Reliable, standardized measurements for cell mechanical properties.

Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Young's modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.

Activation of Nod2 signaling upon norovirus infection enhances antiviral immunity and susceptibility to colitis.

Over 90% of epidemic non-bacterial gastroenteritis are caused by human noroviruses (NoVs), which persist in a substantial subset of people allowing their spread worldwide. This has led to a significant number of endemic cases and up to 70,000 children deaths in developing countries. NoVs are primarily transmitted through the fecal-oral route. To date, studies have focused on the influence of the gut microbiota on enteric viral clearance by mucosal immunity. In this study, the use of mouse norovirus S99 (MNoV_S99) and CR6 (MNoV_CR6), two persistent strains, allowed us to provide evidence that the norovirus-induced exacerbation of colitis severity relied on bacterial sensing by nucleotide-binding oligomerization domain 2 (Nod2). Consequently, Nod2-deficient mice showed reduced levels of gravity of Dextran sodium sulfate (DSS)-induced colitis with both viral strains. And MNoV_CR6 viremia was heightened in Nod2-/- mice in comparison with animals hypomorphic for Atg16l1, which are prone to aggravated inflammation under DSS. Accordingly, the infection of macrophages derived from WT mice promoted the phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and NOD2's expression levels. Higher secretion of Tumor Necrosis Factor alpha (TNFα) following NOD2 activation and better viral clearance were measured in these cells. By contrast, reduced levels of pSTAT1 and blunted downstream secretion of TNFα were found in Nod2-deficient macrophages infected by MNoV_S99. Hence, our results uncover a previously unidentified virus-host-bacterial interplay that may represent a novel therapeutic target for treating noroviral origin gastroenteritis that may be linked with susceptibility to several common illnesses such as Crohn's disease.

Coupled mechanical mapping and interference contrast microscopy reveal viscoelastic and adhesion hallmarks of monocyte differentiation into macrophages.

Monocytes activated by pro-inflammatory signals adhere to the vascular endothelium and migrate from the bloodstream to the tissue ultimately differentiating into macrophages. Cell mechanics and adhesion play a crucial role in macrophage functions during this inflammatory process. However, how monocytes change their adhesion and mechanical properties upon differentiation into macrophages is still not well understood. In this work, we used various tools to quantify the morphology, adhesion, and viscoelasticity of monocytes and differentiatted macrophages. Combination of atomic force microscopy (AFM) high resolution viscoelastic mapping with interference contrast microscopy (ICM) at the single-cell level revealed viscoelasticity and adhesion hallmarks during monocyte differentiation into macrophages. Quantitative holographic tomography imaging revealed a dramatic increase in cell volume and surface area during monocyte differentiation and the emergence of round and spread macrophage subpopulations. AFM viscoelastic mapping showed important stiffening (increase of the apparent Young's modulus, E0) and solidification (decrease of cell fluidity, ß) on differentiated cells that correlated with increased adhesion area. These changes were enhanced in macrophages with a spread phenotype. Remarkably, when adhesion was perturbed, differentiated macrophages remained stiffer and more solid-like than monocytes, suggesting a permanent reorganization of the cytoskeleton. We speculate that the stiffer and more solid-like microvilli and lamellipodia might help macrophages to minimize energy dissipation during mechanosensitive activities. Thus, our results revealed viscoelastic and adhesion hallmarks of monocyte differentiation that may be important for biological function.

Comparison of Rheological Properties of Healthy versus Dupuytren Fibroblasts When Treated with a Cell Contraction Inhibitor by Atomic Force Microscope.

Mechanical properties of healthy and Dupuytren fibroblasts were investigated by atomic force microscopy (AFM). In addition to standard force curves, rheological properties were assessed using an oscillatory testing methodology, in which the frequency was swept from 1 Hz to 1 kHz, and data were analyzed using the structural damping model. Dupuytren fibroblasts showed larger apparent Young's modulus values than healthy ones, which is in agreement with previous results. Moreover, cell mechanics were compared before and after ML-7 treatment, which is a myosin light chain kinase inhibitor (MLCK) that reduces myosin activity and hence cell contraction. We employed two different concentrations of ML-7 inhibitor and could observe distinct cell reactions. At 1 µM, healthy and scar fibroblasts did not show measurable changes in stiffness, but Dupuytren fibroblasts displayed a softening and recovery after some time. When increasing ML-7 concentration (3 µM), the majority of cells reacted, Dupuytren fibroblasts were the most susceptible, not being able to recover from the drug and dying. These results suggested that ML-7 is a potent inhibitor for MLCK and that myosin II is essential for cytoskeleton stabilization and cell survival.

Solid-state NMR molecular snapshots of Aspergillus fumigatus cell wall architecture during a conidial morphotype transition.

While establishing an invasive infection, the dormant conidia of Aspergillus fumigatus transit through swollen and germinating stages, to form hyphae. During this morphotype transition, the conidial cell wall undergoes dynamic remodeling, which poses challenges to the host immune system and antifungal drugs. However, such cell wall reorganization during conidial germination has not been studied so far. Here, we explored the molecular rearrangement of Aspergillus fumigatus cell wall polysaccharides during different stages of germination. We took advantage of magic-angle spinning NMR to investigate the cell wall polysaccharides, without employing any destructive method for sample preparation. The breaking of dormancy was associated with a significant change in the molar ratio between the major polysaccharides ß-1,3-glucan and α-1,3-glucan, while chitin remained equally abundant. The use of various polarization transfers allowed the detection of rigid and mobile polysaccharides; the appearance of mobile galactosaminogalactan was a molecular hallmark of germinating conidia. We also report for the first time highly abundant triglyceride lipids in the mobile matrix of conidial cell walls. Water to polysaccharides polarization transfers revealed an increased surface exposure of glucans during germination, while chitin remained embedded deeper in the cell wall, suggesting a molecular compensation mechanism to keep the cell wall rigidity. We complement the NMR analysis with confocal and atomic force microscopies to explore the role of melanin and RodA hydrophobin on the dormant conidial surface. Exemplified here using Aspergillus fumigatus as a model, our approach provides a powerful tool to decipher the molecular remodeling of fungal cell walls during their morphotype switching.

Study of the persistence and dynamics of recombinant mCherry-producing Yarrowia lipolytica strains in the mouse intestine using fluorescence imaging.

Yarrowia lipolytica is a dimorphic oleaginous non-conventional yeast widely used as a powerful host for expressing heterologous proteins, as well as a promising source of engineered cell factories for various applications. This microorganism has a documented use in Feed and Food and a GRAS (generally recognized as safe) status. Moreover, in vivo studies demonstrated a beneficial effect of this yeast on animal health. However, despite the focus on Y. lipolytica for the industrial manufacturing of heterologous proteins and for probiotic effects, its potential for oral delivery of recombinant therapeutic proteins has seldom been evaluated in mammals. As the first steps towards this aim, we engineered two Y. lipolytica strains, a dairy strain and a laboratory strain, to produce the model fluorescent protein mCherry. We demonstrated that both Y. lipolytica strains transiently persisted for at least 1 week after four daily oral administrations and they maintained the active expression of mCherry in the mouse intestine. We used confocal microscopy to image individual Y. lipolytica cells of freshly collected intestinal tissues. They were found essentially in the lumen and they were rarely in contact with epithelial cells while transiting through the ileum, caecum and colon of mice. Taken as a whole, our results have shown that fluorescent Y. lipolytica strains constitute novel tools to study the persistence and dynamics of orally administered yeasts which could be used in the future as oral delivery vectors for the secretion of active therapeutic proteins in the gut.

PyFMLab: Open-source software for atomic force microscopy microrheology data analysis.

Background: Atomic force microscopy (AFM) is one of the main techniques used to characterize the mechanical properties of soft biological samples and biomaterials at the nanoscale. Despite efforts made by the AFM community to promote open-source data analysis tools, standardization continues to be a significant concern in a field that requires common analysis procedures. AFM-based mechanical measurements involve applying a controlled force to the sample and measure the resulting deformation in the so-called force-distance curves. These may include simple approach and retract or oscillatory cycles at various frequencies (microrheology). To extract quantitative parameters, such as the elastic modulus, from these measurements, AFM measurements are processed using data analysis software. Although open tools exist and allow obtaining the mechanical properties of the sample, most of them only include standard elastic models and do not allow the processing of microrheology data. In this work, we have developed an open-source software package (called PyFMLab, as of python force microscopy laboratory) capable of determining the viscoelastic properties of samples from both conventional force-distance curves and microrheology measurements. Methods: PyFMLab has been written in Python, which provides an accessible syntax and sufficient computational efficiency. The software features were divided into separate, self-contained libraries to enhance code organization and modularity and to improve readability, maintainability, testability, and reusability. To validate PyFMLab, two AFM datasets, one composed of simple force curves and another including oscillatory measurements, were collected on HeLa cells. Results: The viscoelastic parameters obtained on the two datasets analysed using PyFMLab were validated against data processing proprietary software and against validated MATLAB routines developed before obtaining equivalent results. Conclusions: Its open-source nature and versatility makes PyFMLab an open-source solution that paves the way for standardized viscoelastic characterization of biological samples from both force-distance curves and microrheology measurements.

Just like we can test the ripeness of fruit by touching it, we can use our hands to gently touch an object and determine if it is soft or stiff. Doctors use this technique, called palpation, to explore our organs and check for signs of disease. We can think about doing something similar, but on a much smaller scale ­ the nanoscale ­ so small that you cannot even see it with your naked eye. Atomic force microscopy (AFM) allows to apply palpation at the nanoscale. AFM is a powerful tool that allows scientists to examine incredibly small objects, like individual cells or molecules. AFM uses super sensitive "fingers" to touch and explore things that are too small to be seen under a regular microscope. During the European project Phys2BioMed, we explored how to apply AFM to diagnose diseases using nanopalpation. For example, touching samples from biopsies of patients and determining how soft or stiff they are. Here's the catch: there is not a single, standardized method or software that can efficiently process all the data obtained from AFM. It's a bit like having a lot of different languages but no universal translator. Just like a scale or a measuring cup are standardized, scientists need to analyze AFM data accurately and consistently. This is crucial to ensure reliable comparisons between results obtained by different researchers on different instruments, something particularly important when the results are to be used for diagnostic or predictive purposes. To help tackle this problem, we have developed PyFMLab. This software is a reliable and easy-to-use tool that translates AFM data into insights about the tiny structures being studied. By providing a standardized, open-source, modular and accessible way to analyze AFM data, PyFMLab democratizes access to this field of Biophysics, paving the way for clinical applications of AFM.

Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division.

Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signalling to conform cell behavior. Although the contribution of ECM compliance to the control of cell migration or division is extensively studied, little is reported regarding infectious processes. We study this phenomenon with the extraintestinal Escherichia coli pathogen UTI89. We show that UTI89 takes advantage, via its CNF1 toxin, of integrin mechanoactivation to trigger its invasion into cells. We identify the HACE1 E3 ligase-interacting protein Optineurin (OPTN) as a protein regulated by ECM stiffness. Functional analysis establishes a role of OPTN in bacterial invasion and integrin mechanical coupling and for stimulation of HACE1 E3 ligase activity towards the Rac1 GTPase. Consistent with a role of OPTN in cell mechanics, OPTN knockdown cells display defective integrin-mediated traction force buildup, associated with limited cellular invasion by UTI89. Nevertheless, OPTN knockdown cells display strong mechanochemical adhesion signalling, enhanced Rac1 activation and increased cyclin D1 translation, together with enhanced cell proliferation independent of ECM stiffness. Together, our data ascribe a new function to OPTN in mechanobiology.

Surface tension of model tissues during malignant transformation and epithelial-mesenchymal transition.

Epithelial-mesenchymal transition is associated with migration, invasion, and metastasis. The translation at the tissue scale of these changes has not yet been enlightened while being essential in the understanding of tumor progression. Thus, biophysical tools dedicated to measurements on model tumor systems are needed to reveal the impact of epithelial-mesenchymal transition at the collective cell scale. Herein, using an original biophysical approach based on magnetic nanoparticle insertion inside cells, we formed and flattened multicellular aggregates to explore the consequences of the loss of the metastasis suppressor NME1 on the mechanical properties at the tissue scale. Multicellular spheroids behave as viscoelastic fluids, and their equilibrium shape is driven by surface tension as measured by their deformation upon magnetic field application. In a model of breast tumor cells genetically modified for NME1, we correlated tumor invasion, migration, and adhesion modifications with shape maintenance properties by measuring surface tension and exploring both invasive and migratory potential as well as adhesion characteristics.

The emergence of spontaneous coordinated epithelial rotation on cylindrical curved surfaces.

Three-dimensional collective epithelial rotation around a given axis represents a coordinated cellular movement driving tissue morphogenesis and transformation. Questions regarding these behaviors and their relationship with substrate curvatures are intimately linked to spontaneous active matter processes and to vital morphogenetic and embryonic processes. Here, using interdisciplinary approaches, we study the dynamics of epithelial layers lining different cylindrical surfaces. We observe large-scale, persistent, and circumferential rotation in both concavely and convexly curved cylindrical tissues. While epithelia of inverse curvature show an orthogonal switch in actomyosin network orientation and opposite apicobasal polarities, their rotational movements emerge and vary similarly within a common curvature window. We further reveal that this persisting rotation requires stable cell-cell adhesion and Rac-1-dependent cell polarity. Using an active polar gel model, we unveil the different relationships of collective cell polarity and actin alignment with curvatures, which lead to coordinated rotational behavior despite the inverted curvature and cytoskeleton order.

Modulation of designer biomimetic matrices for optimized differentiated intestinal epithelial cultures.

The field of intestinal biology is thirstily searching for different culture methods that complement the limitations of organoids, particularly the lack of a differentiated intestinal compartment. While being recognized as an important milestone for basic and translational biological studies, many primary cultures of intestinal epithelium (IE) rely on empirical trials using hydrogels of various stiffness, whose mechanical impact on epithelial organization remains vague until now. Here, we report the development of hydrogel scaffolds with a range of elasticities and their influence on IE expansion, organization, and differentiation. On stiff substrates (>5 kPa), mouse IE cells adopt a flat cell shape and detach in the short-term. In contrast, on soft substrates (80-500 Pa), they sustain for a long-term, pack into high density, develop columnar shape with improved apical-basal polarity and differentiation marker expression, a phenotype reminiscent of features in vivo mouse IE. We then developed a soft gel molding process to produce 3D Matrigel scaffolds of close-to-nature stiffness, which support and maintain a culture of mouse IE into crypt-villus architecture. Thus, the present work is up-to-date informative for the design of biomaterials for ex vivo intestinal models, offering self-renewal in vitro culture that emulates the mouse IE.

The Alzheimer susceptibility gene BIN1 induces isoform-dependent neurotoxicity through early endosome defects.

The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer's disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.

DHA-containing phospholipids control membrane fusion and transcellular tunnel dynamics.

Metabolic studies and animal knockout models point to the critical role of polyunsaturated docosahexaenoic acid (22:6, DHA)-containing phospholipids (DHA-PLs) in physiology. Here, we investigated the impact of DHA-PLs on the dynamics of transendothelial cell macroapertures (TEMs) triggered by RhoA inhibition-associated cell spreading. Lipidomic analyses showed that human umbilical vein endothelial cells (HUVECs) subjected to a DHA diet undergo a 6-fold enrichment in DHA-PLs at the plasma membrane (PM) at the expense of monounsaturated oleic acid-containing PLs (OA-PLs). Consequently, DHA-PL enrichment at the PM induces a reduction in cell thickness and shifts cellular membranes towards a permissive mode of membrane fusion for transcellular tunnel initiation. We provide evidence that a global homeostatic control of membrane tension and cell cortex rigidity minimizes overall changes of TEM area through a decrease of TEM size and lifetime. Conversely, low DHA-PL levels at the PM lead to the opening of unstable and wider TEMs. Together, this provides evidence that variations of DHA-PL levels in membranes affect cell biomechanical properties.

A noncanonical autophagy is involved in the transfer of Plasmodium-microvesicles to astrocytes.

Cerebral malaria is a neuroinflammatory disease induced by P. falciparum infection. In animal models, the neuro-pathophysiology of cerebral malaria results from the sequestration of infected red blood cells (iRBCs) in microvessels that promotes the activation of glial cells in the brain. This activation provokes an exacerbated inflammatory response characterized by the secretion of proinflammatory cytokines and chemokines, leading to brain infiltration by pathogenic CD8+ T lymphocytes. Astrocytes are a major subtype of brain glial cells that play an important role in maintaining the homeostasis of the central nervous system, the integrity of the brain-blood barrier and in mounting local innate immune responses. We have previously shown that parasitic microvesicles (PbA-MVs) are transferred from iRBCs to astrocytes. The present study shows that an unconventional LC3-mediated autophagy pathway independent of ULK1 is involved in the transfer and degradation of PbA-MVs inside the astrocytes. We further demonstrate that inhibition of the autophagy process by treatment with 3-methyladenine blocks the transfer of PbA-MVs, which remain localized in the astrocytic cell membrane and are not internalized. Moreover, bafilomycin A1, another drug against autophagy promotes the accumulation of PbA-MVs inside the astrocytes by inhibiting the fusion with lysosomes, and prevents ECM in mice infected with PbA. Finally, we establish that RUBCN/rubicon or ATG5 silencing impede astrocyte production in CCL2 and CXCL10 chemokines induced by PbA stimulation. Altogether, our data suggest that a non-canonical autophagy-lysosomal pathway may play a key role in cerebral malaria through regulation of brain neuro-inflammation by astrocytes.

Persistence and dynamics of fluorescent Lactobacillus plantarum in the healthy versus inflamed gut.

The gastrointestinal tract is the main ecological niche in which Lactobacillus strains may provide health benefits in mammals. There is currently a need to characterize host-microbe interactions in space and time by tracking these bacteria in vivo. We combined noninvasive whole-body imaging with ex vivo fluorescence confocal microscopy imaging to monitor the impact of intestinal inflammation on the persistence of orally administered Lactobacillus plantarum NCIMB8826 in healthy and inflamed mouse colons. We developed fluorescent L. plantarum strains and demonstrated that mCherry is the best system for in vivo imaging and ex vivo fluorescence confocal microscopy of these bacteria. We also used whole-body imaging to show that this anti-inflammatory, orally administered strain persists for longer and at higher counts in the inflamed colon than in the healthy colon. We confirmed these results by the ex vivo confocal imaging of colons from mice with experimental colitis for 3 days after induction. Moreover, extended orthogonal view projections enabled us to localize individual L. plantarum in sites that differed for healthy versus inflamed guts. In healthy colons, orally administered bacteria were localized in the lumen (in close contact with commensal bacteria) and sometimes in the crypts (albeit very rarely in contact with intestinal cells). The bacteria were observed within and outside the mucus layer. In contrast, L. plantarum bacteria in the inflamed colon were mostly located in the lumen and (in less inflamed areas) within the mucus layer. In more intensely inflamed areas (i.e., where the colon had undergone structural damage), the L. plantarum were in direct contact with damaged epithelial cells. Taken as a whole, our results show that fluorescently labeled L. plantarum can be used to study the persistence of these bacteria in inflamed guts using both noninvasive whole-body imaging and ex vivo fluorescence confocal microscopy.

Rab11A regulates dense granule transport and secretion during Toxoplasma gondii invasion of host cells and parasite replication.

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells.

Development of a novel multiphysical approach for the characterization of mechanical properties of musculotendinous tissues.

At present, there is a lack of well-validated protocols that allow for the analysis of the mechanical properties of muscle and tendon tissues. Further, there are no reports regarding characterization of mouse skeletal muscle and tendon mechanical properties in vivo using elastography thereby limiting the ability to monitor changes in these tissues during disease progression or response to therapy. Therefore, we sought to develop novel protocols for the characterization of mechanical properties in musculotendinous tissues using atomic force microscopy (AFM) and ultrasound elastography. Given that TIEG1 knockout (KO) mice exhibit well characterized defects in the mechanical properties of skeletal muscle and tendon tissue, we have chosen to use this model system in the present study. Using TIEG1 knockout and wild-type mice, we have devised an AFM protocol that does not rely on the use of glue or chemical agents for muscle and tendon fiber immobilization during acquisition of transversal cartographies of elasticity and topography. Additionally, since AFM cannot be employed on live animals, we have also developed an ultrasound elastography protocol using a new linear transducer, SLH20-6 (resolution: 38 µm, footprint: 2.38 cm), to characterize the musculotendinous system in vivo. This protocol allows for the identification of changes in muscle and tendon elasticities. Such innovative technological approaches have no equivalent to date, promise to accelerate our understanding of musculotendinous mechanical properties and have numerous research and clinical applications.

Stiffness tomography of eukaryotic intracellular compartments by atomic force microscopy.

Precise localization and biophysical characterization of cellular structures is a key to the understanding of biological processes happening both inside the cell and at the cell surface. Atomic force microscopy is a powerful tool to study the cell surface - topography, elasticity, viscosity, interactions - and also the viscoelastic behavior of the underlying cytoplasm, cytoskeleton or the nucleus. Here, we demonstrate the ability of atomic force microscopy to also map and characterize organelles and microorganisms inside cells, at the nanoscale, by combining stiffness tomography with super-resolution fluorescence and electron microscopy. By using this correlative approach, we could both identify and characterize intracellular compartments. The validation of this approach was performed by monitoring the stiffening effect according to the metabolic status of the mitochondria in living cells in real-time.

Targeting Type IV pili as an antivirulence strategy against invasive meningococcal disease.

Bacterial virulence factors are attractive targets for the development of therapeutics. Type IV pili, which are associated with a remarkable array of properties including motility, the interaction between bacteria and attachment to biotic and abiotic surfaces, represent particularly appealing virulence factor targets. Type IV pili are present in numerous bacterial species and are critical for their pathogenesis. In this study, we report that trifluoperazine and related phenothiazines block functions associated with Type IV pili in different bacterial pathogens, by affecting piliation within minutes. Using Neisseria meningitidis as a paradigm of Gram-negative bacterial pathogens that require Type IV pili for pathogenesis, we show that piliation is sensitive to altered activity of the Na+ pumping NADH-ubiquinone oxidoreductase (Na+-NQR) complex and that these compounds probably altered the establishment of the sodium gradient. In vivo, these compounds exert a strong protective effect. They reduce meningococcal colonization of the human vessels and prevent subsequent vascular dysfunctions, intravascular coagulation and overwhelming inflammation, the hallmarks of invasive meningococcal infections. Finally, they reduce lethality. This work provides a proof of concept that compounds with activity against bacterial Type IV pili could beneficially participate in the treatment of infections caused by Type IV pilus-expressing bacteria.

Automated multi-sample acquisition and analysis using atomic force microscopy for biomedical applications.

In the last 20 years, atomic force microscopy (AFM) has emerged as a ubiquitous technique in biological research, allowing the analysis of biological samples under near-physiological conditions from single molecules to living cells. Despite its growing use, the low process throughput remains a major drawback. Here, we propose a solution validated on a device allowing a fully automated, multi-sample analysis. Our approach is mainly designed to study samples in fluid and biological cells. As a proof of concept, we demonstrate its feasibility applied to detect and scan both fixed and living bacteria before completion of data processing. The effect of two distinct treatments (i.e. gentamicin and heating) is then evidenced on physical parameters of fixed Yersinia pseudotuberculosis bacteria. The multi-sample analysis presented allows an increase in the number of scanned samples while limiting the user's input. Importantly, cantilever cleaning and control steps are performed regularly-as part of the automated process-to ensure consistent scanning quality. We discuss how such an approach is paving the way to AFM developments in medical and clinical fields, in which statistical significance of results is a prerequisite.