Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit juices

[EN] A comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against (RS)-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propyl-1,1,2,2-tetrafluoroethyl ether (tetraconazole), 2-(4-triazolyl)benzimidazole (thiabendazole), and (RS)-1-(beta-allyloxy-2,4-dichlorophenylethyl)imidazole (imazalil) were used as a source of immunoglobulin gene fragments for the production of single-chain variable fragment (scFv) and fusion scFv-pIII recombinant antibodies in Escherichia coli. Selected recombinant antibodies displayed cross-reactivity profiles very similar to those of the parent monoclonal antibodies. Imazalil and tetraconazole recombinant antibodies showed one order of magnitude lower affinity than their respective monoclonal antibodies, whereas the thiabendazole recombinant antibodies showed an affinity similar to that of their parent monoclonal antibody. On the other hand, scFv-pIII fusion fragments showed similar analytical properties as, and occasionally better than, scFv recombinant antibodies. Finally, ELISAs developed from each antibody type showed similar analytical performance when applied to the analysis of the target fungicides in fruit juices.This work was funded by Ministerio de Educacion y Ciencia (MEC, Spain, Project AGL2002-03266). E. P. was the recipient of a doctoral fellowship from Conselleria d'Educacio (Generalitat Valenciana, Spain).Moreno Tamarit, MJ.; Plana Andani, E.; Manclus Ciscar, JJ.; Montoya Baides, Á. (2014). Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit juices. Food Analytical Methods. 7(2):481-489. https://doi.org/10.1007/s12161-013-9655-zS48148972Abad A, Manclús JJ, Moreno M, Montoya A (2001) J AOAC Int 84:1–6Alcocer MJC, Doyen C, Lee HA, Morgan MRA (2000) J Agric Food Chem 48:4053–4059Brichta J, Vesela H, Franek M (2003) Vet Med 48:237–247Brichta J, Hnilova M, Viskovic T (2005) Vet Med 50:231–252Charlton K, Harris WJ, Potter AJ (2001) Biosens Bioelec 16:639–646EU Pesticide Database (2013) Pesticide EU-MRLs. http://ec.europa.eu/sanco_pesticides/public/index.cfm . Accessed Jan 2013Ferrer C, Martínez-Bueno MJ, Lozano A, Fernández-Alba AR (2011) Talanta 83:1552–1561Garret SD, Appleford DJA, Wyatt GM, Lee HA, Morgan MRA (1997) J Agric Food Chem 45:4183–4189Graham BM, Porter AJ, Harris WJ (1995) J Chem Technol Biotech 63:279–289Hiemstra M, de Kok A (2007) J Chromatog A 1154:3–25Kipriyanov SM, Moldenhauer G, Little M (1997) J Immunol Meth 200:69–77Kramer K, Hock B (2007) Recombinant antibodies for agrochemicals: Evolutionary optimization. In: Kennedy IR, Solomon KR, Gee SJ, Crossan AN, Wang S, Sánchez-Bayo F (eds) Rational environmental management of agrochemicals: Risk assessment, monitoring, and remedial action. ACS Symposium Series, vol. 966, pp 155−170Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Plückthun A (1997) J Immunol Meth 201:35–55Leong SSJ, Chen WN (2008) Chem Engin Sci 63:1401–1414Li T, Zhang Q, Liu Y, Chen D, Hu B, Blake DA, Liu F (2006) J Agric Food Chem 54:9085–9091Manclús JJ, Moreno M, Plana E, Montoya A (2008) J Agric Food Chem 56:8790–8800Markus V, Janne L, Urpo L (2011) Trends Anal Chem 30:219–226Mersmann M, Schmidt A, Tesar M, Schöneberg A, Welschof M, Kipriyanov S, Terness P, Little M, Pfizenmaier K, Moosmayer D (1998) J Immunol Meth 220:51–58Moreno M, Plana E, Montoya A, Caputo P, Manclús JJ (2007) Food Addit Contam 24:704–712Morozova VS, Levashova AI, Eremin SA (2005) J Anal Chem 60:202–217Nishi K, Imajuku Y, Nakata M, Ohde K, Miyake S, Morimune K, Kawata M, Ohkawa H (2003) J Pest Sci 28:301–309Nishi K, Ishiuchi M, Morimune K, Ohkawa H (2005) J Agric Food Chem 53:5096–5104Scholthof KB, Whang G, Karu AE (1997) J Agric Food Chem 45:1509–1517Sheedy C, MacKenzie CR, Hall JC (2007) Biotech Adv 25:25333–25352Tout NL, Yau KYF, Trevors JT, Lee H, Hall JC (2001) J Agric Food Chem 49:3628–3637Webb SR, Lee H, Hall JC (1997) J Agric Food Chem 45:535–541Yau KYF, Tout NL, Trevors JT, Lee H, Hall JC (1998) J Agric Food Chem 46:4457–4463Yoshioka N, Akiyama Y, Matsuoka T, Mitsuhashi T (2010) Food Control 21:212–21

Pathological Features in Paediatric Patients with TK2 Deficiency

Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype

Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies

Background Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. Results We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. Conclusion Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required

miR-146a rs2431697 identifies myeloproliferative neoplasm patients with higher secondary myelofibrosis progression risk

Myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PV) and essential thrombocythemia (ET), and remarkably shortens survival. Although JAK2V617F and CALR allele burden are the main transformation risk factors, inflammation plays a critical role by driving clonal expansion toward end-stage disease. NF-κB is a key mediator of inflammation-induced carcinogenesis. Here, we explored the involvement of miR-146a, a brake in NF-κB signaling, in MPN susceptibility and progression. rs2910164 and rs2431697, that affect miR-146a expression, were analyzed in 967 MPN (320 PV/333 ET/314 MF) patients and 600 controls. We found that rs2431697 TT genotype was associated with MF, particularly with post-PV/ET MF (HR = 1.5; p < 0.05). Among 232 PV/ET patients (follow-up time=8.5 years), 18 (7.8%) progressed to MF, being MF-free-survival shorter for rs2431697 TT than CC + CT patients (p = 0.01). Multivariate analysis identified TT genotype as independent predictor of MF progression. In addition, TT (vs. CC + CT) patients showed increased plasma inflammatory cytokines. Finally, miR-146a−/− mice showed significantly higher Stat3 activity with aging, parallel to the development of the MF-like phenotype. In conclusion, we demonstrated that rs2431697 TT genotype is an early predictor of MF progression independent of the JAK2V617F allele burden. Low levels of miR-146a contribute to the MF phenotype by increasing Stat3 signaling

Calibration test of PET scanners in a multi-centre clinical trial on breast cancer therapy monitoring using 18F-FLT.

UNLABELLED: A multi-centre trial using PET requires the analysis of images acquired on different systems We designed a multi-centre trial to estimate the value of 18F-FLT-PET to predict response to neoadjuvant chemotherapy in patients with newly diagnosed breast cancer. A calibration check of each PET-CT and of its peripheral devices was performed to evaluate the reliability of the results. MATERIAL AND METHODS: 11 centres were investigated. Dose calibrators were assessed by repeated measurements of a 68Ge certified source. The differences between the clocks associated with the dose calibrators and inherent to the PET systems were registered. The calibration of PET-CT was assessed with an homogeneous cylindrical phantom by comparing the activities per unit of volume calculated from the dose calibrator measurements with that measured on 15 Regions of Interest (ROIs) drawn on 15 consecutive slices of reconstructed filtered back-projection (FBP) images. Both repeatability of activity concentration based upon the 15 ROIs (ANOVA-test) and its accuracy were evaluated. RESULTS: There was no significant difference for dose calibrator measurements (median of difference -0.04%; min = -4.65%; max = +5.63%). Mismatches between the clocks were less than 2 min in all sites and thus did not require any correction, regarding the half life of 18F. For all the PET systems, ANOVA revealed no significant difference between the activity concentrations estimated from the 15 ROIs (median of difference -0.69%; min = -9.97%; max = +9.60%). CONCLUSION: No major difference between the 11 centres with respect to calibration and cross-calibration was observed. The reliability of our 18F-FLT multi-centre clinical trial was therefore confirmed from the physical point of view. This type of procedure may be useful for any clinical trial involving different PET systems

Single hadron response measurement and calorimeter jet energy scale uncertainty with the ATLAS detector at the LHC

The uncertainty on the calorimeter energy response to jets of particles is derived for the ATLAS experiment at the Large Hadron Collider (LHC). First, the calorimeter response to single isolated charged hadrons is measured and compared to the Monte Carlo simulation using proton-proton collisions at centre-of-mass energies of sqrt(s) = 900 GeV and 7 TeV collected during 2009 and 2010. Then, using the decay of K_s and Lambda particles, the calorimeter response to specific types of particles (positively and negatively charged pions, protons, and anti-protons) is measured and compared to the Monte Carlo predictions. Finally, the jet energy scale uncertainty is determined by propagating the response uncertainty for single charged and neutral particles to jets. The response uncertainty is 2-5% for central isolated hadrons and 1-3% for the final calorimeter jet energy scale.Comment: 24 pages plus author list (36 pages total), 23 figures, 1 table, submitted to European Physical Journal

Search for squarks and gluinos in events with isolated leptons, jets and missing transverse momentum at s√=8 TeV with the ATLAS detector

The results of a search for supersymmetry in final states containing at least one isolated lepton (electron or muon), jets and large missing transverse momentum with the ATLAS detector at the Large Hadron Collider are reported. The search is based on proton-proton collision data at a centre-of-mass energy s√=8 TeV collected in 2012, corresponding to an integrated luminosity of 20 fb−1. No significant excess above the Standard Model expectation is observed. Limits are set on supersymmetric particle masses for various supersymmetric models. Depending on the model, the search excludes gluino masses up to 1.32 TeV and squark masses up to 840 GeV. Limits are also set on the parameters of a minimal universal extra dimension model, excluding a compactification radius of 1/R c = 950 GeV for a cut-off scale times radius (ΛR c) of approximately 30

Evidence for the Higgs-boson Yukawa coupling to tau leptons with the ATLAS detector

Results of a search for H → τ τ decays are presented, based on the full set of proton-proton collision data recorded by the ATLAS experiment at the LHC during 2011 and 2012. The data correspond to integrated luminosities of 4.5 fb−1 and 20.3 fb−1 at centre-of-mass energies of √s = 7 TeV and √s = 8 TeV respectively. All combinations of leptonic (τ → `νν¯ with ` = e, µ) and hadronic (τ → hadrons ν) tau decays are considered. An excess of events over the expected background from other Standard Model processes is found with an observed (expected) significance of 4.5 (3.4) standard deviations. This excess provides evidence for the direct coupling of the recently discovered Higgs boson to fermions. The measured signal strength, normalised to the Standard Model expectation, of µ = 1.43 +0.43 −0.37 is consistent with the predicted Yukawa coupling strength in the Standard Model

Measurements of fiducial and differential cross sections for Higgs boson production in the diphoton decay channel at s√=8 TeV with ATLAS

Measurements of fiducial and differential cross sections are presented for Higgs boson production in proton-proton collisions at a centre-of-mass energy of s√=8 TeV. The analysis is performed in the H → γγ decay channel using 20.3 fb−1 of data recorded by the ATLAS experiment at the CERN Large Hadron Collider. The signal is extracted using a fit to the diphoton invariant mass spectrum assuming that the width of the resonance is much smaller than the experimental resolution. The signal yields are corrected for the effects of detector inefficiency and resolution. The pp → H → γγ fiducial cross section is measured to be 43.2 ±9.4(stat.) − 2.9 + 3.2 (syst.) ±1.2(lumi)fb for a Higgs boson of mass 125.4GeV decaying to two isolated photons that have transverse momentum greater than 35% and 25% of the diphoton invariant mass and each with absolute pseudorapidity less than 2.37. Four additional fiducial cross sections and two cross-section limits are presented in phase space regions that test the theoretical modelling of different Higgs boson production mechanisms, or are sensitive to physics beyond the Standard Model. Differential cross sections are also presented, as a function of variables related to the diphoton kinematics and the jet activity produced in the Higgs boson events. The observed spectra are statistically limited but broadly in line with the theoretical expectations

Measurement of χ c1 and χ c2 production with s√ = 7 TeV pp collisions at ATLAS

The prompt and non-prompt production cross-sections for the χ c1 and χ c2 charmonium states are measured in pp collisions at s√ = 7 TeV with the ATLAS detector at the LHC using 4.5 fb−1 of integrated luminosity. The χ c states are reconstructed through the radiative decay χ c → J/ψγ (with J/ψ → μ + μ −) where photons are reconstructed from γ → e + e − conversions. The production rate of the χ c2 state relative to the χ c1 state is measured for prompt and non-prompt χ c as a function of J/ψ transverse momentum. The prompt χ c cross-sections are combined with existing measurements of prompt J/ψ production to derive the fraction of prompt J/ψ produced in feed-down from χ c decays. The fractions of χ c1 and χ c2 produced in b-hadron decays are also measured