bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ranking the ecological relative status of exploited marine ecosystems
Authors: Coll Marta , Shannon Lynne J. , Yemane Dawit , Link Jason S. , Ojaveer Henn , Neira Sergio , Jouffre Didier , Labrosse Pierre , Heymans Johanna J. , Fulton Elizabeth A. , Shin Yunne-Jai 
Published: December 2009

Abstract
Coll, M., Shannon, L. J., Yemane, D., Link, J. S., Ojaveer, H., Neira, S., Jouffre, D., Labrosse, P., Heymans, J. J., Fulton, E. A., and Shin, Y-J. 2010. Ranking the ecological relative status of exploited marine ecosystems. – ICES Journal of Marine Science, 67: 769–786. A set of simple, data-based ecological indicators was used to rank exploited ecosystems regarding fishing impacts with respect to their status, trends, and ecosystem EAF attributes. Expected theoretical changes in indicators with respect to increasing fishing impacts were considered, and ecosystems were compared by examining the mean values of indicators in the most recent three years for which data were available and over time (1980–2005 and 1996–2005). Systems were classified into nine potential categories according to whether they were most, moderately, or least impacted, and whether they were becoming more or less impacted, or remaining stationary. The responses of ecological indicators to additional environmental and socio-economic explanatory factors were tested. Ecosystems ranked using short- and long-term trends and states differed because of differences in trends, underscoring the importance of analysing both states and trends in ecosystem analyses. The number of ecosystems classified as unclear or intermediately impacted has increased recently, the proportion of ecosystems classified as less strongly impacted has been maintained, but more now fall within the category more strongly impacted in terms of long-term trends and states. Ecosystem type, fisheries enforcement, primary production, sea temperature, and fishing type were important variables explaining the ecological indicators. The results reflect different changes and processes in the ecosystems, demonstrating that information on ecological, environmental, and fishery histories is crucial to interpreting indicators correctly, while disentangling the effects of fishing and of the environment.
The Tammar Wallaby,Macropus eugenii: A Model Kangaroo for the Study of Developmental and Reproductive Biology
Authors: Hickford Danielle , Frankenberg Stephen , Renfree Marilyn B. 
Published: December 2009

INTRODUCTIONThe tammar wallaby(Macropus eugenii)is the best-studied marsupial in terms of its reproduction and development. It fares well in captivity, is large enough to take serial blood samples from, and its young are accessible in the pouch for experimental manipulation. These features have made it an excellent model for both physiological and molecular research. It is a seasonal, monovular breeder, but also has a lactational control of its reproduction, as well as embryonic diapause. As in all marsupials, the composition of the milk changes throughout the lengthy lactation, and the dynamic changes that occur in the genes and secreted proteins of the mammary gland have now been characterized. Studies of the tammar have contributed to our understanding of the control of reproduction in all mammals, and more recently, have helped clarify the endocrine and molecular aspects of sexual differentiation. The completion of the tammar wallaby genome will facilitate further study of this marsupial.
The Sea Squirt Ciona intestinalis: Figure 1.
Authors: Christiaen Lionel , Wagner Eileen , Shi Weiyang , Levine Michael 
Published: December 2009

INTRODUCTIONSea squirts (Ciona intestinalis) are tunicates (or urochordates), the closest living relatives of the vertebrates. Although the adults are simple, sessile filter feeders, the embryos and larvae possess clear chordate features including a prominent notochord and dorsal, hollow neural tube. Tail-bud-stage embryos and mature swimming tadpoles are composed of approximately 1000 and 2600 cells, respectively, with complete lineage information. This cellular simplicity is coupled with a streamlined genome that has not undergone the duplications seen in vertebrates. A variety of molecular tools have been applied to understanding Ciona embryogenesis. Comparisons of the C. intestinalis genome and the related but divergent Ciona savignyi genome have facilitated the identification of conserved non-coding DNAs, including regulatory DNAs such as tissue-specific enhancers. Systematic in situ hybridization assays and gene-disruption experiments using specific morpholino antisense oligonucleotides have led to the elaboration of provisional gene regulatory networks underlying the specification of key chordate tissues, including the notochord, neural tube, and beating heart. These networks provide a foundation for understanding the mechanistic basis of more complex cell-specification processes in vertebrates, and for understanding the evolutionary origins of distinctive vertebrate characteristics such as the neural crest. Because tunicates and vertebrates are sister groups, there is every indication that the developmental mechanisms revealed in the simple Ciona model will be applicable to comparable processes in vertebrates.
In Vitro Transcription Using HeLa Cell Extracts and Primer Extension
Authors: Carey Michael F. , Peterson Craig L. , Smale Stephen T. 
Published: December 2009

INTRODUCTIONPrimer extension is a popular method for measuring transcription. Among the methods for start-site mapping in vivo, it is the one most easily adapted for in vitro transcription. Typically, DNA templates bearing activator binding sites are co-incubated with nuclear extracts (which provide the general transcription factors) and the corresponding activators to synthesize mRNA. In some cases, the activator is present in the extract (e.g., Sp1) and templates lacking or containing sites are compared. In the primer extension assay, a 32P-end-labeled DNA oligonucleotide is annealed to a complementary region in the mRNA and then extended by a reverse transcriptase to the 5′ end of the mRNA. The resulting labeled cDNA is then fractionated and detected on a polyacrylamide/urea gel. The amount of the product is a measure of the transcriptional activation by the activators. Generally, the addition of 0.1 pmol of primer to the extension reaction is sufficient because it will be in vast excess over the amount of mRNA, facilitating quantitative primer hybridization and extension.
Performing Surgery on Tammar Wallaby (Macropus eugenii) Adults
Authors: Hickford Danielle , Frankenberg Stephen , Renfree Marilyn B. 
Published: December 2009

INTRODUCTIONAdult tammar wallabies (Macropus eugenii) are very tolerant of surgery if it is conducted with care. Adult females weigh ~4‐5 kg, males ~7‐8 kg. Routine surgery performed in our laboratory and described in this protocol includes castration, laparotomy (to visualize the reproductive tract), ovariectomy, and hysterectomy (for embryo collection). All surgery is conducted under aseptic conditions. For most reproductive surgery on females, the optimum surgical approach is via the inside of the pouch. Any pouch young must be removed before surgery.
Collection, Handling, Fixation, and Processing of Tammar Wallaby (Macropus eugenii) Embryos
Authors: Hickford Danielle , Frankenberg Stephen , Renfree Marilyn B. 
Published: December 2009

INTRODUCTIONThis protocol describes methods for collecting and handling tammar wallaby (Macropus eugenii) conceptuses. Zygotes, cleavage stages, diapausing blastocysts, and unexpanded blastocysts are ~250 μm in diameter and are collected by flushing either the oviduct or uterus. Later stages are collected by opening the uterus. Late preimplantation stages must be handled carefully because their large size (>2 cm in diameter) results in high surface tension within the yolk sac, especially on the delicate avascular area. When handled too roughly, the embryonic vesicle tends to split and then rapidly collapse; such embryos do not reinflate. If embryos are to be cultured, all steps during collection must be carried out aseptically. In all cases, solutions should be prewarmed to 37°C and embryos should be kept warm by use of a heating stage. Standard fixatives, fixation times, and washing protocols used for mouse embryos are also used for tammar embryos. Methods for embedding tammar conceptuses in paraffin wax depend on the size and stage of the conceptus.
Culturing Tammar Wallaby (Macropus eugenii) Pouch Young Gonads
Authors: Hickford Danielle , Frankenberg Stephen , Renfree Marilyn B. 
Published: December 2009

INTRODUCTIONTammar wallaby (Macropus eugenii) pouch young (PY) gonads are ideal for studies on gonadal differentiation and germ cell proliferation because, in contrast to the mouse, both of these events occur postnatally in the tammar. In the male, testicular cords start developing at day 2 postpartum (pp), whereas in the female the ovarian cortex and medulla differentiate from ~day 8 pp. In females, meiosis starts from day 25 pp and is complete in most germ cells by day 50 pp. The gonads can be cultured either in grooves within agar or flat on a membrane. The former arrangement maintains the close contact of the gonad and mesonephros better than culturing them flat on a membrane. This protocol provides methods for isolation and culturing of PY gonads.
Culturing Tammar Wallaby(Macropus eugenii)Peri-gastrulation Stage Embryos
Authors: Hickford Danielle , Frankenberg Stephen , Renfree Marilyn B. 
Published: December 2009

INTRODUCTIONTammar wallaby(Macropus eugenii)peri-gastrulation stage embryos can be cultured in complete medium in 95% oxygen for up to 3 d. There is little apparent difference in terms of development between tammar peri-gastrulation stage embryos cultured in DMEM versus Medium 199 with Earle’s salts, or between the supplementation of complete medium with either 10% fetal calf serum (FCS) or 10% wallaby serum (WS). The peri-gastrulation marsupial conceptus is hollow, which means that some techniques, such as grafting, cannot be performed on embryos to be cultured whole because, once punctured, the embryo collapses and does not reinflate. This protocol describes both whole embryo culture and an alternative method of culture. In the latter method, the embryonic disc and some adjoining vascular yolk sac are isolated, laid flat on a membrane, and cultured as a flat mount. Embryos cultured in this way develop to a similar stage as those cultured whole.
Whole-Mount Immunohistochemical Staining of Tammar Wallaby (Macropus eugenii) Cleavage Stages and Blastocysts
Authors: Hickford Danielle , Frankenberg Stephen , Renfree Marilyn B. 
Published: December 2009

INTRODUCTIONThis protocol describes a method for whole-mount immunofluorescence staining of early stages of tammar wallaby (Macropus eugenii) development, including cleavage and unilaminar blastocysts. Conceptuses of these stages are surrounded by a shell coat, which must be removed before staining to ensure proper penetration of all solutions.
Whole-Mount Immunohistochemical Staining of Tammar Wallaby(Macropus eugenii)Peri-gastrulation Embryos
Authors: Hickford Danielle , Frankenberg Stephen , Renfree Marilyn B. 
Published: December 2009

INTRODUCTIONThis protocol describes a method for whole-mount immunohistochemical staining of tammar wallaby (Macropus eugenii) peri-gastrulation stage embryos using the avidin-biotin complex system. Tammar embryos are ideal for whole-mount immunostaining because of their morphology: They develop in a planar manner on the surface of the embryonic vesicle and are not covered by any extraembryonic membranes until amnion formation (which occurs during mid-somitogenesis). Tammar embryos are surrounded by a transparent shell coat, which must be opened prior to staining to ensure penetration of solutions and reagents.
Biarsenical Labeling of Tetracysteine-Tagged Proteins for Tracking Existing and Newly Synthesized Pools of Proteins
Authors: Pattnaik Asit K. , Panda Debasis 
Published: December 2009

INTRODUCTIONGenetically tagging proteins of interest with fluorescent labels such as green fluorescent protein is a valuable technique for studying protein localization and interactions in live cells. However, the relatively large size of fluorescent fusion proteins can limit the utility of this approach, and sometimes the tag can render the protein of interest nonfunctional. Small genetic tagging with tetracysteine (TC) and subsequent labeling with membrane-permeable biarsenical dyes can overcome these limitations. This protocol describes the method to detect TC-tagged proteins by biarsenical labeling in live cells. A small amino acid tag containing four cysteines (CCPGCC) is introduced to a protein of interest. Cells expressing proteins with this tag are then treated with membrane-permeable dyes (FlAsH-EDT2 or ReAsH-EDT2), and the two arsenic groups of the dyes each bind to the two thiols in the TC motif. Once bound, FlAsH and ReAsH are converted to a fluorescent state (green and red fluorescence at excitation maxima of 508 and 593 nm, respectively) and can be detected by fluorescence microscopy. The method is particularly suitable for investigating protein dynamics that are difficult to study using a fluorescent protein tag. It also is a powerful method for visualizing trafficking of newly synthesized proteins in real time, because existing and newly synthesized pools of proteins can be labeled differentially with the red and green dyes. A method for labeling virally produced proteins is also presented.
Microinjection of Morpholino Oligos and RNAs in Sea Squirt (Ciona) Embryos
Authors: Christiaen Lionel , Wagner Eileen , Shi Weiyang , Levine Michael 
Published: December 2009

INTRODUCTIONThis protocol describes microinjection of morpholino oligos (MOs) and RNAs into sea squirt (Ciona) embryos. This is the method of choice for gene disruption assays. MOs that target the initiating ATG can be used, in addition to those that target splice donor and acceptor sites. The latter method permits the selective inhibition of zygotic mRNAs in cases in which the gene in question is expressed in both the egg and embryo. Although injection is usually performed at the one-cell stage, it is possible to target individual blastomeres, up to the eight-cell stage, thereby permitting lineage-specific knockdown of pleiotropic genes. Injection can also be performed in unfertilized eggs to inhibit maternal genes. Microinjection also permits DiI labeling and lineage tracing.
Isolation of Individual Cells and Tissues from Electroporated Sea Squirt (Ciona) Embryos by Fluorescence-Activated Cell Sorting (FACS)
Authors: Christiaen Lionel , Wagner Eileen , Shi Weiyang , Levine Michael 
Published: December 2009

INTRODUCTIONThis protocol describes the dissociation of electroporated sea squirt (Ciona) embryos and the subsequent isolation of individual cells, cell lineages, and tissues, using fluorescence-activated cell sorting (FACS). Sorted samples can be used for RNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR) or microarray analysis. Alternatively, dissociated samples can be used for the subsequent culturing and/or chemical treatment of isolated embryonic cells. This method permits the identification of effector genes responsible for changes in cell shape, polarity, or motility in a given tissue.
Fluorescent Protein Tracking and Detection: Fluorescent Protein Structure and Color Variants
Authors: Rizzo Mark A. , Davidson Michael W. , Piston David W. 
Published: December 2009

INTRODUCTIONThe rapidly growing arsenal of genetically encoded fluorescent proteins (FPs) obtained from sea creatures has launched and fueled a revolution in live cell imaging. The diverse array of applications benefiting from FPs ranges from markers targeted at organelles and protein fusions designed to monitor intracellular dynamics to reporters of transcriptional regulation and in vivo probes for whole-body imaging and detection of cancer. FPs have enabled the creation of highly specific biosensors to monitor a wide range of intracellular phenomena, including pH and metal-ion concentration, protein kinase activity, apoptosis, membrane voltage, cyclic nucleotide signaling, and tracing neuronal pathways. The purpose of this article is to provide a description of the wide variety of FPs that are currently available.
Computational Image Analysis of Cellular Dynamics: A Case Study Based on Particle Tracking
Authors: Jaqaman Khuloud , Danuser Gaudenz 
Published: December 2009

INTRODUCTIONObtaining quantitative data from live cell images is the key to testing mechanistic hypotheses of molecular and cellular processes. The importance of using computer vision-based methods to accomplish this task is well recognized. However, in practice, investigators often encounter obstacles that render the application of computational image processing in cell biology far from routine. First, it is not always clear which measurements are necessary to characterize a molecular system and whether these measurements are sufficient to characterize the cellular process under investigation. Second, even when the requirements for measurements are well defined, it often is difficult to find a software tool to extract these data. It can be even more challenging to find software tools to answer specific questions raised by the hypotheses underlying the experiments. One solution is for investigators to develop their own software tools. This is feasible for some applications with the assistance of commercial and open-source software packages that support the assembly and integration of custom-designed algorithms, even for users with limited computational expertise. Another solution is for investigators to develop interdisciplinary collaborations with computer scientists. Such collaborations require close interaction between the computer scientists and experimental biologists to optimize the data acquisition and analytical procedures, which must be tightly coupled in any project applying computational analysis to biological image data. This article introduces the basic concepts that make the application of computational image processing to live cell image data successful.
Genome-Wide Association Studies
Authors: Al-Chalabi Ammar 
Published: December 2009

INTRODUCTIONThe goal of association studies is to discover genetic variation that differs in frequency between cases and controls or between individuals with different phenotypic values. Until a few years ago, the only method available for such studies was low-throughput analysis in which a single gene was selected and either genotyped for known genetic variants or sequenced to identify such variants. With the completion of the Human Genome Mapping Project, we have learned that single-nucleotide polymorphisms (SNPs) are frequent in the genome and that variants in physical proximity tend to correlate in genotype. Therefore, a major international effort was started to map this correlation in the form of the International HapMap Project. The concurrent advances in genetic laboratory techniques, statistical methods, and computing power, coupled with the information from the HapMap, have allowed large-scale microchip-based technologies to be used to assay large numbers of SNPs quickly and easily. Thus, truly genome-wide association studies (GWAS) can now be performed, analogous to linkage studies of Mendelian diseases in having no prior hypothesis of the chromosomal location responsible for disease. In this article, we will only discuss case-control studies in which family members are not analyzed, but the principles apply to large-scale family-based association studies as well.
Phylogeny of the large extinct South American Canids (Mammalia, Carnivora, Canidae) using a “total evidence” approach
Authors: Prevosti Francisco J. 
Published: December 2009

AbstractSouth America currently possesses a high diversity of canids, comprising mainly small to medium‐sized omnivorous species, but in the Pleistocene there were large hypercarnivorous taxa that were assigned to Protocyon spp., Theriodictis spp., Canis gezi, Canis nehringi and Canis dirus. These fossils have never been included in phylogenies based on quantitative cladistics, but hand‐constructed cladograms published in the 1980s included some of them in the South American canine clade and others in the Canis clade. In this work, the phylogenetic position of the large extinct South American canids was studied using a large sample of living and extinct canids, as well as different sources of characters (e.g. DNA and 133 osteological characters). The phylogenetic analysis corroborates the inclusion of Theriodictis and Protocyon in the “South American clade”, where C. gezi is also included. In addition, the position of C. dirus as a highly derived Canis species is confirmed. The simultaneous analysis supports hypercarnivory having arisen at least three times in Caninae and once in the “South American clade”. The combination of the phylogenetic analyses, the fossil record and divergence dates estimated in previous works suggests that at least three or four independent lineages of the “South American clade” invaded South America after the establishment of the Panama bridge around 3 million years ago, plus other events corresponding to the immigration of Urocyon and Canis dirus. © The Willi Hennig Society 2009.
Parents' views about pre‐school immunization: an interview study in southern England
Authors: Tickner S. , Leman P. J. , Woodcock A. 
Published: December 2009

AbstractBackground  National Health Service immunization statistics for England indicate that uptake of the first plus second dose of measles, mumps and rubella (MMR) vaccine, and pre‐school booster against diphtheria, tetanus, polio and pertussis, is lower than for the primary course alone. This qualitative study aimed to explore parents' views about pre‐school immunization and to identify possible reasons for lower pre‐school uptake compared with the primary course.Methods  Semi‐structured interviews were conducted with 21 parents of children aged 2–5 years from 19 family units. The parents were recruited from nine playgroups and pre‐schools in three locations in southern England. Data collection and analysis were guided by a modified Grounded Theory approach.Results  Although most parents believed pre‐school immunization to be important and most intended to immunise, a minority questioned whether it was necessary based on their understanding of the duration of protection provided by the primary course. Compared with primary immunization, parents typically received no information about pre‐school doses prior to their invitation to attend and had little or no contact with healthcare professionals. Other barriers included minor illness, apprehension about taking an older child for vaccinations and work or childcare commitments.Conclusions  Parents reported uncertainties, anxieties and time constraints, all of which may contribute to poor attendance for pre‐school immunization. These findings have important implications for providing parents with timely information about boosters and the two‐dose MMR programme. They indicate the potential value of playgroup or pre‐school involvement and the need for improved communication with children about immunization.