Establishment of one-step SYBR green-based real time-PCR assay for rapid detection and quantification of chikungunya virus infection

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus and one of the prevalent re-emerging arbovirus in tropical and subtropical regions of Asia, Africa, and Central and South America. It produces a spectrum of illness ranging from inapparent infection to moderate febrile illness as well as severe arthralgia or arthritis affecting multiple joints. In this study, a quantitative, one-step real-time SYBR Green-based RT-PCR system for the non-structural protein 2 (nsP2) of CHIKV that can quantify a wide range of viral RNA concentrations was developed. Comparisons between the conventional semi-quantitative RT-PCR assay, immunofluorescence detection method and the one-step SYBR Green-based RT-PCR assay in the detection of CHIKV infection revealed much rapid and increase sensitivity of the latter method. Furthermore, this newly developed assay was validated by in vitro experiments in which ribavirin, a well-known RNA virus inhibitor, showed a dose-dependent inhibition of virus replication on cells that was assessed by viral infectivity and viral RNA production. Our results demonstrate the potential of this newly developed one-step SYBR Green I-based RT-PCR assay may be a useful tool in rapid detection of CHIKV and monitoring the extent of viral replication possibly in patients' samples

Diagnostic and mechanistic implications of serum free light chains, albumin and alpha-fetoprotein in hepatocellular carcinoma

Background: Mass spectroscopy analysis suggested low serum albumin and high immunoglobulin free light chain (sFLC) levels may have diagnostic value in hepatocellular carcinoma (HCC). Our aims were to apply quantitative assays to confirm these observations, determine their diagnostic utility, and investigate the mechanisms involved. Methods: Albumin, sFLC, routine liver and renal function tests were measured in patients with chronic liver disease with (n=102) and without (n=113) HCC. The discriminant performance was compared with the current standard serological test alpha-fetoprotein (AFP) using receiver operating characteristic (ROC) and area under the curve (AUC) analyses. Results: sFLC and serum albumin were each confirmed to have discriminatory utility in HCC with AUC values of 0.7 and 0.8, respectively. sFLC were strongly correlated with gammaglobulin levels and both these were inversely related to serum albumin levels. The discriminatory utility of sFLC was retained after adjusting for renal and liver function. Conclusions: Serum levels of sFLC and albumin were strongly associated with HCC as predicted by mass spectroscopy. Discrimination of HCC by AFP was improved by the addition of either albumin or sFLC. Larger prospective studies are required to determine how AFP, sFLC and albumin might be combined in a useful diagnostic approach for HCC

Predicting Infectious ComplicatioNs in Children with Cancer : an external validation study

Background:The aim of this study was to validate the 'Predicting Infectious ComplicatioNs in Children with Cancer' (PICNICC) clinical decision rule (CDR) that predicts microbiologically documented infection (MDI) in children with cancer and fever and neutropenia (FN). We also investigated costs associated with current FN management strategies in Australia.Methods:Demographic, episode, outcome and cost data were retrospectively collected on 650 episodes of FN. We assessed the discrimination, calibration, sensitivity and specificity of the PICNICC CDR in our cohort compared with the derivation data set.Results:Using the original variable coefficients, the CDR performed poorly. After recalibration the PICNICC CDR had an area under the receiver operating characteristic (AUC-ROC) curve of 0.638 (95% CI 0.590-0.685) and calibration slope of 0.24. The sensitivity, specificity, positive predictive value and negative predictive value of the PICNICC CDR at presentation was 78.4%, 39.8%, 28.6% and 85.7%, respectively. For bacteraemia, the sensitivity improved to 85.2% and AUC-ROC to 0.71. Application at day 2, taking into consideration the proportion of MDI known (43%), further improved the sensitivity to 87.7%. Length of stay is the main contributor to cost of FN treatment, with an average cost per day of AUD 2183 in the low-risk group.Conclusions:For prediction of any MDI, the PICNICC rule did not perform as well at presentation in our cohort as compared with the derivation study. However, for bacteraemia, the predictive ability was similar to that of the derivation study, highlighting the importance of recalibration using local data. Performance also improved after an overnight period of observation. Implementation of a low-risk pathway, using the PICNICC CDR after a short period of inpatient observation, is likely to be safe and has the potential to reduce health-care expenditure

Predicting infectious complications in neutropenic children and young people with cancer (IPD protocol)

<p>Abstract</p> <p>Background</p> <p>A common and potentially life-threatening complication of the treatment of childhood cancer is infection, which frequently presents as fever with neutropenia. The standard management of such episodes is the extensive use of intravenous antibiotics, and though it produces excellent survival rates of over 95%, it greatly inconveniences the three-fourths of patients who do not require such aggressive treatment. There have been a number of studies which have aimed to develop risk prediction models to stratify treatment. Individual participant data (IPD) meta-analysis in therapeutic studies has been developed to improve the precision and reliability of answers to questions of treatment effect and recently have been suggested to be used to answer questions regarding prognosis and diagnosis to gain greater power from the frequently small individual studies.</p> <p>Design</p> <p>In the IPD protocol, we will collect and synthesise IPD from multiple studies and examine the outcomes of episodes of febrile neutropenia as a consequence of their treatment for malignant disease. We will develop and evaluate a risk stratification model using hierarchical regression models to stratify patients by their risk of experiencing adverse outcomes during an episode. We will also explore specific practical and methodological issues regarding adaptation of established techniques of IPD meta-analysis of interventions for use in synthesising evidence derived from IPD from multiple studies for use in predictive modelling contexts.</p> <p>Discussion</p> <p>Our aim in using this model is to define a group of individuals at low risk for febrile neutropenia who might be treated with reduced intensity or duration of antibiotic therapy and so reduce the inconvenience and cost of these episodes, as well as to define a group of patients at very high risk of complications who could be subject to more intensive therapies. The project will also help develop methods of IPD predictive modelling for use in future studies of risk prediction.</p

CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development

Charged multivesicular body protein 1A (CHMP1A; also known as chromatin-modifying protein 1A) is a member of the ESCRT-III (endosomal sorting complex required for transport-III) complex but is also suggested to localize to the nuclear matrix and regulate chromatin structure. Here, we show that loss-of-function mutations in human CHMP1A cause reduced cerebellar size (pontocerebellar hypoplasia) and reduced cerebral cortical size (microcephaly). CHMP1A-mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation. Chromatin immunoprecipitation suggests loss of the normal INK4A repression by BMI in these cells. Morpholino-based knockdown of zebrafish chmp1a resulted in brain defects resembling those seen after bmi1a and bmi1b knockdown, which were partially rescued by INK4A ortholog knockdown, further supporting links between CHMP1A and BMI1-mediated regulation of INK4A. Our results suggest that CHMP1A serves as a critical link between cytoplasmic signals and BMI1-mediated chromatin modifications that regulate proliferation of central nervous system progenitor cells

Perinatal asphyxia: current status and approaches towards neuroprotective strategies, with focus on sentinel proteins

Delivery is a stressful and risky event menacing the newborn. The mother-dependent respiration has to be replaced by autonomous pulmonary breathing immediately after delivery. If delayed, it may lead to deficient oxygen supply compromising survival and development of the central nervous system. Lack of oxygen availability gives rise to depletion of NAD+ tissue stores, decrease of ATP formation, weakening of the electron transport pump and anaerobic metabolism and acidosis, leading necessarily to death if oxygenation is not promptly re-established. Re-oxygenation triggers a cascade of compensatory biochemical events to restore function, which may be accompanied by improper homeostasis and oxidative stress. Consequences may be incomplete recovery, or excess reactions that worsen the biological outcome by disturbed metabolism and/or imbalance produced by over-expression of alternative metabolic pathways. Perinatal asphyxia has been associated with severe neurological and psychiatric sequelae with delayed clinical onset. No specific treatments have yet been established. In the clinical setting, after resuscitation of an infant with birth asphyxia, the emphasis is on supportive therapy. Several interventions have been proposed to attenuate secondary neuronal injuries elicited by asphyxia, including hypothermia. Although promising, the clinical efficacy of hypothermia has not been fully demonstrated. It is evident that new approaches are warranted. The purpose of this review is to discuss the concept of sentinel proteins as targets for neuroprotection. Several sentinel proteins have been described to protect the integrity of the genome (e.g. PARP-1; XRCC1; DNA ligase IIIα; DNA polymerase β, ERCC2, DNA-dependent protein kinases). They act by eliciting metabolic cascades leading to (i) activation of cell survival and neurotrophic pathways; (ii) early and delayed programmed cell death, and (iii) promotion of cell proliferation, differentiation, neuritogenesis and synaptogenesis. It is proposed that sentinel proteins can be used as markers for characterising long-term effects of perinatal asphyxia, and as targets for novel therapeutic development and innovative strategies for neonatal care

Search for the Standard Model Higgs boson produced in association with top quarks and decaying into bb¯ in pp collisions at √s=8 TeV with the ATLAS detector

A search for the Standard Model Higgs boson produced in association with a top-quark pair, tt¯H, is presented. The analysis uses 20.3 fb−1 of pp collision data at √s=8TeV, collected with the ATLAS detector at the Large Hadron Collider during 2012. The search is designed for the H→bb¯ decay mode and uses events containing one or two electrons or muons. In order to improve the sensitivity of the search, events are categorised according to their jet and b-tagged jet multiplicities. A neural network is used to discriminate between signal and background events, the latter being dominated by tt¯+jets production. In the single-lepton channel, variables calculated using a matrix element method are included as inputs to the neural network to improve discrimination of the irreducible tt¯+bb¯ background. No significant excess of events above the background expectation is found and an observed (expected) limit of 3.4 (2.2) times the Standard Model cross section is obtained at 95% confidence level. The ratio of the measured tt¯H signal cross section to the Standard Model expectation is found to be μ=1.5±1.1 assuming a Higgs boson mass of 125GeV

Measurement of distributions sensitive to the underlying event in inclusive Z-boson production in pp collisions at √s = 7 TeV with the ATLAS detector

A measurement of charged-particle distributions sensitive to the properties of the underlying event is presented for an inclusive sample of events containing a Z-boson, decaying to an electron or muon pair. The measurement is based on data collected using the ATLAS detector at the LHC in proton–proton collisions at a centre-of-mass energy of 7 TeV with an integrated luminosity of 4.6 fb −¹. Distributions of the charged particle multiplicity and of the charged particle transverse momentum are measured in regions of azimuthal angle defined with respect to the Z-boson direction. The measured distributions are compared to similar distributions measured in jet events, and to the predictions of various Monte Carlo generators implementing different underlying event models