A NEW METHODOLOGY FOR DIAGNOSIS OF FANCONI ANEMIA BASED ON BIOLOGICAL DOSIMETRY

Fanconi Anemia (FA) is a syndrome associated with chromosomal fragility. Current laboratory tests to diagnose this disease are based on the scoring of chromosomal aberrations induced in peripheral blood lymphocytes by clastogenic chemical agents, mainly: diepoxybutane (DEB) or mitomycin C (MMC). This study evaluated an alternative test for the diagnosis of FA, in which ionizing radiation replaces DEB/MMC. Two groups were studied: normal and DEB-sensitive individuals. From each individual, samples of peripheral blood were irradiated using an electron linear accelerator. Following lymphocyte cultures, and slide preparation, metaphases were scored based on the same methodology for biological dosimetry, according to recommendations of the International Atomic Energy Agency. Our results emphasized a pattern of distribution of dicentrics, fragments, as well as abnormal chromosomal arrangements. The methodology of analysis here proposed permitted to distinguish normal from DEB-sensitive subjects

The effect of mixing entire male pigs prior to transport to slaughter on behaviour, welfare and carcass lesions

peer-reviewedData set for article is also provided.Research is needed to validate lesions recorded at meat inspection as indicators of pig welfare on farm. The aims were to determine the influence of mixing pigs on carcass lesions and to establish whether such lesions correlate with pig behaviour and lesions scored on farm. Aggressive and mounting behaviour of pigs in three single sex pens was recorded on Day −5, −2, and −1 relative to slaughter (Day 0). On Day 0 pigs were randomly allocated to 3 treatments (n = 20/group) over 5 replicates: males mixed with females (MF), males mixed with males (MM), and males unmixed (MUM). Aggressive and mounting behaviours were recorded on Day 0 at holding on farm and lairage. Skin/tail lesions were scored according to severity at the farm (Day −1), lairage, and on the carcass (Day 0). Effect of treatment and time on behaviour and lesions were analysed by mixed models. Spearman rank correlations between behaviour and lesion scores and between scores recorded at different stages were determined. In general, MM performed more aggressive behaviour (50.4 ± 10.72) than MUM (20.3 ± 9.55, P < 0.05) and more mounting (30.9 ± 9.99) than MF (11.4 ± 3.76) and MUM (9.8 ± 3.74, P < 0.05). Skin lesion scores increased between farm (Day −1) and lairage (P < 0.001), but this tended to be significant only for MF and MM (P = 0.08). There was no effect of treatment on carcass lesions and no associations were found with fighting/mounting. Mixing entire males prior to slaughter stimulated mounting and aggressive behaviour but did not influence carcass lesion scores. Carcass skin/tail lesions scores were correlated with scores recorded on farm (rskin = 0.21 and rtail = 0.18, P < 0.01) suggesting that information recorded at meat inspection could be used as indicators of pig welfare on farm.This study was part of the PIGWELFIND project funded by the Department of Agriculture, Food and the Marine (DAFM), Ireland

NT-proBNP and Circulating Inflammation Markers in Prediction of a Normal Myocardial Scintigraphy in Patients with Symptoms of Coronary Artery Disease

Myocardial perfusion imaging (MPI) can detect myocardial perfusion abnormalities but many examinations are without pathological findings. This study examines whether circulating biomarkers can be used as screening modality prior to MPI.243 patients with an intermediate risk of CAD or with known CAD with renewed suspicion of ischemia were referred to MPI. Blood samples were analyzed for N-terminal fragment of the prohormone brain natriuretic peptide (NT-proBNP), YKL-40, IL-6, matrix metalloproteinase 9 (MMP-9) and high sensitive C-reactive protein (hsCRP). Patients with myocardial perfusion defects had elevated levels of NT-proBNP (p<0.0001), YKL-40 (p = 0.03) and IL-6 (p = 0.03) but not of hsCRP (p = 0.58) nor of MMP-9 (p = 0.14). The NT-proBNP increase was observed in both genders (p<0.0001), whereas YKL-40 (p = 0.005) and IL-6 (p = 0.02) were elevated only in men. A NT-proBNP cut off-concentration at 25 ng/l predicted a normal MPI with a negative predictive value >95% regardless of existing CAD.20-25% of patients suspected of CAD could have been spared a MPI by using a NT-proBNP cut-off concentration at 25 ng/l with a negative predictive value >95%. NT-proBNP has the potential use of being a screening marker of CAD before referral of the patient to MPI

Dissecting mitosis by RNAi in Drosophila tissue culture cells

Here we describe a detailed methodology to study the function of genes whose products function during mitosis by dsRNA-mediated interference (RNAi) in cultured cells of Drosophila melanogaster. This procedure is particularly useful for the analysis of genes for which genetic mutations are not available or for the dissection of complicated phenotypes derived from the analysis of such mutants. With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved in particular cellular processes. In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells. We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique

BCRP expression does not result in resistance to STX140 in vivo, despite the increased expression of BCRP in A2780 cells in vitro after long-term STX140 exposure

The anti-proliferative and anti-angiogenic properties of the endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2), are enhanced in a series of sulphamoylated derivatives of 2-MeOE2. To investigate possible mechanisms of resistance to these compounds, a cell line, A2780.140, eightfold less sensitive to the 3,17-O,O-bis-sulphamoylated derivative, STX140, was derived from the A2780 ovarian cancer cell line by dose escalation. Other cell lines tested did not develop STX140 resistance. RT–PCR and immunoblot analysis demonstrated that breast cancer resistance protein (BCRP) expression is dramatically increased in A2780.140 cells. The cells are cross-resistant to the most structurally similar bis-sulphamates, and to BCRP substrates, mitoxantrone and doxorubicin; but they remain sensitive to taxol, an MDR1 substrate, and to all other sulphamates tested. Sensitivity can be restored using a BCRP inhibitor, and this pattern of resistance is also seen in a BCRP-expressing MCF-7-derived cell line, MCF-7.MR. In mice bearing wild-type (wt) and BCRP-expressing tumours on either flank, both STX140 and mitoxantrone inhibited the growth of the MCF-7wt xenografts, but only STX140 inhibited growth of the MCF-7.MR tumours. In conclusion, STX140, a promising orally bioavailable anti-cancer agent in pre-clinical development, is highly efficacious in BCRP-expressing xenografts. This is despite an increase in BCRP expression in A2780 cells in vitro after chronic dosing with STX140

Instrumentation for fluorescence lifetime measurement using photon counting

We describe the evolution of HORIBA Jobin Yvon IBH Ltd, and its time-correlated single-photon counting (TCSPC) products, from university research beginnings through to its present place as a market leader in fluorescence lifetime spectroscopy. The company philosophy is to ensure leading-edge research capabilities continue to be incorporated into instruments in order to meet the needs of the diverse range of customer applications, which span a multitude of scientific and engineering disciplines. We illustrate some of the range of activities of a scientific instrument company in meeting this goal and highlight by way of an exemplar the performance of the versatile DeltaFlex instrument in measuring fluorescence lifetimes. This includes resolving fluorescence lifetimes down to 5 ps, as frequently observed in energy transfer, nanoparticle metrology with sub-nanometre resolution and measuring a fluorescence lifetime in as little as 60 μs for the study of transient species and kinetics

Effects of Ploidy and Recombination on Evolution of Robustness in a Model of the Segment Polarity Network

Many genetic networks are astonishingly robust to quantitative variation, allowing these networks to continue functioning in the face of mutation and environmental perturbation. However, the evolution of such robustness remains poorly understood for real genetic networks. Here we explore whether and how ploidy and recombination affect the evolution of robustness in a detailed computational model of the segment polarity network. We introduce a novel computational method that predicts the quantitative values of biochemical parameters from bit sequences representing genotype, allowing our model to bridge genotype to phenotype. Using this, we simulate 2,000 generations of evolution in a population of individuals under stabilizing and truncation selection, selecting for individuals that could sharpen the initial pattern of engrailed and wingless expression. Robustness was measured by simulating a mutation in the network and measuring the effect on the engrailed and wingless patterns; higher robustness corresponded to insensitivity of this pattern to perturbation. We compared robustness in diploid and haploid populations, with either asexual or sexual reproduction. In all cases, robustness increased, and the greatest increase was in diploid sexual populations; diploidy and sex synergized to evolve greater robustness than either acting alone. Diploidy conferred increased robustness by allowing most deleterious mutations to be rescued by a working allele. Sex (recombination) conferred a robustness advantage through “survival of the compatible”: those alleles that can work with a wide variety of genetically diverse partners persist, and this selects for robust alleles

The obesity and inflammatory marker haptoglobin attracts monocytes via interaction with chemokine (C-C motif) receptor 2 (CCR2)

<p>Abstract</p> <p>Background</p> <p>Obesity is a chronic low inflammatory state. In the obesity condition the white adipose tissue (WAT) is massively infiltrated with monocytes/macrophages, and the nature of the signals recruiting these inflammatory cells has yet to be fully elucidated. Haptoglobin (Hp) is an inflammatory marker and its expression is induced in the WAT of obese subjects. In an effort to elucidate the biological significance of Hp presence in the WAT and of its upregulation in obesity we formulated the hypothesis that Hp may serve as a macrophage chemoattractant.</p> <p>Results</p> <p>We demonstrated by chemotaxis assay that Hp is able to attract chemokine (C-C motif) receptor 2 (CCR2)-transfected pre-B lymphocytes and monocytes in a dose-dependent manner. Moreover, Hp-mediated migration of monocytes is impaired by CCR2-specific inhibition or previous cell exposure to monocyte chemoattractant protein 1 (MCP1) (also known as CCR2 ligand or chemokine (C-C motif) ligand 2 (CCL2)). Downstream effects of Hp/CCR2 interaction were also investigated: flow cytometry proved that monocytes treated with Hp show reduced CCR2 expression on their surface; Hp interaction induces calcium release that is reduced upon pretreatment with CCR2 antagonist; extracellular signal-regulated kinase (ERK)1/2, a signal transducer activated by CCR2, is phosphorylated following Hp treatment and this phosphorylation is reduced when cells are pretreated with a specific CCR2 inhibitor. Consistently, blocking the ERK1/2 pathway with U0126, the selective inhibitor of the ERK upstream mitogen-activated protein (MAP)-ERK kinase (MEK), results in a dramatic reduction (by almost 100%) of the capability of Hp to induce monocyte migration.</p> <p>Conclusions</p> <p>Our data show that Hp is a novel monocyte chemoattractant and that its chemotactic potential is mediated, at least in part. by its interaction with CCR2.</p

Surgical treatment for acromioclavicular joint osteoarthritis: patient selection, surgical options, complications, and outcome

Osteoarthritis is one of the most common causes of pain originating from the acromioclavicular (AC) joint. An awareness of appropriate diagnostic techniques is necessary in order to localize clinical symptoms to the AC joint. Initial treatments for AC joint osteoarthritis, which include non-steroidal anti-inflammatory drugs (NSAIDS) and corticosteroids, are recommended prior to surgical interventions. Distal clavicle excision, the main surgical treatment option, can be performed by various surgical approaches, such as open procedures, direct arthroscopic, and indirect arthroscopic techniques. When choosing the best surgical option, factors such as avoidance of AC ligament damage, clavicular instability, and post-operative pain must be considered. This article examines patient selection, complications, and outcomes of surgical treatment options for AC joint osteoarthritis