Tailoring the specificity of the type C feruloyl esterase FoFaeC from Fusarium oxysporum towards methyl sinapate by rational redesign based on small molecule docking simulations

The type C feruloyl esterase FoFaeC from Fusarium oxysporum is a newly discovered enzyme with high potential for use in the hydrolysis of lignocellulosic biomass but it shows low activity towards sinapates. In this work, small molecule docking simulations were employed in order to identify important residues for the binding of the four model methyl esters of hydroxycinnamic acids, methyl ferulate/caffeate/sinapate/p-coumarate, to the predicted structure of FoFaeC. Subsequently rational redesign was applied to the enzyme’ active site in order to improve its specificity towards methyl sinapate. A double mutation (F230H/T202V) was considered to provide hydrophobic environment for stabilization of the methoxy substitution on sinapate and a larger binding pocket. Five mutant clones and the wild type were produced in Pichia pastoris and biochemically characterized. All clones showed improved activity, substrate affinity, catalytic efficiency and turnover rate compared to the wild type against methyl sinapate, with clone P13 showing a 5-fold improvement in catalytic efficiency. Although the affinity of all mutant clones was improved against the four model substrates, the catalytic efficiency and turnover rate decreased for the substrates containing a hydroxyl substitution

Photonic Label-Free Biosensors for Fast and Multiplex Detection of Swine Viral Diseases

[EN] In this paper we present the development of photonic integrated circuit (PIC) biosensors for the label-free detection of six emerging and endemic swine viruses, namely: African Swine Fever Virus (ASFV), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PPRSV), Porcine Parvovirus (PPV), Porcine Circovirus 2 (PCV2), and Swine Influenza Virus A (SIV). The optical biosensors are based on evanescent wave technology and, in particular, on Resonant Rings (RRs) fabricated in silicon nitride. The novel biosensors were packaged in an integrated sensing cartridge that included a microfluidic channel for buffer/sample delivery and an optical fiber array for the optical operation of the PICs. Antibodies were used as molecular recognition elements (MREs) and were selected based on western blotting and ELISA experiments to ensure the high sensitivity and specificity of the novel sensors. MREs were immobilized on RR surfaces to capture viral antigens. Antibody-antigen interactions were transduced via the RRs to a measurable resonant shift. Cell culture supernatants for all of the targeted viruses were used to validate the biosensors. Resonant shift responses were dose-dependent. The results were obtained within the framework of the SWINOSTICS project, contributing to cover the need of the novel diagnostic tools to tackle swine viral diseases.This work was funded by the EU-2020 program under grant agreement Nº 771649-SWINOSTICS project.Gómez-Gómez, MI.; Sánchez, C.; Peransi, S.; Zurita, D.; Bellieres, L.; Recuero, S.; Rodrigo, M.... (2022). Photonic Label-Free Biosensors for Fast and Multiplex Detection of Swine Viral Diseases. Sensors. 22(3):1-14. https://doi.org/10.3390/s2203070811422

Cytokine Expression by Inflammatory Cells Obtained from the Spinal Cords of Lewis Rats with Experimental Autoimmune Encephalomyelitis Induced by Inoculation with Myelin Basic Protein and Adjuvants

Inflammatory cells were obtained from the spinal cords of rats with acute experimental autoimmune encephalomyelitis EAE induced by inoculation with myelin basic protein MBP and adjuvants. Reverse transcriptase-polymerase chain reaction RT-PCR was used to investigate the expression of mRNA for interleukin-2 IL-2 , IL-4, IL-10 and interferon-gamma (IFN-gamma) by cells from groups of rats studied 10-21 days after inoculation. On all days of study, the inflammatory cells, which were predominantly lymphocytes, expressed mRNA for IL-2, IL-4, IL-10 and IFN-gamma. In the mRNA from normal rat spinal cord tissue, there was little expression of cytokine mRNA. Cells from a short-term MBP-reactive T cell line expressed all the cytokines. Densitometry was used to measure the products of PCR, to assess the expression of each cytokine relative to that of beta-actin. IL-2 mRNA was expressed throughout the course of disease and reached a peak on day 18, during late clinical recovery. IFN-gamma was expressed throughout the course of the disease and was also high during late recovery. IL-4 mRNA was present in the spinal cord throughout the course of the disease, with a slight rise during late recovery. Relative expression of IL-10 rose to a peak on days 17-19, during late recovery from clinical disease. This study indicates that IL-2, IL-4, IL-10 and IFN-gamma are expressed by inflammatory cells in the spinal cord in EAE, with the relative expression of all cytokines being high during late clinical recovery

Demonstrator of Time Services based on European GNSS signals: the H2020 DEMETRA Project

During 2015-2016, a European Consortium of 15 partners from 8 different countries, developed the DEMETRA (DEMonstrator of EGNSS services based on Time Reference Architecture), a project funded by the European Union in the frame of the Horizon 2020 program. This project aims at developing and experimenting time dissemination services dedicated to specific users like traffic control, energy distribution, finance, telecommunication, and scientific institutions. Nine services have been developed. These services provide time dissemination with accuracy levels from millisecond to the sub-ns, and also additional services like certification, calibration, or integrity. Five of these services are based on the European GNSS. After a development phase (see PTTI 2016 presentation) the full DEMETRA system has been working during six months for demonstration. The paper will report about the experimentation results, showing performances and limits of the proposed time dissemination services, aiming to foster the exploitation of the European GNSS for timing applications

Searches for lepton-flavour-violating decays of the Higgs boson into eτ and μτ in \sqrt{s} = 13 TeV pp collisions with the ATLAS detector

Abstract This paper presents direct searches for lepton flavour violation in Higgs boson decays, H → eτ and H → μτ, performed using data collected with the ATLAS detector at the LHC. The searches are based on a data sample of proton-proton collisions at a centre-of-mass energy s $$\sqrt{s}$$ = 13 TeV, corresponding to an integrated luminosity of 138 fb−1. Leptonic (τ → ℓνℓντ) and hadronic (τ → hadrons ντ) decays of the τ-lepton are considered. Two background estimation techniques are employed: the MC-template method, based on data-corrected simulation samples, and the Symmetry method, based on exploiting the symmetry between electrons and muons in the Standard Model backgrounds. No significant excess of events is observed and the results are interpreted as upper limits on lepton-flavour-violating branching ratios of the Higgs boson. The observed (expected) upper limits set on the branching ratios at 95% confidence level, B $$\mathcal{B}$$ (H → eτ) < 0.20% (0.12%) and B $$\mathcal{B}$$ (H → μτ ) < 0.18% (0.09%), are obtained with the MC-template method from a simultaneous measurement of potential H → eτ and H → μτ signals. The best-fit branching ratio difference, B $$\mathcal{B}$$ (H → μτ) → B $$\mathcal{B}$$ (H → eτ), measured with the Symmetry method in the channel where the τ-lepton decays to leptons, is (0.25 ± 0.10)%, compatible with a value of zero within 2.5σ

Studies of new Higgs boson interactions through nonresonant HH production in the b¯bγγ fnal state in pp collisions at √s = 13 TeV with the ATLAS detector

A search for nonresonant Higgs boson pair production in the b ¯bγγ fnal state is performed using 140 fb−1 of proton-proton collisions at a centre-of-mass energy of 13 TeV recorded by the ATLAS detector at the CERN Large Hadron Collider. This analysis supersedes and expands upon the previous nonresonant ATLAS results in this fnal state based on the same data sample. The analysis strategy is optimised to probe anomalous values not only of the Higgs (H) boson self-coupling modifer κλ but also of the quartic HHV V (V = W, Z) coupling modifer κ2V . No signifcant excess above the expected background from Standard Model processes is observed. An observed upper limit µHH &lt; 4.0 is set at 95% confdence level on the Higgs boson pair production cross-section normalised to its Standard Model prediction. The 95% confdence intervals for the coupling modifers are −1.4 &lt; κλ &lt; 6.9 and −0.5 &lt; κ2V &lt; 2.7, assuming all other Higgs boson couplings except the one under study are fxed to the Standard Model predictions. The results are interpreted in the Standard Model efective feld theory and Higgs efective feld theory frameworks in terms of constraints on the couplings of anomalous Higgs boson (self-)interactions