Radiotherapy Quality Assurance in the PORTEC-3 (TROG 08.04) Trial.

AIMS: Quality assurance in radiotherapy (QART) is essential to ensure the scientific integrity of a clinical trial. This paper reports the findings of the retrospective QART assessment for all centres that participated in PORTEC-3; a randomised controlled trial that compared pelvic radiotherapy with concurrent chemoradiotherapy to the pelvis followed by adjuvant chemotherapy. The trial showed an overall survival benefit for the addition of the chemotherapy in the management of women with high-risk endometrial cancer. MATERIALS AND METHODS: Clinicians were invited to upload a randomly selected case/s treated at each of the participating sites. Panel reviewers analysed the contours to certify that the target volumes and organ at risk structures were contoured according to guidelines. The results were categorised into acceptable, minor variation, major variation or unevaluable. The radiotherapy plans were dosimetrically evaluated using the well-established Trans-Tasman Radiation Oncology Group (TROG) protocol. RESULTS: Between August 2010 and January 2018, data from 146 patients of 686 consecutively treated patients were retrospectively reviewed. All 16 Australia and New Zealand and 71 of 77 international centres uploaded data for evaluation. In total, 3514 dosimetric and contour variables were reviewed. Of these, 3136 variables were deemed acceptable (89.2%), with 335 minor (9.6%) and 43 major variations (1.2%). Major contour variations included the clinical target volume vaginal vault, clinical target volume parametria and differential planning target volume vault expansion. CONCLUSION: The results of the QART assessment confirmed high uniformity and low rates of both minor and major deviations in contouring and dosimetry in all sites. This supports the safe introduction of the PORTEC-3 treatment protocol into routine clinical practice.

Corticosteroids and risk of gastrointestinal bleeding in critically ill adults: Protocol for a systematic review.

BACKGROUND: Corticosteroids are frequently prescribed to critically ill patients. However, their use may increase the risk of gastrointestinal (GI) bleeding, which is associated with morbidity and mortality. Accordingly, we aim to assess whether continued administration of corticosteroids for >24 hours increases the rate of GI bleeding in adult critically ill patients compared to placebo or no treatment. METHODS/DESIGN: We will conduct a systematic review of randomized clinical trials with meta-analysis and trial sequential analysis. The participants will be adult (as defined in the included trials) critically ill patients. The intervention will be any corticosteroid administered systematically for >24 hours and the comparator will be placebo or no treatment. The primary outcome will be rate of clinically important GI bleeding. We will systematically search EMBASE, MEDLINE, Medline In-Process, Cochrane Library, Epistemonikos and trial registries for relevant literature, as well as perform a hand search. We will follow the recommendations by the Cochrane Collaboration and the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statement. The risk of systematic errors (bias) and random errors will be assessed and the overall quality of evidence will be evaluated using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. DISCUSSION: The risk of GI bleeding in adult critically ill patients treated with corticosteroids is unknown. Hence, there is need for a robust systematic review to assess this risk and provide clinicians with a clearer understanding of the strength and limitations of existing data.

Huckebein repressor activity in Drosophila terminal patterning is mediated by Groucho.

The Groucho corepressor mediates negative transcriptional regulation in association with various DNA-binding proteins in diverse developmental contexts. We have previously implicated Groucho in Drosophila embryonic terminal patterning, showing that it is required to confine tailless and huckebein terminal gap gene expression to the pole regions of the embryo. Here we reveal an additional requirement for Groucho in this developmental process by establishing that Groucho mediates repressor activity of the Huckebein protein. Putative Huckebein target genes are derepressed in embryos lacking maternal groucho activity and biochemical experiments demonstrate that Huckebein physically interacts with Groucho. Using an in vivo repression assay, we identify a functional repressor domain in Huckebein that contains an FRPW tetrapeptide, similar to the WRPW Groucho-recruitment domain found in Hairy-related repressor proteins. Mutations in Huckebein's FRPW motif abolish Groucho binding and in vivo repression activity, indicating that binding of Groucho through the FRPW motif is required for the repressor function of Huckebein. Taken together with our earlier results, these findings show that Groucho-repression regulates sequential aspects of terminal patterning in Drosophila.

Membrane topology of the rat brain Na+-Ca2+ exchanger.

To provide experimental evidence for the topology of the Na+-Ca2+ exchanger protein NCX1 in the membrane, indirect immunofluorescence studies using site specific anti-peptide antibodies and Flag-epitope insertion into chosen locations of the protein were carried out. Anti-peptide antibodies AbO-6 and AbO-8 were raised against peptide segments present in a large hydrophilic loop of about 500 amino acids, which separates the hydrophobic amino terminal part of the protein from the hydrophobic carboxy terminal. AbO-10 was raised against the C-terminal tail of the protein. All three antibodies bound to the exchanger protein expressed in transfected cells, in rat brain synaptic plasma membrane and in dog sarcolemmal preparations. The antibodies bound only to those NCX1 isoforms that contained the epitope against which they were raised. Detection of the exchanger protein in transfected cells in situ required the addition of permeabilizing agents suggesting an intracellular location of the epitopes to which AbO-6, AbO-8 and AbO-10 bind. The Flag epitope was inserted into ten putative extramembraneous segments along the exchanger protein. For topology studies, only the Flag-mutants that retained Na+-Ca2+ exchange activity in whole HeLa cells, were used. Immunofluorescence studies indicated, that the N-terminal of the protein is extracellular, the first hydrophilic loop separating transmembrane helices 1 and 2 as well as the C-terminal, are intracellular.

The putative amino-terminal signal peptide of the cloned rat brain Na(+)-Ca2+ exchanger gene (RBE-1) is not mandatory for functional expression.

The rat brain Na(+)-Ca2+ exchanger (RBE) gene, as well as other isoforms of this protein family, can be organized into 12 transmembrane alpha helices, the first of which was proposed by Durkin et al. (14) to constitute a cleavable signal peptide. We have prepared three amino-terminal mutants, in which 21, 26, and 31 amino acids beyond the initiating methionine were deleted. The deletions include the hydrophobic core of the putative signal peptide (N21), the entire putative signal peptide and parts of the putative signal peptidase cleavage site (N26), and the entire putative signal peptide and putative signal peptidase cleavage site (N31). All three mutant clones were transiently expressed in HeLa cells. The average Na+ gradient-dependent Ca2+ transport activity of the mutant exchangers was 108% (N21), 37.2% (N26), and 60.06% (N31) of the wild-type clone. Mutation of the putative cleavage site by an exchange of Ala-32 --> Asp, resulted in a decrease in Na(+)-Ca2+ exchange activity to 7.7%, relative to the wild-type exchanger. Functional reconstitution of the proteins that were expressed in the transfected cells, resulted in transport activities of: 60.1% (N21), 26.75% (N26), 85.36% (N31), and 31% (Ala-32 --> Asp) relative to the wild-type exchanger. Western blot analysis of the protein profile of RBE-1, N21, N26, N31 and Ala-32 --> Asp-transfected HeLa cells was carried out by using an antipeptide antibody directed against a pentadecapeptide segment derived from the large putative cytoplasmic loop of the cloned rat exchanger gene. In the total cell extract and in the plasma membrane-enriched fraction, in addition to a major protein band of about 125 kDa, which corresponds to the molecular mass of the mature fully processed Na(+)-Ca2+ exchanger, an additional protein of about 135 kDa is revealed in the profile of N21- and N26-transfected cells. This band is not detected in the protein profile of RBE-1, N31, or Ala-32 -->Asp. The amino-terminal truncated mutants of the cloned Na(+)-Ca2+ exchanger could be expressed and processed also in a reticulocyte lysate supplemented with dog microsomes. Our results suggest that the putative signal peptide of the cloned Na(+)-Ca2+ exchanger gene does not play a mandatory role in functional expression of the protein in HeLa cells.

Cloning of two isoforms of the rat brain Na(+)-Ca2+ exchanger gene and their functional expression in HeLa cells.

Two functional isoforms of the rat brain Na(+)-Ca2+ exchanger were isolated from a lambda ZAP hippocampus cDNA library. The open reading frame of clone RBE-1 codes for a protein 935 amino acids long, and that of clone RBE-2 codes for a protein 958 amino acids long. Expression HeLa cells of Na+ gradient dependent Ca2+ transport activity was determined following transfection of the cells with either RBE-1 or RBE-2. Both clones expressed proteins that exchange Na+ with Ca2+ in an electrogenic manner and none of them exhibited a dependency of the antiport on K, since they transported Ca2+ in an Na+ gradient dependent manner in external choline chloride as well.