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Generative machine learning models such as Generative Adversarial Networks (GANs) have been shown to be especially successful in generating realistic synthetic data in image and tabular domains. However, it has been shown that such generative models, as well as the generated synthetic data, can reveal information contained in their privacy-sensitive training data, and therefore must be carefully evaluated before being used. The gold standard method through which such privacy leakage can be estimated is simulating membership inference attacks (MIAs), in which an attacker attempts to learn whether a given sample was part of the training data of a generative model. The state-of-the art MIAs against generative models, however, rely on strong assumptions (knowledge of the exact training dataset size), or require a lot of computational power (to retrain many "surrogate" generative models), which make them hard to use in practice. In this work, we propose a technique for evaluating privacy risks in GANs which exploits the outputs of the discriminator part of the standard GAN architecture. We evaluate our attacks in terms of performance in two synthetic image generation applications in radiology and ophthalmology, showing that our technique provides a more complete picture of the threats by performing worst-case privacy risk estimation and by identifying attacks with higher precision than the prior work.

The first case of hereditary fibrinogen Aα-chain amyloidosis was recognized >20 years ago, but disease mechanisms still remain unknown. Here we report detailed clinical and proteomics studies of a French kindred with a novel amyloidogenic fibrinogen Aα-chain frameshift variant, Phe521Leufs, causing a severe familial form of renal amyloidosis. Next, we focused our investigations to elucidate the molecular basis that render this Aα-chain variant amyloidogenic. We show that a 49-mer peptide derived from the C-terminal part of the Phe521Leufs chain is deposited as fibrils in the patient's kidneys, establishing that only a small portion of Phe521Leufs directly contributes to amyloid formation in vivo. In silico analysis indicated that this 49-mer Aα-chain peptide contained a motif (VLITL), with a high intrinsic propensity for β-aggregation at residues 44 to 48 of human renal fibrils. To experimentally verify the amyloid propensity of VLITL, we generated synthetic Phe521Leufs-derived peptides and compared their capacity for fibril formation in vitro with that of their VLITL-deleted counterparts. We show that VLITL forms typical amyloid fibrils in vitro and is a major signal for cross-β-sheet self-association of the 49-mer Phe521Leufs peptide identified in vivo, whereas its absence abrogates fibril formation. This study provides compelling evidence that VLITL confers amyloidogenic properties to Aα-chain frameshift variants, yielding a previously unknown molecular basis for the pathogenesis of Aα-chain amyloidosis., (© 2017 by The American Society of Hematology.)

Elastic properties of cells are mainly derived from the actin cytoskeleton. However, intermediate filaments are emerging as major contributors to the mechanical properties of cells. Using atomic force microscopy, we studied the elasticity of mouse myoblasts expressing a mutant form of the gene encoding for desmin intermediate filaments, p.D399Y. This variant produces desmin aggregates, the main pathological symptom of myofibrillar myopathies. Here we show that desmin-mutated cells display a 39% increased median elastic modulus compared to wild-type cells. Desmin-mutated cells required higher forces than wild-type cells to reach high indentation depths, where desmin intermediate filaments are typically located. In addition, heat-shock treatment increased the proportion of cells with aggregates and induced a secondary peak in the distribution of Young's moduli. By performing atomic force microscopy mechanical mapping combined with fluorescence microscopy, we show that higher Young's moduli were measured where desmin aggregates were located, indicating that desmin aggregates are rigid. Therefore, we provide evidence that p.D399Y stiffens mouse myoblasts. Based on these results, we suggest that p.D399Y-related myofibrillar myopathy is at least partly due to altered mechanical properties at the single-cell scale, which are propagated to the tissue scale., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Mutations within the human desmin gene are responsible for a subcategory of myofibrillar myopathies called desminopathies. However, a single inherited mutation can produce different phenotypes within a family, suggesting that environmental factors influence disease states. Although several mouse models have been used to investigate organ-specific desminopathies, a more general mechanistic perspective is required to advance our knowledge toward patient treatment. To improve our understanding of disease pathology, we have developed cellular models to observe desmin behaviour in early stages of disease pathology, e.g., upon formation of cytoplasmic desmin aggregates, within an isogenic background. We cloned the wildtype and three mutant desmin cDNAs using a Tet-On Advanced® expression system in C2C12 cells. Mutations were selected based on positioning within desmin and capacity to form aggregates in transient experiments, as follows: DesS46Y (head domain; low aggregation), DesD399Y (central rod domain; high aggregation), and DesS460I (tail domain; moderate aggregation). Introduction of these proteins into a C2C12 background permitted us to compare between desmin variants as well as to determine the role of external stress on aggregation. Three different types of stress, likely encountered during muscle activity, were introduced to the cell models-thermal (heat shock), redox-associated (H2O2 and cadmium chloride), and mechanical (stretching) stresses-after which aggregation was measured. Cells containing variant DesD399Y were more sensitive to stress, leading to marked cytoplasmic perinuclear aggregations. We then evaluated the capacity of biochemical compounds to prevent this aggregation, applying dexamethasone (an inducer of heat shock proteins), fisetin or N-acetyl-L-cysteine (antioxidants) before stress induction. Interestingly, N-acetyl-L-cysteine pre-treatment prevented DesD399Y aggregation during most stress. N-acetyl-L-cysteine has recently been described as a promising antioxidant in myopathies linked to selenoprotein N or ryanodin receptor defects. Our findings indicate that this drug warrants further study in animal models to speed its potential development as a therapy for DesD399Y-linked desminopathies.

Amyloidoses are increasingly recognized as a major public health concern in Western countries. All amyloidoses share common morphological, structural, and tinctorial properties. These consist of staining by specific dyes, a fibrillar aspect in electron microscopy and a typical cross-β folding in x-ray diffraction patterns. Most studies that aim at deciphering the amyloid structure rely on fibers generated in vitro or extracted from tissues using protocols that may modify their intrinsic structure. Therefore, the fine details of the in situ architecture of the deposits remain unknown. Here, we present to our knowledge the first data obtained on ex vivo human renal tissue sections using x-ray microdiffraction. The typical cross-β features from fixed paraffin-embedded samples are similar to those formed in vitro or extracted from tissues. Moreover, the fiber orientation maps obtained across glomerular sections reveal an intrinsic texture that is correlated with the glomerulus morphology. These results are of the highest importance to understanding the formation of amyloid deposits and are thus expected to trigger new incentives for tissue investigation. Moreover, the access to intrinsic structural parameters such as fiber size and orientation using synchrotron x-ray microdiffraction, could provide valuable information concerning in situ mechanisms and deposit formation with potential benefits for diagnostic and therapeutic purposes., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A characteristic feature of the dense phases formed by fiber-shaped molecules is their organization into parallel rods packed in a hexagonal or pseudo-hexagonal lateral network. This is typically the case for the collagen triple helices inside fibrils, as confirmed by recent X-ray diffraction experiments carried out on highly crystallized fibers obtained by immersing the freshly extracted fibers in a salt-controlled medium. However such diffraction patterns also generally exhibit additional features in the form of diffuse scattering, which is a clear signature of a low degree of lateral ordering. Only few studies have analyzed and modeled the lateral packing of collagen triple helices when the structure is disordered. Some authors have used the concept of short-range order but this approach does not contain any echo of a hexagonal order. In this study, we use an analytical expression derived from the paracrystal model which retains the hexagonal symmetry information and leads to a good agreement with the experimental data in the medium-angle region. This method is quite sensitive to the degree of disorder and to the inter-object distance. One clear result is that the shift in peak positions, generally attributed to variations in intermolecular distances, can also arise from a change in the degree of ordering without any significant modification of the distances. This underlines the importance of evaluating the degree of ordering before attributing a shift in peak position to a change in the unit-cell. This method is generic and can be applied to any system composed of rod-shaped molecules., (Copyright © 2010 Elsevier Inc. All rights reserved.)

X-rays interact strongly with biological organisms. Synchrotron radiation sources deliver very intense X-ray photon fluxes within micro- or submicro cross-section beams, resulting in doses larger than the MGy. The relevance of synchrotron radiation analyses of biological materials is therefore questionable since such doses, million times higher than the ones used in radiotherapy, can cause huge damages in tissues, with regard to not only DNA, but also proteic and lipid organizations. Very few data concerning the effect of very high X-ray doses in tissues are available in the literature. We present here an analysis of the structural phenomena which occur when the model tissue of human hair is irradiated by a synchrotron X-ray micro-beam. The choice of hair is supported by its hierarchical and partially ordered keratin structure which can be analysed inside the tissue by X-ray diffraction. To assess the damages caused by hard X-ray micro-beams (1 microm(2) cross-section), short exposure time scattering SAXS/WAXS patterns have been recorded at beamline ID13 (ESRF) after various irradiation times. Various modifications of the scattering patterns are observed, they provide fine insight of the radiation damages at various hierarchical levels and also unexpectedly provide information about the stability of the various hierarchical structural levels. It appears that the molecular level, i.e. the alpha helices which are stabilized by hydrogen bonds and the alpha-helical coiled coils which are stabilized by hydrophobic interactions, is more sensitive to radiation than the supramolecular architecture of the keratin filament and the filament packing within the keratin associated proteins matrix, which is stabilized by disulphide bonds., ((c) 2009 Elsevier Inc. All rights reserved.)

Several aspects of the intermediate filaments' molecular architecture remain mysterious despite decades of study. The growth process and the final architecture may depend on the physical, chemical, and biochemical environment. Aiming at clarifying this issue, we have revisited the structure of the human hair follicle by means of X-ray microdiffraction. We conclude that the histology-based growth zones along the follicle are correlated to the fine architecture of the filaments deduced from X-ray microdiffraction. Our analysis reveals the existence of two major polymorph intermediate filament architectures. Just above the bulb, the filaments are characterized by a diameter of 100 Angstroms and a low-density core. The following zone upwards is characterized by the lateral aggregation of the filaments into a compact network of filaments, by a contraction of their diameter (to 75 Angstroms) and by the setting up of a long-range longitudinal ordering. In the upper zone, the small structural change associated with the tissue hardening likely concerns the terminal domains. The architecture of the intermediate filament in the upper zones could be specific to hard alpha-keratin whilst the other architecture found in the lower zone could be representative for intermediate filaments in a different environment.

A structural model of the murine PrP small beta-sheet was obtained by synthesizing the RGYMLGSADPNGNQVYYRG peptide comprising the two beta-strands 127-133 and 159-164 linked by a four-residue sequence of high turn propensity. The DPNG turn sequence is a "short circuit" replacing the original protein sequence between the two strands. This 19-residue peptide spontaneously forms very long single fibrils as observed by electron microscopy. The X-ray diffraction patterns of a partially oriented sample reveals an average arrangement of the hairpin peptides into a structure which can be geometrically approximated by an empty-core cylinder. The hairpins are oriented perpendicular to the cylinder axis and a 130 A helix period is observed. Based on X-ray diffraction constraints and on more indirect general protein structure considerations, a precise and consistent fibril model was built. The structure consists of two beta-sheet ribbons wound around a cylinder and assembled into a single fibril with a hairpin orientation perpendicular to the fibril axis. Subsequent implicit and explicit solvent molecular dynamics simulations provided the final structure at atomic resolution and further insights into the stabilizing interactions. Particularly important are the zipper-like network of polar interactions between the edges of the two ribbons, including the partially buried water molecules. The hydrophobic core is not optimally compact explaining the low density of this region seen by X-ray diffraction. The present findings provide also a simple model for further investigating the sequence-stability relationship using a mutational approach with a quasi-independent consideration of the polar and apolar interactions.

Despite investigation since the 1950s, the molecular architecture of intermediate filaments has not yet been fully elucidated. Reliable information about the longitudinal organization of the molecules within the filaments and about the lateral interfilament packing is now available, which is not the case for the transverse architecture. Interesting results were recently obtained from in vitro microscopy observations and cross-linking of keratin, desmin, and vimentin analyses. The structural features that emerge from these analyses could not be fully representative of the in vivo architecture because intermediate filaments are subject to polymorphism. To bring new light to the transverse intermediate filament architecture, we have analyzed the x-ray scattering equatorial profile of human hair. Its comparison with simulated profiles from atomic models of a real sequence has allowed results to be obtained that are representative of hard alpha-keratin intermediate filaments under in vivo conditions. In short, the alpha-helical coiled coils, which are characteristic of the central rod of intermediate filament dimers, are straight and not supercoiled into oligomers; the radial density across the intermediate filament section is fairly uniform; the coiled coils are probably assembled into tetrameric oligomers, and finally the oligomer positions and orientations are not regularly ordered. These features are discussed in terms of filament self-assembling and structural variability.

The [URE3] phenotype in the yeast Saccharomyces cerevisiae is inherited by a prion mechanism involving self-propagating Ure2p aggregates. It is believed that assembly of intact Ure2p into fibrillar polymers that bind Congo Red and show yellow-green birefringence upon staining and are resistant to proteolysis is the consequence of a major change in the conformation of the protein. We recently dissected the assembly process of Ure2p and showed the protein to retain its native alpha-helical structure upon assembly into protein fibrils that are similar to amyloids in that they are straight, bind Congo red and show green-yellow birefringence and have an increased resistance to proteolysis (). Here we further show using specific ligand binding, FTIR spectroscopy and X-ray fiber diffraction that Ure2p fibrils assembled under physiologically relevant conditions are devoid of a cross-beta core. The X-ray fiber diffraction pattern of these fibrils reveals their well-defined axial supramolecular order. By analyzing the effect of heat-treatment on Ure2p fibrils we bring evidences for a large conformational change that occurs within the fibrils with the loss of the ligand binding capacity, decrease of the alpha helicity, the formation of a cross-beta core and the disappearance of the axial supramolecular order. The extent of the conformational change suggests that it is not limited to the N-terminal part of Ure2p polypeptide chain. We show that the heat-treated fibrils that possess a cross-beta core are unable to propagate their structural characteristic while native-like fibrils are. Finally, the potential evolution of native-like fibrils into amyloid fibrils is discussed.

We describe a combined use of experimental and simulation techniques to configure side chains in a coiled coil structure. As already demonstrated in a previous work, x-ray diffraction patterns from hard alpha-keratin fibers in the 5.15 A meridian zone reflect the global configuration of the chi(1) dihedral angle of the coiled coil side chains. Molecular simulations, such as energy minimization and molecular dynamics, and rotameric representation in the PDB, are used here on a heterodimeric coiled coil to investigate the dihedral angle distribution along the sequence. Different procedures have been used to build the structure, the quality assessment was based on the agreement between the simulated diffraction patterns and the experimental ones in the fingerprint region of coiled coils (5.15 A). The best one for building a realistic coiled coil structure consists of placing the side chains using molecular dynamics (MD) simulations, followed by side chain positioning using SMD or SCWRL procedures. The side chains and the backbone are equilibrated during the MD until they reach an equilibrium state for the t/g(+) ratio. Positioning the side chains on the resulting backbone, using the above procedures, gives rise to a well-defined 5.15 A meridian reflection.

Transformations of proteins secondary and tertiary structures are generally studied in globular proteins in solution. In fibrous proteins, such as hard alpha-keratin, that contain long and well-defined double stranded alpha-helical coiled coil domains, such study can be directly done on the native fibrous tissue. In order to assess the structural behavior of the coiled coil domains under an axial mechanical stress, wide angle x-ray scattering and small angle x-ray scattering experiments have been carried out on stretched horse hair fibers at relative humidity around 30%. Our observations of the three major axial spacings as a function of the applied macroscopic strain have shown two rates. Up to 4% macroscopic strain the coiled coils were slightly distorted but retained their overall conformation. Above 4% the proportion of coiled coil domains progressively decreased. The main and new result of our study is the observation of the transition from alpha-helical coiled coils to disordered chains instead of the alpha-helical coiled coil to beta-sheet transition that occurs in wet fibers.

A combined approach, using synchrotron radiation-based diffraction and infrared microspectrometry, has been used to study the structure and molecular composition of hair samples. These methods allowed us to get an insight at different structural scales into the composition and structure of hair. Firstly, information about the configuration of amino-acid residues was obtained at atomic scale, secondly, a model was presented for the geometry and the packing of the microfibrils at medium scale and finally different structural zones were evidenced by microdiffraction at macroscopic scale. We also showed that the two main components of hair--proteins and lipids--are not evenly distributed within the fiber. In addition, these two components exhibit different structure, depending upon their location. Moreover, diffraction and microdiffraction data indicate that the cuticle zone is mainly composed of lipid granules, whereas the cortex and the medulla zones are composed primarily of alpha-keratin. Infrared microspectroscopy, using an enhanced lateral resolution thanks to synchrotron radiation, indicates, on one hand, that the protein structure between the cuticle and cortex are different, and on the other hand, that the concentration of lipids, inside the medulla, is much higher than everywhere else. This work emphasizes the complementarity between both techniques, and highlights the potentialities they can offer in the case of various other studies in biology.

The origin of the 5.15-A meridional reflection on hard alpha-keratin X-ray diffraction patterns is discussed in terms of side-chains conformations. We show it to reveal specific configurations of the side chains which are common to all two-stranded alpha-helical coiled coils. Combining literature data on crystallised coiled coil pieces and molecular dynamics results with our X-ray diffraction pattern simulations, we propose rules for the attribution of chi1 torsion angles for coiled coils involved in fibres whose structure cannot be resolved at atomic resolution: in a (a b c d e f g) heptad repeat, a and d residues, respectively, adopt mean t and g+ configurations, whereas statistical rules are given for the other residues., (Copyright 1999 Academic Press.)

Low-angle X-ray scattering patterns of hard alpha-keratin fibers have been studied for more than 50 years but a completely convincing modelling has never been presented. The models which have been proposed so far are specific to the sample and cannot be adapted to others, mainly because they do not use a parametric analytical expression of the distribution function describing the relative positions of the microfibrils. Our new approach is based on a paracrystal distribution function. In addition, a huge background originating from a non-ordered matrix is taken into account. Various hard alpha-keratins from different origins have been studied using our approach. From the rather good modellings obtained, it appears that the diameter of the microfibril is not origin dependent (7.4 nm) whereas the distances between microfibrils and their electron density profiles are. Hair microfibrils can be reasonably approximated by a solid cylinder but a core and an outer ring are necessary for porcupine. Our method is of course not limited to keratin microfibrils; it can be used for modelling equatorial X-ray scattering profiles of all types of hexagonal fibrillar assemblies, which are in fact widely found in biological tissues.