Inter-laboratory comparison of methods to measure androstenone in pork fat

Today, different analytical methods are used by different laboratories to quantify androstenone in fat tissue. This study shows the comparison of methods used routinely in different laboratories for androstenone quantification: Time-resolved fluoroimmunoassay in Norwegian School of Veterinary Science (NSVS; Norway), gas chromatography coupled to mass spectrometry in Co-operative Central Laboratory (CCL; The Netherlands) and in Institut de Recerca i Tecnologia Agroalimentàries (IRTA; Spain), and high-pressure liquid chromatography in Agroscope Liebefeld-Posieux Research Station (ALP; Switzerland). In a first trial, a set of adipose tissue (AT) samples from 53 entire males was sent to CCL, IRTA and NSVS for determination of androstenone concentration. The average androstenone concentration (s.d.) was 2.47 (2.10) μg/g at NSVS, 1.31 (0.98) μg/g at CCL and 0.62 (0.52) μg/g at IRTA. Despite the large differences in absolute values, inter-laboratory correlations were high, ranging from 0.82 to 0.92. A closer look showed differences in the preparation step. Indeed, different matrices were used for the analysis: pure fat at NSVS, melted fat at CCL and AT at IRTA. A second trial was organised in order to circumvent the differences in sample preparation. Back fat samples from 10 entire males were lyophilised at the ALP labortary in Switzerland and were sent to the other laboratories for androstenone concentration measurement. The average concentration (s.d.) of androstenone in the freeze-dried AT samples was 0.87 (0.52), 1.03 (0.55), 0.84 (0.46) and 0.99 (0.67) μg/g at NSVS, CCL, IRTA and ALP, respectively, and the pairwise correlations between laboratories ranged from 0.92 to 0.97. Thus, this study shows the influence of the different sample preparation protocols, leading to major differences in the results, although still allowing high inter-laboratory correlations. The results further highlight the need for method standardisation and inter-laboratory ring tests for the determination of androstenone. This standardisation is especially relevant when deriving thresholds of consumer acceptance, whereas the ranking of animals for breeding purposes will be less affected due to the high correlations between method

Pre-clinical Evaluation of a Cyanine-Based SPECT Probe for Multimodal Tumor Necrosis Imaging

Purpose: Recently we showed that a number of carboxylated near-infrared fluorescent (NIRF) cyanine dyes possess strong necrosis avid properties in vitro as well as in different mouse models of spontaneous and therapy-induced tumor necrosis, indicating their potential use for cancer diagnostic- and prognostic purposes. In the previous study, the detection of the cyanines was achieved by whole body optical imaging, a technique that, due to the limited penetration of near-infrared light, is not suitable for investigations deeper than 1 cm within the human body. Therefore, in order to facilitate clinical translation, the purpose of the present study was to generate a necrosis avid cyanine-based NIRF probe that could also be used for single photon emission computed tomography (SPECT). For this, the necrosis avid NIRF cyanine HQ4 was radiolabeled with 111indium, via the chelate diethylene triamine pentaacetic acid (DTPA). Procedures: The necrosis avid properties of the radiotracer [111In]DTPA-HQ4 were examined in vitro and in vivo in different breast tumor models in mice using SPECT and optical imaging. Moreover, biodistribution studies were performed to examine the pharmacokinetics of the probe in vivo. Results: Using optical imaging and radioactivity measurements, in vitro, we showed selective accumulation of [111In]DTPA-HQ4 in dead cells. Using SPECT and in biodistribution studies, the necrosis avidity of the radiotracer was confirmed in a 4T1 mouse breast cancer model of spontaneous tumor necrosis and in a MCF-7 human breast cancer model of chemotherapy-induced tumor necrosis. Conclusions: The radiotracer [111In]DTPA-HQ4 possessed strong and selective necrosis avidity in vitro and in various mouse models of tumor necrosis in vivo, indicating its potential to be clinically applied for diagnostic purposes and to monitor anti-cancer treatment efficacy

Perlecan Maintains microvessel integrity in vivo and modulates their formation in vitro

Perlecan is a heparan sulfate proteoglycan assembled into the vascular basement membranes (BMs) during vasculogenesis. In the present study we have investigated vessel formation in mice, teratomas and embryoid bodies (EBs) in the absence of perlecan. We found that perlecan was dispensable for blood vessel formation and maturation until embryonic day (E) 12.5. At later stages of development 40% of mutant embryos showed dilated microvessels in brain and skin, which ruptured and led to severe bleedings. Surprisingly, teratomas derived from perlecan-null ES cells showed efficient contribution of perlecan-deficient endothelial cells to an apparently normal tumor vasculature. However, in perlecan-deficient EBs the area occupied by an endothelial network and the number of vessel branches were significantly diminished. Addition of FGF-2 but not VEGF165 rescued the in vitro deficiency of the mutant ES cells. Furthermore, in the absence of perlecan in the EB matrix lower levels of FGFs are bound, stored and available for cell surface presentation. Altogether these findings suggest that perlecan supports the maintenance of brain and skin subendothelial BMs and promotes vasculo- and angiogenesis by modulating FGF-2 function

Multi-centred mixed-methods PEPFAR HIV care & support public health evaluation: study protocol

BACKGROUND: A public health response is essential to meet the multidimensional needs of patients and families affected by HIV disease in sub-Saharan Africa. In order to appraise current provision of HIV care and support in East Africa, and to provide evidence-based direction to future care programming, and Public Health Evaluation was commissioned by the PEPFAR programme of the US Government. METHODS/DESIGN: This paper described the 2-Phase international mixed methods study protocol utilising longitudinal outcome measurement, surveys, patient and family qualitative interviews and focus groups, staff qualitative interviews, health economics and document analysis. Aim 1) To describe the nature and scope of HIV care and support in two African countries, including the types of facilities available, clients seen, and availability of specific components of care [Study Phase 1]. Aim 2) To determine patient health outcomes over time and principle cost drivers [Study Phase 2]. The study objectives are as follows. 1) To undertake a cross-sectional survey of service configuration and activity by sampling 10% of the facilities being funded by PEPFAR to provide HIV care and support in Kenya and Uganda (Phase 1) in order to describe care currently provided, including pharmacy drug reviews to determine availability and supply of essential drugs in HIV management. 2) To conduct patient focus group discussions at each of these (Phase 1) to determine care received. 3) To undertake a longitudinal prospective study of 1200 patients who are newly diagnosed with HIV or patients with HIV who present with a new problem attending PEPFAR care and support services. Data collection includes self-reported quality of life, core palliative outcomes and components of care received (Phase 2). 4) To conduct qualitative interviews with staff, patients and carers in order to explore and understand service issues and care provision in more depth (Phase 2). 5) To undertake document analysis to appraise the clinical care procedures at each facility (Phase 2). 6) To determine principle cost drivers including staff, overhead and laboratory costs (Phase 2). DISCUSSION: This novel mixed methods protocol will permit transparent presentation of subsequent dataset results publication, and offers a substantive model of protocol design to measure and integrate key activities and outcomes that underpin a public health approach to disease management in a low-income setting

Somatic Mutagenesis with a Sleeping Beauty Transposon System Leads to Solid Tumor Formation in Zebrafish

Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700–6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers

Robust detection of translocations in lymphoma FFPE samples using targeted locus capture-based sequencing

Preservation of cancer biopsies by FFPE introduces DNA fragmentation, hindering analysis of rearrangements. Here the authors introduce FFPE Targeted Locus Capture for identification of translocations in preserved samples.In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Clinical characteristics of women captured by extending the definition of severe postpartum haemorrhage with 'refractoriness to treatment': a cohort study

Background: The absence of a uniform and clinically relevant definition of severe postpartum haemorrhage hampers comparative studies and optimization of clinical management. The concept of persistent postpartum haemorrhage, based on refractoriness to initial first-line treatment, was proposed as an alternative to common definitions that are either based on estimations of blood loss or transfused units of packed red blood cells (RBC). We compared characteristics and outcomes of women with severe postpartum haemorrhage captured by these three types of definitions. Methods: In this large retrospective cohort study in 61 hospitals in the Netherlands we included 1391 consecutive women with postpartum haemorrhage who received either ≥4 units of RBC or a multicomponent transfusion. Clinical characteristics and outcomes of women with severe postpartum haemorrhage defined as persistent postpartum haemorrhage were compared to definitions based on estimated blood loss or transfused units of RBC within 24 h following birth. Adverse maternal outcome was a composite of maternal mortality, hysterectomy, arterial embolisation and intensive care unit admission. Results: One thousand two hundred sixty out of 1391 women (90.6%) with postpartum haemorrhage fulfilled the definition of persistent postpartum haemorrhage. The majority, 820/1260 (65.1%), fulfilled this definition within 1 h following birth, compared to 819/1391 (58.7%) applying the definition of ≥1 L blood loss and 37/845 (4.4%) applying the definition of ≥4 units of RBC. The definition persistent postpartum haemorrhage captured 430/471 adverse maternal outcomes (91.3%), compared to 471/471 (100%) for ≥1 L blood loss and 383/471 (81.3%) for ≥4 units of RBC. Persistent postpartum haemorrhage did not capture all adverse outcomes because of missing data on timing of initial, first-line treatment. Conclusion: The definition persistent postpartum haemo