bluuebunny/crossref_metadata_2025_split huggingface dataset:
Automated Extraction of Viral-Pathogen RNA and DNA for High-Throughput Quantitative Real-Time PCR
Authors: Beuselinck Kurt , van Ranst Marc , van Eldere J. 
Published: November 2005

ABSTRACT

The performance of the
m
1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput “in-house” quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples (hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTA-blood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 ± 0.06 for HCV and 0.97 ± 0.03 for HBV, indicating a linear extraction from 100 to 1.0 × 10
5
HCV IU/ml and from 100 to 1.0 × 10
6
HBV IU/ml. Intra- and interrun variability was below 0.23 log
10
IU/ml for 2.98 to 5.28 log
10
HCV IU/ml and 2.70 to 5.20 log
10
HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log
10
HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log
10
HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log
10
copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log
10
copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the
m
1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.
Development of a General-Primer-PCR-Reverse-Line-Blotting System for Detection of Beta and Gamma Cutaneous Human Papillomaviruses
Authors: Brink Antoinette A. T. P. , Lloveras Belen , Nindl Ingo , Heideman Daniëlle A. M. , Kramer Debbie , Pol René , Fuente M. Jose , Meijer Chris J. L. M. , Snijders Peter J. F. 
Published: November 2005

ABSTRACT

The beta and gamma genera of papillomaviruses consist of epidermodysplasia verruciformis-related human papillomaviruses (HPVs) and phylogenetically related cutaneous HPVs. Here, we have developed a consensus primer PCR assay and reverse line blot typing system coupled thereto (referred to as beta and gamma cutaneous HPV PCR [BGC-PCR]) for detection and typing of 24 beta and gamma HPVs (HPV types 4, 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 48, 49, 50, 60, and 65). Because the HPV-specific PCR products are only 72 bp in size, the system is suitable for formalin-fixed, paraffin-embedded specimens and other samples in which the DNA is of suboptimal quality. This system was able to detect and type as little as 100 ag to 1 fg HPV DNA per reaction (depending on the HPV type) in a background of 100 ng human DNA without any cross-reactivity between the tested types. Beta and gamma HPVs were detected in DNA extracted from plucked eyebrow hairs of 31 of 34 renal transplant recipients. In addition, formalin-fixed, paraffin-embedded specimens from nonmelanoma skin tumors of renal transplant recipients (
n
= 25) and immunocompetent individuals (
n
= 15) scored BGC-PCR positive in 21 and 6 cases, respectively, with HPV type 5 (HPV5) and HPV8 being the predominant types. The data indicate that this method can be a valuable, user-friendly tool for the detection and typing of cutaneous HPV in clinical specimens and may have implications for future monitoring of vaccines or alternative treatment modalities for diseases caused by these cutaneous HPVs.
Population Structure and Properties of
Candida albicans
, as Determined by Multilocus Sequence Typing
Authors: Tavanti Arianna , Davidson Amanda D. , Fordyce Mark J. , Gow Neil A. R. , Maiden Martin C. J. , Odds Frank C. 
Published: November 2005

ABSTRACT

We submitted a panel of 416 isolates of
Candida albicans
from separate sources to multilocus sequence typing (MLST). The data generated determined a population structure in which four major clades of closely related isolates were delineated, together with eight minor clades comprising five or more isolates. By Fisher's exact test, a statistically significant association was found between particular clades and the anatomical source, geographical source, ABC genotype, decade of isolation, and homozygosity versus heterozygosity at the mating type-like locus (MTL) of the isolates in the clade. However, these associations may have been influenced by confounding variables, since in a univariate analysis of variance, only the clade associations with ABC type and anatomical source emerged as statistically significant, providing the first indication of possible differences between
C. albicans
strain type clades and their propensity to infect or colonize different anatomical locations. There were no significant differences between clades with respect to distributions of isolates resistant to fluconazole, itraconazole, or flucytosine. However, the majority of flucytosine-resistant isolates belonged to clade 1, and these isolates, but not flucytosine-resistant isolates in other clades, bore a unique mutation in the
FUR1
gene that probably accounts for their resistance. A significantly higher proportion of isolates resistant to fluconazole, itraconazole, and flucytosine were homozygous at the MTL, suggesting that antifungal pressure may trigger a common mechanism that leads both to resistance and to MTL homozygosity. The utility of MLST for determining clade assignments of clinical isolates will form the basis for strain selection for future research into
C. albicans
virulence.
Fast Ligation-Mediated PCR, a Fast and Reliable Method for IS
6110
-Based Typing of
Mycobacterium tuberculosis
Complex
Authors: Reisig Florian , Kremer Kristin , Amthor Beate , van Soolingen Dick , Haas Walter H. 
Published: November 2005

ABSTRACT

IS
6110
restriction fragment length polymorphism (RFLP) analysis is the most widely applied method for strain differentiation of
Mycobacterium tuberculosis
complex. We have previously described mixed-linker PCR, an IS
6110
-based PCR method that favorably compared with other typing methods for
M. tuberculosis
complex according to reproducibility and ability to differentiate between strains. Here we report the further development of this method, called fast ligation-mediated PCR (FLiP), which allows analysis of strains within one working day and starting from less than 1 ng of mycobacterial DNA or a crude cell lysate. Blinded analysis of a standard set of 131
M. tuberculosis
complex and nontuberculous isolates showed the ability to differentiate 81 types among 90
M. tuberculosis
complex isolates with 84 different IS
6110
RFLP fingerprint patterns and detected 97% of the 31 duplicate samples. We suggest that FLiP can serve to rapidly detect chains of transmission prior to starting high-throughput analysis or standard IS
6110
RFLP. It may as well serve as a secondary typing technique for other, non-IS
6110
-based methods.
Characterization of Clonal Complexity in Tuberculosis by Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing
Authors: García de Viedma Darío , Alonso Rodriguez Noelia , Andrés Sandra , Ruiz Serrano Maria Jesús , Bouza Emilio 
Published: November 2005

ABSTRACTIn recent years, the application of molecular tools has shown us that clonal complexity in infection byMycobacterium tuberculosisis not anecdotal. Exogenous reinfections, mixed infections, compartmentalization, and microevolution are different aspects of this issue. The detection and characterization of clonal variants ofM. tuberculosisby standard genotyping methods is laborious and frequently requires expertise. Our aim was to evaluate a new genotyping PCR-based method forM. tuberculosis, mycobacterial interspersed repetitive unit-variable-number tandem repeat typing (MIRU-VNTR), as a potential tool to simplify and optimize the clonal analysis of tuberculosis. MIRU-VNTR was able to detect mixed clonal variants in vitro, even for clones at low ratios (1:99). This technique was prospectively applied to search for cases infected by more than one clone. Clonal variants within the same host were detected in 3 out of 115 cases (2.6%), including cases with clones which were indistinguishable by restriction fragment length polymorphism or spoligotyping. In one case, coinfecting clonal variants differed in antibiotic susceptibilities. MIRU-VNTR was applied to cases with proven polyclonal infection, and it succeeded in detecting the coinfecting strains and proved useful in confirming cases of compartmentalized infection. MIRU-VNTR is a simple, rapid, and sensitive method which could facilitate and optimize the identification and characterization of clonal complexity inM. tuberculosisinfection.
Rapid and Accurate Genotyping of YMDD Motif Variants in the Hepatitis B Virus Genome by an Improved Reverse Dot Blot Method
Authors: Ou Zhi-Ying , Liu Na , Chen Chang-Jie , Cheng Gang , He Yun-Shao 
Published: November 2005

ABSTRACT
By using the “flow-through hybridization” principle, we developed a new, rapid and accurate reverse dot blot (RDB) method to detect lamivudine resistance-associated YMDD motif variants in hepatitis B virus (HBV) genome. The improved RDB method was very fast at simultaneously detecting HBV YMDD wild-type and mutant motifs. In a blind analysis, 100 samples previously genotyped by DNA clonal sequencing analysis were used to evaluate the sensitivity and specificity of this assay. Conventional restriction fragment length polymorphism (RFLP) data were also used to test our method. In blind experiments, our improved RDB method had an accuracy and specificity of 100%, which was much higher than RFLP, which had an accuracy and specificity of only 83.0%. In clinical detection practice, 49 patients highly suspected of lamivudine resistance were successfully diagnosed by this method. Our improved RDB assay is a simple, rapid, cheap, semiautomatic, accurate, sensitive, and contamination-proof method of detecting lamivudine resistance-associated mutants in the human hepatitis B virus genome.
Human Infection with an Avian H9N2 Influenza A Virus in Hong Kong in 2003
Authors: Butt K. M. , Smith Gavin J. D. , Chen Honglin , Zhang L. J. , Leung Y. H. Connie , Xu K. M. , Lim Wilina , Webster Robert G. , Yuen K. Y. , Peiris J. S. Malik , Guan Yi 
Published: November 2005

ABSTRACT
Avian H9N2 influenza A virus has caused repeated human infections in Asia since 1998. Here we report that an H9N2 influenza virus infected a 5-year-old child in Hong Kong in 2003. To identify the possible source of the infection, the human isolate and other H9N2 influenza viruses isolated from Hong Kong poultry markets from January to October 2003 were genetically and antigenically characterized. The findings of this study show that the human H9N2 influenza virus, A/Hong Kong/2108/03, is of purely avian origin and is closely related to some viruses circulating in poultry in the markets of Hong Kong. The continued presence of H9N2 influenza viruses in poultry markets in southern China increases the likelihood of avian-to-human interspecies transmission.
Use of a Real-Time PCR TaqMan Assay for Rapid Identification and Differentiation of
Burkholderia pseudomallei
and
Burkholderia mallei
Authors: U'Ren Jana M. , Van Ert Matthew N. , Schupp James M. , Easterday W. Ryan , Simonson Tatum S. , Okinaka Richard T. , Pearson Talima , Keim Paul 
Published: November 2005

ABSTRACT

A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype
Burkholderia pseudomallei
and
Burkholderia mallei
isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as
Burkholderia thailandensis
and
Burkholderia cepacia
.
Distribution of Strain Families ofMycobacterium tuberculosisCausing Pulmonary and Extrapulmonary Disease in Hospitalized Children in Cape Town, South Africa
Authors: Nicol Mark P. , Sola Christophe , February Bradley , Rastogi Nalin , Steyn Lafras , Wilkinson Robert J. 
Published: November 2005

ABSTRACTWe studied the association between strain family and extrapulmonary tuberculosis among 285 children presenting to a pediatric hospital. Extrapulmonary disease occurred in 56% of children without known human immunodeficiency virus infection, with meningitis accounting for 22% of the cases. Two strain families, LAM3/F11 and W-Beijing, predominated; but there was no overall association with extrapulmonary disease.
Phylogenetic Analysis of the p24-p7 Region of the Human Immunodeficiency Virus Type 1
gag
Gene To Determine Subtype Distribution among Female Sex Workers in Calcutta, India
Authors: Sengupta Satarupa , Jana Smarajit , Roy Pratim , Sarkar Kamalesh , Bhattacharya Sujit K. , Chakrabarti Sekhar 
Published: November 2005

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) subtype C, based on the envelope region, has been reported to be predominant in India. We sequenced the p24-p7
gag
region from 51 HIV-1 seropositive female sex workers in Calcutta, India, for more-detailed molecular characterization. Subtype C was found to be prevalent, although no strong monophyletic cluster was observed.
Emergence of a
Streptococcus pneumoniae
Clinical Isolate Highly Resistant to Telithromycin and Fluoroquinolones
Authors: Faccone Diego , Andres Patricia , Galas Marcelo , Tokumoto Marta , Rosato Adriana , Corso Alejandra 
Published: November 2005

ABSTRACT

Streptococcus pneumoniae
is a major pathogen causing community-acquired pneumonia and acute bronchitis. Macrolides, fluoroquinolones (FQs), and, recently, telithromycin (TEL) constitute primary therapeutic options, and rare cases of resistance have been reported. In this report, we describe the emergence of an
S. pneumoniae
clinical isolate with high-level TEL resistance (MIC, 256 μg/ml) and simultaneous resistance to FQs. Ongoing studies are oriented to elucidate the precise mechanism of resistance to TEL.
The Law of International Organizations: A Subject which needs Exploration and Analysis
Authors:   Amerasinghe
Published: November 2005

AbstractThe law of international organizations, including the institutional law, has been somewhat neglected in the past, even though, or perhaps because, international organizations are creations largely of the 20th century. In my treatise on Principles of the Institutional Law of International Organizations, first published in late 1996 and going now, at the request of the publisher, into a second edition, I directed attention, perhaps in a seminal way, to this institutional law, its importance and its qualification to be considered a specific category not only of general international law but also of international organizational law. In my view there is ample room for further thorough study of various aspects particularly of this law without neglecting the functional international law of international organizations. Apart from principle, their application or non-application in practice may usefully be studied. This by itself justifies a law journal devoted to the subject of international organizational law in general. Moreover, the justification is further reinforced by the fact that now international organizations have become a feature of everyday life in the world. Here, at the risk of repeating what I have said in my book referred to above, because such repetition can only emphasize the importance of the subject matter, I shall concentrate on four aspects which are relevant to international organizational law, to its importance as a part of international law and to its influence on international relations: (i) the pervasiveness of international organizations; (ii) the concept of international institutional law; (iii) its nature; and (iv) its sources.
International Organizations and their Members
Authors: Blokker Niels 
Published: November 2005

AbstractThe law of international organizations is a field of study full of themes with variations. With respect to a number of key chapters in this field, such as the legal status of international organizations, their institutional structure, powers, decision-making and decisions, sanctions, fundamental principles exist or are being developed. These principles or themes have a large number of variations in the rules and practice of individual organizations. Like in music, the variations in themselves may be interesting as such, but they can only be fully appreciated by not losing sight of the theme. Like in music, the themes in themselves may be interesting as such, but they can only be fully appreciated by also listening carefully to the multicoloured variations. This contribution is devoted to a central theme of the law of international organizations: the relationship between an international organization and its members. Already in the early days of existence of international organizations, this topic has given rise to numerous questions. Many of these have now been answered, but sometimes resurface in contemporary variations. In addition there are new questions. It is useful to first explore briefly the meaning of the word "members". The English word "member" and the French "membre" are both derived from the Latin word "membrum", which means "part of the body". This meaning is important because it indicates clearly that members are part of a whole – a fact which takes us straight to one of the core questions addressed in this article. Members of an international organization are not just members, after all – like the members of a bridge club or a gardening club. In most cases, the members of international organizations are states. How then can members of an international organization be members of a whole when in most cases those members are sovereign states, even if the concept of sovereignty of states is no longer as absolute at it used to be? This is one of the major questions within the field of the law of international organizations. In order to examine this question, this contribution is divided into two parts. Section I will concentrate on the role of members vis-à-vis international organizations. In Section II the focus will shift to the whole of which the members are part; this Section will briefly look at some of the questions involved in giving a certain amount of autonomy to this whole.
On Deploying Scientific Software within the Grid-TLSE Project
Authors: Caron Eddy , Desprez Frédéric , Daydé Michel , Hurault Aurélie , Pantel Marc 
Published: November 2005

The main goal of the Grid-TLSE project is to design an expert site that provides an easy access to a number of sparse matrix solver packages allowing their comparative analysis on user-submitted problems, as well as on matrices from collections also available on the site. The site provide user assistance in choosing the right solver for its problems and appropriate values for the solver parameters. A computational Grid is used to deal with all the runs arising from user requests.After an overview of the project, we discuss on some of the main difficulties we face in Grid- TLSE: considering the amount of softwares that will be available, facilitating to facilitate their deployment and their exploitation over a Grid is crucial. We make use of an abstract meta-data based description of solvers and have introduced the concept of scenario, i.e. a high-level user assistance description that provides ways of discovering automatically the most appropriate solvers based on their description. The scenarios are used for generating the dynamic workflows that will be executed over the Grid and take advantage of some of the features available in the DIET Grid middleware. We also report on the introduction of a semantic-based component model for efficient service trading.
Sequential analysis of surfactant, lung function and inflammation in cystic fibrosis patients
Authors: Griese Matthias , Essl Robert , Schmidt Reinhold , Ballmann Manfred , Paul Karl , Rietschel Ernst , Ratjen Felix ,   the Beat Study Group
Published: November 2005

AbstractBackgroundIn a cross-sectional analysis of cystic fibrosis (CF) patients with mild lung disease, reduced surfactant activity was correlated to increased neutrophilic airway inflammation, but not to lung function. So far, longitudinal measurements of surfactant function in CF patients are lacking and it remains unclear how these alterations relate to the progression of airway inflammation as well as decline in pulmonary function over time.MethodsAs part of the BEAT trial, a longitudinal study to assess the course of airway inflammation in CF, we studied lung function, surfactant function and endobronchial inflammation using bronchoalveolar lavage fluid from 20 CF patients with normal pulmonary function (median FEV194% of predicted) at three times over a three year period.ResultsThere was a progressive loss of surfactant function, assessed as minimal surface tension. The decline in surfactant function was negatively correlated to an increase in neutrophilic inflammation and a decrease in lung function, assessed by FEV1, MEF75/25%VC, and MEF25%VC. The concentrations of the surfactant specific proteins A, C and D did not change, whereas SP-B increased during this time period.ConclusionOur findings suggest a link between loss of surfactant function driven by progressive airway inflammation and loss of small airway function in CF patients with limited lung disease.
Identification of clustered microRNAs using an ab initio prediction method
Authors: Sewer Alain , Paul Nicodème , Landgraf Pablo , Aravin Alexei , Pfeffer Sébastien , Brownstein Michael J , Tuschl Thomas , van Nimwegen Erik , Zavolan Mihaela 
Published: November 2005

Abstract
Background
MicroRNAs (miRNAs) are endogenous 21 to 23-nucleotide RNA molecules that regulate protein-coding gene expression in plants and animals via the RNA interference pathway. Hundreds of them have been identified in the last five years and very recent works indicate that their total number is still larger. Therefore miRNAs gene discovery remains an important aspect of understanding this new and still widely unknown regulation mechanism. Bioinformatics approaches have proved to be very useful toward this goal by guiding the experimental investigations.

Results
In this work we describe our computational method for miRNA prediction and the results of its application to the discovery of novel mammalian miRNAs. We focus on genomic regions around already known miRNAs, in order to exploit the property that miRNAs are occasionally found in clusters. Starting with the known human, mouse and rat miRNAs we analyze 20 kb of flanking genomic regions for the presence of putative precursor miRNAs (pre-miRNAs). Each genome is analyzed separately, allowing us to study the species-specific identity and genome organization of miRNA loci. We only use cross-species comparisons to make conservative estimates of the number of novel miRNAs. Our ab initio method predicts between fifty and hundred novel pre-miRNAs for each of the considered species. Around 30% of these already have experimental support in a large set of cloned mammalian small RNAs. The validation rate among predicted cases that are conserved in at least one other species is higher, about 60%, and many of them have not been detected by prediction methods that used cross-species comparisons. A large fraction of the experimentally confirmed predictions correspond to an imprinted locus residing on chromosome 14 in human, 12 in mouse and 6 in rat. Our computational tool can be accessed on the world-wide-web.

Conclusion
Our results show that the assumption that many miRNAs occur in clusters is fruitful for the discovery of novel miRNAs. Additionally we show that although the overall miRNA content in the observed clusters is very similar across the three considered species, the internal organization of the clusters changes in evolution.