Detection of azaspiracids in mussels using electrochemical immunosensors for fast screening in monitoring programs

Given the widespread occurrence of azaspiracids (AZAs), it is clearly necessary to advance in simple and low-cost methods for the rapid detection of these marine toxins in order to protect seafood consumers. To address this need, electrochemical immunosensors for the detection of AZAs based on a competitive direct immunoassay using peroxidase-labelled AZA as a tracer were developed. An anti-AZA polyclonal antibody was immobilised in a controlled and stable manner on protein G or avidin-coated electrodes. Experimental conditions were first optimised using colorimetric immunoassays on microtitre plates, providing intermediate products already applicable to the accurate detection of AZAs. Then, transfer of the protein G and avidin–biotin interaction-based immunoassays to 8-electrode arrays provided compact and miniaturised devices for the high-throughput detection of AZAs. The low amounts of immunoreagents required as well as the potential for reusability of the avidin–biotin interaction-based immunosensors represented significant economic savings as well as a contribution to sustainability. The electrochemical immunosensors enabled the quantification of all regulated AZAs below the regulatory limit, as well as a broad range of other toxic AZA analogues (from 63 ± 3 to 2841 ± 247 μg AZA-1 equiv./kg for the protein G-based immunosensor and from 46 ± 2 to 3079 ± 358 μg AZA-1 equiv./kg for the avidin–biotin interaction-based immunosensor). The good agreement between the results obtained by the immunosensors and LC–MS/MS in the analysis of naturally contaminated mussel samples demonstrated the easy implementation of electrochemical immunosensors for routine analysis of AZAs in food safety monitoring programs.info:eu-repo/semantics/acceptedVersio

Colloquium: Mechanical formalisms for tissue dynamics

The understanding of morphogenesis in living organisms has been renewed by tremendous progressin experimental techniques that provide access to cell-scale, quantitative information both on theshapes of cells within tissues and on the genes being expressed. This information suggests that ourunderstanding of the respective contributions of gene expression and mechanics, and of their crucialentanglement, will soon leap forward. Biomechanics increasingly benefits from models, which assistthe design and interpretation of experiments, point out the main ingredients and assumptions, andultimately lead to predictions. The newly accessible local information thus calls for a reflectionon how to select suitable classes of mechanical models. We review both mechanical ingredientssuggested by the current knowledge of tissue behaviour, and modelling methods that can helpgenerate a rheological diagram or a constitutive equation. We distinguish cell scale ("intra-cell")and tissue scale ("inter-cell") contributions. We recall the mathematical framework developpedfor continuum materials and explain how to transform a constitutive equation into a set of partialdifferential equations amenable to numerical resolution. We show that when plastic behaviour isrelevant, the dissipation function formalism appears appropriate to generate constitutive equations;its variational nature facilitates numerical implementation, and we discuss adaptations needed in thecase of large deformations. The present article gathers theoretical methods that can readily enhancethe significance of the data to be extracted from recent or future high throughput biomechanicalexperiments.Comment: 33 pages, 20 figures. This version (26 Sept. 2015) contains a few corrections to the published version, all in Appendix D.2 devoted to large deformation

Bioaccessibility of contaminants of emerging concern in raw and cooked commercial seafood species: insights for food safety risk assessment.

Autoria na publicação: FABIOLA FOGAÇA

Dynamic stability of spindles controlled by molecular motor kinetics

We analyze the role of the force-dependent kinetics of motor proteins in the stability of antiparallel arrays of polar filaments, such as those in the mitotic spindle. We determine the possible stable structures and show that there exists an instability associated to the collective behavior of motors that leads to the collapse of the structure. The agreement of our results and several experimental observations in eukaryotic cell division suggests an important role of kinesin-5 motors and microtubule bundles in the stability of the mitotic spindle

Geometrical characterization of fluorescently labelled surfaces from noisy 3D microscopy data

Modern fluorescence microscopy enables fast 3D imaging of biological and inert systems alike. In many studies, it is important to detect the surface of objects and quantitatively characterize its local geometry, including its mean curvature. We present a fully automated algorithm to determine the location and curvatures of an object from 3D fluorescence images, such as those obtained using confocal or light-sheet microscopy. The algorithm aims at reconstructing surface labelled objects with spherical topology and mild deformations from the spherical geometry with high accuracy, rather than reconstructing arbitrarily deformed objects with lower fidelity. Using both synthetic data with known geometrical characteristics and experimental data of spherical objects, we characterize the algorithm's accuracy over the range of conditions and parameters typically encountered in 3D fluorescence imaging. We show that the algorithm can detect the location of the surface and obtain a map of local mean curvatures with relative errors typically below 2% and 20%, respectively, even in the presence of substantial levels of noise. Finally, we apply this algorithm to analyse the shape and curvature map of fluorescently labelled oil droplets embedded within multicellular aggregates and deformed by cellular forces

Dynamic kinesin-1 clustering on microtubules due to mutually attractive interactions

Molecular motors often work collectively inside the cell. While the properties of individual motors have been extensively studied over the last decade, much less is known on how motors coordinate their action when working in ensembles. The motor collective behaviour in conditions where they contact each other, as in intracellular transport, may strongly depend on their mutual interactions. In particular, mutual interactions may result in motor clustering without the need of additional proteins. Here we study the interactions between kinesin-1 molecules by analysing their attachment/detachment kinetics on microtubules in the absence of motor motion. Our in vitro experiments show that kinesins-1 remain longer attached to the microtubule in the presence of neighbouring motors, resulting in the formation of motor clusters. Numerical simulations of the motor attachment/detachment dynamics show that the presence of attractive interactions between motors quantitatively accounts for the experimental observations. From the comparison of the numerical results and the experimental data we estimate the interaction energy between kinesin-1 molecules to be 1.6 +/- 0.5K(B)T. The existence of attractive interactions between kinesins-1 provides a new insight into the coordination mechanism between motor proteins and may be crucial to understand the large scale traffic in cells

The kitchen as a physics classroom

Cooking is a tangible, familiar, and delicious tool for teaching physics, which is easy to implement in a university setting. Through our courses at Harvard and UCLA, each year we are engaging hundreds of undergraduate students, primarily non-science majors, in science concepts and the scientific research process. We find that weekly lectures by chefs and professors, paired with edible lab experiments, generate enthusiasm and provide strong motivation for students to learn physics. By the end of the course, students are able to conduct independent scientific research and present their results in a final science fair. Given the considerable broad appeal of food and cooking, the topic could be adapted to other post-secondary as well as secondary-level courses

Hybrid Antibody–Aptamer Assay for Detection of Tetrodotoxin in Pufferfish

The marine toxin tetrodotoxin (TTX) poses a great risk to public health safety due to its severe paralytic effects after ingestion. Seafood poisoning caused by the consumption of contaminated marine species like pufferfish due to its expansion to nonendemic areas has increased the need for fast and reliable detection of the toxin to effectively implement prevention strategies. Liquid chromatography-mass spectrometry is considered the most accurate method, although competitive immunoassays have also been reported. In this work, we sought to develop an aptamer-based assay for the rapid, sensitive, and cost-effective detection of TTX in pufferfish. Using capture-SELEX combined with next-generation sequencing, aptamers were identified, and their binding properties were evaluated. Finally, a highly sensitive and user-friendly hybrid antibody–aptamer sandwich assay was developed with superior performance compared to several assays reported in the literature and commercial immunoassay kits. The assay was successfully applied to the quantification of TTX in pufferfish extracts, and the results obtained correlated very well with a competitive magnetic bead-based immunoassay performed in parallel for comparison. This is one of the very few works reported in the literature of such hybrid assays for small-molecule analytes whose compatibility with field samples is also demonstrated.info:eu-repo/semantics/publishedVersio