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

Development of functionalized SYBR green II related cyanine dyes for viral RNA detection

Fluorescent probes for sensing nucleic acids have found widespread use in the field of cell and molecular biology. However, probes combined with potential for post-synthetic conjugation, e.g. for intra-endosomal measurements of RNA, are unavailable. Herein we developed cyanine dyes that can be conjugated to viral capsid or other targets. First, we solved the crystal structure of SYBR Green II. The structural elucidation of this commonly used RNA probe provided the basis for synthesizing similar molecules with much desired function for post-synthetic conjugation. To address this need, cyanine dyes were prepared using an alternative synthesis protocol. All studied compounds showed considerable brightness upon binding to nucleic acids. However, regardless of the common chromophore on the dyes, the observed fluorescence emission intensities varied significantly, where methyl-substituted dye 1 gave values higher than SYBR Green II, whereas compounds 2–5 containing undecyl spacers had lower values. Studying the structure-activity relationship revealed the longer alkyl chains to induce slight perturbation in dye intercalation, as well as demanding larger binding area on the nucleic acid lattice, explaining these differences. To study the potential biological use of the dyes, the RNA genome of enterovirus echovirus 1 was studied in vitro with the probes. A novel method employing the low binding space requirement of 1 was developed to determine the single-to-double-stranded RNA ratio of a sample, whereas compound 4 was covalently bound to the viral capsid and used successfully to monitor the viral RNA release from within the capsid. The presented results open new possibilities for preparation and use of SYBR Green-based nucleic acid probes to further apply these compounds for increasingly demanding targeting in biological contexts.peerReviewe

Artificial intelligence-enhanced care pathway planning and scheduling system:content validity assessment of required functionalities

Abstract Background: Artificial intelligence (AI) and machine learning are transforming the optimization of clinical and patient workflows in healthcare. There is a need for research to specify clinical requirements for AI-enhanced care pathway planning and scheduling systems to improve human–AI interaction in machine learning applications. The aim of this study was to assess content validity and prioritize the most relevant functionalities of an AI-enhanced care pathway planning and scheduling system. Methods: A prospective content validity assessment was conducted in five university hospitals in three different countries using an electronic survey. The content of the survey was formed from clinical requirements, which were formulated into generic statements of required AI functionalities. The relevancy of each statement was evaluated using a content validity index. In addition, weighted ranking points were calculated to prioritize the most relevant functionalities of an AI-enhanced care pathway planning and scheduling system. Results: A total of 50 responses were received from clinical professionals from three European countries. An item-level content validity index ranged from 0.42 to 0.96. 45% of the generic statements were considered good. The highest ranked functionalities for an AI-enhanced care pathway planning and scheduling system were related to risk assessment, patient profiling, and resources. The highest ranked functionalities for the user interface were related to the explainability of machine learning models. Conclusion: This study provided a comprehensive list of functionalities that can be used to design future AI-enhanced solutions and evaluate the designed solutions against requirements. The relevance of statements concerning the AI functionalities were considered somewhat relevant, which might be due to the low level or organizational readiness for AI in healthcare

Liquid‐phase hydrodeoxygenation of 4‐propylphenol to propylbenzene:reducible supports for Pt catalysts

Abstract Pyrolysis and liquefaction biocrudes obtained from lignocellulose are rich in phenolic compounds that can be converted to renewable aromatics. In this study, Pt catalysts on reducible metal oxide supports (Nb₂O₅, TiO₂), along with non‐reducible ZrO₂ as a reference, were investigated in the liquid‐phase hydrodeoxygenation (HDO) of 4‐propylphenol (350 °C, 20 bar H₂, organic solvent). The most active catalyst was Pt/Nb₂O₅, which led to the molar propylbenzene selectivity of 77%, and a yield of 75% (98% conversion). Reducible metal oxide supports provided an increased activity and selectivity to the aromatic product compared to ZrO₂, and the obtained results are among the best reported in liquid phase. The reusability of the spent catalysts was also studied. The spent Pt/Nb₂O₅ catalyst provided the lowest conversion, while the product distribution of the spent Pt/ZrO₂ catalyst changed towards oxygenates. The results highlight the potential of pyrolysis or liquefaction biocrudes as a source of aromatic chemicals

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 (1.5 μg/kg) and an IC50 value of 0.36 ng/mL. 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.MSIU for a FPU contractDepto. de Química AnalíticaFac. de Ciencias QuímicasTRUEpu