bluuebunny/crossref_metadata_2025_split huggingface dataset:
Imaging Action Potentials with Calcium Indicators: Figure 1.
Authors: MacLean Jason N. , Yuste Rafael 
Published: November 2009

INTRODUCTIONThe understanding of neuronal circuits has been, and will continue to be, greatly advanced by the simultaneous imaging of action potentials in neuronal ensembles. This protocol describes “bulk” loading of brain slices with acetoxymethyl (AM) ester calcium indicators in order to monitor action potential activity in large populations of neurons simultaneously. The imaging of calcium influx into neurons provides an indirect, but accurate, measure of action potential generation in individual neurons. Single-cell resolution, and thus the easy identification of every active cell, is the key advantage of the technique.
Indirect Immunofluorescence Labeling in the Yeast Saccharomyces cerevisiae
Authors: Silver Pamela 
Published: November 2009

INTRODUCTIONThe budding yeast Saccharomyces cerevisiae is an ideal model system for the initial characterization of novel genes and their corresponding gene products. Genetic analysis is straightforward, and sophisticated cell biological methods are available. Furthermore, only in a genetically tractable organism is it possible to use epitope-tagged proteins to the best advantage. In S. cerevisiae, it is a trivial matter to determine whether a modified version of a protein is functional. This is accomplished by testing whether the tagged protein can complement a null phenotype or rescue a conditional phenotype. Methods such as indirect immunofluorescence and the use of green fluorescent protein (GFP) allow the investigator to correlate the intracellular localization of the protein with its function in vivo. This article includes a detailed protocol for performing indirect immunofluorescence with S. cerevisiae. Cells are grown exponentially and then fixed. After fixation, an enzyme is used to remove the cell wall, and the cells are adhered to slides. Subsequent steps involve the application of primary and secondary antibodies and the final processing of the slide.
Bimolecular Affinity Purification (BAP): Tandem Affinity Purification Using Two Protein Baits: Figure 1.
Authors: Maine Gabriel N. , Gluck Nathan , Zaidi Iram W. , Burstein Ezra 
Published: November 2009

INTRODUCTIONThe tandem affinity purification (TAP) procedure was pioneered in yeast for the purpose of purifying and characterizing protein complexes. While affinity purification is relatively easy to perform, nonspecific protein interactions can plague the identification of true interacting partners of the given bait utilized in the purification. To alleviate this problem, two sequential affinity purification steps are employed in the TAP procedure. Since its inception in yeast, TAP has gone through many adaptations and has been employed multiple times in diverse organisms, including mammalian systems. In all these approaches, two out of many possible affinity moieties are employed and are usually expressed as a fusion polypeptide in the amino or carboxyl-terminal region of the protein bait. In this protocol, we describe a variation on the TAP procedure in which the affinity moieties are placed on two different proteins of a molecular complex to isolate or detect components present in the complex. This variation, which we refer to as bimolecular affinity purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known components.
Preparation of Genomic DNA from Hawaiian Bobtail Squid (Euprymna scolopes) Tissue by Cesium Chloride Gradient Centrifugation
Authors: Lee Patricia N. , McFall-Ngai Margaret J. , Callaerts Patrick , de Couet H. Gert 
Published: November 2009

INTRODUCTIONThis procedure describes the extraction of genomic DNA from adult bobtail squid (Euprymna scolopes) tissues by cesium chloride (CsCl) gradient centrifugation. There are numerous generic methods and commercial kits for the preparation of genomic DNA based on proteolytic digestion of chromatin components, followed by selective binding of nucleic acids to ion-exchange affinity media, but many of these do not yield DNA that can be readily restricted. Also, molluscan tissues contain mucopolysaccharides, which tend to copurify with DNA under certain conditions. Although nucleic acids prepared this way can serve as a template for polymerase chain reaction (PCR), other enzymatic modifications of nucleic acids are inhibited by these contaminants. The method described here yields high-molecular-weight DNA that can be readily restricted for Southern hybridization. The procedure uses brain tissue under the assumption that its genome is unlikely to be rearranged in any way, has a high nucleic acid:protein ratio, and avoids potential sources of enzymatic contaminants and parasites from the intestinal sac. However, the method can be applied to other tissue sources and works well with other species. The purification of DNA by gradient centrifugation is an established method based on the specific buoyant density of double-stranded nucleic acids and the ability of CsCl solutions to form a salt gradient in a centrifugal field. It can also be adapted to the purification of RNA, which has a higher buoyant density than DNA. Unfortunately, this method is somewhat involved and expensive and produces large amounts of ethidium bromide waste.
Whole-Mount In Situ Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos with DIG-Labeled Riboprobes: I. DNA Template Preparation and In Vitro Transcription of Riboprobes
Authors: Lee Patricia N. , McFall-Ngai Margaret J. , Callaerts Patrick , de Couet H. Gert 
Published: November 2009

INTRODUCTIONWhole-mount in situ hybridization is a technique used to localize and visualize specific gene transcripts in whole embryos by hybridizing labeled RNA probes complementary to the sequence of interest. A digoxigenin (DIG)-labeled riboprobe synthesized during in vitro transcription through the incorporation of a DIG-labeled UTP is hybridized to the target sequence under stringent conditions, and excess, unhybridized probe is removed during a series of washes. The location of the labeled riboprobe, and thus the mRNA sequence of interest, is then visualized by immunohistochemistry. This protocol outlines the techniques for preparing RNA probes for whole-mount in situ hybridization in Hawaiian bobtail squid (Euprymna scolopes) embryos from linearized plasmid DNA or polymerase chain reaction (PCR) products.
Whole-Mount In Situ Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos with DIG-Labeled Riboprobes: II. Embryo Preparation, Hybridization, Washes, and Immunohistochemistry
Authors: Lee Patricia N. , McFall-Ngai Margaret J. , Callaerts Patrick , de Couet H. Gert 
Published: November 2009

INTRODUCTIONWhole-mount in situ hybridization is a technique used to localize and visualize specific gene transcripts in whole embryos by hybridizing labeled RNA probes complementary to the sequence of interest. A digoxigenin (DIG)-labeled riboprobe synthesized during in vitro transcription through the incorporation of DIG-labeled UTP is hybridized to the target sequence under stringent conditions, and excess unhybridized probe is removed during a series of washes. The location of the labeled riboprobe, and thus the mRNA sequence of interest, is then visualized by immunohistochemistry. This protocol outlines the steps involved in preparing Hawaiian bobtail squid (Euprymna scolopes) embryos, hybridizing a DIG-labeled riboprobe in whole-mount embryos, and visualizing the labeled RNA colorimetrically using an alkaline-phosphatase-conjugated anti-DIG antibody.
Culture of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos and Observation of Normal Development
Authors: Lee Patricia N. , Callaerts Patrick , de Couet H. Gert 
Published: November 2009

INTRODUCTIONThe ability to rear Hawaiian bobtail squid (Euprymna scolopes) embryos under controlled environmental conditions is a basic and necessary tool for developmental studies. It negates the need to know when fertilization occurred, allows correlation of the phase of development with the time of development (thereby facilitating collection of embryos at specific stages), and allows comparisons between cephalopod species. Embryonic development in E. scolopes is robust over a range of temperatures, is relatively rapid (~21 d), and proceeds normally under laboratory conditions at ambient temperature (27°C-29°C). Here we present methods for maintaining E. scolopes embryos in culture from cleavage through hatching, as well as observing and recording live or freshly fixed embryos under a dissecting microscope.
Yam (Dioscorea) Husbandry: Cultivating Yams in the Field or Greenhouse
Authors: Mignouna Hodeba D. , Abang Mathew M. , Asiedu Robert , Geeta R. 
Published: November 2009

INTRODUCTIONThis protocol describes how to cultivate yams (Dioscorea) in the field or greenhouse. It refers especially to the tropical food species but it will also work for temperate species. The tropical food species of Dioscorea grow in warm, sunny climates with temperatures between 25ºC and 30ºC. Short days of 10-11 h result in tuber formation, while days longer than 12 h favor vine growth. Yams require deep, loose, textured loamy soil that is rich in organic matter. They are best planted at the beginning of the rainy season. Mulch around the planted sets protects them from excessive heat and desiccation, especially in areas with hot temperatures and dry weather. It also adds organic matter to the soil, prevents soil erosion, preserves water in the soil, and increases microbial activity in the soil. Yams do not tolerate waterlogged conditions. It is important to stake the plants to allow full exposure of their leaves to light for photosynthetic activity and to reduce disease.
Post-Flask Management of Yam (Dioscorea) Plantlets
Authors: Mignouna Hodeba D. , Abang Mathew M. , Asiedu Robert , Geeta R. 
Published: November 2009

INTRODUCTIONThis protocol describes how to harden or acclimatize tissue culture-derived yam (Dioscorea) plantlets before transplanting them in the soil. Briefly, the plantlets are first put in pots with a vermiculite and peat moss mixture, and the pots are placed in a humidity chamber, where they are sprayed and watered daily. After 2 wk, the plants are transplanted to pots or plastic bags with ordinary or sterile soil and watered daily. After another 2 wk, they are transplanted into the field.
Producing Yam (Dioscorea) Seeds through Controlled Crosses
Authors: Mignouna Hodeba D. , Abang Mathew M. , Asiedu Robert , Geeta R. 
Published: November 2009

INTRODUCTIONThis protocol describes how to produce yam (Dioscorea) seeds through controlled pollination of flowers on female plants. Pollination by flower thrips takes place naturally in normal yam-growing environments in the presence of flowering male and female genotypes, although it is not efficient. This protocol leads to the production of hybrid seeds of known parentage for genetic studies and use in breeding programs.
Nuclear Run-On Assay: Figure 1.
Authors: Smale Stephen T. 
Published: November 2009

INTRODUCTIONThe nuclear run-on assay was developed as a method for establishing that the transcription initiation rate contributes to the regulated expression of mammalian genes. The difference between monitoring gene expression by the nuclear run-on assay versus most other assays is that the nuclear run-on assay provides a measure of the frequency of transcription initiation and is largely independent of the effects of RNA stability. It can also be used to determine whether polymerase pausing or attenuation contributes to gene regulation. Briefly, the nuclear run-on assay begins with samples of cells that contain different steady-state amounts of the mRNA or protein of interest. The cells are chilled, and the plasmid membranes are permeabilized or lysed. These steps result in polymerase pausing. The nuclei are then incubated for a short time at 37°C in the presence of nucleoside triphosphates (NTPs) and radiolabeled uridine 5′-triphosphate (UTP). New transcripts are not initiated during this incubation, but the radiolabeled nucleotide becomes incorporated into transcripts that were being synthesized when the cells were first chilled and lysed. The number of nascent transcripts on the gene at the time of chilling is thought to be proportional to the frequency of transcription initiation. To determine the relative number of nascent transcripts in each sample, the radiolabeled RNA is purified and hybridized to a membrane containing immobilized DNA from the gene of interest. The amount of radioactivity that hybridizes to the membrane is approximately proportional to the number of nascent transcripts.
Assessing Human Variation Data for Signatures of Natural Selection
Authors: Bamshad Mike , Stephens J. Claiborne 
Published: November 2009

INTRODUCTIONIn this article, we highlight some of the different types of natural selection, their effects on patterns of DNA variation, and some of the statistical tests that are commonly used to detect such effects. We also explain some of the relative strengths and weaknesses of different strategies that can be used to detect signatures of natural selection at individual loci. These strategies are illustrated by their application to empirical data from gene variants that are often associated with differences in disease susceptibility. We briefly outline some of the methods proposed to scan the genome for evidence of selection. Finally, we discuss some of the problems associated with identifying signatures of selection and with making inferences about the nature of the selective process.
Laboratory Methods for High-Throughput Genotyping
Authors: Edenberg Howard J. , Liu Yunlong 
Published: November 2009

INTRODUCTIONThe genetics of complex diseases has been given a tremendous boost in recent years by the introduction of high-throughput laboratory methods that make it possible to approach larger questions in larger populations and to cover the genome more comprehensively. The ability to determine genotypes of many individuals accurately and efficiently has allowed genetic studies that cover more of the variation within individual genes, instead of focusing only on one or a few coding variants, and to do so in study samples of reasonable power. Chip-based genotyping assays, combined with knowledge of the patterns of coinheritance of markers (linkage disequilibrium [LD]), have stimulated genome-wide association studies (GWAS) of complex diseases. Recent successes of GWAS in identifying specific genes that affect risk for common diseases are dramatic illustrations of how improved technology can lead to scientific breakthroughs. A key issue in high-throughput genotyping is to choose the appropriate technology for your goals and for the stage of your experiment, being cognizant of your sample numbers and resources. This article introduces some of the commonly used methods of high-throughput single-nucleotide polymorphism (SNP) genotyping for different stages of genetic studies and briefly reviews some of the high-throughput sequencing methods just coming into use. It also mentions some recent developments in “next-generation” sequencing that will enable other kinds of studies. This article is not intended to be comprehensive, and because technology in this area is rapidly changing, our comments should be taken as a starting point for further investigation.
Laying the foundations for a new classification of Agaonidae (Hymenoptera: Chalcidoidea), a multilocus phylogenetic approach
Authors: Cruaud Astrid , Jabbour‐Zahab Roula , Genson Gwenaëlle , Cruaud Corinne , Couloux Arnaud , Kjellberg Finn , Van Noort Simon , Rasplus Jean‐Yves 
Published: November 2009

AbstractA phylogeny of the Agaonidae (Chalcidoidea) in their restricted sense, pollinators ofFicusspecies (Moraceae), is estimated using 4182 nucleotides from six genes, obtained from 101 species representing 19 of the 20 recognized genera, and four outgroups. Data analysed by parsimony and Bayesian inference methods demonstrate that Agaonidae are monophyletic and that the previous classification is not supported. Agaonidae are partitioned into four groups: (i)Tetrapus, (ii)Ceratosolen + Kradibia, (iii) someBlastophaga + Wiebesiaspecies, and (iv) all genera associated with monoecious figs and a fewBlastophagaandWiebesia. The latter group is subdivided into subgroups: (i)Pleistodontes, (ii)Blastophaga psenesand neocaledonianDolichoris, (iii) someBlastophagaandWiebesiaspecies, and (iv)Platyscapa, all afrotropical genera and all genera associated with sectionConosycea. Eleven genera were recovered as monophyletic, six were para‐ or polyphyletic, and two cannot be tested with our data set. Based on our phylogeny we propose a new classification for the Agaonidae. Two new subfamilies are proposed: Tetrapusiinae for the genusTetrapus, and Kradibiinae forCeratosolen + Kradibia.Liporrhopalumis synonymized withKradibiaand the subgenusValisiaofBlastophagais elevated to generic rank. These changes resulted in 36 new combinations. Finally, we discuss the hypothesis of co‐speciation between the pollinators and their host species by comparing the two phylogenies. © The Willi Hennig Society 2009.
Insensitive blueberries: a total‐evidence analysis of Disterigma s.l. (Ericaceae) exploring transformation costs
Authors: Pedraza‐Peñalosa Paola 
Published: November 2009

Abstract Disterigma sensu lato (s.l.) is a polyphyletic genus of neotropical blueberries that comprises 37 species found in Central and South America, mostly in Andean cloud forests and páramos. The present phylogenetic analysis, based on molecular and morphological data for 84% of the species of Disterigma s.l., addressed the monophyly of the genus and identified the putative morphological synapomorphies of the main clades. The sensitivity of the phylogenetic hypotheses to the choice of analytical parameters was addressed in detail, by varying all possible parameters and applying different treatments for indel coding. A total of 29 transformation cost matrices were investigated and all these analyses were performed in POY 4, using direct optimization. A diagnosable Disterigma sensu stricto (s.s.) clade was recovered in all analyses, despite changes in the transformation costs. Additionally, none of the analyses refuted the segregation of D. trimerum, D. ulei, D. pentandrum, D. rimbachii, and D. bracteatum from Disterigma s.s. Although measurements of congruence are used to choose among alternative analytical parameter sets, the behaviour of the RILD index (rescaled incongruence length difference) in this study suggested that it may not be a good measure of congruence and that further studies are necessary to better understand its performance. © The Willi Hennig Society 2009.
Evaluating paediatric brain injury services in NSW
Authors: Badge H. , Hancock J. , Waugh M.‐C. 
Published: November 2009

AbstractBackground  Rehabilitation professionals strive to provide high‐quality evidence‐based services for children. Developing systems to measure and monitor the benefits of our services, and health outcomes for children is complex and challenging. The Community Outcome Project aims to introduce systematic outcome measurement across the network of paediatric community‐based brain injury services within the New South Wales Brain Injury Rehabilitation Program (BIRP) to support clinical practice and service evaluation.Methods  A literature review informed the development of the evaluative framework and identified available paediatric outcome measures which may be appropriate. Extensive consultation with clinicians supported project planning and identified clinical priorities that the outcome measures needed to capture. Outcome measures were shortlisted by matching them to identified clinical priorities, and then trialled in clinical practice. Qualitative feedback regarding clinical utility and feasibility was obtained from clinical staff. The process has utilized change management strategies to ensure the success of the project and keep staff engaged.Results  The process identified the three main clinical priorities for outcome measurement – family functioning, school performance and participation. Three outcome measures were chosen for the pilot project that is currently underway. They are Family Burden of Injury Interview, Academic Competence and Evaluation Scales and Child and Adolescent Scale of Participation. Plans for analyses of outcome data within the paediatric BIRP services are discussed.Conclusions  Extensive preparation is required to optimize staff engagement in a project that systematically introduces outcome measures that are useful to clinicians, clients and service providers. Managing the change required is a key focus of the project. Benefits and costs to clinicians and services will be discussed.
Surfactant protein D inhibits mite‐induced alveolar macrophage and dendritic cell activations through TLR signalling and DC‐SIGN expression
Authors: Liu C‐F , Rivere M. , Huang H‐J , Puzo G. , Wang J‐Y 
Published: November 2009

SummaryBackground
Surfactant protein D (SP‐D), a secreted pattern recognition molecule associated with pulmonary innate immunity, has been shown to mediate the clearance of pathogens in multiple ways. However, how SP‐D interacts with alveolar macrophages (AMs) and dendritic cells (DCs) during allergen exposure remains unclear.Objective
This study was performed to characterize the immunomodulatory effects of SP‐D on mite allergen (Dermatophagoides pteronyssinus, Der p)‐induced inflammatory signalling in AMs and DCs.Methods
Murine AM, alveolar macrophage cell line derived from BALB/c mice (MH‐S cells), and human monocyte‐derived dendritic cells (MDDC) were used as model systems. The production of nitric oxide (NO) and TNF‐α, expression of surface Toll‐like receptors (TLRs), and expression of the C‐type lectin receptor known as dendritic cell (DC)‐specific ICAM‐grabbing non‐integrin (DC‐SIGN) were measured as a function of pretreatment with SP‐D and subsequent exposure to Der p. Der p‐dependent cellular activations that were modified by SP‐D in these model systems were then identified.Results
Pretreatment of MH‐S cells with SP‐D reduced Der p‐dependent production of NO, TNF‐α, and the downstream activations of IL‐1 receptor‐associated kinase, mitogen activated protein kinase (MAPK) kinase, and nuclear factor‐κB. SP‐D interacted with CD14 such that CD14 binding to Der p was inhibited and Der p‐induced signalling via TLRs was blocked. DC‐SIGN expression was suppressed by Der p in MH‐S and MDDC; this down‐regulation of DC‐SIGN expression was prevented by pretreatment with SP‐D.Conclusions
These results indicated that the inhibition of Der p‐induced activation of MH‐S and MDDC by SP‐D is mediated through suppression of the CD14/TLR signalling pathway and maintenance of DC‐SIGN expression, which may protect allergen‐induced airway inflammation. Cite this as: C‐F Liu, M. Rivere, H‐J Huang, G. Puzo and J‐Y Wang, Clinical & Experimental Allergy, 2010 (40) 111–122.