bluuebunny/crossref_metadata_2025_split huggingface dataset:
Insights into the role of Val45 and Gln182 of Escherichia coli MutY in DNA substrate binding and specificity
Authors: Chang Po-Wen , Madabushi Amrita , Lu A-Lien 
Published: June 2009

Abstract

Background

Escherichia coli MutY (EcMutY) reduces mutagenesis by removing adenines paired with guanines or 7,8-dihydro-8-oxo-guanines (8-oxoG). V45 and Q182 of EcMutY are considered to be the key determinants of adenine specificity. Both residues are spatially close to each other in the active site and are conserved in MutY family proteins but not in Methanobacterium thermoautotrophicum Mig.MthI T/G mismatch DNA glycosylase (A50 and L187 at the corresponding respective positions).

Results
Targeted mutagenesis study was performed to determine the substrate specificities of V45A, Q182L, and V45A/Q182L double mutant of EcMutY. All three mutants had significantly lower binding and glycosylase activities for A/G and A/8-oxoG mismatches than the wild-type enzyme. The double mutant exhibited an additive reduction in binding to both the A/G and A/GO in comparison to the single mutants. These mutants were also tested for binding and glycosylase activities with T/G- or T/8-oxoG-containing DNA. Both V45A and Q182L mutants had substantially increased affinities towards T/G, however, they did not exhibit any T/G or T/8-oxoG glycosylase activity. Surprisingly, the V45A/Q182L double mutant had similar binding affinities to T/G as the wild-type EcMutY. V45A, Q182L, and V45A/Q182L EcMutY mutants could not reduce the G:C to T:A mutation frequency of a mutY mutant. Expression of the V45A mutant protein caused a dominant negative phenotype with an increased G:C to A:T mutation frequency.

Conclusion
The substrate specificities are altered in V45A, Q182L, and V45A/Q182L EcMutY mutants. V45A and Q182L mutants had reduced binding and glycosylase activities for A/G and A/8-oxoG mismatches and increased affinities towards T/G mismatch. However, in contrast to a previous report that Mig.MthI thymine DNA glycosylase can be converted to a MutY-like adenine glycosylase by replacing two residues (A50V and L187Q), both V45A and Q182L EcMutY mutants did not exhibit any T/G or T/8-oxoG glycosylase activity. The dominant negative phenotype of V45A EcMutY mutant protein is probably caused by its increased binding affinity to T/G mismatch and thus inhibiting other repair pathways.
Improved results in proteomics by use of local and peptide-class specific false discovery rates
Authors: Sennels Lau , Bukowski-Wills Jimi-Carlo , Rappsilber Juri 
Published: June 2009

Abstract

Background
Proteomic protein identification results need to be compared across laboratories and platforms, and thus a reliable method is needed to estimate false discovery rates. The target-decoy strategy is a platform-independent and thus a prime candidate for standardized reporting of data. In its current usage based on global population parameters, the method does not utilize individual peptide scores optimally.

Results
Here we show that proteomic analyses largely benefit from using separate treatment of peptides matching to proteins alone or in groups based on locally estimated false discovery rates. Our implementation reduces the number of false positives and simultaneously increases the number of proteins identified. Importantly, single peptide identifications achieve defined confidence and the sequence coverage of proteins is optimized. As a result, we improve the number of proteins identified in a human serum analysis by 58% without compromising identification confidence.

Conclusion
We show that proteins can reliably be identified with a single peptide and the sequence coverage for multi-peptide proteins can be increased when using an improved estimation of false discovery rates.
The expression of the β-defensins hBD-2 and hBD-3 is differentially regulated by NF-κB and MAPK/AP-1 pathways in an in vitro model of Candida esophagitis
Authors: Steubesand Nadine , Kiehne Karlheinz , Brunke Gabriele , Pahl Rene , Reiss Karina , Herzig Karl-Heinz , Schubert Sabine , Schreiber Stefan , Fölsch Ulrich R , Rosenstiel Philip , Arlt Alexander 
Published: June 2009

AbstractBackgroundCandida albicansresides on epithelial surfaces as part of the physiological microflora. However, under certain conditions it may cause life-threatening infections likeCandidasepsis. Human β-defensins (hBDs) are critical components of host defense at mucosal surfaces and we have recently shown that hBD-2 and hBD-3 are upregulated inCandidaesophagitis. We therefore studied the role ofCandidate signalling pathways in order to understand the mechanisms involved in regulation of hBD-expression byC. albicans. We used the esophageal cell line OE21 and analysed the role of paracrine signals from polymorphonuclear leukocytes (PMN) in anin vitromodel of esophageal candidiasis.ResultsSupernatants ofC. albicansor indirect coculture withC. albicansinduces upregulation of hBD-2 and hBD-3 expression. PMNs strongly amplifiesC. albicans-mediated induction of hBDs. By EMSA we demonstrate thatC. albicansactivates NF-κB and AP-1 in OE21 cells. Inhibition of these pathways revealed that hBD-2 expression is synergistically regulated by both NF-κB and AP-1. In contrast hBD-3 expression is independent of NF-κB and relies solely on an EGFR/MAPK/AP-1-dependent pathway.ConclusionOur analysis of signal transduction events demonstrate a functional interaction of epithelial cells with PMNs in response toCandidainfection involving divergent signalling events that differentially govern hBD-2 and hBD-3 expression.
Coordinated surface activities in Variovorax paradoxus EPS
Authors: Jamieson W David , Pehl Michael J , Gregory Glenn A , Orwin Paul M 
Published: June 2009

Abstract

Background

Variovorax paradoxus is an aerobic soil bacterium frequently associated with important biodegradative processes in nature. Our group has cultivated a mucoid strain of Variovorax paradoxus for study as a model of bacterial development and response to environmental conditions. Colonies of this organism vary widely in appearance depending on agar plate type.

Results
Surface motility was observed on minimal defined agar plates with 0.5% agarose, similar in nature to swarming motility identified in Pseudomonas aeruginosa PAO1. We examined this motility under several culture conditions, including inhibition of flagellar motility using Congo Red. We demonstrated that the presence of a wetting agent, mineral, and nutrient content of the media altered the swarming phenotype. We also demonstrated that the wetting agent reduces the surface tension of the agar. We were able to directly observe the presence of the wetting agent in the presence and absence of Congo Red, and found that incubation in a humidified chamber inhibited the production of wetting agent, and also slowed the progression of the swarming colony. We observed that swarming was related to both carbon and nitrogen sources, as well as mineral salts base. The phosphate concentration of the mineral base was critical for growth and swarming on glucose, but not succinate. Swarming on other carbon sources was generally only observed using M9 salts mineral base. Rapid swarming was observed on malic acid, d-sorbitol, casamino acids, and succinate. Swarming at a lower but still detectable rate was observed on glucose and sucrose, with weak swarming on maltose. Nitrogen source tests using succinate as carbon source demonstrated two distinct forms of swarming, with very different macroscopic swarm characteristics. Rapid swarming was observed when ammonium ion was provided as nitrogen source, as well as when histidine, tryptophan, or glycine was provided. Slower swarming was observed with methionine, arginine, or tyrosine. Large effects of mineral content on swarming were seen with tyrosine and methionine as nitrogen sources. Biofilms form readily under various culture circumstances, and show wide variance in structure under different conditions. The amount of biofilm as measured by crystal violet retention was dependent on carbon source, but not nitrogen source. Filamentous growth in the biofilm depends on shear stress, and is enhanced by continuous input of nutrients in chemostat culture.

Conclusion
Our studies have established that the beta-proteobacterium Variovorax paradoxus displays a number of distinct physiologies when grown on surfaces, indicative of a complex response to several growth parameters. We have identified a number of factors that drive sessile and motile surface phenotypes. This work forms a basis for future studies using this genetically tractable soil bacterium to study the regulation of microbial development on surfaces.
Lessons from the Pacific programme to eliminate lymphatic filariasis: a case study of 5 countries
Authors: Huppatz Clare , Capuano Corinne , Palmer Kevin , Kelly Paul M , Durrheim David N 
Published: June 2009

Abstract

Background
Lymphatic Filariasis (LF) is an important Neglected Tropical Disease, being a major cause of disability worldwide. The Global Programme to Eliminate Lymphatic Filariasis aims to eliminate LF as a public health problem by the year 2020, primarily through repeated Mass Drug Administration (MDA). The Pacific region programme commenced in 1999. By June 2007, five of the eleven countries classified as endemic had completed five MDA campaigns and post-MDA prevalence surveys to assess their progress. We review available programme data and discuss their implications for other LF elimination programs in developing countries.

Methods
Reported MDA coverage and results from initial surveys and post-MDA surveys of LF using the immunochromatographic test (ICT) from these five Pacific Island countries (Tonga, Niue, Vanuatu, Samoa and Cook Islands) were analysed to provide an understanding of their quality and programme progress towards LF elimination. Denominator data reported by each country programme for 2001 was compared to official sources to assess the accuracy of MDA coverage data.

Results
Initial survey results from these five countries revealed an ICT prevalence of between 2.7 and 8.6 percent in individuals tested prior to commencement of the programme. Country MDA coverage results varied depending on the source of denominator data. Of the five countries in this case study, three countries (Tonga, Niue and Vanuatu) reached the target prevalence of <1% antigenaemia following five rounds of MDA. However, endpoint data could not be reliably compared to baseline data as survey methodology varied.

Conclusion
Accurate and representative baseline and post-campaign prevalence data is crucial for determining program effectiveness and the factors contributing to effectiveness. This is emphasised by the findings of this case study. While three of the five Pacific countries reported achieving the target prevalence of <1% antigenaemia, limitations in the data preclude identification of key determinants of this achievement.
Homozygosity and risk of childhood death due to invasive bacterial disease
Authors: Lyons Emily J , Amos William , Berkley James A , Mwangi Isaiah , Shafi Mohammed , Williams Thomas N , Newton Charles R , Peshu Norbert , Marsh Kevin , Scott J Anthony G , Hill Adrian VS 
Published: June 2009

AbstractBackgroundGenetic heterozygosity is increasingly being shown to be a key predictor of fitness in natural populations, both through inbreeding depression, inbred individuals having low heterozygosity, and also through chance linkage between a marker and a gene under balancing selection. One important component of fitness that is often highlighted is resistance to parasites and other pathogens. However, the significance of equivalent loci in human populations remains unclear. Consequently, we performed a case-control study of fatal invasive bacterial disease in Kenyan children using a genome-wide screen with microsatellite markers.Methods148 cases, comprising children aged <13 years who died of invasive bacterial disease, (variously, bacteraemia, bacterial meningitis or neonatal sepsis) and 137 age-matched, healthy children were sampled in a prospective study conducted at Kilifi District Hospital, Kenya. Samples were genotyped for 134 microsatellite markers using the ABI LD20 marker set and analysed for an association between homozygosity and mortality.ResultsAt five markers homozygosity was strongly associated with mortality (odds ratio range 4.7 – 12.2) with evidence of interactions between some markers. Mortality was associated with different non-overlapping marker groups in Gram positive and Gram negative bacterial disease. Homozygosity at susceptibility markers was common (prevalence 19–49%) and, with the large effect sizes, this suggests that bacterial disease mortality may be strongly genetically determined.ConclusionBalanced polymorphisms appear to be more widespread in humans than previously appreciated and play a critical role in modulating susceptibility to infectious disease. The effect sizes we report, coupled with the stochasticity of exposure to pathogens suggests that infection and mortality are far from random due to a strong genetic basis.
Molecular analysis of the APC and MUTYH genes in Galician and Catalonian FAP families: a different spectrum of mutations?
Authors: Gómez-Fernández Nuria , Castellví-Bel Sergi , Fernández-Rozadilla Ceres , Balaguer Francesc , Muñoz Jenifer , Madrigal Irene , Milà Montserrat , Graña Begoña , Vega Ana , Castells Antoni , Carracedo Ángel , Ruiz-Ponte Clara 
Published: June 2009

Abstract

Background
Familial adenomatous polyposis (FAP) is an autosomal dominant-inherited colorectal cancer syndrome, caused by germline mutations in the APC gene. Recently, biallelic mutations in MUTYH have also been identified in patients with multiple colorectal adenomas and in APC-negative patients with FAP. The aim of this work is therefore to determine the frequency of APC and MUTYH mutations among FAP families from two Spanish populations.

Methods
Eighty-two unrelated patients with classical or attenuated FAP were screened for APC germline mutations. MUTYH analysis was then conducted in those APC-negative families and in 9 additional patients from a previous study. Direct sequencing, SSCP analysis and TaqMan genotyping were used to identify point and frameshift mutations, meanwhile large rearrangements in the APC gene were screened by multiplex ligation-dependent probe amplification (MLPA).

Results

APC germline mutations were found in 39% of the patients and, despite the great number of genetic variants described so far in this gene, seven new mutations were identified. The two hotspots at codons 1061 and 1309 of the APC gene accounted for 9,4% of the APC-positive families, although they were underrepresented in Galician samples. The deletion at codon 1061 was not found in 19 APC-positive Galician patients but represented 23% of the Catalonian positive families (p = 0,058). The same trend was observed at codon 1309, even though statistical analysis showed no significance between populations. Twenty-four percent of the APC-negative patients carried biallelic MUTYH germline mutations, and showed an attenuated polyposis phenotype generally without extracolonic manifestations. New genetic variants were found, as well as the two hotspots already reported (p.Tyr165Cys and p.Gly382Asp).

Conclusion
The results we present indicate that in Galician patients the frequency of the hotspot at codon 1061 in APC differs significantly from the Catalonian and also other Caucasian populations. Similar results had already been obtained in a previous study and could be due to the genetic isolation of the Galician population. MUTYH analysis is also recommended for all APC-negative families, even if a recessive inheritance is not confirmed.
Variations in the NBN/NBS1 gene and the risk of breast cancer in non-BRCA1/2French Canadian families with high risk of breast cancer
Authors: Desjardins Sylvie , Beauparlant Joly Charles , Labrie Yvan , Ouellette Geneviève ,   INHERIT BRCAs, Durocher Francine 
Published: June 2009

AbstractBackgroundThe Nijmegen Breakage Syndrome is a chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency, and increased frequency of cancers. Familial studies on relatives of these patients indicated that they also appear to be at increased risk of cancer.MethodsIn a candidate gene study aiming at identifying genetic determinants of breast cancer susceptibility, we undertook the full sequencing of theNBNgene in our cohort of 97 high-risk non-BRCA1and -BRCA2breast cancer families, along with 74 healthy unrelated controls, also from the French Canadian population.In silicoprograms (ESEfinder, NNSplice, Splice Site Finder and MatInspector) were used to assess the putative impact of the variants identified. The effect of the promoter variant was further studied by luciferase gene reporter assay in MCF-7, HEK293, HeLa and LNCaP cell lines.ResultsTwenty-four variants were identified in our case series and their frequency was further evaluated in healthy controls. The potentially deleterious p.Ile171Val variant was observed in one case only. The p.Arg215Trp variant, suggested to impair NBN binding to histone γ-H2AX, was observed in one breast cancer case and one healthy control. A promoter variant c.-242-110delAGTA displayed a significant variation in frequency between both sample sets. Luciferase reporter gene assay of the promoter construct bearing this variant did not suggest a variation of expression in the MCF-7 breast cancer cell line, but indicated a reduction of luciferase expression in both the HEK293 and LNCaP cell lines.ConclusionOur analysis ofNBNsequence variations indicated that potentialNBNalterations are present, albeit at a low frequency, in our cohort of high-risk breast cancer cases. Further analyses will be needed to fully ascertain the exact impact of those variants on breast cancer susceptibility, in particular for variants located inNBNpromoter region.
High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization
Authors: Kato Tatsuya , Manoha Suganthi Lavender , Tanaka Shigeyasu , Park Enoch Y 
Published: June 2009

Abstract

Background
Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR) that displayed human (pro)renin receptor (hPRR) connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC).

Results
A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR). A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 108 plaque forming unit (pfu) in hemolymph, which was 2.8 × 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i.), but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa) to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i..

Conclusion
The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.
Substantial reduction of inappropriate tablet splitting with computerised decision support: a prospective intervention study assessing potential benefit and harm
Authors: Quinzler Renate , Schmitt Simon PW , Pritsch Maria , Kaltschmidt Jens , Haefeli Walter E 
Published: June 2009

Abstract

Background
Currently ambulatory patients break one in four tablets before ingestion. Roughly 10% of them are not suitable for splitting because they lack score lines or because enteric or modified release coating is destroyed impairing safety and effectiveness of the medication. We assessed impact and safety of computerised decision support on the inappropriate prescription of split tablets.

Methods
We performed a prospective intervention study in a 1680-bed university hospital. Over a 15-week period we evaluated all electronically composed medication regimens and determined the fraction of tablets and capsules that demanded inappropriate splitting. In a subsequent intervention phase of 15 weeks duration for 10553 oral drugs divisibility characteristics were indicated in the system. In addition, an alert was generated and displayed during the prescription process whenever the entered dosage regimen demanded inappropriate splitting (splitting of capsules, unscored tablets, or scored tablets unsuitable for the intended fragmentation).

Results
During the baseline period 12.5% of all drugs required splitting and 2.7% of all drugs (257/9545) required inappropriate splitting. During the intervention period the frequency of inappropriate splitting was significantly reduced (1.4% of all drugs (146/10486); p = 0.0008). In response to half of the alerts (69/136) physicians adjusted the medication regimen. In the other half (67/136) no corrections were made although a switch to more suitable drugs (scored tablets, tablets with lower strength, liquid formulation) was possible in 82% (55/67).

Conclusion
This study revealed that computerised decision support can immediately reduce the frequency of inappropriate splitting without introducing new safety hazards.