bluuebunny/crossref_metadata_2025_split huggingface dataset:
Staining of Tissue Slices for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos
Authors: Stoeckli Esther T. 
Published: September 2006

INTRODUCTIONThis protocol describes a method to stain nerve fibers in tissue slices of avian embryos using an antibody against the 160-kD subunit of neurofilaments. This allows the comparison of the branching pattern of motor and sensory neurons between control and experimental embryos. The tissue is cut in slices using a vibratome or tissue slicer. The protocol is suitable for older embryos after approximately stage 33 and regions that are not accessible by whole-mount analysis. Slices can also be used for tracing axon tracts with lipophilic dyes rather than immunohistochemical detection of all neurites with anti-neurofilament antibodies.
Immunoprecipitation: Lysing Yeast Cells Using Glass Beads
Authors: Harlow Ed , Lane David 
Published: September 2006

INTRODUCTIONFor yeast, the preferred method of lysis is mechanical shearing by vortexing the cells in the presence of glass beads as described in this protocol. This is a powerful lysis method but imparts extensive energy into the lysis procedure and results in considerable denaturation of the samples. For this reason, yeast immunoprecipitations are not as powerful a technique as similar studies in mammalian cells and should not be relied upon for determination of protein properties that require authentic protein structure. The lysate prepared in this procedure is ready for preclearing.
Immunoprecipitation: Denaturing Lysis
Authors: Harlow Ed , Lane David 
Published: September 2006

INTRODUCTIONFor many sources of antigens, one useful method of lysis is to treat cells with harsh, denaturing solutions to release most of the protein antigens, as described here. The lysates are then diluted to reduce the denaturing conditions to levels that are suitable for the formation of antibody-antigen complexes. The resulting solution is precleared prior to immunoprecipitation. The major consideration in choosing this lysis method is that all noncovalent protein-protein interactions will be lost and that denaturation-sensitive epitopes will be destroyed. Therefore, this is not an appropriate method for many types of final assays, but it is a good method for quantitation of the antigen, careful identification of the polypeptide band recognized by the antibody, and display of denaturation-resistant epitopes.
Immunoprecipitation: Preclearing the Lysate
Authors: Harlow Ed , Lane David 
Published: September 2006

INTRODUCTIONTo reduce backgrounds and to improve the signal-to-noise ratio, an antibody that does not recognize the antigen being studied can be added to the lysate and processed as for a normal immunoprecipitation. Any nonspecific proteins that might contaminate the final immunoprecipitation step are presumably removed with this irrelevant antibody. Rabbit serum collected from a healthy adult animal that has not been previously immunized is used because it provides a complex mixture of different antibody types. Fixed Staphylococcus aureus Cowan I (SAC), a relatively inexpensive, concentrated source of protein A is used to collect the rabbit antibodies. Precleared samples prepared according to this protocol are ready for the final immunoprecipitation step.
SDS-Polyacrylamide Gel Electrophoresis of Proteins
Authors: Sambrook Joseph , Russell David W. 
Published: September 2006

INTRODUCTIONThis protocol describes the separation of proteins by SDS-polyacrylamide gel electrophoresis. SDS is used with a reducing agent and heat to dissociate the proteins. SDS-polypeptide complexes form and migrate through the gels according to the size of the polypeptide. By using markers of known molecular weight, the molecular weight of the polypeptide chain(s) can be estimated.
Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Cloning by Homologous Recombination and High-Throughput Transformation
Authors: Hazbun Tony R. , Miller John P. 
Published: September 2006

INTRODUCTIONAlthough described here as a means of producing baits and preys, the generation of an array of ORFs fused to any moiety can be achieved using this protocol. This procedure capitalizes on the asexual/sexual reproductive cycle of yeast cells. Laboratory yeast strains can be maintained indefinitely by asexual reproduction as one of two haploid mating types, MATa or MATα. However, overlaying MATa and MATα cells allows them to fuse and form diploid cells. Hence, the strategy involves transforming MATα cells with the Gal4 BD (bait) fusion plasmids and transforming MATa cells with the Gal4 AD (prey) fusion plasmids. The transformants are then mixed on a plate, or in liquid, and diploids are selected by plating them onto media lacking the marker nutrients for both of the plasmids. In this way, the prey transformations need only be performed once, and each bait protein can then be tested against the array of prey-containing strains using only one transformation per bait. The protocol is easily adapted to small-scale transformation of single microcentrifuge tubes for bait transformations.
Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Storage, Maintenance, and Working with Living Arrays
Authors: Hazbun Tony R. , Miller John P. 
Published: September 2006

INTRODUCTIONThe most difficult aspect of using an array approach for a high-throughput study is maintaining the array’s viability while avoiding contamination of the plates. This protocol describes three different storage methods for the array. Frozen stocks are the most permanent method and are an absolute requirement for maintaining a record of the array. Unfortunately, because thawing is necessary to access the strains from a 96-well format, the repeated use of the frozen stocks as a source is expected to eventually lead to loss of viability of the stock. (Generating frozen stocks of the array strains individually, as well as in the arrayed format, is essential.) Maintenance of a water stock provides a source that is more convenient and (relative to yeast cells in media) semi-permanent (~6 months). However, the water stock is fairly precarious because cross-transfer of strains between wells can occur quite easily if the plates are dropped or disturbed. The third method of storage, that of maintaining extra copies of the solid medium array plates, is the most convenient, but it is also the least stable. In short, the best option is to make several frozen stocks. For greater convenience in repeated use of the array, generate water stocks and additional copies of the solid medium plates.
Hop stunt viroid is processed and translocated in transgenic Nicotiana benthamiana plants
Authors: GÓMEZ GUSTAVO , PALLÁS VICENTE 
Published: September 2006

SUMMARYViroids are small, single‐stranded, circular, pathogenic RNAs that do not code for proteins and thus depend on host factors for their replication and movement. They induce diseases in plants by direct interaction with host factors through a mechanism as yet unidentified. Hop stunt viroid (HSVd) has been found in a wide range of herbaceous and woody hosts. Nicotiana benthamiana is one of the most frequently used experimental systems in the study of plant–virus interactions. However, this plant is a non‐host for HSVd. To go further in the study of the requirements of a plant to be a host for viroids N. benthamiana was transformed with cDNA expressing dimeric (+) HSVd. Correct processing to the circular (+) monomers was always observed, demonstrating that N. benthamiana has the appropriate machinery to cleave and circularize (+) HSVd molecules. Additionally, N. benthamiana plants agroinoculated with the dimeric (–) HSVd accumulated the circular and linear (+) monomers, indicating that (–) HSVd transcripts can be used as template for the RNA–RNA transcription. Grafting assays showed that HSVd is able to move to distal plant parts. In addition, alterations in the normal flower development were observed. Taken together, these results indicate that deficiencies in the interaction with host factors related either to the early steps of the replication process or to cell to cell movement appear to be the factors that limit infectivity of HSVd in N. benthamiana. These transgenic plants can be an useful tool to study the HSVd–host factor interactions.
Plant species as indicators of the extent of desertification in four sandy rangelands
Authors: An Ping , Inanaga Shinobu , Zhu Nanwen , Li Xiangjun , Fadul Hassan M. , Mars Messaoud 
Published: September 2006

AbstractWe examined the distribution of plants in sandy rangelands with different degrees of desertification in (i) Horqin and Mu Us sandy lands in north China, (ii) Darfur district in north‐west Sudan and (iii) southern Tunisia. The severity of desertification in these areas was classified as light, moderate, severe or extreme. The following vegetation changes were observed with increasing severity of desertification: a decrease in the number of plant species, an increase in drought‐tolerant plants, a decrease in the proportion of palatable grasses and the occurrence of some species in only one category of desertification, whereas other species occurred in several categories. Plant species with unique niche requirements were identified and recommended as indicators of the degree of desertification for particular areas. We discuss the characteristics of species in areas with severe desertification and the method of using plant species as indicators of the extent of desertification.
Oxidative stress and inflammation in iron‐overloaded patients with β‐thalassaemia or sickle cell disease
Authors: Walter Patrick B. , Fung Ellen B. , Killilea David W. , Jiang Qing , Hudes Mark , Madden Jacqueline , Porter John , Evans Patricia , Vichinsky Elliott , Harmatz Paul 
Published: September 2006

SummaryBlood transfusion therapy is life‐saving for patients with β‐thalassaemia and sickle cell disease (SCD), but often results in severe iron overload. This pilot study examined whether the biomarkers of tissue injury or inflammation differ in these two diseases. Plasma malondialdehyde (MDA) was significantly increased 1·8‐fold in thalassaemia relative to control patients. In contrast, MDA in SCD was not significantly different from controls. In multivariate analysis, the strongest predictors of elevated MDA were liver iron concentration (P < 0·001) and specific diagnosis (P = 0·019). A significant 2‐fold elevation of non‐transferrin bound iron (NTBI) was observed in thalassaemia relative to SCD. NTBI was not a significant predictor of high MDA in multivariate analysis. SCD patients showed a significant 2·2‐fold elevation of the inflammatory marker interleukin (IL)‐6 relative to controls, and a 3·6‐ and 1·7‐fold increase in IL‐5 and IL‐10 relative to thalassaemia. Although α‐tocopherol was significantly decreased by at least 32% in both thalassaemia and SCD, indicating ongoing oxidant stress and antioxidant consumption, γ‐tocopherol, a nitric oxide‐selective antioxidant, was increased 36% in SCD relative to thalassaemia. These results demonstrate that thalassaemia patients have increased MDA and circulating NTBI relative to SCD patients and lower levels of some cytokines and γ‐tocopherol. This supports the hypothesis that the biology of SCD may show increased inflammation and increased levels of protective antioxidants compared with thalassaemia.
Outcome in patients with Wiskott–Aldrich syndrome following stem cell transplantation: an analysis of 57 patients in Japan
Authors: Kobayashi Ryoji , Ariga Tadashi , Nonoyama Shigeaki , Kanegane Hirokazu , Tsuchiya Shigeru , Morio Tomohiro , Yabe Hiromasa , Nagatoshi Yoshihisa , Kawa Keisei , Tabuchi Ken , Tsuchida Masahiro , Miyawaki Toshio , Kato Shunichi 
Published: September 2006

SummaryA total of 57 patients with Wiskott–Aldrich syndrome (WAS) were studied after undergoing stem cell transplantation (SCT) in Japan between January 1985 and December 2004. Eleven patients received transplants from human leucocyte antigen (HLA)‐matched related donors, 10 from HLA‐mismatched related donors, 21 from unrelated bone marrow donors, and 15 from unrelated cord blood donors. Nine of the 57 patients rejected the initial graft. The overall 5‐year survival rate was 73·7% and the 5‐year failure‐free survival rate was 65·7% (failure was defined as rejection or death). The overall 5‐year survival rates for patients receiving bone marrow and cord blood from unrelated donors were both 80·0%. Based on univariate analysis, the factors associated with poor survival were: transplantation from an HLA‐mismatched related donor, patient age of more than 5 years at the time of transplantation, and a conditioning regimen other than busulfan and cyclophosphamide (BU‐CY) or busulfan, cyclophosphamide and antithymocyte globulin (BU‐CY‐ATG). In a multivariate analysis, a conditioning regimen other than BU‐CY and BU‐CY‐ATG was the only independent factor associated with transplantation failure. Given the improved outcome for WAS patients following transplantation from an unrelated donor, we conclude that patients with WAS should receive SCT as soon as possible after diagnosis.
Cytology is useful in breast screening: results and long‐term follow‐up of the Singapore Breast Screening Pilot Project
Authors: Chew I. , Tan Y. , Tan P. H. 
Published: September 2006

Objective: The Singapore Breast Screening Pilot Project (SBSPP) was embarked upon (1994–1997) to determine if mammography was useful in early breast cancer detection among Asian women.Patients and measurements: Of 28 231 women screened, fine needle aspiration cytology (FNAC) was performed in 232 individuals as part of the triple assessment.Results: Absolute and complete sensitivities for the diagnosis of carcinoma were 46.7% and 82.2%, respectively, based on the results of FNAC. Specificity was 63.3%. The inadequate rate was 31%. Five women who were considered cancer‐free on triple assessment and, in two cases open diagnostic biopsy during the SBSPP, subsequently developed breast cancer after a median follow‐up of 6 years.Conclusion: Although our FNAC results compared relatively well with international standards, they reflect a small cohort, and may face additional difficulties in a larger programme.
Mastopathic fibroadenoma of the breast: a pitfall of aspiration cytology
Authors: Mori I. , Han B. , Wang X. , Taniguchi E. , Nakamura M. , Nakamura Y. , Bai Y. , Kakudo K. 
Published: September 2006

Objective:  To assess the correlation between cytological diagnoses and histological subtypes of fibroadenoma (FA) and to clarify the cytological features of a specific group of FA displaying variable features similar to fibrocystic disease (mastopathic type, MFA), and to evaluate the significance of this subtype in cytological diagnosis.Methods:  A review of 141 cases of histologically proven FA was performed. We re‐classified them into four subtypes according to Kinoshita's criteria [Jpn J Breast Cancer6 (1991) 377] and further selected 92 cases for which both fine needle aspiration (FNA) smears and histological specimens were available. Among them, 18 cases of MFA and their cytological smears were selected for the present study.Results:  There was significant correlation between MFA and cytological diagnosis of ‘indeterminate’ or ‘suspicious for malignancy’ (P < 0.01). Although no false‐positive diagnosis was experienced in our series, 56% of the MFAs (10/18) had cytological diagnoses of indeterminate or were included in the category ‘suspicious for malignancy’. Smears from MFA revealed high cellularity (9/18 smears had more than 10 epithelial clusters each composed of more than 50 cells), presence of cellular discohesiveness (13/18, 72.2%), but only mild nuclear atypia (5/18, 27.8%). Anisonucleosis was present in fewer than half the cases and no apparent condensed chromatin was identified.Conclusion:  We highlight the significance of subclassification of MFA in aspiration cytology of breast. MFA had a significantly higher chance of falling into the ‘suspicious for malignancy’ or ‘indeterminate’ diagnostic category in aspiration cytology. It might be a diagnostic challenge for cytopathologist to identify this subtype of FA in FNA smears.
Body size changes in ground beetle assemblages – a reanalysis of Braunet al. (2004)’s data
Authors: LÖVEI GÁBOR L. , MAGURA TIBOR 
Published: September 2006

Abstract1. Data inBraunet al.’s (2004)recent paper on size trends in ground beetles (Carabidae) are re‐analysed, after re‐classifying the species according to feeding categories.2. When ground beetles are classified as predatory species, mixed feeders or herbivores, and evaluated separately, the size trends indicate group‐specific differences. Most of these do not support the efficiency‐specialisation hypothesis.3. Taxonomic groups rarely fit into one ecological category and ecological analyses should explicitly consider this hurdle.
The origin of the savanna biome
Authors: BEERLING DAVID J. , OSBORNE COLIN P. 
Published: September 2006

AbstractSavannas are a major terrestrial biome, comprising of grasses with the C4 photosynthetic pathway and trees with the C3 type. This mixed grass–tree biome rapidly appeared on the ecological stage 8 million years ago with the near‐synchronous expansion of C4 grasses around the world. We propose a new hypothesis for this global event based on a systems analysis that integrates recent advances in how fire influences cloud microphysics, climate and savanna ecology in a low carbon dioxide (CO2) world. We show that fire accelerates forest loss and C4 grassland expansion through multiple positive feedback loops that each promote drought and more fire. A low CO2 atmosphere amplifies this cycle by limiting tree recruitment, allowing the ingress of C4 grasses to greatly increase ecosystem flammability. Continued intensification of land use could enhance or moderate the network of feedbacks that have initiated, promoted and sustained savannas for millions of years. We suggest these alterations will overprint the effects of anthropogenic atmospheric change in coming decades.
Regulation of surfactant protein and defensin mRNA expression in cultured ovine type II pneumocytes by all‐trans retinoic acid and VEGF
Authors: Grubor B. , Meyerholz D. K. , Lazic T. , DeMacedo M. M. , Derscheid R. J. , Hostetter J. M. , Gallup J. M. , DeMartini J. C. , Ackermann M. R. 
Published: September 2006

SummaryBeta‐defensins and surfactant proteins are components of the pulmonary innate immune system. Their gene expression is regulated by development, hormones, growth and immunoregulatory factors. It was our hypothesis that growth and differentiation factors such as all‐trans retinoic acid (RA) and vascular endothelial growth factor (VEGF) may affect expression of selected innate immune genes by respiratory epithelial cells. Ovine JS7 cells (alveolar type II pneumocytes) were incubated in serum‐free Dulbecco's modified Eagle's medium (DMEM) complete media that contained: no treatment (negative control), RA (500 nM), or VEGF (100 ng/ml) for 6, 12 or 24 h incubation. Total RNA was isolated, cDNA synthesized, and relative mRNA levels of surfactant protein A (SP‐A) and SP‐D, and sheep beta‐defensin‐1 (SBD‐1) were determined by real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR). Cells had significantly increased expression of SP‐D mRNA at 6 h and 24 h, decreased expression of SP‐A mRNA at 12 h, and unchanged levels of SBD‐1 mRNA after the treatment with RA compared with their respective negative controls. VEGF did not alter the expression of the three innate immune genes. These findings suggest that SP‐A and SP‐D have different transcription regulation pathways, and that expression of SBD‐1 is not inducible by RA similar to its human homolog HBD‐1. The lack of changes induced by VEGF treatment suggests that VEGF does not have a direct effect on epithelial cells, but may affect gene expression indirectly.
Percutaneous endoscopic gastrostomies: the burden of treatment from a patient perspective
Authors: Jordan Sue , Philpin Sue , Warring Joanne , Cheung Wai Yee , Williams John 
Published: September 2006

Aim.  This paper reports a study of patients’ experiences of this relatively new technology, and explores possible strategies for defining, delineating and addressing patients’ concerns, problems and needs.Background.  The number of patients in the community relying on percutaneous endoscopic gastrostomy for their nutritional needs is increasing, but percutaneous endoscopic gastrostomy feeding is not problem‐free. However, few studies report on its impact from a patient perspective.Methods.  Twenty adults with long‐term percutaneous endoscopic gastrostomies were interviewed in their own homes, using both semi‐structured and structured approaches. Qualitative data were analysed thematically and related to a symptom checklist and an established quality of life measure, the Short Form‐12.Findings.  Most participants felt that insertion of a percutaneous endoscopic gastrostomy had been life‐saving, but found that percutaneous endoscopic gastrostomy feeding came to dominate their lives and was associated with an appreciable ‘burden of treatment’. Some, but not all, of the problems recounted were unavoidable: percutaneous endoscopic gastrostomy feeding inevitably imposed physical restrictions on mobility; 17/20 participants had experienced serious technical problems with their percutaneous endoscopic gastrostomy tubes. On structured questioning, all interviewees had some, potentially treatable, gastrointestinal symptoms, and eight had continuous problems. In some cases, the burden of treatment was exaggerated by being unanticipated and, for 13 patients, difficulties with care provided by non‐specialist services. Describing this ‘burden of treatment’ for each patient required both a narrative and a structured approach: without the symptom checklists, some important and treatable problems were missed. Both physical and mental health Short Form‐12 scores were low, and mental health scores were congruent with the interview data.Conclusions.  It might be possible to reduce the ‘burden of treatment’ identified by incorporating a structured approach to patient monitoring and by reviewing the occupational territories of specialist and non‐specialist practitioners. Further research with larger numbers of participants is needed to explore the integration of narrative and quantitative data when determining patients’ clinical needs.
Factors contributing to frailty: literature review
Authors: Levers Merry‐Jo , Estabrooks Carole A. , Ross Kerr Janet C. 
Published: September 2006

Aim.  This paper presents a review of theoretical and research literature in order to identify the factors contributing to frailty.Background.  Frailty is a multifaceted gerontological concept that lacks a clear definition, but may result from an identifiable homogeneous cluster of bio–psycho–social–spiritual factors.Method.  A total of 134 articles were identified through a search of the MEDLINE (1966 to July 2004), CINAHL (1982 to July 2004), PsychInfo (1985 to July 2004) and Ageline (1995 to July 2004) databases. Each article was reviewed to determine its fit with inclusion/exclusion criteria. Seven research and 11 theoretical articles were retained and further reviewed for methodological quality using a validity tool.Findings.  Seventeen different definitions of frailty were identified. Regardless of the differing definitions, common contributing factors could be identified. Physical, cognitive/psychological, nutritional and social factors, as well as ageing and disease, were evident in both the theoretical and research literature.Conclusions.  Although there is strong agreement that a relationship exists between a cluster of factors and frailty, designation of the factors as contributors or outcomes of frailty differs. Without a clear explanatory theory of the path from contributors to frailty to outcomes, research will continue to produce confusing results. A theoretical framework that includes bio–psycho–social–spiritual factors as contributors to frailty is recommended as the most useful framework for gerontological nursing.