bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phenotypic Analysis of Arabidopsis Mutants: Trypan Blue Stain for Fungi, Oomycetes, and Dead Plant Cells
Authors: van Wees Saskia 
Published: August 2008

INTRODUCTIONTrypan blue stains vasculature, dead plant cells, and fungal and oomycete hyphae. It is useful for assessing the extent of colonization of tissue, and for detecting microlesions present in certain lesion-mimic mutants. Trypan blue staining requires chloral hydrate for destaining, which is inconvenient, because it is a controlled substance. The chloral hydrate can be replaced with 1:2 lactophenol:ethanol, but the background staining will be higher than it is when chloral hydrate is used.
Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version B: Figure 1.
Authors: Tiscornia Gustavo , Singer Oded , Verma Inder M. 
Published: August 2008

INTRODUCTIONThis protocol describes the use of lentiviral vectors to deliver small interfering RNA (siRNA)-mediated silencing cassettes. The combination of these two technologies allows for the development of a powerful tool to achieve long-term down-regulation of specific target genes both in vitro and in vivo. It combines the specificity of RNA interference with the versatility of lentiviral vectors to stably transduce a wide range of cell types. In this method, a small hairpin (shRNA) is cloned initially into an entry vector (pENTR/U6) immediately downstream from an hU6 promoter. The silencing cassette is flanked by recombination sites from bacteriophage λ (attL1 and attL2). Once an effective shRNA is obtained, it can be transferred to the destination vector. The destination vector is a lentiviral vector carrying a marker (green fluorescent protein [GFP] or a selection marker) with a destination cassette cloned upstream of the marker (attR1 and attR2 flanking a ccdB toxic gene). Thus, the silencing cassette can be transferred from the entry vector to the destination vector in a simple Gateway LR cloning reaction. The positioning of the silencing cassette upstream of the marker expression cassette avoids down-regulation of the marker.
Analyzing DNA Replication II: Fixation and Processing of Tissues and Cells Labeled with Bromodeoxyuridine (BrdU)
Authors: Jackson Dean , Cook Peter R. 
Published: August 2008

INTRODUCTIONThe number of cells traversing the cell cycle and the rate of progression through it provide important indices of cell growth and tumorigenicity. S-phase cells can also be identified by their high content of DNA polymerase and proliferating cell nuclear antigen, a component of the leading-strand polymerase. Although both these markers can be detected rapidly and conveniently using the appropriate antibodies, neither are found exclusively in S-phase cells. Immunolabeling after incorporation of modified DNA precursors (e.g., 5-bromodeoxyuridine [BrdU, bromodeoxyuridine]) allows more rapid and precise detection of cells in S-phase of the cell cycle. BrdU is phosphorylated by cells to give BrdUTP, and this precursor is incorporated into DNA instead of deoxythimidine triphosphate. In living cells, BrdU is incorporated into replication sites that can then be detected using fluorochrome or enzyme-coupled antibodies. Alternatively, DNA synthesis sites can be labeled at high resolution by incubating cells with analogs of the natural precursors of DNA. The cells are then fixed and the incorporation sites are detected using fluorochrome- or enzyme-tagged antibodies. Formaldehyde- or alcohol-based fixation and paraffin embedding are used frequently. Formaldehyde, a mild protein cross-linking reagent, preserves cell structure well, and washing with a nonionic detergent or organic solvent permeabilizes membranes and allows antibodies access to the interior of the cell. This protocol describe the methods necessary to fix and process tissue and cell samples for subsequent antibody detection of loaded DNA precursors.
Phage-Display Methodology for the Study of Protein-Protein Interactions: Overview
Authors: Goodyear Carl S. , Silverman Gregg J. 
Published: August 2008

INTRODUCTIONIn recent years, phage display has evolved into a powerful tool providing opportunities to define natural protein-protein interactions and to mold novel ligand receptors. The essential advantages of phage-display approaches originate in the incorporation of the protein and genetic components into a single phage particle. By providing a direct physical link between the expressed protein and the encoding genetic information, clones with desirable functional capacities can be efficiently subjected to iterative rounds of selection, followed by amplification of the selected sublibrary. Hence, during library selection (or panning), specific phage clones are progressively enriched on the basis of their specificity and affinity for ligand. Thus, relatively rare ligand-binding clones can be rescued rapidly and efficiently from large libraries. As these expression cloning systems have matured, versatile selection methods have been reported that are based on the functional properties of displayed proteins in diverse immunochemical and biological settings. This article summarizes phage-display methodology, including relevant biology, nucleotide-doping strategies, and considerations for library design.
Advances in understanding of pathogenesis of aHUS and HELLP
Authors: Fang Celia J. , Richards Anna , Liszewski M. Kathryn , Kavanagh David , Atkinson John P. 
Published: August 2008

SummaryBoth atypical haemolytic uraemic syndrome (aHUS) and the HELLP syndrome (haemolytic anaemia, elevated liver enzymes, and low platelets) are thrombotic microangiopathies characterized by microvascular endothelial activation, cell injury and thrombosis. aHUS is a disease of complement dysregulation, specifically a gain of function of the alternative pathway, due to mutations in complement regulatory proteins and activating components. Recently, the same complement mutation identified in multiple patients with aHUS was found in a patient with the HELLP syndrome. The pathogeneses of both diseases are reviewed focusing on the role of the complement system and how its dysfunction could result in a thrombotic microangiopathy in the kidney in the case of aHUS and in the liver in the case of the HELLP syndrome.
Influence of smoking on airway inflammation and remodelling in asthma
Authors: St‐Laurent J. , Bergeron C. , Pagé N. , Couture C. , Laviolette M. , Boulet L‐P. 
Published: August 2008

SummaryBackground
Although exposure to tobacco smoke has been associated with increased morbidity and mortality, cigarette smoking is still common in the asthmatic population. Induced sputum neutrophilia has been observed in asthmatic smokers, but the effects of regular smoking on their bronchial mucosa morphology remain to be defined. This study documents the inflammatory and remodelling features in bronchial biopsies of smoking compared with non‐smoking asthmatics.Methods
We analysed bronchial biopsies from 24 steroid‐naïve young subjects with mild asthma: 12 non‐smoking and 12 currently smoking subjects. In addition to airway morphology assessment, inflammation and remodelling were analysed by immunohistochemistry using antibodies against CD3, CD68, major basic protein, neutrophil elastase, and tryptase. Expression of the cytokines IL‐4, IL‐5, IL‐8, IFN‐γ, transforming growth factor‐β, and TNF was determined by in situ hybridization.Results
Compared with non‐smoking asthmatic subjects, smoking asthmatics' bronchial mucosa showed squamous cell metaplasia, in addition to increased expression of subepithelial neutrophil elastase, IFN‐γ, and intraepithelial IL‐8.Conclusions
Smoking status modifies morphological and inflammatory processes in young subjects with mild asthma. The changes may possibly affect asthma treatment responses and clinical outcomes.
A cross‐sectional study on the prevalence of food allergy to eggplant (Solanum melongena L.) reveals female predominance
Authors: Harish Babu B. N. , Mahesh P. A. , Venkatesh Y. P. 
Published: August 2008

SummaryBackground
Only a few case reports of allergy to eggplant (Solanum melongena) have been reported. A relatively large number of individuals appear to experience food‐related symptoms to eggplant in India.Objective
The major aims of this study are to assess the prevalence of food allergy to eggplant and analyse the age and gender distribution.Methods
Seven hundred and forty‐one subjects (age range: 5–60 years) randomly selected from rural and urban areas of Mysore city were analysed for the prevalence of eggplant allergy based on case history, skin prick test (SPT) with eggplant extracts and allergen‐specific IgE. The age and gender distribution for the prevalence of eggplant allergy and its association with other atopic conditions were assessed.Results
Sixty‐eight (9.2%) subjects reported adverse reactions to ingestion of eggplant, of which 32 (4.3%) subjects had positive history/positive SPT and 36 (4.9%) had positive history/negative SPT. Sixteen (2.2%) subjects had negative history/positive SPT. Ten subjects (1.4%) experienced allergic symptoms in <2 h. Sensitization to eggplant by SPT was more in atopic (16.7%) compared with non‐atopic subjects (3.8%). All the SPT‐positive subjects (n=48) underwent evaluation for eggplant allergen‐specific IgE, which was detected in 6 subjects (0.8%). Majority of the subjects sensitized to eggplant were in the age groups 16–45 years, and females were twice as likely to be sensitized as males. Female predominance (4 : 1) is more in the 16–30 year group.Conclusions
Many subjects experience adverse reactions to the ingestion of eggplant, possibly due to the pharmacologic action of histamine and other non‐protein components, rather than to specific protein allergen(s). The prevalence of IgE‐mediated eggplant allergy is estimated at ∼0.8%, with higher rates of sensitization in females.
Health‐related quality of life in long‐term follow‐up of patients with cured TNM Stage I or II differentiated thyroid carcinoma
Authors: Pelttari Hanna , Sintonen Harri , Schalin‐Jäntti Camilla , Välimäki Matti J. 
Published: August 2008

SummaryObjective  The purpose of this study was to assess the impact of cured low‐risk differentiated thyroid carcinoma (DTC) on health‐related quality of life (HRQoL) after long‐term follow‐up.Design  A cross‐sectional study including an age‐ and gender‐standardized sample of the general population.Patients and methods  HRQoL was assessed by the 15D in 341 DTC patients, whose initial treatment for stage I or II DTC was performed 12·4 years (range 5–20) earlier. The results were compared to those of an age‐ and gender‐standardized sample of the general population (n= 6001). Determinants of HRQoL in DTC patients were assessed by the Tobit model.Results  Mean total 15D scores did not differ between patients and control subjects. In single dimensions, DTC patients were significantly worse off with regards to sleeping, speech and distress (P= 0·001, 0·002 and 0·012, respectively), but better off with regards to discomfort and symptoms (P < 0·001). Within the patient group, the only significant independent predictor of HRQoL was age at the time of the initial treatment (P < 0·001).Conclusions  After long‐term follow‐up, overall HRQoL in DTC patients is comparable to that of the general population. DTC patients demonstrate an age‐related decline in HRQoL, similar to that seen in the population in general.
Impact of elevated ozone concentration on growth, physiology, and yield of wheat (Triticum aestivum L.): a meta‐analysis
Authors: FENG ZHAOZHONG , KOBAYASHI KAZUHIKO , AINSWORTH ELIZABETH A. 
Published: August 2008

AbstractWe quantitatively evaluated the effects of elevated concentration of ozone (O3) on growth, leaf chemistry, gas exchange, grain yield, and grain quality relative to carbon‐filtered air (CF) by means of meta‐analysis of published data. Our database consisted of 53 peer‐reviewed studies published between 1980 and 2007, taking into account wheat type, O3 fumigation method, rooting environment, O3 concentration ([O3]), developmental stage, and additional treatments such as drought and elevated carbon dioxide concentration ([CO2]). The results suggested that elevated [O3] decreased wheat grain yield by 29% (CI: 24–34%) and aboveground biomass by 18% (CI: 13–24%), where CI is the 95% confidence interval. Even in studies where the [O3] range was between 31 and 59 ppb (average 43 ppb), there was a significant decrease in the grain yield (18%) and biomass (16%) relative to CF. Despite the increase in the grain protein content (6.8%), elevated [O3] significantly decreased the grain protein yield (−18%). Relative to CF, elevated [O3] significantly decreased photosynthetic rates (−20%), Rubisco activity (−19%), stomatal conductance (−22%), and chlorophyll content (−40%). For the whole plant, rising [O3] induced a larger decrease in belowground (−27%) biomass than in aboveground (−18%) biomass. There was no significant response difference between spring wheat and winter wheat. Wheat grown in the field showed larger decreases in leaf photosynthesis parameters than wheat grown in < 5 L pots. Open‐top chamber fumigation induced a larger reduction than indoor growth chambers, when plants were exposed to elevated [O3]. The detrimental effect was progressively greater as the average daily [O3] increased, with very few exceptions. The impact of O3 increased with developmental stages, with the largest detrimental impact during grain filling. Both drought and elevated [CO2] significantly ameliorated the detrimental effects of elevated [O3], which could be explained by a significant decrease in O3 uptake resulting from decreased stomatal conductance.
An assessment of the variation in the prevalence of renal myxosporidiosis and hepatitis in wild brown trout, Salmo trutta L., within and between rivers in South‐West England
Authors: Peeler E J , Feist S W , Longshaw M , Thrush M A , St‐Hilaire S 
Published: August 2008

AbstractThe prevalence of renal myxosporidiosis in wild brown trout, Salmo trutta, in seven river catchments in South‐West England was investigated. Three hundred and twenty‐seven fish were sampled from 16 sites, of which 54 (16.5%) were found, by histological examination of the kidney, to be infected with Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease. No T. bryosalmonae infected fish were found in one river catchment, in other catchments the prevalence ranged from 2.5% to 36%. Hepatitis was strongly associated with the presence of T. bryosalmonae (odds ratio = 20.2, P < 0.001). Chloromyxum schurovi was found in 25% of fish and in six of seven river catchments, where the prevalence ranged from 2.4% to 63%. There was a strong negative association between the presence of T. bryosalmonae and C. schurovi (odds ratio = 0.10, P < 0.001). A hierarchical binomal model of the variance indicated that for T. bryosalmonae most of the variance existed at the site level, whereas for C. schurovi most variance existed at the river catchment level, suggesting that prevalence of T. bryosalmonae infection is determined largely by site level factors (e.g. presence of alternate host). The intraclass correlation coefficients (ICC) were 0.2 and 0.4 for T. bryosalmonae and C. schurovi, respectively, indicating the latter has higher effective transmission because of a higher level of infectiousness and/or abundance of alternate oligochaete hosts. These values can be used in future studies to estimate the sample sizes required to generate prevalence estimates with the required precision.
Diagnosis of bacterial kidney disease by detection of Renibacterium salmoninarum by real‐time PCR
Authors: Jansson E , Lindberg L , Säker E , Aspán A 
Published: August 2008

AbstractBacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), is a serious threat to salmon in aquaculture as well as to wild populations. We have developed a real‐time polymerase chain reaction (PCR) for detection of Rs in kidney samples. The PCR is based on detection of unique parts of the 16S rRNA gene of Rs and DNA equivalent to 1–10 Rs genomes was detected per reaction. No cross‐reactivity with other fish pathogenic or related bacteria could be demonstrated. Analysis of individual kidney samples collected from BKD classified populations identified 39.9% of the fish as positive by real‐time PCR compared with 28.0% by polyclonal enzyme‐linked immunosorbent assay (ELISA). The real‐time PCR assay was found to be well suited for complementary use with ELISA for diagnosis of BKD, with the ability to detect clinical as well as covert Rs infections. The infection level determined by the polyclonal ELISA and by real‐time PCR was significantly correlated.
Important discoveries from analysing bacterial phenotypes
Authors: Bochner Barry R. , Giovannetti Luciana , Viti Carlo 
Published: August 2008

SummaryThe ability to test hundreds to thousands of cellular phenotypes in a single experiment has opened up new avenues of investigation and exploration and led to important discoveries in very diverse applications of microbiological research and development. The information provided by global phenotyping is complementary to, and often more easily interpretable than information provided by global molecular analytical methods such as gene chips and proteomics. This report summarizes advances presented by scientists brought together to share their experiences and knowledge gained with high‐throughput phenotyping.
The cia operon of Streptococcus mutans encodes a unique component required for calcium‐mediated autoregulation
Authors: He Xuesong , Wu Chenggang , Yarbrough Daniel , Sim Lucy , Niu Guoqing , Merritt Justin , Shi Wenyuan , Qi Fengxia 
Published: August 2008

Summary Streptococcus mutans is a primary pathogen for dental caries in humans. CiaR and CiaH of S. mutans comprise a two‐component signal transduction system (TCS) involved in regulating various virulent factors. However, the signal that triggers the CiaRH response remains unknown. In this study, we show that calcium is a signal for regulation of the ciaRH operon, and that a double‐glycine‐containing small peptide encoded within the ciaRH operon (renamed ciaX) mediates this regulation. CiaX contains a serine + aspartate (SD) domain that is shared by calcium‐binding proteins. A markerless in‐frame deletion of ciaX reduced ciaRH operon expression and diminished the calcium repression of operon transcription. Point mutations of the SD domain resulted in the same phenotype as the in‐frame deletion, indicating that the SD domain is required for CiaX function. Further characterization of ciaX demonstrated that it is involved in calcium‐mediated biofilm formation. Furthermore, inactivation of ciaR or ciaH led to the same phenotype as the in‐frame deletion of ciaX, suggesting that all three genes are involved in the same regulatory pathway. Sequence analysis and real‐time RT‐PCR identified a putative CiaR binding site upstream of ciaX. We conclude that the ciaXRH operon is a three‐component, self‐regulatory system modulating cellular functions in response to calcium.
Four million neonatal deaths: counting and attribution of cause of death
Authors: Lawn Joy E. , Osrin David , Adler Alma , Cousens Simon 
Published: August 2008

SummaryEach year there are an estimated four million neonatal deaths and at least 3.2 million stillbirths. Three‐quarters of the world's neonatal deaths are counted only through five‐yearly retrospective household surveys. Without these surveys we would have no data, but limitations remain particularly in detecting deaths on the first day of life. Comparable reliable neonatal cause of death data through vital registration are available for less than 5% of the world's neonatal deaths, necessitating modelled estimates for the majority of the world. Improving the quantity, quality and frequency of data for numbers and causes of neonatal deaths is essential to effectively guide the increasing investments to reduce these deaths. Advancing the data requires general investment in information systems and specific improvements of tools and methods for both household surveys and verbal autopsy, particularly the use of consistent case definitions and hierarchical attribution of cause of death. An important paradigm shift is from historical categories for cause of death (‘perinatal causes’) to programmatic categories which are consistent with the International Classification of Diseases. If neonatal deaths remain uncounted, they cannot count in policy and in programmes.
Aetiology of stillbirths and neonatal deaths in rural Ghana: implications for health programming in developing countries
Authors: Edmond Karen M. , Quigley Maria A. , Zandoh Charles , Danso Samuel , Hurt Chris , Agyei Seth Owusu , Kirkwood Betty R. 
Published: August 2008

SummaryIn developing countries many stillbirths and neonatal deaths occur at home and cause of death is not recorded by national health information systems. A community‐level verbal autopsy tool was used to obtain data on the aetiology of stillbirths and neonatal deaths in rural Ghana. Objectives were to describe the timing and distribution of causes of stillbirths and neonatal deaths according to site of death (health facility or home). Data were collected from 1 January 2003 to 30 June 2004; 20 317 deliveries, 696 stillbirths and 623 neonatal deaths occurred over that time.Most deaths occurred in the antepartum period (28 weeks gestation to the onset of labour) (33.0%). However, the highest risk periods were during labour and delivery (intrapartum period) and the first day of life. Infections were a major cause of death in the antepartum (10.1%) and neonatal (40.3%) periods. The most important cause of intrapartum death was obstetric complications (59.3%). There were significantly fewer neonatal deaths resulting from birth asphyxia in the home than in the health facilities and more deaths from infection. Only 59 (20.7%) mothers of neonates who died at home reported that they sought care from an appropriate health care provider (doctor, nurse or health facility) during their baby's illness. The results from this study highlight the importance of studying community‐level data in developing countries and the high risk of intrapartum stillbirths and infectious diseases in the rural African mother and neonate. Community‐level interventions are urgently needed, especially interventions that reduce intrapartum deaths and infection rates in the mother and infant.