Methods and Protocol for Single-Cell Motility Assays under Topological Constraints.

The extracellular environment plays a crucial role in many physiological and pathological processes involving cell motility, such as metastatic invasion in cancer development, by heavily impacting the migration strategies adopted by the cells. The study of how mechanical constraints affect the dynamics of cell migration may be relevant to gain more insight into such processes, and it may prove to be a powerful tool in the hands of biologists. In this chapter, we describe the methods used to investigate the ability of neoplastic cells to migrate through narrowing, rigid microstructures upon chemoattractant stimulation.

Structured-light-sheet imaging in an integrated optofluidic platform.

Heterogeneity investigation at the single-cell level reveals morphological and phenotypic characteristics in cell populations. In clinical research, heterogeneity has important implications in the correct detection and interpretation of prognostic markers and in the analysis of patient-derived material. Among single-cell analysis, imaging flow cytometry allows combining information retrieved by single cell images with the throughput of fluidic platforms. Nevertheless, these techniques might fail in a comprehensive heterogeneity evaluation because of limited image resolution and bidimensional analysis. Light sheet fluorescence microscopy opened new ways to study in 3D the complexity of cellular functionality in samples ranging from single-cells to micro-tissues, with remarkably fast acquisition and low photo-toxicity. In addition, structured illumination microscopy has been applied to single-cell studies enhancing the resolution of imaging beyond the conventional diffraction limit. The combination of these techniques in a microfluidic environment, which permits automatic sample delivery and translation, would allow exhaustive investigation of cellular heterogeneity with high throughput image acquisition at high resolution. Here we propose an integrated optofluidic platform capable of performing structured light sheet imaging flow cytometry (SLS-IFC). The system encompasses a multicolor directional coupler equipped with a thermo-optic phase shifter, cylindrical lenses and a microfluidic network to generate and shift a patterned light sheet within a microchannel. The absence of moving parts allows a stable alignment and an automated fluorescence signal acquisition during the sample flow. The platform enables 3D imaging of an entire cell in about 1 s with a resolution enhancement capable of revealing sub-cellular features and sub-diffraction limit details.

On the robustness of machine learning algorithms toward microfluidic distortions for cell classification via on-chip fluorescence microscopy.

Single-cell imaging and sorting are critical technologies in biology and clinical applications. The power of these technologies is increased when combined with microfluidics, fluorescence markers, and machine learning. However, this quest faces several challenges. One of these is the effect of the sample flow velocity on the classification performances. Indeed, cell flow speed affects the quality of image acquisition by increasing motion blur and decreasing the number of acquired frames per sample. We investigate how these visual distortions impact the final classification task in a real-world use-case of cancer cell screening, using a microfluidic platform in combination with light sheet fluorescence microscopy. We demonstrate, by analyzing both simulated and experimental data, that it is possible to achieve high flow speed and high accuracy in single-cell classification. We prove that it is possible to overcome the 3D slice variability of the acquired 3D volumes, by relying on their 2D sum z-projection transformation, to reach an efficient real time classification with an accuracy of 99.4% using a convolutional neural network with transfer learning from simulated data. Beyond this specific use-case, we provide a web platform to generate a synthetic dataset and to investigate the effect of flow speed on cell classification for any biological samples and a large variety of fluorescence microscopes (https://www.creatis.insa-lyon.fr/site7/en/MicroVIP).

Microfluidic Lab-on-a-Chip for Studies of Cell Migration under Spatial Confinement.

Understanding cell migration is a key step in unraveling many physiological phenomena and predicting several pathologies, such as cancer metastasis. In particular, confinement has been proven to be a key factor in the cellular migration strategy choice. As our insight in the field improves, new tools are needed in order to empower biologists' analysis capabilities. In this framework, microfluidic devices have been used to engineer the mechanical and spatial stimuli and to investigate cellular migration response in a more controlled way. In this work, we will review the existing technologies employed in the realization of microfluidic cellular migration assays, namely the soft lithography of PDMS and hydrogels and femtosecond laser micromachining. We will give an overview of the state of the art of these devices, focusing on the different geometrical configurations that have been exploited to study specific aspects of cellular migration. Our scope is to highlight the advantages and possibilities given by each approach and to envisage the future developments in in vitro migration studies under spatial confinement in microfluidic devices.

Structural variation in the linkage region of pharmaceutical heparin arising from oxidative treatments during manufacture.

During the manufacture of pharmaceutical heparin, a range of treatments are applied to sanitize, decolourise and reduce the pyrogenic properties of the samples. The structural effects of bleaching, an oxidative process, are examined. Among 1H and 13C NMR signals ascribable to the tetrasaccharide linkage region of heparin, samples of porcine mucosal heparin frequently display characteristic signals at chemical shift values of 4.5 and 106 ppm respectively, which have not been explained previously. Fractions enriched with material reporting this signal were isolated from heparinase digested porcine mucosal heparin samples and subjected to analysis using mass spectrometry and NMR spectroscopy. A novel structure, ΔU-Gal-Gal-Xyl-CH2-CONH2, was identified by mass fragmentation experiments and further interesting structural motifs emerged following evaluation by mass spectrometry of longer oligosaccharide chains biosynthesized away from the linker tetrasaccharide, GlcA-Gal-Gal-Xyl. The carbohydrate-protein linkage region is thus affected by the bleaching step involved in the manufacturing process of heparin. The discovery of specific modifications that reflect the extent of the oxidation treatment adopted is relevant to the monitoring of inadvertent damage to the heparin structure during manufacture that contributes to sample variation and which could also lead to reduced drug quality.

Strategies for improved temporal response of glass-based optical switches.

We present an optimization of the dynamics of integrated optical switches based on thermal phase shifters. These devices have been fabricated in the volume of glass substrates by femtosecond laser micromachining and are constituted by an integrated Mach-Zehnder interferometer and a superficial heater. Simulations, surface micromachining and innovative layouts allowed us to improve the temporal response of the optical switches down to a few milliseconds. In addition, taking advantage of an electrical pulse shaping approach where an optimized voltage signal is applied to the heater, we proved a switching time as low as 78 µs, about two orders of magnitude shorter with respect to the current state of the art of thermally-actuated optical switches in glass.

Enisamium Inhibits SARS-CoV-2 RNA Synthesis.

Pandemic SARS-CoV-2 causes a mild to severe respiratory disease called coronavirus disease 2019 (COVID-19). While control of the SARS-CoV-2 spread partly depends on vaccine-induced or naturally acquired protective herd immunity, antiviral strategies are still needed to manage COVID-19. Enisamium is an inhibitor of influenza A and B viruses in cell culture and clinically approved in countries of the Commonwealth of Independent States. In vitro, enisamium acts through metabolite VR17-04 and inhibits the activity of the influenza A virus RNA polymerase. Here we show that enisamium can inhibit coronavirus infections in NHBE and Caco-2 cells, and the activity of the SARS-CoV-2 RNA polymerase in vitro. Docking and molecular dynamics simulations provide insight into the mechanism of action and indicate that enisamium metabolite VR17-04 prevents GTP and UTP incorporation. Overall, these results suggest that enisamium is an inhibitor of SARS-CoV-2 RNA synthesis in vitro.

Use of Chènevotte, a Valuable Co-Product of Industrial Hemp Fiber, as Adsorbent for Pollutant Removal. Part I: Chemical, Microscopic, Spectroscopic and Thermogravimetric Characterization of Raw and Modified Samples.

FINEAU (2021-2024) is a trans-disciplinary research project involving French, Serbian, Italian, Portuguese and Romanian colleagues, a French agricultural cooperative and two surface-treatment industries, intending to propose chènevotte, a co-product of the hemp industry, as an adsorbent for the removal of pollutants from polycontaminated wastewater. The first objective of FINEAU was to prepare and characterize chènevotte-based materials. In this study, the impact of water washing and treatments (KOH, Na2CO3 and H3PO4) on the composition and structure of chènevotte (also called hemp shives) was evaluated using chemical analysis, X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, X-ray computed nanotomography (nano-CT), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, solid state NMR spectroscopy and thermogravimetric analysis. The results showed that all these techniques are complementary and useful to characterize the structure and morphology of the samples. Before any chemical treatment, the presence of impurities with a compact unfibrillated structure on the surfaces of chènevotte samples was found. Data indicated an increase in the crystallinity index and significant changes in the chemical composition of each sample after treatment as well as in surface morphology and roughness. The most significant changes were observed in alkaline-treated samples, especially those treated with KOH.

Identification of Compounds Targeting HuD. Another Brick in the Wall of Neurodegenerative Disease Treatment.

ELAV-like (ELAVL) RNA-binding proteins play a pivotal role in post-transcriptional processes, and their dysregulation is involved in several pathologies. This work was focused on HuD (ELAVL4), which is specifically expressed in nervous tissues, and involved in differentiation and synaptic plasticity mechanisms. HuD represents a new, albeit unexplored, candidate target for the treatment of several relevant neurodegenerative diseases. The aim of this pioneering work was the identification of new molecules able to recognize and bind HuD, thus interfering with its activity. We combined virtual screening, molecular dynamics (MD), and STD-NMR techniques. Starting from around 51 000 compounds, four promising hits eventually provided experimental evidence of their ability to bind HuD. Among the selected best hits, folic acid was found to be the most interesting one, being able to well recognize the HuD binding site. Our results provide a basis for the identification of new HuD interfering compounds which may be useful against neurodegenerative syndromes.

Rapid Prototyping of 3D Biochips for Cell Motility Studies Using Two-Photon Polymerization.

The study of cellular migration dynamics and strategies plays a relevant role in the understanding of both physiological and pathological processes. An important example could be the link between cancer cell motility and tumor evolution into metastatic stage. These strategies can be strongly influenced by the extracellular environment and the consequent mechanical constrains. In this framework, the possibility to study the behavior of single cells when subject to specific topological constraints could be an important tool in the hands of biologists. Two-photon polymerization is a sub-micrometric additive manufacturing technique that allows the fabrication of 3D structures in biocompatible resins, enabling the realization of ad hoc biochips for cell motility analyses, providing different types of mechanical stimuli. In our work, we present a new strategy for the realization of multilayer microfluidic lab-on-a-chip constructs for the study of cell motility which guarantees complete optical accessibility and the possibility to freely shape the migration area, to tailor it to the requirements of the specific cell type or experiment. The device includes a series of micro-constrictions that induce different types of mechanical stress on the cells during their migration. We show the realization of different possible geometries, in order to prove the versatility of the technique. As a proof of concept, we present the use of one of these devices for the study of the motility of murine neuronal cancer cells under high physical confinement, highlighting their peculiar migration mechanisms.

Effects of Thermal Annealing on Femtosecond Laser Micromachined Glass Surfaces.

Femtosecond laser micromachining (FLM) of fused silica allows for the realization of three-dimensional embedded optical elements and microchannels with micrometric feature size. The performances of these components are strongly affected by the machined surface quality and residual roughness. The polishing of 3D buried structures in glass was demonstrated using different thermal annealing processes, but precise control of the residual roughness obtained with this technique is still missing. In this work, we investigate how the FLM irradiation parameters affect surface roughness and we characterize the improvement of surface quality after thermal annealing. As a result, we achieved a strong roughness reduction, from an average value of 49 nm down to 19 nm. As a proof of concept, we studied the imaging performances of embedded mirrors before and after thermal polishing, showing the capacity to preserve a minimum feature size of the reflected image lower than µ5µm. These results allow for us to push forward the capabilities of this enabling fabrication technology, and they can be used as a starting point to improve the performances of more complex optical elements, such as hollow waveguides or micro-lenses.

Automatic imaging of Drosophila embryos with light sheet fluorescence microscopy on chip.

We present a microscope on chip for automated imaging of Drosophila embryos by light sheet fluorescence microscopy. This integrated device, constituted by both optical and microfluidic components, allows the automatic acquisition of a 3D stack of images for specimens diluted in a liquid suspension. The device has been fully optimized to address the challenges related to the specimens under investigation. Indeed, the thickness and the high ellipticity of Drosophila embryos can degrade the image quality. In this regard, optical and fluidic optimization has been carried out to implement dual-sided illumination and automatic sample orientation. In addition, we highlight the dual color investigation capabilities of this device, by processing two sample populations encoding different fluorescent proteins. This work was made possible by the versatility of the used fabrication technique, femtosecond laser micromachining, which allows straightforward fabrication of both optical and fluidic components in glass substrates.

High-throughput 3D imaging of single cells with light-sheet fluorescence microscopy on chip.

Single-cell analysis techniques are fundamental to study the heterogeneity of cellular populations, which is the basis to understand several biomedical mechanisms. Light-sheet fluorescence microscopy is a powerful technique for obtaining high-resolution imaging of individual cells, but the complexity of the setup and the sample mounting procedures limit its overall throughput. In our work, we present an optofluidic microscope-on-chip with integrated microlenses for light-sheet shaping and with a fluidic microchannel that allows the automatic and continuous delivery of samples of a few tens of microns in size. The device is used to perform dual-color fluorescence analysis and 3D reconstruction of xenograft-derived mouse breast cancer cells.

Evaluación endoscópica de la deglución. Resultados en pacientes con tumores benignos y malignos de laringe y hipofaringe, tratados con cirugía transoral con láser de CO2 / Functional endoscopic evaluation of swallowing. Results inpatients with benign and malignant tumors of the larynx and hypopharynx treated with transoral CO2 laser surgery / A avaliação endoscópica da deglutição. Os resultados em pacientes com tumores benignos e malignos da laringe e hipofaringe tratados com cirurgia transoral laser de CO2

La utilización del LASER de CO2 en laringología comenzó en la década del ‘80, permitiendo la exéresis de tumores por vía transoral. Su indicación se basa en los excelentes resultados oncológicos. La función deglutoria postquirúrgica es un factor importante en la calidad de vida de los pacientes con cáncer del tracto aerodigestivo superior. Los resultados de la deglución son relevantes para elegir la modalidad terapéutica, la cual debe ser no sólo efectiva en controlar el cáncer sino también en preservar la función del órgano. El grado y el tipo de alteración deglutoria deben ser determinados en forma precisa, para establecer el tratamiento postoperatorio adecuado. Esto es posible mediante estudios como la videodeglución y la evaluación endoscópica de la deglución (FEESST)...

The CO2 laser have been used for treatment of laryngeal cancers since 1980, with excellent oncological results. The swallowing plays an important roll in superior aerodigestive cáncer patient. Swallowing results are relevant to choose the therapeutic modality, which must be effective in controlling not only cancer but also in preserving organ function. The degree and type of swallowing impairment must be determined precisely, to establish the appropriate postoperative treatment. This is possible through studies like videodeglucion and endoscopic evaluation of swallowing (FEESST)...

O uso do laser de CO2 laringologia começou nos anos 80, permitindo a excisão tumor transoral. A indicação baseia-se nos excelentes resultados oncológicos. Função de deglutição pós-cirúrgico é uma importante qualidade de vida de pacientes com câncer do fator trato aerodigestivo superior. Engolindo resultados são relevantes para escolher a modalidade terapêutica, que deve ser eficaz no controle não só do câncer, mas também em preservar a função do órgão. O grau e tipo de comprometimento da deglutição deve ser determinada com precisão, para estabelecer o tratamento pós-operatório adequado. Isso é possível através de estudos como videodeglucion e avaliação endoscópica da deglutição (FEESST)...

Isolation and characterisation of impurities in adapalene.

Three impurities of structure 2-4 were isolated and characterised during the optimisation of a synthetic procedure to adapalene. Impurity 1 was a by-product of the Friedel-Crafts reaction of adamantanol with 4-bromoanisole. Impurities 3 and 4 were due to side reactions of the final Negishi coupling.