Publications by authors named "Jean-Christophe Olivo-Marin"

119 Publications

Bioimage Analysis and Cell Motility.

Patterns (N Y) 2021 Jan 8;2(1):100170. Epub 2021 Jan 8.

Institut Pasteur, Bioimage Analysis Unit, 25 rue du Dr. Roux, Paris Cedex 15 75724, France.

Bioimage analysis (BIA) has historically helped study and cells move; biological experiments evolved in intimate feedback with the most classical image processing techniques because they contribute objectivity and reproducibility to an eminently qualitative science. Cell segmentation, tracking, and morphology descriptors are all discussed here. Using ameboid motility as a case study, these methods help us illustrate how proper quantification can augment biological data, for example, by choosing mathematical representations that amplify initially subtle differences, by statistically uncovering general laws or by integrating physical insight. More recently, the non-invasive nature of quantitative imaging is fertilizing two blooming fields: mechanobiology, where many biophysical measurements remain inaccessible, and microenvironments, where the quest for physiological relevance has exploded data size. From relief to remedy, this trend indicates that BIA is to become a main vector of biological discovery as human visual analysis struggles against ever more complex data.
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http://dx.doi.org/10.1016/j.patter.2020.100170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815951PMC
January 2021

Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

PLoS One 2021 11;16(1):e0243712. Epub 2021 Jan 11.

ESPCI PSL, CBI, IPGG, Paris, France.

To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT-PCR (Reverse transcription-Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT-LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross-reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called "COVIDISC" to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243712PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799764PMC
January 2021

, One Uninucleate Species with Disparate Offspring.

J Fungi (Basel) 2021 Jan 6;7(1). Epub 2021 Jan 6.

Unité des Aspergillus, Institut Pasteur, 75015 Paris, France.

Establishment of a fungal infection due to relies on the efficient germination of the airborne conidia once they penetrate the respiratory tract. However, the features of conidial germination have been poorly explored and understood in this fungal species as well as in other species of filamentous fungi. We show here that the germination of is asynchronous. If the nutritional environment and extensive gene deletions can modify the germination parameters for , the asynchrony is maintained in all germinative conditions tested. Even though the causes for this asynchrony of conidial germination remain unknown, asynchrony is essential for the completion of the biological cycle of this filamentous fungus.
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http://dx.doi.org/10.3390/jof7010030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825634PMC
January 2021

Missing Self-Induced Activation of NK Cells Combines with Non-Complement-Fixing Donor-Specific Antibodies to Accelerate Kidney Transplant Loss in Chronic Antibody-Mediated Rejection.

J Am Soc Nephrol 2021 Feb 25;32(2):479-494. Epub 2020 Nov 25.

International Center of Infectiology research (CIRI), French Institute of Health and Medical Research (INSERM) Unit 1111, Claude Bernard University Lyon I, National Center for Scientific Research (CNRS) Mixed University Unit (UMR) 5308, Ecole Normale Supérieure de Lyon, University of Lyon, Lyon, France

Background: Binding of donor-specific antibodies (DSAs) to kidney allograft endothelial cells that does not activate the classic complement cascade can trigger the recruitment of innate immune effectors, including NK cells. Activated NK cells contribute to microvascular inflammation leading to chronic antibody-mediated rejection (AMR). Recipient NK cells can also trigger antibody-independent microvascular inflammation by sensing the absence of self HLA class I molecules ("missing self") on allograft endothelial cells. This translational study investigated whether the condition of missing self amplifies DSA-dependent NK cell activation to worsen chronic AMR.

Methods And Results: Among 1682 kidney transplant recipients who underwent an allograft biopsy at Lyon University Hospital between 2004 and 2017, 135 fulfilled the diagnostic criteria for AMR and were enrolled in the study. Patients with complement-fixing DSAs identified by a positive C3d binding assay (=73, 54%) had a higher risk of transplant failure (=0.002). Among the remaining patients with complement-independent chronic AMR (=62, 46%), those in whom missing self was identified through donor and recipient genotyping exhibited worse allograft survival (=0.02). In multivariable analysis, only proteinuria (HR: 7.24; =0.01) and the presence of missing self (HR: 3.57; =0.04) were independent predictors for transplant failure following diagnosis of chronic AMR. Cocultures of human NK cells and endothelial cells confirmed that addition of missing self to DSA-induced NK cell activation increased endothelial damage.

Conclusions: The assessment of missing self at the time of diagnosis of chronic AMR identifies patients at higher risk for kidney transplant failure.
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http://dx.doi.org/10.1681/ASN.2020040433DOI Listing
February 2021

Evaluating the Stability of Spatial Keypoints via Cluster Core Correspondence Index.

IEEE Trans Image Process 2021 23;30:386-401. Epub 2020 Nov 23.

Detection and analysis of informative keypoints is a fundamental problem in image analysis and computer vision. Keypoint detectors are omnipresent in visual automation tasks, and recent years have witnessed a significant surge in the number of such techniques. Evaluating the quality of keypoint detectors remains a challenging task owing to the inherent ambiguity over what constitutes a good keypoint. In this context, we introduce a reference based keypoint quality index which is based on the theory of spatial pattern analysis. Unlike traditional correspondence-based quality evaluation which counts the number of feature matches within a specified neighborhood, we present a rigorous mathematical framework to compute the statistical correspondence of the detections inside a set of salient zones (cluster cores) defined by the spatial distribution of a reference set of keypoints. We leverage the versatility of the level sets to handle hypersurfaces of arbitrary geometry, and develop a mathematical framework to estimate the model parameters analytically to reflect the robustness of a feature detection algorithm. Extensive experimental studies involving several keypoint detectors tested under different imaging scenarios demonstrate efficacy of our method to evaluate keypoint quality for generic applications in computer vision and image analysis.
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http://dx.doi.org/10.1109/TIP.2020.3036759DOI Listing
November 2020

E-cadherin focuses protrusion formation at the front of migrating cells by impeding actin flow.

Nat Commun 2020 10 26;11(1):5397. Epub 2020 Oct 26.

Institute of Cell Biology, Center for Molecular Biology of Inflammation, University of Münster, 48149, Münster, Germany.

The migration of many cell types relies on the formation of actomyosin-dependent protrusions called blebs, but the mechanisms responsible for focusing this kind of protrusive activity to the cell front are largely unknown. Here, we employ zebrafish primordial germ cells (PGCs) as a model to study the role of cell-cell adhesion in bleb-driven single-cell migration in vivo. Utilizing a range of genetic, reverse genetic and mathematical tools, we define a previously unknown role for E-cadherin in confining bleb-type protrusions to the leading edge of the cell. We show that E-cadherin-mediated frictional forces impede the backwards flow of actomyosin-rich structures that define the domain where protrusions are preferentially generated. In this way, E-cadherin confines the bleb-forming region to a restricted area at the cell front and reinforces the front-rear axis of migrating cells. Accordingly, when E-cadherin activity is reduced, the bleb-forming area expands, thus compromising the directional persistence of the cells.
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http://dx.doi.org/10.1038/s41467-020-19114-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588466PMC
October 2020

Cell Tracking Profiler - a user-driven analysis framework for evaluating 4D live-cell imaging data.

J Cell Sci 2020 11 23;133(22). Epub 2020 Nov 23.

Centre for Craniofacial and Regenerative Biology, King's College London, Guy's Hospital, London SE1 9RT, UK

Accurate measurements of cell morphology and behaviour are fundamentally important for understanding how disease, molecules and drugs affect cell function Here, by using muscle stem cell (muSC) responses to injury in zebrafish as our biological paradigm, we established a 'ground truth' for muSC behaviour. This revealed that segmentation and tracking algorithms from commonly used programs are error-prone, leading us to develop a fast semi-automated image analysis pipeline that allows user-defined parameters for segmentation and correction of cell tracking. Cell Tracking Profiler (CTP) is a package that runs two existing programs, HK Means and Phagosight within the Icy image analysis suite, to enable user-managed cell tracking from 3D time-lapse datasets to provide measures of cell shape and movement. We demonstrate how CTP can be used to reveal changes to cell behaviour of muSCs in response to manipulation of the cell cytoskeleton by small-molecule inhibitors. CTP and the associated tools we have developed for analysis of outputs thus provide a powerful framework for analysing complex cell behaviour from 4D datasets that are not amenable to straightforward analysis.
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http://dx.doi.org/10.1242/jcs.241422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710012PMC
November 2020

Human intestinal models to study interactions between intestine and microbes.

Open Biol 2020 10 21;10(10):200199. Epub 2020 Oct 21.

Institut Pasteur, Unité d'Analyse d'Images Biologiques, 25 Rue du Dr Roux, 75015 Paris, France.

Implementations of suitable cell culture systems of the human intestine have been essential tools in the study of the interaction among organs, commensal microbiota, pathogens and parasites. Due to the great complexity exhibited by the intestinal tissue, researchers have been developing / systems to diminish the gap between conventional cell culture models and the human intestine. These models are able to reproduce different structures and functional aspects of the tissue. In the present review, information is recapitulated on the most used models, such as cell culture, intestinal organoids, scaffold-based three-dimensional models, and organ-on-a-chip and their use in studying the interaction between human intestine and microbes, and their advantages and limitations are also discussed.
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http://dx.doi.org/10.1098/rsob.200199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653360PMC
October 2020

Transcriptional Changes in Kidney Allografts with Histology of Antibody-Mediated Rejection without Anti-HLA Donor-Specific Antibodies.

J Am Soc Nephrol 2020 09 8;31(9):2168-2183. Epub 2020 Jul 8.

Department of Microbiology, Immunology and Transplantation, Nephrology and Renal Transplantation Research Group, Katholieke Universiteit (KU) Leuven, Leuven, Belgium

Background: Circulating donor-specific anti-HLA antibodies (HLA-DSAs) are often absent in serum of kidney allograft recipients whose biopsy specimens demonstrate histology of antibody-mediated rejection (ABMR). It is unclear whether cases involving ABMR histology without detectable HLA-DSAs represent a distinct clinical and molecular phenotype.

Methods: In this multicenter cohort study, we integrated allograft microarray analysis with extensive clinical and histologic phenotyping from 224 kidney transplant recipients between 2011 and 2017. We used the term ABMR histology for biopsy specimens that fulfill the first two Banff 2017 criteria for ABMR, irrespective of HLA-DSA status.

Results: Of 224 biopsy specimens, 56 had ABMR histology; 26 of these (46.4%) lacked detectable serum HLA-DSAs. Biopsy specimens with ABMR histology showed overexpression of transcripts mostly related to IFN-induced pathways and activation of natural killer cells and endothelial cells. HLA-DSA-positive and HLA-DSA-negative biopsy specimens with ABMR histology displayed similar upregulation of pathways and enrichment of infiltrating leukocytes. Transcriptional heterogeneity observed in biopsy specimens with ABMR histology was not associated with HLA-DSA status but was caused by concomitant T cell-mediated rejection. Compared with cases lacking ABMR histology, those with ABMR histology and HLA-DSA had higher allograft failure risk (hazard ratio [HR], 7.24; 95% confidence interval [95% CI], 3.04 to 17.20) than cases without HLA-DSA (HR, 2.33; 95% CI, 0.85 to 6.33), despite the absence of transcriptional differences.

Conclusions: ABMR histology corresponds to a robust intragraft transcriptional signature, irrespective of HLA-DSA status. Outcome after ABMR histology is not solely determined by the histomolecular presentation but is predicted by the underlying etiologic factor. It is important to consider this heterogeneity in further research and in treatment decisions for patients with ABMR histology.
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http://dx.doi.org/10.1681/ASN.2020030306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461663PMC
September 2020

Insights into amebiasis using a human 3D-intestinal model.

Cell Microbiol 2020 08 20;22(8):e13203. Epub 2020 Apr 20.

Institut Pasteur, Paris, France.

Entamoeba histolytica is the causative agent of amebiasis, an infectious disease targeting the intestine and the liver in humans. Two types of intestinal infection are caused by this parasite: silent infection, which occurs in the majority of cases, and invasive disease, which affects 10% of infected persons. To understand the intestinal pathogenic process, several in vitro models, such as cell cultures, human tissue explants or human intestine xenografts in mice, have been employed. Nevertheless, our knowledge on the early steps of amebic intestinal infection and the molecules involved during human-parasite interaction is scarce, in part due to limitations in the experimental settings. In the present work, we took advantage of tissue engineering approaches to build a three-dimensional (3D)-intestinal model that is able to replicate the general characteristics of the human colon. This system consists of an epithelial layer that develops tight and adherens junctions, a mucus layer and a lamina propria-like compartment made up of collagen containing macrophages and fibroblast. By means of microscopy imaging, omics assays and the evaluation of immune responses, we show a very dynamic interaction between E. histolytica and the 3D-intestinal model. Our data highlight the importance of several virulence markers occurring in patients or in experimental models, but they also demonstrate the involvement of under described molecules and regulatory factors in the amoebic invasive process.
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http://dx.doi.org/10.1111/cmi.13203DOI Listing
August 2020

Tracking and line integration of diffuse cellular subdomains by mesh advection.

Annu Int Conf IEEE Eng Med Biol Soc 2019 Jul;2019:6018-6021

Active molecular transport ensures a purposeful spatiotemporal distribution of cellular proteins and is therefore key to a wide range of processes such as morphogenesis, homeostasis or migration. However, redistributions of molecules in bulk are seldom quantified because the regions involved are too diffuse to be segmented consistently. To bridge this gap, we propose a Laplace-corrected Runge-Kutta advection that is based on mesh triangulation. Our framework can follow the movement and deformation of multiple parts of a diffuse region at once and offers a seamless combination with spatiotemporal line integration in Lagrangian coordinates. This allows the flexibility to taylor specific measures to the question at hand, e.g. mechanical work, bringing long-established physics concepts into biology grounds. We exemplify our approach by quantifying how the isotropy of intracellular protein distributions changes during cargo transport.
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http://dx.doi.org/10.1109/EMBC.2019.8857425DOI Listing
July 2019

Missing self triggers NK cell-mediated chronic vascular rejection of solid organ transplants.

Nat Commun 2019 11 25;10(1):5350. Epub 2019 Nov 25.

CIRI, INSERM U1111, Université Claude Bernard Lyon I, CNRS UMR5308, Ecole Normale Supérieure de Lyon, Univ. Lyon, 21, avenue Tony Garnier, 69007, Lyon, France.

Current doctrine is that microvascular inflammation (MVI) triggered by a transplant -recipient antibody response against alloantigens (antibody-mediated rejection) is the main cause of graft failure. Here, we show that histological lesions are not mediated by antibodies in approximately half the participants in a cohort of 129 renal recipients with MVI on graft biopsy. Genetic analysis of these patients shows a higher prevalence of mismatches between donor HLA I and recipient inhibitory killer cell immunoglobulin-like receptors (KIRs). Human in vitro models and transplantation of β2-microglobulin-deficient hearts into wild-type mice demonstrates that the inability of graft endothelial cells to provide HLA I-mediated inhibitory signals to recipient circulating NK cells triggers their activation, which in turn promotes endothelial damage. Missing self-induced NK cell activation is mTORC1-dependent and the mTOR inhibitor rapamycin can prevent the development of this type of chronic vascular rejection.
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http://dx.doi.org/10.1038/s41467-019-13113-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877588PMC
November 2019

Treatment of Breast Cancer With Gonadotropin-Releasing Hormone Analogs.

Front Oncol 2019 1;9:943. Epub 2019 Oct 1.

Unidad de Investigación Médica en Medicina Reproductiva, IMSS, Unidad Médica de Alta Especialidad No. 4, Mexico City, Mexico.

Although significant progress has been made in the implementation of new breast cancer treatments over the last three decades, this neoplasm annually continues to show high worldwide rates of morbidity and mortality. In consequence, the search for novel therapies with greater effectiveness and specificity has not come to a stop. Among the alternative therapeutic targets, the human gonadotropin-releasing hormone type I and type II (hGnRH-I and hGnRH-II, respectively) and its receptor, the human gonadotropin-releasing hormone receptor type I (hGnRHR-I), have shown to be powerful therapeutic targets to decrease the adverse effects of this disease. In the present review, we describe how the administration of GnRH analogs is able to reduce circulating concentrations of estrogen in premenopausal women through their action on the hypothalamus-pituitary-ovarian axis, consequently reducing the growth of breast tumors and disease recurrence. Also, it has been mentioned that, regardless of the suppression of synthesis and secretion of ovarian steroids, GnRH agonists exert direct anticancer action, such as the reduction of tumor growth and cell invasion. In addition, we discuss the effects on breast cancer of the hGnRH-I and hGnRH-II agonist and antagonist, non-peptide GnRH antagonists, and cytotoxic analogs of GnRH and their implication as novel adjuvant therapies as antitumor agents for reducing the adverse effects of breast cancer. In conclusion, we suggest that the hGnRH/hGnRHR system is a promising target for pharmaceutical development in the treatment of breast cancer, especially for the treatment of advanced states of this disease.
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http://dx.doi.org/10.3389/fonc.2019.00943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779786PMC
October 2019

Real-time analysis of the behaviour of groups of mice via a depth-sensing camera and machine learning.

Nat Biomed Eng 2019 11 20;3(11):930-942. Epub 2019 May 20.

Institut Pasteur, BioImage Analysis Unit, CNRS UMR 3691, Paris, France.

Preclinical studies of psychiatric disorders use animal models to investigate the impact of environmental factors or genetic mutations on complex traits such as decision-making and social interactions. Here, we introduce a method for the real-time analysis of the behaviour of mice housed in groups of up to four over several days and in enriched environments. The method combines computer vision through a depth-sensing infrared camera, machine learning for animal and posture identification, and radio-frequency identification to monitor the quality of mouse tracking. It tracks multiple mice accurately, extracts a list of behavioural traits of both individuals and the groups of mice, and provides a phenotypic profile for each animal. We used the method to study the impact of Shank2 and Shank3 gene mutations-mutations that are associated with autism-on mouse behaviour. Characterization and integration of data from the behavioural profiles of Shank2 and Shank3 mutant female mice revealed their distinctive activity levels and involvement in complex social interactions.
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http://dx.doi.org/10.1038/s41551-019-0396-1DOI Listing
November 2019

Fluid dynamics during bleb formation in migrating cells in vivo.

PLoS One 2019 26;14(2):e0212699. Epub 2019 Feb 26.

Institute of Cell Biology, ZMBE, Münster, Germany.

Blebs are cellular protrusions observed in migrating cells and in cells undergoing spreading, cytokinesis, and apoptosis. Here we investigate the flow of cytoplasm during bleb formation and the concurrent changes in cell volume using zebrafish primordial germ cells (PGCs) as an in vivo model. We show that bleb inflation occurs concomitantly with cytoplasmic inflow into it and that during this process the total cell volume does not change. We thus show that bleb formation in primordial germ cells results primarily from redistribution of material within the cell rather than being driven by flow of water from an external source.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212699PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391022PMC
November 2019

Compressed sensing-enabled phase-sensitive swept-source optical coherence tomography.

Opt Express 2019 Jan;27(2):855-871

Here we present a novel phase-sensitive swept-source optical coherence tomography (PhS-SS-OCT) system. The simultaneously recorded calibration signal, which is commonly used in SS-OCT to stabilize the phase, is randomly sub-sampled during the acquisition, and it is later reconstructed based on the Compressed Sensing (CS) theory. We first mathematically investigated the method, and verified it through computer simulations. We then conducted a vibrational frequency test and a flow velocity measurement in phantoms to demonstrate the system's capability of handling phase-sensitive tasks. The proposed scheme shows excellent phase stability with greatly discounted data bandwidth compared with conventional procedures. We further showcased the usefulness of the system in biological samples by detecting the blood flow in ex vivo swine left marginal artery. The proposed system is compatible with most of the existing SS-OCT systems and could be a preferred solution for future high-speed phase-sensitive applications.
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http://dx.doi.org/10.1364/OE.27.000855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410915PMC
January 2019

Stress-induced brain activation: buffering role of social behavior and neuronal nicotinic receptors.

Brain Struct Funct 2018 Dec 8;223(9):4259-4274. Epub 2018 Sep 8.

Neuroscience Paris-Saclay Institute (Neuro-PSI), UMR CNRS 9197, Université Paris-Sud, Bâtiment 446, 15 Bd Clémenceau, 91405, Orsay Cedex, France.

Social behavior and stress responses both rely on activity in the prefrontal cortex (PFC) and amygdala. We previously reported that acute stress exposure impoverishes social repertoire and triggers behavioral rigidity, and that both effects are modulated by β2-containing nicotinic receptors. We, therefore, hypothesized that the activity of brain regions associated with the integration of social cues will be modulated by stress exposure. We mapped the expression of c-fos protein in all subregions of the PFC and basolateral (BLA) and central (Ce) areas of the amygdala in C57BL/6J (B6) and β2 mice. We show altered brain activity and differences in functional connectivity between the two genotypes after stress: the PFC and BLA were hyperactivated in B6 and hypo-activated in β2 mice, showing that the impact of stress on brain activity and functional organization depends on the nicotinic system. We also show that the effects of the opportunity to explore a novel environment or interact socially after acute stress depended on genotype: exploration induced only marginal PFC activation in both genotypes relative to stress alone, excluding a major role for β2 receptors in this process. However, social interaction following stress only activated the rostral and caudolateral areas of the PFC in B6 mice, while it induced a kindling of activation in all PFC and amygdalar areas in β2 mice. These results indicate that acute stress triggers important PFC-amygdala plasticity, social interaction has a buffering role during stress-induced brain activation, and β2 receptors contribute to the effects of social interaction under stress.
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http://dx.doi.org/10.1007/s00429-018-1745-7DOI Listing
December 2018

Anisotropic organization of circumferential actomyosin characterizes hematopoietic stem cells emergence in the zebrafish.

Elife 2018 08 22;7. Epub 2018 Aug 22.

Department of Developmental and Stem Cell Biology, Institut Pasteur, Paris, France.

Hematopoiesis leads to the formation of blood and immune cells. Hematopoietic stem cells emerge during development, from vascular components, via a process called the endothelial-to-hematopoietic transition (EHT). Here, we reveal essential biomechanical features of the EHT, using the zebrafish embryo imaged at unprecedented spatio-temporal resolution and an algorithm to unwrap the aorta into 2D-cartography. We show that the transition involves anisotropic contraction along the antero-posterior axis, with heterogenous organization of contractile circumferential actomyosin. The biomechanics of the contraction is oscillatory, with unusually long periods in comparison to other apical constriction mechanisms described so far in morphogenesis, and is supported by the anisotropic reinforcement of junctional contacts. Finally, we show that abrogation of blood flow impairs the actin cytoskeleton, the morphodynamics of EHT cells, and the orientation of the emergence. Overall, our results underline the peculiarities of the EHT biomechanics and the influence of the mechanical forces exerted by blood flow.
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http://dx.doi.org/10.7554/eLife.37355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105311PMC
August 2018

Morphodynamics of the Actin-Rich Cytoskeleton in .

Front Cell Infect Microbiol 2018 29;8:179. Epub 2018 May 29.

Cell Biology of Parasitism Unit, Institut Pasteur, Paris, France.

is the anaerobic protozoan parasite responsible for human amoebiasis, the third most deadly parasitic disease worldwide. This highly motile eukaryotic cell invades human tissues and constitutes an excellent experimental model of cell motility and cell shape deformation. The absence of extranuclear microtubules in means that the actin-rich cytoskeleton takes on a crucial role in not only amoebic motility but also other processes sustaining pathogenesis, such as the phagocytosis of human cells and the parasite's resistance of host immune responses. Actin is highly conserved among eukaryotes, although diverse isoforms exist in almost all organisms studied to date. However, has a single actin protein, the structure of which differs significantly from those of its human homologs. Here, we studied the expression, structure and dynamics of actin in . We used molecular and cellular approaches to evaluate actin gene expression during intestinal invasion by trophozoites. Based on a three-dimensional structural bioinformatics analysis, we characterized protein domains differences between amoebic actin and human actin. Fine-tuned molecular dynamics simulations enabled us to examine protein motion and refine the three-dimensional structures of both actins, including elements potentially accounting for differences changes in the affinity properties of amoebic actin and deoxyribonuclease I. The dynamic, multifunctional nature of the amoebic cytoskeleton prompted us to examine the pleiotropic forms of actin structures within live cells; we observed the cortical cytoskeleton, stress fibers, "dot-like" structures, adhesion plates, and macropinosomes. In line with these data, a proteomics study of actin-binding proteins highlighted the Arp2/3 protein complex as a crucial element for the development of macropinosomes and adhesion plaques.
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http://dx.doi.org/10.3389/fcimb.2018.00179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986921PMC
June 2019

Denoising of Microscopy Images: A Review of the State-of-the-Art, and a New Sparsity-Based Method.

IEEE Trans Image Process 2018 Aug;27(8):3842-3856

This paper reviews the state-of-the-art in denoising methods for biological microscopy images and introduces a new and original sparsity-based algorithm. The proposed method combines total variation (TV) spatial regularization, enhancement of low-frequency information, and aggregation of sparse estimators and is able to handle simple and complex types of noise (Gaussian, Poisson, and mixed), without any a priori model and with a single set of parameter values. An extended comparison is also presented, that evaluates the denoising performance of the thirteen (including ours) state-of-the-art denoising methods specifically designed to handle the different types of noises found in bioimaging. Quantitative and qualitative results on synthetic and real images show that the proposed method outperforms the other ones on the majority of the tested scenarios.
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http://dx.doi.org/10.1109/TIP.2018.2819821DOI Listing
August 2018

Mapping molecular assemblies with fluorescence microscopy and object-based spatial statistics.

Nat Commun 2018 02 15;9(1):698. Epub 2018 Feb 15.

Institut Pasteur, BioImage Analysis Unit. CNRS UMR 3691. 25 rue du Docteur Roux, 75724, Paris Cedex 15, France.

Elucidating protein functions and molecular organisation requires to localise precisely single or aggregated molecules and analyse their spatial distributions. We develop a statistical method SODA (Statistical Object Distance Analysis) that uses either micro- or nanoscopy to significantly improve on standard co-localisation techniques. Our method considers cellular geometry and densities of molecules to provide statistical maps of isolated and associated (coupled) molecules. We use SODA with three-colour structured-illumination microscopy (SIM) images of hippocampal neurons, and statistically characterise spatial organisation of thousands of synapses. We show that presynaptic synapsin is arranged in asymmetric triangle with the 2 postsynaptic markers homer and PSD95, indicating a deeper localisation of homer. We then determine stoichiometry and distance between localisations of two synaptic vesicle proteins with 3D-STORM. These findings give insights into the protein organisation at the synapse, and prove the efficiency of SODA to quantitatively assess the geometry of molecular assemblies.
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http://dx.doi.org/10.1038/s41467-018-03053-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5814551PMC
February 2018

Quantitative and Statistical Study of the Dynamics of Clathrin-Dependent and -Independent Endocytosis Reveal a Differential Role of EndophilinA2.

Cell Rep 2018 02;22(6):1574-1588

Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, INSERM U1202. 28, rue du Docteur Roux, 75015 Paris, France. Electronic address:

Eukaryotic cells internalize cargos specifically through clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE). EndophilinA2 was shown as preferentially implicated in CIE, although initially involved in CME. Here, we investigated the native interplay of endophilinA2 and dynamin2 during CME as compared to CIE. We developed an unbiased integrative approach based on genome engineering, robust tracking methodology, and advanced analytics. We statistically identified CME and CIE subpopulations corresponding to abortive, active, and static endocytic events. Depletion of dynamin2 strongly affected active CME and CIE events, whereas the absence of endophilinA2 impacted only CIE. Accordingly, we demonstrated that endophilinA2 is needed for dynamin2 recruitment during CIE, but not in CME. Despite these differences, endophilinA2 and dynamin2 acted at the latest stage of endocytosis within a similar stoichiometry in both mechanisms. Thus, we propose a conserved function of dynamin2 and endophilinA2 in vesicle scission, but a differential regulation of their recruitment during CME and CIE.
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http://dx.doi.org/10.1016/j.celrep.2018.01.039DOI Listing
February 2018

Crosstalk between Entamoeba histolytica and the human intestinal tract during amoebiasis.

Parasitology 2019 08 7;146(9):1140-1149. Epub 2017 Dec 7.

Cell Biology of Parasitism Unit,Institut Pasteur,Paris,France.

The protozoan parasite Entamoeba histolytica is the microbial agent of amoebiasis - an infection that is endemic worldwide and is associated with high morbidity and mortality rates. As the disease develops, virulent E. histolytica deplete the mucus layer, interact with the intestinal epithelium, and then degrade the colonic mucosa and disrupt the extracellular matrix (ECM). Our research demonstrated that virulent parasites with an invasive phenotype display rapid, highly specific changes in their transcriptome (notably for essential factors involved in carbohydrate metabolism and the processing of glycosylated residues). Moreover, combined activation of parasite and host lytic enzymes leads to the destruction of the intestinal parenchyma. Together, these enzymes degrade the mucus layer and the ECM, and trigger the inflammatory response essential to the development of amoebiasis.
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http://dx.doi.org/10.1017/S0031182017002190DOI Listing
August 2019

An objective comparison of cell-tracking algorithms.

Nat Methods 2017 Dec 30;14(12):1141-1152. Epub 2017 Oct 30.

CIBERONC, IDISNA and Program of Solid Tumors and Biomarkers, Center for Applied Medical Research, University of Navarra, Pamplona, Spain.

We present a combined report on the results of three editions of the Cell Tracking Challenge, an ongoing initiative aimed at promoting the development and objective evaluation of cell segmentation and tracking algorithms. With 21 participating algorithms and a data repository consisting of 13 data sets from various microscopy modalities, the challenge displays today's state-of-the-art methodology in the field. We analyzed the challenge results using performance measures for segmentation and tracking that rank all participating methods. We also analyzed the performance of all of the algorithms in terms of biological measures and practical usability. Although some methods scored high in all technical aspects, none obtained fully correct solutions. We found that methods that either take prior information into account using learning strategies or analyze cells in a global spatiotemporal video context performed better than other methods under the segmentation and tracking scenarios included in the challenge.
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http://dx.doi.org/10.1038/nmeth.4473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777536PMC
December 2017

Legionella pneumophila Modulates Mitochondrial Dynamics to Trigger Metabolic Repurposing of Infected Macrophages.

Cell Host Microbe 2017 Sep 31;22(3):302-316.e7. Epub 2017 Aug 31.

Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris 75724, France; CNRS UMR 3525, Paris 75724, France. Electronic address:

The intracellular bacteria Legionella pneumophila encodes a type IV secretion system (T4SS) that injects effector proteins into macrophages in order to establish and replicate within the Legionella-containing vacuole (LCV). Once generated, the LCV interacts with mitochondria through unclear mechanisms. We show that Legionella uses both T4SS-independent and T4SS-dependent mechanisms to respectively interact with mitochondria and induce mitochondrial fragmentation that ultimately alters mitochondrial metabolism. The T4SS effector MitF, a Ran GTPase activator, is required for fission of the mitochondrial network. These effects of MitF occur through accumulation of mitochondrial DNM1L, a GTPase critical for fission. Furthermore mitochondrial respiration is abruptly halted in a T4SS-dependent manner, while T4SS-independent upregulation of cellular glycolysis remains elevated. Collectively, these alterations in mitochondrial dynamics promote a Warburg-like phenotype in macrophages that favors bacterial replication. Hence the rewiring of cellular bioenergetics to create a replication permissive niche in host cells is a virulence strategy of L. pneumophila.
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http://dx.doi.org/10.1016/j.chom.2017.07.020DOI Listing
September 2017

BioFlow: a non-invasive, image-based method to measure speed, pressure and forces inside living cells.

Sci Rep 2017 08 23;7(1):9178. Epub 2017 Aug 23.

Institut Pasteur, Bioimage Analysis Unit, Paris, France.

Cell motility is governed by a complex molecular machinery that converts physico-chemical cues into whole-cell movement. Understanding the underlying biophysical mechanisms requires the ability to measure physical quantities inside the cell in a simple, reproducible and preferably non-invasive manner. To this end, we developed BioFlow, a computational mechano-imaging method and associated software able to extract intracellular measurements including pressure, forces and velocity everywhere inside freely moving cells in two and three dimensions with high spatial resolution in a non-invasive manner. This is achieved by extracting the motion of intracellular material observed using fluorescence microscopy, while simultaneously inferring the parameters of a given theoretical model of the cell interior. We illustrate the power of BioFlow in the context of amoeboid cell migration, by modelling the intracellular actin bulk flow of the parasite Entamoeba histolytica using fluid dynamics, and report unique experimental measures that complement and extend both theoretical estimations and invasive experimental measures. Thanks to its flexibility, BioFlow is easily adaptable to other theoretical models of the cell, and alleviates the need for complex or invasive experimental conditions, thus constituting a powerful tool-kit for mechano-biology studies. BioFlow is open-source and freely available via the Icy software.
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http://dx.doi.org/10.1038/s41598-017-09240-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569094PMC
August 2017

Activation of human gonadotropin-releasing hormone receptor promotes down regulation of ARHGAP18 and regulates the cell invasion of MDA-MB-231 cells.

Mol Cell Endocrinol 2018 01 11;460:94-103. Epub 2017 Jul 11.

Institut Pasteur, Unité d'Analyse d'Images Biologiques, 25 Rue du Dr Roux, F-75015 Paris, France; Centre National de la Recherche Scientifique, CNRS UMR3691, 25 Rue du Dr Roux, F-75015 Paris, France.

The Gonadotropin-Releasing Hormone Receptor (GnRHR) is expressed mainly in the gonadotrope membrane of the adenohypophysis and its natural ligand, the Gonadotropin-Releasing Hormone (GnRH), is produced in anterior hypothalamus. Furthermore, both molecules are also present in the membrane of cells derived from other reproductive tissues such as the breast, endometrium, ovary, and prostate, as well as in tumors derived from these tissues. The functions of GnRH receptor and its hormone in malignant cells have been related with the decrease of proliferation and the invasiveness of those tumors however, little is known about the molecules associated with the signaling pathways regulated by both molecules in malignant cells. To further analyze the potential mechanisms employed by the GnRHR/GnRH system to reduce the tumorigenesis of the highly invasive breast cancer cell line MDA-MB-231, we performed microarrays experiments to evaluated changes in genes expression and validate these modifications by functional assays. We show that activation of human GnRHR is able to diminish the expression and therefore functions of the Rho GTPase-Activating Protein 18 (ARHGAP18). Decrease of this GAP following GnRHR activation, correlates to the higher of cell adhesion and also with reduction of tumor cell invasion, supporting the notion that GnRHR triggers intracellular signaling pathways that acts through ARHGAP18. On the contrary, although a decline of cellular proliferation was observed during GnRHR activation in MDA-MB-231, this was independent of ARHGAP18 showing the complex system in which is involved the signaling pathways regulated by the GnRHR/GnRH system.
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http://dx.doi.org/10.1016/j.mce.2017.07.009DOI Listing
January 2018

Deciphering tissue morphodynamics using bioimage informatics.

Philos Trans R Soc Lond B Biol Sci 2017 May;372(1720)

Institut Pasteur, Bioimage Analysis Unit, 25-28 rue du Docteur Roux, Paris, France

In recent years developmental biology has greatly benefited from the latest advances in fluorescence microscopy techniques. Consequently, quantitative and automated analysis of this data is becoming a vital first step in the quest for novel insights into the various aspects of development. Here we present an introductory overview of the various image analysis methods proposed for developmental biology images, with particular attention to openly available software packages. These tools, as well as others to come, are rapidly paving the way towards standardized and reproducible bioimaging studies at the whole-tissue level. Reflecting on these achievements, we discuss the remaining challenges and the future endeavours lying ahead in the post-image analysis era.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'.
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http://dx.doi.org/10.1098/rstb.2015.0512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379021PMC
May 2017

Imaging tissue-mimic with light sheet microscopy: A comparative guideline.

Sci Rep 2017 03 21;7:44939. Epub 2017 Mar 21.

ITAV, Université de Toulouse, CNRS, UPS, France.

Tissue mimics (TMs) on the scale of several hundred microns provide a beneficial cell culture configuration for in vitro engineered tissue and are currently under the spotlight in tissue engineering and regenerative medicine. Due to the cell density and size, TMs are fairly inaccessible to optical observation and imaging within these samples remains challenging. Light Sheet Fluorescence Microscopy (LSFM)- an emerging and attractive technique for 3D optical sectioning of large samples- appears to be a particularly well-suited approach to deal with them. In this work, we compared the effectiveness of different light sheet illumination modalities reported in the literature to improve resolution and/or light exposure for complex 3D samples. In order to provide an acute and fair comparative assessment, we also developed a systematic, computerized benchmarking method. The outcomes of our experiment provide meaningful information for valid comparisons and arises the main differences between the modalities when imaging different types of TMs.
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http://dx.doi.org/10.1038/srep44939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381005PMC
March 2017

Computer-assisted topological analysis of renal allograft inflammation adds to risk evaluation at diagnosis of humoral rejection.

Kidney Int 2017 07 18;92(1):214-226. Epub 2017 Mar 18.

Hospices Civils de Lyon, Hôpital Edouard Herriot, Service de Transplantation, Néphrologie et Immunologie Clinique, Lyon, France; INSERM U1111, Centre International de Recherche en Infectiologie (CIRI), Ecole Normale Supérieure de Lyon, Lyon, France; Université Lyon 1, Faculté de medecine Lyon-Est, Lyon, France. Electronic address:

Antibody-mediated rejection is associated with heterogeneous kidney allograft outcomes. Accurate evaluation of risk for graft loss at time of diagnosis is necessary to offer personalized treatment. In contrast with serological and molecular assessment, morpho-histological evaluation of antibody-mediated rejection lesions has not significantly evolved. This relies on Banff classifications designed to be of diagnostic discriminatory power rather than prognostic and face quantitative and qualitative limitations. Here we developed a method of Computer-assisted Analysis of Graft Inflammation (CAGI) to improve the classification of allograft inflammation. Digitization of immunostained biopsy sections, image processing and algorithm-driven analysis allowed quantification of macrophages, T cells, B cells, and granulocytes per unit surface of interstitium, capillaries or glomeruli. CAGI was performed on biopsy specimens of 52 patients with extensively phenotyped antibody-mediated rejection. Macrophage numbers in capillaries and interstitium, but not Banff scores or the amount of other immune cell subsets, correlated with donor-specific antibody (DSA) mean fluorescence intensity and DSA-C3d status. The quantity of macrophages in the interstitium and DSA-C3d status were the only independent predictors for significant allograft loss at the time of antibody-mediated rejection diagnosis (hazard ratio 3.71 and 2.34, respectively). A significant strategy integrating the DSA-C3d assay and the quantification of interstitial macrophages allowed identification of three groups with distinct renal prognosis: DSA-C3d, DSA-C3d/macrophages-low and DSAC3d/macrophages-high. Thus, CAGI brings a missing piece to the antibody-mediated rejection puzzle by identifying morpho-histological processes that bridge in vitro parameters of DSA pathogenicity and graft loss. Hence, this approach could be useful in future integrated strategies of risk evaluation.
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http://dx.doi.org/10.1016/j.kint.2017.01.011DOI Listing
July 2017