter deals with the basic principles of TLC and describes methods for the analysis and isolation of natural products. Examples of methods for isolation of several classes of natural product are detailed and protocols for TLC bioassays are given.

Reprogramming retinal neurons and standardized quantification of their differentiation in 3-dimensional retinal cultures

PubMed Central

Hiler, Daniel J.; Barabas, Marie E.; Griffiths, Lyra M.; Dyer, Michael A.

2017-01-01

Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. Other protocols to reprogram postmitotic neurons have required the inactivation of the p53 tumor suppressor. We describe a method that does not require p53 inactivation and induces reprogramming in cells purified from the retinae of reprogrammable mice in aggregates with wild-type retinal cells. After the first 10 days of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantitated using this protocol. Reprogramming retinal neurons with this protocol will take 56 days, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 days. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantitates eye field specification, optic cup formation, and retinal differentiation in 3-dimensional cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol. PMID:27658012

Of taps and toilets: quasi-experimental protocol for evaluating community-demand-driven projects.

PubMed

Pattanayak, Subhrendu K; Poulos, Christine; Yang, Jui-Chen; Patil, Sumeet R; Wendland, Kelly J

2009-09-01

Sustainable and equitable access to safe water and adequate sanitation are widely acknowledged as vital, yet neglected, development goals. Water supply and sanitation (WSS) policies are justified because of the usual efficiency criteria, but also major equity concerns. Yet, to date there are few scientific impact evaluations showing that WSS policies are effective in delivering social welfare outcomes. This lack of an evaluation culture is partly because WSS policies are characterized by diverse mechanisms, broad goals and the increasing importance of decentralized delivery, and partly because programme administrators are unaware of appropriate methods. We describe a protocol for a quasi-experimental evaluation of a community-demand-driven programme for water and sanitation in rural India, which addresses several evaluation challenges. After briefly reviewing policy and implementation issues in the sector, we describe key features of our protocol, including control group identification, pre-post measurement, programme theory, sample sufficiency and robust indicators. At its core, our protocol proposes to combine propensity score matching and difference-in-difference estimation. We conclude by briefly summarizing how quasi-experimental impact evaluations can address key issues in WSS policy design and when such evaluations are needed.

Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.

PubMed

Bertolde, F Z; Almeida, A-A F; Silva, F A C; Oliveira, T M; Pirovani, C P

2014-07-04

Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots.

Novel methods to estimate antiretroviral adherence: protocol for a longitudinal study.

PubMed

Saberi, Parya; Ming, Kristin; Legnitto, Dominique; Neilands, Torsten B; Gandhi, Monica; Johnson, Mallory O

2018-01-01

There is currently no gold standard for assessing antiretroviral (ARV) adherence, so researchers often resort to the most feasible and cost-effective methods possible (eg, self-report), which may be biased or inaccurate. The goal of our study was to evaluate the feasibility and acceptability of innovative and remote methods to estimate ARV adherence, which can potentially be conducted with less time and financial resources in a wide range of clinic and research settings. Here, we describe the research protocol for studying these novel methods and some lessons learned. The 6-month pilot study aimed to examine the feasibility and acceptability of a remotely conducted study to evaluate the correlation between: 1) text-messaged photographs of pharmacy refill dates for refill-based adherence; 2) text-messaged photographs of pills for pill count-based adherence; and 3) home-collected hair sample measures of ARV concentration for pharmacologic-based adherence. Participants were sent monthly automated text messages to collect refill dates and pill counts that were taken and sent via mobile telephone photographs, and hair collection kits every 2 months by mail. At the study end, feasibility was calculated by specific metrics, such as the receipt of hair samples and responses to text messages. Participants completed a quantitative survey and qualitative exit interviews to examine the acceptability of these adherence evaluation methods. The relationship between the 3 novel metrics of adherence and self-reported adherence will be assessed. Investigators conducting adherence research are often limited to using either self-reported adherence, which is subjective, biased, and often overestimated, or other more complex methods. Here, we describe the protocol for evaluating the feasibility and acceptability of 3 novel and remote methods of estimating adherence, with the aim of evaluating the relationships between them. Additionally, we note the lessons learned from the protocol implementation to date. We expect that these novel measures will be feasible and acceptable. The implications of this research will be the identification and evaluation of innovative and accurate metrics of ARV adherence for future implementation.

Formative research in a school-based obesity prevention program for Native American school children (Pathways)

PubMed Central

Gittelsohn, Joel; Evans, Marguerite; Helitzer, Deborah; Anliker, Jean; Story, Mary; Metcalfe, Lauve; Davis, Sally; Cloud, Patty Iron

2016-01-01

This paper describes how formative research was developed and implemented to produce obesity prevention interventions among school children in six different Native American nations that are part of the Pathways study. The formative assessment work presented here was unique in several ways: (1) it represents the first time formative research methods have been applied across multiple Native American tribes; (2) it is holistic, including data collection from parents, children, teachers, administrators and community leaders; and (3) it was developed by a multi-disciplinary group, including substantial input from Native American collaborators. The paper describes the process of developing the different units of the protocol, how data collection was implemented and how analyses were structured around the identification of risk behaviors. An emphasis is placed on describing which units of the formative assessment protocol were most effective and which were less effective. PMID:10181023

Developments in fiber optics for distribution automation

NASA Technical Reports Server (NTRS)

Kirkham, H.; Friend, H.; Jackson, S.; Johnston, A.

1991-01-01

An optical fiber based communications system of unusual design is described. The system consists of a network of optical fibers overlaid on the distribution system. It is configured as a large number of interconnected rings, with some spurs. Protocols for access to and control of the network are described. Because of the way they function, the protocols are collectively called AbNET, in commemoration of the microbiologists' abbreviation Ab for antibody. Optical data links that could be optically powered are described. There are two versions, each of which has a good frequency response and minimal filtering requirements. In one, a conventional FM pulse train is used at the transmitter, and a novel form of phase-locked loop is used as demodulator. In the other, the FM transmitter is replaced with a pulse generator arranged so that the period between pulses represents the modulating signal. Transmitter and receiver designs, including temperature compensation methods, are presented. Experimental results are given.

A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies.

PubMed

Ng, Elizabeth S; Davis, Richard; Stanley, Edouard G; Elefanty, Andrew G

2008-01-01

In order to promote the uniform and reproducible differentiation of human embryonic stem cells (HESCs) in response to exogenously added growth factors, we have developed a method (spin embryoid bodies (EBs)) that uses a recombinant protein-based, animal product-free medium in which HESCs are aggregated by centrifugation to form EBs. In this protocol we describe the formulation of this medium, denoted APEL (Albumin Polyvinylalcohol Essential Lipids), and its use in spin EB differentiation of HESCs. We also describe a more economical variant, BPEL (Bovine Serum Albumin (BSA) Polyvinylalchohol Essential Lipids), in which BSA replaces the recombinant human albumin. The integration of a medium that includes only defined and recombinant components with a defined number of cells to initiate EB formation results in a generally applicable, robust platform for growth factor-directed HESC differentiation.

21 CFR 58.35 - Quality assurance unit.

Code of Federal Regulations, 2013 CFR

2013-04-01

... the corrective actions taken. (5) Determine that no deviations from approved protocols or standard operating procedures were made without proper authorization and documentation. (6) Review the final study report to assure that such report accurately describes the methods and standard operating procedures, and...

21 CFR 58.35 - Quality assurance unit.

Code of Federal Regulations, 2010 CFR

2010-04-01

... the corrective actions taken. (5) Determine that no deviations from approved protocols or standard operating procedures were made without proper authorization and documentation. (6) Review the final study report to assure that such report accurately describes the methods and standard operating procedures, and...

21 CFR 58.35 - Quality assurance unit.

Code of Federal Regulations, 2012 CFR

2012-04-01

... the corrective actions taken. (5) Determine that no deviations from approved protocols or standard operating procedures were made without proper authorization and documentation. (6) Review the final study report to assure that such report accurately describes the methods and standard operating procedures, and...

21 CFR 58.35 - Quality assurance unit.

Code of Federal Regulations, 2011 CFR

2011-04-01

... the corrective actions taken. (5) Determine that no deviations from approved protocols or standard operating procedures were made without proper authorization and documentation. (6) Review the final study report to assure that such report accurately describes the methods and standard operating procedures, and...

21 CFR 58.35 - Quality assurance unit.

Code of Federal Regulations, 2014 CFR

2014-04-01

... the corrective actions taken. (5) Determine that no deviations from approved protocols or standard operating procedures were made without proper authorization and documentation. (6) Review the final study report to assure that such report accurately describes the methods and standard operating procedures, and...

Decade of Change.

ERIC Educational Resources Information Center

Hunter, Leslie Gene

1995-01-01

Discusses advancements in the field of history-related computer-assisted instruction and research. Describes the components of Historiography and Methods of Research, a class that introduces history students to such practical applications as the World Wide Web (WWW), File Transfer Protocol (FTP), listservs, archival access, and others. Briefly…

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM).

PubMed

Wang, Yilin; Kanchanawong, Pakorn

2016-12-01

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and > 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Fluorescent in situ hybridisation to amphioxus chromosomes.

PubMed

Castro, Luis Filipe Costa; Holland, Peter William Harold

2002-12-01

We describe an efficient protocol for mapping genes and other DNA sequences to amphioxus chromosomes using fluorescent in situ hybridisation. We apply this method to identify the number and location of ribosomal DNA gene clusters and telomere sequences in metaphase spreads of Branchiostoma floridae. We also describe how the locations of two single copy genes can be mapped relative to each other, and demonstrate this by mapping an amphioxus Pax gene relative to a homologue of the Notch gene. These methods have great potential for performing comparative genomics between amphioxus and vertebrates.

Large-Scale Culture and Genetic Modification of Human Natural Killer Cells for Cellular Therapy.

PubMed

Lapteva, Natalia; Parihar, Robin; Rollins, Lisa A; Gee, Adrian P; Rooney, Cliona M

2016-01-01

Recent advances in methods for the ex vivo expansion of human natural killer (NK) cells have facilitated the use of these powerful immune cells in clinical protocols. Further, the ability to genetically modify primary human NK cells following rapid expansion allows targeting and enhancement of their immune function. We have successfully adapted an expansion method for primary NK cells from peripheral blood mononuclear cells or from apheresis products in gas permeable rapid expansion devices (G-Rexes). Here, we describe an optimized protocol for rapid and robust NK cell expansion as well as a method for highly efficient retroviral transduction of these ex vivo expanded cells. These methodologies are good manufacturing practice (GMP) compliant and could be used for clinical-grade product manufacturing.

Realist theory construction for a mixed method multilevel study of neighbourhood context and postnatal depression.

PubMed

Eastwood, John G; Kemp, Lynn A; Jalaludin, Bin B

2016-01-01

We have recently described a protocol for a study that aims to build a theory of neighbourhood context and postnatal depression. That protocol proposed a critical realist Explanatory Theory Building Method comprising of an: (1) emergent phase, (2) construction phase, and (3) confirmatory phase. A concurrent triangulated mixed method multilevel cross-sectional study design was described. The protocol also described in detail the Theory Construction Phase which will be presented here. The Theory Construction Phase will include: (1) defining stratified levels; (2) analytic resolution; (3) abductive reasoning; (4) comparative analysis (triangulation); (5) retroduction; (6) postulate and proposition development; (7) comparison and assessment of theories; and (8) conceptual frameworks and model development. The stratified levels of analysis in this study were predominantly social and psychological. The abductive analysis used the theoretical frames of: Stress Process; Social Isolation; Social Exclusion; Social Services; Social Capital, Acculturation Theory and Global-economic level mechanisms. Realist propositions are presented for each analysis of triangulated data. Inference to best explanation is used to assess and compare theories. A conceptual framework of maternal depression, stress and context is presented that includes examples of mechanisms at psychological, social, cultural and global-economic levels. Stress was identified as a necessary mechanism that has the tendency to cause several outcomes including depression, anxiety, and health harming behaviours. The conceptual framework subsequently included conditional mechanisms identified through the retroduction including the stressors of isolation and expectations and buffers of social support and trust. The meta-theory of critical realism is used here to generate and construct social epidemiological theory using stratified ontology and both abductive and retroductive analysis. The findings will be applied to the development of a middle range theory and subsequent programme theory for local perinatal child and family interventions.

Health care access for rural youth on equal terms? A mixed methods study protocol in northern Sweden.

PubMed

Goicolea, Isabel; Carson, Dean; San Sebastian, Miguel; Christianson, Monica; Wiklund, Maria; Hurtig, Anna-Karin

2018-01-11

The purpose of this paper is to propose a protocol for researching the impact of rural youth health service strategies on health care access. There has been no published comprehensive assessment of the effectiveness of youth health strategies in rural areas, and there is no clearly articulated model of how such assessments might be conducted. The protocol described here aims to gather information to; i) Assess rural youth access to health care according to their needs, ii) Identify and understand the strategies developed in rural areas to promote youth access to health care, and iii) Propose actions for further improvement. The protocol is described with particular reference to research being undertaken in the four northernmost counties of Sweden, which contain a widely dispersed and diverse youth population. The protocol proposes qualitative and quantitative methodologies sequentially in four phases. First, to map youth access to health care according to their health care needs, including assessing horizontal equity (equal use of health care for equivalent health needs,) and vertical equity (people with greater health needs should receive more health care than those with lesser needs). Second, a multiple case study design investigates strategies developed across the region (youth clinics, internet applications, public health programs) to improve youth access to health care. Third, qualitative comparative analysis of the 24 rural municipalities in the region identifies the best combination of conditions leading to high youth access to health care. Fourth, a concept mapping study involving rural stakeholders, care providers and youth provides recommended actions to improve rural youth access to health care. The implementation of this research protocol will contribute to 1) generating knowledge that could contribute to strengthening rural youth access to health care, as well as to 2) advancing the application of mixed methods to explore access to health care.

Straight Talk About Birth Control: A Contraceptive Education Protocol for Home Care.

PubMed

Schoenberg, Leslie

Home healthcare providers play a critical role in the prevention of unintended pregnancies by providing evidence-based contraception education during home visits. This article describes an innovative and comprehensive contraception protocol that was developed for Nurse-Family Partnership to improve contraception education for home healthcare patients. The protocol focused on increasing uptake of long-acting reversible contraception (LARC) for high-risk prenatal and postpartum home healthcare patients. The protocol was designed to reduce early subsequent pregnancies and thereby improve outcomes for mothers and their infants. An evidence-based translation project was designed and piloted in three California counties. The protocol consisted of a contraception education module for nurses and a patient education toolkit. The toolkit included an interactive patient education workbook emphasizing LARC methods for nurses to complete with their patients along with other teaching tools. The project was evaluated using pre- and posttest surveys that measured changes in nurses' knowledge, attitudes, and practice before, after, and 2 months after implementation. Outcomes revealed the following statistically significant results: (a) nurses' knowledge doubled at the first posttest and persisted at 2 months, (b) nurses' attitudes improved on two of the three measures, and (c) there was a 17.7% increase in the frequency of LARC birth control education 2 months after implementation. An evidence-based contraception protocol can promote acceptance of LARC methods and improve home healthcare clinician comfort with and frequency of birth control education.

The Viability of Portraiture for Science Education Research: Learning from Portraits of Two Science Classrooms

ERIC Educational Resources Information Center

Quigley, Cassie; Trauth-Nare, Amy; Beeman-Cadwallader, Nicole

2015-01-01

The purpose of this paper is to describe the relevance of a qualitative methodology called portraiture for science education. Portraiture is a method of inquiry that blends art and science by combining the empirical aspects of inquiry with beauty and aesthetic properties. This method encompasses all aspects of a research study, including protocol,…

Biolistic- and Agrobacterium-mediated transformation protocols for wheat.

PubMed

Tamás-Nyitrai, Cecília; Jones, Huw D; Tamás, László

2012-01-01

After rice, wheat is considered to be the most important world food crop, and the demand for high-quality wheat flour is increasing. Although there are no GM varieties currently grown, wheat is an important target for biotechnology, and we anticipate that GM wheat will be commercially available in 10-15 years. In this chapter, we summarize the main features and challenges of wheat transformation and then describe detailed protocols for the production of transgenic wheat plants both by biolistic and Agrobacterium-mediated DNA-delivery. Although these methods are used mainly for bread wheat (Triticum aestivum L.), they can also be successfully applied, with slight modifications, to tetraploid durum wheat (T. turgidum L. var. durum). The appropriate size and developmental stage of explants (immature embryo-derived scutella), the conditions to produce embryogenic callus tissues, and the methods to regenerate transgenic plants under increasing selection pressure are provided in the protocol. To illustrate the application of herbicide selection system, we have chosen to describe the use of the plasmid pAHC25 for biolistic transformation, while for Agrobacterium-mediated transformation the binary vector pAL156 (incorporating both the bar gene and the uidA gene) has been chosen. Beside the step-by-step methodology for obtaining stably transformed and normal fertile plants, procedures for screening and testing transgenic wheat plants are also discussed.

MetaboTools: A comprehensive toolbox for analysis of genome-scale metabolic models

DOE PAGES

Aurich, Maike K.; Fleming, Ronan M. T.; Thiele, Ines

2016-08-03

Metabolomic data sets provide a direct read-out of cellular phenotypes and are increasingly generated to study biological questions. Previous work, by us and others, revealed the potential of analyzing extracellular metabolomic data in the context of the metabolic model using constraint-based modeling. With the MetaboTools, we make our methods available to the broader scientific community. The MetaboTools consist of a protocol, a toolbox, and tutorials of two use cases. The protocol describes, in a step-wise manner, the workflow of data integration, and computational analysis. The MetaboTools comprise the Matlab code required to complete the workflow described in the protocol. Tutorialsmore » explain the computational steps for integration of two different data sets and demonstrate a comprehensive set of methods for the computational analysis of metabolic models and stratification thereof into different phenotypes. The presented workflow supports integrative analysis of multiple omics data sets. Importantly, all analysis tools can be applied to metabolic models without performing the entire workflow. Taken together, the MetaboTools constitute a comprehensive guide to the intra-model analysis of extracellular metabolomic data from microbial, plant, or human cells. In conclusion, this computational modeling resource offers a broad set of computational analysis tools for a wide biomedical and non-biomedical research community.« less

The characteristics of national health initiatives promoting earlier cancer diagnosis among adult populations: a systematic review protocol

PubMed Central

Calanzani, Natalia; Weller, David; Campbell, Christine

2017-01-01

Introduction The increasing burden of cancer morbidity and mortality has led to the development of national health initiatives to promote earlier cancer diagnosis and improve cancer survival. This protocol describes a systematic review aiming to identify the evidence about such initiatives among the adult population. We will describe their components, stakeholders and target populations, and summarise their outcomes. Methods and analysis We will search databases and websites for peer-reviewed publications and grey literature on national health initiatives in high-income countries as defined by the World Bank. Quantitative, qualitative and mixed-methods studies will be included and assessed for their methodological quality. Study selection, quality assessment and data extraction will be carried out independently by two reviewers. Narrative synthesis will be used to analyse the findings. Ethics and dissemination This systematic review analyses secondary data and ethical approval is not required. Review findings will be helpful to researchers, policy makers, governments and other key stakeholders developing similar initiatives and assessing cancer outcomes. The results will be submitted to a peer-reviewed journal in order to reach a diverse group of healthcare professionals, researchers and policy makers. This systematic review protocol is registered at PROSPERO (CRD42016047233). PMID:28698336

A Practicum for Oral Cancer Teaching.

ERIC Educational Resources Information Center

Barr, Charles E.; Goldberg, Marshal D.

1983-01-01

A hospital-based method for teaching general practice dental residents, involving patients for whom the residents are responsible, is described. Residents present prepared cases of dental patients according to a predetermined protocol: a talk, complete documentation of clinical history and laboratory findings, and discussion of therapy and…

Feeder-free reprogramming of human fibroblasts with messenger RNA.

PubMed

Warren, Luigi; Wang, Jiwu

2013-11-13

This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols. Copyright © 2013 John Wiley & Sons, Inc.

Orchids (Cymbidium spp., Oncidium, and Phalaenopsis).

PubMed

Chan, Ming-Tsair; Chan, Yuan-Li; Sanjaya

2006-01-01

Recent advances in genetic engineering have made the transformation and regeneration of plants into a powerful tool for orchid improvement. This chapter presents a simple and reproducible Agrobacterium tumefaciens-mediated transformation protocol and molecular screening technique of transgenics for two orchid species, Oncidium and Phalaenopsis. The target tissues for gene transfer were protocorm-like bodies (PLBs) derived from protocorms, into which constructed foreign genes were successfully introduced. To establish stable transformants, two stages of selection were applied on the PLBs co-cultivated with A. tumefaciens. About 10% transformation efficiency was achieved in Oncidium orchid, as 108 antibiotic resistant independent PLBs were proliferated from 1000 infected PLBs. In Phalaenopsis orchid about 11 to 12% of transformation efficiency was achieved by using the present protocol. Different molecular methods and GUS-staining used to screen putative transgenic plants to confirm the integration of foreign DNA into the orchid genome were also described in detail. The methods described would also be useful for transformation of desired genes into other orchid species.

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

Bioindicators in the MIDUS National Study: Protocol, Measures, Sample, and Comparative Context

PubMed Central

Love, Gayle Dienberg; Seeman, Teresa E.; Weinstein, Maxine; Ryff, Carol D.

2010-01-01

Objectives MIDUS is a national study of health and aging among individuals aged 25 to 74 at baseline(1995/96). Longitudinal survey assessments (2004/05), were followed by biological assessments on a subsample aged 35–85. To facilitate public use, we describe the protocol, measures, and sample. Methods Respondents traveled to clinics for a two-day data collection protocol that included fasting blood specimens, 12-hour urine specimen, medical history, physical exam, bone densitometry, a laboratory challenge (heart rate variability, blood pressure, respiration, salivary cortisol). Results Response rates for the biological protocol (N = 1,255) were 39.3%, or 43.1% (adjusting for those who could not be located or contacted). Reasons for non-participation were travel, family obligations, and being too busy. Respondents were comparable to the recruitment pool on most demographic characteristics and health assessments. Discussion Strengths of the protocol vis-à-vis other similar studies include opportunities to link biological factors with diverse content from other MIDUS projects. PMID:20876364

Analysis of Nuclear RNA Interference (RNAi) in Human Cells by Subcellular Fractionation and Argonaute Loading

PubMed Central

Gagnon, Keith T.; Li, Liande; Janowski, Bethany A.; Corey, David R.

2014-01-01

RNA interference (RNAi) is well known for its ability to regulate gene expression in the cytoplasm of mammalian cells. In mammalian cell nuclei, however, the impact of RNAi has remained more controversial. A key technical hurdle has been a lack of optimized protocols for the isolation and analysis of cell nuclei. Here we describe a simplified protocol for nuclei isolation from cultured cells that incorporates a method for obtaining nucleoplasmic and chromatin fractions and removing cytoplasmic contamination. Cell fractions can then be used to detect the presence and activity of RNAi factors in the nucleus. We present a protocol for investigating an early step in RNAi, Argonaute protein loading with small RNAs, which is enabled by our improved extract preparations. These protocols facilitate characterization of nuclear RNAi and can be applied to the analysis of other nuclear proteins and pathways. From cellular fractionation to analysis of Argonaute loading results, this protocol takes 4–6 d to complete. PMID:25079428

Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation

PubMed Central

Aboulhouda, Soufiane; Di Santo, Rachael; Therizols, Gabriel; Weinberg, David

2017-01-01

The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to determine the number of ribosomes associated with an mRNA molecule, normalized to the length of the coding sequence. The primary method for this analysis of individual mRNAs is sucrose gradient fractionation, which physically separates mRNAs based on the number of bound ribosomes. Here, we describe a streamlined protocol for accurate analysis of mRNA association with ribosomes. Compared to previous protocols, our method incorporates internal controls and improved buffer conditions that together reduce artifacts caused by non-specific mRNA–ribosome interactions. Moreover, our direct-from-fraction qRT-PCR protocol eliminates the need for RNA purification from gradient fractions, which greatly reduces the amount of hands-on time required and facilitates parallel analysis of multiple conditions or gene targets. Additionally, no phenol waste is generated during the procedure. We initially developed the protocol to investigate the translationally repressed state of the HAC1 mRNA in S. cerevisiae, but we also detail adapted procedures for mammalian cell lines and tissues. PMID:29170751

Design of durability test protocol for vehicular fuel cell systems operated in power-follow mode based on statistical results of on-road data

NASA Astrophysics Data System (ADS)

Xu, Liangfei; Reimer, Uwe; Li, Jianqiu; Huang, Haiyan; Hu, Zunyan; Jiang, Hongliang; Janßen, Holger; Ouyang, Minggao; Lehnert, Werner

2018-02-01

City buses using polymer electrolyte membrane (PEM) fuel cells are considered to be the most likely fuel cell vehicles to be commercialized in China. The technical specifications of the fuel cell systems (FCSs) these buses are equipped with will differ based on the powertrain configurations and vehicle control strategies, but can generally be classified into the power-follow and soft-run modes. Each mode imposes different levels of electrochemical stress on the fuel cells. Evaluating the aging behavior of fuel cell stacks under the conditions encountered in fuel cell buses requires new durability test protocols based on statistical results obtained during actual driving tests. In this study, we propose a systematic design method for fuel cell durability test protocols that correspond to the power-follow mode based on three parameters for different fuel cell load ranges. The powertrain configurations and control strategy are described herein, followed by a presentation of the statistical data for the duty cycles of FCSs in one city bus in the demonstration project. Assessment protocols are presented based on the statistical results using mathematical optimization methods, and are compared to existing protocols with respect to common factors, such as time at open circuit voltage and root-mean-square power.

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples.

PubMed

Rehimi, Rizwan; Bartusel, Michaela; Solinas, Francesca; Altmüller, Janine; Rada-Iglesias, Alvaro

2017-08-29

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 10 4 - 5 x 10 5 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Intervention mapping protocol for developing a theory-based diabetes self-management education program.

PubMed

Song, Misoon; Choi, Suyoung; Kim, Se-An; Seo, Kyoungsan; Lee, Soo Jin

2015-01-01

Development of behavior theory-based health promotion programs is encouraged with the paradigm shift from contents to behavior outcomes. This article describes the development process of the diabetes self-management program for older Koreans (DSME-OK) using intervention mapping (IM) protocol. The IM protocol includes needs assessment, defining goals and objectives, identifying theory and determinants, developing a matrix to form change objectives, selecting strategies and methods, structuring the program, and planning for evaluation and pilot testing. The DSME-OK adopted seven behavior objectives developed by the American Association of Diabetes Educators as behavioral outcomes. The program applied an information-motivation-behavioral skills model, and interventions were targeted to 3 determinants to change health behaviors. Specific methods were selected to achieve each objective guided by IM protocol. As the final step, program evaluation was planned including a pilot test. The DSME-OK was structured as the 3 determinants of the IMB model were intervened to achieve behavior objectives in each session. The program has 12 weekly 90-min sessions tailored for older adults. Using the IM protocol in developing a theory-based self-management program was beneficial in terms of providing a systematic guide to developing theory-based and behavior outcome-focused health education programs.

Protocols for Image Processing based Underwater Inspection of Infrastructure Elements

NASA Astrophysics Data System (ADS)

O'Byrne, Michael; Ghosh, Bidisha; Schoefs, Franck; Pakrashi, Vikram

2015-07-01

Image processing can be an important tool for inspecting underwater infrastructure elements like bridge piers and pile wharves. Underwater inspection often relies on visual descriptions of divers who are not necessarily trained in specifics of structural degradation and the information may often be vague, prone to error or open to significant variation of interpretation. Underwater vehicles, on the other hand can be quite expensive to deal with for such inspections. Additionally, there is now significant encouragement globally towards the deployment of more offshore renewable wind turbines and wave devices and the requirement for underwater inspection can be expected to increase significantly in the coming years. While the merit of image processing based assessment of the condition of underwater structures is understood to a certain degree, there is no existing protocol on such image based methods. This paper discusses and describes an image processing protocol for underwater inspection of structures. A stereo imaging image processing method is considered in this regard and protocols are suggested for image storage, imaging, diving, and inspection. A combined underwater imaging protocol is finally presented which can be used for a variety of situations within a range of image scenes and environmental conditions affecting the imaging conditions. An example of detecting marine growth is presented of a structure in Cork Harbour, Ireland.

Post-trial follow-up methodology in large randomized controlled trials: a systematic review protocol.

PubMed

Llewellyn-Bennett, Rebecca; Bowman, Louise; Bulbulia, Richard

2016-12-15

Clinical trials typically have a relatively short follow-up period, and may both underestimate potential benefits of treatments investigated, and fail to detect hazards, which can take much longer to emerge. Prolonged follow-up of trial participants after the end of the scheduled trial period can provide important information on both efficacy and safety outcomes. This protocol describes a systematic review to qualitatively compare methods of post-trial follow-up used in large randomized controlled trials. A systematic search of electronic databases and clinical trial registries will use a predefined search strategy. All large (more than 1000 adult participants) randomized controlled trials will be evaluated. Two reviewers will screen and extract data according to this protocol with the aim of 95% concordance of papers checked and discrepancies will be resolved by a third reviewer. Trial methods, participant retention rates and prevalence of missing data will be recorded and compared. The potential for bias will be evaluated using the Cochrane Risk of Bias tool (applied to the methods used during the in-trial period) with the aim of investigating whether the quality of the post-trial follow-up methodology might be predicted by the quality of the methods used for the original trial. Post-trial follow-up can provide valuable information about the long-term benefits and hazards of medical interventions. However, it can be logistically challenging and costly. The aim of this systematic review is to describe how trial participants have been followed-up post-trial in order to inform future post-trial follow-up designs. Not applicable for PROSPERO registration.

Compression and Transmission of RF Signals for Telediagnosis

NASA Astrophysics Data System (ADS)

Seko, Toshihiro; Doi, Motonori; Oshiro, Osamu; Chihara, Kunihiro

2000-05-01

Health care is a critical issue nowadays. Much emphasis is given to quality care for all people. Telediagnosis has attracted public attention. We propose a new method of ultrasound image transmission for telediagnosis. In conventional methods, video image signals are transmitted. In our method, the RF signals which are acquired by an ultrasound probe, are transmitted. The RF signals can be transformed to color Doppler images or high-resolution images by a receiver. Because a stored form is adopted, the proposed system can be realized with existent technology such as hyper text transfer protocol (HTTP) and file transfer protocol (FTP). In this paper, we describe two lossless compression methods which specialize in the transmission of RF signals. One of the methods uses the characteristics of the RF signal. In the other method, the amount of the data is reduced. Measurements were performed in water targeting an iron block and triangular Styrofoam. Additionally, abdominal fat measurement was performed. Our method achieved a compression rate of 13% with 8 bit data.

ASCOT: a text mining-based web-service for efficient search and assisted creation of clinical trials

PubMed Central

2012-01-01

Clinical trials are mandatory protocols describing medical research on humans and among the most valuable sources of medical practice evidence. Searching for trials relevant to some query is laborious due to the immense number of existing protocols. Apart from search, writing new trials includes composing detailed eligibility criteria, which might be time-consuming, especially for new researchers. In this paper we present ASCOT, an efficient search application customised for clinical trials. ASCOT uses text mining and data mining methods to enrich clinical trials with metadata, that in turn serve as effective tools to narrow down search. In addition, ASCOT integrates a component for recommending eligibility criteria based on a set of selected protocols. PMID:22595088

Peach (Prunus persica L.).

PubMed

Sabbadini, Silvia; Pandolfini, Tiziana; Girolomini, Luca; Molesini, Barbara; Navacchi, Oriano

2015-01-01

Until now, the application of genetic transformation techniques in peach has been limited by the difficulties in developing efficient regeneration and transformation protocols. Here we describe an efficient regeneration protocol for the commercial micropropagation of GF677 rootstock (Prunus persica × Prunus amygdalus). The method is based on the production, via organogenesis, of meristematic bulk tissues characterized by a high competence for shoot regeneration. This protocol has also been used to obtain GF677 plants genetically engineered with an empty hairpin cassette (hereafter indicated as hp-pBin19), through Agrobacterium tumefaciens-mediated transformation. After 7-8 months of selection on media containing kanamycin, we obtained two genetically modified GF677 lines. PCR and Southern blot analyses were performed to confirm the genetic status.

ASCOT: a text mining-based web-service for efficient search and assisted creation of clinical trials.

PubMed

Korkontzelos, Ioannis; Mu, Tingting; Ananiadou, Sophia

2012-04-30

Clinical trials are mandatory protocols describing medical research on humans and among the most valuable sources of medical practice evidence. Searching for trials relevant to some query is laborious due to the immense number of existing protocols. Apart from search, writing new trials includes composing detailed eligibility criteria, which might be time-consuming, especially for new researchers. In this paper we present ASCOT, an efficient search application customised for clinical trials. ASCOT uses text mining and data mining methods to enrich clinical trials with metadata, that in turn serve as effective tools to narrow down search. In addition, ASCOT integrates a component for recommending eligibility criteria based on a set of selected protocols.

General Approach to Quantum Channel Impossibility by Local Operations and Classical Communication.

PubMed

Cohen, Scott M

2017-01-13

We describe a general approach to proving the impossibility of implementing a quantum channel by local operations and classical communication (LOCC), even with an infinite number of rounds, and find that this can often be demonstrated by solving a set of linear equations. The method also allows one to design a LOCC protocol to implement the channel whenever such a protocol exists in any finite number of rounds. Perhaps surprisingly, the computational expense for analyzing LOCC channels is not much greater than that for LOCC measurements. We apply the method to several examples, two of which provide numerical evidence that the set of quantum channels that are not LOCC is not closed and that there exist channels that can be implemented by LOCC either in one round or in three rounds that are on the boundary of the set of all LOCC channels. Although every LOCC protocol must implement a separable quantum channel, it is a very difficult task to determine whether or not a given channel is separable. Fortunately, prior knowledge that the channel is separable is not required for application of our method.

Proposal for the Development of a Standardized Protocol for Assessing the Economic Costs of HIV Prevention Interventions

PubMed Central

Pinkerton, Steven D.; Pearson, Cynthia R.; Eachus, Susan R.; Berg, Karina M.; Grimes, Richard M.

2008-01-01

Summary Maximizing our economic investment in HIV prevention requires balancing the costs of candidate interventions against their effects and selecting the most cost-effective interventions for implementation. However, many HIV prevention intervention trials do not collect cost information, and those that do use a variety of cost data collection methods and analysis techniques. Standardized cost data collection procedures, instrumentation, and analysis techniques are needed to facilitate the task of assessing intervention costs and to ensure comparability across intervention trials. This article describes the basic elements of a standardized cost data collection and analysis protocol and outlines a computer-based approach to implementing this protocol. Ultimately, the development of such a protocol would require contributions and “buy-in” from a diverse range of stakeholders, including HIV prevention researchers, cost-effectiveness analysts, community collaborators, public health decision makers, and funding agencies. PMID:18301128

Use of Medaka in Toxicity Testing

PubMed Central

Cowden, John; Hinton, David E.; Johnson, Rodney; Flynn, Kevin; Hardman, Ronald C.; Yuen, Bonny; Law, Sheran; Kullman, Seth W.; Au, Doris W.T.

2015-01-01

Small aquarium fishes are increasingly used as animal models, and one of these, Japanese Medaka (Oryzias latipes), is frequently utilized for toxicity testing. While these vertebrates have many similarities with their terrestrial counterparts, there are differences that must be considered if these organisms are to be used to their highest potential. Testing commonly may employ either the developing embryo or adults; both are easy to use and to work with. We present here three main protocols to illustrate the utility and breadth of toxicity testing possible using medaka fish. The first protocol assesses neurotoxicity in developing embryos. The second protocol describes the sexual genotyping of medaka to evaluate toxicant effects on sexual phenotype after treatment with endocrine disrupting chemicals. The third protocol assesses hepatotoxicity in adult fish after treatment with a model hepatotoxicant. The methods run the gamut from immunohistology through PCR to basic histological techniques. PMID:20922755

A protocol for isolating insect mitochondrial genomes: a case study of NUMT in Melipona flavolineata (Hymenoptera: Apidae).

PubMed

Françoso, Elaine; Gomes, Fernando; Arias, Maria Cristina

2016-07-01

Nuclear mitochondrial DNA insertions (NUMTs) are mitochondrial DNA sequences that have been transferred into the nucleus and are recognized by the presence of indels and stop codons. Although NUMTs have been identified in a diverse range of species, their discovery was frequently accidental. Here, our initial goal was to develop and standardize a simple method for isolating NUMTs from the nuclear genome of a single bee. Subsequently, we tested our new protocol by determining whether the indels and stop codons of the cytochrome c oxidase subunit I (COI) sequence of Melipona flavolineata are of nuclear origin. The new protocol successfully demonstrated the presence of a COI NUMT. In addition to NUMT investigations, the protocol described here will also be very useful for studying mitochondrial mutations related to diseases and for sequencing complete mitochondrial genomes with high read coverage by Next-Generation technology.

BioBlocks: Programming Protocols in Biology Made Easier.

PubMed

Gupta, Vishal; Irimia, Jesús; Pau, Iván; Rodríguez-Patón, Alfonso

2017-07-21

The methods to execute biological experiments are evolving. Affordable fluid handling robots and on-demand biology enterprises are making automating entire experiments a reality. Automation offers the benefit of high-throughput experimentation, rapid prototyping, and improved reproducibility of results. However, learning to automate and codify experiments is a difficult task as it requires programming expertise. Here, we present a web-based visual development environment called BioBlocks for describing experimental protocols in biology. It is based on Google's Blockly and Scratch, and requires little or no experience in computer programming to automate the execution of experiments. The experiments can be specified, saved, modified, and shared between multiple users in an easy manner. BioBlocks is open-source and can be customized to execute protocols on local robotic platforms or remotely, that is, in the cloud. It aims to serve as a de facto open standard for programming protocols in Biology.

Development of a manualized protocol of massage therapy for clinical trials in osteoarthritis.

PubMed

Ali, Ather; Kahn, Janet; Rosenberger, Lisa; Perlman, Adam I

2012-10-04

Clinical trial design of manual therapies may be especially challenging as techniques are often individualized and practitioner-dependent. This paper describes our methods in creating a standardized Swedish massage protocol tailored to subjects with osteoarthritis of the knee while respectful of the individualized nature of massage therapy, as well as implementation of this protocol in two randomized clinical trials. The manualization process involved a collaborative process between methodologic and clinical experts, with the explicit goals of creating a reproducible semi-structured protocol for massage therapy, while allowing some latitude for therapists' clinical judgment and maintaining consistency with a prior pilot study. The manualized protocol addressed identical specified body regions with distinct 30- and 60-min protocols, using standard Swedish strokes. Each protocol specifies the time allocated to each body region. The manualized 30- and 60-min protocols were implemented in a dual-site 24-week randomized dose-finding trial in patients with osteoarthritis of the knee, and is currently being implemented in a three-site 52-week efficacy trial of manualized Swedish massage therapy. In the dose-finding study, therapists adhered to the protocols and significant treatment effects were demonstrated. The massage protocol was manualized, using standard techniques, and made flexible for individual practitioner and subject needs. The protocol has been applied in two randomized clinical trials. This manualized Swedish massage protocol has real-world utility and can be readily utilized both in the research and clinical settings. Clinicaltrials.gov NCT00970008 (18 August 2009).

High-throughput process development: II. Membrane chromatography.

PubMed

Rathore, Anurag S; Muthukumar, Sampath

2014-01-01

Membrane chromatography is gradually emerging as an alternative to conventional column chromatography. It alleviates some of the major disadvantages associated with the latter including high pressure drop across the column bed and dependence on intra-particle diffusion for the transport of solute molecules to their binding sites within the pores of separation media. In the last decade, it has emerged as a method of choice for final polishing of biopharmaceuticals, in particular monoclonal antibody products. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today. This protocol describes the steps involved in performing HTPD of a membrane chromatography step. It describes operation of a commercially available device (AcroPrep™ Advance filter plate with Mustang S membrane from Pall Corporation). This device is available in 96-well format with 7 μL membrane in each well. We discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that is gathered from such a platform. A case study involving use of the protocol for examining ion exchange chromatography of Granulocyte Colony Stimulating Factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that is representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.99). We think that this protocol will be of significant value to those involved in performing high-throughput process development of membrane chromatography.

Developing Multimedia Courseware for the Internet's Java versus Shockwave.

ERIC Educational Resources Information Center

Majchrzak, Tina L.

1996-01-01

Describes and compares two methods for developing multimedia courseware for use on the Internet: an authoring tool called Shockwave, and an object-oriented language called Java. Topics include vector graphics, browsers, interaction with network protocols, data security, multithreading, and computer languages versus development environments. (LRW)

Blueberry (Vaccinium corymbosum L.).

PubMed

Song, Guo-Qing; Sink, Kenneth C

2006-01-01

Recent advances in plant biotechnology have led to a reliable and reproductive method for genetic transformation of blueberry. These efforts built on previous attempts at transient and stable transformation of blueberry that demonstrated the potential of Agrobacterium tumefaciens-mediated transformation, and as well, the difficulties of selecting and regenerating transgenic plants. As a prerequisite for successful stable transformation, efficient regeneration systems were required despite many reports on factors controlling shoot regeneration from leaf explants. The A. tumefaciens-mediated transformation protocol described in this chapter is based on combining efficient regeneration methods and the results of A. tumefaciens-mediated transient transformation studies to optimize selected parameters for gene transfer. The protocol has led to successful regeneration of transgenic plants of four commercially important highbush blueberry cultivars.

Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction.

PubMed

Sankaranarayanan, Krithivasan; Timofeeff, Michael N; Spathis, Rita; Lowenstein, Tim K; Lum, J Koji

2011-01-01

Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds.

PubMed

de-Souza-Silva, Calliandra Maria; Guilhelmelli, Fernanda; Zamith-Miranda, Daniel; de Oliveira, Marco Antônio; Nosanchuk, Joshua Daniel; Silva-Pereira, Ildinete; Albuquerque, Patrícia

2018-02-14

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Indication-Based Ordering: A New Paradigm for Glycemic Control in Hospitalized Inpatients

PubMed Central

Lee, Joshua; Clay, Brian; Zelazny, Ziband; Maynard, Gregory

2008-01-01

Background Inpatient glycemic control is a constant challenge. Institutional insulin management protocols and structured order sets are commonly advocated but poorly studied. Effective and validated methods to integrate algorithmic protocol guidance into the insulin ordering process are needed. Methods We introduced a basic structured set of computerized insulin orders (Version 1), and later introduced a paper insulin management protocol, to assist users with the order set. Metrics were devised to assess the impact of the protocol on insulin use, glycemic control, and hypoglycemia using pharmacy data and point of care glucose tests. When incremental improvement was seen (as described in the results), Version 2 of the insulin orders was created to further streamline the process. Results The percentage of regimens containing basal insulin improved with Version 1. The percentage of patient days with hypoglycemia improved from 3.68% at baseline to 2.59% with Version 1 plus the paper insulin management protocol, representing a relative risk for hypoglycemic day of 0.70 [confidence interval (CI) 0.62, 0.80]. The relative risk of an uncontrolled (mean glucose over 180 mg/dl) patient stay was reduced to 0.84 (CI 0.77, 0.91) with Version 1 and was reduced further to 0.73 (CI 0.66, 0.81) with the paper protocol. Version 2 used clinician-entered patient parameters to guide protocol-based insulin ordering and simultaneously improved the flexibility and ease of ordering over Version 1. Conclusion Patient parameter and protocol-based clinical decision support, added to computerized provider order entry, has a track record of improving glycemic control indices. This justifies the incorporation of these algorithms into online order management. PMID:19885198

A standard protocol for describing individual-based and agent-based models

USGS Publications Warehouse

Grimm, Volker; Berger, Uta; Bastiansen, Finn; Eliassen, Sigrunn; Ginot, Vincent; Giske, Jarl; Goss-Custard, John; Grand, Tamara; Heinz, Simone K.; Huse, Geir; Huth, Andreas; Jepsen, Jane U.; Jorgensen, Christian; Mooij, Wolf M.; Muller, Birgit; Pe'er, Guy; Piou, Cyril; Railsback, Steven F.; Robbins, Andrew M.; Robbins, Martha M.; Rossmanith, Eva; Ruger, Nadja; Strand, Espen; Souissi, Sami; Stillman, Richard A.; Vabo, Rune; Visser, Ute; DeAngelis, Donald L.

2006-01-01

Simulation models that describe autonomous individual organisms (individual based models, IBM) or agents (agent-based models, ABM) have become a widely used tool, not only in ecology, but also in many other disciplines dealing with complex systems made up of autonomous entities. However, there is no standard protocol for describing such simulation models, which can make them difficult to understand and to duplicate. This paper presents a proposed standard protocol, ODD, for describing IBMs and ABMs, developed and tested by 28 modellers who cover a wide range of fields within ecology. This protocol consists of three blocks (Overview, Design concepts, and Details), which are subdivided into seven elements: Purpose, State variables and scales, Process overview and scheduling, Design concepts, Initialization, Input, and Submodels. We explain which aspects of a model should be described in each element, and we present an example to illustrate the protocol in use. In addition, 19 examples are available in an Online Appendix. We consider ODD as a first step for establishing a more detailed common format of the description of IBMs and ABMs. Once initiated, the protocol will hopefully evolve as it becomes used by a sufficiently large proportion of modellers.

Adsorptive Stripping Voltammetry of Environmental Indicators: Determination of Zinc in Algae

ERIC Educational Resources Information Center

Collado-Sanchez, C.; Hernandez-Brito, J. J.; Perez-Pena, J.; Torres-Padron, M. E.; Gelado-Caballero, M. D.

2005-01-01

A method for sample preparation and for the determination of average zinc content in algae using adsorptive stripping voltammetry are described. The students gain important didactic advantages through metal determination in environmental matrices, which include carrying out clean protocols for sampling and handling, and digesting samples using…

Developing Recruitment Methods for Vulnerable, Traumatized Adolescents: A Feminist Evaluation Approach

ERIC Educational Resources Information Center

Campbell, Rebecca; Greeson, Megan R.; Fehler-Cabral, Giannina

2014-01-01

This article describes the process by which we created a recruitment protocol for engaging adolescent sexual assault victims in a qualitative evaluation study. Working in collaboration with forensic nurses, rape victim advocates, adolescent rape survivors, and our institutional review board (IRB), we created a prospective recruitment method…

Measurement of cell viability in in vitro cultures.

PubMed

Castro-Concha, Lizbeth A; Escobedo, Rosa María; Miranda-Ham, María de Lourdes

2006-01-01

An overview of the methods for assessing cell viability in in vitro cultures is presented. The protocols of four of the most commonly used assays are described in detail, so the readers may be able to determine which assay is suitable for their own projects using plant cell cultures.

Production of a maternal-zygotic medaka mutant using hybrid sterility.

PubMed

Shimada, Atsuko; Takeda, Hiroyuki

2008-08-01

Taking advantage of the characteristics that make hybrids between Japanese and Chinese medaka grow well, albeit sterile, we have developed a method of germ-line replacement in which these hybrids are used as hosts for the production of a maternal-zygotic mutant. The protocol is described herein.

The Missing Link: Peer Conferencing in Civics Education

ERIC Educational Resources Information Center

Anderson, Derek L.; Lubig, Joe

2012-01-01

This article describes a method--Collaborative Civics Conference Protocol (3CP)--that teachers can use with any civics education program to engage students in meaningful collaborative assessment of each others' thinking and writing and to make connections between civics activities and essential social studies content. Borrowing from the Writer's…

Self-organization of human embryonic stem cells on micropatterns

PubMed Central

Deglincerti, Alessia; Etoc, Fred; Guerra, M. Cecilia; Martyn, Iain; Metzger, Jakob; Ruzo, Albert; Simunovic, Mijo; Yoney, Anna; Brivanlou, Ali H.; Siggia, Eric; Warmflash, Aryeh

2018-01-01

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate to specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. While embryoid bodies and organoids can exhibit some spatial organization of differentiated cells, these methods do not yield consistent and fully reproducible results. Here, we describe a micropatterning approach where human embryonic stem cells are confined to disk-shaped, sub-millimeter colonies. After 42 hours of BMP4 stimulation, cells form self-organized differentiation patterns in concentric radial domains, which express specific markers associated with the embryonic germ layers, reminiscent of gastrulating embryos. Our protocol takes 3 days; it uses commercial microfabricated slides (CYTOO), human laminin-521 (LN-521) as extra-cellular matrix coating, and either conditioned or chemically-defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation. PMID:27735934

Single-cell transcriptome conservation in cryopreserved cells and tissues.

PubMed

Guillaumet-Adkins, Amy; Rodríguez-Esteban, Gustavo; Mereu, Elisabetta; Mendez-Lago, Maria; Jaitin, Diego A; Villanueva, Alberto; Vidal, August; Martinez-Marti, Alex; Felip, Enriqueta; Vivancos, Ana; Keren-Shaul, Hadas; Heath, Simon; Gut, Marta; Amit, Ido; Gut, Ivo; Heyn, Holger

2017-03-01

A variety of single-cell RNA preparation procedures have been described. So far, protocols require fresh material, which hinders complex study designs. We describe a sample preservation method that maintains transcripts in viable single cells, allowing one to disconnect time and place of sampling from subsequent processing steps. We sequence single-cell transcriptomes from >1000 fresh and cryopreserved cells using 3'-end and full-length RNA preparation methods. Our results confirm that the conservation process did not alter transcriptional profiles. This substantially broadens the scope of applications in single-cell transcriptomics and could lead to a paradigm shift in future study designs.

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.

Communication and protocol compliance and their relation to the quality of cardiopulmonary resuscitation (CPR): A mixed-methods study of simulated telephone-assisted CPR.

PubMed

Nord-Ljungquist, Helena; Brännström, Margareta; Bohm, Katarina

2015-07-01

In the event of a cardiac arrest, emergency medical dispatchers (EMDs) play a critical role by providing telephone-assisted cardiopulmonary resuscitation (T-CPR) to laypersons. The aim of our investigation was to describe compliance with the T-CPR protocol, the performance of the laypersons in a simulated T-CPR situation, and the communication between laypersons and EMDs during these actions. We conducted a retrospective observational study by analysing 20 recorded video and audio files. In a simulation, EMDs provided laypersons with instructions following T-CPR protocols. These were then analysed using a mixed method with convergent parallel design. If the EMDs complied with the T-CPR protocol, the laypersons performed the correct procedures in 71% of the actions. The single most challenging instruction of the T-CPR protocol, for both EMDs and laypersons, was airway control. Mean values for compression depth and frequency did not reach established guideline goals for CPR. Proper application of T-CPR protocols by EMDs resulted in better performance by laypersons in CPR. The most problematic task for EMDs as well for laypersons was airway management. The study results did not establish that the quality of communication between EMDs and laypersons performing CPR in a cardiac arrest situation led to statistically different outcomes, as measured by the quality and effectiveness of the CPR delivered. Copyright © 2014 Elsevier Ltd. 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.

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

PubMed Central

Nguyen, Nga T.; McInturf, Samuel A.; Mendoza-Cózatl, David G.

2016-01-01

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition. This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient. The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements. PMID:27500800

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements.

PubMed

Nguyen, Nga T; McInturf, Samuel A; Mendoza-Cózatl, David G

2016-07-13

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition. This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient. The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Optimal early active mobilisation protocol after extensor tendon repairs in zones V and VI: A systematic review of literature.

PubMed

Collocott, Shirley Jf; Kelly, Edel; Ellis, Richard F

2018-03-01

Early mobilisation protocols after repair of extensor tendons in zone V and VI provide better outcomes than immobilisation protocols. This systematic review investigated different early active mobilisation protocols used after extensor tendon repair in zone V and VI. The purpose was to determine whether any one early active mobilisation protocol provides superior results. An extensive literature search was conducted to identify articles investigating the outcomes of early active mobilisation protocols after extensor tendon repair in zone V and VI. Databases searched were AMED, Embase, Medline, Cochrane and CINAHL. Studies were included if they involved participants with extensor tendon repairs in zone V and VI in digits 2-5 and described a post-operative rehabilitation protocol which allowed early active metacarpophalangeal joint extension. Study designs included were randomised controlled trials, observational studies, cohort studies and case series. The Structured Effectiveness Quality Evaluation Scale was used to evaluate the methodological quality of the included studies. Twelve articles met the inclusion criteria. Two types of early active mobilisation protocols were identified: controlled active motion protocols and relative motion extension splinting protocols. Articles describing relative motion extension splinting protocols were more recent but of lower methodological quality than those describing controlled active motion protocols. Participants treated with controlled active motion and relative motion extension splinting protocols had similar range of motion outcomes, but those in relative motion extension splinting groups returned to work earlier. The evidence reviewed suggested that relative motion extension splinting protocols may allow an earlier return to function than controlled active motion protocols without a greater risk of complication.

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.

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.

Efficient regeneration and improved sonication-assisted Agrobacterium transformation (SAAT) method for Catharanthus roseus.

PubMed

Alam, Pravej; Khan, Zainul Abdeen; Abdin, Malik Zainul; Khan, Jawaid A; Ahmad, Parvaiz; Elkholy, Shereen F; Sharaf-Eldin, Mahmoud A

2017-05-01

Catharanthus roseus is an important medicinal plant known for its pharmacological qualities such as antimicrobial, anticancerous, antifeedant, antisterility, antidiabetic activities. More than 130 bioactive compounds like vinblastine, vindoline and vincristine have been synthesized in this plant. Extensive studies have been carried out for optimization regeneration and transformation protocols. Most of the protocol described are laborious and time-consuming. Due to sophisticated protocol of regeneration and genetic transformation, the production of these bioactive molecules is less and not feasible to be commercialized worldwide. Here we have optimized the efficient protocol for regeneration and transformation to minimize the time scale and enhance the transformation frequency through Agrobacterium and sonication-assisted transformation (SAAT) method. In this study, hypocotyl explants responded best for maximal production of transformed shoots. The callus percentage were recorded 52% with 1.0 mg L -1 (BAP) and 0.5 mg L -1 (NAA) while 80% shoot percentage obtained with 4.0 mg L -1 (BAP) and 0.05 mg L -1 (NAA). The microscopic studies revealed that the expression of GFP was clearly localized in leaf tissue of the C. roseus after transformation of pRepGFP0029 construct. Consequently, transformation efficiency was revealed on the basis of GFP localization. The transformation efficiency of SAAT method was 6.0% comparable to 3.5% as conventional method. Further, PCR analysis confirmed the integration of the nptII gene in the transformed plantlets of C. roseus.

Elder Abuse Identification in the Prehospital Setting: An Examination of State Emergency Medical Services Protocols.

PubMed

Namboodri, Brooke L; Rosen, Tony; Dayaa, Joseph A; Bischof, Jason J; Ramadan, Nadeem; Patel, Mehul D; Grover, Joseph; Brice, Jane H; Platts-Mills, Timothy F

2018-03-22

To describe statewide emergency medical service (EMS) protocols relating to identification, management, and reporting of elder abuse in the prehospital setting. Cross-sectional analysis. Statewide EMS protocols in the United States. Publicly available statewide EMS protocols identified from published literature, http://EMSprotocols.org, and each state's public health website. Protocols were reviewed to determine whether elder abuse was mentioned, elder abuse was defined, potential indicators of elder abuse were listed, management of older adults experiencing abuse was described, and instructions regarding reporting were provided. EMS protocols for child abuse were reviewed in the same manner for the purpose of comparison. Of the 35 publicly available statewide EMS protocols, only 14 (40.0%) mention elder abuse. Of protocols that mention elder abuse, 6 (42.9%) define elder abuse, 10 (71.4%) describe indicators of elder abuse, 8 (57.1%) provide instruction regarding management, and 12 (85.7%) provide instruction regarding reporting. Almost twice as many states met each of these metrics for child abuse. Statewide EMS protocols for elder abuse vary in regard to identification, management, and reporting, with the majority of states having no content on this subject. Expansion and standardization of protocols may increase the identification of elder abuse. © 2018, Copyright the Authors Journal compilation © 2018, The American Geriatrics Society.

Advanced tools for the analysis of protein phosphorylation in yeast mitochondria.

PubMed

Walter, Corvin; Gonczarowska-Jorge, Humberto; Sickmann, Albert; Zahedi, René P; Meisinger, Chris; Schmidt, Oliver

2018-05-24

The biochemical analysis of protein phosphorylation in mitochondria lags behind that of cytosolic signaling events. One reason is the poor stability of many phosphorylation sites during common isolation procedures for mitochondria. We present here an optimized, fast protocol for the purification of yeast mitochondria that greatly increases recovery of phosphorylated mitochondrial proteins. Moreover, we describe improved protocols for the biochemical analysis of mitochondrial protein phosphorylation by Zn 2+ -Phos-tag electrophoresis under both denaturing and - for the first time - native conditions, and demonstrate that they outperform previously applied methods. Copyright © 2018. Published by Elsevier Inc.

Cryopreservation of Human Pluripotent Stem Cells in Defined Medium

PubMed Central

Liu, Weiwei; Chen, Guokai

2014-01-01

This protocol describes a cryopreservation procedure using an enzyme-free dissociation method to harvest cells and preserve cells in albumin-free chemically defined E8 medium for human pluripotent stem cells (hPSCs). The dissociation by EDTA/PBS produces small cell aggregates that allow high survival efficiency in passaging and cryopreservation. The preservation in E8 medium eliminates serum or other animal products, and is suitable for the increasing demand for high quality hPSCs in translational research. In combination with the special feature of EDTA/PBS dissociation, this protocol allows efficient cryopreservation in more time-saving manner. PMID:25366897

A Modified Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines.

PubMed

de Lima, Júlio César; Füller, Thanise Nogueira; de Costa, Fernanda; Rodrigues-Corrêa, Kelly C S; Fett-Neto, Arthur G

2016-01-01

RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes through high-throughput sequencing technologies, microarray, and also northern blots, RT-PCR, and RTqPCR. Even though many specific protocols designed for plants with high content of secondary metabolites have been developed, these are often expensive, time consuming, and not suitable for a wide range of tissues. Here we present a modification of the method previously described using the commercially available Concert™ Plant RNA Reagent (Invitrogen) buffer for field-grown adult pine trees with high oleoresin content.

Empirical Examinations of Modifications and Adaptations to Evidence-Based Psychotherapies: Methodologies, Impact, and Future Directions.

PubMed

Stirman, Shannon Wiltsey; Gamarra, Jennifer; Bartlett, Brooke; Calloway, Amber; Gutner, Cassidy

2017-12-01

This review describes methods used to examine the modifications and adaptations to evidence-based psychological treatments (EBPTs), assesses what is known about the impact of modifications and adaptations to EBPTs, and makes recommendations for future research and clinical care. One hundred eight primary studies and three meta-analyses were identified. All studies examined planned adaptations, and many simultaneously investigated multiple types of adaptations. With the exception of studies on adding or removing specific EBPT elements, few studies compared adapted EBPTs to the original protocols. There was little evidence that adaptations in the studies were detrimental, but there was also limited consistent evidence that adapted protocols outperformed the original protocols, with the exception of adding components to EBPTs. Implications for EBPT delivery and future research are discussed.

A 96-well-plate-based optical method for the quantitative and qualitative evaluation of Pseudomonas aeruginosa biofilm formation and its application to susceptibility testing.

PubMed

Müsken, Mathias; Di Fiore, Stefano; Römling, Ute; Häussler, Susanne

2010-08-01

A major reason for bacterial persistence during chronic infections is the survival of bacteria within biofilm structures, which protect cells from environmental stresses, host immune responses and antimicrobial therapy. Thus, there is concern that laboratory methods developed to measure the antibiotic susceptibility of planktonic bacteria may not be relevant to chronic biofilm infections, and it has been suggested that alternative methods should test antibiotic susceptibility within a biofilm. In this paper, we describe a fast and reliable protocol for using 96-well microtiter plates for the formation of Pseudomonas aeruginosa biofilms; the method is easily adaptable for antimicrobial susceptibility testing. This method is based on bacterial viability staining in combination with automated confocal laser scanning microscopy. The procedure simplifies qualitative and quantitative evaluation of biofilms and has proven to be effective for standardized determination of antibiotic efficiency on P. aeruginosa biofilms. The protocol can be performed within approximately 60 h.

A Protocol for Lifetime Energy and Environmental Impact Assessment of Building Insulation Materials

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

Shrestha, Som S; Biswas, Kaushik; Desjarlais, Andre Omer

This article describes a proposed protocol that is intended to provide a comprehensive list of factors to be considered in evaluating the direct and indirect environmental impacts of building insulation materials, as well as detailed descriptions of standardized calculation methodologies to determine those impacts. The energy and environmental impacts of insulation materials can generally be divided into two categories: (1) direct impact due to the embodied energy of the insulation materials and other factors, and (2) indirect or environmental impacts avoided as a result of reduced building energy use due to addition of insulation. Standards and product category rules existmore » that provide guidelines about the life cycle assessment (LCA) of materials, including building insulation products. However, critical reviews have suggested that these standards fail to provide complete guidance to LCA studies and suffer from ambiguities regarding the determination of the environmental impacts of building insulation and other products. The focus of the assessment protocol described here is to identify all factors that contribute to the total energy and environmental impacts of different insulation products and, more importantly, provide standardized determination methods that will allow comparison of different insulation material types. Further, the intent is not to replace current LCA standards but to provide a well-defined, easy-to-use comparison method for insulation materials using existing LCA guidelines.« less

Detection of EEG electrodes in brain volumes.

PubMed

Graffigna, Juan P; Gómez, M Eugenia; Bustos, José J

2010-01-01

This paper presents a method to detect 128 EEG electrodes in image study and to merge with the Nuclear Magnetic Resonance volume for better diagnosis. First we propose three hypotheses to define a specific acquisition protocol in order to recognize the electrodes and to avoid distortions in the image. In the second instance we describe a method for segmenting the electrodes. Finally, registration is performed between volume of the electrodes and NMR.

Synthesis of aza-fused polycyclic quinolines through copper-catalyzed cascade reactions.

PubMed

Cai, Qian; Li, Zhengqiu; Wei, Jiajia; Fu, Liangbin; Ha, Chengyong; Pei, Duanqing; Ding, Ke

2010-04-02

A new and efficient method for the synthesis of aza-fused polycyclic quinolines (e.g., benzimidazo[1,2-a]quinolines) is described. This protocol includes an intermolecular condensation followed by a copper-catalyzed intramolecular C-N coupling reaction. The method is applied to a wide range of 2-iodo, 2-bromo, and 2-chloro aryl aldehyde substrates to yield the aza-fused polycyclic quinolines in good yields.

An Advanced Approach to Simultaneous Monitoring of Multiple Bacteria in Space

NASA Technical Reports Server (NTRS)

Eggers, Mitch

1998-01-01

This interim report describes progress on the development of spacecraft-compatible methods of nucleic acid preparation and cleanup. Grantee found that performance of a widely-known protocol could be greatly enhanced by the addition of a sizing step to eliminate acids. Testing of alternative adsorptive methods is underway. Grantee also confirmed and extended observation of precipitation of DNA by condensing agents such a spermine at low ionic strength.

Micropropagation and organogenesis of Anthurium andreanum Lind cv Rubrun.

PubMed

Maira, Oropeza; Alexander, Mejías; Vargas, Teresa Edith

2010-01-01

Tissue culture techniques are routinely used for mass propagation and the establishment of disease free stock material. Virtually all pot type Anthuriums available in the market today are produced by tissue culture. In this chapter, we describe an efficient protocol to obtain Anthurium andreanum cv Rubrun vitro plants through micropropagation and organogenesis. Seeds from plant spadixes were germinated on MS medium supplemented with 0.5 mg/L BA. Micro-cuttings from in vitro germinated seedlings were subcultured on MS medium containing 2 mg/L BA and 0.5 mg/L NAA. Four-week-old in vitro plants obtained from microcuttings, showed callus proliferation at the stem base. The development of shoots and plantlets was observed from callus tissue. We also describe a detailed method for the histological analysis of callus tissue and a vitro plants acclimatization protocol.

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

Assessment of the treatment protocol described in the guidelines for Trichophyton tonsurans infection.

PubMed

Shiraki, Yumi; Hiruma, Masataro; Sugita, Takashi; Ikeda, Shigaku

2008-01-01

Infection with the anthropophilic fungus Trichophyton tonsurans has spread among members of combat sports clubs and has become a serious public health problem in Japan and other countries. Infection usually provokes only a weak inflammatory response, and treatment compliance tends to be poor. To evaluate the hairbrush method and the treatment protocol described in the guidelines for T. tonsurans infection. The study subjects were 69 individuals with positive hairbrush culture from among 327 members of 12 judo clubs participating in the survey. (a) Subjects with no more than 4 colonies by the hairbrush method were treated with miconazole nitrate shampoo. (b) Subjects with 5 or more colonies were treated with (1) itraconazole at a dose of 100 mg/day for 6 weeks or at a dose of 400 mg/day for 1 week, or (2) terbinafine at a dose of 125 mg/day for 6 weeks or at a dose of 500 mg/day for 1 week. Treatment efficacy was monitored by the hairbrush method at 1.5 and 3 months after treatment. Of the 46 subjects with 5 or more colonies isolated by the hairbrush method, 32 (69.6%) took itraconazole or terbinafine in compliance with their treatment schedules and were negative for T. tonsurans after treatment. Of the 23 subjects with 4 or fewer colonies, 15 (65.2%) were negative for T. tonsurans after treatment with miconazole nitrate shampoo. The treatment protocol seems promising, but poor compliance is a problem with the oral treatment regimens. The shampoo therapy is only partially effective, with 35% of subjects remaining positive for T. tonsurans after this therapy. In order to eradicate this disease, we have renewed the guidelines for T. tonsurans infection.

Administration of medicines by emergency nurse practitioners according to protocols in an accident and emergency department.

PubMed Central

Marshall, J; Edwards, C; Lambert, M

1997-01-01

OBJECTIVE: To present the legal and professional issues related to nurse administration of drugs according to protocols, and describe the implementation and initial audit findings of such a scheme. SETTING: Accident and emergency (A&E) department of a district general hospital. METHODS: Analysis of legal and professional opinion. Protocols acceptable to the medical, nursing, and pharmacy professions were developed across a wide range of drugs appropriate for administration by accident and emergency nurse practitioners (ENPs). The first six months of the scheme were audited. Audit initially addressed general compliance with protocols and later the specific areas of tetanus immunisation and emergency contraception. RESULTS: ENPs assessed 2925 patients in six months (10.9% of all new patients); 455 patients (15.5% of the ENP patients) were given drugs according to protocols. There were no breaches of the protocols. Subsequent audit of tetanus immunisation showed 94-100% compliance with protocol standards and 71-100% compliance for emergency contraception. CONCLUSIONS: There are no legal or professional obstacles to the development of protocols for the administration of drugs to patients by nurses without reference to a doctor, providing the protocols meet all the requirements of the UKCC and have the support of consultant medical staff. Such a system must be subject to regular audit to promote a dynamic approach to protocols and training. The system safely enhanced the quality of care of patients treated by ENPs in A&E. Images Figure 1 PMID:9248912

Examining a New Method to Studying Velopharyngeal Structures in a Child With 22q11.2 Deletion Syndrome.

PubMed

Kollara, Lakshmi; Schenck, Graham; Jaskolka, Michael; Perry, Jamie L

2017-04-14

To date, no studies have imaged the velopharynx in children with 22q11.2 deletion syndrome (22q11.2 DS) without the use of sedation. Dysmorphology in velopharyngeal structures has been shown to have significant negative implications on speech among these individuals. This single case study was designed to assess the feasibility of a child-friendly magnetic resonance imaging (MRI) scanning protocol in this clinically challenging population and to determine the utility of this MRI protocol for future work in this area. One 6-year-old White girl diagnosed with 22q11.2 DS was imaged using a child-friendly, nonsedated MRI protocol. Quantitative and qualitative measures of the velopharyngeal area and associated structures were evaluated, and comparisons were made to age-matched control subjects with normal velopharyngeal anatomy. MRI data were successfully obtained using the child-friendly scanning protocol in the subject in the present study. Quantitative and qualitative differences of the levator muscle and associated velopharyngeal structures were noted. Using these MRI and structural analyses methods, insights related to muscle morphology can be obtained and considered as part of the research and clinical examination of children with 22q11.2 DS. The imaging protocol described in this study presents an effective means to counteract difficulties in imaging young children.

Phonological outcome of laryngeal framework surgery by different anesthesia protocols: a single-surgeon experience.

PubMed

Kanazawa, Takeharu; Watanabe, Yusuke; Komazawa, Daigo; Indo, Kanako; Misawa, Kiyoshi; Nagatomo, Takafumi; Shimada, Mari; Iino, Yukiko; Ichimura, Keiichi

2014-02-01

Similar to combined arytenoid adduction and medialization laryngoplasty (i.e. combined surgery) under local anesthesia, general anesthesia by intubation or by the laryngeal mask airway (LMA) method significantly improves phonological outcome. Thus, laryngeal framework surgery under general anesthesia is a promising surgical approach for selected patients with unilateral vocal cord paralysis (UVCP). The advantages of laryngeal framework surgery under local anesthesia have been described, but no studies exist concerning the difference in phonological outcome of laryngeal framework surgery performed under general anesthesia. To add new information, we retrospectively investigated the phonological outcome of the combined surgery performed under three different anesthesia protocols. Thirty-nine consecutive patients with severe UVCP underwent the combined surgery under three anesthesia protocols performed by a single surgeon: (1) under general anesthesia by intubation, (2) under general anesthesia using LMA, and (3) under local anesthesia. Under all anesthesia protocols, the vocal cords of most patients could be positioned such that the best vocal outcome could be expected. Statistical analyses demonstrated improved maximum phonation time and mean airflow rate, and grade, roughness, breathiness, asthenia, and strain (GRBAS) scale in all patients, regardless of their anesthesia protocol. Furthermore, of the three protocols, local anesthesia had the shortest operation time.

A New Cellular Architecture for Information Retrieval from Sensor Networks through Embedded Service and Security Protocols

PubMed Central

Shahzad, Aamir; Landry, René; Lee, Malrey; Xiong, Naixue; Lee, Jongho; Lee, Changhoon

2016-01-01

Substantial changes have occurred in the Information Technology (IT) sectors and with these changes, the demand for remote access to field sensor information has increased. This allows visualization, monitoring, and control through various electronic devices, such as laptops, tablets, i-Pads, PCs, and cellular phones. The smart phone is considered as a more reliable, faster and efficient device to access and monitor industrial systems and their corresponding information interfaces anywhere and anytime. This study describes the deployment of a protocol whereby industrial system information can be securely accessed by cellular phones via a Supervisory Control And Data Acquisition (SCADA) server. To achieve the study goals, proprietary protocol interconnectivity with non-proprietary protocols and the usage of interconnectivity services are considered in detail. They support the visualization of the SCADA system information, and the related operations through smart phones. The intelligent sensors are configured and designated to process real information via cellular phones by employing information exchange services between the proprietary protocol and non-proprietary protocols. SCADA cellular access raises the issue of security flaws. For these challenges, a cryptography-based security method is considered and deployed, and it could be considered as a part of a proprietary protocol. Subsequently, transmission flows from the smart phones through a cellular network. PMID:27314351

A New Cellular Architecture for Information Retrieval from Sensor Networks through Embedded Service and Security Protocols.

PubMed

Shahzad, Aamir; Landry, René; Lee, Malrey; Xiong, Naixue; Lee, Jongho; Lee, Changhoon

2016-06-14

Substantial changes have occurred in the Information Technology (IT) sectors and with these changes, the demand for remote access to field sensor information has increased. This allows visualization, monitoring, and control through various electronic devices, such as laptops, tablets, i-Pads, PCs, and cellular phones. The smart phone is considered as a more reliable, faster and efficient device to access and monitor industrial systems and their corresponding information interfaces anywhere and anytime. This study describes the deployment of a protocol whereby industrial system information can be securely accessed by cellular phones via a Supervisory Control And Data Acquisition (SCADA) server. To achieve the study goals, proprietary protocol interconnectivity with non-proprietary protocols and the usage of interconnectivity services are considered in detail. They support the visualization of the SCADA system information, and the related operations through smart phones. The intelligent sensors are configured and designated to process real information via cellular phones by employing information exchange services between the proprietary protocol and non-proprietary protocols. SCADA cellular access raises the issue of security flaws. For these challenges, a cryptography-based security method is considered and deployed, and it could be considered as a part of a proprietary protocol. Subsequently, transmission flows from the smart phones through a cellular network.

Virioplankton 'pegylation': use of PEG (polyethylene glycol) to concentrate and purify viruses in pelagic ecosystems.

PubMed

Colombet, J; Robin, A; Lavie, L; Bettarel, Y; Cauchie, H M; Sime-Ngando, T

2007-12-01

We have described the use of Polyethylene glycol (PEG) for the precipitation of natural communities of aquatic viruses, and its comparison with the usual concentration method based on ultracentrifugation. Experimental samples were obtained from different freshwater ecosystems whose trophic status varied. Based on transmission electron microscope observations and counting of phage-shaped particles, our results showed that the greatest recovery efficiency for all ecosystems was obtained when we used the PEG protocol. On average, this protocol allowed the recovery of >2-fold more viruses, compared to ultracentrifugation. In addition, the diversity of virioplankton, based on genomic size profiling using pulsed field gel electrophoresis, was higher and better discriminated when we used the PEG method. We conclude that pegylation offers a valid, simple and cheaper alternative method to ultracentrifugation, for the concentration and the purification of pelagic viruses.

Protocol for the use of a rapid real-time PCR method for the detection of HIV-1 proviral DNA using double-stranded primer.

PubMed

Pau, Chou-Pong; Wells, Susan K; Granade, Timothy C

2012-01-01

This chapter describes a real-time PCR method for the detection of HIV-1 proviral DNA in whole blood samples using a novel double-stranded primer system. The assay utilizes a simple commercially available DNA extraction method and a rapid and easy-to-perform real-time PCR protocol to consistently detect a minimum of four copies of HIV-1 group M proviral DNA in as little as 90 min after sample (whole blood) collection. Co-amplification of the human RNase P gene serves as an internal control to monitor the efficiency of both the DNA extraction and amplification. Once the assay is validated properly, it may be suitable as an alternative confirmation test for HIV-1 infections in a variety of HIV testing venues including the mother-to-child transmission testing sites, clinics, and diagnostic testing centers.

Modeling of Receptor Tyrosine Kinase Signaling: Computational and Experimental Protocols.

PubMed

Fey, Dirk; Aksamitiene, Edita; Kiyatkin, Anatoly; Kholodenko, Boris N

2017-01-01

The advent of systems biology has convincingly demonstrated that the integration of experiments and dynamic modelling is a powerful approach to understand the cellular network biology. Here we present experimental and computational protocols that are necessary for applying this integrative approach to the quantitative studies of receptor tyrosine kinase (RTK) signaling networks. Signaling by RTKs controls multiple cellular processes, including the regulation of cell survival, motility, proliferation, differentiation, glucose metabolism, and apoptosis. We describe methods of model building and training on experimentally obtained quantitative datasets, as well as experimental methods of obtaining quantitative dose-response and temporal dependencies of protein phosphorylation and activities. The presented methods make possible (1) both the fine-grained modeling of complex signaling dynamics and identification of salient, course-grained network structures (such as feedback loops) that bring about intricate dynamics, and (2) experimental validation of dynamic models.

Whole-genome multiple displacement amplification from single cells.

PubMed

Spits, Claudia; Le Caignec, Cédric; De Rycke, Martine; Van Haute, Lindsey; Van Steirteghem, André; Liebaers, Inge; Sermon, Karen

2006-01-01

Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization.

Pre-Steady-State Kinetic Analysis of Single-Nucleotide Incorporation by DNA Polymerases

PubMed Central

Su, Yan; Guengerich, F. Peter

2016-01-01

Pre-steady-state kinetic analysis is a powerful and widely used method to obtain multiple kinetic parameters. This protocol provides a step-by-step procedure for pre-steady-state kinetic analysis of single-nucleotide incorporation by a DNA polymerase. It describes the experimental details of DNA substrate annealing, reaction mixture preparation, handling of the RQF-3 rapid quench-flow instrument, denaturing polyacrylamide DNA gel preparation, electrophoresis, quantitation, and data analysis. The core and unique part of this protocol is the rationale for preparation of the reaction mixture (the ratio of the polymerase to the DNA substrate) and methods for conducting pre-steady-state assays on an RQF-3 rapid quench-flow instrument, as well as data interpretation after analysis. In addition, the methods for the DNA substrate annealing and DNA polyacrylamide gel preparation, electrophoresis, quantitation and analysis are suitable for use in other studies. PMID:27248785

Labeling proteins on live mammalian cells using click chemistry.

PubMed

Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

2015-05-01

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

An alternative method for processing northern blots after capillary transfer.

PubMed

Nilsen, Timothy W

2015-03-02

Different laboratories use different methods for the prehybridization, hybridization, and washing steps of the northern blotting procedure. In this protocol, a northern blot is pretreated with Church and Gilbert hybridization buffer to block nonspecific probe-binding sites. The immobilized RNA is then hybridized to a DNA probe specific for the RNA of interest. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. The solutions and conditions described here may be ideal for those who prefer to use fewer ingredients in their solutions. This protocol is designed to achieve the same goals as other northern blotting approaches. It minimizes background (nonspecific adherence of probe to membrane and nonspecific hybridization) and maximizes specific hybridization to RNAs immobilized on a membrane. © 2015 Cold Spring Harbor Laboratory Press.

Directed differentiation of embryonic stem cells using a bead-based combinatorial screening method.

PubMed

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.

A duplex PCR-based assay for measuring the amount of bacterial contamination in a nucleic acid extract from a culture of free-living protists.

PubMed

Marron, Alan O; Akam, Michael; Walker, Giselle

2013-01-01

Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell. Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample. The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient to develop qPCR protocols.

Breadth in Design Problem Scoping: Using Insights from Experts to Investigate Student Processes. Research Brief

ERIC Educational Resources Information Center

Morozov, Andrew; Kilgore, Deborah; Atman, Cynthia

2007-01-01

In this study, the authors used two methods for analyzing expert data: verbal protocol analysis (VPA) and narrative analysis. VPA has been effectively used to describe the design processes employed by engineering students, expert designers, and expert-novice comparative research. VPA involves asking participants to "think aloud" while…

Direct amination of γ-halo-β-ketoesters with anilines

PubMed Central

Zhang, Yinan; Silverman, Richard B.

2012-01-01

The direct amination of α-haloacetoacetates with anilines is described. Compared to existing methods, this simple protocol provides an attractive strategy to prepare diverse γ-anilino-β-ketoesters in one step. Good to excellent yields of the amination products were obtained under robust conditions, providing versatile and useful scaffolds. PMID:22390154

A Tool for Estimating Variability in Wood Preservative Treatment Retention

Treesearch

Patricia K. Lebow; Adam M. Taylor; Timothy M. Young

2015-01-01

Composite sampling is standard practice for evaluation of preservative retention levels in preservative-treated wood. Current protocols provide an average retention value but no estimate of uncertainty. Here we describe a statistical method for calculating uncertainty estimates using the standard sampling regime with minimal additional chemical analysis. This tool can...

Simple Heuristic Approach to Introduction of the Black-Scholes Model

ERIC Educational Resources Information Center

Yalamova, Rossitsa

2010-01-01

A heuristic approach to explaining of the Black-Scholes option pricing model in undergraduate classes is described. The approach draws upon the method of protocol analysis to encourage students to "think aloud" so that their mental models can be surfaced. It also relies upon extensive visualizations to communicate relationships that are…

Development of a Single Locus Sequence Typing (SLST) Scheme for Typing Bacterial Species Directly from Complex Communities.

PubMed

Scholz, Christian F P; Jensen, Anders

2017-01-01

The protocol describes a computational method to develop a Single Locus Sequence Typing (SLST) scheme for typing bacterial species. The resulting scheme can be used to type bacterial isolates as well as bacterial species directly from complex communities using next-generation sequencing technologies.

Validating visual disturbance types and classes used for forest soil monitoring protocols

Treesearch

D. S. Page-Dumroese; A. M. Abbott; M. P. Curran; M. F. Jurgensen

2012-01-01

We describe several methods for validating visual soil disturbance classes used during forest soil monitoring after specific management operations. Site-specific vegetative, soil, and hydrologic responses to soil disturbance are needed to identify sensitive and resilient soil properties and processes; therefore, validation of ecosystem responses can provide information...

The Rules of the Game: Properties of a Database of Expository Language Samples

ERIC Educational Resources Information Center

Heilmann, John; Malone, Thomas O.

2014-01-01

Purpose: The authors created a database of expository oral language samples with the aims of describing the nature of students' expository discourse and providing benchmark data for typically developing preteen and teenage students. Method: Using a favorite game or sport protocol, language samples were collected from 235 typically developing…

Rapid deletion production in fungi via Agrobacterium mediated transformation of OSCAR deletion contructs.

USDA-ARS?s Scientific Manuscript database

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene’s function in the living organism. In this protocol we describe the OSCAR method of precise and rapid deletion plasmid construction. OSCAR relies on t...

An Instrument to Assess the Obesogenic Environment of Child Care Centers

ERIC Educational Resources Information Center

Ward, Dianne; Hales, Derek; Haverly, Katie; Marks, Julie; Benjamin, Sara; Ball, Sarah; Trost, Stewart

2008-01-01

Objectives: To describe protocol and interobserver agreements of an instrument to evaluate nutrition and physical activity environments at child care. Methods: Interobserver data were collected from 9 child care centers, through direct observation and document review (17 observer pairs). Results: Mean agreement between observer pairs was 87.26%…

Quantification of protein carbonylation.

PubMed

Wehr, Nancy B; Levine, Rodney L

2013-01-01

Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is most often measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent 2,4 dinitrophenylhydrazine (DNPH). We present protocols for the derivatization and quantification of protein carbonylation with these two methods, including a newly described dot blot with greatly increased sensitivity.

Assessing Information on the Internet: Toward Providing Library Services for Computer-Mediated Communication.

ERIC Educational Resources Information Center

Dillon, Martin; And Others

1992-01-01

Describes a project that examined textual information available on the Internet and potential means of providing access to this information. Highlights include an overview of Internet resources, a profile of a document sample, description of FTP (File Transfer Protocol) sites, and an automated method of categorizing files. (MES)

Plaque assay for African swine fever virus on swine macrophages.

PubMed

Bustos, M J; Nogal, M L; Revilla, Y; Carrascosa, A L

2002-07-01

A plaque assay developed to detect the infection of African Swine Fever Virus on swine macrophages is described. Plaques were generated by all of the virus isolates tested. The method is suitable not only for virus titration but also for the selection of clones in protocols for isolation/purification of recombinant viruses.

Resident-Directed Long-Term Care: Staff Provision of Choice during Morning Care

ERIC Educational Resources Information Center

Simmons, Sandra F.; Rahman, Annie; Beuscher, Linda; Jani, Victoria; Durkin, Daniel W.; Schnelle, John F.

2011-01-01

Purpose: To develop an observational protocol to assess the quality of staff-resident communication relevant to choice and describe staff-resident interactions as preliminary evidence of the usefulness of the tool to assess current nursing home practices related to offering choice during morning care provision. Design and Methods: This study…

40 CFR 63.788 - Recordkeeping and reporting requirements.

Code of Federal Regulations, 2014 CFR

2014-07-01

... containers meet the standards as described in § 63.783(b)(2); and (vii) The results of any Method 24 of appendix A to 40 CFR part 60 or approved VOHAP measurement test conducted on individual containers of... summary of the number and total duration of incidents that the monitoring protocol failed to perform in...

A Qualitative Analysis of an Advanced Practice Nurse-Directed Transitional Care Model Intervention

ERIC Educational Resources Information Center

Bradway, Christine; Trotta, Rebecca; Bixby, M. Brian; McPartland, Ellen; Wollman, M. Catherine; Kapustka, Heidi; McCauley, Kathleen; Naylor, Mary D.

2012-01-01

Purpose: The purpose of this study was to describe barriers and facilitators to implementing a transitional care intervention for cognitively impaired older adults and their caregivers lead by advanced practice nurses (APNs). Design and Methods: APNs implemented an evidence-based protocol to optimize transitions from hospital to home. An…

Semi-high throughput screening for potential drought-tolerance in lettuce (Lactuca sativa) germplasm collections

USDA-ARS?s Scientific Manuscript database

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of th...

Combined Heat and Power Protocol for Uniform Methods Project | Advanced

Science.gov Websites

Manufacturing Research | NREL Combined Heat and Power Protocol for Uniform Methods Project Combined Heat and Power Protocol for Uniform Methods Project NREL developed a protocol that provides a ; is consistent with the scope and other protocols developed for the Uniform Methods Project (UMP

Dual-view plane illumination microscopy for rapid and spatially isotropic imaging

PubMed Central

Kumar, Abhishek; Wu, Yicong; Christensen, Ryan; Chandris, Panagiotis; Gandler, William; McCreedy, Evan; Bokinsky, Alexandra; Colón-Ramos, Daniel A; Bao, Zhirong; McAuliffe, Matthew; Rondeau, Gary; Shroff, Hari

2015-01-01

We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and Caenorhabditis elegans embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ~6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3–8 h, depending on the size of the data. PMID:25299154

The Deployment of IPv6 in an IPv4 World and Transition Strategies.

ERIC Educational Resources Information Center

Bouras, C.; Ganos, P.; Karaliotas, A.

2003-01-01

The current version of the IP protocol, IPv4, is the most widely used protocol in computer networks. This article describes mechanisms that can be used to facilitate the transition to the new version of the IP protocol, IPv6, and examines usability, usefulness and manageability. Describes how some of these mechanisms were applied to the Greek…

Framework and indicator testing protocol for developing and piloting quality indicators for the UK quality and outcomes framework.

PubMed

Campbell, Stephen M; Kontopantelis, Evangelos; Hannon, Kerin; Burke, Martyn; Barber, Annette; Lester, Helen E

2011-08-10

Quality measures should be subjected to a testing protocol before being used in practice using key attributes such as acceptability, feasibility and reliability, as well as identifying issues derived from actual implementation and unintended consequences. We describe the methodologies and results of an indicator testing protocol (ITP) using data from proposed quality indicators for the United Kingdom Quality and Outcomes Framework (QOF). The indicator testing protocol involved a multi-step and methodological process: 1) The RAND/UCLA Appropriateness Method, to test clarity and necessity, 2) data extraction from patients' medical records, to test technical feasibility and reliability, 3) diaries, to test workload, 4) cost-effectiveness modelling, and 5) semi-structured interviews, to test acceptability, implementation issues and unintended consequences. Testing was conducted in a sample of representative family practices in England. These methods were combined into an overall recommendation for each tested indicator. Using an indicator testing protocol as part of piloting was seen as a valuable way of testing potential indicators in 'real world' settings. Pilot 1 (October 2009-March 2010) involved thirteen indicators across six clinical domains and twelve indicators passed the indicator testing protocol. However, the indicator testing protocol identified a number of implementation issues and unintended consequences that can be rectified or removed prior to national roll out. A palliative care indicator is used as an exemplar of the value of piloting using a multiple attribute indicator testing protocol - while technically feasible and reliable, it was unacceptable to practice staff and raised concerns about potentially causing actual patient harm. This indicator testing protocol is one example of a protocol that may be useful in assessing potential quality indicators when adapted to specific country health care settings and may be of use to policy-makers and researchers worldwide to test the likely effect of implementing indicators prior to roll out. It builds on and codifies existing literature and other testing protocols to create a field testing methodology that can be used to produce country specific quality indicators for pay-for-performance or quality improvement schemes.

Construction of a system for single-cell transgene induction in Caenorhabditis elegans using a pulsed infrared laser

PubMed Central

Churgin, Matthew A.; He, Liping; Murray, John I.; Fang-Yen, Christopher

2014-01-01

The spatial and temporal control of transgene expression is an important tool in C. elegans biology. We previously described a method for evoking gene expression in arbitrary cells by using a focused pulsed infrared laser to induce a heat shock response (Churgin et al 2013). Here we describe detailed methods for building and testing a system for performing single-cell heat shock. Steps include setting up the laser and associated components, coupling the laser beam to a microscope, and testing heat shock protocols. All steps can be carried out using readily available off-the-shelf components. PMID:24835576

Fully automated spectrometric protocols for determination of antioxidant activity: advantages and disadvantages.

PubMed

Sochor, Jiri; Ryvolova, Marketa; Krystofova, Olga; Salas, Petr; Hubalek, Jaromir; Adam, Vojtech; Trnkova, Libuse; Havel, Ladislav; Beklova, Miroslava; Zehnalek, Josef; Provaznik, Ivo; Kizek, Rene

2010-11-29

The aim of this study was to describe behaviour, kinetics, time courses and limitations of the six different fully automated spectrometric methods--DPPH, TEAC, FRAP, DMPD, Free Radicals and Blue CrO5. Absorption curves were measured and absorbance maxima were found. All methods were calibrated using the standard compounds Trolox® and/or gallic acid. Calibration curves were determined (relative standard deviation was within the range from 1.5 to 2.5%). The obtained characteristics were compared and discussed. Moreover, the data obtained were applied to optimize and to automate all mentioned protocols. Automatic analyzer allowed us to analyse simultaneously larger set of samples, to decrease the measurement time, to eliminate the errors and to provide data of higher quality in comparison to manual analysis. The total time of analysis for one sample was decreased to 10 min for all six methods. In contrary, the total time of manual spectrometric determination was approximately 120 min. The obtained data provided good correlations between studied methods (R=0.97-0.99).

TAPRegExt: a VOResource Schema Extension for Describing TAP Services Version 1.0

NASA Astrophysics Data System (ADS)

Demleitner, Markus; Dowler, Patrick; Plante, Ray; Rixon, Guy; Taylor, Mark; Demleitner, Markus

2012-08-01

This document describes an XML encoding standard for metadata about services implementing the table access protocol TAP [TAP], referred to as TAPRegExt. Instance documents are part of the service's registry record or can be obtained from the service itself. They deliver information to both humans and software on the languages, output formats, and upload methods supported by the service, as well as data models implemented by the exposed tables, optional language features, and certain limits enforced by the service.

Analysis of In Vivo Chromatin and Protein Interactions of Arabidopsis Transcript Elongation Factors.

PubMed

Pfab, Alexander; Antosz, Wojciech; Holzinger, Philipp; Bruckmann, Astrid; Griesenbeck, Joachim; Grasser, Klaus D

2017-01-01

A central step to elucidate the function of proteins commonly comprises the analysis of their molecular interactions in vivo. For nuclear regulatory proteins this involves determining protein-protein interactions as well as mapping of chromatin binding sites. Here, we present two protocols to identify protein-protein and chromatin interactions of transcript elongation factors (TEFs) in Arabidopsis. The first protocol (Subheading 3.1) describes protein affinity-purification coupled to mass spectrometry (AP-MS) that utilizes suspension cultured cells as experimental system. This approach provides an unbiased view of proteins interacting with epitope-tagged TEFs. The second protocol (Subheading 3.2) depicts details about a chromatin immunoprecipitation (ChIP) procedure to characterize genomic binding sites of TEFs. These methods should be valuable tools for the analysis of a broad variety of nuclear proteins.

21 CFR 312.30 - Protocol amendments.

Code of Federal Regulations, 2011 CFR

2011-04-01

.... (b) Changes in a protocol. (1) A sponsor shall submit a protocol amendment describing any change in a Phase 1 protocol that significantly affects the safety of subjects or any change in a Phase 2 or 3... forth the provisions under which new protocols may be submitted and changes in previously submitted...

21 CFR 312.30 - Protocol amendments.

Code of Federal Regulations, 2010 CFR

2010-04-01

.... (b) Changes in a protocol. (1) A sponsor shall submit a protocol amendment describing any change in a Phase 1 protocol that significantly affects the safety of subjects or any change in a Phase 2 or 3... forth the provisions under which new protocols may be submitted and changes in previously submitted...

Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes

PubMed Central

Robert, Catherine; Pascalis, Hervé; Michelle, Caroline; Jardot, Priscilla; Charrel, Rémi; Raoult, Didier; Desnues, Christelle

2015-01-01

Background Metagenomic analyses have been widely used in the last decade to describe viral communities in various environments or to identify the etiology of human, animal, and plant pathologies. Here, we present a simple and standardized protocol that allows for the purification and sequencing of RNA viromes from complex biological samples with an important reduction of host DNA and RNA contaminants, while preserving the infectivity of viral particles. Principal Findings We evaluated different viral purification steps, random reverse transcriptions and sequence-independent amplifications of a pool of representative RNA viruses. Viruses remained infectious after the purification process. We then validated the protocol by sequencing the RNA virome of human body lice engorged in vitro with artificially contaminated human blood. The full genomes of the most abundant viruses absorbed by the lice during the blood meal were successfully sequenced. Interestingly, random amplifications differed in the genome coverage of segmented RNA viruses. Moreover, the majority of reads were taxonomically identified, and only 7–15% of all reads were classified as “unknown”, depending on the random amplification method. Conclusion The protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins. Our protocol allows further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses. PMID:26431175

A model-guided symbolic execution approach for network protocol implementations and vulnerability detection.

PubMed

Wen, Shameng; Meng, Qingkun; Feng, Chao; Tang, Chaojing

2017-01-01

Formal techniques have been devoted to analyzing whether network protocol specifications violate security policies; however, these methods cannot detect vulnerabilities in the implementations of the network protocols themselves. Symbolic execution can be used to analyze the paths of the network protocol implementations, but for stateful network protocols, it is difficult to reach the deep states of the protocol. This paper proposes a novel model-guided approach to detect vulnerabilities in network protocol implementations. Our method first abstracts a finite state machine (FSM) model, then utilizes the model to guide the symbolic execution. This approach achieves high coverage of both the code and the protocol states. The proposed method is implemented and applied to test numerous real-world network protocol implementations. The experimental results indicate that the proposed method is more effective than traditional fuzzing methods such as SPIKE at detecting vulnerabilities in the deep states of network protocol implementations.

An Action-Based Fine-Grained Access Control Mechanism for Structured Documents and Its Application

PubMed Central

Su, Mang; Li, Fenghua; Tang, Zhi; Yu, Yinyan; Zhou, Bo

2014-01-01

This paper presents an action-based fine-grained access control mechanism for structured documents. Firstly, we define a describing model for structured documents and analyze the application scenarios. The describing model could support the permission management on chapters, pages, sections, words, and pictures of structured documents. Secondly, based on the action-based access control (ABAC) model, we propose a fine-grained control protocol for structured documents by introducing temporal state and environmental state. The protocol covering different stages from document creation, to permission specification and usage control are given by using the Z-notation. Finally, we give the implementation of our mechanism and make the comparisons between the existing methods and our mechanism. The result shows that our mechanism could provide the better solution of fine-grained access control for structured documents in complicated networks. Moreover, it is more flexible and practical. PMID:25136651

An action-based fine-grained access control mechanism for structured documents and its application.

PubMed

Su, Mang; Li, Fenghua; Tang, Zhi; Yu, Yinyan; Zhou, Bo

2014-01-01

This paper presents an action-based fine-grained access control mechanism for structured documents. Firstly, we define a describing model for structured documents and analyze the application scenarios. The describing model could support the permission management on chapters, pages, sections, words, and pictures of structured documents. Secondly, based on the action-based access control (ABAC) model, we propose a fine-grained control protocol for structured documents by introducing temporal state and environmental state. The protocol covering different stages from document creation, to permission specification and usage control are given by using the Z-notation. Finally, we give the implementation of our mechanism and make the comparisons between the existing methods and our mechanism. The result shows that our mechanism could provide the better solution of fine-grained access control for structured documents in complicated networks. Moreover, it is more flexible and practical.

Creating fractional quantum Hall states with atomic clusters using light-assisted insertion of angular momentum

NASA Astrophysics Data System (ADS)

Zhang, Junyi; Beugnon, Jerome; Nascimbene, Sylvain

We describe a protocol to prepare clusters of ultracold bosonic atoms in strongly interacting states reminiscent of fractional quantum Hall states. Our scheme consists in injecting a controlled amount of angular momentum to an atomic gas using Raman transitions carrying orbital angular momentum. By injecting one unit of angular momentum per atom, one realizes a single-vortex state, which is well described by mean-field theory for large enough particle numbers. We also present schemes to realize fractional quantum Hall states, namely, the bosonic Laughlin and Moore-Read states. We investigate the requirements for adiabatic nucleation of such topological states, in particular comparing linear Landau-Zener ramps and arbitrary ramps obtained from optimized control methods. We also show that this protocol requires excellent control over the isotropic character of the trapping potential. ERC-Synergy Grant UQUAM, ANR-10-IDEX-0001-02, DIM NanoK Atocirc project.

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis.

PubMed

Bozec, Aline; Hannemann, Nicole

2016-06-03

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1α protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes.

Tackling reliability and construct validity: the systematic development of a qualitative protocol for skill and incident analysis.

PubMed

Savage, Trevor Nicholas; McIntosh, Andrew Stuart

2017-03-01

It is important to understand factors contributing to and directly causing sports injuries to improve the effectiveness and safety of sports skills. The characteristics of injury events must be evaluated and described meaningfully and reliably. However, many complex skills cannot be effectively investigated quantitatively because of ethical, technological and validity considerations. Increasingly, qualitative methods are being used to investigate human movement for research purposes, but there are concerns about reliability and measurement bias of such methods. Using the tackle in Rugby union as an example, we outline a systematic approach for developing a skill analysis protocol with a focus on improving objectivity, validity and reliability. Characteristics for analysis were selected using qualitative analysis and biomechanical theoretical models and epidemiological and coaching literature. An expert panel comprising subject matter experts provided feedback and the inter-rater reliability of the protocol was assessed using ten trained raters. The inter-rater reliability results were reviewed by the expert panel and the protocol was revised and assessed in a second inter-rater reliability study. Mean agreement in the second study improved and was comparable (52-90% agreement and ICC between 0.6 and 0.9) with other studies that have reported inter-rater reliability of qualitative analysis of human movement.

Predicting Silk Fiber Mechanical Properties through Multiscale Simulation and Protein Design.

PubMed

Rim, Nae-Gyune; Roberts, Erin G; Ebrahimi, Davoud; Dinjaski, Nina; Jacobsen, Matthew M; Martín-Moldes, Zaira; Buehler, Markus J; Kaplan, David L; Wong, Joyce Y

2017-08-14

Silk is a promising material for biomedical applications, and much research is focused on how application-specific, mechanical properties of silk can be designed synthetically through proper amino acid sequences and processing parameters. This protocol describes an iterative process between research disciplines that combines simulation, genetic synthesis, and fiber analysis to better design silk fibers with specific mechanical properties. Computational methods are used to assess the protein polymer structure as it forms an interconnected fiber network through shearing and how this process affects fiber mechanical properties. Model outcomes are validated experimentally with the genetic design of protein polymers that match the simulation structures, fiber fabrication from these polymers, and mechanical testing of these fibers. Through iterative feedback between computation, genetic synthesis, and fiber mechanical testing, this protocol will enable a priori prediction capability of recombinant material mechanical properties via insights from the resulting molecular architecture of the fiber network based entirely on the initial protein monomer composition. This style of protocol may be applied to other fields where a research team seeks to design a biomaterial with biomedical application-specific properties. This protocol highlights when and how the three research groups (simulation, synthesis, and engineering) should be interacting to arrive at the most effective method for predictive design of their material.

A robust mass spectrometry method for rapid profiling of erythrocyte ghost membrane proteomes.

PubMed

Fye, Haddy K S; Mrosso, Paul; Bruce, Lesley; Thézénas, Marie-Laëtitia; Davis, Simon; Fischer, Roman; Rwegasira, Gration L; Makani, Julie; Kessler, Benedikt M

2018-01-01

Red blood cell (RBC) physiology is directly linked to many human disorders associated with low tissue oxygen levels or anemia including chronic obstructive pulmonary disease, congenital heart disease, sleep apnea and sickle cell anemia. Parasites such as Plasmodium spp. and phylum Apicomplexa directly target RBCs, and surface molecules within the RBC membrane are critical for pathogen interactions. Proteomics of RBC membrane 'ghost' fractions has therefore been of considerable interest, but protocols described to date are either suboptimal or too extensive to be applicable to a larger set of clinical cohorts. Here, we describe an optimised erythrocyte isolation protocol from blood, tested for various storage conditions and explored using different fractionation conditions for isolating ghost RBC membranes. Liquid chromatography mass spectrometry (LC-MS) analysis on a Q-Exactive Orbitrap instrument was used to profile proteins isolated from the comparative conditions. Data analysis was run on the MASCOT and MaxQuant platforms to assess their scope and diversity. The results obtained demonstrate a robust method for membrane enrichment enabling consistent MS based characterisation of > 900 RBC membrane proteins in single LC-MS/MS analyses. Non-detergent based membrane solubilisation methods using the tissue and supernatant fractions of isolated ghost membranes are shown to offer effective haemoglobin removal as well as diverse recovery including erythrocyte membrane proteins of high and low abundance. The methods described in this manuscript propose a medium to high throughput framework for membrane proteome profiling by LC-MS of potential applicability to larger clinical cohorts in a variety of disease contexts.

SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs

PubMed Central

Shaikh, Tanvir R; Gao, Haixiao; Baxter, William T; Asturias, Francisco J; Boisset, Nicolas; Leith, Ardean; Frank, Joachim

2009-01-01

This protocol describes the reconstruction of biological molecules from the electron micrographs of single particles. Computation here is performed using the image-processing software SPIDER and can be managed using a graphical user interface, termed the SPIDER Reconstruction Engine. Two approaches are described to obtain an initial reconstruction: random-conical tilt and common lines. Once an existing model is available, reference-based alignment can be used, a procedure that can be iterated. Also described is supervised classification, a method to look for homogeneous subsets when multiple known conformations of the molecule may coexist. PMID:19180078

Annotating Mutational Effects on Proteins and Protein Interactions: Designing Novel and Revisiting Existing Protocols.

PubMed

Li, Minghui; Goncearenco, Alexander; Panchenko, Anna R

2017-01-01

In this review we describe a protocol to annotate the effects of missense mutations on proteins, their functions, stability, and binding. For this purpose we present a collection of the most comprehensive databases which store different types of sequencing data on missense mutations, we discuss their relationships, possible intersections, and unique features. Next, we suggest an annotation workflow using the state-of-the art methods and highlight their usability, advantages, and limitations for different cases. Finally, we address a particularly difficult problem of deciphering the molecular mechanisms of mutations on proteins and protein complexes to understand the origins and mechanisms of diseases.

Isolation of zymogen granules from rat pancreas.

PubMed

Rindler, Michael J

2006-01-01

This unit describes methods for preparing zymogen granules from rat pancreas. Zymogen granules are storage organelles in pancreatic acinar cells containing digestive enzymes that are released into the pancreatic duct. The protocols in this unit take advantage of the large size (up to 1 microm diameter) and high density (>1.20 g/cm(3) on sucrose gradients) of the granules as compared to other cellular organelles. They use a combination of differential sedimentation and density gradient separation to accomplish the purification. Similar procedures can be used to isolate zymogen granules from mouse pancreas and canine pancreas. A protocol for preparing zymogen granules from dog pancreas is also included.

Single-step method for β-galactosidase assays in Escherichia coli using a 96-well microplate reader.

PubMed

Schaefer, Jorrit; Jovanovic, Goran; Kotta-Loizou, Ioly; Buck, Martin

2016-06-15

Historically, the lacZ gene is one of the most universally used reporters of gene expression in molecular biology. Its activity can be quantified using an artificial substrate, o-nitrophenyl-ß-d-galactopyranoside (ONPG). However, the traditional method for measuring LacZ activity (first described by J. H. Miller in 1972) can be challenging for a large number of samples, is prone to variability, and involves hazardous compounds for lysis (e.g., chloroform, toluene). Here we describe a single-step assay using a 96-well microplate reader with a proven alternative cell permeabilization method. This modified protocol reduces handling time by 90%. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

Phytoplankton IF-FISH: Species-specific labeling of cellular proteins by immunofluorescence (IF) with simultaneous species identification by fluorescence immunohybridization (FISH).

PubMed

Meek, Megan E; Van Dolah, Frances M

2016-05-01

Phytoplankton rarely occur as unialgal populations. Therefore, to study species-specific protein expression, indicative of physiological status in natural populations, methods are needed that will both assay for a protein of interest and identify the species expressing it. Here we describe a protocol for IF-FISH, a dual labeling procedure using immunofluorescence (IF) labeling of a protein of interest followed by fluorescence in situ hybridization (FISH) to identify the species expressing that protein. The protocol was developed to monitor expression of the cell cycle marker proliferating cell nuclear antigen (PCNA) in the red tide dinoflagellate, Karenia brevis, using a large subunit (LSU) rRNA probe to identify K. brevis in a mixed population of morphologically similar Karenia species. We present this protocol as proof of concept that IF-FISH can be successfully applied to phytoplankton cells. This method is widely applicable for the analysis of single-cell protein expression of any protein of interest within phytoplankton communities. Copyright © 2016 Elsevier B.V. All rights reserved.

Sacrificial adhesive bonding: a powerful method for fabrication of glass microchips

PubMed Central

Lima, Renato S.; Leão, Paulo A. G. C.; Piazzetta, Maria H. O.; Monteiro, Alessandra M.; Shiroma, Leandro Y.; Gobbi, Angelo L.; Carrilho, Emanuel

2015-01-01

A new protocol for fabrication of glass microchips is addressed in this research paper. Initially, the method involves the use of an uncured SU-8 intermediate to seal two glass slides irreversibly as in conventional adhesive bonding-based approaches. Subsequently, an additional step removes the adhesive layer from the channels. This step relies on a selective development to remove the SU-8 only inside the microchannel, generating glass-like surface properties as demonstrated by specific tests. Named sacrificial adhesive layer (SAB), the protocol meets the requirements of an ideal microfabrication technique such as throughput, relatively low cost, feasibility for ultra large-scale integration (ULSI), and high adhesion strength, supporting pressures on the order of 5 MPa. Furthermore, SAB eliminates the use of high temperature, pressure, or potential, enabling the deposition of thin films for electrical or electrochemical experiments. Finally, the SAB protocol is an improvement on SU-8-based bondings described in the literature. Aspects such as substrate/resist adherence, formation of bubbles, and thermal stress were effectively solved by using simple and inexpensive alternatives. PMID:26293346

In vivo imaging and analysis of cerebrovascular hemodynamic responses and tissue oxygenation in the mouse brain.

PubMed

Kisler, Kassandra; Lazic, Divna; Sweeney, Melanie D; Plunkett, Shane; El Khatib, Mirna; Vinogradov, Sergei A; Boas, David A; Sakadži, Sava; Zlokovic, Berislav V

2018-06-01

Cerebrovascular dysfunction has an important role in the pathogenesis of multiple brain disorders. Measurement of hemodynamic responses in vivo can be challenging, particularly as techniques are often not described in sufficient detail and vary between laboratories. We present a set of standardized in vivo protocols that describe high-resolution two-photon microscopy and intrinsic optical signal (IOS) imaging to evaluate capillary and arteriolar responses to a stimulus, regional hemodynamic responses, and oxygen delivery to the brain. The protocol also describes how to measure intrinsic NADH fluorescence to understand how blood O 2 supply meets the metabolic demands of activated brain tissue, and to perform resting-state absolute oxygen partial pressure (pO 2 ) measurements of brain tissue. These methods can detect cerebrovascular changes at far higher resolution than MRI techniques, although the optical nature of these techniques limits their achievable imaging depths. Each individual procedure requires 1-2 h to complete, with two to three procedures typically performed per animal at a time. These protocols are broadly applicable in studies of cerebrovascular function in healthy and diseased brain in any of the existing mouse models of neurological and vascular disorders. All these procedures can be accomplished by a competent graduate student or experienced technician, except the two-photon measurement of absolute pO 2 level, which is better suited to a more experienced, postdoctoral-level researcher.

Test Protocols for Advanced Inverter Interoperability Functions – Main Document

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

Johnson, Jay Dean; Gonzalez, Sigifredo; Ralph, Mark E.

2013-11-01

Distributed energy resources (DER) such as photovoltaic (PV) systems, when deployed in a large scale, are capable of influencing significantly the operation of power systems. Looking to the future, stakeholders are working on standards to make it possible to manage the potentially complex interactions between DER and the power system. In 2009, the Electric Power Research Institute (EPRI), Sandia National Laboratories (SNL) with the U.S. Department of Energy (DOE), and the Solar Electric Power Association (SEPA) initiated a large industry collaborative to identify and standardize definitions for a set of DER grid support functions. While the initial effort concentrated onmore » grid-tied PV inverters and energy storage systems, the concepts have applicability to all DER. A partial product of this on-going effort is a reference definitions document (IEC TR 61850-90-7, Object models for power converters in distributed energy resources (DER) systems) that has become a basis for expansion of related International Electrotechnical Commission (IEC) standards, and is supported by US National Institute of Standards and Technology (NIST) Smart Grid Interoperability Panel (SGIP). Some industry-led organizations advancing communications protocols have also embraced this work. As standards continue to evolve, it is necessary to develop test protocols to independently verify that the inverters are properly executing the advanced functions. Interoperability is assured by establishing common definitions for the functions and a method to test compliance with operational requirements. This document describes test protocols developed by SNL to evaluate the electrical performance and operational capabilities of PV inverters and energy storage, as described in IEC TR 61850-90-7. While many of these functions are not currently required by existing grid codes or may not be widely available commercially, the industry is rapidly moving in that direction. Interoperability issues are already apparent as some of these inverter capabilities are being incorporated in large demonstration and commercial projects. The test protocols are intended to be used to verify acceptable performance of inverters within the standard framework described in IEC TR 61850-90-7. These test protocols, as they are refined and validated over time, can become precursors for future certification test procedures for DER advanced grid support functions.« less

Test Protocols for Advanced Inverter Interoperability Functions - Appendices

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

Johnson, Jay Dean; Gonzalez, Sigifredo; Ralph, Mark E.

2013-11-01

Distributed energy resources (DER) such as photovoltaic (PV) systems, when deployed in a large scale, are capable of influencing significantly the operation of power systems. Looking to the future, stakeholders are working on standards to make it possible to manage the potentially complex interactions between DER and the power system. In 2009, the Electric Power Research Institute (EPRI), Sandia National Laboratories (SNL) with the U.S. Department of Energy (DOE), and the Solar Electric Power Association (SEPA) initiated a large industry collaborative to identify and standardize definitions for a set of DER grid support functions. While the initial effort concentrated onmore » grid-tied PV inverters and energy storage systems, the concepts have applicability to all DER. A partial product of this on-going effort is a reference definitions document (IEC TR 61850-90-7, Object models for power converters in distributed energy resources (DER) systems) that has become a basis for expansion of related International Electrotechnical Commission (IEC) standards, and is supported by US National Institute of Standards and Technology (NIST) Smart Grid Interoperability Panel (SGIP). Some industry-led organizations advancing communications protocols have also embraced this work. As standards continue to evolve, it is necessary to develop test protocols to independently verify that the inverters are properly executing the advanced functions. Interoperability is assured by establishing common definitions for the functions and a method to test compliance with operational requirements. This document describes test protocols developed by SNL to evaluate the electrical performance and operational capabilities of PV inverters and energy storage, as described in IEC TR 61850-90-7. While many of these functions are not now required by existing grid codes or may not be widely available commercially, the industry is rapidly moving in that direction. Interoperability issues are already apparent as some of these inverter capabilities are being incorporated in large demonstration and commercial projects. The test protocols are intended to be used to verify acceptable performance of inverters within the standard framework described in IEC TR 61850-90-7. These test protocols, as they are refined and validated over time, can become precursors for future certification test procedures for DER advanced grid support functions.« less

Mesenchymal-epithelial interaction techniques

PubMed Central

Baskin, Lawrence

2016-01-01

This paper reviews the importance of mesenchymal-epithelial interactions in development and gives detailed technical protocols for investigating these interactions. Successful analysis of mesenchymal-epithelial interactions requires knowing the ages in which embryonic, neonatal and adult organs can be separated into mesenchymal and epithelial tissues. Methods for separation of mesenchymal and epithelial and preparation of tissue recombinants are described. PMID:26610327

A Robust New Method for Analzing Community Change and an Example using 83 years of Avian Response to Forest Succession

EPA Science Inventory

This manuscript describes a novel statistical analysis technique developed by the authors for use in combining survey data carried out under different field protocols. We apply the technique to 83 years of survey data on avian songbird populations in northern lower Michigan to de...

Developing and Applying a Protocol for a Systematic Review in the Social Sciences

ERIC Educational Resources Information Center

Campbell, Allison; Taylor, Brian; Bates, Jessica; O'Connor-Bones, Una

2018-01-01

The article reports on a systematic method of undertaking a literature search on the educational impact of being a young carer (16-24 years old). The search methodology applied and described in detail will be of value to academic librarians and to other education researchers who undertake systematic literature searches. Seven bibliographic…

Determination of Titratable Acidity in Wine Using Potentiometric, Conductometric, and Photometric Methods

ERIC Educational Resources Information Center

Volmer, Dietrich A.; Curbani, Luana; Parker, Timothy A.; Garcia, Jennifer; Schultz, Linda D.; Borges, Endler Marcel

2017-01-01

This experiment describes a simple protocol for teaching acid-base titrations using potentiometry, conductivity, and/or photometry to determine end points without an added indicator. The chosen example examines the titratable acidity of a red wine with NaOH. Wines contain anthocyanins, the colors of which change with pH. Importantly, at the…

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Bruni, Renato

2014-01-01

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

Examining the ethical and social issues of health technology design through the public appraisal of prospective scenarios: a study protocol describing a multimedia-based deliberative method

PubMed Central

2014-01-01

Background The design of health technologies relies on assumptions that affect how they will be implemented, such as intended use, complexity, impact on user autonomy, and appropriateness. Those who design and implement technologies make several ethical and social assumptions on behalf of users and society more broadly, but there are very few tools to examine prospectively whether such assumptions are warranted and how the public define and appraise the desirability of health innovations. This study protocol describes a three-year study that relies on a multimedia-based prospective method to support public deliberations that will enable a critical examination of the social and ethical issues of health technology design. Methods The first two steps of our mixed-method study were completed: relying on a literature review and the support of our multidisciplinary expert committee, we developed scenarios depicting social and technical changes that could unfold in three thematic areas within a 25-year timeframe; and for each thematic area, we created video clips to illustrate prospective technologies and short stories to describe their associated dilemmas. Using this multimedia material, we will: conduct four face-to-face deliberative workshops with members of the public (n = 40) who will later join additional participants (n = 25) through an asynchronous online forum; and analyze and integrate three data sources: observation, group deliberations, and a self-administered participant survey. Discussion This study protocol will be of interest to those who design and assess public involvement initiatives and to those who examine the implementation of health innovations. Our premise is that using user-friendly tools in a deliberative context that foster participants’ creativity and reflexivity in pondering potential technoscientific futures will enable our team to analyze a range of normative claims, including some that may prove problematic and others that may shed light over potentially more valuable design options. This research will help fill an important knowledge gap; intervening earlier in technological development could help reduce undesirable effects and inform the design and implementation of more appropriate innovations. PMID:24952582

The best of both worlds: Building on the COPUS and RTOP observation protocols to easily and reliably measure various levels of reformed instructional practice.

PubMed

Lund, Travis J; Pilarz, Matthew; Velasco, Jonathan B; Chakraverty, Devasmita; Rosploch, Kaitlyn; Undersander, Molly; Stains, Marilyne

2015-01-01

Researchers, university administrators, and faculty members are increasingly interested in measuring and describing instructional practices provided in science, technology, engineering, and mathematics (STEM) courses at the college level. Specifically, there is keen interest in comparing instructional practices between courses, monitoring changes over time, and mapping observed practices to research-based teaching. While increasingly common observation protocols (Reformed Teaching Observation Protocol [RTOP] and Classroom Observation Protocol in Undergraduate STEM [COPUS]) at the postsecondary level help achieve some of these goals, they also suffer from weaknesses that limit their applicability. In this study, we leverage the strengths of these protocols to provide an easy method that enables the reliable and valid characterization of instructional practices. This method was developed empirically via a cluster analysis using observations of 269 individual class periods, corresponding to 73 different faculty members, 28 different research-intensive institutions, and various STEM disciplines. Ten clusters, called COPUS profiles, emerged from this analysis; they represent the most common types of instructional practices enacted in the classrooms observed for this study. RTOP scores were used to validate the alignment of the 10 COPUS profiles with reformed teaching. Herein, we present a detailed description of the cluster analysis method, the COPUS profiles, and the distribution of the COPUS profiles across various STEM courses at research-intensive universities. © 2015 T. J. Lund et al. CBE—Life Sciences Education © 2015 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

PubMed Central

Crites, Travis J.; Maddox, Michael; Padhan, Kartika; Muller, James; Eigsti, Calvin; Varma, Rajat

2015-01-01

Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell-cell interfaces, in particular, T cell–antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene-expression analysis. Finally, we describe how to design and prepare transmembrane-anchored protein-laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane-anchored proteins may have many novel applications in cell biology and immunology. PMID:26331983

Patient-Centred Innovations for Persons with Multimorbidity: funded evaluation protocol.

PubMed

Stewart, Moira; Fortin, Martin

2017-05-09

The high prevalence of multimorbidity necessitates rethinking of the health care system. The overarching goal of the Patient-Centred Innovations for Persons with Multimorbidity program is to build on existing structures and find and evaluate patient-centred innovations relevant to multimorbidity. We describe the protocol for a proposed multijurisdictional (Quebec and Ontario) concurrent triangulation mixed-methods study. In both provinces, a qualitative descriptive study will be used to explore innovations in patient-centred multimorbidity care. Two randomized controlled trials, 1 in either province, will evaluate the innovations in a wait-list-controlled design using patient-reported outcomes. An additional control group, matched on age, sex, enrolment/index date (± 3 mo) and propensity score, will be created with the use of health administrative data. Patients will be 18-80 years of age and will have 3 or more chronic conditions. The innovations will have elements of relevance to multimorbidity care, patient-centred partnerships and integration of care. The primary outcome measures will be 2 patient-reported outcomes: patient education and self-efficacy. Secondary outcomes will include patient-reported health status, quality of life, psychological distress and health behaviours, and costs of care. This protocol describes a mixed-method study in 2 jurisdictions. The studies will answer the questions of what innovations work and how they work for patients, health care professionals and policy-makers. Trial registration: ClinicalTrials.gov, no NCT02789800 (Quebec Trial), NCT02742597 (Ontario Trial). Copyright 2017, Joule Inc. or its licensors.

Methods to study the biogenesis of membrane proteins in yeast mitochondria.

PubMed

Weckbecker, Daniel; Herrmann, Johannes M

2013-01-01

The biogenesis of mitochondrial membrane proteins is an intricate process that relies on the import and submitochondrial sorting of nuclear-encoded preproteins and on the synthesis of mitochondrial translation products in the matrix. Subsequently, these polypeptides need to be inserted into the outer and the inner membranes of the organelle where many of them assemble into multisubunit complexes. In this chapter we provide established protocols to study these different processes experimentally using mitochondria of budding yeast. In particular, methods are described in detail to purify mitochondria, to study mitochondrial protein synthesis, to follow the import of radiolabeled preproteins into isolated mitochondria, and to assess membrane association and the aggregation of mitochondrial proteins by fractionation. These protocols and a list of dos and don'ts shall enable beginners and experienced scientists to address the targeting and assembly of mitochondrial membrane proteins.

Directed Differentiation of Embryonic Stem Cells Using a Bead-Based Combinatorial Screening Method

PubMed Central

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J.; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported. PMID:25251366

Spectrophotometric determination of protein concentration.

PubMed

Grimsley, Gerald R; Pace, C Nick

2004-11-01

The concentration of a purified protein in solution is most conveniently and accurately measured using absorbance spectroscopy. The absorbance, A, is a linear function of the molar concentration, C, according to the Beer-Lambert law: A = epsilon x l x c, where e is the molar absorption coefficient and l is the cell path length. This unit provides protocols for calculation of epsilon for a folded or unfolded protein, making use of the average epsilon values for the three contributing chromophores in proteins (the side chains of Trp, Tyr, and Cys). A basic protocol describes how to measure the concentration of a protein using the calculated epsilon and the Beer-Lambert law. A sensitive method is provided for measuring the concentration of proteins that contain few if any tryptophan or tyrosine residues, and a simple method is provided for estimating total protein concentration in crude extracts.

Realist explanatory theory building method for social epidemiology: a protocol for a mixed method multilevel study of neighbourhood context and postnatal depression.

PubMed

Eastwood, John G; Jalaludin, Bin B; Kemp, Lynn A

2014-01-01

A recent criticism of social epidemiological studies, and multi-level studies in particular has been a paucity of theory. We will present here the protocol for a study that aims to build a theory of the social epidemiology of maternal depression. We use a critical realist approach which is trans-disciplinary, encompassing both quantitative and qualitative traditions, and that assumes both ontological and hierarchical stratification of reality. We describe a critical realist Explanatory Theory Building Method comprising of an: 1) emergent phase, 2) construction phase, and 3) confirmatory phase. A concurrent triangulated mixed method multilevel cross-sectional study design is described. The Emergent Phase uses: interviews, focus groups, exploratory data analysis, exploratory factor analysis, regression, and multilevel Bayesian spatial data analysis to detect and describe phenomena. Abductive and retroductive reasoning will be applied to: categorical principal component analysis, exploratory factor analysis, regression, coding of concepts and categories, constant comparative analysis, drawing of conceptual networks, and situational analysis to generate theoretical concepts. The Theory Construction Phase will include: 1) defining stratified levels; 2) analytic resolution; 3) abductive reasoning; 4) comparative analysis (triangulation); 5) retroduction; 6) postulate and proposition development; 7) comparison and assessment of theories; and 8) conceptual frameworks and model development. The strength of the critical realist methodology described is the extent to which this paradigm is able to support the epistemological, ontological, axiological, methodological and rhetorical positions of both quantitative and qualitative research in the field of social epidemiology. The extensive multilevel Bayesian studies, intensive qualitative studies, latent variable theory, abductive triangulation, and Inference to Best Explanation provide a strong foundation for Theory Construction. The study will contribute to defining the role that realism and mixed methods can play in explaining the social determinants and developmental origins of health and disease.

Effects of a peer support programme for youth social services employees experiencing potentially traumatic events: a protocol for a prospective cohort study

PubMed Central

Guay, Stephane; Tremblay, Nicole; Goncalves, Jane; Bilodeau, Henriette; Geoffrion, Steve

2017-01-01

Introduction The use of peer support programmes to help workers experiencing potentially traumatic events (PTE) has increased in high-risk organisations in the last decades. However, the scientific evidence of its effectiveness is still very limited. This paper aims to describe the protocol of a prospective cohort study that assesses the efficacy of a peer support programme among youth social services employees exposed to a PTE at work on psychological well-being, work functioning and needs of support. Methods and analysis This is a mixed-methods prospective study that will examine workers' evolution four times over a 12-month period in Canada. This study involves: (1) quantitative data obtained through self-administrated questionnaires among 222 workers, and (2) qualitative in-depth interviews with a subsample of 45 workers. This study will compare findings from a cohort who received the support of a peer following a PTE (peer support–experimental protocol) as part of the experimental protocol of the Montreal Youth Social Services-University Institute (MYSS-UI), the second group of workers did not ask for the peer support (no peer support-experimental protocol) but was part of MYSS-UI, and the third group received standard organisational support from the Monteregie Youth Social Services (MYSS) (standard organisational protocol). Ethics and dissemination The protocol and informed consent form complied with the ethics guidelines of the MYSS-UI. The Research Ethics Board of MYSS-UI and MYSS reviewed and accepted the protocol as required. The results of the study will be published in peer-reviewed journals, presented at research and general public conferences, disseminated via a public report for the institute that funded the project and for all workers. Results of this study will influence decision making regarding intervention policies following PTE and peer support interventions may be expanded throughout the youth social services in Canada and worldwide. PMID:28647721

High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.

PubMed

Liu, Guangbo; Lanham, Clayton; Buchan, J Ross; Kaplan, Matthew E

2017-01-01

Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

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

Miquel Guennoc, Cora; Rose, Christophe; Guinnet, Frédéric

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells. While laser scanning confocal microscopy (LSCM)more » is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). Furthermore, this report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.« less

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

DOE PAGES

Miquel Guennoc, Cora; Rose, Christophe; Guinnet, Frédéric; ...

2017-01-01

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells. While laser scanning confocal microscopy (LSCM)more » is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). Furthermore, this report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.« less

Dual sensory loss: development of a dual sensory loss protocol and design of a randomized controlled trial

PubMed Central

2013-01-01

Background Dual sensory loss (DSL) has a negative impact on health and wellbeing and its prevalence is expected to increase due to demographic aging. However, specialized care or rehabilitation programs for DSL are scarce. Until now, low vision rehabilitation does not sufficiently target concurrent impairments in vision and hearing. This study aims to 1) develop a DSL protocol (for occupational therapists working in low vision rehabilitation) which focuses on optimal use of the senses and teaches DSL patients and their communication partners to use effective communication strategies, and 2) describe the multicenter parallel randomized controlled trial (RCT) designed to test the effectiveness and cost-effectiveness of the DSL protocol. Methods/design To develop a DSL protocol, literature was reviewed and content was discussed with professionals in eye/ear care (interviews/focus groups) and DSL patients (interviews). A pilot study was conducted to test and confirm the DSL protocol. In addition, a two-armed international multi-center RCT will evaluate the effectiveness and cost-effectiveness of the DSL protocol compared to waiting list controls, in 124 patients in low vision rehabilitation centers in the Netherlands and Belgium. Discussion This study provides a treatment protocol for rehabilitation of DSL within low vision rehabilitation, which aims to be a valuable addition to the general low vision rehabilitation care. Trial registration Netherlands Trial Register (NTR) identifier: NTR2843 PMID:23941667

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

Precise, High-throughput Analysis of Bacterial Growth.

PubMed

Kurokawa, Masaomi; Ying, Bei-Wen

2017-09-19

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

A genotyping protocol for multiple tissue types from the polyploid tree species Sequoia sempervirens (Cupressaceae).

PubMed

Narayan, Lakshmi; Dodd, Richard S; O'Hara, Kevin L

2015-03-01

Identifying clonal lineages in asexually reproducing plants using microsatellite markers is complicated by the possibility of nonidentical genotypes from the same clonal lineage due to somatic mutations, null alleles, and scoring errors. We developed and tested a clonal identification protocol that is robust to these issues for the asexually reproducing hexaploid tree species coast redwood (Sequoia sempervirens). Microsatellite data from four previously published and two newly developed primers were scored using a modified protocol, and clones were identified using Bruvo genetic distances. The effectiveness of this clonal identification protocol was assessed using simulations and by genotyping a test set of paired samples of different tissue types from the same trees. Data from simulations showed that our protocol allowed us to accurately identify clonal lineages. Multiple test samples from the same trees were identified correctly, although certain tissue type pairs had larger genetic distances on average. The methods described in this paper will allow for the accurate identification of coast redwood clones, facilitating future studies of the reproductive ecology of this species. The techniques used in this paper can be applied to studies of other clonal organisms as well.

PROTOCOL TO EVALUATE THE MOISTURE DURABILITY OF ENERGY-EFFICIENT WALLS

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

Boudreaux, Philip R; Pallin, Simon B; Hun, Diana E

Walls account for about 8% of the energy used in residential buildings. This energy penalty can be reduced with higher insulation levels and increased airtightness. However, these measures can compromise the moisture durability and long-term performance of wall assemblies because they can lead to lower moisture tolerance due to reduced drying potential. To avert these problems, a moisture durability protocol was developed to evaluate the probability that an energy-efficient wall design will experience mold growth. This protocol examines the effects of moisture sources in walls through a combination of simulations and lab experiments, uses the mold growth index as themore » moisture durability indicator, and is based on a probabilistic approach that utilizes stochastically varying input parameters. The simulation tools used include a new validated method for taking into account the effects of air leakage in wall assemblies This paper provides an overview of the developed protocol, discussion of the probabilistic simulation approach and describes results from the evaluation of two wall assemblies in Climate Zones 2, 4, and 6. The protocol will be used to supply builders with wall designs that are energy efficient, moisture durable and cost-effective.« less

Arthroscopic management of the painful total elbow arthroplasty

PubMed Central

Bain, Gregory I

2015-01-01

Background Failure of total elbow arthroplasty is more common than after other major joint arthroplasties and is often a result of aseptic loosening, peri-prosthetic infection, fracture and instability. Infection can be a devastating complication, yet there are no established guidelines for the pre-operative diagnosis of total elbow peri-prosthetic infection. This is because pre-operative clinical, radiographic and biochemical tests are often unreliable. Methods Using three case examples, a standardized protocol for the clinical and arthroscopic assessment of the painful total elbow arthroplasty is described. This is used to provide a mechanical and microbiological diagnosis of the patient’s pain. Results There have been no complications resulting from the use of this technique in the three patients described, nor in any other patient to date. Conclusions The staged protocol described in the present study, utilizing arthroscopic assessment, has refined the approach to the painful total elbow arthroplasty because it directly influences the definitive surgical management of the patient. It is recommended that other surgeons follow the principles outlined in the present study when faced with this challenging problem. PMID:27583000

Application of SEM and EDX in studying biomineralization in plant tissues.

PubMed

He, Honghua; Kirilak, Yaowanuj

2014-01-01

This chapter describes protocols using formalin-acetic acid-alcohol (FAA) to fix plant tissues for studying biomineralization by means of scanning electron microscopy (SEM) and qualitative energy-dispersive X-ray microanalysis (EDX). Specimen preparation protocols for SEM and EDX mainly include fixation, dehydration, critical point drying (CPD), mounting, and coating. Gold-coated specimens are used for SEM imaging, while gold- and carbon-coated specimens are prepared for qualitative X-ray microanalyses separately to obtain complementary information on the elemental compositions of biominerals. During the specimen preparation procedure for SEM, some biominerals may be dislodged or scattered, making it difficult to determine their accurate locations, and light microscopy is used to complement SEM studies. Specimen preparation protocols for light microscopy generally include fixation, dehydration, infiltration and embedding with resin, microtome sectioning, and staining. In addition, microwave processing methods are adopted here to speed up the specimen preparation process for both SEM and light microscopy.

The Isolation of Pure Populations of Neurons by Laser Capture Microdissection: Methods and Application in Neuroscience.

PubMed

Morris, Renée; Mehta, Prachi

2018-01-01

In mammals, the central nervous system (CNS) is constituted of various cellular elements, posing a challenge to isolating specific cell types to investigate their expression profile. As a result, tissue homogenization is not amenable to analyses of motor neurons profiling as these represent less than 10% of the total spinal cord cell population. One way to tackle the problem of tissue heterogeneity and obtain meaningful genomic, proteomic, and transcriptomic profiling is to use laser capture microdissection technology (LCM). In this chapter, we describe protocols for the capture of isolated populations of motor neurons from spinal cord tissue sections and for downstream transcriptomic analysis of motor neurons with RT-PCR. We have also included a protocol for the immunological confirmation that the captured neurons are indeed motor neurons. Although focused on spinal cord motor neurons, these protocols can be easily optimized for the isolation of any CNS neurons.

A protocol for the creation of useful geometric shape metrics illustrated with a newly derived geometric measure of leaf circularity.

PubMed

Krieger, Jonathan D

2014-08-01

I present a protocol for creating geometric leaf shape metrics to facilitate widespread application of geometric morphometric methods to leaf shape measurement. • To quantify circularity, I created a novel shape metric in the form of the vector between a circle and a line, termed geometric circularity. Using leaves from 17 fern taxa, I performed a coordinate-point eigenshape analysis to empirically identify patterns of shape covariation. I then compared the geometric circularity metric to the empirically derived shape space and the standard metric, circularity shape factor. • The geometric circularity metric was consistent with empirical patterns of shape covariation and appeared more biologically meaningful than the standard approach, the circularity shape factor. The protocol described here has the potential to make geometric morphometrics more accessible to plant biologists by generalizing the approach to developing synthetic shape metrics based on classic, qualitative shape descriptors.

A reference protocol for comparing the biocidal properties of gas plasma generating devices

NASA Astrophysics Data System (ADS)

Shaw, A.; Seri, P.; Borghi, C. A.; Shama, G.; Iza, F.

2015-12-01

Growing interest in the use of non-thermal, atmospheric pressure gas plasmas for decontamination purposes has resulted in a multiplicity of plasma-generating devices. There is currently no universally approved method of comparing the biocidal performance of such devices and in the work described here spores of the Gram positive bacterium Bacillus subtilis (ATCC 6633) are proposed as a suitable reference biological agent. In order to achieve consistency in the form in which the biological agent in question is presented to the plasma, a polycarbonate membrane loaded with a monolayer of spores is proposed. The advantages of the proposed protocol are evaluated by comparing inactivation tests in which an alternative microorganism (methicillin resistant Staphylococcus aureus—MRSA) and the widely-used sample preparation technique of directly pipetting cell suspensions onto membranes are employed. In all cases, inactivation tests with either UV irradiation or plasma exposure were more reproducible when the proposed protocol was followed.

Laboratory procedures to generate viral metagenomes.

PubMed

Thurber, Rebecca V; Haynes, Matthew; Breitbart, Mya; Wegley, Linda; Rohwer, Forest

2009-01-01

This collection of laboratory protocols describes the steps to collect viruses from various samples with the specific aim of generating viral metagenome sequence libraries (viromes). Viral metagenomics, the study of uncultured viral nucleic acid sequences from different biomes, relies on several concentration, purification, extraction, sequencing and heuristic bioinformatic methods. No single technique can provide an all-inclusive approach, and therefore the protocols presented here will be discussed in terms of hypothetical projects. However, care must be taken to individualize each step depending on the source and type of viral-particles. This protocol is a description of the processes we have successfully used to: (i) concentrate viral particles from various types of samples, (ii) eliminate contaminating cells and free nucleic acids and (iii) extract, amplify and purify viral nucleic acids. Overall, a sample can be processed to isolate viral nucleic acids suitable for high-throughput sequencing in approximately 1 week.

Analytical Measurement of Discrete Hydrogen Sulfide Pools in Biological Specimens

PubMed Central

Shen, Xinggui; Peter, Elvis A.; Bir, Shyamal; Wang, Rui; Kevil, Christopher G.

2015-01-01

Hydrogen sulfide (H2S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors including pharmaco-therapeutic manipulation. Amongst the challenges facing the field is the accurate measurement of biologically active H2S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bonafide H2S. The complexity of analytical H2S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in different forms, including a free form, an acid labile pool and as bound sulfane sulfur. Here we describe a new protocol to discretely measure specific H2S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping and derivatization of H2S. Acid-labile H2S is released by incubating the sample in an acidic solution (pH 2.6) of 100 mM phosphate buffer with 0.1 mM DTPA, in an enclosed system to contain volatilized H2S. Volatilized H2S is then trapped in 100 mM Tris-HCl (pH 9.5, 0.1 mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of bound sulfane sulfur pool was measured by incubating the sample with 1 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent to reduce disulfide bonds, in 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA), and H2S measurement performed in an analogous manner to the one described above. The acid labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H2S measurement from the acid liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method protocol allows very sensitive and accurate measurement of the three primary biological pools of H2S including free, acid labile, and bound sulfane sulfur in various biological specimens. PMID:22561703

Towards the development of universal, fast and highly accurate docking/scoring methods: a long way to go

PubMed Central

Moitessier, N; Englebienne, P; Lee, D; Lawandi, J; Corbeil, C R

2008-01-01

Accelerating the drug discovery process requires predictive computational protocols capable of reducing or simplifying the synthetic and/or combinatorial challenge. Docking-based virtual screening methods have been developed and successfully applied to a number of pharmaceutical targets. In this review, we first present the current status of docking and scoring methods, with exhaustive lists of these. We next discuss reported comparative studies, outlining criteria for their interpretation. In the final section, we describe some of the remaining developments that would potentially lead to a universally applicable docking/scoring method. PMID:18037925

Parallel production and verification of protein products using a novel high-throughput screening method.

PubMed

Tegel, Hanna; Yderland, Louise; Boström, Tove; Eriksson, Cecilia; Ukkonen, Kaisa; Vasala, Antti; Neubauer, Peter; Ottosson, Jenny; Hober, Sophia

2011-08-01

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 μL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Technical note: a novel method for routine genotyping of horse coat color gene polymorphisms.

PubMed

Royo, L J; Fernández, I; Azor, P J; Alvarez, I; Pérez-Pardal, L; Goyache, F

2008-06-01

The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype using a macro freely available at http://www.igijon.com/personales/fgoyache/software_i.htm (last accessed February 26, 2007). The performance of the method is demonstrated on 156 Spanish Purebred horses.

Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging-drop and mass culture methods.

PubMed

Fuegemann, Christopher J; Samraj, Ajoy K; Walsh, Stuart; Fleischmann, Bernd K; Jovinge, Stefan; Breitbach, Martin

2010-12-01

Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes. © 2010 by John Wiley & Sons, Inc.

The reliable multicast protocol application programming interface

NASA Technical Reports Server (NTRS)

Montgomery , Todd; Whetten, Brian

1995-01-01

The Application Programming Interface for the Berkeley/WVU implementation of the Reliable Multicast Protocol is described. This transport layer protocol is implemented as a user library that applications and software buses link against.

A Simple XML Producer-Consumer Protocol

NASA Technical Reports Server (NTRS)

Smith, Warren; Gunter, Dan; Quesnel, Darcy; Biegel, Bryan (Technical Monitor)

2001-01-01

There are many different projects from government, academia, and industry that provide services for delivering events in distributed environments. The problem with these event services is that they are not general enough to support all uses and they speak different protocols so that they cannot interoperate. We require such interoperability when we, for example, wish to analyze the performance of an application in a distributed environment. Such an analysis might require performance information from the application, computer systems, networks, and scientific instruments. In this work we propose and evaluate a standard XML-based protocol for the transmission of events in distributed systems. One recent trend in government and academic research is the development and deployment of computational grids. Computational grids are large-scale distributed systems that typically consist of high-performance compute, storage, and networking resources. Examples of such computational grids are the DOE Science Grid, the NASA Information Power Grid (IPG), and the NSF Partnerships for Advanced Computing Infrastructure (PACIs). The major effort to deploy these grids is in the area of developing the software services to allow users to execute applications on these large and diverse sets of resources. These services include security, execution of remote applications, managing remote data, access to information about resources and services, and so on. There are several toolkits for providing these services such as Globus, Legion, and Condor. As part of these efforts to develop computational grids, the Global Grid Forum is working to standardize the protocols and APIs used by various grid services. This standardization will allow interoperability between the client and server software of the toolkits that are providing the grid services. The goal of the Performance Working Group of the Grid Forum is to standardize protocols and representations related to the storage and distribution of performance data. These standard protocols and representations must support tasks such as profiling parallel applications, monitoring the status of computers and networks, and monitoring the performance of services provided by a computational grid. This paper describes a proposed protocol and data representation for the exchange of events in a distributed system. The protocol exchanges messages formatted in XML and it can be layered atop any low-level communication protocol such as TCP or UDP Further, we describe Java and C++ implementations of this protocol and discuss their performance. The next section will provide some further background information. Section 3 describes the main communication patterns of our protocol. Section 4 describes how we represent events and related information using XML. Section 5 describes our protocol and Section 6 discusses the performance of two implementations of the protocol. Finally, an appendix provides the XML Schema definition of our protocol and event information.

Developing a guideline for clinical trial protocol content: Delphi consensus survey

PubMed Central

2012-01-01

Background Recent evidence has highlighted deficiencies in clinical trial protocols, having implications for many groups. Existing guidelines for randomized clinical trial (RCT) protocol content vary substantially and most do not describe systematic methodology for their development. As one of three prespecified steps for the systematic development of a guideline for trial protocol content, the objective of this study was to conduct a three-round Delphi consensus survey to develop and refine minimum content for RCT protocols. Methods Panellists were identified using a multistep iterative approach, met prespecified minimum criteria and represented key stakeholders who develop or use clinical trial protocols. They were asked to rate concepts for importance in a minimum set of items for RCT protocols. The main outcome measures were degree of importance (scale of 1 to 10; higher scores indicating higher importance) and level of consensus for items. Results were presented as medians, interquartile ranges, counts and percentages. Results Ninety-six expert panellists participated in the Delphi consensus survey including trial investigators, methodologists, research ethics board members, funders, industry, regulators and journal editors. Response rates were between 88 and 93% per round. Overall, panellists rated 63 of 88 concepts of high importance (of which 50 had a 25th percentile rating of 8 or greater), 13 of moderate importance (median 6 or 7) and 12 of low importance (median less than or equal to 5) for minimum trial protocol content. General and item-specific comments and subgroup results provided valuable insight for further discussions. Conclusions This Delphi process achieved consensus from a large panel of experts from diverse stakeholder groups on essential content for RCT protocols. It also highlights areas of divergence. These results, complemented by other empirical research and consensus meetings, are helping guide the development of a guideline for protocol content. PMID:23006145

A technique for setting analytical thresholds in massively parallel sequencing-based forensic DNA analysis

PubMed Central

2017-01-01

Amplicon (targeted) sequencing by massively parallel sequencing (PCR-MPS) is a potential method for use in forensic DNA analyses. In this application, PCR-MPS may supplement or replace other instrumental analysis methods such as capillary electrophoresis and Sanger sequencing for STR and mitochondrial DNA typing, respectively. PCR-MPS also may enable the expansion of forensic DNA analysis methods to include new marker systems such as single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) that currently are assayable using various instrumental analysis methods including microarray and quantitative PCR. Acceptance of PCR-MPS as a forensic method will depend in part upon developing protocols and criteria that define the limitations of a method, including a defensible analytical threshold or method detection limit. This paper describes an approach to establish objective analytical thresholds suitable for multiplexed PCR-MPS methods. A definition is proposed for PCR-MPS method background noise, and an analytical threshold based on background noise is described. PMID:28542338

A technique for setting analytical thresholds in massively parallel sequencing-based forensic DNA analysis.

PubMed

Young, Brian; King, Jonathan L; Budowle, Bruce; Armogida, Luigi

2017-01-01

Amplicon (targeted) sequencing by massively parallel sequencing (PCR-MPS) is a potential method for use in forensic DNA analyses. In this application, PCR-MPS may supplement or replace other instrumental analysis methods such as capillary electrophoresis and Sanger sequencing for STR and mitochondrial DNA typing, respectively. PCR-MPS also may enable the expansion of forensic DNA analysis methods to include new marker systems such as single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) that currently are assayable using various instrumental analysis methods including microarray and quantitative PCR. Acceptance of PCR-MPS as a forensic method will depend in part upon developing protocols and criteria that define the limitations of a method, including a defensible analytical threshold or method detection limit. This paper describes an approach to establish objective analytical thresholds suitable for multiplexed PCR-MPS methods. A definition is proposed for PCR-MPS method background noise, and an analytical threshold based on background noise is described.

Potato (Solanum tuberosum L.).

PubMed

Chetty, Venkateswari J; Narváez-Vásquez, Javier; Orozco-Cárdenas, Martha L

2015-01-01

Agrobacterium-mediated transformation is the most common method for the incorporation of foreign genes into the genome of potato as well as many other species in the Solanaceae family. This chapter describes protocols for the genetic transformation of three species of potato: Solanum tuberosum subsp. tuberosum (Desiréé), S. tuberosum subsp. andigenum (Blue potato), and S. tuberosum subsp. andigena using internodal segments as explants.

Resident Choice and the Survey Process: The Need for Standardized Observation and Transparency

ERIC Educational Resources Information Center

Schnelle, John F.; Bertrand, Rosanna; Hurd, Donna; White, Alan; Squires, David; Feuerberg, Marvin; Hickey, Kelly; Simmons, Sandra F.

2009-01-01

Purpose: To describe a standardized observation protocol to determine if nursing home (NH) staff offer choice to residents during 3 morning activities of daily living (ADL) and compare the observational data with deficiency statements cited by state survey staff. Design and Methods: Morning ADL care was observed in 20 NHs in 5 states by research…

An Undergraduate Laboratory Experiment that Utilizes a Glass Fiber Filter Assay to Determine the Steroid Specificity and Equilibrium Binding Properties of Glucocorticoid Receptors.

ERIC Educational Resources Information Center

John, Nancy J.; Firestone, Gary L.

1987-01-01

Describes two complementary laboratory exercises that use the glass fiber assay to assess receptor specificity and hormone binding affinity in rat liver cytoplasmic extracts. Details the methods, materials and protocol of the experiments. Discusses the basic concepts illustrated and the feasibility of using the experiments at the undergraduate…

Transcript patterns of Phanerochaete chrysosporium genes in organopollutant contaminated soils and in wood

Treesearch

Amber Vanden Wymelenberg; Bernard Janse; Jill Gaskell; Diane Dietrich; Marcelo Vallim; Dan Cullen

1998-01-01

We describe here recent methods for quantitative assessment of specific P. chrysosporium mRNAs in organopollutant contaminated soils and in Aspen wood chips. Magnetic capture techniques were used to rapidly purify poly(A)-RNA, and quantitative RT-PCR protocols were developed for all known lignin peroxidase (lip) and cellobiohydrolase (cbh1) genes. The methodology is...

Missouri Ozark Forest Ecosystem Project: site history, soils, landforms, woody and herbaceous vegetation, down wood, and inventory methods for the landscape experiment.

Treesearch

Stephen R. Shifley; Brian L., eds. Brookshire

2000-01-01

Describes vegetation and physical site conditions at the initiation (1991-1995) of the Missouri Ozark Forest Ecosystem Project (MOFEP) in the southeastern Missouri Ozarks. Provides detailed information on sampling protocols and summarizes initial conditions of the landscape experiment prior to harvest treatments. Summaries are by plot, by ~800-acre...

How to Improve Pupils' Literacy? A Cost-Effectiveness Analysis of a French Educational Project

ERIC Educational Resources Information Center

Massoni, Sebastien; Vergnaud, Jean-Christophe

2012-01-01

The "Action Lecture" program is an innovative teaching method run in some nursery and primary schools in Paris and designed to improve pupils' literacy. We report the results of an evaluation of this program. We describe the experimental protocol that was built to estimate the program's impact on several types of indicators. Data were…

Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.

PubMed

Chamma, Ingrid; Rossier, Olivier; Giannone, Grégory; Thoumine, Olivier; Sainlos, Matthieu

2017-04-01

Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.

Development of a real-time PCR protocol for the species origin confirmation of isolated animal particles detected by NIRM.

PubMed

Fumière, O; Marien, A; Fernández Pierna, J A; Baeten, V; Berben, G

2010-08-01

At present, European legislation prohibits totally the use of processed animal proteins in feed for all farmed animals (Commission Regulation (EC) No. 1234/2003-extended feed ban). A softening of the feed ban for non-ruminants would nevertheless be considered if alternative methods could be used to gain more information concerning the species origin of processed animal proteins than that which can be provided by classical optical microscopy. This would allow control provisions such as the ban of feeding animals with proteins from the same species or intra-species recycling (Regulation (EC) No. 1774/2002). Two promising alternative methods, near-infrared microscopy (NIRM) and real-time polymerase chain reaction (PCR), were combined to authenticate, at the species level, the presence of animal particles. The paper describes the improvements of the real-time PCR method made to the DNA extraction protocol, allowing five PCR analyses to be performed with the DNA extracted from a single particle.

Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

PubMed Central

Young, Jonathan W; Locke, James C W; Altinok, Alphan; Rosenfeld, Nitzan; Bacarian, Tigran; Swain, Peter S; Mjolsness, Eric; Elowitz, Michael B

2014-01-01

Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1–2 d for progressing through the analysis procedure. PMID:22179594

Formation of Stomach Tissue by Organoid Culture Using Mouse Embryonic Stem Cells.

PubMed

Noguchi, Taka-Aki K; Kurisaki, Akira

2017-01-01

In this chapter, we describe a method for the induction of stomach organoids from mouse embryonic stem (ES) cells. We used an embryoid body-based differentiation method to induce gastric primordial epithelium covered with mesenchyme and further differentiate it in Matrigel by 3D culture. The differentiated organoid contains both corpus- and antrum-specific mature gastric tissue cells. This protocol may be useful for a variety of studies in developmental biology and disease modeling of the stomach.

Chapter 4: Small Commercial and Residential Unitary and Split System HVAC Heating and Cooling Equipment-Efficiency Upgrade Evaluation Protocol. The Uniform Methods Project: Methods for Determining Energy Efficiency Savings for Specific Measures

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

Kurnik, Charles W; Jacobson, David; Metoyer, Jarred

The specific measure described here involves improving the overall efficiency in air-conditioning systems as a whole (compressor, evaporator, condenser, and supply fan). The efficiency rating is expressed as the energy efficiency ratio (EER), seasonal energy efficiency ratio (SEER), and integrated energy efficiency ratio (IEER). The higher the EER, SEER or IEER, the more efficient the unit is.

Using Environmental DNA for Invasive Species Surveillance and Monitoring.

PubMed

Mahon, Andrew R; Jerde, Christopher L

2016-01-01

The method employed for environmental DNA (eDNA) surveillance for detection and monitoring of rare species in aquatic systems has evolved dramatically since its first large-scale applications. Both active (targeted) and passive (total diversity) surveillance methods provide helpful information for management groups, but each has a suite of techniques that necessitate proper equipment training and use. The protocols described in this chapter represent some of the latest iterations in eDNA surveillance being applied in aquatic and marine systems.

A practical guide for the fabrication of microfluidic devices using glass and silicon

PubMed Central

Iliescu, Ciprian; Taylor, Hayden; Avram, Marioara; Miao, Jianmin; Franssila, Sami

2012-01-01

This paper describes the main protocols that are used for fabricating microfluidic devices from glass and silicon. Methods for micropatterning glass and silicon are surveyed, and their limitations are discussed. Bonding methods that can be used for joining these materials are summarized and key process parameters are indicated. The paper also outlines techniques for forming electrical connections between microfluidic devices and external circuits. A framework is proposed for the synthesis of a complete glass/silicon device fabrication flow. PMID:22662101

Quality-assurance plan for the analysis of fluvial sediment by laboratories of the U.S. Geological Survey

USGS Publications Warehouse

Matthes, Wilbur J.; Sholar, Clyde J.; George, John R.

1992-01-01

This report describes procedures used by the Iowa District sediment laboratory of the U.S. Geological Survey to assure the quality of sediment-laboratory data. These procedures can be used by other U.S. Geological Survey laboratories regardless of size and type of operation for quality assurance and quality control of specific sediment-laboratory processes. Also described are the equipment, specifications, calibration and maintenance, and the protocol for methods used in the analyses of fluvial sediment for concentration or particle size.

Serious gaming during multidisciplinary rehabilitation for patients with complex chronic pain or fatigue complaints: study protocol for a controlled trial and process evaluation

PubMed Central

Joosen, Margot C W; Mert, Agali; Zedlitz, Aglaia; Vrijhoef, Hubertus J M

2017-01-01

Introduction Many individuals suffer from chronic pain or functional somatic syndromes and face boundaries for diminishing functional limitations by means of biopsychosocial interventions. Serious gaming could complement multidisciplinary interventions through enjoyment and independent accessibility. A study protocol is presented for studying whether, how, for which patients and under what circumstances, serious gaming improves patient health outcomes during regular multidisciplinary rehabilitation. Methods and analysis A mixed-methods design is described that prioritises a two-armed naturalistic quasi-experiment. An experimental group is composed of patients who follow serious gaming during an outpatient multidisciplinary programme at two sites of a Dutch rehabilitation centre. Control group patients follow the same programme without serious gaming in two similar sites. Multivariate mixed-modelling analysis is planned for assessing how much variance in 250 patient records of routinely monitored pain intensity, pain coping and cognition, fatigue and psychopathology outcomes is attributable to serious gaming. Embedded qualitative methods include unobtrusive collection and analyses of stakeholder focus group interviews, participant feedback and semistructured patient interviews. Process analyses are carried out by a systematic approach of mixing qualitative and quantitative methods at various stages of the research. Ethics and dissemination The Ethics Committee of the Tilburg School of Social and Behavioural Sciences approved the research after reviewing the protocol for the protection of patients’ interests in conformity to the letter and rationale of the applicable laws and research practice (EC 2016.25t). Findings will be presented in research articles and international scientific conferences. Trial registration number A prospective research protocol for the naturalistic quasi-experimental outcome evaluation was entered in the Dutch trial register (registration number: NTR6020; Pre-results). PMID:28600377

Sampling protocol for monitoring abiotic and biotic characteristics of mountain ponds and lakes

USGS Publications Warehouse

Hoffman, Robert L.; Tyler, Torrey J.; Larson, Gary L.; Adams, Michael J.; Wente, Wendy; Galvan, Stephanie

2005-01-01

This document describes field techniques and procedures used for sampling mountain ponds and lakes. These techniques and procedures will be used primarily to monitor, as part of long-term programs in National Parks and other protected areas, the abiotic and biotic characteristics of naturally occurring permanent montane lentic systems up to 75 ha in surface area. However, the techniques and procedures described herein also can be used to sample temporary or ephemeral montane lentic sites. Each Standard Operating Procedure (SOP) section addresses a specific component of the limnological investigation, and describes in detail field sampling methods pertaining to parameters to be measured for each component.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

PubMed Central

Lucanic, Mark; Garrett, Theo; Gill, Matthew S.; Lithgow, Gordon J.

2018-01-01

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening. PMID:29630057

Isolation of Eosinophils from the Lamina Propria of the Murine Small Intestine.

PubMed

Berek, Claudia; Beller, Alexander; Chu, Van Trung

2016-01-01

Only recently has it become apparent that eosinophils play a crucial role in mucosal immune homeostasis. Although eosinophils are the main cellular component of the lamina propria of the gastrointestinal tract, they have often been overlooked because they express numerous markers, which are normally used to characterize macrophages and/or dendritic cells. To study their function in mucosal immunity, it is important to isolate them with high purity and viability. Here, we describe a protocol to purify eosinophils from the lamina propria of the murine small intestine. The method involves preparation of the small intestine, removal of epithelial cells and digestion of the lamina propria to release eosinophils. A protocol to sort eosinophils is included.

How to Prepare SMC and BMC-like Compounds to Perform Relevant Rheological Experiments?

NASA Astrophysics Data System (ADS)

Guiraud, Olivier; Dumont, Pierre J. J.; Orgéas, Laurent

2013-04-01

The study of the rheology of injected or compression moulded compounds like SMC or BMC is made particularly difficult by the high content and the intricate arrangement of their fibrous reinforcement. For these two types of compounds, inappropriate rheological testing protocols and rheometers are often used, which leads to a very large scatter of the experimental data. This study describes specific sampling and specimen's preparation methods, as well as dedicated rheometry devices to test their rheology. Following the proposed protocols, it is possible to obtain rheological measurements showing low scatter of the recorded stress values: about ±10% for SMC and about ±15% for BMC-like compounds.

National protocol framework for the inventory and monitoring of bees

USGS Publications Warehouse

Droege, Sam; Engler, Joseph D.; Sellers, Elizabeth A.; Lee O'Brien,

2016-01-01

This national protocol framework is a standardized tool for the inventory and monitoring of the approximately 4,200 species of native and non-native bee species that may be found within the National Wildlife Refuge System (NWRS) administered by the U.S. Fish and Wildlife Service (USFWS). However, this protocol framework may also be used by other organizations and individuals to monitor bees in any given habitat or location. Our goal is to provide USFWS stations within the NWRS (NWRS stations are land units managed by the USFWS such as national wildlife refuges, national fish hatcheries, wetland management districts, conservation areas, leased lands, etc.) with techniques for developing an initial baseline inventory of what bee species are present on their lands and to provide an inexpensive, simple technique for monitoring bees continuously and for monitoring and evaluating long-term population trends and management impacts. The latter long-term monitoring technique requires a minimal time burden for the individual station, yet can provide a good statistical sample of changing populations that can be investigated at the station, regional, and national levels within the USFWS’ jurisdiction, and compared to other sites within the United States and Canada. This protocol framework was developed in cooperation with the United States Geological Survey (USGS), the USFWS, and a worldwide network of bee researchers who have investigated the techniques and methods for capturing bees and tracking population changes. The protocol framework evolved from field and lab-based investigations at the USGS Bee Inventory and Monitoring Laboratory at the Patuxent Wildlife Research Center in Beltsville, Maryland starting in 2002 and was refined by a large number of USFWS, academic, and state groups. It includes a Protocol Introduction and a set of 8 Standard Operating Procedures or SOPs and adheres to national standards of protocol content and organization. The Protocol Narrative describes the history and need for the protocol framework and summarizes the basic elements of objectives, sampling design, field methods, training, data management, analysis, and reporting. The SOPs provide more detail and specific instructions for implementing the protocol framework. A central database, for managing all the resulting data is under development. We welcome use of this protocol framework by our partners, as appropriate for their bee inventory and monitoring objectives.

OpenFlow arbitrated programmable network channels for managing quantum metadata

DOE PAGES

Dasari, Venkat R.; Humble, Travis S.

2016-10-10

Quantum networks must classically exchange complex metadata between devices in order to carry out information for protocols such as teleportation, super-dense coding, and quantum key distribution. Demonstrating the integration of these new communication methods with existing network protocols, channels, and data forwarding mechanisms remains an open challenge. Software-defined networking (SDN) offers robust and flexible strategies for managing diverse network devices and uses. We adapt the principles of SDN to the deployment of quantum networks, which are composed from unique devices that operate according to the laws of quantum mechanics. We show how quantum metadata can be managed within a software-definedmore » network using the OpenFlow protocol, and we describe how OpenFlow management of classical optical channels is compatible with emerging quantum communication protocols. We next give an example specification of the metadata needed to manage and control quantum physical layer (QPHY) behavior and we extend the OpenFlow interface to accommodate this quantum metadata. Here, we conclude by discussing near-term experimental efforts that can realize SDN’s principles for quantum communication.« less

OpenFlow arbitrated programmable network channels for managing quantum metadata

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

Dasari, Venkat R.; Humble, Travis S.

Quantum networks must classically exchange complex metadata between devices in order to carry out information for protocols such as teleportation, super-dense coding, and quantum key distribution. Demonstrating the integration of these new communication methods with existing network protocols, channels, and data forwarding mechanisms remains an open challenge. Software-defined networking (SDN) offers robust and flexible strategies for managing diverse network devices and uses. We adapt the principles of SDN to the deployment of quantum networks, which are composed from unique devices that operate according to the laws of quantum mechanics. We show how quantum metadata can be managed within a software-definedmore » network using the OpenFlow protocol, and we describe how OpenFlow management of classical optical channels is compatible with emerging quantum communication protocols. We next give an example specification of the metadata needed to manage and control quantum physical layer (QPHY) behavior and we extend the OpenFlow interface to accommodate this quantum metadata. Here, we conclude by discussing near-term experimental efforts that can realize SDN’s principles for quantum communication.« less

Evaluation of Cyanea capillata Sting Management Protocols Using Ex Vivo and In Vitro Envenomation Models

PubMed Central

Headlam, Jasmine L.; MacLoughlin, Eoin

2017-01-01

Lion’s mane jellyfish (Cyanea capillata) stings cause severe pain and can lead to dangerous systemic effects, including Irukandji-like syndrome. As is the case for most cnidarian stings, recommended medical protocols in response to such stings lack rigorous scientific support. In this study, we sought to evaluate potential first aid care protocols using previously described envenomation models that allow for direct measurements of venom activity. We found that seawater rinsing, the most commonly recommended method of tentacle removal for this species, induced significant increases in venom delivery, while rinsing with vinegar or Sting No More® Spray did not. Post-sting temperature treatments affected sting severity, with 40 min of hot-pack treatment reducing lysis of sheep’s blood (in agar plates), a direct representation of venom load, by over 90%. Ice pack treatment had no effect on sting severity. These results indicate that sting management protocols for Cyanea need to be revised immediately to discontinue rinsing with seawater and include the use of heat treatment. PMID:28686221

Respiratory analysis of coupled mitochondria in cryopreserved liver biopsies.

PubMed

García-Roche, Mercedes; Casal, Alberto; Carriquiry, Mariana; Radi, Rafael; Quijano, Celia; Cassina, Adriana

2018-07-01

The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at -80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Blueberry (Vaccinium corymbosum L.).

PubMed

Song, Guo-Qing

2015-01-01

Vaccinium consists of approximately 450 species, of which highbush blueberry (Vaccinium corymbosum) is one of the three major Vaccinium fruit crops (i.e., blueberry, cranberry, and lingonberry) domesticated in the twentieth century. In blueberry the adventitious shoot regeneration using leaf explants has been the most desirable regeneration system to date; Agrobacterium tumefaciens-mediated transformation is the major gene delivery method and effective selection has been reported using either the neomycin phosphotransferase II gene (nptII) or the bialaphos resistance (bar) gene as selectable markers. The A. tumefaciens-mediated transformation protocol described in this chapter is based on combining the optimal conditions for efficient plant regeneration, reliable gene delivery, and effective selection. The protocol has led to successful regeneration of transgenic plants from leaf explants of four commercially important highbush blueberry cultivars for multiple purposes, providing a powerful approach to supplement conventional breeding methods for blueberry by introducing genes of interest.

Expression microdissection adapted to commercial laser dissection instruments

PubMed Central

Hanson, Jeffrey C; Tangrea, Michael A; Kim, Skye; Armani, Michael D; Pohida, Thomas J; Bonner, Robert F; Rodriguez-Canales, Jaime; Emmert-Buck, Michael R

2016-01-01

Laser-based microdissection facilitates the isolation of specific cell populations from clinical or animal model tissue specimens for molecular analysis. Expression microdissection (xMD) is a second-generation technology that offers considerable advantages in dissection capabilities; however, until recently the method has not been accessible to investigators. This protocol describes the adaptation of xMD to commonly used laser microdissection instruments and to a commercially available handheld laser device in order to make the technique widely available to the biomedical research community. The method improves dissection speed for many applications by using a targeting probe for cell procurement in place of an operator-based, cell-by-cell selection process. Moreover, xMD can provide improved dissection precision because of the unique characteristics of film activation. The time to complete the protocol is highly dependent on the target cell population and the number of cells needed for subsequent molecular analysis. PMID:21412274

Revealing of photon-number splitting attack on quantum key distribution system by photon-number resolving devices

NASA Astrophysics Data System (ADS)

Gaidash, A. A.; Egorov, V. I.; Gleim, A. V.

2016-08-01

Quantum cryptography allows distributing secure keys between two users so that any performed eavesdropping attempt would be immediately discovered. However, in practice an eavesdropper can obtain key information from multi-photon states when attenuated laser radiation is used as a source of quantum states. In order to prevent actions of an eavesdropper, it is generally suggested to implement special cryptographic protocols, like decoy states or SARG04. In this paper, we describe an alternative method based on monitoring photon number statistics after detection. We provide a useful rule of thumb to estimate approximate order of difference of expected distribution and distribution in case of attack. Formula for calculating a minimum value of total pulses or time-gaps to resolve attack is shown. Also formulas for actual fraction of raw key known to Eve were derived. This method can therefore be used with any system and even combining with mentioned special protocols.

Transformation of the rodent malaria parasite Plasmodium chabaudi.

PubMed

Spence, Philip J; Cunningham, Deirdre; Jarra, William; Lawton, Jennifer; Langhorne, Jean; Thompson, Joanne

2011-04-01

The rodent malaria parasite Plasmodium chabaudi chabaudi shares many features with human malaria species, including P. falciparum, and is the in vivo model of choice for many aspects of malaria research in the mammalian host, from sequestration of parasitized erythrocytes, to antigenic variation and host immunity and immunopathology. This protocol describes an optimized method for the transformation of mature blood-stage P.c. chabaudi and a description of a vector that targets efficient, single crossover integration into the P.c. chabaudi genome. Transformed lines are reproducibly generated and selected within 14-20 d, and show stable long-term protein expression even in the absence of drug selection. This protocol, therefore, provides the scientific community with a robust and reproducible method to generate transformed P.c. chabaudi parasites expressing fluorescent, bioluminescent and model antigens that can be used in vivo to dissect many of the fundamental principles of malaria infection.

Transformation of the rodent malaria parasite Plasmodium chabaudi

PubMed Central

Spence, Philip J; Cunningham, Deirdre; Jarra, William; Lawton, Jennifer

2014-01-01

The rodent malaria parasite Plasmodium chabaudi chabaudi shares many features with human malaria species, including P. falciparum, and is the in vivo model of choice for many aspects of malaria research in the mammalian host, from sequestration of parasitized erythrocytes, to antigenic variation and host immunity and immunopathology. this protocol describes an optimized method for the transformation of mature blood-stage P.c. chabaudi and a description of a vector that targets efficient, single crossover integration into the P.c. chabaudi genome. Transformed lines are reproducibly generated and selected within 4–20 d, and show stable long-term protein expression even in the absence of drug selection. this protocol, therefore, provides the scientific community with a robust and reproducible method to generate transformed P.c. chabaudi parasites expressing fluorescent, bioluminescent and model antigens that can be used in vivo to dissect many of the fundamental principles of malaria infection. PMID:21455190

The touchscreen operant platform for testing learning and memory in rats and mice

PubMed Central

Horner, Alexa E.; Heath, Christopher J.; Hvoslef-Eide, Martha; Kent, Brianne A.; Kim, Chi Hun; Nilsson, Simon R. O.; Alsiö, Johan; Oomen, Charlotte A.; Holmes, Andrew; Saksida, Lisa M.; Bussey, Timothy J.

2014-01-01

Summary An increasingly popular method of assessing cognitive functions in rodents is the automated touchscreen platform, on which a number of different cognitive tests can be run in a manner very similar to touchscreen methods currently used to test human subjects. This methodology is low stress (using appetitive, rather than aversive reinforcement), has high translational potential, and lends itself to a high degree of standardisation and throughput. Applications include the study of cognition in rodent models of psychiatric and neurodegenerative diseases (e.g., Alzheimer’s disease, schizophrenia, Huntington’s disease, frontotemporal dementia), and characterisation of the role of select brain regions, neurotransmitter systems and genes in rodents. This protocol describes how to perform four touchscreen assays of learning and memory: Visual Discrimination, Object-Location Paired-Associates Learning, Visuomotor Conditional Learning and Autoshaping. It is accompanied by two further protocols using the touchscreen platform to assess executive function, working memory and pattern separation. PMID:24051959

Diagnosing a Failed Proof in Fault-Tolerance: A Disproving Challenge Problem

NASA Technical Reports Server (NTRS)

Pike, Lee; Miner, Paul; Torres-Pomales, Wilfredo

2006-01-01

This paper proposes a challenge problem in disproving. We describe a fault-tolerant distributed protocol designed at NASA for use in a fly-by-wire system for next-generation commercial aircraft. An early design of the protocol contains a subtle bug that is highly unlikely to be caught in fault injection testing. We describe a failed proof of the protocol's correctness in a mechanical theorem prover (PVS) with a complex unfinished proof conjecture. We use a model checking suite (SAL) to generate a concrete counterexample to the unproven conjecture to demonstrate the existence of a bug. However, we argue that the effort required in our approach is too high and propose what conditions a better solution would satisfy. We carefully describe the protocol and bug to provide a challenging but feasible case study for disproving research.

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

Automated monitoring of medical protocols: a secure and distributed architecture.

PubMed

Alsinet, T; Ansótegui, C; Béjar, R; Fernández, C; Manyà, F

2003-03-01

The control of the right application of medical protocols is a key issue in hospital environments. For the automated monitoring of medical protocols, we need a domain-independent language for their representation and a fully, or semi, autonomous system that understands the protocols and supervises their application. In this paper we describe a specification language and a multi-agent system architecture for monitoring medical protocols. We model medical services in hospital environments as specialized domain agents and interpret a medical protocol as a negotiation process between agents. A medical service can be involved in multiple medical protocols, and so specialized domain agents are independent of negotiation processes and autonomous system agents perform monitoring tasks. We present the detailed architecture of the system agents and of an important domain agent, the database broker agent, that is responsible of obtaining relevant information about the clinical history of patients. We also describe how we tackle the problems of privacy, integrity and authentication during the process of exchanging information between agents.

Methods for a multicenter randomized trial for mixed urinary incontinence: rationale and patient-centeredness of the ESTEEM trial.

PubMed

Sung, Vivian W; Borello-France, Diane; Dunivan, Gena; Gantz, Marie; Lukacz, Emily S; Moalli, Pamela; Newman, Diane K; Richter, Holly E; Ridgeway, Beri; Smith, Ariana L; Weidner, Alison C; Meikle, Susan

2016-10-01

Mixed urinary incontinence (MUI) can be a challenging condition to manage. We describe the protocol design and rationale for the Effects of Surgical Treatment Enhanced with Exercise for Mixed Urinary Incontinence (ESTEEM) trial, designed to compare a combined conservative and surgical treatment approach versus surgery alone for improving patient-centered MUI outcomes at 12 months. ESTEEM is a multisite, prospective, randomized trial of female participants with MUI randomized to a standardized perioperative behavioral/pelvic floor exercise intervention plus midurethral sling versus midurethral sling alone. We describe our methods and four challenges encountered during the design phase: defining the study population, selecting relevant patient-centered outcomes, determining sample size estimates using a patient-reported outcome measure, and designing an analysis plan that accommodates MUI failure rates. A central theme in the design was patient centeredness, which guided many key decisions. Our primary outcome is patient-reported MUI symptoms measured using the Urogenital Distress Inventory (UDI) score at 12 months. Secondary outcomes include quality of life, sexual function, cost-effectiveness, time to failure, and need for additional treatment. The final study design was implemented in November 2013 across eight clinical sites in the Pelvic Floor Disorders Network. As of 27 February 2016, 433 total/472 targeted participants had been randomized. We describe the ESTEEM protocol and our methods for reaching consensus for methodological challenges in designing a trial for MUI by maintaining the patient perspective at the core of key decisions. This trial will provide information that can directly impact patient care and clinical decision making.

Mouse Embryo Cryopreservation by Rapid Cooling.

PubMed

Shaw, Jillian

2018-05-01

Embryo cryopreservation has been used to archive mouse strains. Protocols have evolved over this time and now vary considerably in terms of cryoprotectant solution, cooling and warming rates, methods to add and remove cryoprotectant, container or carrier type, volume of cryoprotectant, the stage of preimplantation development, and the use of additional treatments such as blastocyst puncture and microinjection. The rapid cooling methods use concentrated solutions of cryoprotectants to reduce the water content of the cell before cooling commences, thus preventing the formation of ice crystals. Embryos are equilibrated with the cryoprotectants, loaded into a carrier, and then rapidly cooled (e.g., by being plunged directly into LN 2 or onto a surface cooled in LN 2 ). The rapid cooling methods eliminate the need for controlled-rate freezers and seeding procedures. However, they are much more sensitive to minor variations when performing the steps. The rapid-cooling protocol described here is suitable for use with plastic insemination straws. Because it uses relatively large volumes, it is less technically demanding than some other methods that use minivolume devices. © 2018 Cold Spring Harbor Laboratory Press.

An optimized high quality male DNA extraction from spermatophores in open thelycum shrimp species.

PubMed

Planella, Laia; Heras, Sandra; Vera, Manuel; García-Marín, José-Luis; Roldán, María Inés

2017-09-01

The crucial step of most of the current genetic studies is the extraction of DNA of sufficient quantity and quality. Several genomic DNA isolation methods have been described to successfully obtain male DNA from shrimp species. However, all current protocols require invasive handling methods with males for DNA isolation. Using Aristeus antennatus as a model we tested a reliable non-invasive differential DNA extraction method to male DNA isolation from spermatophores attached to female thelycum. The present protocol provides high quality and quantity DNA for polymerase chain reaction amplification and male genotyping. This new approach could be useful to experimental shrimp culture to select sires with relevant genetic patterns for selective breeding programs. More importantly, it can be applied to identify the mating pairs and male structure in wild populations of species as A. antennatus, where males are often difficult to capture. Our method could be also valuable for biological studies on other spermatophore-using species, such as myriapods, arachnids and insects. © 2016 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.

A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq.

PubMed

Logan, Grace; Freimanis, Graham L; King, David J; Valdazo-González, Begoña; Bachanek-Bankowska, Katarzyna; Sanderson, Nicholas D; Knowles, Nick J; King, Donald P; Cottam, Eleanor M

2014-09-30

Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template. The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5' genomic termini and area immediately flanking the poly(C) region. We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

Rapid Sampling of Hydrogen Bond Networks for Computational Protein Design.

PubMed

Maguire, Jack B; Boyken, Scott E; Baker, David; Kuhlman, Brian

2018-05-08

Hydrogen bond networks play a critical role in determining the stability and specificity of biomolecular complexes, and the ability to design such networks is important for engineering novel structures, interactions, and enzymes. One key feature of hydrogen bond networks that makes them difficult to rationally engineer is that they are highly cooperative and are not energetically favorable until the hydrogen bonding potential has been satisfied for all buried polar groups in the network. Existing computational methods for protein design are ill-equipped for creating these highly cooperative networks because they rely on energy functions and sampling strategies that are focused on pairwise interactions. To enable the design of complex hydrogen bond networks, we have developed a new sampling protocol in the molecular modeling program Rosetta that explicitly searches for sets of amino acid mutations that can form self-contained hydrogen bond networks. For a given set of designable residues, the protocol often identifies many alternative sets of mutations/networks, and we show that it can readily be applied to large sets of residues at protein-protein interfaces or in the interior of proteins. The protocol builds on a recently developed method in Rosetta for designing hydrogen bond networks that has been experimentally validated for small symmetric systems but was not extensible to many larger protein structures and complexes. The sampling protocol we describe here not only recapitulates previously validated designs with performance improvements but also yields viable hydrogen bond networks for cases where the previous method fails, such as the design of large, asymmetric interfaces relevant to engineering protein-based therapeutics.

The Interlibrary Loan Protocol: An OSI Solution to ILL Messaging.

ERIC Educational Resources Information Center

Turner, Fay

1990-01-01

Discusses the interlibrary loan (ILL) protocol, a standard based on the principles of the Open Systems Interconnection (OSI) Reference Model. Benefits derived from protocol use are described, the status of the protocol as an international standard is reviewed, and steps taken by the National Library of Canada to facilitate migration to an ILL…

Atlas-based analysis of cardiac shape and function: correction of regional shape bias due to imaging protocol for population studies.

PubMed

Medrano-Gracia, Pau; Cowan, Brett R; Bluemke, David A; Finn, J Paul; Kadish, Alan H; Lee, Daniel C; Lima, Joao A C; Suinesiaputra, Avan; Young, Alistair A

2013-09-13

Cardiovascular imaging studies generate a wealth of data which is typically used only for individual study endpoints. By pooling data from multiple sources, quantitative comparisons can be made of regional wall motion abnormalities between different cohorts, enabling reuse of valuable data. Atlas-based analysis provides precise quantification of shape and motion differences between disease groups and normal subjects. However, subtle shape differences may arise due to differences in imaging protocol between studies. A mathematical model describing regional wall motion and shape was used to establish a coordinate system registered to the cardiac anatomy. The atlas was applied to data contributed to the Cardiac Atlas Project from two independent studies which used different imaging protocols: steady state free precession (SSFP) and gradient recalled echo (GRE) cardiovascular magnetic resonance (CMR). Shape bias due to imaging protocol was corrected using an atlas-based transformation which was generated from a set of 46 volunteers who were imaged with both protocols. Shape bias between GRE and SSFP was regionally variable, and was effectively removed using the atlas-based transformation. Global mass and volume bias was also corrected by this method. Regional shape differences between cohorts were more statistically significant after removing regional artifacts due to imaging protocol bias. Bias arising from imaging protocol can be both global and regional in nature, and is effectively corrected using an atlas-based transformation, enabling direct comparison of regional wall motion abnormalities between cohorts acquired in separate studies.

The preparation of Drosophila embryos for live-imaging using the hanging drop protocol.

PubMed

Reed, Bruce H; McMillan, Stephanie C; Chaudhary, Roopali

2009-03-13

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

Attacks on quantum key distribution protocols that employ non-ITS authentication

NASA Astrophysics Data System (ADS)

Pacher, C.; Abidin, A.; Lorünser, T.; Peev, M.; Ursin, R.; Zeilinger, A.; Larsson, J.-Å.

2016-01-01

We demonstrate how adversaries with large computing resources can break quantum key distribution (QKD) protocols which employ a particular message authentication code suggested previously. This authentication code, featuring low key consumption, is not information-theoretically secure (ITS) since for each message the eavesdropper has intercepted she is able to send a different message from a set of messages that she can calculate by finding collisions of a cryptographic hash function. However, when this authentication code was introduced, it was shown to prevent straightforward man-in-the-middle (MITM) attacks against QKD protocols. In this paper, we prove that the set of messages that collide with any given message under this authentication code contains with high probability a message that has small Hamming distance to any other given message. Based on this fact, we present extended MITM attacks against different versions of BB84 QKD protocols using the addressed authentication code; for three protocols, we describe every single action taken by the adversary. For all protocols, the adversary can obtain complete knowledge of the key, and for most protocols her success probability in doing so approaches unity. Since the attacks work against all authentication methods which allow to calculate colliding messages, the underlying building blocks of the presented attacks expose the potential pitfalls arising as a consequence of non-ITS authentication in QKD post-processing. We propose countermeasures, increasing the eavesdroppers demand for computational power, and also prove necessary and sufficient conditions for upgrading the discussed authentication code to the ITS level.

A survey on routing protocols for large-scale wireless sensor networks.

PubMed

Li, Changle; Zhang, Hanxiao; Hao, Binbin; Li, Jiandong

2011-01-01

With the advances in micro-electronics, wireless sensor devices have been made much smaller and more integrated, and large-scale wireless sensor networks (WSNs) based the cooperation among the significant amount of nodes have become a hot topic. "Large-scale" means mainly large area or high density of a network. Accordingly the routing protocols must scale well to the network scope extension and node density increases. A sensor node is normally energy-limited and cannot be recharged, and thus its energy consumption has a quite significant effect on the scalability of the protocol. To the best of our knowledge, currently the mainstream methods to solve the energy problem in large-scale WSNs are the hierarchical routing protocols. In a hierarchical routing protocol, all the nodes are divided into several groups with different assignment levels. The nodes within the high level are responsible for data aggregation and management work, and the low level nodes for sensing their surroundings and collecting information. The hierarchical routing protocols are proved to be more energy-efficient than flat ones in which all the nodes play the same role, especially in terms of the data aggregation and the flooding of the control packets. With focus on the hierarchical structure, in this paper we provide an insight into routing protocols designed specifically for large-scale WSNs. According to the different objectives, the protocols are generally classified based on different criteria such as control overhead reduction, energy consumption mitigation and energy balance. In order to gain a comprehensive understanding of each protocol, we highlight their innovative ideas, describe the underlying principles in detail and analyze their advantages and disadvantages. Moreover a comparison of each routing protocol is conducted to demonstrate the differences between the protocols in terms of message complexity, memory requirements, localization, data aggregation, clustering manner and other metrics. Finally some open issues in routing protocol design in large-scale wireless sensor networks and conclusions are proposed.

A Survey on Routing Protocols for Large-Scale Wireless Sensor Networks

PubMed Central

Li, Changle; Zhang, Hanxiao; Hao, Binbin; Li, Jiandong

2011-01-01

With the advances in micro-electronics, wireless sensor devices have been made much smaller and more integrated, and large-scale wireless sensor networks (WSNs) based the cooperation among the significant amount of nodes have become a hot topic. “Large-scale” means mainly large area or high density of a network. Accordingly the routing protocols must scale well to the network scope extension and node density increases. A sensor node is normally energy-limited and cannot be recharged, and thus its energy consumption has a quite significant effect on the scalability of the protocol. To the best of our knowledge, currently the mainstream methods to solve the energy problem in large-scale WSNs are the hierarchical routing protocols. In a hierarchical routing protocol, all the nodes are divided into several groups with different assignment levels. The nodes within the high level are responsible for data aggregation and management work, and the low level nodes for sensing their surroundings and collecting information. The hierarchical routing protocols are proved to be more energy-efficient than flat ones in which all the nodes play the same role, especially in terms of the data aggregation and the flooding of the control packets. With focus on the hierarchical structure, in this paper we provide an insight into routing protocols designed specifically for large-scale WSNs. According to the different objectives, the protocols are generally classified based on different criteria such as control overhead reduction, energy consumption mitigation and energy balance. In order to gain a comprehensive understanding of each protocol, we highlight their innovative ideas, describe the underlying principles in detail and analyze their advantages and disadvantages. Moreover a comparison of each routing protocol is conducted to demonstrate the differences between the protocols in terms of message complexity, memory requirements, localization, data aggregation, clustering manner and other metrics. Finally some open issues in routing protocol design in large-scale wireless sensor networks and conclusions are proposed. PMID:22163808

Detecting and removing multiplicative spatial bias in high-throughput screening technologies.

PubMed

Caraus, Iurie; Mazoure, Bogdan; Nadon, Robert; Makarenkov, Vladimir

2017-10-15

Considerable attention has been paid recently to improve data quality in high-throughput screening (HTS) and high-content screening (HCS) technologies widely used in drug development and chemical toxicity research. However, several environmentally- and procedurally-induced spatial biases in experimental HTS and HCS screens decrease measurement accuracy, leading to increased numbers of false positives and false negatives in hit selection. Although effective bias correction methods and software have been developed over the past decades, almost all of these tools have been designed to reduce the effect of additive bias only. Here, we address the case of multiplicative spatial bias. We introduce three new statistical methods meant to reduce multiplicative spatial bias in screening technologies. We assess the performance of the methods with synthetic and real data affected by multiplicative spatial bias, including comparisons with current bias correction methods. We also describe a wider data correction protocol that integrates methods for removing both assay and plate-specific spatial biases, which can be either additive or multiplicative. The methods for removing multiplicative spatial bias and the data correction protocol are effective in detecting and cleaning experimental data generated by screening technologies. As our protocol is of a general nature, it can be used by researchers analyzing current or next-generation high-throughput screens. The AssayCorrector program, implemented in R, is available on CRAN. makarenkov.vladimir@uqam.ca. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

PubMed

Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

2017-07-01

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

Calcium dependent current recordings in Xenopus laevis oocytes in microgravity

NASA Astrophysics Data System (ADS)

Wuest, Simon L.; Roesch, Christian; Ille, Fabian; Egli, Marcel

2017-12-01

Mechanical unloading by microgravity (or weightlessness) conditions triggers profound adaptation processes at the cellular and organ levels. Among other mechanisms, mechanosensitive ion channels are thought to play a key role in allowing cells to transduce mechanical forces. Previous experiments performed under microgravity have shown that gravity affects the gating properties of ion channels. Here, a method is described to record a calcium-dependent current in native Xenopus laevis oocytes under microgravity conditions during a parabolic flight. A 3-voltage-step protocol was applied to provoke a calcium-dependent current. This current increased with extracellular calcium concentration and could be reduced by applying extracellular gadolinium. The custom-made ;OoClamp; hardware was validated by comparing the results of the 3-voltage-step protocol to results obtained with a well-established two-electrode voltage clamp (TEVC). In the context of the 2nd Swiss Parabolic Flight Campaign, we tested the OoClamp and the method. The setup and experiment protocol worked well in parabolic flight. A tendency that the calcium-dependent current was smaller under microgravity than under 1 g condition could be observed. However, a conclusive statement was not possible due to the small size of the data base that could be gathered.

How to Study Biofilms after Microbial Colonization of Materials Used in Orthopaedic Implants.

PubMed

Drago, Lorenzo; Agrappi, Serse; Bortolin, Monica; Toscano, Marco; Romanò, Carlo Luca; De Vecchi, Elena

2016-02-26

Over the years, various techniques have been proposed for the quantitative evaluation of microbial biofilms. Spectrophotometry after crystal violet staining is a widespread method for biofilm evaluation, but several data indicate that it does not guarantee a good specificity, although it is rather easy to use and cost saving. Confocal laser microscopy is one of the most sensitive and specific tools to study biofilms, and it is largely used for research. However, in some cases, no quantitative measurement of the matrix thickness or of the amount of embedded microorganisms has been performed, due to limitation in availability of dedicated software. For this reason, we have developed a protocol to evaluate the microbial biofilm formed on sandblasted titanium used for orthopaedic implants, that allows measurement of biomass volume and the amount of included cells. Results indicate good reproducibility in terms of measurement of biomass and microbial cells. Moreover, this protocol has proved to be applicable for evaluation of the efficacy of different anti-biofilm treatments used in the orthopaedic setting. Summing up, the protocol here described is a valid and inexpensive method for the study of microbial biofilm on prosthetic implant materials.

Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography.

PubMed

Roiban, Lucian; Sorbier, Loïc; Hirlimann, Charles; Ersen, Ovidiu

2018-06-09

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a nanometric scale. EFTEM tomography can separate chemical elements that are very difficult to distinguish using other imaging techniques. The experimental protocol described here shows how to create 3D chemical maps to understand the chemical distribution and morphology of a material. Sample preparation steps for data segmentation are presented. This protocol permits the 3D distribution analysis of chemical elements in a nanometric sample. However, it should be noted that currently, the 3D chemical maps can only be generated for samples that are not beam sensitive, since the recording of filtered images requires long exposure times to an intense electron beam. The protocol was applied to quantify the chemical distribution of the components of two different heterogeneous catalyst supports. In the first study, the chemical distribution of aluminum and titanium in titania-alumina supports was analyzed. The samples were prepared using the swing-pH method. In the second, the chemical distribution of aluminum and silicon in silica-alumina supports that were prepared using the sol-powder and mechanical mixture methods was examined.

Spatial transcriptomic analysis of cryosectioned tissue samples with Geo-seq.

PubMed

Chen, Jun; Suo, Shengbao; Tam, Patrick Pl; Han, Jing-Dong J; Peng, Guangdun; Jing, Naihe

2017-03-01

Conventional gene expression studies analyze multiple cells simultaneously or single cells, for which the exact in vivo or in situ position is unknown. Although cellular heterogeneity can be discerned when analyzing single cells, any spatially defined attributes that underpin the heterogeneous nature of the cells cannot be identified. Here, we describe how to use Geo-seq, a method that combines laser capture microdissection (LCM) and single-cell RNA-seq technology. The combination of these two methods enables the elucidation of cellular heterogeneity and spatial variance simultaneously. The Geo-seq protocol allows the profiling of transcriptome information from only a small number cells and retains their native spatial information. This protocol has wide potential applications to address biological and pathological questions of cellular properties such as prospective cell fates, biological function and the gene regulatory network. Geo-seq has been applied to investigate the spatial transcriptome of mouse early embryo, mouse brain, and pathological liver and sperm tissues. The entire protocol from tissue collection and microdissection to sequencing requires ∼5 d, Data analysis takes another 1 or 2 weeks, depending on the amount of data and the speed of the processor.

Left ventricular assist device malfunction: a systematic approach to diagnosis.

PubMed

Horton, Steven C; Khodaverdian, Reza; Powers, Amanda; Revenaugh, James; Renlund, Dale G; Moore, Stephanie A; Rasmusson, Brad; Nelson, Karl E; Long, James W

2004-05-05

A protocol was designed to diagnose the common malfunctions of a left ventricular assist device (LVAD). Mechanical circulatory support, primarily with an LVAD, is increasingly used for treatment of advanced heart failure (HF). Left ventricular assist device dysfunction is a recognized complication; but heretofore, a systematic method to accurately diagnose LVAD dysfunction has not been thoroughly described. We developed a catheter-based protocol designed to characterize a normally functioning LVAD and diagnose multiple types of dysfunction. A total of 15 studies of 10 patients supported with an LVAD were reviewed. All patients had been evaluated due to concerns regarding LVAD dysfunction. Of 15 examinations performed, 11 documented severe LVAD inflow valve regurgitation. One of these cases proved to have coexistent severe mitral valve regurgitation. One case was diagnosed with distortion of the LVAD outflow graft. One case of suspected embolization from the pumping chamber excluded the outflow graft as the source of emboli. One study had aortic insufficiency. As LVAD use for treatment of end-stage HF becomes widespread and durations of support are extended, dysfunction will be increasingly prevalent. This catheter-based protocol provided a practical method to diagnose multiple causes of LVAD dysfunction.

Design of a functional cyclic HSV1-TK reporter and its application to PET imaging of apoptosis

PubMed Central

Wang, Zhe; Wang, Fu; Hida, Naoki; Kiesewetter, Dale O; Tian, Jie; Niu, Gang; Chen, Xiaoyuan

2017-01-01

Positron emission tomography (PET) is a sensitive and noninvasive imaging method that is widely used to explore molecular events in living subjects. PET can precisely and quantitatively evaluate cellular apoptosis, which has a crucial role in various physiological and pathological processes. In this protocol, we describe the design and use of an engineered cyclic herpes simplex virus 1–thymidine kinase (HSV1-TK) PET reporter whose kinase activity is specifically switched on by apoptosis. The expression of cyclic TK (cTK) in healthy cells leads to inactive product, whereas the activation of apoptosis through the caspase-3 pathway cleaves cTK, thus restoring its activity and enabling PET imaging. In addition to detailing the design and construction of the cTK plasmid in this protocol, we include assays for evaluating the function and specificity of the cTK reporter in apoptotic cells, such as assays for measuring the cell uptake of PET tracer in apoptotic cells, correlating doxorubicin (Dox)-induced cell apoptosis to cTK function recovery, and in vivo PET imaging of cancer cell apoptosis, and we also include corresponding data acquisition methods. The time to build the entire cTK reporter is ~2–3 weeks. The selection of a stable cancer cell line takes ~4–6 weeks. The time to implement assays regarding cTK function in apoptotic cells and the in vivo imaging varies depending on the experiment. The cyclization strategy described in this protocol can also be adapted to create other reporter systems for broad biomedical applications. PMID:25927390

The Dust Storm Index (DSI): A method for monitoring broadscale wind erosion using meteorological records

NASA Astrophysics Data System (ADS)

O'Loingsigh, T.; McTainsh, G. H.; Tews, E. K.; Strong, C. L.; Leys, J. F.; Shinkfield, P.; Tapper, N. J.

2014-03-01

Wind erosion of soils is a natural process that has shaped the semi-arid and arid landscapes for millennia. This paper describes the Dust Storm Index (DSI); a methodology for monitoring wind erosion using Australian Bureau of Meteorology (ABM) meteorological observational data since the mid-1960s (long-term), at continental scale. While the 46 year length of the DSI record is its greatest strength from a wind erosion monitoring perspective, there are a number of technical challenges to its use because when the World Meteorological Organisation (WMO) recording protocols were established the use of the data for wind erosion monitoring was never intended. Data recording and storage protocols are examined, including the effects of changes to the definition of how observers should interpret and record dust events. A method is described for selecting the 180 long-term ABM stations used in this study and the limitations of variable observation frequencies between stations are in part resolved. The rationale behind the DSI equation is explained and the examples of temporal and spatial data visualisation products presented include; a long term national wind erosion record (1965-2011), continental DSI maps, and maps of the erosion event types that are factored into the DSI equation. The DSI is tested against dust concentration data and found to provide an accurate representation of wind erosion activity. As the ABM observational records used here were collected according to WMO protocols, the DSI methodology could be used in all countries with WMO-compatible meteorological observation and recording systems.

Method to grow Actinobacillus pleuropneumoniae biofilm on a biotic surface.

PubMed

Tremblay, Yannick D N; Lévesque, Cynthia; Segers, Ruud P A M; Jacques, Mario

2013-10-20

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and a member of the Pasteurellaceae family. This bacterium is the causative agent of porcine pleuropneumonia, which is a highly contagious respiratory disease causing important economical losses to the worldwide pig industry. It has been shown that A. pleuropneumoniae can form biofilms on abiotic surfaces (plastic and glass). Although in vitro models are extremely useful to gain information on biofilm formation, these models may not be representative of the conditions found at the mucosal surface of the host, which is the natural niche of A. pleuropneumoniae. In this paper, we describe a method to grow A. pleuropneumoniae biofilms on the SJPL cell line, which represents a biotic surface. A non-hemolytic, non-cytotoxic mutant of A. pleuropneumoniae was used in our assays and this allowed the SJPL cell monolayers to be exposed to A. pleuropneumoniae for longer periods. This resulted in the formation of biofilms on the cell monolayer after incubations of 24 and 48 h. The biofilms can be stained with fluorescent probes, such as a lectin against the polymer of N-acetyl-D-glucosamine present in the biofilm matrix, and easily observed by confocal laser scanning microscopy. This is the first protocol that describes the formation of an A. pleuropneumoniae biofilm on a biotic surface. The advantage of this protocol is that it can be used to study biofilm formation in a context of host-pathogen interactions. The protocol could also be adapted to evaluate biofilm inhibitors or the efficacy of antibiotics in the presence of biofilms.

Point-to-Point Multicast Communications Protocol

NASA Technical Reports Server (NTRS)

Byrd, Gregory T.; Nakano, Russell; Delagi, Bruce A.

1987-01-01

This paper describes a protocol to support point-to-point interprocessor communications with multicast. Dynamic, cut-through routing with local flow control is used to provide a high-throughput, low-latency communications path between processors. In addition multicast transmissions are available, in which copies of a packet are sent to multiple destinations using common resources as much as possible. Special packet terminators and selective buffering are introduced to avoid a deadlock during multicasts. A simulated implementation of the protocol is also described.

A Deconstructive Pedagogy

ERIC Educational Resources Information Center

Uba, Laura

2008-01-01

This article describes a four-pronged pedagogical protocol for teaching undergraduates how to deconstruct statements, including a list of deconstructive considerations students learn to apply. The protocol also encourages oral participation in class discussions. Three assessment measures demonstrate the protocol's effectiveness: (1) a qualitative…

Gigabit Ethernet: A Technical Assessment.

ERIC Educational Resources Information Center

Axner, David

1997-01-01

Describes gigabit ethernet for LAN (local area network) technology that will expand ethernet bandwidth. Technical details are discussed, including protocol stacks, optical fiber, deployment strategy for performance improvement, ATM (Asynchronous Transfer Mode), real-time protocol, reserve reservation protocol, and standards. (LRW)

The Phyre2 web portal for protein modelling, prediction and analysis

PubMed Central

Kelley, Lawrence A; Mezulis, Stefans; Yates, Christopher M; Wass, Mark N; Sternberg, Michael JE

2017-01-01

Summary Phyre2 is a suite of tools available on the web to predict and analyse protein structure, function and mutations. The focus of Phyre2 is to provide biologists with a simple and intuitive interface to state-of-the-art protein bioinformatics tools. Phyre2 replaces Phyre, the original version of the server for which we previously published a protocol. In this updated protocol, we describe Phyre2, which uses advanced remote homology detection methods to build 3D models, predict ligand binding sites, and analyse the effect of amino-acid variants (e.g. nsSNPs) for a user’s protein sequence. Users are guided through results by a simple interface at a level of detail determined by them. This protocol will guide a user from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain composition and model quality. A range of additional available tools is described to find a protein structure in a genome, to submit large number of sequences at once and to automatically run weekly searches for proteins difficult to model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2. A typical structure prediction will be returned between 30mins and 2 hours after submission. PMID:25950237

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography.

PubMed

Gicquel, Yannig; Schubert, Robin; Kapis, Svetlana; Bourenkov, Gleb; Schneider, Thomas; Perbandt, Markus; Betzel, Christian; Chapman, Henry N; Heymann, Michael

2018-04-24

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 µm grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing.

PubMed

Marshall, Owen J; Southall, Tony D; Cheetham, Seth W; Brand, Andrea H

2016-09-01

This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.

A short protocol using dexamethasone and monophosphoryl lipid A generates tolerogenic dendritic cells that display a potent migratory capacity to lymphoid chemokines

PubMed Central

2013-01-01

Background Generation of tolerogenic dendritic cells (TolDCs) for therapy is challenging due to its implications for the design of protocols suitable for clinical applications, which means not only using safe products, but also working at defining specific biomarkers for TolDCs identification, developing shorter DCs differentiation methods and obtaining TolDCs with a stable phenotype. We describe here, a short-term protocol for TolDCs generation, which are characterized in terms of phenotypic markers, cytokines secretion profile, CD4+ T cell-stimulatory ability and migratory capacity. Methods TolDCs from healthy donors were generated by modulation with dexamethasone plus monophosphoryl lipid A (MPLA-tDCs). We performed an analysis of MPLA-tDCs in terms of yield, viability, morphology, phenotypic markers, cytokines secretion profile, stability, allogeneic and antigen-specific CD4+ T-cell stimulatory ability and migration capacity. Results After a 5-day culture, MPLA-tDCs displayed reduced expression of costimulatory and maturation molecules together to an anti-inflammatory cytokines secretion profile, being able to maintain these tolerogenic features even after the engagement of CD40 by its cognate ligand. In addition, MPLA-tDCs exhibited reduced capabilities to stimulate allogeneic and antigen-specific CD4+ T cell proliferation, and induced an anti-inflammatory cytokine secretion pattern. Among potential tolerogenic markers studied, only TLR-2 was highly expressed in MPLA-tDCs when compared to mature and immature DCs. Remarkable, like mature DCs, MPLA-tDCs displayed a high CCR7 and CXCR4 expression, both chemokine receptors involved in migration to secondary lymphoid organs, and even more, in an in vitro assay they exhibited a high migration response towards CCL19 and CXCL12. Conclusion We describe a short-term protocol for TolDC generation, which confers them a stable phenotype and migratory capacity to lymphoid chemokines, essential features for TolDCs to be used as therapeutics for autoimmunity and prevention of graft rejection. PMID:23706017

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.

Automated generation of patient-tailored electronic care pathways by translating computer-interpretable guidelines into hierarchical task networks.

PubMed

González-Ferrer, Arturo; ten Teije, Annette; Fdez-Olivares, Juan; Milian, Krystyna

2013-02-01

This paper describes a methodology which enables computer-aided support for the planning, visualization and execution of personalized patient treatments in a specific healthcare process, taking into account complex temporal constraints and the allocation of institutional resources. To this end, a translation from a time-annotated computer-interpretable guideline (CIG) model of a clinical protocol into a temporal hierarchical task network (HTN) planning domain is presented. The proposed method uses a knowledge-driven reasoning process to translate knowledge previously described in a CIG into a corresponding HTN Planning and Scheduling domain, taking advantage of HTNs known ability to (i) dynamically cope with temporal and resource constraints, and (ii) automatically generate customized plans. The proposed method, focusing on the representation of temporal knowledge and based on the identification of workflow and temporal patterns in a CIG, makes it possible to automatically generate time-annotated and resource-based care pathways tailored to the needs of any possible patient profile. The proposed translation is illustrated through a case study based on a 70 pages long clinical protocol to manage Hodgkin's disease, developed by the Spanish Society of Pediatric Oncology. We show that an HTN planning domain can be generated from the corresponding specification of the protocol in the Asbru language, providing a running example of this translation. Furthermore, the correctness of the translation is checked and also the management of ten different types of temporal patterns represented in the protocol. By interpreting the automatically generated domain with a state-of-art HTN planner, a time-annotated care pathway is automatically obtained, customized for the patient's and institutional needs. The generated care pathway can then be used by clinicians to plan and manage the patients long-term care. The described methodology makes it possible to automatically generate patient-tailored care pathways, leveraging an incremental knowledge-driven engineering process that starts from the expert knowledge of medical professionals. The presented approach makes the most of the strengths inherent in both CIG languages and HTN planning and scheduling techniques: for the former, knowledge acquisition and representation of the original clinical protocol, and for the latter, knowledge reasoning capabilities and an ability to deal with complex temporal and resource constraints. Moreover, the proposed approach provides immediate access to technologies such as business process management (BPM) tools, which are increasingly being used to support healthcare processes. Copyright © 2012 Elsevier B.V. All rights reserved.

An optimized method for high quality DNA extraction from microalga Prototheca wickerhamii for genome sequencing.

PubMed

Jagielski, Tomasz; Gawor, Jan; Bakuła, Zofia; Zuchniewicz, Karolina; Żak, Iwona; Gromadka, Robert

2017-01-01

The complex cell wall structure of algae often precludes efficient extraction of their genetic material. The purpose of this study was to design a next-generation sequencing-suitable DNA isolation method for unicellular, achlorophyllous, yeast-like microalgae of the genus Prototheca , the only known plant pathogens of both humans and animals. The effectiveness of the newly proposed scheme was compared with five other, previously described methods, commonly used for DNA isolation from plants and/or yeasts, available either as laboratory-developed, in-house assays, based on liquid nitrogen grinding or different enzymatic digestion, or as commercially manufactured kits. All five, previously described, isolation assays yielded DNA concentrations lower than those obtained with the new method, averaging 16.15 ± 25.39 vs 74.2 ± 0.56 ng/µL, respectively. The new method was also superior in terms of DNA purity, as measured by A260/A280 (-0.41 ± 4.26 vs 2.02 ± 0.03), and A260/A230 (1.20 ± 1.12 vs 1.97 ± 0.07) ratios. Only the liquid nitrogen-based method yielded DNA of comparable quantity (60.96 ± 0.16 ng/µL) and quality (A260/A280 = 2.08 ± 0.02; A260/A230 = 2.23 ± 0.26). Still, the new method showed higher integrity, which was best illustrated upon electrophoretic analysis. Genomic DNA of Prototheca wickerhamii POL-1 strain isolated with the protocol herein proposed was successfully sequenced on the Illumina MiSeq platform. A new method for DNA isolation from Prototheca algae is described. The method, whose protocol involves glass beads pulverization and cesium chloride (CsCl) density gradient centrifugation, was demonstrated superior over the other common assays in terms of DNA quantity and quality. The method is also the first to offer the possibility of preparation of DNA template suitable for whole genome sequencing of Prototheca spp.

Microfluidic, marker-free isolation of circulating tumor cells from blood samples

PubMed Central

Karabacak, Nezihi Murat; Spuhler, Philipp S; Fachin, Fabio; Lim, Eugene J; Pai, Vincent; Ozkumur, Emre; Martel, Joseph M; Kojic, Nikola; Smith, Kyle; Chen, Pin-i; Yang, Jennifer; Hwang, Henry; Morgan, Bailey; Trautwein, Julie; Barber, Thomas A; Stott, Shannon L; Maheswaran, Shyamala; Kapur, Ravi; Haber, Daniel A; Toner, Mehmet

2014-01-01

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen–independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2–5 d. PMID:24577360

Site-specific protein labeling with PRIME and chelation-assisted Click chemistry

PubMed Central

Uttamapinant, Chayasith; Sanchez, Mateo I.; Liu, Daniel S.; Yao, Jennifer Z.; White, Katharine A.; Grecian, Scott; Clarke, Scott; Gee, Kyle R.; Ting, Alice Y.

2016-01-01

This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: PRobe Incorporation Mediated by Enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase site-specifically attaches a picolyl azide derivative to a 13-amino acid recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing picolyl azide are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize picolyl azide, perform PRIME labeling, and achieve CuAAC derivatization of picolyl azide on live cells, fixed cells, and purified proteins. Reagent preparations, including synthesis of picolyl azide probes and expression of lipoic acid ligase, take 12 d, while the procedure to perform site-specific picolyl azide ligation and CuAAC on cells or on purified proteins takes 40 min-3 h. PMID:23887180

A novel assay to identify the trafficking proteins that bind to specific vesicle populations

PubMed Central

Bentley, Marvin; Banker, Gary

2016-01-01

Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin–binding domain (FRB)–tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule-organizing center and a specialized protocol designed to detect protein-vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled. PMID:26621371

Perceptual attributes for the comparison of head-related transfer functions.

PubMed

Simon, Laurent S R; Zacharov, Nick; Katz, Brian F G

2016-11-01

The benefit of using individual head-related transfer functions (HRTFs) in binaural audio is well documented with regards to improving localization precision. However, with the increased use of binaural audio in more complex scene renderings, cognitive studies, and virtual and augmented reality simulations, the perceptual impact of HRTF selection may go beyond simple localization. In this study, the authors develop a list of attributes which qualify the perceived differences between HRTFs, providing a qualitative understanding of the perceptual variance of non-individual binaural renderings. The list of attributes was designed using a Consensus Vocabulary Protocol elicitation method. Participants followed an Individual Vocabulary Protocol elicitation procedure, describing the perceived differences between binaural stimuli based on binauralized extracts of multichannel productions. This was followed by an automated lexical reduction and a series of consensus group meetings during which participants agreed on a list of relevant attributes. Finally, the proposed list of attributes was then evaluated through a listening test, leading to eight valid perceptual attributes for describing the perceptual dimensions affected by HRTF set variations.

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

PubMed Central

Bozec, Aline; Hannemann, Nicole

2016-01-01

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1α protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes. PMID:27284940

A Quantitative Microscopy Technique for Determining the Number of Specific Proteins in Cellular Compartments

PubMed Central

Mutch, Sarah A.; Gadd, Jennifer C.; Fujimoto, Bryant S.; Kensel-Hammes, Patricia; Schiro, Perry G.; Bajjalieh, Sandra M.; Chiu, Daniel T.

2013-01-01

This protocol describes a method to determine both the average number and variance of proteins in the few to tens of copies in isolated cellular compartments, such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number but lack information on the variance or are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling the cellular compartment with fluorescent primary-secondary antibody complexes, TIRF (total internal reflection fluorescence) microscopy imaging of the cellular compartment, digital image analysis, and deconvolution of the fluorescence intensity data. A minimum of 2.5 days is required to complete the labeling, imaging, and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes. PMID:22094731

Cyclotide isolation and characterization.

PubMed

Craik, David J; Henriques, Sonia Troeira; Mylne, Joshua S; Wang, Conan K

2012-01-01

Cyclotides are disulfide-rich cyclic peptides produced by plants with the presumed natural function of defense agents against insect pests. They are present in a wide range of plant tissues, being ribosomally synthesized via precursor proteins that are posttranslationally processed to produce mature peptides with a characteristic cyclic backbone and cystine knot motif associated with their six conserved cysteine residues. Their processing is not fully understood but involves asparaginyl endoproteinase activity. In addition to interest in their defense roles and their unique topologies, cyclotides have attracted attention as potential templates in peptide-based drug design applications. This chapter provides protocols for the isolation of cyclotides from plants, their detection and sequencing by mass spectrometry, and their structural analysis by NMR, as well as describing methods for the isolation of nucleic acid sequences that encode their precursor proteins. Assays to assess their membrane-binding interactions are also described. These protocols provide a "starter kit" for researchers entering the cyclotide field. Copyright © 2012 Elsevier Inc. All rights reserved.

Forest Soil Disturbance Monitoring Protocol: Volume I: Rapid assessment

Treesearch

Deborah S. Page-Dumroese; Ann M. Abbott; Thomas M. Rice

2009-01-01

This volume of the Forest Soil Disturbance Monitoring Protocol (FSDMP) describes how to monitor forest sites before and after ground disturbing management activities for physical attributes that could influence site resilience and long-term sustainability. The attributes describe surface conditions that affect site sustainability and hydrologic function. Monitoring the...

Nonbibliographic Applications of Z39.50.

ERIC Educational Resources Information Center

Kunze, John A.

1992-01-01

Describes the use of the Z39.50 information retrieval protocol as the basis for Infocal, a read-only, client/server-based campus information system. Technical considerations in adapting the protocol to nonbibliographic data, including semantic modules, dynamic attribute sets, and dynamic record syntax, are described in detail. (Contains 11…

Methods for Acquiring Structural MRI Data in Very Young Children with Autism without the Use of Sedation

ERIC Educational Resources Information Center

Nordahl, Christine Wu; Simon, Tony J.; Zierhut, Cynthia; Solomon, Marjorie; Rogers, Sally J.; Amaral, David G.

2008-01-01

We describe a protocol with which we achieved a 93% success rate in acquiring high quality MRI scans without the use of sedation in 2.5-4.5 year old children with autism, developmental delays, and typical development. Our main strategy was to conduct MRIs during natural nocturnal sleep in the evenings after the child's normal bedtime.…

Examining the ethical and social issues of health technology design through the public appraisal of prospective scenarios: a study protocol describing a multimedia-based deliberative method.

PubMed

Lehoux, Pascale; Gauthier, Philippe; Williams-Jones, Bryn; Miller, Fiona A; Fishman, Jennifer R; Hivon, Myriam; Vachon, Patrick

2014-06-21

The design of health technologies relies on assumptions that affect how they will be implemented, such as intended use, complexity, impact on user autonomy, and appropriateness. Those who design and implement technologies make several ethical and social assumptions on behalf of users and society more broadly, but there are very few tools to examine prospectively whether such assumptions are warranted and how the public define and appraise the desirability of health innovations. This study protocol describes a three-year study that relies on a multimedia-based prospective method to support public deliberations that will enable a critical examination of the social and ethical issues of health technology design. The first two steps of our mixed-method study were completed: relying on a literature review and the support of our multidisciplinary expert committee, we developed scenarios depicting social and technical changes that could unfold in three thematic areas within a 25-year timeframe; and for each thematic area, we created video clips to illustrate prospective technologies and short stories to describe their associated dilemmas. Using this multimedia material, we will: conduct four face-to-face deliberative workshops with members of the public (n=40) who will later join additional participants (n=25) through an asynchronous online forum; and analyze and integrate three data sources: observation, group deliberations, and a self-administered participant survey. This study protocol will be of interest to those who design and assess public involvement initiatives and to those who examine the implementation of health innovations. Our premise is that using user-friendly tools in a deliberative context that foster participants' creativity and reflexivity in pondering potential technoscientific futures will enable our team to analyze a range of normative claims, including some that may prove problematic and others that may shed light over potentially more valuable design options. This research will help fill an important knowledge gap; intervening earlier in technological development could help reduce undesirable effects and inform the design and implementation of more appropriate innovations.

Miniaturization of the Clonogenic Assay Using Confluence Measurement

PubMed Central

Mayr, Christian; Beyreis, Marlena; Dobias, Heidemarie; Gaisberger, Martin; Pichler, Martin; Ritter, Markus; Jakab, Martin; Neureiter, Daniel; Kiesslich, Tobias

2018-01-01

The clonogenic assay is a widely used method to study the ability of cells to ‘infinitely’ produce progeny and is, therefore, used as a tool in tumor biology to measure tumor-initiating capacity and stem cell status. However, the standard protocol of using 6-well plates has several disadvantages. By miniaturizing the assay to a 96-well microplate format, as well as by utilizing the confluence detection function of a multimode reader, we here describe a new and modified protocol that allows comprehensive experimental setups and a non-endpoint, label-free semi-automatic analysis. Comparison of bright field images with confluence images demonstrated robust and reproducible detection of clones by the confluence detection function. Moreover, time-resolved non-endpoint confluence measurement of the same well showed that semi-automatic analysis was suitable for determining the mean size and colony number. By treating cells with an inhibitor of clonogenic growth (PTC-209), we show that our modified protocol is suitable for comprehensive (broad concentration range, addition of technical replicates) concentration- and time-resolved analysis of the effect of substances or treatments on clonogenic growth. In summary, this protocol represents a time- and cost-effective alternative to the commonly used 6-well protocol (with endpoint staining) and also provides additional information about the kinetics of clonogenic growth. PMID:29510509

Unification of quantum information theory

NASA Astrophysics Data System (ADS)

Abeyesinghe, Anura

We present the unification of many previously disparate results in noisy quantum Shannon theory and the unification of all of noiseless quantum Shannon theory. More specifically we deal here with bipartite, unidirectional, and memoryless quantum Shannon theory. We find all the optimal protocols and quantify the relationship between the resources used, both for the one-shot and for the ensemble case, for what is arguably the most fundamental task in quantum information theory: sharing entangled states between a sender and a receiver. We find that all of these protocols are derived from our one-shot superdense coding protocol and relate nicely to each other. We then move on to noisy quantum information theory and give a simple, direct proof of the "mother" protocol, or rather her generalization to the Fully Quantum Slepian-Wolf protocol (FQSW). FQSW simultaneously accomplishes two goals: quantum communication-assisted entanglement distillation, and state transfer from the sender to the receiver. As a result, in addition to her other "children," the mother protocol generates the state merging primitive of Horodecki, Oppenheim, and Winter as well as a new class of distributed compression protocols for correlated quantum sources, which are optimal for sources described by separable density operators. Moreover, the mother protocol described here is easily transformed into the so-called "father" protocol, demonstrating that the division of single-sender/single-receiver protocols into two families was unnecessary: all protocols in the family are children of the mother.

Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method.

PubMed

Honda, Shozo; Morichika, Keisuke; Kirino, Yohei

2016-03-01

RNA digestions catalyzed by many ribonucleases generate RNA fragments that contain a 2',3'-cyclic phosphate (cP) at their 3' termini. However, standard RNA-seq methods are unable to accurately capture cP-containing RNAs because the cP inhibits the adapter ligation reaction. We recently developed a method named cP-RNA-seq that is able to selectively amplify and sequence cP-containing RNAs. Here we describe the cP-RNA-seq protocol in which the 3' termini of all RNAs, except those containing a cP, are cleaved through a periodate treatment after phosphatase treatment; hence, subsequent adapter ligation and cDNA amplification steps are exclusively applied to cP-containing RNAs. cP-RNA-seq takes ∼6 d, excluding the time required for sequencing and bioinformatics analyses, which are not covered in detail in this protocol. Biochemical validation of the existence of cP in the identified RNAs takes ∼3 d. Even though the cP-RNA-seq method was developed to identify angiogenin-generating 5'-tRNA halves as a proof of principle, the method should be applicable to global identification of cP-containing RNA repertoires in various transcriptomes.

Pyrrole-Imidazole Polyamides: Manual Solid-Phase Synthesis.

PubMed

Pauff, Steven M; Fallows, Andrew J; Mackay, Simon P; Su, Wu; Cullis, Paul M; Burley, Glenn A

2015-12-01

Pyrrole-imidazole polyamides (PAs) are a family of DNA-binding peptides that bind in the minor groove of double-stranded DNA (dsDNA) in a sequence-selective, programmable fashion. This protocol describes a detailed manual procedure for the solid-phase synthesis of this family of compounds. The protocol entails solution-phase synthesis of the Boc-protected pyrrole (Py) and imidazole (Im) carboxylic acid building blocks. This unit also describes the importance of choosing the appropriate condensing agent to form the amide linkages between each building block. Finally, a monomeric coupling protocol and a fragment-based approach are described that delivers PAs in 13% to 30% yield in 8 days. Copyright © 2015 John Wiley & Sons, Inc.

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

Design of the multicenter standardized supervised exercise training intervention for the claudication: exercise vs endoluminal revascularization (CLEVER) study.

PubMed

Bronas, Ulf G; Hirsch, Alan T; Murphy, Timothy; Badenhop, Dalynn; Collins, Tracie C; Ehrman, Jonathan K; Ershow, Abby G; Lewis, Beth; Treat-Jacobson, Diane J; Walsh, M Eileen; Oldenburg, Niki; Regensteiner, Judith G

2009-11-01

The CLaudication: Exercise Vs Endoluminal Revascularization (CLEVER) study is the first randomized, controlled, clinical, multicenter trial that is evaluating a supervised exercise program compared with revascularization procedures to treat claudication. In this report, the methods and dissemination techniques of the supervised exercise training intervention are described. A total of 217 participants are being recruited and randomized to one of three arms: (1) optimal medical care; (2) aortoiliac revascularization with stent; or (3) supervised exercise training. Of the enrolled patients, 84 will receive supervised exercise therapy. Supervised exercise will be administered according to a protocol designed by a central CLEVER exercise training committee based on validated methods previously used in single center randomized control trials. The protocol will be implemented at each site by an exercise committee member using training methods developed and standardized by the exercise training committee. The exercise training committee reviews progress and compliance with the protocol of each participant weekly. In conclusion, a multicenter approach to disseminate the supervised exercise training technique and to evaluate its efficacy, safety and cost-effectiveness for patients with claudication due to peripheral arterial disease (PAD) is being evaluated for the first time in CLEVER. The CLEVER study will further establish the role of supervised exercise training in the treatment of claudication resulting from PAD and provide standardized methods for use of supervised exercise training in future PAD clinical trials as well as in clinical practice.

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.

Gender-Specific Combination HIV Prevention for Youth in High-Burden Settings: The MP3 Youth Observational Pilot Study Protocol

PubMed Central

Agot, Kawango

2017-01-01

Background Nearly three decades into the epidemic, sub-Saharan Africa (SSA) remains the region most heavily affected by human immunodeficiency virus (HIV), with nearly 70% of the 34 million people living with HIV globally residing in the region. In SSA, female and male youth (15 to 24 years) are at a disproportionately high risk of HIV infection compared to adults. As such, there is a need to target HIV prevention strategies to youth and to tailor them to a gender-specific context. This protocol describes the process for the multi-staged approach in the design of the MP3 Youth pilot study, a gender-specific, combination, HIV prevention intervention for youth in Kenya. Objective The objective of this multi-method protocol is to outline a rigorous and replicable methodology for a gender-specific combination HIV prevention pilot study for youth in high-burden settings, illustrating the triangulated methods undertaken to ensure that age, sex, and context are integral in the design of the intervention. Methods The mixed-methods, cross-sectional, longitudinal cohort pilot study protocol was developed by first conducting a systematic review of the literature, which shaped focus group discussions around prevention package and delivery options, and that also informed age- and sex- stratified mathematical modeling. The review, qualitative data, and mathematical modeling created a triangulated evidence base of interventions to be included in the pilot study protocol. To design the pilot study protocol, we convened an expert panel to select HIV prevention interventions effective for youth in SSA, which will be offered in a mobile health setting. The goal of the pilot study implementation and evaluation is to apply lessons learned to more effective HIV prevention evidence and programming. Results The combination HIV prevention package in this protocol includes (1) offering HIV testing and counseling for all youth; (2) voluntary medical circumcision and condoms for males; (3) pre-exposure prophylaxis (PrEP), conditional cash transfer (CCT), and contraceptives for females; and (4) referrals for HIV care among those identified as HIV-positive. The combination package platform selected is mobile health teams in an integrated services delivery model. A cross-sectional analysis will be conducted to determine the uptake of the interventions. To determine long-term impact, the protocol outlines enrolling selected participants in mutually exclusive longitudinal cohorts (HIV-positive, PrEP, CCT, and HIV-negative) followed by using mobile phone text messages (short message service, SMS) and in-person surveys to prospectively assess prevention method uptake, adherence, and risk compensation behaviors. Cross-sectional and sub-cohort analyses will be conducted to determine intervention packages uptake. Conclusions The literature review, focus groups, and modeling indicate that offering age- and gender- specific combination HIV prevention interventions that include biomedical, behavioral, and structural interventions can have an impact on HIV risk reduction. Implementing this protocol will show the feasibility of delivering these services at scale. The MP3 Youth study is one of the few combination HIV prevention intervention protocols incorporating youth- and gender-specific interventions in one delivery setting. Lessons learned from the design of the protocol can be incorporated into the national guidance for combination HIV prevention for youth in Kenya and other high-burden SSA settings. Trial Registration ClinicalTrials.gov NCT01571128; http://clinicaltrials.gov/ct2/show/NCT01571128?term=MP3+youth&rank=1 (Archived by WebCite at http://www.webcitation.org/6nmioPd54) PMID:28274904

I Move: systematic development of a web-based computer tailored physical activity intervention, based on motivational interviewing and self-determination theory

PubMed Central

2014-01-01

Background This article describes the systematic development of the I Move intervention: a web-based computer tailored physical activity promotion intervention, aimed at increasing and maintaining physical activity among adults. This intervention is based on the theoretical insights and practical applications of self-determination theory and motivational interviewing. Methods/design Since developing interventions in a systemically planned way increases the likelihood of effectiveness, we used the Intervention Mapping protocol to develop the I Move intervention. In this article, we first describe how we proceeded through each of the six steps of the Intervention Mapping protocol. After that, we describe the content of the I Move intervention and elaborate on the planned randomized controlled trial. Discussion By integrating self-determination theory and motivational interviewing in web-based computer tailoring, the I Move intervention introduces a more participant-centered approach than traditional tailored interventions. Adopting this approach might enhance computer tailored physical activity interventions both in terms of intervention effectiveness and user appreciation. We will evaluate this in an randomized controlled trial, by comparing the I Move intervention to a more traditional web-based computer tailored intervention. Trial registration NTR4129 PMID:24580802

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

PubMed Central

Kranzfelder, Petra; Anderson, Alyssa M.; Egan, Alexander T.; Mazack, Jane E.; Bouchard,, R. William; Rufer, Moriya M.; Ferrington,, Leonard C.

2015-01-01

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water’s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males. PMID:26274889

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies.

PubMed

Kranzfelder, Petra; Anderson, Alyssa M; Egan, Alexander T; Mazack, Jane E; Bouchard, R William; Rufer, Moriya M; Ferrington, Leonard C

2015-07-24

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water's surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

The Alzheimer's Disease Neuroimaging Initiative (ADNI): MRI Methods

PubMed Central

Jack, Clifford R.; Bernstein, Matt A.; Fox, Nick C.; Thompson, Paul; Alexander, Gene; Harvey, Danielle; Borowski, Bret; Britson, Paula J.; Whitwell, Jennifer L.; Ward, Chadwick; Dale, Anders M.; Felmlee, Joel P.; Gunter, Jeffrey L.; Hill, Derek L.G.; Killiany, Ron; Schuff, Norbert; Fox-Bosetti, Sabrina; Lin, Chen; Studholme, Colin; DeCarli, Charles S.; Krueger, Gunnar; Ward, Heidi A.; Metzger, Gregory J.; Scott, Katherine T.; Mallozzi, Richard; Blezek, Daniel; Levy, Joshua; Debbins, Josef P.; Fleisher, Adam S.; Albert, Marilyn; Green, Robert; Bartzokis, George; Glover, Gary; Mugler, John; Weiner, Michael W.

2008-01-01

The Alzheimer's Disease Neuroimaging Initiative (ADNI) is a longitudinal multisite observational study of healthy elders, mild cognitive impairment (MCI), and Alzheimer's disease. Magnetic resonance imaging (MRI), (18F)-fluorode-oxyglucose positron emission tomography (FDG PET), urine serum, and cerebrospinal fluid (CSF) biomarkers, as well as clinical/psychometric assessments are acquiredat multiple time points. All data will be cross-linked and made available to the general scientific community. The purpose of this report is to describe the MRI methods employed in ADNI. The ADNI MRI core established specifications thatguided protocol development. A major effort was devoted toevaluating 3D T1-weighted sequences for morphometric analyses. Several options for this sequence were optimized for the relevant manufacturer platforms and then compared in a reduced-scale clinical trial. The protocol selected for the ADNI study includes: back-to-back 3D magnetization prepared rapid gradient echo (MP-RAGE) scans; B1-calibration scans when applicable; and an axial proton density-T2 dual contrast (i.e., echo) fast spin echo/turbo spin echo (FSE/TSE) for pathology detection. ADNI MRI methods seek to maximize scientific utility while minimizing the burden placed on participants. The approach taken in ADNI to standardization across sites and platforms of the MRI protocol, postacquisition corrections, and phantom-based monitoring of all scanners could be used as a model for other multisite trials. PMID:18302232

Protocol vulnerability detection based on network traffic analysis and binary reverse engineering.

PubMed

Wen, Shameng; Meng, Qingkun; Feng, Chao; Tang, Chaojing

2017-01-01

Network protocol vulnerability detection plays an important role in many domains, including protocol security analysis, application security, and network intrusion detection. In this study, by analyzing the general fuzzing method of network protocols, we propose a novel approach that combines network traffic analysis with the binary reverse engineering method. For network traffic analysis, the block-based protocol description language is introduced to construct test scripts, while the binary reverse engineering method employs the genetic algorithm with a fitness function designed to focus on code coverage. This combination leads to a substantial improvement in fuzz testing for network protocols. We build a prototype system and use it to test several real-world network protocol implementations. The experimental results show that the proposed approach detects vulnerabilities more efficiently and effectively than general fuzzing methods such as SPIKE.

Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds.

PubMed

Milles, J; Krämer, J; Prange, A

2007-12-01

In general, proteome studies compare different states of metabolism to investigate external or internal influences on protein expression. In the context of mycotoxin production the method could open another view on this complex and could be helpful to gain knowledge about proteins which are involved in metabolism (enzymes, transporters). In this short technical report, we describe a new protocol suitable for protein preparation for whole proteome analysis ofFusarium graminearum. Cell lysis was performed by grinding the mycelium with liquid nitrogen. Proteins were extracted with TCA/acetone and then cleaned; the isolated proteins were separated in a 2D-gel electrophoresis system (BioRad) using different pH gradients. The protocol established seems also generally applicable for other mycotoxin producing fungi.

Chemokine receptor binding and signal transduction in native cells of the central nervous system.

PubMed

Davis, Christopher N; Chen, Shuzhen; Boehme, Stefen A; Bacon, Kevin B; Harrison, Jeffrey K

2003-04-01

Chemokine receptors belong to the superfamily of seven-transmembrane-spanning, G-protein-coupled receptors, and their expression by central nervous system cells is clearly documented. As this gene family has become the target of novel therapeutic development, the analysis of these receptors requires radioligand binding techniques as well as methods that entail assessing receptor stimulation of signal transduction pathways. Herein, we describe specific protocols for measuring radiolabeled chemokine binding to their cognate receptors on cultured glial cells as well as to receptors expressed in heterologous cell systems. Multiple downstream signaling pathways, including intracellular calcium influx and receptor-dependent kinase activation, are associated with chemokine receptor stimulation. Protocols for measuring these signaling events in chemokine-receptor-expressing cells are also presented.

Proteomic approaches and their application to plant gravitropism.

PubMed

Basu, Proma; Luesse, Darron R; Wyatt, Sarah E

2015-01-01

Proteomics is a powerful technique that allows researchers a window into how an organism responds to a mutation, a specific environment, or at a distinct point during development by quantifying relative protein abundance and posttranslational modifications. Here, we describe methods for the proteomic analysis of Arabidopsis thaliana tissue. Extraction protocols are provided for isolation of soluble, plasma membrane, and tonoplast proteins. In addition, basic analysis and quality metrics for MS/MS data are discussed. The protocols outlined have the potential to unlock new avenues of research that are not possible through basic genetics or transcriptomic approaches. By combining proteomic information with known gene regulatory patterns, researchers can gain a complete picture of how molecular pathways, such as those required for gravitropism, are initiated, regulated, and terminated.

A novel protocol for generating intact, whole-head spider cephalothorax tissue sections.

PubMed

Long, Skye M

2018-04-01

The diversity of spider behavior and sensory systems provides an excellent opportunity for comparative studies of the relationship between the brain and behavior. However, the morphology of spiders poses a challenge for histologists since the spider cephalothorax contains heterogeneous tissues and has both tough external and internal sclerotized regions. Unlike the heads of insects, the cephalothorax is highly pressurized, which can cause tissues to shift during processing and can reduce tissue cohesion in thick sections. This work describes a novel protocol for producing thick whole-head sections for morphological study by softening the exoskeleton and stabilizing friable tissue, without freezing or dehydration. It also presents an effective whole-head DiI staining method that uses minimal dehydration and highlights neural structures.

Comprehensive protocols for CRISPR/Cas9-based gene editing in human pluripotent stem cells

PubMed Central

Santos, David P.; Kiskinis, Evangelos; Eggan, Kevin; Merkle, Florian T.

2016-01-01

Application of the CRISPR/Cas9 system to edit the genomes of human pluripotent stem cells (hPSCs) has the potential to revolutionize hPSC-based disease modeling, drug screening, and transplantation therapy. Here, we aim to provide a single resource to enable groups, even those with limited experience with hPSC culture or the CRISPR/Cas9 system, to successfully perform genome editing. The methods are presented in detail and are supported by a theoretical framework to allow for the incorporation of inevitable improvements in the rapidly evolving gene-editing field. We describe protocols to generate hPSC lines with gene-specific knock-outs, small targeted mutations, or knock-in reporters. PMID:27532820

A Public-Use, Full-Screen Interface for SPIRES Databases.

ERIC Educational Resources Information Center

Kriz, Harry M.

This paper describes the techniques for implementing a full-screen, custom SPIRES interface for a public-use library database. The database-independent protocol that controls the system is described in detail. Source code for an entire working application using this interface is included. The protocol, with less than 170 lines of procedural code,…

GoActive: a protocol for the mixed methods process evaluation of a school-based physical activity promotion programme for 13-14year old adolescents.

PubMed

Jong, Stephanie T; Brown, Helen Elizabeth; Croxson, Caroline H D; Wilkinson, Paul; Corder, Kirsten L; van Sluijs, Esther M F

2018-05-21

Process evaluations are critical for interpreting and understanding outcome trial results. By understanding how interventions function across different settings, process evaluations have the capacity to inform future dissemination of interventions. The complexity of Get others Active (GoActive), a 12-week, school-based physical activity intervention implemented in eight schools, highlights the need to investigate how implementation is achieved across a variety of school settings. This paper describes the mixed methods GoActive process evaluation protocol that is embedded within the outcome evaluation. In this detailed process evaluation protocol, we describe the flexible and pragmatic methods that will be used for capturing the process evaluation data. A mixed methods design will be used for the process evaluation, including quantitative data collected in both the control and intervention arms of the GoActive trial, and qualitative data collected in the intervention arm. Data collection methods will include purposively sampled, semi-structured interviews and focus group interviews, direct observation, and participant questionnaires (completed by students, teachers, older adolescent mentors, and local authority-funded facilitators). Data will be analysed thematically within and across datasets. Overall synthesis of findings will address the process of GoActive implementation, and through which this process affects outcomes, with careful attention to the context of the school environment. This process evaluation will explore the experience of participating in GoActive from the perspectives of key groups, providing a greater understanding of the acceptability and process of implementation of the intervention across the eight intervention schools. This will allow for appraisal of the intervention's conceptual base, inform potential dissemination, and help optimise post-trial sustainability. The process evaluation will also assist in contextualising the trial effectiveness results with respect to how the intervention may or may not have worked and, if it was found to be effective, what might be required for it to be sustained in the 'real world'. Furthermore, it will offer suggestions for the development and implementation of future initiatives to promote physical activity within schools. ISRCTN, ISRCTN31583496 . Registered on 18 February 2014.

Novel Method for Quantitative Estimation of Biofilms.

PubMed

Syal, Kirtimaan

2017-10-01

Biofilm protects bacteria from stress and hostile environment. Crystal violet (CV) assay is the most popular method for biofilm determination adopted by different laboratories so far. However, biofilm layer formed at the liquid-air interphase known as pellicle is extremely sensitive to its washing and staining steps. Early phase biofilms are also prone to damage by the latter steps. In bacteria like mycobacteria, biofilm formation occurs largely at the liquid-air interphase which is susceptible to loss. In the proposed protocol, loss of such biofilm layer was prevented. In place of inverting and discarding the media which can lead to the loss of the aerobic biofilm layer in CV assay, media was removed from the formed biofilm with the help of a syringe and biofilm layer was allowed to dry. The staining and washing steps were avoided, and an organic solvent-tetrahydrofuran (THF) was deployed to dissolve the biofilm, and the absorbance was recorded at 595 nm. The protocol was tested for biofilm estimation of E. coli, B. subtilis and M. smegmatis, and compared with the traditional CV assays. Isoniazid drug molecule, a known inhibitor of M. smegmatis biofilm, was tested and its inhibitory effects were quantified by the proposed protocol. For ease in referring, this method has been described as the Syal method for biofilm quantification. This new method was found to be useful for the estimation of early phase biofilm and aerobic biofilm layer formed at the liquid-air interphase. The biofilms formed by all three tested bacteria-B. subtilis, E. coli and M. smegmatis, were precisely quantified.

The development, deployment, and impact of the virtual observatory, Part II

NASA Astrophysics Data System (ADS)

Hanisch, R. J.

2015-06-01

This is the second special issue of Astronomy and Computing devoted to the Virtual Observatory, and we again see a combination of papers covering various aspects of the VO, from infrastructure to applications to programmatics. The critical role of data models is described by Louys, and the method by which applications communicate amongst each other through the Simple Applications Messaging Protocol (SAMP) is described by Taylor et al. Demleitner et al. explain the client interfaces to the VO registry, that is, how applications developers can query the registry for information about VO-compliant data collections and services.1

Wheat (Triticum aestivum L.) transformation using mature embryos.

PubMed

Medvecká, Eva; Harwood, Wendy A

2015-01-01

In most protocols for the Agrobacterium-mediated transformation of wheat, the preferred target tissues are immature embryos. However, transformation methods relying on immature embryos require the growth of plants under controlled conditions to provide a continuous supply of good-quality target tissue. The use of mature embryos as a target tissue has the advantage of only requiring good-quality seed as the starting material. Here we describe a transformation method based on the Agrobacterium-mediated transformation of callus cultures derived from mature wheat embryos of the genotype Bobwhite S56.

Systematic Review Protocol to Assess the Effectiveness of Usability Questionnaires in mHealth App Studies

PubMed Central

Bao, Jie; Parmanto, Bambang

2017-01-01

Background Usability questionnaires have a wide use in mobile health (mHealth) app usability studies. However, no systematic review has been conducted for assessing the effectiveness of these questionnaires. Objective This paper describes a protocol for conducting a systematic review of published questionnaire-based mHealth app usability studies. Methods In this systematic review, we will select recently published (2008-2017) articles from peer-reviewed journals and conferences that describe mHealth app usability studies and implement at least one usability questionnaire. The search strategy will include terms such as “mobile app” and “usability.” Multiple databases such as PubMed, CINAHL, IEEE Xplore, ACM Digital Library, and INSPEC will be searched. There will be 2 independent reviewers in charge of screening titles and abstracts as well as determining those articles that should be included for a full-text review. The third reviewer will act as a mediator between the other 2 reviewers. Moreover, a data extraction form will be created and used during the full article data analysis. Notably, the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) guidelines will be followed in reporting this protocol. Results A preliminary search produced 1271 articles, 40 of which are duplicate records. The inclusion-exclusion criteria are being strictly followed in performing the ongoing study selection. Conclusions Usability questionnaires are an important tool in mHealth app usability studies. This review will summarize the usability questionnaires used in published research articles while assessing the efficacy of these questionnaires in determining the usability of mHealth apps. PMID:28765101

A review of protocols for 308 nm excimer laser phototherapy in psoriasis.

PubMed

Mudigonda, Tejaswi; Dabade, Tushar S; Feldman, Steven R

2012-01-01

308 nm excimer laser phototherapy is efficacious in the treatment of localized psoriasis. Different approaches regarding dose fluency, number of treatments, and maintenance have been utilized, and there is yet to be a consensus on standard protocol. To characterize treatment parameters for 308 nm excimer laser phototherapy. We performed a PubMed search for studies describing excimer laser treatment protocol with particular attention to dosage determination, dose adjustment, dose fluency, number of treatments, and maintenance. Seven prospective studies were found describing the excimer efficacy for psoriasis. All studies determined the initial treatment dose using either the minimal erythema dose (MED) or induration. Fluency ranged from 0.5 MED (low) to 16 MED (high); one study demonstrated that medium to high fluencies yielded better improvement in fewer number of treatments. Fluency adjustments during the course of treatment were important to minimize phototherapy-associated side effects. The use of higher fluencies was reported to result in higher occurrences of blistering. One study implemented a maintenance tapering of dose-frequency phase to better manage psoriasis flare-ups. The 308 nm excimer laser is an effective therapy for psoriasis regardless of the method used to determine initial dosage, dose fluency, or number of treatments. As its usage as a targeted monotherapy increases, future trials should consider evaluating and modifying these parameters to determine the most optimal management of localized psoriasis. Based on our reviewed studies, there is no consensus for a single excimer laser therapy protocol and as a result, patient preferences should continue to be an important consideration for phototherapy regimen planning.

Network Upgrade for the SLC: Control System Modifications

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

Crane, M.; Mackenzie, R.; Sass, R.

2011-09-09

Current communications between the SLAC Linear Collider control system central host and the SLCmicros is built upon the SLAC developed SLCNET communication hardware and protocols. We will describe how the Internet Suite of protocols (TCP/IP) are used to replace the SLCNET protocol interface. The major communication pathways and their individual requirements are described. A proxy server is used to reduce the number of total system TCP/IP connections. The SLCmicros were upgraded to use Ethernet and TCP/IP as well as SLCNET. Design choices and implementation experiences are addressed.

SITE-SPECIFIC PROTOCOL FOR MEASURING SOIL RADON POTENTIALS FOR FLORIDA HOUSES

EPA Science Inventory

The report describes a protocol for site-specific measurement of radon potentials for Florida houses that is consistent with existing residential radon protection maps. The protocol gives further guidance on the possible need for radon-protective house construction features. In a...

Post-trial period surveillance for randomised controlled cardiovascular studies: submitted protocols, consent forms and the role of the ethics board.

PubMed

Zia, Mohammad I; Heslegrave, Ronald; Newton, Gary E

2011-12-01

The post-trial period is the time period after the end of study drug administration. It is unclear whether post-trial arrangements for patient surveillance are routinely included in study protocols and consents, and whether research ethics boards (REB) consider the post-trial period. The objective was to determine whether trial protocols and consent forms reviewed by the REB describe procedures for post-trial period surveillance. An observational study of protocols of randomised trials of chronic therapies for cardiac conditions, approved by the REB of two academic institutions affiliated with the University of Toronto in Canada (University Health Network and Mount Sinai Hospital) from 1995 to 2007. Plans for patient surveillance in the post-trial period described in the protocol or in the consent form before and after REB approval were recorded. 42 studies were identified including 18 heart failure and 15 coronary artery disease trials. Only four studies planned a clinical visit after trial termination, and an additional three planned a telephone contact after trial completion. Five trials submitted consent forms to the REB with a discussion of the post-trial period. The majority of protocols and consent forms did not discuss plans for post-trial period surveillance. The post-trial period and the REB approval process could be improved by systematic follow-up being described in the protocol and consent form. The small number of trial protocols evaluated in the study may impair the degree to which the results can be generalised.

Informal trail monitoring protocols: Denali National Park and Preserve. Final Report, October 2011

USGS Publications Warehouse

Marion, Jeffrey L.; Wimpey, Jeremy F.

2011-01-01

Managers at Alaska?s Denali National Park and Preserve (DENA) sponsored this research to assess and monitor visitor-created informal trails (ITs). DENA is located in south-central Alaska and managed as a six million acre wilderness park. This program of research was guided by the following objectives: (1) Investigate alternative methods for monitoring the spatial distribution, aggregate lineal extent, and tread conditions of informal (visitor-created) trails within the park. (2) In consultation with park staff, develop, pilot test, and refine cost-effective and scientifically defensible trail monitoring procedures that are fully integrated with the park?s Geographic Information System. (3) Prepare a technical report that compiles and presents research results and their management implications. This report presents the protocol development and field testing process, illustrates the types of data produced by their application, and provides guidance for their application and use. The protocols described provide managers with an efficient means to document and monitor IT conditions in settings ranging from pristine to intensively visited.

In Vitro Mass Propagation of Cymbopogon citratus Stapf., a Medicinal Gramineae.

PubMed

Quiala, Elisa; Barbón, Raúl; Capote, Alina; Pérez, Naivy; Jiménez, Elio

2016-01-01

Cymbopogon citratus (D.C.) Stapf. is a medicinal plant source of lemon grass oils with multiple uses in the pharmaceutical and food industry. Conventional propagation in semisolid culture medium has become a fast tool for mass propagation of lemon grass, but the production cost must be lower. A solution could be the application of in vitro propagation methods based on liquid culture advantages and automation. This chapter provides two efficient protocols for in vitro propagation via organogenesis and somatic embryogenesis of this medicinal plant. Firstly, we report the production of shoots using a temporary immersion system (TIS). Secondly, a protocol for somatic embryogenesis using semisolid culture for callus formation and multiplication, and liquid culture in a rotatory shaker and conventional bioreactors for the maintenance of embryogenic culture, is described. Well-developed plants can be achieved from both protocols. Here we provide a fast and efficient technology for mass propagation of this medicinal plant taking the advantage of liquid culture and automation.

Design of Light-Controlled Protein Conformations and Functions.

PubMed

Ritterson, Ryan S; Hoersch, Daniel; Barlow, Kyle A; Kortemme, Tanja

2016-01-01

In recent years, interest in controlling protein function with light has increased. Light offers a number of unique advantages over other methods, including spatial and temporal control and high selectivity. Here, we describe a general protocol for engineering a protein to be controllable with light via reaction with an exogenously introduced photoisomerizable small molecule and illustrate our protocol with two examples from the literature: the engineering of the calcium affinity of the cell-cell adhesion protein cadherin, which is an example of a protein that switches from a native to a disrupted state (Ritterson et al. J Am Chem Soc (2013) 135:12516-12519), and the engineering of the opening and closing of the chaperonin Mm-cpn, an example of a switch between two functional states (Hoersch et al.: Nat Nanotechn (2013) 8:928-932). This protocol guides the user from considering which proteins may be most amenable to this type of engineering, to considerations of how and where to make the desired changes, to the assays required to test for functionality.

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.

Design and Construction of a Multi-wavelength, Micromirror Total Internal Reflectance Fluorescence Microscope

PubMed Central

Larson, Joshua; Kirk, Matt; Drier, Eric A.; O’Brien, William; MacKay, James F.; Friedman, Larry; Hoskins, Aaron

2015-01-01

Colocalization Single Molecule Spectroscopy (CoSMoS) has proven to be a useful method for studying the composition, kinetics, and mechanisms of complex cellular machines. Key to the technique is the ability to simultaneously monitor multiple proteins and/or nucleic acids as they interact with one another. Here we describe a protocol for constructing a CoSMoS micromirror Total Internal Reflection Fluorescence Microscope (mmTIRFM). Design and construction of a scientific microscope often requires a number of custom components and a significant time commitment. In our protocol, we have streamlined this process by implementation of a commercially available microscopy platform designed to accommodate the optical components necessary for a mmTIRFM. The mmTIRF system eliminates the need for machining custom parts by the end-user and facilitates optical alignment. Depending on the experience-level of the microscope builder, these time-savings and the following protocol can enable mmTIRF construction to be completed within two months. PMID:25188633

Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope.

PubMed

Larson, Joshua; Kirk, Matt; Drier, Eric A; O'Brien, William; MacKay, James F; Friedman, Larry J; Hoskins, Aaron A

2014-10-01

Colocalization single-molecule spectroscopy (CoSMoS) has proven to be a useful method for studying the composition, kinetics and mechanisms of complex cellular machines. Key to the technique is the ability to simultaneously monitor multiple proteins and/or nucleic acids as they interact with one another. Here we describe a protocol for constructing a CoSMoS micromirror total internal reflection fluorescence microscope (mmTIRFM). Design and construction of a scientific microscope often requires a number of custom components and a substantial time commitment. In our protocol, we have streamlined this process by implementation of a commercially available microscopy platform designed to accommodate the optical components necessary for an mmTIRFM. The mmTIRF system eliminates the need for machining custom parts by the end user and facilitates optical alignment. Depending on the experience level of the microscope builder, these time savings and the following protocol can enable mmTIRF construction to be completed within 2 months.

Commercial Building Energy Asset Score Program Overview and Technical Protocol (Version 1.1)

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

Wang, Na; Goel, Supriya; Makhmalbaf, Atefe

The U.S. Department of Energy (DOE) is developing a voluntary national scoring system for commercial buildings to help building owners and managers assess a building’s energy-related systems independent of operations. The goal of the score is to facilitate cost-effective investment in energy efficiency improvements of commercial buildings. The system, known as the Commercial Building Energy Asset Score, will allow building owners and managers to compare their building infrastructure against peers and track building upgrades over time. The system will also help other building stakeholders (e.g., building investors, tenants, financiers, and appraisers) understand the relative efficiency of different buildings in amore » way that is independent from operations and occupancy. This report outlines the technical protocol used to generate the energy asset score, explains the scoring methodology, and provides additional details regarding the energy asset scoring tool. The alternative methods that were considered prior to developing the current approach are described in the Program Overview and Technical Protocol Version 1.0.« less

Reproducible and controllable light induction of in vitro fruiting of the white-rot basidiomycete Pleurotus ostreatus.

PubMed

Arjona, Davinia; Aragón, Carlos; Aguilera, José Antonio; Ramírez, Lucía; Pisabarro, Antonio G

2009-05-01

Fruiting is a crucial developmental process in basidiomycetes yet the genetic and molecular factors that control it are not yet fully understood. The search for fruiting inducers is of major relevance for both basic research and for their use in industrial applications. In this paper, an efficient and reproducible protocol for controlled fruiting induction of Pleurotus ostreatus growing on synthetic medium is described. The protocol is based on the control of light intensity and photoperiod and permits the life cycle for this fungus to be completed in less than two weeks. The fruiting bodies produced by this method release fertile spores after 4-5 d of culture. Our results indicate that fruiting induction is solely dependent on the illumination regime and that it occurs long before the available nutrients are depleted in the culture. This protocol will greatly facilitate molecular and developmental biology research in this fungus as it avoids the need for complex culture media based on lignocellulosic materials or the use of chemical inducers.

Synthesis of immunotargeted magneto-plasmonic nanoclusters.

PubMed

Wu, Chun-Hsien; Sokolov, Konstantin

2014-08-22

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture and detection of circulating tumor cells (CTCs), and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

CON4EI: CONsortium for in vitro Eye Irritation testing strategy - EpiOcular™ time-to-toxicity (EpiOcular ET-50) protocols for hazard identification and labelling of eye irritating chemicals.

PubMed

Kandarova, Helena; Letasiova, Silvia; Adriaens, Els; Guest, Robert; Willoughby, Jamin A; Drzewiecka, Agnieszka; Gruszka, Katarzyna; Alépée, Nathalie; Verstraelen, Sandra; Van Rompay, An R

2018-06-01

Assessment of acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. The objective of the CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was to develop tiered testing strategies for eye irritation assessment for all drivers of classification. A set of 80 reference chemicals (38 liquids and 42 solids) was tested with eight different alternative methods. Here, the results obtained with reconstructed human cornea-like epithelium (RhCE) EpiOcular™ in the EpiOcular time-to-toxicity Tests (Neat and Dilution ET-50 protocols) are presented. The primary aim of this study was to evaluate whether test methods can discriminate chemicals not requiring classification for serious eye damage/eye irritancy (No Category) from chemicals requiring classification and labelling for Category 1 and Category 2. In addition, the predictive capacity in terms of in vivo drivers of classification was investigated. The chemicals were tested in two independent runs by MatTek In Vitro Life Science Laboratories. Results of this study demonstrate very high specificity of both test protocols. With the existing prediction models described in the SOPs, the specificity of the Neat and Dilution method was 87% and 100%, respectively. The Dilution method was able to correctly predicting 66% of GHS Cat 2 chemicals, however, prediction of GHS Cat 1 chemicals was only 47%-55% using the current protocols. In order to achieve optimal prediction for all three classes, a testing strategy was developed which combines the most predictive time-points of both protocols and for tests liquids and solids separately. Using this new testing strategy, the sensitivity for predicting GHS Cat 1 and GHS Cat 2 chemicals was 73% and 64%, respectively and the very high specificity of 97% was maintained. None of the Cat 1 chemicals was underpredicted as GHS No Category. Further combination of the EpiOcular time-to-toxicity protocols with other validated in vitro systems evaluated in this project, should enable significant reduction and even possible replacement of the animal tests for the final assessment of the irritation potential in all of the GHS classes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fast protocol for radiochromic film dosimetry using a cloud computing web application.

PubMed

Calvo-Ortega, Juan-Francisco; Pozo, Miquel; Moragues, Sandra; Casals, Joan

2017-07-01

To investigate the feasibility of a fast protocol for radiochromic film dosimetry to verify intensity-modulated radiotherapy (IMRT) plans. EBT3 film dosimetry was conducted in this study using the triple-channel method implemented in the cloud computing application (Radiochromic.com). We described a fast protocol for radiochromic film dosimetry to obtain measurement results within 1h. Ten IMRT plans were delivered to evaluate the feasibility of the fast protocol. The dose distribution of the verification film was derived at 15, 30, 45min using the fast protocol and also at 24h after completing the irradiation. The four dose maps obtained per plan were compared using global and local gamma index (5%/3mm) with the calculated one by the treatment planning system. Gamma passing rates obtained for 15, 30 and 45min post-exposure were compared with those obtained after 24h. Small differences respect to the 24h protocol were found in the gamma passing rates obtained for films digitized at 15min (global: 99.6%±0.9% vs. 99.7%±0.5%; local: 96.3%±3.4% vs. 96.3%±3.8%), at 30min (global: 99.5%±0.9% vs. 99.7%±0.5%; local: 96.5%±3.2% vs. 96.3±3.8%) and at 45min (global: 99.2%±1.5% vs. 99.7%±0.5%; local: 96.1%±3.8% vs. 96.3±3.8%). The fast protocol permits dosimetric results within 1h when IMRT plans are verified, with similar results as those reported by the standard 24h protocol. Copyright © 2017 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

The BIMDA shuttle flight mission - A low cost MPS payload

NASA Technical Reports Server (NTRS)

Holemans, Jaak; Cassanto, John M.; Morrison, Dennis; Rose, Alan; Luttges, Marvin

1990-01-01

The design, operation, and experimental protocol of the Bioserve-ITA Materials Dispersion Apparatus Payload (BIMDA) to be flown on the Space Shuttle on STS-37 are described. The aim of BIMDA is to investigate the methods and commercial potential of biomedical and fluid science applications in the microgravity environment. The BIMDA payload operations are diagrammed, and the payload components and experiments are listed, including the investigators and sponsoring institutions.

An Extensible NetLogo Model for Visualizing Message Routing Protocols

DTIC Science & Technology

2017-08-01

the hard sciences to the social sciences to computer-generated art. NetLogo represents the world as a set of...describe the model is shown here; for the supporting methods , refer to the source code. Approved for public release; distribution is unlimited. 4 iv...if ticks - last-inject > time-to-inject [inject] if run# > #runs [stop] end Next, we present some basic statistics collected for the

Optimized efflux assay for the NorA multidrug efflux pump in Staphylococcus aureus.

PubMed

Zimmermann, Saskia; Tuchscherr, Lorena; Rödel, Jürgen; Löffler, Bettina; Bohnert, Jürgen A

2017-11-01

Real-time fluorescent efflux assays are commonly used for measuring the efflux of bacterial pumps. Here we describe an optimized protocol for the NorA efflux pump in S. aureus using DiOC 3 instead of ethidium bromide. Glucose and sodium formate were tested as energy carriers. This novel method is fast and reproducible. Copyright © 2017 Elsevier B.V. All rights reserved.

Maize (Zea mays L.).

PubMed

Frame, Bronwyn; Warnberg, Katey; Main, Marcy; Wang, Kan

2015-01-01

Agrobacterium tumefaciens-mediated transformation is an effective method for introducing genes into maize. In this chapter, we describe a detailed protocol for genetic transformation of the maize genotype Hi II. Our starting plant material is immature embryos cocultivated with an Agrobacterium strain carrying a standard binary vector. In addition to step-by-step laboratory transformation procedures, we include extensive details in growing donor plants and caring for transgenic plants in the greenhouse.

Normalization of cortical thickness measurements across different T1 magnetic resonance imaging protocols by novel W-Score standardization.

PubMed

Chung, Jinyong; Yoo, Kwangsun; Lee, Peter; Kim, Chan Mi; Roh, Jee Hoon; Park, Ji Eun; Kim, Sang Joon; Seo, Sang Won; Shin, Jeong-Hyeon; Seong, Joon-Kyung; Jeong, Yong

2017-10-01

The use of different 3D T1-weighted magnetic resonance (T1 MR) imaging protocols induces image incompatibility across multicenter studies, negating the many advantages of multicenter studies. A few methods have been developed to address this problem, but significant image incompatibility still remains. Thus, we developed a novel and convenient method to improve image compatibility. W-score standardization creates quality reference values by using a healthy group to obtain normalized disease values. We developed a protocol-specific w-score standardization to control the protocol effect, which is applied to each protocol separately. We used three data sets. In dataset 1, brain T1 MR images of normal controls (NC) and patients with Alzheimer's disease (AD) from two centers, acquired with different T1 MR protocols, were used (Protocol 1 and 2, n = 45/group). In dataset 2, data from six subjects, who underwent MRI with two different protocols (Protocol 1 and 2), were used with different repetition times, echo times, and slice thicknesses. In dataset 3, T1 MR images from a large number of healthy normal controls (Protocol 1: n = 148, Protocol 2: n = 343) were collected for w-score standardization. The protocol effect and disease effect on subjects' cortical thickness were analyzed before and after the application of protocol-specific w-score standardization. As expected, different protocols resulted in differing cortical thickness measurements in both NC and AD subjects. Different measurements were obtained for the same subject when imaged with different protocols. Multivariate pattern difference between measurements was observed between the protocols. Classification accuracy between two protocols was nearly 90%. After applying protocol-specific w-score standardization, the differences between the protocols substantially decreased. Most importantly, protocol-specific w-score standardization reduced both univariate and multivariate differences in the images while maintaining the AD disease effect. Compared to conventional regression methods, our method showed the best performance for in terms of controlling the protocol effect while preserving disease information. Protocol-specific w-score standardization effectively resolved the concerns of conventional regression methods. It showed the best performance for improving the compatibility of a T1 MR post-processed feature, cortical thickness. Copyright © 2017 Elsevier Inc. All rights reserved.

Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification.

PubMed Central

Coleman, S S; Melanson, D M; Biosca, E G; Oliver, J D

1996-01-01

DNA extraction procedures and PCR conditions to detect Vibrio vulnificus cells naturally occurring in oysters were developed. In addition, PCR amplification of V. vulnificus from oysters seeded with biotype 1 cells was demonstrated. By the methods described, V. vulnificus cells on a medium (colistin-polymyxin B-cellobiose agar) selective for this pathogen were detectable in oysters harvested in January and March, containing no culturable cells (< 67 CFU/g), as well as in oysters harvested in May and June, containing culturable cells. It was possible to complete DNA extraction, PCR, and gel electrophoresis within 10 h by using the protocol described for oysters. V. vulnificus biotype 2 cells were also detected in eel tissues that had been infected with this strain and subsequently preserved in formalin. The protocol used for detection of V. vulnificus cells in eels required less than 5 h to complete. Optimum MgCl2 concentrations for the PCR of V. vulnificus from oysters and eels were different, although the same primer pair was used for both. This is the first report on the detection of cells of V. vulnificus naturally present in shellfish and represents a potentially powerful method for monitoring this important human and eel pathogen. PMID:8919800

Analysis of simultaneous MEG and intracranial LFP recordings during Deep Brain Stimulation: a protocol and experimental validation

PubMed Central

Oswal, Ashwini; Jha, Ashwani; Neal, Spencer; Reid, Alphonso; Bradbury, David; Aston, Peter; Limousin, Patricia; Foltynie, Tom; Zrinzo, Ludvic; Brown, Peter; Litvak, Vladimir

2016-01-01

Background Deep Brain Stimulation (DBS) is an effective treatment for several neurological and psychiatric disorders. In order to gain insights into the therapeutic mechanisms of DBS and to advance future therapies a better understanding of the effects of DBS on large-scale brain networks is required. New method In this paper, we describe an experimental protocol and analysis pipeline for simultaneously performing DBS and intracranial local field potential (LFP) recordings at a target brain region during concurrent magnetoencephalography (MEG) measurement. Firstly we describe a phantom setup that allowed us to precisely characterise the MEG artefacts that occurred during DBS at clinical settings. Results Using the phantom recordings we demonstrate that with MEG beamforming it is possible to recover oscillatory activity synchronised to a reference channel, despite the presence of high amplitude artefacts evoked by DBS. Finally, we highlight the applicability of these methods by illustrating in a single patient with Parkinson's disease (PD), that changes in cortical-subthalamic nucleus coupling can be induced by DBS. Comparison with existing approaches To our knowledge this paper provides the first technical description of a recording and analysis pipeline for combining simultaneous cortical recordings using MEG, with intracranial LFP recordings of a target brain nucleus during DBS. PMID:26698227

Implementation of AICAR analysis by GC-C-IRMS for anti-doping purposes.

PubMed

Buisson, C; Frelat, C; Mongongu, C; Martinat, N; Audran, M

2017-11-01

AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), is a naturally occurring substance which is part to the World Anti-Doping Agency (WADA) Prohibited List. It is claimed to improve physical performance when administered as a supplement. As for other endogenous compounds such as steroids, the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis remains an efficient tool to differentiate endogenous substances from exogenous ones. A protocol was described in the literature for the analysis of AICAR by GC-C-IRMS. The aim of the present study was to implement this protocol in our laboratory and to propose solutions to avoid the difficulties encountered. The first point discussed in this study is the derivatization step. Due to the structure of the AICAR molecule, conventional derivatization for GC-C-IRMS such as acetylation could not be applied and silylation was preferred. The improvement of the derivatives stability was achieved thanks to several derivatization conditions tested. This adjustment led to a reproducible derivatization pattern with the 3-TMS form as major derivative product. The second point discussed in this study is the diminution of extracts' background noise. Indeed, the implementation of the published protocol was not easy due to high performance liquid chromatography (HPLC) problems encountered when concentrated urine was injected into our system. Also, too many interferences in the endogenous reference compound fractions were observed. The addition of both a wash step before the HPLC purification and a HPLC purification step for the endogenous reference compound (ERC) fraction allowed us to increase the robustness of the method. This study presents the modified protocol compared to the original protocol as well as the evaluation of the whole method performances. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

A step-by-step protocol for assaying protein carbonylation in biological samples.

PubMed

Colombo, Graziano; Clerici, Marco; Garavaglia, Maria Elisa; Giustarini, Daniela; Rossi, Ranieri; Milzani, Aldo; Dalle-Donne, Isabella

2016-04-15

Protein carbonylation represents the most frequent and usually irreversible oxidative modification affecting proteins. This modification is chemically stable and this feature is particularly important for storage and detection of carbonylated proteins. Many biochemical and analytical methods have been developed during the last thirty years to assay protein carbonylation. The most successful method consists on protein carbonyl (PCO) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and consequent spectrophotometric assay. This assay allows a global quantification of PCO content due to the ability of DNPH to react with carbonyl giving rise to an adduct able to absorb at 366 nm. Similar approaches were also developed employing chromatographic separation, in particular HPLC, and parallel detection of absorbing adducts. Subsequently, immunological techniques, such as Western immunoblot or ELISA, have been developed leading to an increase of sensitivity in protein carbonylation detection. Currently, they are widely employed to evaluate change in total protein carbonylation and eventually to highlight the specific proteins undergoing selective oxidation. In the last decade, many mass spectrometry (MS) approaches have been developed for the identification of the carbonylated proteins and the relative amino acid residues modified to carbonyl derivatives. Although these MS methods are much more focused and detailed due to their ability to identify the amino acid residues undergoing carbonylation, they still require too expensive equipments and, therefore, are limited in distribution. In this protocol paper, we summarise and comment on the most diffuse protocols that a standard laboratory can employ to assess protein carbonylation; in particular, we describe step-by-step the different protocols, adding suggestions coming from our on-bench experience. Copyright © 2015 Elsevier B.V. All rights reserved.

Geochemical and mineralogical data for soils of the conterminous United States

USGS Publications Warehouse

Smith, David B.; Cannon, William F.; Woodruff, Laurel G.; Solano, Federico; Kilburn, James E.; Fey, David L.

2013-01-01

In 2007, the U.S. Geological Survey initiated a low-density (1 site per 1,600 square kilometers, 4,857 sites) geochemical and mineralogical survey of soils of the conterminous United States as part of the North American Soil Geochemical Landscapes Project. Sampling and analytical protocols were developed at a workshop in 2003, and pilot studies were conducted from 2004 to 2007 to test and refine these recommended protocols. The final sampling protocol for the national-scale survey included, at each site, a sample from a depth of 0 to 5 centimeters, a composite of the soil A horizon, and a deeper sample from the soil C horizon or, if the top of the C horizon was at a depth greater than 1 meter, from a depth of approximately 80–100 centimeters. The <2-millimeter fraction of each sample was analyzed for a suite of 45 major and trace elements by methods that yield the total or near-total elemental content. The major mineralogical components in the samples from the soil A and C horizons were determined by a quantitative X-ray diffraction method using Rietveld refinement. Sampling in the conterminous United States was completed in 2010, with chemical and mineralogical analyses completed in May 2013. The resulting dataset provides an estimate of the abundance and spatial distribution of chemical elements and minerals in soils of the conterminous United States and represents a baseline for soil geochemistry and mineralogy against which future changes may be recognized and quantified. This report (1) describes the sampling, sample preparation, and analytical methods used; (2) gives details of the quality control protocols used to monitor the quality of chemical and mineralogical analyses over approximately six years; and (3) makes available the soil geochemical and mineralogical data in downloadable tables.

Generation of Oligodendrogenic Spinal Neural Progenitor Cells From Human Induced Pluripotent Stem Cells.

PubMed

Khazaei, Mohamad; Ahuja, Christopher S; Fehlings, Michael G

2017-08-14

This unit describes protocols for the efficient generation of oligodendrogenic neural progenitor cells (o-NPCs) from human induced pluripotent stem cells (hiPSCs). Specifically, detailed methods are provided for the maintenance and differentiation of hiPSCs, human induced pluripotent stem cell-derived neural progenitor cells (hiPS-NPCs), and human induced pluripotent stem cell-oligodendrogenic neural progenitor cells (hiPSC-o-NPCs) with the final products being suitable for in vitro experimentation or in vivo transplantation. Throughout, cell exposure to growth factors and patterning morphogens has been optimized for both concentration and timing, based on the literature and empirical experience, resulting in a robust and highly efficient protocol. Using this derivation procedure, it is possible to obtain millions of oligodendrogenic-NPCs within 40 days of initial cell plating which is substantially shorter than other protocols for similar cell types. This protocol has also been optimized to use translationally relevant human iPSCs as the parent cell line. The resultant cells have been extensively characterized both in vitro and in vivo and express key markers of an oligodendrogenic lineage. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

Bank robberies: A psychological protocol of intervention in financial institutions and principal effects.

PubMed

Tabanelli, Maria Carla; Bonfiglioli, Roberta; Violante, Francesco S

2013-03-26

BACKGROUND: Robbery in workplaces represents a potentially traumatic experience for workers. OBJECTIVES: This article describes the set up and evaluation of a comprehensive psychological intervention designed to help to reduce the adverse consequences of bank robberies. PARTICIPANTS: The study population was selected among the employees of two Italian banks.METHODS: The psychological protocol was designed according to the results of a comprehensive non-systematic review of the scientific literature and it was evaluated at work site. RESULTS: The protocol consists of a "pre-event" formative intervention and "post-event" psychological support. The qualitative data collected allowed us to understand that the reactions after a robbery can differ depending on the phase during which the workers were exposed to the robbery. We noted that the main consequences can be classified in emotional/sentimental reactions, behavioral reactions, physiological reactions and experiences during the event; emotions/feelings following the robbery and psycho/physical state and emotions/feelings in the following days.CONCLUSIONS: In a working environment, the chance to take advantage of a specific protocol for the traumatic event of a bank robbery offers both the company and the workers important tools for well-being, including post-robbery psychological support and classroom instructions.

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...

Applying the WHO recommendations on health-sector response to violence against women to assess the Spanish health system. A mixed methods approach.

PubMed

Goicolea, Isabel; Vives-Cases, Carmen; Minvielle, Fauhn; Briones-Vozmediano, Erica; Ohman, Ann

2014-01-01

This methodological note describes the development and application of a mixed-methods protocol to assess the responsiveness of Spanish health systems to violence against women in Spain, based on the World Health Organization (WHO) recommendations. Five areas for exploration were identified based on the WHO recommendations: policy environment, protocols, training, accountability/monitoring, and prevention/promotion. Two data collection instruments were developed to assess the situation of 17 Spanish regional health systems (RHS) with respect to these areas: 1) a set of indicators to guide a systematic review of secondary sources, and 2) an interview guide to be used with 26 key informants at the regional and national levels. We found differences between RHSs in the five areas assessed. The progress of RHSs on the WHO recommendations was notable at the level of policies, moderate in terms of health service delivery, and very limited in terms of preventive actions. Using a mixed-methods approach was useful for triangulation and complementarity during instrument design, data collection and interpretation. Copyright © 2013 SESPAS. Published by Elsevier Espana. All rights reserved.

Sizing Single Cantilever Beam Specimens for Characterizing Facesheet/Core Peel Debonding in Sandwich Structure

NASA Technical Reports Server (NTRS)

Ratcliffe, James G.

2010-01-01

This technical publication details part of an effort focused on the development of a standardized facesheet/core peel debonding test procedure. The purpose of the test is to characterize facesheet/core peel in sandwich structure, accomplished through the measurement of the critical strain energy release rate associated with the debonding process. Following an examination of previously developed tests and a recent evaluation of a selection of these methods, a single cantilever beam (SCB) specimen was identified as being a promising candidate for establishing such a standardized test procedure. The objective of the work described here was to begin development of a protocol for conducting a SCB test that will render the procedure suitable for standardization. To this end, a sizing methodology was developed to ensure appropriate SCB specimen dimensions are selected for a given sandwich system. Application of this method to actual sandwich systems yielded SCB specimen dimensions that would be practical for use. This study resulted in the development of a practical SCB specimen sizing method, which should be well-suited for incorporation into a standardized testing protocol.

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

PubMed Central

Blackburn, Jessica S; Liu, Sali; Raimondi, Aubrey R; Ignatius, Myron S; Salthouse, Christopher D; Langenau, David M

2011-01-01

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis. PMID:21293462

Quality-assurance procedures: Method 5G determination of particulate emissions from wood heaters from a dilution tunnel sampling location

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

Ward, T.E.; Hartman, M.W.; Olin, R.C.

1989-06-01

Quality-assurance procedures are contained in this comprehensive document intended to be used as an aid for wood-heater manufacturers and testing laboratories in performing particulate matter sampling of wood heaters according to EPA protocol, Method 5G. These procedures may be used in research and development, and as an aid in auditing and certification testing. A detailed, step-by-step quality assurance guide is provided to aid in the procurement and assembly of testing apparatus, to clearly describe the procedures, and to facilitate data collection and reporting. Suggested data sheets are supplied that can be used as an aid for both recordkeeping and certificationmore » applications. Throughout the document, activity matrices are provided to serve as a summary reference. Checklists are also supplied that can be used by testing personnel. Finally, for the purposes of ensuring data quality, procedures are outlined for apparatus operation, maintenance, and traceability. These procedures combined with the detailed description of the sampling and analysis protocol will help ensure the accuracy and reliability of Method 5G emission-testing results.« less

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis.

PubMed

Salinas-Jazmín, Nohemi; González-González, Edith; Vásquez-Bochm, Luz X; Pérez-Tapia, Sonia M; Velasco-Velázquez, Marco A

2017-05-04

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

Assessing the detection, reporting and investigation of adverse events in clinical trial protocols implemented in Cameroon: a documentary review of clinical trial protocols.

PubMed

Ebile, Akoh Walter; Ateudjieu, Jerome; Yakum, Martin Ndinakie; Djuidje, Marceline Ngounoue; Watcho, Pierre

2015-09-29

International guidelines recommend ethical and scientific quality standards for managing and reporting adverse events occurring during clinical trials to competent research ethics committees and regulatory authorities. The purpose of this study was to determine whether clinical trial protocols in Cameroon are developed in line with national requirements and international guidelines as far as detecting, reporting and investigating of adverse events is concerned. It was a documentary review of all approved clinical trial protocols that were submitted at the Cameroon National Ethics Committee for evaluation from 1997 through 2012. Data were extracted using a preconceived and validated grid. Protocol review process targeted the title, abstract, objectives, methodology, resources, and the chapter on safety. In total, 106 (4.9 %) clinical trial protocols were identified from 2173 protocols seen in the archive and 104 (4.8 %) included for review. Seventy six (73.1 %) trials did not include the surveillance of adverse events as part of their objective. A total of 91 (87.5 %) protocols did not budget for adverse event surveillance, 76 (73.1 %) did not have a data safety management board (DSMB), 11(10.6 %) included insurance for participants, 47 (45.2 %) did not include a case definition for serious adverse events, 33 (31.7 %) described procedures to detect adverse events, 33 (31.7 %) described procedure for reporting and 22 (21.2 %) described procedure for investigating adverse events. Most clinical trial protocols in Cameroon are developed to focus on benefits and pay little attention to harms. The development of national guidelines can improve the surveillance of adverse events in clinical trial research conducted in Cameroon. Adverse events surveillance tools and a budget are critical for an adequate planning for adverse event surveillance when developing trial protocols. Clinical trial protocols submitted in the Cameroon National Ethics Committee do not adequately plan to assess adverse events in clinical trial protocols. In order to improve on the safety of participants and marketed drug, there is a need to develop national guidelines for clinical trials by the government, and to improve evaluation procedures and monitoring of ongoing trials by the ethics committee.

Landbird Monitoring Protocol for National Parks in the North Coast and Cascades Network

USGS Publications Warehouse

Siegel, Rodney B.; Wilkerson, Robert L.; Jenkins, Kurt J.; Kuntz, Robert C.; Boetsch, John R.; Schaberl, James P.; Happe, Patricia J.

2007-01-01

This protocol narrative outlines the rationale, sampling design and methods for monitoring landbirds in the North Coast and Cascades Network (NCCN) during the breeding season. The NCCN, one of 32 networks of parks in the National Park System, comprises seven national park units in the Pacific Northwest, including three large, mountainous, natural area parks (Mount Rainier [MORA] and Olympic [OLYM] National Parks, North Cascades National Park Service Complex [NOCA]), and four small historic cultural parks (Ebey's Landing National Historical Reserve [EBLA], Lewis and Clark National Historical Park [LEWI], Fort Vancouver National Historical Park [FOVA], and San Juan Island National Historical Park [SAJH]). The protocol reflects decisions made by the NCCN avian monitoring group, which includes NPS representatives from each of the large parks in the Network as well as personnel from the U.S. Geological Survey Forest and Rangeland Ecosystem Science Center (USGS-FRESC) Olympic Field Station, and The Institute for Bird Populations, at meetings held between 2000 (Siegel and Kuntz, 2000) and 2005. The protocol narrative describes the monitoring program in relatively broad terms, and its structure and content adhere to the outline and recommendations developed by Oakley and others (2003) and adopted by NPS. Finer details of the methodology are addressed in a set of standard operating procedures (SOPs) that accompany the protocol narrative. We also provide appendixes containing additional supporting materials that do not clearly belong in either the protocol narrative or the standard operating procedures.

Intelligent routing protocol for ad hoc wireless network

NASA Astrophysics Data System (ADS)

Peng, Chaorong; Chen, Chang Wen

2006-05-01

A novel routing scheme for mobile ad hoc networks (MANETs), which combines hybrid and multi-inter-routing path properties with a distributed topology discovery route mechanism using control agents is proposed in this paper. In recent years, a variety of hybrid routing protocols for Mobile Ad hoc wireless networks (MANETs) have been developed. Which is proactively maintains routing information for a local neighborhood, while reactively acquiring routes to destinations beyond the global. The hybrid protocol reduces routing discovery latency and the end-to-end delay by providing high connectivity without requiring much of the scarce network capacity. On the other side the hybrid routing protocols in MANETs likes Zone Routing Protocol still need route "re-discover" time when a route between zones link break. Sine the topology update information needs to be broadcast routing request on local zone. Due to this delay, the routing protocol may not be applicable for real-time data and multimedia communication. We utilize the advantages of a clustering organization and multi-routing path in routing protocol to achieve several goals at the same time. Firstly, IRP efficiently saves network bandwidth and reduces route reconstruction time when a routing path fails. The IRP protocol does not require global periodic routing advertisements, local control agents will automatically monitor and repair broke links. Secondly, it efficiently reduces congestion and traffic "bottlenecks" for ClusterHeads in clustering network. Thirdly, it reduces significant overheads associated with maintaining clusters. Fourthly, it improves clusters stability due to dynamic topology changing frequently. In this paper, we present the Intelligent Routing Protocol. First, we discuss the problem of routing in ad hoc networks and the motivation of IRP. We describe the hierarchical architecture of IRP. We describe the routing process and illustrate it with an example. Further, we describe the control manage mechanisms, which are used to control active route and reduce the traffic amount in the route discovery procedure. Finial, the numerical experiments are given to show the effectiveness of IRP routing protocol.

MTP: An atomic multicast transport protocol

NASA Technical Reports Server (NTRS)

Freier, Alan O.; Marzullo, Keith

1990-01-01

Multicast transport protocol (MTP); a reliable transport protocol that utilizes the multicast strategy of applicable lower layer network architectures is described. In addition to transporting data reliably and efficiently, MTP provides the client synchronization necessary for agreement on the receipt of data and the joining of the group of communicants.

Ex vitro composite plants: an inexpensive, rapid method for root biology.

PubMed

Collier, Ray; Fuchs, Beth; Walter, Nathalie; Kevin Lutke, William; Taylor, Christopher G

2005-08-01

Plant transformation technology is frequently the rate-limiting step in gene function analysis in non-model plants. An important tool for root biologists is the Agrobacterium rhizogenes-derived composite plant, which has made possible genetic analyses in a wide variety of transformation recalcitrant dicotyledonous plants. The novel, rapid and inexpensive ex vitro method for producing composite plants described in this report represents a significant advance over existing composite plant induction protocols, which rely on expensive and time-consuming in vitro conditions. The utility of the new system is validated by expression and RNAi silencing of GFP in transgenic roots of composite plants, and is bolstered further by experimental disruption, via RNAi silencing, of endogenous plant resistance to the plant parasitic nematode Meloidogyne incognita in transgenic roots of Lycopersicon esculentum cv. Motelle composite plants. Critical parameters of the method are described and discussed herein.

Microfluidic "Pouch" Chips for Immunoassays and Nucleic Acid Amplification Tests.

PubMed

Mauk, Michael G; Liu, Changchun; Qiu, Xianbo; Chen, Dafeng; Song, Jinzhao; Bau, Haim H

2017-01-01

Microfluidic cassettes ("chips") for processing and analysis of clinical specimens and other sample types facilitate point-of-care (POC) immunoassays and nucleic acid based amplification tests. These single-use test chips can be self-contained and made amenable to autonomous operation-reducing or eliminating supporting instrumentation-by incorporating laminated, pliable "pouch" and membrane structures for fluid storage, pumping, mixing, and flow control. Materials and methods for integrating flexible pouch compartments and diaphragm valves into hard plastic (e.g., acrylic and polycarbonate) microfluidic "chips" for reagent storage, fluid actuation, and flow control are described. We review several versions of these pouch chips for immunoassay and nucleic acid amplification tests, and describe related fabrication techniques. These protocols thus offer a "toolbox" of methods for storage, pumping, and flow control functions in microfluidic devices.

Analysis of the Proteome of Hair-Cell Stereocilia by Mass Spectrometry

PubMed Central

Krey, Jocelyn F.; Wilmarth, Philip A.; David, Larry L.; Barr-Gillespie, Peter G.

2017-01-01

Characterization of proteins that mediate mechanotransduction by hair cells, the sensory cells of the inner ear, is hampered by the scarcity of these cells and their sensory organelle, the hair bundle. Mass spectrometry, with its high sensitivity and identification precision, is the ideal method for determining which proteins are present in bundles and what proteins they interact with. We describe here the isolation of mouse hair bundles, as well as preparation of bundle-protein samples for mass spectrometry. We also describe protocols for data-dependent (shotgun) and parallel-reaction-monitoring (targeted) mass spectrometry that allow us to identify and quantify proteins of the hair bundle. These sensitive methods are particularly useful for comparing proteomes of wild-type and mice with deafness mutations affecting hair-bundle proteins. (120 words; maximum 250) PMID:28109437

Constructing Tissue Microarrays: Protocols and Methods Considering Potential Advantages and Disadvantages for Downstream Use.

PubMed

Bingle, Lynne; Fonseca, Felipe P; Farthing, Paula M

2017-01-01

Tissue microarrays were first constructed in the 1980s but were used by only a limited number of researchers for a considerable period of time. In the last 10 years there has been a dramatic increase in the number of publications describing the successful use of tissue microarrays in studies aimed at discovering and validating biomarkers. This, along with the increased availability of both manual and automated microarray builders on the market, has encouraged even greater use of this novel and powerful tool. This chapter describes the basic techniques required to build a tissue microarray using a manual method in order that the theory behind the practical steps can be fully explained. Guidance is given to ensure potential disadvantages of the technique are fully considered.

The infiltration-centrifugation technique for extraction of apoplastic fluid from plant leaves using Phaseolus vulgaris as an example.

PubMed

O'Leary, Brendan M; Rico, Arantza; McCraw, Sarah; Fones, Helen N; Preston, Gail M

2014-12-19

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

PubMed Central

O'Leary, Brendan M.; Rico, Arantza; McCraw, Sarah; Fones, Helen N.; Preston, Gail M.

2014-01-01

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition. PMID:25549068

Wear measurement of dental tissues and materials in clinical studies: A systematic review.

PubMed

Wulfman, C; Koenig, V; Mainjot, A K

2018-06-01

This study aims to systematically review the different methods used for wear measurement of dental tissues and materials in clinical studies, their relevance and reliability in terms of accuracy and precision, and the performance of the different steps of the workflow taken independently. An exhaustive search of clinical studies related to wear of dental tissues and materials reporting a quantitative measurement method was conducted. MedLine, Embase, Scopus, Cochrane Library and Web of Science databases were used. Prospective studies, pilot studies and case series (>10 patients), as long as they contained a description of wear measurement methodology. Only studies published after 1995 were considered. After duplicates' removal, 495 studies were identified, and 41 remained for quantitative analysis. Thirty-four described wear-measurement protocols, using digital profilometry and superimposition, whereas 7 used alternative protocols. A specific form was designed to analyze the risk of bias. The methods were described in terms of material analyzed; study design; device used for surface acquisition; matching software details and settings; type of analysis (vertical height-loss measurement vs volume loss measurement); type of area investigated (entire occlusal area or selective areas); and results. There is a need of standardization of clinical wear measurement. Current methods exhibit accuracy, which is not sufficient to monitor wear of restorative materials and tooth tissues. Their performance could be improved, notably limiting the use of replicas, using standardized calibration procedures and positive controls, optimizing the settings of scanners and matching softwares, and taking into account unusable data. Copyright © 2018 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.

Chapter 11: Sample Design Cross-Cutting Protocol. The Uniform Methods Project: Methods for Determining Energy Efficiency Savings for Specific Measures

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

Kurnik, Charles W; Khawaja, M. Sami; Rushton, Josh

Evaluating an energy efficiency program requires assessing the total energy and demand saved through all of the energy efficiency measures provided by the program. For large programs, the direct assessment of savings for each participant would be cost-prohibitive. Even if a program is small enough that a full census could be managed, such an undertaking would almost always be an inefficient use of evaluation resources. The bulk of this chapter describes methods for minimizing and quantifying sampling error. Measurement error and regression error are discussed in various contexts in other chapters.

Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors.

PubMed

Thakore, Pratiksha I; Gersbach, Charles A

2016-01-01

Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.

An Acupuncture Research Protocol Developed from Historical Writings by Mathematical Reflections: A Rational Individualized Acupoint Selection Method for Immediate Pain Relief

PubMed Central

Schroeder, Sven; Meyer-Hamme, Gesa; Zhang, Jianwei; Epplée, Susanne; Friedemann, Thomas; Hu, Weiguo

2013-01-01

While balancing yin and yang is one basic principle of Chinese medicine, balancing methods for combination of meridians and acupoints had been described throughout the history of Chinese medicine. We have identified six historical systems for combinations of acupuncture points in historical writings. All of them represent symmetrical combinations which are defined by the steps in the Chinese Clock. Taking the historical systems as a basis, we calculated the possible combinations that fit into these systems they revealed, leading to a total of 19 systems offering new balancing combinations. Merging the data of these 19 systems, there are 7 combinatorial options for every meridian. On the basis of this data, we calculated 4-meridian combinations with an ideal balance pattern, which is given when all meridians balance each other. We identified 5 of these patterns for every meridian, so we end up with 60 patterns for all the 12 meridians but we find multiple overlapping. Finally, 15 distinct patterns remain. By combining this theoretical concept with the Image and Mirror Concept, we developed an acupuncture research protocol. This protocol potentially solves some problems of acupuncture trials because it represents a rational reproducible procedure independent of examiner experience, but the resulting treatment is individualized. PMID:23431334

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation.

PubMed

D'Aiuto, Leonardo; Zhi, Yun; Kumar Das, Dhanjit; Wilcox, Madeleine R; Johnson, Jon W; McClain, Lora; MacDonald, Matthew L; Di Maio, Roberto; Schurdak, Mark E; Piazza, Paolo; Viggiano, Luigi; Sweet, Robert; Kinchington, Paul R; Bhattacharjee, Ayantika G; Yolken, Robert; Nimgaonka, Vishwajit L; Nimgaonkar, Vishwajit L

2014-01-01

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.

High throughput functional assays for P2X receptors.

PubMed

Namovic, Marian T; Jarvis, Michael F; Donnelly-Roberts, Diana

2012-06-01

Adenosine triphosphate (ATP) activates two receptor superfamilies, metabotropic P2Y and ionotropic P2X receptors. The P2X receptors are nonselective cation channels that are widely expressed on excitable cells including neurons, glia, and smooth muscle cells. The protocols in this unit are useful for evaluating ligands that interact with P2X receptors on native cells or that are cloned and expressed in recombinant heterologous cell systems. Calcium imaging methods are described for the pharmacological characterization of fast or slowly desensitizing P2X receptors. While these methods are readily applicable to a wide variety of ligand-gated ion channels, the protocols provided herein detail how they can be used to measure activation of homomeric P2X3 (fast desensitizing) and heteromeric P2X2/3 (slowly desensitizing) receptors. Appropriate agonists and/or calcium dyes can be substituted to assess activity at other P2X receptor subtypes. An additional protocol is provided for measuring P2X7 receptor-mediated pore formation in THP-1, a native human acute monocytic leukemia cell line that can be used to study homomeric P2X7 (non-desensitizing) receptors that are expressed on macrophages and microglial cells. © 2012 by John Wiley & Sons, Inc.

A Streamlined Protocol for Molecular Testing of the DMD Gene within a Diagnostic Laboratory: A Combination of Array Comparative Genomic Hybridization and Bidirectional Sequence Analysis

PubMed Central

Marquis-Nicholson, Renate; Lai, Daniel; Love, Jennifer M.; Love, Donald R.

2013-01-01

Purpose. The aim of this study was to develop a streamlined mutation screening protocol for the DMD gene in order to confirm a clinical diagnosis of Duchenne or Becker muscular dystrophy in affected males and to clarify the carrier status of female family members. Methods. Sequence analysis and array comparative genomic hybridization (aCGH) were used to identify mutations in the dystrophin DMD gene. We analysed genomic DNA from six individuals with a range of previously characterised mutations and from eight individuals who had not previously undergone any form of molecular analysis. Results. We successfully identified the known mutations in all six patients. A molecular diagnosis was also made in three of the four patients with a clinical diagnosis who had not undergone prior genetic screening, and testing for familial mutations was successfully completed for the remaining four patients. Conclusion. The mutation screening protocol described here meets best practice guidelines for molecular testing of the DMD gene in a diagnostic laboratory. The aCGH method is a superior alternative to more conventional assays such as multiplex ligation-dependent probe amplification (MLPA). The combination of aCGH and sequence analysis will detect mutations in 98% of patients with the Duchenne or Becker muscular dystrophy. PMID:23476807

RNA interference by feeding in vitro synthesized double-stranded RNA to planarians: methodology and dynamics

PubMed Central

Rouhana, Labib; Weiss, Jennifer A.; Forsthoefel, David J.; Lee, Hayoung; King, Ryan S.; Inoue, Takeshi; Shibata, Norito; Agata, Kiyokazu; Newmark, Phillip A.

2013-01-01

Background The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced via injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. Results We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time, and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. Conclusions This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. PMID:23441014