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To detect cellular activities deep within the body using magnetic resonance platforms, magnetosomes are the ideal model of genetically-encoded nanoparticles. These membrane-bound iron biominerals produced by magnetotactic bacteria are highly regulated by approximately 30 genes; however, the number of magnetosome genes that are essential and/or constitute the root structure upon which biominerals form is largely undefined. To examine the possibility that key magnetosome genes may interact in a foreign environment, we expressed mamI and mamL as fluorescent fusion proteins in mammalian cells. Localization and potential protein-protein interaction(s) were investigated using confocal microscopy and fluorescence correlation spectroscopy (FCS). Enhanced green fluorescent protein (EGFP)-MamI and the red fluorescent Tomato-MamL displayed distinct intracellular localization, with net-like and punctate fluorescence, respectively. Remarkably, co-expression revealed co-localization of both fluorescent fusion proteins in the same punctate pattern. An interaction between MamI and MamL was confirmed by co-immunoprecipitation. In addition, changes in EGFP-MamI distribution were accompanied by acquisition of intracellular mobility which all Tomato-MamL structures displayed. Analysis of extracts from these cells by FCS was consistent with an interaction between fluorescent fusion proteins, including an increase in particle radius. Co-localization and interaction of MamI and MamL demonstrate that select magnetosome proteins may associate in mammalian cells., (© 2024. The Author(s).)

Transcription factors play an essential role in pattern formation during early embryo development, generating a strikingly fast and precise transcriptional response that results in sharp gene expression boundaries. To characterize the steps leading up to transcription, we performed a side-by-side comparison of the nuclear dynamics of two morphogens, a transcriptional activator, Bicoid (Bcd), and a transcriptional repressor, Capicua (Cic), both involved in body patterning along the anterior-posterior axis of the early Drosophila embryo. We used a combination of fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and single-particle tracking to access a wide range of dynamical timescales. Despite their opposite effects on gene transcription, we find that Bcd and Cic have very similar nuclear dynamics, characterized by the coexistence of a freely diffusing monomer population with a number of oligomeric clusters, which range from low stoichiometry and high mobility clusters to larger, DNA-bound hubs. Our observations are consistent with the inclusion of both Bcd and Cic into transcriptional hubs or condensates, while putting constraints on the mechanism by which these form. These results fit in with the recent proposal that many transcription factors might share a common search strategy for target gene regulatory regions that makes use of their large unstructured regions, and may eventually help explain how the transcriptional response they elicit can be at the same time so fast and so precise., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Helical-shaped magnetotactic bacteria provide a rare opportunity to precisely measure both the translational and rotational friction coefficients of micron-sized chiral particles. The possibility to align these cells with a uniform magnetic field allows clearly separating diffusion along and perpendicular to their longitudinal axis. Meanwhile, their corkscrew shape allows detecting rotations around their longitudinal axis, after which orientation correlation analysis can be used to retrieve rotational diffusion coefficients in the two principal directions. Using light microscopy, we measured the four principal friction coefficients of deflagellated Magnetospirillum magneticum cells, and compared our results to that expected for cylinders of comparable size. We show that for rotational motions, the overall dimensions of the cell body are what matters most, while the exact body shape has a larger influence on translational motions. To obtain a full characterization of the friction matrix of these elongated chiral particles, we also quantified the coupling between the rotation around and translation along the longitudinal axis of the cell. Our results suggest that for this bacterial species cell body rotation could significantly contribute to cellular propulsion.

For over 40 years, the Bicoid- hunchback (Bcd- hb ) system in the fruit fly embryo has been used as a model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation. By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the early hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoid's partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate., Competing Interests: GF, HT, MA, YD, CP, CF, MC, ND No competing interests declared, AW Senior editor, eLife, (© 2022, Fernandes et al.)

Biological hydrogels play important physiological roles in the body. These hydrogels often contain ordered subdomains that provide mechanical toughness and other tissue-specific functionality. Filamentous bacteriophages are nanofilaments with a high aspect ratio that can self-assemble into liquid crystalline domains that could be designed to mimic ordered biological hydrogels and can thus find applications in biomedical engineering. We have previously reported hydrogels of pure cross-linked liquid crystalline filamentous phage formed at very high concentrations exhibiting a tightly packed microstructure and high stiffness. In this work, we report a method for inducing self-assembly of filamentous phage into liquid crystalline hydrogels at concentrations that are several orders of magnitude below that of lyotropic liquid crystal formation, thus creating structural order but a less densely packed microstructure. Hybrid hydrogels of M13 phage and bovine serum albumin (0.25 w/v%) were formed and shown to adsorb up to 16× their weight in water. Neither component gelled on its own at the low concentrations used, suggesting synergistic action between the two components in the formation of the hydrogel. The hybrid hydrogels exhibited repetitive self-healing under physiological conditions and at room temperature, autofluorescence in three channels, and antibacterial activity toward Escherichia coli host cells. Furthermore, the hybrid hydrogels exhibited a more than 2× higher ability to pack water compared to BSA-only hydrogels and 2× lower compression modulus compared to tightly packed M13-only hydrogels, suggesting that our method could be used to create hydrogels with tunable mechanical properties and pore structure through the addition of globular proteins, while maintaining bioactivity and microscale structural order.

The impact of dietary advanced glycation end products (dAGEs) on human health has been discussed in many studies but, to date, no consensual pathophysiological process has been demonstrated. The intestinal absorption pathways which have so far been described for dAGEs, the passive diffusion of free AGE adducts and transport of glycated di-tripeptides by the peptide transporter 1 (PEPT-1), are not compatible with certain pathophysiological processes described. To get new insight into the intestinal absorption pathways and the pathophysiological mechanisms of dAGEs, we initiated an in vivo study with a so-called simple animal model with a complete digestive tract, Caenorhabditis elegans . Dietary bacteria were chemically modified with glyoxylic acid to mainly produce Nε -carboxymethyllysine (CML) and used to feed the worms. We performed different immunotechniques using an anti-CML antibody for the relative quantification of ingested CML and localization of this AGE in the worms' intestine. The relative expression of genes encoding different biological processes such as response to stresses and intestinal digestion were determined. The physiological development of the worms was verified. All the results were compared with those obtained with the control bacteria. The results revealed a new route for the intestinal absorption of dietary CML (dCML), endocytosis, which could be mediated by scavenger receptors. The exposure of worms to dCML induced a reproductive defect and a transcriptional response reflecting oxidative, carbonyl and protein folding stresses. These data, in particular the demonstration of endocytosis of dCML by enterocytes, open up new perspectives to better characterize the pathophysiological mechanisms of dAGEs.

Quantitative cell biology requires precise and accurate concentration measurements, resolved both in space and time. Fluorescence correlation spectroscopy (FCS) has been held as a promising technique to perform such measurements because the fluorescence fluctuations it relies on are directly dependent on the absolute number of fluorophores in the detection volume. However, the most interesting applications are in cells, where autofluorescence and confinement result in strong background noise and important levels of photobleaching. Both noise and photobleaching introduce systematic bias in FCS concentration measurements and need to be corrected for. Here, we propose to make use of the photobleaching inevitably occurring in confined environments to perform series of FCS measurements at different fluorophore concentration, which we show allows a precise in situ measurement of both background noise and molecular brightness. Such a measurement can then be used as a calibration to transform confocal intensity images into concentration maps. The power of this approach is first illustrated with in vitro measurements using different dye solutions, then its applicability for in vivo measurements is demonstrated in Drosophila embryos for a model nuclear protein and for two morphogens, Bicoid and Capicua., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The adherent-invasive Escherichia coli (AIEC) pathotype has been implicated in the pathogenesis of inflammatory bowel diseases in general and in Crohn's disease (CD) in particular. AIEC strains are primarily characterized by their ability to adhere to and invade intestinal epithelial cells. However, the genetic and phenotypic features of AIEC isolates vary greatly as a function of the strain's clonality, host factors, and the gut microenvironment. It is thus essential to identify the determinants of AIEC pathogenicity and understand their role in intestinal epithelial barrier dysfunction and inflammation. We reasoned that soil nematode Caenorhabditis elegans (a simple but powerful model of host-bacterium interactions) could be used to study the virulence of AIEC vs. non- AIEC E. coli strains. Indeed, we found that the colonization of C. elegans (strain N2) by E. coli impacted survival in a strain-specific manner. Moreover, the AIEC strains' ability to invade cells in vitro was linked to the median lifespan in C. elegans (strain PX627). However, neither the E. coli intrinsic invasiveness (i.e., the fact for an individual strain to be characterized as invasive or not) nor AIEC's virulence levels (i.e., the intensity of invasion, established in % from the infectious inoculum) in intestinal epithelial cells was correlated with C. elegans ' lifespan in the killing assay. Nevertheless, AIEC longevity of C. elegans might be a relevant model for screening anti-adhesion drugs and anti-invasive probiotics.

Even though transcriptional repressors are studied with ever-increasing molecular resolution, the temporal aspects of gene repression remain poorly understood. Here, we address the dynamics of transcriptional repression by Capicua (Cic), which is essential for normal development and is commonly mutated in human cancers and neurodegenerative diseases. 1 , 2 We report the speed limit for Cic-dependent gene repression based on live imaging and optogenetic perturbations in the early Drosophila embryo, where Cic was originally discovered. 3 Our measurements of Cic concentration and intranuclear mobility, along with real-time monitoring of the activity of Cic target genes, reveal remarkably fast transcriptional repression within minutes of removing an optogenetic de-repressive signal. In parallel, quantitative analyses of transcriptional bursting of Cic target genes support a repression mechanism providing a fast-acting brake on burst generation. This work sets quantitative constraints on potential mechanisms for gene regulation by Cic., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6μm-2 to ≃0.1μm-2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.

Candida albicans mannan consists of a large repertoire of oligomannosides with different types of mannose linkages and chain lengths, which act as individual epitopes with more or less overlapping antibody specificities. Although anti-C. albicans mannan antibody levels are monitored for diagnostic purposes nothing is known about the qualitative distribution of these antibodies in terms of epitope specificity. We addressed this question using a bank of previously synthesized biotin sulfone tagged oligomannosides (BSTOs) of α and β anomery complemented with a synthetic β-mannotriose described as a protective epitope. The reactivity of these BSTOs was analyzed with IgM isotype monoclonal antibodies (MAbs) of known specificity, polyclonal sera from patients colonized or infected with C. albicans, and mannose binding lectin (MBL). Surface plasmon resonance (SPR) and multiple analyte profiling (MAP) were used. Both methods confirmed the usual reactivity of MAbs against either α or β linkages, excepted for MAb B6.1 (protective epitope) reacting with β-Man whereas the corresponding BSTO reacted with anti-α-Man. These results were confirmed in western blots with native C. albicans antigens. Using patients' sera in MAP, a significant correlation was observed between the detection of anti-mannan antibodies recognizing β- and α-Man epitopes and detection of antibodies against β-linked mannotriose suggesting that this epitope also reacts with human polyclonal antibodies of both specificities. By contrast, the reactivity of human sera with other α- and β-linked BSTOs clearly differed according to their colonized or infected status. In these cases, the establishment of an α/β ratio was extremely discriminant. Finally SPR with MBL, an important lectin of innate immunity to C. albicans, classically known to interact with α-mannose, also interacted in an unexpected way with the protective epitope. These cumulative data suggest that structure/activity investigations of the finely tuned C. albicans anti-mannose immune response are worthwhile to increase our basic knowledge and for translation in medicine.

Hypothesis: The formation of porous nanostructures on surfaces and the control of their size and shape is fundamental for various applications. The creation of nanotubes is particularly difficult to implement without the aid of hard and rigid templates. Recently, methods that form nanotubular structures in a straightforward manner and without direct templating, e.g. soft templating, have been highly sought after. Here we propose the use of "soft templating" via self-assembly of conducting monomers during electropolymerization in organic solvents as a mean to form porous, nanotubular features., Experiments: Naphtho[2,3-b]thieno[3,4-e][1,4]dioxine (NaphDOT) is employed as monomer for electropolymerizations conducted in dichloromethane and chloroform containing varying amounts of water. SEM analyses of the resulting surfaces confirms the strong capacity of NaphDOT to form vertically aligned nanotubes. Polymerization solutions analyzed by DLS and TEM reveal the presence of micelles prior to electropolymerization, and the size of the micelles correlates with the inner diameter of the nanotubes formed., Findings: We show that micelles in polymerization solutions are stabilized by both monomers and electrolytes. We propose a mechanism where reverse micelles are forming a soft-template responsible for the formation of porous nanostructures during electropolymerization in organic, non-polar solvents. In this mechanism, the monomer and electrolyte assume the role of surfactant in the reverse micelle system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hypothesis: Controlling the size and the shape of nanostructures on surfaces is fundamental for various applications while the formation of porous structures such as nanotubes is particularly difficult. The templateless electropolymerization is a choice process that not only forms nanostructured surfaces, but also can tune their morphologies using different monomers., Experiments: In this work, we used this soft-template and surfactant free electropolymerization in organic solvent to deposit for the first time carbazole-based monomers. Five different conjugated carbazole-based monomers are tested here., Findings: We show that the shape of surfaces nanostructures is highly dependent on the amount of water present in the organic solvent and on the molecular structure of the carbazole monomers. Different morphologies are obtained from fibers to vertically aligned nanotubes and even porous membranes, depending on the monomer and on the electropolymerization method. The nanostructured surfaces reach superhydrophobic properties and their dynamical non-wetting behavior varies with the monomer and the electrochemical parameters., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Collective motion is found at all scales in biological and artificial systems, and extensive research is devoted to describing the interplay between interactions and external cues in collective dynamics. Magnetotactic bacteria constitute a remarkable example of living organisms for which motion can be easily controlled remotely. Here, we report a new type of collective motion where a uniform distribution of magnetotactic bacteria is rendered unstable by a magnetic field. A new state of "bacterial magneto-convection" results, wherein bacterial plumes emerge spontaneously perpendicular to an interface and develop into self-sustained flow convection cells. While there are similarities to gravity driven bioconvection and the Rayleigh-Bénard instability, these rely on a density mismatch between layers of the fluids. Remarkably, here no external forces are applied on the fluid and the magnetic field only exerts an external torque aligning magnetotactic bacteria with the field. Using a theoretical model based on hydrodynamic singularities, we capture quantitatively the instability and the observed long-time growth. Bacterial magneto-convection represents a new class of collective behaviour resulting only from the balance between hydrodynamic interactions and external alignment.

Current models of apoptosis regulation by the Bcl-2 family of proteins postulate that heterodimeric interactions between family members determine whether Bax and Bak are activated to trigger cell death. Thus, the relative abundance and binding affinities between pro- and anti-apoptotic proteins determines the outcome of these interactions. Examination of these interactions using purified mitochondria and liposomes with full-length recombinant proteins revealed that Bcl-x L inhibits apoptosis as a higher-order complex that binds multiple BH3 proteins. Allosteric regulation of this complex by the BH3 sensitizer Bad confers switch-like activity to the indirect activation of Bax. The BH3 activator cBid sequestered by Bcl-x L complexes changes from an inactive to an active form while bound to a Bcl-x L complex only when Bad is also bound. Bcl-x L complexes enable Bad to function as a non-competitive inhibitor of Bcl-x L and allosterically activate cBid, dramatically enhancing the pro-apoptotic potency of Bad., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

While most quantitative studies of the motion of magnetotactic bacteria rely on the premise that the cells' magnetic dipole moment is aligned with their direction of motility, this assumption has so far rarely been challenged. Here we use phase contrast microscopy to detect the rotational diffusion of non-motile cells of Magnetospirillum magneticum AMB-1 around their magnetic moment, showing that in this species the magnetic dipole moment is, in fact, not exactly aligned with the cell body axis. From the cell rotational trajectories, we are able to infer the misalignment between cell magnetic moment and body axis with a precision of better than 1°, showing that it is, on average, 6°, and can be as high as 20°. We propose a method to correct for this misalignment, and perform a non-biased measurement of the magnetic moment of single cells based on the analysis of their orientation distribution. Using this correction, we show that magnetic moment strongly correlates with cell length. The existence of a range of misalignments between magnetic moment and cell axis in a population implies that the orientation and trajectories of magnetotactic bacteria placed in external magnetic fields is more complex than generally assumed, and might show some important cell-to-cell differences.

In nature, wetting phenomena are present nearly everywhere and are a source of inspiration for liquid transportation. A good understanding of the underlying dynamic phenomena that governs wettability is therefore extremely important for researchers involved in bio-inspired surfaces. Herein, we study the adhesive behavior with water of mesh substrates modified with structured copolymers in order to tune the surfaces from parahydrophobic states (high water adhesion) to superhydrophobic states (low water adhesion). Using the ejection test method (ETM), a new technique that consists of the ejection of water droplets deposited onto a substrate with the aid of a catapult system, we experimentally demonstrate that the elasticity of the mesh substrate can be exploited for efficient vertical actuation of droplets., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Two enveloped viral vectors, vesicular stomatitis virus and influenza virus, and a non-enveloped viral vector, human adenovirus type 5, were encapsulated by spray drying to enhance thermal stability.Results with these candidates led to the hypothesis that stability performance of chosen excipients may be less virus-specific, as previously postulated in the literature, and more differentiated based on whether the virus has a lipid envelope. Spray dried samples were characterized for their thermal properties, RNA viability and in vitro viral activity after storage at 37 °C for up to 30 days or at 45 °C for up to 3 days. The enveloped viral vectors, as a group, were more thermally stable in trehalose while the non-enveloped viral vector showed higher activity with mannitol as the primary excipient in blends. Trehalose shows strong hydrogen bonds with the envelope's lipid membrane than the other carbohydrates, more effectively replacing water molecules while maintaining the fluidity of the membrane. Conversely, the small size of mannitol molecules was attributed to the more effective hydrogen bonding between water and the protein capsid of non-enveloped viral vectors. In all cases, a matrix with high glass transition temperature contributed to thermal stabilization through vitrification. This work suggests that carbohydrate stabilizer selection may be more dependent on the envelope rather than the specific viral vector, which, if universally true, will provide a guideline for future formulation development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pro-aging effects of endogenous advanced glycation end-products (AGEs) have been reported, and there is increasing interest in the pro-inflammatory and -fibrotic effects of their binding to RAGE (the main AGE receptor). The role of dietary AGEs in aging remains ill-defined, but the predominantly renal accumulation of dietary carboxymethyllysine (CML) suggests the kidneys may be particularly affected. We studied the impact of RAGE invalidation and a CML-enriched diet on renal aging. Two-month-old male, wild-type (WT) and RAGE -/- C57Bl/6 mice were fed a control or a CML-enriched diet (200 μg CML/g food ) for 18 months. Compared to controls, we observed higher CML levels in the kidneys of both CML WT and CML RAGE -/- mice, with a predominantly tubular localization. The CML-rich diet had no significant impact on the studied renal parameters, whereby only a trend to worsening glomerular sclerosis was detected. Irrespective of diet, RAGE -/- mice were significantly protected against nephrosclerosis lesions (hyalinosis, tubular atrophy, fibrosis and glomerular sclerosis) and renal senile apolipoprotein A-II (ApoA-II) amyloidosis (p < 0.001). A positive linear correlation between sclerosis score and ApoA-II amyloidosis score (r = 0.92) was observed. Compared with old WT mice, old RAGE -/- mice exhibited lower expression of inflammation markers and activation of AKT, and greater expression of Sod2 and SIRT1. Overall, nephrosclerosis lesions and senile amyloidosis were significantly reduced in RAGE -/- mice, indicating a protective effect of RAGE deletion with respect to renal aging. This could be due to reduced inflammation and oxidative stress in RAGE -/- mice, suggesting RAGE is an important receptor in so-called inflamm-aging., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Using fluorescence correlation spectroscopy (FCS) to distinguish between different types of diffusion processes is often a perilous undertaking because the analysis of the resulting autocorrelation data is model dependant. Two recently introduced strategies, however, can help move toward a model-independent interpretation of FCS experiments: 1) the obtention of correlation data at different length scales and 2) their inversion to retrieve the mean-squared displacement associated with the process under study. We use computer simulations to examine the signature of several biologically relevant diffusion processes (simple diffusion, continuous-time random walk, caged diffusion, obstructed diffusion, two-state diffusion, and diffusing diffusivity) in variable-length-scale FCS. We show that, when used in concert, length-scale variation and data inversion permit us to identify non-Gaussian processes and, regardless of Gaussianity, to retrieve their mean-squared displacement over several orders of magnitude in time. This makes unbiased discrimination between different classes of diffusion models possible., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

With the advent of polymyxin B (PmB) resistance in bacteria, the mechanisms for mcr -1 resistance are of crucial importance in the design of novel therapeutics. The mcr -1 phenotype is known to decrease membrane charge and increase membrane packing by modification of the bacterial outer membrane. We used X-ray diffraction, Molecular Dynamics simulations, electrochemistry, and leakage assays to determine the location of PmB in different membranes and assess membrane damage. By varying membrane charge and lipid tail packing independently, we show that increasing membrane surface charge promotes penetration of PmB and membrane damage, whereas increasing lipid packing decreases penetration and damage. The penetration of the PmB molecules is well described by a phenomenological model that relates an attractive electrostatic and a repulsive force opposing insertion due to increased membrane packing. The model applies well to several gram-negative bacterial strains and may be used to predict resistance strength., Competing Interests: The authors declare no competing interests.

Background: The link between Candida phenotypical characteristics and invasive candidiasis (IC) prognosis is still partially unknown. Methods: Candida strains isolated during the AmarCAND2 study were centrally analyzed for species identification, antifungal susceptibility, biofilm formation, and expression of surface and glycoconjugate mannosides. Correlation between these phenotypical features and patient outcome was sought using a multivariable Cox survival model. Results: Candida albicans was predominant (65.4%, n = 285), with a mortality rate significantly lower than that in patients with non- albicans strains [HR 0.67 (0.46-1.00), p = 0.048]. The rate of fluconazole-resistant strains was low ( C. albicans and Candida glabrata : 3.5 and 6.2%, respectively) as well as caspofungin-resistant ones (1 and 3.1%, respectively). Early biofilm formation was less frequent among C. albicans (45.4%) than among non- albicans (81.2%). While the strains of C. albicans showed variable levels of surface mannosides expression, strains isolated from candidemia exhibited a high expression of β-man, which was correlated with an increased mortality ( p = 0.02). Conclusion: Candida albicans IC were associated with lower mortality, and with strains that exhibited less frequently early biofilm formation than non- albicans strains. A high expression of β-man was associated with increased IC mortality. Further studies are warranted to confirm this data and to evaluate other virulence factors in yeasts.

Morphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of the establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the major Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to distinguish subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on the cooperativity between the 6 known Bicoid binding sites in the hb promoter region, assuming rate limiting concentrations of the Bicoid transcription factor at the boundary, is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that a simple model based only on the cooperative binding of Bicoid is not sufficient to describe the spatiotemporal dynamics of early hb expression., Competing Interests: The authors have declared that no competing interests exist.

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Fly development amazes us by the precision and reproducibility of gene expression, especially since the initial expression patterns are established during very short nuclear cycles. Recent live imaging of hunchback promoter dynamics shows a stable steep binary expression pattern established within the three minute interphase of nuclear cycle 11. Considering expression models of different complexity, we explore the trade-off between the ability of a regulatory system to produce a steep boundary and minimize expression variability between different nuclei. We show how a limited readout time imposed by short developmental cycles affects the gene's ability to read positional information along the embryo's anterior posterior axis and express reliably. Comparing our theoretical results to real-time monitoring of the hunchback transcription dynamics in live flies, we discuss possible regulatory strategies, suggesting an important role for additional binding sites, gradients or non-equilibrium binding and modified transcription factor search strategies., Competing Interests: The authors have declared that no competing interests exist.

Aging is a physiological and complex process associated with increasing age of living organisms. Simple model organisms have brought significant advances in our understanding of the aging process. Caenorhabditis elegans, a nematode originally used to establish the genetic and molecular basis of development, has become one of the leading model organisms for research on aging. This invertebrate has allowed identifying a connection between cellular signaling pathways and longevity. Although C. elegans is not suitable for analysis of the complete process of human aging, it remains a model of choice to analyze specific aging mechanisms and phenotypes., (© 2018 médecine/sciences – Inserm.)

We present the LiveFly toolbox for quantitative analysis of transcription dynamics in live Drosophila embryos. The toolbox allows users to process two-color 3D confocal movies acquired using nuclei-labeling and the fluorescent RNA-tagging system described in the previous chapter and export the nuclei's position as a function of time, their lineages and the intensity traces of the active loci. The toolbox, which is tailored for the context of Drosophila early development, is semiautomatic, and requires minimal user intervention. It also includes a tool to combine data from multiple movies and visualize several features of the intensity traces and the expression pattern.

Live imaging has been used in recent years for the understanding of dynamic processes in biology, such as embryo development. This was made possible by a combination of advancements in microscopy, leading to improved signal-to-noise ratios and better spatial and temporal resolutions, and by the development of new fluorescence markers, allowing for the quantification of protein expression and transcriptional dynamics in vivo. Here we describe a general protocol, which can be used in standard confocal microscopes to image early Drosophila melanogaster embryos, in order to learn about the transcriptional dynamics of a fluorescently labeled RNA.

Morphogens are proteins that form concentration gradients in embryos and developing tissues, where they act as postal codes, providing cells with positional information and allowing them to behave accordingly. Bicoid was the first discovered morphogen, and remains one of the most studied. It regulates segmentation in flies, forming a striking exponential gradient along the anterior-posterior axis of early Drosophila embryos, and activating the transcription of multiple target genes in a concentration-dependent manner. In this review, the work done by us and by others to characterize the mobility of Bicoid in D. melanogaster embryos is presented. The central role played by the diffusion of Bicoid in both the establishment of the gradient and the activation of target genes is discussed, and placed in the context of the need for these processes to be all at once rapid, precise and robust. The Bicoid system, and morphogen gradients in general, remain amongst the most amazing examples of the coexistence, often observed in living systems, of small-scale disorder and large-scale spatial order. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The simultaneous expression of the hunchback gene in the numerous nuclei of the developing fly embryo gives us a unique opportunity to study how transcription is regulated in living organisms. A recently developed MS2-MCP technique for imaging nascent messenger RNA in living Drosophila embryos allows us to quantify the dynamics of the developmental transcription process. The initial measurement of the morphogens by the hunchback promoter takes place during very short cell cycles, not only giving each nucleus little time for a precise readout, but also resulting in short time traces of transcription. Additionally, the relationship between the measured signal and the promoter state depends on the molecular design of the reporting probe. We develop an analysis approach based on tailor made autocorrelation functions that overcomes the short trace problems and quantifies the dynamics of transcription initiation. Based on live imaging data, we identify signatures of bursty transcription initiation from the hunchback promoter. We show that the precision of the expression of the hunchback gene to measure its position along the anterior-posterior axis is low both at the boundary and in the anterior even at cycle 13, suggesting additional post-transcriptional averaging mechanisms to provide the precision observed in fixed embryos., Competing Interests: The authors have declared that no competing interests exist.

The diffusion of macromolecules in cells and in complex fluids is often found to deviate from simple Fickian diffusion. One explanation offered for this behavior is that molecular crowding renders diffusion anomalous, where the mean-squared displacement of the particles scales as 〈r(2)〉∝t(α) with α < 1. Unfortunately, methods such as fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) probe diffusion only over a narrow range of lengthscales and cannot directly test the dependence of the mean-squared displacement (MSD) on time. Here we show that variable-lengthscale FCS (VLS-FCS), where the volume of observation is varied over several orders of magnitude, combined with a numerical inversion procedure of the correlation data, allows retrieving the MSD for up to five decades in time, bridging the gap between diffusion experiments performed at different lengthscales. In addition, we show that VLS-FCS provides a way to assess whether the propagator associated with the diffusion is Gaussian or non-Gaussian. We used VLS-FCS to investigate two systems where anomalous diffusion had been previously reported. In the case of dense cross-linked agarose gels, the measured MSD confirmed that the diffusion of small beads was anomalous at short lengthscales, with a cross-over to simple diffusion around ≈1 μm, consistent with a caged diffusion process. On the other hand, for solutions crowded with marginally entangled dextran molecules, we uncovered an apparent discrepancy between the MSD, found to be linear, and the propagators at short lengthscales, found to be non-Gaussian. These contradicting features call to mind the "anomalous, yet Brownian" diffusion observed in several biological systems, and the recently proposed "diffusing diffusivity" model.

Despite the fact that Candida albicans is an important human fungal pathogen and Dectin-2 is a major pattern recognition receptor for fungi, our knowledge regarding the role of Dectin-2 for the host defense against disseminated candidiasis is limited. Dectin-2 deficient (Dectin-2(-/-)) mice were more susceptible to systemic candidiasis, and the susceptibility was mirrored by an elevated fungal load in the kidneys that correlated with the presence of large inflammatory foci. Phagocytosis of Candida by the macrophages lacking the Dectin-2 receptor was moderately decreased, while production of most of the macrophage-derived cytokines from Dectin-2(-/-) mice with systemic candidiasis was decreased. No striking differences among several Candida mutants defective in mannans could be detected between naïve wild-type and Dectin-2(-/-) mice, apart from the β-mannan-deficient bmt1Δ/bmt2Δ/bmt5Δ triple mutant, suggesting that β-mannan may partially mask α-mannan detection, which is the major fungal structure recognized by Dectin-2. Deciphering the mechanisms responsible for host defense against the majority of C. albicans strains represents an important step in understanding the pathophysiology of systemic candidiasis, which might lead to the development of novel immunotherapeutic strategies.

We describe for the first time the chemical synthesis of a tetramannoside, containing both α (1→2) and β (1→2) linkages. Dodecylthio (lauryl) glycosides were prepared from odorless dodecyl thiol and used as donors for the glycosylation steps. This tetramannoside, was coupled to a mantyl group, and revealed to be a perfect substrate of β-mannosyltransferase Bmt3, confirming the proposed specificity and allowing the preparation of a pentamannoside sequence (β Man (1,2) β Man (1,2) α Man (1,2) α Man (1,2) α Man) usable as a novel substrate for further elongation studies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

β-1,2-Linked mannosides are expressed on numerous cell-wall glycoconjugates of the opportunistic pathogen yeast Candida albicans. Several studies evidenced their implication in the host-pathogen interaction and virulence mechanisms. In the present study, we characterized the in vitro activity of CaBmt3, a β-1,2-mannosyltransferase involved in the elongation of β-1,2-oligomannosides oligomers onto the cell-wall polymannosylated N-glycans. A recombinant soluble enzyme Bmt3p was produced in Pichia pastoris and its enzyme activity was investigated using natural and synthetic oligomannosides as potential acceptor substrates. Bmt3p was shown to exhibit an exquisite enzymatic specificity by adding a single terminal β-mannosyl residue to α-1,2-linked oligomannosides capped by a Manβ1-2Man motif. Furthermore, we demonstrated that the previously identified CaBmt1 and CaBmt3 efficiently act together to generate Manβ1-2Manβ1-2[Manα1-2]n sequence from α-1,2-linked oligomannosides onto exogenous and endogenous substrates., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Diffusion in lipid membranes is an essential component of many cellular process and fluorescence a method of choice to study membrane dynamics. The goal of this work was to directly compare two common fluorescence methods, line-scanning fluorescence correlation spectroscopy and single-particle tracking, to observe the diffusion of a fluorescent lipophilic dye, DiD, in a complex five-component mitochondria-like solid-supported lipid bilayer. We measured diffusion coefficients of DFCS ~ 3 um2 * s-1 and DSPT ~ 2 um2 * s-1, respectively. These comparable, yet statistically different values are used to highlight the main message of the paper, namely that the two considered methods give access to distinctly different dynamic ranges: D sup or approximatively 1um2 * s-1 for FCS and D inf or approximatively 5 um2 s-1 for SPT (with standard imaging conditions). In the context of membrane diffusion, this means that FCS allows studying lipid diffusion in fluid membranes, as well as the diffusion of loosely-bound proteins hovering above the membrane. SPT, on the other hand, is ideal to study the motions of membrane-inserted proteins, especially those presenting different conformations, but only allows studying lipid diffusion in relatively viscous membranes, such as supported lipid bilayers and cell membranes.

Cellular membranes have a complex phospholipid composition that varies greatly depending on the organism, cell type and function. In spite of this complexity, most structural data available for phospholipid bilayers concern model systems containing only one or two different phospholipids. Here, we examine the effect of cholesterol on the structure of a complex membrane reflecting the lipid composition of mitochondrial membranes, with five different types of headgroups (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL)) and a variety of hydrocarbon tails. This particular system was chosen because elevated cholesterol contents in mitochondrial membranes have been linked to a breaking down of Bax-mediated membrane permeabilization and resistance to cancer treatments. High resolution electron density profiles were determined by X-ray reflectivity, while the area per phospholipid chain, Apc, and the chain order parameter, SX-ray, were determined by wide-angle X-ray scattering (WAXS). We show that chain order increases upon the addition of cholesterol, resulting in both a thickening of the lipid bilayer and a reduction in the average surface area per phospholipid chain. This effect, well known as cholesterol's condensation effect, is similar, but not as pronounced as for single-component phospholipid membranes. We conclude by discussing the relevance of these findings for the insertion of the pro-apoptotic protein Bax in mitochondrial membranes with elevated cholesterol content.

β-1,2-mannosylation of Candida albicans glycoconjugates has been investigated through the identification of enzymes involved in the addition of β-1,2-oligomannosides (β-Mans) to phosphopeptidomannan and phospholipomannan. β-1,2-oligomannosides are supposed to have virulence properties that they confer to these glycoconjugates. In a previous study, we showed that cell wall mannoproteins (CWMPs) harbor β-Mans in their O-mannosides; therefore, we analyzed their biosynthesis and impact on virulence. In this study, we demonstrate that O-mannans are heterogeneous and that α-mannosylated O-mannosides, which are biosynthesized by Mnt1 and Mnt2 α-1,2-mannosyltransferases, can be modified with β-Mans but only at the nonreducing end of α-1,2-mannotriose. β-1,2-mannosylation of this O-mannotriose depends on growth conditions, and it involves 2 β-1,2-mannosyltransferases, Bmt1 and Bmt3. These Bmts are essential for β-1,2-mannosylation of CWMPs and expression of β-Mans on germ tubes. A bmt1Δ mutant and a mutant expressing no β-Mans unexpectedly disseminated more in BALB/c mice, whereas they had neither attenuated nor enhanced virulence in C57BL/6 mice. In galectin (Gal)3 knockout mice, the reference strain was more virulent than in C57BL/6 mice, suggesting that the β-Mans innate receptor Gal3 is involved in C. albicans fitness during infection.

After exposure to stressful stimuli, apoptotic signals can be relayed to mitochondria by pro-apoptotic activator proteins, tBid (truncated Bid/p15) and Bim (Bcl-2 interacting mediator), which activate Bax (Bcl-2 associated X protein) and or Bak (Bcl-2 antagonist/killer) to induce mitochondrial outer membrane (MOM) permeabilization (MOMP). These protein-protein and protein-membrane interactions are critical for apoptosis regulation, since MOMP irreversibly leads to cell death. Whereas the distinct roles of tBid and Bim as sensors of different types of stress are well recognized, it is not known whether the molecular mechanisms whereby they initiate MOMP are the same. In the present study, we compare membrane permeabilization by Bax activated by either cBid [cleaved Bid (p7 and p15)] or Bim and we examine the role of membrane lipids in the recruitment and activation of these three Bcl-2 (B-cell lymphoma 2) pro-apoptotic proteins. We employ fluorescently-labelled proteins and liposomes to quantify the effects of specific lipids on each of the well-characterized steps in Bax-mediated membrane permeabilization. We show that high levels of cholesterol in the membrane inhibit permeabilization by categorically identifying the recruitment of Bax by the activators and Bax insertion in the membrane as the steps being hindered by cholesterol. Furthermore, we show that binding of both cBid and Bim to membranes is facilitated by electrostatic interactions with anionic phospholipids. However, whereas Bim does not require any particular anionic lipids, the conformational change in tBid depends on cardiolipin (CL). This suggests that CL can activate tBid in a similar manner to Mtch2 (mitochondrial carrier homologue 2). Thus, lipids modify multiple aspects of Bax-mediated membrane permeabilization.

Objective: The protein Hwp1, expressed on the pathogenic phase of Candida albicans, presents sequence analogy with the gluten protein gliadin and is also a substrate for transglutaminase. This had led to the suggestion that C. albicans infection (CI) may be a triggering factor for Celiac disease (CeD) onset. We investigated cross-immune reactivity between CeD and CI., Methods: Serum IgG levels against recombinant Hwp1 and serological markers of CeD were measured in 87 CeD patients, 41 CI patients, and 98 healthy controls (HC). IgA and IgG were also measured in 20 individuals from each of these groups using microchips sensitized with 38 peptides designed from the N-terminal of Hwp1., Results: CI and CeD patients had higher levels of anti-Hwp1 (p=0.0005 and p=0.004) and anti-gliadin (p=0.002 and p=0.0009) antibodies than HC but there was no significant difference between CeD and CI patients. CeD and CI patients had higher levels of anti-transglutaminase IgA than HC (p=0.0001 and p=0.0039). During CI, the increase in anti-Hwp1 paralleled the increase in anti-gliadin antibodies. Microchip analysis showed that CeD patients were more reactive against some Hwp1 peptides than CI patients, and that some deamidated peptides were more reactive than their native analogs. Binding of IgG from CeD patients to Hwp1 peptides was inhibited by γIII gliadin peptides., Conclusions: Humoral cross-reactivity between Hwp1 and gliadin was observed during CeD and CI. Increased reactivity to Hwp1 deamidated peptide suggests that transglutaminase is involved in this interplay. These results support the hypothesis that CI may trigger CeD onset in genetically-susceptible individuals.

Fungal cell walls contain several types of glycans, which play important roles in the pathogenesis of fungal infection and host immune response. Among them, glycosphingolipids have attracted much attention lately since they contribute actively to the fungi development and fungal-induced pathogenesis. Although glycosphingolipids are present in pathogenic and non-pathogenic fungi, pathogenic strains exhibit distinct glycan structures on their sphingolipids, which contribute to the regulatory processes engaged in inflammatory response. In Candida albicans, phospholipomannan (PLM) represents a prototype of these sphingolipids. Through its glycan and lipid moieties, PLM induces activation of host signaling pathways involved in the initial recognition of fungi, causing immune system disorder and persistent fungal disease. In this review, first we describe the general aspects of C. albicans sphingolipids synthesis with a special emphasize on PLM synthesis and its insertion into the cell wall. Then, we discuss the role of PLM glycosylation in regulating immune system activation and its contribution to the chronic persistent inflammation found in Candida infections and chronic inflammatory diseases.

The resolution of stimulated emission depletion (STED) microscopes is ultimately limited by the quality of the doughnut-shaped illumination profile of the STED erase beam. We show here that in the focal plane this illumination profile is well approximated by an analytical expression - a difference of Gaussian functions, which tends towards a first order Laguerre-Gaussian profile in the case of a well aligned beam with a true zero-intensity central minimum. We further show that along the optical axis the maximum intensity profile is reasonably approximated by a Gaussian decay away from the focal plane. The result is a fully Gaussian analytical approximation of the three-dimensional point-spread function of STED erase beams. This allows the derivation of an analytical form for the autocorrelation function of the fluorescence generated by fluorophore diffusion through the STED depletion volume. We verified this form to be correct by performing fluorescence correlation spectroscopy (FCS) experiments in solutions of the dye Alexa Fluor 532. Since the quality of the illumination profile is reflected in the shape of the autocorrelation function, we propose that fluctuation analysis can be used as a tool to assess the quality of STED erase beams.

Glycosylation is a crucial step in the modification of proteins or sphingolipids that then play a prominent role in fungal biology. Glycosylation controls the structure and plasticity of the fungal cell wall and fungi-host interactions. Non-pathogenic and pathogenic yeasts, such as Saccharomyces cerevisiae and Candida albicans, respectively, have been useful models for analyzing the mannosylation of proteins and sphingolipids, which mainly takes place in the Golgi apparatus. Studies of these yeasts have identified different mannosyltransferases that belong to separate families of glycosyltransferases. The characterization of mannosyltransferases and their activities is essential for deciphering cell wall biogenesis, for identifying mannosides involved in virulence and for designing inhibitors that target specific mannosylation processes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)