Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

<p>Abstract</p> <p>Background</p> <p>Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated V_H/V_Linterfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1.</p> <p>Results</p> <p>Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either <it>Discosoma </it>or <it>Aequorea </it>in-between the variable regions of anti-p185^HER2-ECDantibody 4D5-8 resulted in optimal V_H/V_Linterface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly₄Ser)₃linker precipitated at physiological pH 7.4.</p> <p>Conclusions</p> <p>This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative <it>in situ, in vivo </it>and <it>ex vivo </it>imaging, cell sorting and cell trafficking studies. Assembling the single chain antibody with monomeric fluorescent protein linker facilitates optimal variable domain pairing and alters the isoelectric point of the recombinant 4D5-8 protein conferring solubility at physiological pH 7.4. The efficient intracellular expression of these functional molecules opens up the possibility of developing an alternative approach for tagging intracellular targets with fluorescent proteins for a range of molecular cell biology imaging studies.</p

Bioinformatics analysis of the locus for enterocyte effacement provides novel insights into type-III secretion

BACKGROUND: Like many other pathogens, enterohaemorrhagic and enteropathogenic strains of Escherichia coli employ a type-III secretion system to translocate bacterial effector proteins into host cells, where they then disrupt a range of cellular functions. This system is encoded by the locus for enterocyte effacement. Many of the genes within this locus have been assigned names and functions through homology with the better characterised Ysc-Yop system from Yersinia spp. However, the functions and homologies of many LEE genes remain obscure. RESULTS: We have performed a fresh bioinformatics analysis of the LEE. Using PSI-BLAST we have been able to identify several novel homologies between LEE-encoded and Ysc-Yop-associated proteins: Orf2/YscE, Orf5/YscL, rORF8/EscI, SepQ/YscQ, SepL/YopN-TyeA, CesD2/LcrR. In addition, we highlight homology between EspA and flagellin, and report many new homologues of the chaperone CesT. CONCLUSION: We conclude that the vast majority of LEE-encoded proteins do indeed possess homologues and that homology data can be used in combination with experimental data to make fresh functional predictions

Easyfig: a genome comparison visualizer

Summary: Easyfig is a Python application for creating linear comparison figures of multiple genomic loci with an easy-to-use graphical user interface. BLAST comparisons between multiple genomic regions, ranging from single genes to whole prokaryote chromosomes, can be generated, visualized and interactively coloured, enabling a rapid transition between analysis and the preparation of publication quality figures

Secretion of flagellin by the LEE-encoded type III secretion system of enteropathogenic Escherichia coli

Background: Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen that possesses a type III secretion system (T3SS) encoded within the locus of enterocyte effacement (LEE). The LEE is essential for A/E lesion formation and directs the secretion and translocation of multiple LEE-encoded and non-LEE encoded effector proteins into the cytosol of infected cells. In this study we used proteomics to compare proteins exported to the culture supernatant by wild type EPEC E2348/69, a Delta espADB mutant and a Delta escF T3SS mutant

Performance of externally validated enhanced computer-aided versions of the National Early Warning Score in predicting mortality following an emergency admission to hospital in England: a cross-sectional study

YesOBJECTIVES: In the English National Health Service, the patient's vital signs are monitored and summarised into a National Early Warning Score (NEWS) to support clinical decision making, but it does not provide an estimate of the patient's risk of death. We examine the extent to which the accuracy of NEWS for predicting mortality could be improved by enhanced computer versions of NEWS (cNEWS). DESIGN: Logistic regression model development and external validation study. SETTING: Two acute hospitals (YH-York Hospital for model development; NH-Northern Lincolnshire and Goole Hospital for external model validation). PARTICIPANTS: Adult (≥16 years) medical admissions discharged over a 24-month period with electronic NEWS (eNEWS) recorded on admission are used to predict mortality at four time points (in-hospital, 24 hours, 48 hours and 72 hours) using the first electronically recorded NEWS (model M0) versus a cNEWS model which included age+sex (model M1) +subcomponents of NEWS (including diastolic blood pressure) (model M2). RESULTS: The risk of dying in-hospital following emergency medical admission was 5.8% (YH: 2080/35 807) and 5.4% (NH: 1900/35 161). The c-statistics for model M2 in YH for predicting mortality (in-hospital=0.82, 24 hours=0.91, 48 hours=0.88 and 72 hours=0.88) was higher than model M0 (in-hospital=0.74, 24 hours=0.89, 48 hours=0.86 and 72 hours=0.85) with higher Positive Predictive Value (PPVs) for in-hospital mortality (M2 19.3% and M0 16.6%). Similar findings were seen in NH. Model M2 performed better than M0 in almost all major disease subgroups. CONCLUSIONS: An externally validated enhanced computer-aided NEWS model (cNEWS) incrementally improves on the performance of a NEWS only model. Since cNEWS places no additional data collection burden on clinicians and is readily automated, it may now be carefully introduced and evaluated to determine if it can improve care in hospitals that have eNEWS systems.This research was supported by the Health Foundation. The Health Foundation is an independent charity working to improve the quality of healthcare in the UK. This research was also supported by the National Institute for Health Research (NIHR) Yorkshire and Humberside Patient Safety Translational Research Centre (YHPSTRC)

Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species

Background: Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii

Macroinvertebrate responses to flow and stream temperature variability across regulated and non-regulated rivers

Flow regulation via impoundments threatens lotic ecosystems and the services they provide globally. Impoundments drastically alter flow and stream temperature variability within fluvial environments, but efforts to quantify ecohydrological and ecothermal responses to flow regulation in conjunction have been sparsely explored to date. This study examined macroinvertebrate community responses to river flow (discharge) and stream temperature variability across paired regulated and non-regulated systems associated with three reservoirs located in adjacent catchments. Community abundances, functional traits and biomonitoring indices were examined and ecological differences between non-regulated and regulated sites were quantified, with the most sensitive faunal response being correlated against a suite of flow and thermal indices. Regulated sites exhibited reduced low-flow variability and rapid increases in discharge during peak flows that regularly exceeded those conveyed by non-regulated sites, whilst stream temperature variability was highly congruent between sites. Macroinvertebrate functional traits were particularly sensitive to flow regulation and incorporating biomonitoring indices marginally improved the ecological discrimination between regulated and non-regulated sites. Unlike community abundances, functional traits did not vary spatially between catchments, highlighting that such information could guide the implementation of regionally uniform environmental flows. Macroinvertebrate communities responded significantly to various hydrological parameters, particularly those associated with the timing of extreme flows, but were less sensitive to thermal controls. Future research should explore ecological responses to antecedent hydrological and stream temperature variability associated with flow regulation to provide a better understanding of the underlying mechanisms driving biotic alterations, which could guide future environmental flow methodologies

The inclusion of delirium in version 2 of the National Early Warning Score will substantially increase the alerts for escalating levels of care: findings from a retrospective database study of emergency medical admissions in two hospitals

YesBackground The National Early Warning Score (NEWS) is being replaced with NEWS2 which adds 3 points for new confusion or delirium. We estimated the impact of adding delirium on the number of medium/high level alerts that are triggers to escalate care. Methods Analysis of emergency medical admissions in two acute hospitals (York Hospital (YH) and Northern Lincolnshire and Goole NHS Foundation Trust hospitals (NH)) in England. Twenty per cent were randomly assigned to have delirium. Results The number of emergency admissions (YH: 35584; NH: 35795), mortality (YH: 5.7%; NH: 5.5%), index NEWS (YH: 2.5; NH: 2.1) and numbers of NEWS recorded (YH: 879193; NH: 884072) were similar in each hospital. The mean number of patients with medium level alerts per day increased from 55.3 (NEWS) to 69.5 (NEWS2), a 25.7% increase in YH and 64.1 (NEWS) to 77.4 (NEWS2), a 20.7% increase in NH. The mean number of patients with high level alerts per day increased from 27.3 (NEWS) to 34.4 (NEWS2), a 26.0% increase in YH and 29.9 (NEWS) to 37.7 (NEWS2), a 26.1% increase in NH. Conclusions The addition of delirium in NEWS2 will have a substantial increase in medium and high level alerts in hospitalised emergency medical patients. Rigorous evaluation of NEWS2 is required before widespread implementation because the extent to which staff can cope with this increase without adverse consequences remains unknown

An Interprofessional End of Life Simulation Using a Movie/Discussion Format

An Interprofessional End of Life Simulation Using a Movie/Discussion Format Sigma Theta Tau International Nursing Research Congress Cape Town, South Africa July 25, 2016 Mary Val Palumbo, DNP, MSN, BS, APRN, GNP-BC1 Christina S. Melvin, MS, BS, PHCNS, BC, CHPN1 Nancy P. LeMieux, MSN, BSEd, RN1 Deborah A. O\u27Rouke, PhD, MA, MClSc, BSc, PT2 Jean Beatson, EdD, MS, RN3 Patricia A. Prelock, PhD, MA, BS, CCC-SLP, BCS-CL4 Kelly A. Melekis, PhD, MSW, LMSW5 Mary Alice Favro, MA, BS, CCC-SLP6 Vicki Hart, PhD7 (1)Department of Nursing, University of Vermont, Burlington, VT (2)Dept. of Rehabilitation and Movement Science, University of Vermont, Burlington, VT (3)Vermont Interdisciplinary Leadership Education for Health Professionals Program, University Of Vermont, Burlington, VT (4)Department of Communication Sciences & Disorders, University of Vermont, Burlington, VT (5)Department of Social Work, University of Vermont, Burlington, VT (6)Department of Communication Sciences and Disorders, University of Vermont, Burlington, VT, VT (7)Office of Health Promotion Research, University of Vermont, Burlington, VT Purpose: The interprofessional team’s role in end of life care provides a unique opportunity for an educational simulation. Novel ways to teach this content to a large number of students were needed. This research evaluated a palliative care interprofessional educational activity for healthcare students from six disciplines using an in-person movie/discussion format. Methods: Healthcare provider assessment visits and a death scene were pilot tested in actual simulations using twenty four students with standardized patients portraying a patient with ALS and his husband. Scripts were written from recordings of these simulations and were used in the dialogue of the movie which had standardized patient actors playing the patient, husband, sister and the student healthcare providers. The movie portrayed a patient with ALS and his husband in five scenes: assessment of patient and family needs by nursing, physical therapy, social work and speech language pathology; and a death scene. The movie was then edited to be used for discussion by large student groups. An evaluation survey was developed with 14 questions scored on a Likert scale and related to Interprofessional Competency Domains of 1. Values/Ethics, 2. Roles/Responsibilities, 3. Interprofessional communication, and 4. Teams/teamwork. These competencies provided a structure for the evaluation questions, and a review by faculty in each discipline also provided content validity. Reliability of the survey instrument was not tested or established. Graduate nurse practitioner, medical, communication sciences and disorders, social work and physical therapy students as well as undergraduate nursing students participated together in end of life movie discussions in four sessions held in a classroom equipped with tables of six. Survey responses were compared across disciplines and across the four interprofessional competency domains using a Chi-square test and adjusting for multiple comparisons. Significant differences were established at p Results: Student evaluations of the end of life simulations were consistently positive. Eighty percent of the participants (n=162) strongly agreed that they recognize the necessity of utilizing a healthcare team when providing care for patients at the end of life. All participants agreed that they would continue to forge relationships with other healthcare professionals to improve care for elders. Physical therapy students were consistently had the most positive responses; however, significant differences were only observed in three of the 14 questions. There were no significant differences between disciplines across the four interprofessional competency domains. A summary of qualitative data from the open ended questions will be also be presented. Conclusion: Interprofessional end of life care can be simulated in a carefully planned activity. The value of the experience was expressed by all students. Nurse practitioner students valued being able to practice supervising a palliative care team and facilitating the team discussion. Other students appreciated a safe place to discuss how to care for a dying patient and their family. Regarding the evaluation, small sample sizes in some disciplines may have limited the ability to detect significant differences between groups. Production of a movie and the discussion of the different scenes helped lower the cost of the simulation and scale up the activity for use with large groups of students. Interprofessional education requires innovative pedagogy that must be evaluated and disseminated. Consistent evaluation of interprofessional practice competency domains must be included in all educational activities

The co-transcriptome of uropathogenic Escherichia coli-infected mouse macrophages reveals new insights into host-pathogen interactions

© 2014 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd. Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic Escherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional programme associated with intramacrophage survival, we performed host-pathogen co-transcriptome analyses using RNA sequencing. Mouse bone marrow-derived macrophages (BMMs) were challenged over a 24h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated and quantified the mammalian and bacterial transcriptomes. Bone marrow-derived macrophages responded to the two UPEC strains with a broadly similar gene expression programme. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes up-regulated at 24h post-infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co-cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment