Development of a DNA probe colony hybridization hydrophobic grid membrane filter method for detection of Escherichia coli isolates carrying virulence and antimicrobial resistance genes in the pig intestinal microflora

The objective of this study was to develop a DNA probe hybridization procedure using hydrophobic-grid membrane filters (HGMFs) for the detection of E. coli isolates carrying specific virulence or antimicrobial resistance genes. Genes were detected using digoxigenin-labelled probes and an alkaline-phosphatase substrate. Specifically, the eft, estB, and faeG genes encoding the enterotoxins LT, STb, and fimbrial adhesin F4 were targeted. The hybridization procedure was optimized using E. coli strain ECL 7805 which possesses the three targeted genes, and rectal swabs from weaned pigs excreting E. coli positive for these genes, as demonstrated by enrichment PCR

Rethinking Industrial Relations revisited

This article traces the origins and consequences of the book Rethinking Industrial Relations and then explores some of the conceptual and empirical issues raised since its publication

Recruiting women with ductal carcinoma in situ to a randomised controlled trial: lessons from the LORIS study

BackgroundThe LOw RISk DCIS (LORIS) study was set up to compare conventional surgical treatment with active monitoring in women with ductal carcinoma in situ (DCIS). Recruitment to trials with a surveillance arm is known to be challenging, so strategies to maximise patient recruitment, aimed at both patients and recruiting centres, were implemented.MethodsWomen aged ≥ 46 years with a histologically confirmed diagnosis of non-high-grade DCIS were eligible for 1:1 randomisation to either surgery or active monitoring. Prior to randomisation, all eligible women were invited to complete: (1) the Clinical Trials Questionnaire (CTQ) examining reasons for or against participation, and (2) interviews exploring in depth opinions about the study information sheets and film. Women agreeing to randomisation completed validated questionnaires assessing health status, physical and mental health, and anxiety levels. Hospital site staff were invited to communication workshops and refresher site initiation visits to support recruitment. Their perspectives on LORIS recruitment were collected via surveys and interviews.ResultsEighty percent (181/227) of eligible women agreed to be randomised. Over 40% of participants had high anxiety levels at baseline. On the CTQ, the most frequent most important reasons for accepting randomisation were altruism and belief that the trial offered the best treatment, whilst worries about randomisation and the influences of others were the most frequent most important reasons for declining. Most women found the study information provided clear and useful. Communication workshops for site staff improved knowledge and confidence but only about half said they themselves would join LORIS if eligible. The most common recruitment barriers identified by staff were low numbers of eligible patients and patient preference.ConclusionsRecruitment to LORIS was challenging despite strategies aimed at both patients and site staff. Ensuring that recruiting staff support the study could improve recruitment in similar future trials

Addressing overtreatment of screen detected DCIS; the LORIS trial

Abstract Overdiagnosis, and thus overtreatment, are inevitable consequences of most screening programmes; identification of ways of minimising the impact of overdiagnosis demands new prospective research, in particular the need to separate clinically relevant lesions that require active treatment from those that can be safely left alone or monitored and only need treated if they change characteristics. Breast cancer screening has led to a large increase in ductal carcinoma in situ (DCIS) diagnoses. This is a widely heterogeneous disease and most DCIS detected through screening is of high cytonuclear grade and therefore likely to be important clinically. However, the historic practice of surgical treatment for all DCIS is unlikely to be optimal for lower risk patients. A clearer understanding of how to manage DCIS is required. This article describes the background and development of ‘The low risk’ DCIS trial (LORIS), a phase III trial of surgery versus active monitoring. LORIS will determine if it is appropriate to manage women with screen detected or asymptomatic, low grade and intermediate grade DCIS with low grade features, by active monitoring rather than by surgical treatment

SNPs Occur in Regions with Less Genomic Sequence Conservation

Rates of SNPs (single nucleotide polymorphisms) and cross-species genomic sequence conservation reflect intra- and inter-species variation, respectively. Here, I report SNP rates and genomic sequence conservation adjacent to mRNA processing regions and show that, as expected, more SNPs occur in less conserved regions and that functional regions have fewer SNPs. Results are confirmed using both mouse and human data. Regions include protein start codons, 3′ splice sites, 5′ splice sites, protein stop codons, predicted miRNA binding sites, and polyadenylation sites. Throughout, SNP rates are lower and conservation is higher at regulatory sites. Within coding regions, SNP rates are highest and conservation is lowest at codon position three and the fewest SNPs are found at codon position two, reflecting codon degeneracy for amino acid encoding. Exon splice sites show high conservation and very low SNP rates, reflecting both splicing signals and protein coding. Relaxed constraint on the codon third position is dramatically seen when separating exonic SNP rates based on intron phase. At polyadenylation sites, a peak of conservation and low SNP rate occurs from 30 to 17 nt preceding the site. This region is highly enriched for the sequence AAUAAA, reflecting the location of the conserved polyA signal. miRNA 3′ UTR target sites are predicted incorporating interspecies genomic sequence conservation; SNP rates are low in these sites, again showing fewer SNPs in conserved regions. Together, these results confirm that SNPs, reflecting recent genetic variation, occur more frequently in regions with less evolutionarily conservation

Protocol for a national cohort study to explore the long-term clinical and patient-reported outcomes and cost-effectiveness of implant-based and autologous breast reconstruction after mastectomy for breast cancer: The Brighter Study

Introduction Breast reconstruction (BR) is offered to improve quality of life for women with breast cancer undergoing mastectomy. As most women will be long-Term breast cancer survivors, high-quality information regarding the long-Term outcomes of different BR procedures is essential to support informed decision-making. As different techniques vary considerably in cost, policymakers also require high-quality cost-effectiveness evidence to inform care. The Brighter study aims to explore the long-Term clinical and patient-reported outcomes (PROs) of implant-based and autologous BR and use health economic modelling to compare the long-Term cost-effectiveness of different reconstructive techniques. Methods and analysis Women undergoing mastectomy and/or BR following a diagnosis of breast cancer between 1 January 2008 and 31 March 2009 will be identified from hospital episode statistics (HES). Surviving women will be contacted and invited to complete validated PRO measures including the BREAST-Q, EQ-5D-5L and ICECAP-A, or opt out of having their data included in the HES analysis. Long-Term clinical outcomes will be explored using HES data. The primary outcome will be rates of revisional surgery between implant-based and autologous procedures. Secondary outcomes will include rates of secondary reconstruction and reconstruction failure. The long-Term PROs of implant-based and autologous reconstruction will be compared using BREAST-Q, EQ-5D-5L and ICECAP-A scores. Multivariable regression will be used to examine the relationship between long-Term outcomes, patient comorbidities, sociodemographic and treatment factors. A Markov model will be developed using HES and PRO data and published literature to compare the relative long-Term cost-effectiveness of implant-based and autologous BR. Ethics and dissemination The Brighter study has been approved by the South-West-Central Bristol Research Ethics Committee (20/SW/0020), and the Confidentiality Advisory Group (20/CAG/0021). Results will be published in peer-reviewed journals and presented at national meetings. We will work with the professional associations, charities and patient groups to disseminate the results

Features generated for computational splice-site prediction correspond to functional elements

<p>Abstract</p> <p>Background</p> <p>Accurate selection of splice sites during the splicing of precursors to messenger RNA requires both relatively well-characterized signals at the splice sites and auxiliary signals in the adjacent exons and introns. We previously described a feature generation algorithm (FGA) that is capable of achieving high classification accuracy on human 3' splice sites. In this paper, we extend the splice-site prediction to 5' splice sites and explore the generated features for biologically meaningful splicing signals.</p> <p>Results</p> <p>We present examples from the observed features that correspond to known signals, both core signals (including the branch site and pyrimidine tract) and auxiliary signals (including GGG triplets and exon splicing enhancers). We present evidence that features identified by FGA include splicing signals not found by other methods.</p> <p>Conclusion</p> <p>Our generated features capture known biological signals in the expected sequence interval flanking splice sites. The method can be easily applied to other species and to similar classification problems, such as tissue-specific regulatory elements, polyadenylation sites, promoters, etc.</p

Erythrocyte and Porcine Intestinal Glycosphingolipids Recognized by F4 Fimbriae of Enterotoxigenic Escherichia coli

Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAcα3GalNAcß3Galß4Glcß1Cer and GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO3-3Galß1Cer), sulf-lactosylceramide (SO3-3Galß4Glcß1Cer), and globotriaosylceramide (Galα4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Galα3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer)

Passerine Exposure to Primarily PCDFs and PCDDs in the River Floodplains Near Midland, Michigan, USA

House wren (Troglodytes aedon), tree swallow (Tachycineta bicolor), and eastern bluebird (Sialia sialis) tissues collected in study areas (SAs) downstream of Midland, Michigan (USA) contained concentrations of polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzo-p-dioxins (PCDDs) greater than in upstream reference areas (RAs) in the region. The sum of concentrations of PCDD/DFs (ΣPCDD/DFs) in eggs of house wrens and eastern bluebirds from SAs were 4- to 22-fold greater compared to those from RAs, whereas concentrations in tree swallow eggs were similar among areas. Mean concentrations of ΣPCDD/DFs and sum 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (ΣTEQsWHO-Avian), based on 1998 WHO avian toxic equivalency factors, in house wren and eastern bluebird eggs ranged from 860 (430) to 1500 (910) ng/kg wet weight (ww) and 470 (150) to 1100 (510) ng/kg ww, respectively, at the most contaminated study areas along the Tittabawassee River, whereas mean concentrations in tree swallow eggs ranged from 280 (100) to 760 (280) ng/kg ww among all locations. Concentrations of ΣPCDD/DFs in nestlings of all studied species at SAs were 3- to 50-fold greater compared to RAs. Mean house wren, tree swallow, and eastern bluebird nestling concentrations of ΣPCDD/DFs and ΣTEQsWHO-Avian ranged from 350 (140) to 610 (300) ng/kg ww, 360 (240) to 1100 (860) ng/kg ww, and 330 (100) to 1200 (690) ng/kg ww, respectively, at SAs along the Tittabawassee River. Concentrations of ΣTEQsWHO-Avian were positively correlated with ΣPCDD/DF concentrations in both eggs and nestlings of all species studied. Profiles of relative concentrations of individual congeners were dominated by furan congeners (69–84%), primarily 2,3,7,8-tetrachlorodibenzofuran and 2,3,4,7,8-pentachlorodibenzofuran, for all species at SAs on the Tittabawassee and Saginaw rivers but were dominated by dioxin congeners at upstream RAs