Identification of protein carbonyls in serum of the fetal and neonatal pig

Oxidation of serum proteins leads to non-reversible carbonyl formation which alters their function and is associated with stress-related disease processes. The primary objective of this study was to quantify and identify oxidized serum proteins in fetal and newborn piglets. Protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and quantified spectrophotometrically. For identification, serum protein carbonyls were derivatized with biotin hydrazide, separated by 2D PAGE and stained with FITCavidin. Biotin-labeled proteins were excised from gels and identified by mass spectrometry. At birth, carbonyls were determined to be ∼600 pmole/mg serum protein. Fetuses at 50 and 100 days of gestation had similar levels of protein carbonyls as newborns. Carbonyl levels were also similar for control and runt (birth) piglets between 1 and 21 days of age; however, distribution of many proteins varied by age and was also influenced by birth weight. Major oxidized proteins identified in fetal (f) and newborn (n) pigs included; albumin (f, n), transferrin (f, n), fetuin-A (f, n) alpha fetoprotein (f, n), plasminogen (f, n), fetuin-B (f), alpha-1-antitrypsin (f, n) alpha-1-acid glycoprotein (f) and immunoglobulins (n). While abundance and distribution of oxidized proteins changed over time, these changes appear to primarily reflect relative amounts of those proteins in serum

Ultraviolet Emission Line Ratios of Cataclysmic Variables

We present a statistical analysis of the ultraviolet emission lines of cataclysmic variables (CVs) based on $\approx 430$ ultraviolet spectra of 20 sources extracted from the International Ultraviolet Explorer Uniform Low Dispersion Archive. These spectra are used to measure the emission line fluxes of N V, Si IV, C IV, and He II and to construct diagnostic flux ratio diagrams. We investigate the flux ratio parameter space populated by individual CVs and by various CV subclasses (e.g., AM Her stars, DQ Her stars, dwarf novae, nova-like variables). For most systems, these ratios are clustered within a range of $\sim 1$ decade for log Si IV/C IV $\approx -0.5$ and log He II/C IV $\approx -1.0$ and $\sim 1.5$ decades for log N V/C IV $\approx -0.25$. These ratios are compared to photoionization and collisional ionization models to constrain the excitation mechanism and the physical conditions of the line-emitting gas. We find that the collisional models do the poorest job of reproducing the data. The photoionization models reproduce the Si IV/C IV line ratios for some shapes of the ionizing spectrum, but the predicted N V/C IV line ratios are simultaneously too low by typically $\sim 0.5$ decades. Worse, for no parameters are any of the models able to reproduce the observed He II/C IV line ratios; this ratio is far too small in the collisional and scattering models and too large by typically $\sim 0.5$ decades in the photoionization models.Comment: LaTeX format, uses aaspp4.sty, 28 pages, 11 Postscript figures, accepted for publication in The Astrophysical Journal 10/16/9

‘Intensive care unit survivorship’ - a constructivist grounded theory of surviving critical illness

Aims & objectiveTo theorise ICU survivorship after a critical illness based on longitudinal qualitative data.BackgroundIncreasingly patients survive episodes of critical illness. However, the short and long term impact of critical illness include physical, psychological, social and economic challenges long after hospital discharge. An appreciation is emerging that care needs to extend beyond critical illness to enable patients to reclaim their lives post-discharge with the term ‘Survivorship’ being increasingly used in this context. What constitutes critical illness survivorship has, to date, not been theoretically explored.DesignLongitudinal-qualitative and constructivist Grounded Theory. Interviews (n = 46) with 17 participants were conducted at four time points: (1) before discharge from hospital, (2) 4-6 weeks post-discharge, (3) 6 months and (4) 12 months post-discharge across two adult intensive care setting.MethodIndividual face-to-face interviews. Data analysis followed the principles of Charmaz's Constructivist Grounded Theory. ‘ICU survivorship’ emerged as the core category and was theorised using concepts such as Status Passages, Liminality and Temporality to understand the various transitions participants made post-critical illness.FindingsIntensive care survivorship describes the unscheduled status passage of falling critically ill and being taken to the threshold of life and the journey to a life post-critical illness. Surviving critical illness goes beyond recovery; surviving means ‘moving on’ to life post-critical illness. ‘Moving on’ incorporates a re-definition of self that incorporates any lingering intensive care legacies and being in control of one's life again.Relevance to clinical practiceFor healthcare professionals and policy makers it is important to realise that recovery and transitioning through to survivorship happens within an individual's time frame, not a schedule imposed by the healthcare system. Currently there are no care pathways or policies in place for critical illness survivors that would support ICU survivors and their families in the transitions to survivorship

A longitudinal qualitative exploration of healthcare and informal support needs among survivors of critical illness: the RELINQUISH protocol

Introduction and background: Survival following critical illness is associated with a significant burden of physical, emotional and psychosocial morbidity. Recovery can be protracted and incomplete, with important and sustained effects upon everyday life, including family life, social participation and return to work. In stark contrast with other critically ill patient groups (eg, those following cardiothoracic surgery), there are comparatively few interventional studies of rehabilitation among the general intensive care unit patient population. This paper outlines the protocol for a sub study of the RECOVER study: a randomised controlled trial evaluating a complex intervention of enhanced ward-based rehabilitation for patients following discharge from intensive care. Methods and analysis: The RELINQUISH study is a nested longitudinal, qualitative study of family support and perceived healthcare needs among RECOVER participants at key stages of the recovery process and at up to 1 year following hospital discharge. Its central premise is that recovery is a dynamic process wherein patients’ needs evolve over time. RELINQUISH is novel in that we will incorporate two parallel strategies into our data analysis: (1) a pragmatic health services-oriented approach, using an a priori analytical construct, the ‘Timing it Right’ framework and (2) a constructivist grounded theory approach which allows the emergence of new themes and theoretical understandings from the data. We will subsequently use Qualitative Health Needs Assessment methodology to inform the development of timely and responsive healthcare interventionsthroughout the recovery process.Ethics and dissemination: The protocol has been approved by the Lothian Research Ethics Committee (protocol number HSRU011). The study has been added to the UK Clinical Research Network Database(study ID. 9986). The authors will disseminate the findings in peer reviewed publications and to relevant critical care stakeholder groups

Global utilization of low-dose corticosteroids in severe sepsis and septic shock: a report from the PROGRESS registry

The benefits and use of low-dose corticosteroids (LDCs) in severe sepsis and septic shock remain controversial. Surviving sepsis campaign guidelines suggest LDC use for septic shock patients poorly responsive to fluid resuscitation and vasopressor therapy. Their use is suspected to be wide-spread, but paucity of data regarding global practice exists. The purpose of this study was to compare baseline characteristics and clinical outcomes of patients treated or not treated with LDC from the international PROGRESS (PROmoting Global Research Excellence in Severe Sepsis) cohort study of severe sepsis.Journal ArticleMulticenter StudyResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

POT1 mutations predispose to familial melanoma

Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases, and rare variants in CDK4, BRCA2, BAP1 and the promoter of TERT have also been linked to the disease. Here we set out to identify new high-penetrance susceptibility genes by sequencing 184 melanoma cases from 105 pedigrees recruited in the UK, The Netherlands and Australia that were negative for variants in known predisposition genes. We identified families where melanoma cosegregates with loss-of-function variants in the protection of telomeres 1 gene (POT1), with a proportion of family members presenting with an early age of onset and multiple primary tumors. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding and leading to increased telomere length. These findings suggest that POT1 variants predispose to melanoma formation via a direct effect on telomeres.D.J.A., C.D.R.-E., Z.D., J.Z.L., J.C.T., M.P. and T.M.K. were supported by Cancer Research UK and the Wellcome Trust (WT098051). C.D.R.-E. was also supported by the Consejo Nacional de Ciencia y Tecnología of Mexico. K.A.P. and A.M.D. were supported by Cancer Research UK (grants C1287/A9540 and C8197/A10123) and by the Isaac Newton Trust. N.K.H. was supported by a fellowship from the National Health and Medical Research Council of Australia (NHMRC). L.G.A. was supported by an Australia and New Zealand Banking Group Limited Trustees PhD scholarship. A.L.P. is supported by Cure Cancer Australia. The work was funded in part by the NHMRC and Cancer Council Queensland. The work of N.A.G. was in part supported by the Dutch Cancer Society (UL 2012-5489). M.H., J.A.N.-B. and D.T.B. were supported by Cancer Research UK (programme awards C588/A4994 and C588/A10589 and the Genomics Initiative). C.L.-O., A.J.R. and V.Q. are funded by the Spanish Ministry of Economy and Competitiveness through the Instituto de Salud Carlos III (ISCIII), the Red Temática de Investigación del Cáncer (RTICC) del ISCIII and the Consolider-Ingenio RNAREG Consortium. C.L.-O. is an investigator with the Botín Foundation.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ng.294

A randomised controlled trial evaluating a rehabilitation complex intervention for patients following intensive care discharge: the RECOVER study

Introduction: Patients who survive an intensive care unit admission frequently suffer physical and psychological morbidity for many months after discharge. Current rehabilitation pathways are often fragmented and little is known about the optimum method of promoting recovery. Many patients suffer reduced quality of life.Methods and analysis: The authors plan a multicentre randomised parallel group complex intervention trial with concealment of group allocation from outcome assessors. Patients who required more than 48 h of mechanical ventilation and are deemed fit for intensive care unit discharge will be eligible. Patients with primary neurological diagnoses will be excluded. Participants will be randomised into one of the two groups: the intervention group will receive standard ward-based care delivered by the NHS service with additional treatment by a specifically trained generic rehabilitation assistant during ward stay and via telephone contact after hospital discharge and the control group will receive standard ward-based care delivered by the current NHS service. The intervention group will also receive additional information about their critical illness and access to a critical care physician. The total duration of the intervention will be from randomisation to 3 months postrandomisation. The total duration of follow-up will be 12 months from randomisation for both groups. The primary outcome will be the Rivermead Mobility Index at 3 months. Secondary outcomes will include measures of physical and psychological morbidity and function, quality of lifeand survival over a 12-month period. A health economic evaluation will also be undertaken. Groups will be compared in relation to primary and secondary outcomes; quantitative analyses will be supplemented by focus groups with patients, carers and healthcare workers.Ethics and dissemination: Consent will be obtained from patients and relatives according to patient capacity. Data will be analysed accordingto a predefined analysis plan

A rehabilitation intervention to promote physical recovery following intensive care: a detailed description of construct development, rationale and content together with proposed taxonomy to capture processes in a randomised controlled trial.

Background: increasing numbers of patients are surviving critical illness, but survival may be associated with aconstellation of physical and psychological sequelae that can cause on going disability and reduced health-relatedquality of life. Limited evidence currently exists to guide the optimum structure, timing, and content of rehabilitationprogrammes. There is a need to both develop and evaluate interventions to support and expedite recovery during the post-ICU discharge period. This paper describes the construct development for a complex rehabilitationintervention intended to promote physical recovery following critical illness. The intervention is currently beingevaluated in a randomised trial (ISRCTN09412438; funder Chief Scientists Office, Scotland).Methods: the intervention was developed using the Medical Research Council (MRC) framework for developingcomplex healthcare interventions. We ensured representation from a wide variety of stakeholders includingcontent experts from multiple specialties, methodologists, and patient representation. The intervention constructwas initially based on literature review, local observational and audit work, qualitative studies with ICU survivors,and brainstorming activities. Iterative refinement was aided by the publication of a National Institute for Healthand Care Excellence guideline (No. 83), publicly available patient stories (Healthtalkonline), a stakeholder event incollaboration with the James Lind Alliance, and local piloting. Modelling and further work involved a feasibility trial and development of a novel generic rehabilitation assistant (GRA) role. Several rounds of external peer review during successive funding applications also contributed to development.Results: the final construct for the complex intervention involved a dedicated GRA trained to pre-definedcompetencies across multiple rehabilitation domains (physiotherapy, dietetics, occupational therapy, and speech/language therapy), with specific training in post-critical illness issues. The intervention was from ICU discharge to 3 months post-discharge, including inpatient and post-hospital discharge elements. Clear strategies to provide information to patients/families were included. A detailed taxonomy was developed to define and describe the processes undertaken, and capture them during the trial. The detailed process measure description, together with a range of patient, health service, and economic outcomes were successfully mapped on to the modifiedCONSORT recommendations for reporting non-pharmacologic trial interventions.Conclusions: the MRC complex intervention framework was an effective guide to developing a novel post-ICUrehabilitation intervention. Combining a clearly defined new healthcare role with a detailed taxonomy of process and activity enabled the intervention to be clearly described for the purpose of trial delivery and reporting. These data will be useful when interpreting the results of the randomised trial, will increase internal and external trial validity, and help others implement the intervention if the intervention proves clinically and cost effective

Robust, reversible gene knockdown using a single lentiviral short hairpin RNA vector

Manipulation of gene expression is an invaluable tool to study gene function in vitro and in vivo. The application of small inhibitory RNAs to knock down gene expression provides a relatively simple, elegant, but transient approach to study gene function in many cell types as well as in whole animals. Short hairpin structures (shRNAs) are a logical advance as they can be expressed continuously and are hence suitable for stable gene knockdown. Drug-inducible systems have now been developed; however, application of the technology has been hampered by persistent problems with low or transient expression, leakiness or poor inducibility of the short hairpin, and lack of reversibility. We have developed a robust, versatile, single lentiviral vector tool that delivers tightly regulated, fully reversible, doxycycline-responsive knockdown of target genes (FOXP3 and MYB), using single short hairpin RNAs. To demonstrate the capabilities of the vector we targeted FOXP3 because it plays a critical role in the development and function of regulatory T cells. We also targeted MYB because of its essential role in hematopoiesis and implication in breast cancer progression. The versatility of this vector is hence demonstrated by knockdown of distinct genes in two biologically separate systems.Cheryl Y. Brown, Timothy Sadlon, Tessa Gargett, Elizabeth Melville, Rui Zhang, Yvette Drabsch, Michael Ling, Craig A. Strathdee, Thomas J. Gonda, and Simon C. Barr