Publications

2023

• DNA Quadruplex Structure with a Unique Cation Dependency
  
  • Gajarsky Martin
  • Stadlbauer Petr
  • Sponer Jiri
  • Cucchiarini Anne
  • Dobrovolna Michaela
  • Brazda Vaclav
  • Mergny Jean-Louis
  • Trantirek Lukas
  • Lenarcic Zivkovic Martina
  Angewandte Chemie International Edition, Wiley-VCH Verlag, 2023. DNA quadruplex structures provide an additional layer of regulatory control in genome maintenance and gene expression and are widely used in nanotechnology. We report the discovery of a novel tetrastranded structure formed from a native G‐rich DNA sequence originating from the telomeric region of Caenorhabditis elegans. The structure is defined by multiple properties that distinguish it from all other known DNA quadruplexes. Most notably, the formation of a stable so‐called KNa‐quadruplex (KNaQ) requires concurrent coordination of K+ and Na+ ions at two distinct binding sites. This structure provides novel insight into G‐rich DNA folding under ionic conditions relevant to eukaryotic cell physiology and the structural evolution of telomeric DNA. It highlights the differences between the structural organization of human and nematode telomeric DNA, which should be considered when using C. elegans as a model in telomere biology, particularly in drug screening applications. Additionally, the absence/presence of KNaQ motifs in the host/parasite introduces an intriguing possibility of exploiting the KNaQ fold as a plausible antiparasitic drug target. The structure's unique shape and ion dependency and the possibility of controlling its folding by using low‐molecular‐weight ligands can be exploited for the design or discovery of novel recognition DNA elements and sensors. (10.1002/anie.202313226)
  DOI : 10.1002/anie.202313226
• Strong and selective interactions of palmatine with G-rich sequences in TRF2 promoter; experimental and computational studies
  
  • Fazelifar Pegah
  • Cucchiarini Anne
  • Khoshbin Zahra
  • Mergny Jean-Louis
  • Kazemi Noureini Sakineh
  Journal of Biomolecular Structure and Dynamics, Taylor & Francis: STM, Behavioural Science and Public Health Titles, 2023, pp.in press. Background: G-rich sequences have the potential to fold into G-quadruplexes (GQs). G-quadruplexes, particularly those positioned in the regulatory regions of proto-oncogenes, have recently garnered attention in anti-cancer drug design. Methods: A thermal FRET assay was employed to conduct preliminary screening of various alkaloids, aiming to identify stronger interactions with a specific set of G-rich double-labeled oligonucleotides in both K+ and Na+ buffers. These oligonucleotides were derived from regions associated with Kit, Myc, Ceb, Bcl2, human telomeres, and potential G-quadruplex forming sequences found in the Nrf2 and Trf2 promoters. Palmatine generally increased the stability of different G-rich sequences into their folded GQ structures, more or less in a concentration dependent manner. The thermal stability and interaction of palmatine was further studied using transition FRET (t-FRET), CD and UV-visible spectroscopy and molecular dynamics simulation methods. Results: Palmatine showed the strongest interaction with TRF2 in both K+ and Na+ buffers even at equimolar concentration ratio. T-FRET studies revealed that palmatine has the potential to disrupt double-strand formation by the TRF2 sequence in the presence of its complementary strand. Palmatine exhibits a stronger interaction with G-rich strand DNA, promoting its folding into G-quadruplex structures. It is noteworthy that palmatine exhibits the strongest interaction with TRF2, which is the shortest sequence among the G-rich oligonucleotides studied, featuring only one nucleotide for two of its loops (10.1080/07391102.2023.2292793)
  DOI : 10.1080/07391102.2023.2292793
• In-vivo tomographic imaging and shear wave elastography using a single-shot off-axis FF-OCT (SO-FF-OCT) approach
  
  • Martins Seromenho Emmanuel
  • Dufour Nina
  • Legrand Maud
  • Schiffler Jesse
  • Maioli Vincent
  • Facca Sybille
  • Bahlouli Nadia
  • Nahas Amir
  , 2023.
• Development of an opto-microfluidic assay, to probe kidney glomerulus function and signaling on-chip
  
  • Mauviel Maxime
  , 2023. Glomerulonephritis (RPGN) and glomerulosclerosis (FSGS) are diseases resulting from damage to the glomerulus, causing activation of glomerular parietal epithelial cells (PECs), which can lead ultimately to renal insufficiency. We developed an organ-on-chip microfluidic system that emulates the structure and cell types of the glomerulus. This system allows for the controlled co-culture of three cell types mimicking the glomerular organization (glomerular endothelial cells GEnCs, podocytes, and PECs), and retained filtration functions of an actual glomerulus. EGF exposure led to increased PECs proliferation and decreased glomerular filtration capabilities. The characteristics observed in our chip closely mirror those of PECs in RPGN and FSGS progression in vivo.We found through super-resolution (dSTORM) that CD9-rich nano-domains are primary sites for EGFR activation upon HB-EGF stimulations in PECs. Furthermore, in CD9-KO PECs, the ROS production induced by EGFR activation by HB-EGF, quantitatively tracked using luminescent nanoparticles, was significantly reduced. CD9 thus organizes EGFR and its regulates signaling.By combining single molecule imaging techniques and Organ-on-chip, our technology offers a bridge between in vitro and in vivo studies, potentially limiting the need for animal testing.
• Harnessing G-quadruplex ligands for lung cancer treatment: A comprehensive overview
  
  • Figueiredo Joana
  • Djavaheri-Mergny Mojgan
  • Ferret Lucille
  • Mergny Jean-Louis
  • Cruz Carla
  Drug Discovery Today, Elsevier, 2023, 28 (12), pp.103808. Lung cancer (LC) remains a leading cause of mortality worldwide, and new therapeutic strategies are urgently needed. One such approach revolves around the utilization of four-stranded nucleic acid secondary structures, known as G-quadruplexes (G4), which are formed by G-rich sequences. G4 structures constitute enticing targets for therapeutic intervention. Ligands that bind selectively to G4 structures, present a promising strategy for regulating crucial cellular processes involved in the progression of LC, rendering them potent agents for lung cancer treatment. In this review, we offer a summary of the recent advancements in the development of ligands capable of targeting specific genes associated with the development and progression of lung cancer. (10.1016/j.drudis.2023.103808)
  DOI : 10.1016/j.drudis.2023.103808
• The future of CRISPR in Mycobacterium tuberculosis infection
  
  • Zein Eddine Rima
  • Refrégier Guislaine
  • Cervantes Jorge
  • Yokobori Noemí Kaoru
  Journal of Biomedical Science, BioMed Central, 2023, 30 (1), pp.34. Clustered Regularly Interspaced Short Palindromic repeats (CRISPR)-Cas systems rapidly raised from a bacterial genetic curiosity to the most popular tool for genetic modifications which revolutionized the study of microbial physiology. Due to the highly conserved nature of the CRISPR locus in Mycobacterium tuberculosis , the etiological agent of one of the deadliest infectious diseases globally, initially, little attention was paid to its CRISPR locus, other than as a phylogenetic marker. Recent research shows that M. tuberculosis has a partially functional Type III CRISPR, which provides a defense mechanism against foreign genetic elements mediated by the ancillary RNAse Csm6. With the advent of CRISPR-Cas based gene edition technologies, our possibilities to explore the biology of M. tuberculosis and its interaction with the host immune system are boosted. CRISPR-based diagnostic methods can lower the detection threshold to femtomolar levels, which could contribute to the diagnosis of the still elusive paucibacillary and extrapulmonary tuberculosis cases. In addition, one-pot and point-of-care tests are under development, and future challenges are discussed. We present in this literature review the potential and actual impact of CRISPR-Cas research on human tuberculosis understanding and management. Altogether, the CRISPR-revolution will revitalize the fight against tuberculosis with more research and technological developments. (10.1186/s12929-023-00932-4)
  DOI : 10.1186/s12929-023-00932-4
• Filming DNA repair at the atomic level
  
  • Vos Marten
  Science, American Association for the Advancement of Science (AAAS), 2023, 382, pp.996-997. Dissection of multistep catalysis by a photoenzyme could inspire green chemistry applications (10.1126/science.adl3002)
  DOI : 10.1126/science.adl3002
• Fluorescence to measure light intensity
  
  • Lahlou Aliénor
  • Tehrani Hessam Sepasi
  • Coghill Ian
  • Shpinov Yuriy
  • Mandal Mrinal
  • Plamont Marie-Aude
  • Aujard Isabelle
  • Niu Yuxi
  • Nedbal Ladislav
  • Lazár Dusan
  • Mahou Pierre
  • Supatto Willy
  • Beaurepaire Emmanuel
  • Eisenmann Isabelle
  • Desprat Nicolas
  • Croquette Vincent
  • Jeanneret Raphaël
  • Le Saux Thomas
  • Jullien Ludovic
  Nature Methods, Nature Publishing Group, 2023, 20, pp.1930–1938. Abstract Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources. (10.1038/s41592-023-02063-y)
  DOI : 10.1038/s41592-023-02063-y
• MiniBAR/KIAA0355 is a dual Rac and Rab effector required for ciliogenesis
  
  • Shaughnessy Ronan
  • Serres Murielle
  • Escot Sophie
  • Hammich Hussein
  • Cuvelier Frédérique
  • Salles Audrey
  • Rocancourt Murielle
  • Verdon Quentin
  • Gaffuri Anne-Lise
  • Sourigues Yannick
  • Malherbe Gilles
  • Velikovsky Leonid
  • Chardon Florian
  • Tinevez Jean-Yves
  • Callebaut Isabelle
  • Formstecher Etienne
  • Houdusse Anne
  • David Nicolas
  • Pylypenko Olena
  • Echard Arnaud
  , 2023. Cilia protrude from the cell surface and play critical roles in in-tracellular signaling, environmental sensing and development. Actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1-and Rab35-binding protein with a truncated BAR domain that we named MiniBAR (aka KIAA0355/GARRE) which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles traffick-ing to the ciliary base and exhibits remarkable fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia resulting from abnormal Rac-GTP/Rho-GTP levels, increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hall-marks of ciliopathies including left-right asymmetry defects. Thus, MiniBAR is a unique dual Rac and Rab effector that con-trols both actin cytoskeleton and membrane trafficking for cili-ogenesis. (10.1101/2023.07.24.550339)
  DOI : 10.1101/2023.07.24.550339
• Structuring effects of archaeal replication origins
  
  • Mottez Clémence
  • Puech Romain
  • Flament Didier
  • Myllykallio Hannu
  , 2023. Abstract Archaea use eukaryotic-like DNA replication proteins to duplicate circular chromosomes similar to those of bacteria. Although archaeal replication origins have been maintained during the evolution, they are non-essential under laboratory conditions. Here we propose the local deviations from Chargaff’s second parity rule of archaeal chromosomes result from the biased gene orientation and not from mutational biases. Our computational and experimental analyses indicate that the archaeal replication origins prevent head-to-head collisions of replication and transcription complexes as well as participate in coordination of the transfer of genetic information. Our results therefore suggest that the archaeal replication origins have alternative functions not related to their role in initiation of DNA replication. (10.1101/2023.11.15.567178)
  DOI : 10.1101/2023.11.15.567178
• A Versatile G‐quadruplex (G4)‐coated Upconverted Metal‐Organic Framework for Hypoxic Tumor Therapy
  
  • Mao Xuanxiang
  • Zhang Xiaobo
  • Chao Zhicong
  • Qiu Dehui
  • Wei Shijiong
  • Luo Rengan
  • Chen Desheng
  • Zhang Yue
  • Chen Yun
  • Yang Yuanjiao
  • Monchaud David
  • Ju Huangxian
  • Mergny Jean‐louis
  • Lei Jianping
  • Zhou Jun
  Advanced Healthcare Materials, Wiley, 2023, 12 (28), pp.2300561. Given the complexity of the tumor microenvironment, multiple strategies are being explored to tackle hypoxic tumors. One of the most efficient strategies combines several therapeutic modalities and typically requires the development of multifunctional nanocomposites through sophisticated synthetic procedures. Here, the G-quadruplex (G4)-forming sequence AS1411-A (d-(G2T)4TG(TG2)4A) was designed and used for its anti-tumor and biocatalytic properties, such as increasing the production of O2 ca. 2fold as compared to the parent AS1411 sequence. Subsequently, the AS1411-A/hemin complex (GH) was grafted on the surface and pores of a core-shell upconverted metalorganic framework (UMOF) to generate a UMGH nanoplatform. Compared with UMOF, UMGH exhibited enhanced colloidal stability, increased targeting of tumor cells and improved O2 production (8.5-fold) in situ. When irradiated with near-infrared (NIR) light, the UMGH antitumor properties were bolstered by photodynamic therapy (PDT), thanks to its ability to convert O2 into singlet oxygen (1 O2). Combined with the antiproliferative activity of AS1411-A, this novel approach herein lays the foundation for a new type of G4-based nanomedicine. (10.1002/adhm.202300561)
  DOI : 10.1002/adhm.202300561
• Microscopie multiphoton multicolore grand volume appliquée à l’imagerie du cerveau de souris
  
  • Blanc Hugo
  , 2023. L’architecture cellulaire du cerveau est en grande partie définie au cours du développement précoce. Les rôles respectifs des différents mécanismes biologiques menant à cette organisation sont encore mal compris, à cause de la grande complexité du tissu cérébral. L'analyse expérimentale de cette complexité nécessite de pouvoir imager le tissu cérébral en trois dimensions avec une résolution cellulaire, ce qui pose des défis considérables à la fois en microscopie (hétérogénéité optique du tissu cérébral) et en gestion de données (plusieurs teraoctets/acquisition).Ce travail de thèse a porté sur le développement d'une méthode de microscopie grand volume couleurs permettant de cartographier de façon robuste des cerveaux de souris ex vivo avec une résolution micrométrique, quel que soit leur stade de développement (ChroMS-2). La méthode repose sur la microscopie multiphotonique couleurs en coupes sériées, ou ChroMS (chromatic multiphoton serial microscopy), optimisée pour imager des tissus marqués avec des combinaisons de protéines fluorescentes de différentes couleurs ('Brainbow').Nous présentons d'abord le fonctionnement et l'optimisation du microscope ChroMS, de façon à accélérer le débit d’acquisition, améliorer la qualité des données, et assurer la fiabilité du microscope. Ce travail a notamment permis de réaliser un gain de temps de deux ordres de grandeur pour l'acquisition volumique par rapport au prototype, permettant ainsi de cartographier un millimètre cube de tissus en quelques minutes, ou un cerveau de souris adulte en quelques jours.Ensuite, nous détaillons la mise en place d'un pipeline automatisé de traitement et reconstruction de ces grandes images. En particulier, nous discutons les problèmes liés à la présence de déformations dans les images et leur traitement pour la reconstruction des volumes.Enfin, nous présentons l'application de ce nouveau système d'imagerie pour différents projets. Nous discutons la mise au point d'un protocole de préparation et d’acquisition permettant de cartographier le tissu cérébral de souris à différents stades de développement, et présentons les images obtenues. Nous illustrons également le caractère versatile de la plateforme ChroMS et son utilisation possible sur d'autres organes (cœur, moelle épinière), ou espèces (poulet) et avec d’autres sources de contraste que la fluorescence (génération d'harmoniques).
• Giulia & la dégradation des parchemins
  
  • Galante Giulia
  • Elouin Amélie
  , 2023. Cette semaine Amélie reçoit la doctorante Giulia Galante ! Dans cet épisode Giulia nous parlera de son sujet de thèse : la dégradation du parchemin par analyses multiphoton... beaucoup de mots compliqués mais pas de panique, on t'explique tout ! Bon voyage ! Voix'Yageuse : Amélie Elouin, Laboratoire d'Optique et Biosciences, LOB (CNRS, INSERM, École polytechnique, Institut Polytechnique de Paris) Invité : Giulia Galante, Laboratoire d'Optique et Biosciences, LOB (CNRS, INSERM, Institut Polytechnique de Paris) et Centre de Recherche sur la Conservation, CRC (CNRS, MNHN, Ministère de la Culture)
• Chromatically Corrected Multicolor Multiphoton Microscopy
  
  • Blanc Hugo
  • Kaddour Gabriel
  • David Nicolas B
  • Supatto Willy
  • Livet Jean
  • Beaurepaire Emmanuel
  • Mahou Pierre
  ACS photonics, American Chemical Society, 2023. Simultaneous imaging of multiple labels in tissues is key to studying complex biological processes. Although strategies for color multiphoton excitation have been established, chromatic aberration remains a major problem when multiple excitation wavelengths are used in a scanning microscope. Chromatic aberration introduces a spatial shift between the foci of beams of different wavelengths that varies across the field of view, severely degrading the performance of color imaging. In this work, we propose an adaptive correction strategy that solves this problem in two-beam microscopy techniques. Axial chromatic aberration is corrected by a refractive phase mask that introduces pure defocus into one beam, while lateral chromatic aberration is corrected by a piezoelectric mirror that dynamically compensates for lateral shifts during scanning. We show that this lightefficient approach allows seamless chromatic correction over the entire field of view of different multiphoton objectives without compromising spatial and temporal resolution and that the effective area for beam-mixing processes can be increased by more than 1 order of magnitude. We illustrate this approach with simultaneous three-color, two-photon imaging of developing zebrafish embryos and fixed Brainbow mouse brain slices over large areas. These results establish a robust and efficient method for chromatically corrected multiphoton imaging. (10.1021/acsphotonics.3c01104)
  DOI : 10.1021/acsphotonics.3c01104
• Synthesis and Antimalarial Evaluation of New 1,3,5-tris[(4-(Substituted-aminomethyl)phenyl)methyl]benzene Derivatives: A Novel Alternative Antiparasitic Scaffold
  
  • Albenque-Rubio Sandra
  • Guillon Jean
  • Cohen Anita
  • Damiani Céline
  • Mustière Romain
  • Agnamey Patrice
  • Savrimoutou Solène
  • Moreau Stéphane
  • Mergny Jean-Louis
  • Ronga Luisa
  • Kanavos Ioannis
  • Moukha Serge
  • Dozolme Pascale
  • Sonnet Pascal
  , 2023.
• Structuring effects of archaeal replication origins
  
  • Mottez Clémence
  • Puech Romain
  • Flament Didier
  • Myllykallio Hannu
  , 2023. Archaea use eukaryotic-like DNA replication proteins to duplicate circular chromosomes similar to those of bacteria. Although archaeal replication origins have been maintained during the evolution, they are non-essential under laboratory conditions. Here we propose the local deviations from Chargaff’s second parity rule of archaeal chromosomes result from the biased gene orientation and not from mutational biases. Our computational and experimental analyses indicate that the archaeal replication origins prevent head-to-head collisions of replication and transcription complexes as well as participate in coordination of the transfer of genetic information. Our results therefore suggest that the archaeal replication origins have alternative functions not related to their role in initiation of DNA replication. (10.1101/2023.11.15.567178)
  DOI : 10.1101/2023.11.15.567178
• Versatile machine learning for neurodevelopmental imaging
  
  • Lim Seongbin
  , 2023. Recent advances in microscopy allow us to acquire large-volume images that can be used to address novel questions in neurodevelopmental biology. However, these projects are facing difficulty in analyzing massive amount of image data they yield. We will propose a deep learning approach, which proved itself effective in solving various computer vision tasks, to automate analysis and preprocessing of images. This work includes developing deep filters, automated axon tracing, and semantic data visualization, which will be essential to analyze the images, along with building a data infrastructure to efficiently store data and to enable easy access to data.
• Nonlinear optical microscopy of nervous tissue : lipid and metabolism imaging
  
  • Asadipour Bahar
  , 2023. This thesis concerns the development of imaging methods for high-resolution imaging of nervous tissue, based on the combination of nonlinear, label-free optical contrasts. The main objective is to probe myelin distribution and metabolism with sub-cellular resolution in healthy and pathological tissue models. Chapter I introduces the main cellular components of the central nervous system (CNS), namely neurons and glial cells, as well as the structure of myelin, the lipid sheath that enables signals to be transmitted along neurons. The chapter also introduces multiple sclerosis (MS), a demyelinating pathology, and the metabolic processes involved during MS. Finally, the chapter discusses the various morphological and functional imaging techniques available for visualizing the CNS, so as to motivate the development of new high-resolution multimodal imaging methods. Chapter II introduces the fundamentals of nonlinear optical (NLO) microscopy and the contrast modalities used in this study, namely two-photon excited fluorescence (2PEF), two-photon fluorescence lifetime imaging (2P-FLIM), second- and third-harmonic generation (SHG and THG), and the associated technology. Chapter III presents the development of a method based on FLIM / THG contrasts, enabling the joint probing of microglia metabolism and myelin distribution in sections of cultured mouse cerebellum. This work has highlighted the correlations at the cellular level between myelin and metabolism during experimentally-induced stages of demyelination and remyelination. Chapter IV deals with THG imaging of post-mortem human brain samples from MS patients. Given the large size of the samples, part of the work involved the implementation of robust mosaic acquisition strategies. In addition, we implemented an approach for increasing the dynamic range of detection, so as to be able to image this type of heterogeneous sample while maintaining a good signal-to-noise ratio for both weak and strong signals, without having to compromise acquisition speed. Finally, Chapter V explores the use of polarization-resolved second harmonic generation (pSHG) to distinguish different structural proteins in the zebrafish embryo in vivo. By setting up an automated analysis pipeline and optimizing acquisition conditions, we are able to detect and identify axon tubulin, collagen, and myofilament assemblies within the same organism. A numerical framework for predicting the pSHG response in these different macromolecular arrangements is presented, and validated by experimental results.
• Extracellular matrix mediated regulation of immunity during wound healing
  
  • Rousselle Patricia
  • Gross Viktoriia
  • Chessel Anatole
  • Ragaru Bernard
  • Herbage Benjamin
  • Schanne-Klein Marie-Claire
  • Pin Didier
  , 2023. The extracellular matrix is an integral component of all organs and plays a central role in tissue homeostasis and repair. To restore the functional integrity of the skin after injury, a complex and tightly regulated wound healing process takes place that depends on the interaction of many cells, including immune cells. Scarless healing is generally characterised by low levels of inflammation, whereas uncontrolled and persistently high levels of inflammation promote pathological scarring. The role of the extracellular matrix in controlling the immune response is still poorly understood, but is increasingly coming into focus because of the promising prospects it offers. In particular, the basement membranes and the proteolytic maturation processes to which their components are subjected during the epidermal regeneration process may have physiological consequences beyond their sole ultrastructural purpose. In particular, the heparin-binding domains on laminins that are cleaved during epidermal cell stabilisation could play an important role in terminating the inflammatory process. They are known to be involved in the interaction with growth factors, cytokines and even heparan sulphate proteoglycans, but their function is not yet definitively understood. We conducted a study on acute wound healing in pigs using different microscopy approaches and performed quantitative proteomics on primary fibroblasts treated with laminin domains. We demonstrated that the heparin-binding domain of the globular LG4 domain of laminin-332 affects immunity and dramatically reduces inflammation and granulation tissue thickness of porcine excisional wounds. Wounds treated with the LG4 domain surprisingly exhibited thinner granulation tissue populated by CD163+ and CD206+ macrophages; accompanied by decreased fibroblast expression of αSMA. Our results suggest that the maturation process of laminin-332, which coincides with the formation of hemidesmosomes, governs the termination of the inflammatory process in the dermis.
• A new method for in vivo assessment of corneal transparency using spectral-domain OCT
  
  • Vilbert Maëlle
  • Bocheux Romain
  • Georgeon Cristina
  • Borderie Vincent
  • Pernot Pascal
  • Irsch Kristina
  • Plamann Karsten
  PLoS ONE, Public Library of Science, 2023, 18 (10), pp.e0291613. Corneal transparency is essential to provide a clear view into and out of the eye, yet clinical means to assess such transparency are extremely limited and usually involve a subjective grading of visible opacities by means of slit-lamp biomicroscopy. Here, we describe an automated algorithm allowing extraction of quantitative corneal transparency parameters with standard clinical spectral-domain optical coherence tomography (SD-OCT). Our algorithm employs a novel pre-processing procedure to standardize SD-OCT image analysis and to numerically correct common instrumental artifacts before extracting mean intensity stromal-depth ( z ) profiles over a 6-mm-wide corneal area. The z -profiles are analyzed using our previously developed objective method that derives quantitative transparency parameters directly related to the physics of light propagation in tissues. Tissular heterogeneity is quantified by the Birge ratio B r and the photon mean-free path ( l s ) is determined for homogeneous tissues (i.e., B r ~1 ). SD-OCT images of 83 normal corneas (ages 22–50 years) from a standard SD-OCT device (RTVue-XR Avanti, Optovue Inc.) were processed to establish a normative dataset of transparency values. After confirming stromal homogeneity ( B r <10), we measured a median l s of 570 μm (interdecile range: 270–2400 μm). By also considering corneal thicknesses, this may be translated into a median fraction of transmitted (coherent) light T coh(stroma) of 51% (interdecile range: 22–83%). Excluding images with central saturation artifact raised our median T coh(stroma) to 73% (interdecile range: 34–84%). These transparency values are slightly lower than those previously reported, which we attribute to the detection configuration of SD-OCT with a relatively small and selective acceptance angle. No statistically significant correlation between transparency and age or thickness was found. In conclusion, our algorithm provides robust and quantitative measurements of corneal transparency from standard SD-OCT images with sufficient quality (such as ‘Line’ and ‘CrossLine’ B-scan modes without central saturation artifact) and addresses the demand for such an objective means in the clinical setting. (10.1371/journal.pone.0291613)
  DOI : 10.1371/journal.pone.0291613
• Rapport de Prospectives du CSI-INP 2019-2023
  
  • Crépin Claudine
  • Paineau-Lanone Erwan
  • Dimarcq Noel
  • Ramos Laurence
  • Ronda Antoine
  • Abadias Gregory
  • Bove Livia
  • Ceresole Anna
  • Chaud Xavier
  • Crassous Jérôme
  • Debarre Delphine
  • Douarche Carine
  • Duc Fabienne
  • Dugourd Philippe
  • Favero Ivan
  • Fusil Stéphane
  • Garcia Vincent
  • Ingold Gert-Ludwig
  • Maitre Agnès
  • Mambrini Yann
  • Sindzingre Philippe
  • Vaienti Sandro
  • Vézian Stéphane
  • Victor Jean-Marc
  , 2023.
• MiniBAR/GARRE1 is a dual Rac and Rab effector required for ciliogenesis
  
  • Serres Murielle
  • Shaughnessy Ronan
  • Escot Sophie
  • Hammich Hussein
  • Cuvelier Frédérique
  • Salles Audrey
  • Rocancourt Murielle
  • Verdon Quentin
  • Gaffuri Anne-Lise
  • Sourigues Yannick
  • Malherbe Gilles
  • Velikovsky Leonid
  • Chardon Florian
  • Sassoon Nathalie
  • Tinevez Jean-Yves
  • Callebaut Isabelle
  • Formstecher Etienne
  • Houdusse Anne
  • David Nicolas
  • Pylypenko Olena
  • Echard Arnaud
  Developmental Cell, Elsevier, 2023, 58, pp.1-18. Cilia protrude from the cell surface and play critical roles in intracellular signaling, environmental sensing, and development. Reduced actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1- and Rab35-binding protein with a truncated BAR (Bin/amphiphysin/Rvs) domain that we named MiniBAR (also known as KIAA0355/GARRE1), which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles trafficking to the ciliary base and exhibits fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia, resulting from abnormal Rac-GTP/Rho-GTP levels and increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hallmarks of ciliopathies, including left-right asymmetry defects. Thus, MiniBAR is a dual Rac and Rab effector that controls both actin cytoskeleton and membrane trafficking for ciliogenesis. (10.1016/j.devcel.2023.09.010)
  DOI : 10.1016/j.devcel.2023.09.010
• Kidney Stone Classification Using Multimodal Multiphoton Microscopy
  
  • Gleeson Matthew
  • Morizet Joséphine
  • Mahou Pierre
  • Daudon Michel
  • Bazin Dominique
  • Stringari Chiara
  • Schanne-Klein Marie-Claire
  • Beaurepaire Emmanuel
  ACS photonics, American Chemical Society, 2023, 10 (10), pp.3594–3604. Kidney stones are a common form of nephrolithiasis, affecting up to 15% of the world’s population with a high probability of recurrence. These stones exhibit various chemical compositions and crystalline forms associated with different etiologies. Classification of the stones’ components is necessary to optimize treatment and suggest lifestyle changes to reduce the risk of recurrence. Current characterization methods usually require extensive sample preparation or are too detailed for the needs of a high-throughput laboratory. In this article, we present a kidney stone component classification scheme based on the multiphoton response of crushed samples that is label-free, requires minimal sample amounts, and simple preparation. We measure two-photon excited fluorescence, which is sensitive to protein content, second-harmonic generation, which is sensitive to crystalline symmetry, and polarization-resolved third-harmonic generation (pTHG), which is sensitive to crystal heterogeneity and birefringence. The combination of these three contrast modes can distinguish different materials, specifically calcium oxalate in monohydrate (COM), dihydrate (COD), or amorphous forms, cystine, and carbonate apatite. In addition, pTHG images have the potential to distinguish between COM and COD fragments and to provide information on the submicron organization of carbonate apatite fragments. (10.1021/acsphotonics.3c00651)
  DOI : 10.1021/acsphotonics.3c00651
• Real-time in vivo ROS monitoring with luminescent nanoparticles reveals skin inflammation dynamics
  
  • Abdesselem M.
  • Pétri N.
  • Kuhner R.
  • Mousseau F.
  • Rouffiac V.
  • Gacoin T.
  • Laplace-Builhé C.
  • Alexandrou A.
  • Bouzigues C I
  Biomedical optics express, Optical Society of America - OSA Publishing, 2023, 14 (10), pp.5392. Reactive oxygen species (ROS) are key regulators in numerous pathological contexts, including cancer or inflammation. Their role is complex, which justifies the need for methods enabling their quantitative and time-resolved monitoring in vivo, in the perspective to profile tissues of individual patients. However, current ROS detection methods do not provide these features. Here, we propose a new method based on the imaging of lanthanide-ion nanoparticles (GdVO 4 :Eu), whose photoluminescence is modulated by the surrounding ROS concentration. We monitored their luminescence after intradermic injection in a mouse ear submitted to an inflammation-inducing topical stimulus. Based on this approach, we quantified the ROS concentration after inflammation induction and identified a two-step kinetics of ROS production, which may be attributed to the response of resident immune cells and their further recruitment at the inflammation locus. (10.1364/boe.501914)
  DOI : 10.1364/boe.501914
• Multititration: The New Method for Implementing Ultrasensitive and Quantitative Multiplexed In-Field Immunoassays Despite Cross-Reactivity?
  
  • Mousseau Fanny
  • Féraudet Tarisse Cécile
  • Simon Stéphanie
  • Gacoin Thierry
  • Alexandrou Antigoni
  • Bouzigues Cédric Ismael
  Analytical Chemistry, American Chemical Society, 2023, 95 (36), pp.13509-13518. The accurate in-field titration of multiple pathogens is essential to efficiently describe and monitor environmental or biological contamination, isolate, act, and treat adequately. This underscores the requirement of portable, fast, quantitative, and multiplexed detection technologies, which, however, have not been properly developed so far, notably because it has been hindered by the phenomenon of cross-reactivity. In this work, we proposed a new analytical method based on the imaging through a portable device of lanthanide-based nanoparticles (YVO 4 :Eu) for spatially multiplexed detection, relying on a multiparameter analysis, i.e., a simultaneous analysis of all of the luminescence signals through the comparison to a calibration surface built in the presence of multiple analytes of interest. We then demonstrated the possibility to simultaneously quantify by multiplexed lateral flow assay (xLFA) the three enterotoxins SEG, SEH, and SEI in unknown mixtures, over two concentration decades (from a dozen of pg•mL −1 to few ng•mL −1). Assays were performed in less than an hour (25 min of strip migration followed by 30 min of drying at room temperature), the time during which the presence of the operator was not required for more than 5 min, in order to dip the strip and have it imaged by the reader. The concepts of nominal concentration recovery, coefficient of variation (CV), limit of blank (LOB), and limit of detection (LOD) were discussed in detail in the context of multiplexed assays. With our new definitions, quantitative results demonstrated a high recovery of the nominal concentrations (115%), reliability (CV = 20%), and sensitivity (LOBs of 3, 27, and 6 pg•mL −1 for SEG, SEH, and SEI respectively, and LODs of 6, 48, and 11 pg•mL −1 for SEG, SEH, and SEI, respectively). Based on this method, we observed an increase in sensitivity of 100 compared to the other multiplexed LFA labeled with gold particles and we approached the sensitivity of the simplex enzyme-linked immunosorbent assay (ELISA) performed with the same capture and detection antibodies. To conclude, our results, which are applicable to virtually any kind of multiplexed test, pave the way to the next generation of in-field analytical immunoassays by providing fast, quantitative, and highly sensitive multiplexed detection of biomarkers or pathogens. (10.1021/acs.analchem.3c01846)
  DOI : 10.1021/acs.analchem.3c01846