Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL-1 (1.5 μg kg-1) and an IC50 value of 0.36 ng mL-1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.This work has been funded by the Ministry of Science, Innovation and Universities (MSIU) (RTI2018-096410-B-C21, PID2021-127457OB-C21 and PID2019-105237 GB-I00). FP acknowledges the MSIU for an FPU contract.S

Signatures of the sources in the gravitational waves of a perturbed Schwarzschild black hole

The explicit form of perturbation equation for the $\Psi_4$ Weyl scalar, containing the matter source terms, is derived for general type D spacetimes. It is described in detail the particular case of the Schwarzschild spacetime using in-going penetrating coordinates. As a practical application, we focused on the emission of gravitational waves when a black hole is perturbed by a surrounding dust-like fluid matter. The symmetries of the spacetime and the simplicity of the matter source allow, by means of a spherical harmonic decomposition, to study the problem by means of a one dimensional numerical code.Comment: 17 pages, 8 figure

US Cosmic Visions: New Ideas in Dark Matter 2017: Community Report

This white paper summarizes the workshop "U.S. Cosmic Visions: New Ideas in Dark Matter" held at University of Maryland on March 23-25, 2017.Comment: 102 pages + reference

US Cosmic Visions: New Ideas in Dark Matter 2017: Community Report

This white paper summarizes the workshop "U.S. Cosmic Visions: New Ideas in Dark Matter" held at University of Maryland on March 23-25, 2017

Allicin Induces Calcium and Mitochondrial Dysregulation Causing Necrotic Death in Leishmania

BACKGROUND: Allicin has shown antileishmanial activity in vitro and in vivo. However the mechanism of action underlying its antiproliferative effect against Leishmania has been virtually unexplored. In this paper, we present the results obtained in L.infantum and a mechanistic basis is proposed. METHODOLOGY/PRINCIPAL FINDING: Exposure of the parasites to allicin led to high Ca2+ levels and mitochondrial reactive oxygen species (ROS), collapse of the mitochondrial membrane potential, reduced production of ATP and elevation of cytosolic ROS. The incubation of the promastigotes with SYTOX Green revealed that decrease of ATP was not associated with plasma membrane permeabilization. Annexin V and propidium iodide (PI) staining indicated that allicin did not induce phospholipids exposure on the plasma membrane. Moreover, DNA agarose gel electrophoresis and TUNEL analysis demonstrated that allicin did not provoke DNA fragmentation. Analysis of the cell cycle with PI staining showed that allicin induced cell cycle arrest in the G2/M phase. CONCLUSIONS/SIGNIFICANCE: We conclude that allicin induces dysregulation of calcium homeostasis and oxidative stress, uncontrolled by the antioxidant defense of the cell, which leads to mitochondrial dysfunction and a bioenergetic catastrophe leading to cell necrosis and cell cycle arrest in the premitotic phase.This work was supported by Ministerio de Economía y Competitividad, Comisión Interministerial de Ciencia yTecnología, Grant AGL2009-13009, www.mineco.gob.es (JMA), and European Commission COST Action CM1307.www.cost.eu/COST_Actions/cmst/Actions/CM1307 (JMA, MJC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Hierarchically Imprinted Polymer for Peptide Tag Recognition Based on an Oriented Surface Epitope Approach

FLAG tag (DYKDDDDK) is a short peptide commonly used for the purification of recombinant proteins. The high price of the affinity columns and their limited reusability are a shortcoming for their widespread use in biotechnology applications. Molecularly imprinted polymers (MIPs) can circumvent some of the limitations of bioaffinity columns for such applications, including long-term stability, reusability, and cost. We report herein the synthesis of MIPs selective to the FLAG tag by hierarchical imprinting. Using the epitope imprinting approach, a 5-amino acid peptide DYKDC was selected as a template and was covalently immobilized on the surface of microporous silica beads, previously functionalized with different aminosilanes, namely, 3-(2-aminoethylamino)propyldimethoxymethylsilane, AEAPMS, and N-(2-aminoethyl)-2,2,4-trimethyl-1-aza-2-silacyclopentane, AETAZS. We investigated the effect of the type of silane on the production of homogeneous silane-grafted layers with the highest extent of silanol condensation as possible using 29Si CP/MAS NMR. We observed that the right orientation of the imprinted cavities can substantially improve analyte recoveries from the MIP. After template and silica removal, the DYKDC-MIPs were used as sorbents for solid-phase extraction (molecularly imprinted solid-phase extraction) of the FLAG peptide, showing that the polymer prepared with AETAZS-bound silica beads contained binding sites more selective to the tag (RMIP-AZA = 87.4% vs RNIP-AZA = 4.1%, n = 3, RSD ≤ 4.2%) than those prepared using AEAPMS (RMIP-DM = 73.4% vs RNIP-DM = 23.2%, n = 3, RSD ≤ 4.0%) as a functionalization agent. An extensive computational molecular modeling study was also conducted, shedding some light on the interaction mechanism between the FLAG peptide and the imprinted template in the binding cavities.This work was supported by the Spanish Ministry of Economy and Competitiveness (grant RTI2018-096410-B-C21). Wethank Prof. M. J. Torralvo for assistance with the N2adsorption experiments and the National Centre for Electron Microscopy and the Centre for Elemental Microanalysis(UCM, Madrid) for the technical support. N.G. thanks theSpanish Ministry of Economy and Competitiveness(MAT2016-81001-P project) and Dr. LevíLópez (Character-ization Service of ICTP-CSIC) for performing solid-stateNMR spectroscopy. Y.R. thanks Dr. Mihaly Mezei (Depart-ment of Pharmacological Sciences, Icahn School of Medicineat Mount Sinai) for critically reading the computational part ofthis manuscript and suggesting improvements. Computationswere supported in part through the computational resourcesand staffexpertise provided by the Scientific ComputingFacility at the Icahn School of Medicine at Mount Sinai