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Researchers showed that microplastics from our waste are dispersed in water by proteins adsorbed to their surface. This discovery opens the way to low-tech strategies for collecting them. Plastic waste scattered in nature breaks up into invisible particles of micrometric or nanometric size. Because scientists do not yet know their physical and chemical properties and their biological interactions, they cannot fully assess the environmental impact of this diffuse pollution. In nature, microplastics quickly become coated with molecules of biological origin. How do these biomolecules modify their behaviour? To find out, research teams from CEA, IMMM, UPC, and Soleil Synchrotron in France, have studied microplastics in water to understand the role of the protein corona on their stability. The most persistent microplastics in the environment, polyethylene and polypropylene (5 and 10 µm in diameter), were chosen for this study. In the absence of proteins, buoyancy and capillary forces tend to gather the microplastics towards the surface ('creaming') and very few particles remain in solution. In the presence of proteins, hydrophobic microplastics become hydrophilic and can diffuse in water. In addition, this protein corona prevents agglomeration and stabilises microplastics suspensions in water. Taking advantage of this effect, size fractions of stabilized microplastics could be prepared for toxicological screening using a protein corona formed with a well-known protein, serum albumin. Alternatively, it is also possible to destabilise the protein corona surrounding the microplastics, either by adding a salt, which induces protein precipitation, or by heating, which results in protein aggregation. In both cases, 95% of the microparticles were removed from water by creaming after 24 hours. Heat treatment could be used to purify water to make it drinkable, where industrial depollution facilities are lacking

Meningioma is the most common primary intracranial extra-axial tumor. Total surgical removal is the standard therapeutic method to treat this type of brain tumors. However, the risk of recurrence depends on the tumor grade and the extent of the resection including the infiltrated dura mater and, if necessary, the infiltrated bone. Therefore, proper resection of all invasive tumor borders without touching eloquent areas is of primordial in order to decrease the risk of recurrence. Nowadays, none of the intraoperative used tools is able to provide a precise real-time histopathological information on the tumor surrounding areas to help the surgeon to achieve a gross total removal. To respond to this problem, our team is developing a multimodal two-photon fluorescence endomicroscope, compatible with the surgeon tool, to better delimitate tumor boundaries, relying on the endogenous fluorescence of brain tissues. In this context, we are building a tissue database in order to specify each brain tissue, whether healthy or tumoral, with its specific optical signature. In this study, we present a multimodal and multiscale optical measurements on non-tumoral control brain tissue obtained in epilepsy surgery patients and several meningioma grades. We investigated tissue auto-fluorescence to track the molecular changes associated with the tumor grade from deep ultra-violet (DUV) to near infrared (NIR) excitation. Micro-spectroscopy, fluorescence lifetime imaging, two-photon fluorescence imaging and Second Harmonic Generation (SHG) imaging were performed. Several optically derived parameters such as collagen crosslinks fluorescence in DUV, SHG emission in NIR and long lifetime intensity fraction of Nicotinamide Adenine Dinucleotide and Flavins were correlated to discriminate cancerous tissue from control one. While collagen response managed to discriminate meningioma grades from control samples with a 100% sensitivity and 90% specificity through a 3D discriminative algorithm.

International audience; Fourier transform infrared (FTIR) spectroscopy has proven to be a non-invasive tool to analyse cells without the hurdle of employing exogenous dyes or probes. Nevertheless, the study of single live bacteria in their aqueous environment has long remained a big challenge, due to the strong infrared absorption of water and the small size of bacteria compared to the micron-range infrared wavelengths of the probing photons. To record infrared spectra of bacteria in an aqueous environment, at different spatial resolutions, two setups were developed. A custom-built attenuated total reflection inverted microscope was coupled to a synchrotron-based FTIR spectrometer, using a germanium hemisphere. With such a setup, a projected spot size of 1 × 1 μm2 was achieved, which allowed spectral acquisition at the single-cell level in the 1800–1300 cm−1 region. The second setup used a demountable liquid micro-chamber with a thermal source-powered FTIR microscope, in transmission geometry, for probing clusters of a few thousands of live cells in the mid-IR region (4000–975 cm−1). Both setups were applied for studying two strains of a model lactic acid bacterium exhibiting different cryo-resistances. The two approaches allowed the discrimination of both strains and revealed population heterogeneity among bacteria at different spatial resolutions. The multivariate analysis of spectra indicated that the cryo-sensitive cells presented the highest cell heterogeneity and the highest content of proteins with the α-helix structure. Furthermore, the results from clusters of bacterial cells evidenced phosphate and peptidoglycan vibrational bands associated with the cell envelope, as potential markers of resistance to environmental conditions.

Flax fibres have been used by humans for approximately 10,000 years. With time, the geographic area of production and cultivation has changed, as have the applications of flax fibres; from clothing to sails and paintings from antiquity, to automotive, fashion, and design applications in the contemporary era. The degradation process of flax fibres is the same for both ancient and modern objects made from this polysaccharidic material. This review, focusing on the cultural heritage field, after a brief description of flax plants and fibres, retraces the history of their use through Europe and the Near East, and discusses the evolution of extraction methods with human progress. Furthermore, the most important mechanisms of flax fibre degradation and the characterisation techniques currently in use are described. This study highlights the constructive interchange between engineering and cultural heritage that can be realised through a continuous comparison of antiquity and the contemporary era.

International audience; Flax (Linum usitatissimum) lodging is an issue of great interest for industrial producers due to its economic impact; despite a strong varietal selection over around one century, this plant remains sensitive to lodging which represents a main technico-economic issue. To better understand lodging effects at the cell wall and stem scale, the cell wall composition dynamics during cell wall development and after a 90 degrees tilt bending stress is reported. Deep-UltraViolet (DUV) fluorescence emission dynamics recorded at the Synchrotron SOLEIL-DISCO beamline by multichannel autofluorescence imaging is addressed for five cellular wall types of flax stems after an artificially induced gravitropic reaction. The quantitative fluorescent profile intensities were computed after image analysis, and compared to the control flax stems, we reported a systematically higher average intensity fluorescence (probability >95%) for the 90 degrees tilted plants. Moreover, the average stem fluorescence intensities were significantly different among the 3 developmental stages, with the youngest stage (VS) exhibiting on average 30% and 20% less fluorescence than the medium (FG) and mature (M) stages, respectively. The flax stem response to tilt impacted the xylem cellular type, while the bast fibres were arguably less affected by the protein, and hydroxycinnamate contents. A complementary investigation was carried out on bast fibres by infrared microspectroscopy to explore the polysaccharide components not detected in DUV fluorescence, and significant modifications were monitored.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the gene encoding dystrophin. It leads to repeated cycles of muscle fiber necrosis and regeneration and progressive replacement of fibers by fibrotic and adipose tissue, with consequent muscle weakness and premature death. Fibrosis and, in particular, collagen accumulation are important pathological features of dystrophic muscle. A better understanding of the development of fibrosis is crucial to enable better management of DMD. Three-dimensional (3D) characterization of collagen organization by second harmonic generation (SHG) microscopy has already proven a highly informative means of studying the fibrotic network in tissue.Here, we combine for the first-time tissue clearing with SHG microscopy to characterize in depth the 3D cardiac fibrosis network from DMDThe current work highlights the interest to combine multiphoton SHG microscopy and tissue clearing for 3D fibrosis network characterization in label free organ. It could be a relevant tool to characterize the fibrotic tissue remodeling in relation to the disease progression and/or to evaluate the efficacy of therapeutic strategies in preclinical studies in DMD model or others fibrosis-related cardiomyopathies diseases.

International audience; Flax has a long and fascinating history. This plant was domesticated around 8,000 bce1 in the Fertile Crescent area2, first for its seeds and then for its fibres1,3. Although its uses existed long before domestication, residues of flax yarn dated 30,000 years ago have been found in the Caucasus area4. However, Ancient Egypt laid the foundations for the cultivation of flax as a textile fibre crop5. Today flax fibres are used in high-value textiles and in natural actuators6 or reinforcements in composite materials7. Flax is therefore a bridge between ages and civilizations. For several decades, the development of non- or micro-destructive analysis techniques has led to numerous works on the conservation of ancient textiles. Non-destructive methods, such as optical microscopy8 or vibrational techniques9,10, have been largely used to investigate archaeological textiles, principally to evaluate their degradation mechanisms and state of conservation. Vibrational spectroscopy studies can now benefit from synchrotron radiation11 and X-ray diffraction measurement in the archaeometric study of historical textiles12,13. Conservation of mechanical performance and the ultrastructural differences between ancient and modern flax varieties have not been examined thus far. Here we examine the morphological, ultrastructural and mechanical characteristics of a yarn from an Egyptian mortuary linen dating from the early Middle Kingdom (Eleventh Dynasty, ca. 2033–1963 bce) and compare them with a modern flax yarn to assess the quality and durability of ancient flax fibres and relate these to their processing methods. Advanced microscopy techniques, such as nano-tomography, multiphoton excitation microscopy and atomic force microscopy were used. Our findings reveal the cultural know-how of this ancient civilization in producing high-fineness fibres, as well as the exceptional durability of flax, which is sometimes questioned, demonstrating their potential as reinforcements in high-technology composites.

International audience; PLA-flax non-woven composites are promising materials, coupling high performance and possible degradation at their end of life. To explore their ageing mechanisms during garden composting, microstructural investigations were carried out through scanning electron microscopy (SEM) and atomic force microscopy (AFM). We observe that flax fibres preferentially degrade ‘inwards’ from the edge to the core of the composite. In addition, progressive erosion of the cell walls occurs within the fibres themselves, ‘outwards’ from the central lumen to the periphery primary wall. This preferential degradation is reflected in the decrease in indentation modulus from around 23 GPa for fibres located in the preserved core of the composite to 3–4 GPa for the remaining outer-most cell wall crowns located at the edge of the sample that is in contact with the compost. Ageing of the PLA matrix is less drastic with a relatively stable indentation modulus. Nevertheless, a change in the PLA morphology, a significant decrease in its roughness and increase of porosity, can be observed towards the edge of the sample, in comparison to the core. This work highlights the important role of intrinsic fibre porosity, called lumen, which is suspected to be a major variable of the compost ageing process, providing pathways of entry for moisture and microorganisms that are involved in cell wall degradation.

International audience; Currently, the effects of global warming are one of the most important topics on the agendas of all governments and international economic and scientific organisations on the planet. Temperatures and rainfall will be especially subjected to increasing deregulation, and thus crop yields will be affected according to geographic location. Cellulosic materials, such as bast fibres, are considered one solution to decrease human environmental impact: they are a renewable resource, biodegradable and have a lower carbon emission than synthetic materials. However, their quality, yield and mechanical properties depend on environmental conditions during plant growth. In this paper, we explored the possible impact of seasonal drought linked to future climate change on flax plants and fibre quality. Two batches of the same textile flax plant cultivar were grown under two different field environmental conditions in the same year, one taken as a control under regular climatic conditions and the second one grown under drought stress. Carbon isotopic discrimination reveal an increase in water stress plant of the fibre δ13C, reflecting that plants are indeed suffering from drought stress from a physiological point of view. We characterized the mechanical properties, biochemistry and morphology parameters at both the stem and technical fibre scales. Our results showed that the plants of the two batches were morphologically different and that the drought-stressed plants were smaller, mainly in terms of the height of the stem (−28%) and diameter (−16%). Biochemical analyses highlighted a contrasting lignin content between the two batches. A difference in protein content was also measured, with an increased amount in stressed flax plants, with contrasting distributions revealed by tyrosine and tryptophan monitored by synchrotron UV fluorescence. In addition, polysaccharide composition was also quantified with an increase in mannose and an important decrease in glucose in the drought-stressed technical fibres. Surprisingly, despite the difference in biochemistry composition and morphological parameters, the mechanical properties of elementary flax fibres extracted from the two batches were not significantly different. This suggests that drought can affect the yield and biochemistry of the extracted technical flax fibres but does not necessarily impact the longitudinal mechanical performance of single fibres.

Flax lodging is an issue of great interest for producers due to its economic impact. To better understand its effects at the cell wall and stem scale, new knowledge regarding the cell wall composition dynamics during cell wall development and after a 90° tilt bending stress is reported. Deep-Ultra Violet fluorescence emission (DUV) dynamics recorded at the Synchrotron SOLEIL-DISCO beamline by multichannel autofluorescence imaging is reported for five cellular wall types of flax stems after an artificially induced gravitropic reaction. Three flax growth development stages, namely, the vegetative stage (VS), the fast growth (FG) and the mature stage (MS), were selected in normal plants, referred to as the control plants, or in gravitropic-induced response plants, referred to as 90° tilted plants.

International audience; A variety of techniques were used to investigate flax yarns sampled from four selected Italian paintings on canvas dated between the 17th and 18th centuries and compare them with a modern flax yarn. The goal was to establish their state of preservation and highlight the critical issues thanks to a combined approach that used SEM, atomic force microscopy, nanoindentation, nuclear magnetic resonance, second harmonic generation imaging microscopy and FTIR spectroscopy. The condition of the flax fibres was assessed, and the results showed that two of the four canvases were in a generally good state of preservation, while the other two exhibited signs of biological attack and brittleness due to a previous relining intervention.

International audience; This Letter reports the first characterization of nonfluorescent halloysite nanotubes (HNTs) by multiphoton microscopy via their endogenously generated second-harmonic (SH) resonance. The unveiled discovery that HNTs generate a SH signal in the nanoscale regime is supported by studies of the signal's propagation, polarization, and coherence properties and, finally, is directed toward the understanding of the origin of signal's generation from the nanoparticle's aluminosilicate skeleton. The study is significant, since it sheds light on halloysite nanotube technological applications in various domains, including alternative bioimaging, biosensing, and noninvasive biomedicine, as well as possibly nonlinear spectroscopy, optoelectronics, and many others.

International audience; Over the last decades, bio-based composite materials have been developed as an ecological alternative to synthetic fibre-reinforced composites, and flax fibres are one of the most commonly used fibres for this purpose. The secondary cell wall (S2) and the microfibril angle (MFA) of plant fibres are the main factors responsible for the mechanical behaviour of the fibres and, consequently, for the properties of the final biocomposite material. However, the MFA values reported in the literature are obtained through heavy, time-consuming methods and often without resolution at the scale of the elementary fibres. In the present paper, for the first time, the MFAs of elementary flax fibres are measured with the alternative method of second-harmonic generation imaging under controlled polarised light (P-SHG); cotton trichomes are also investigated as a homogeneous and well-known cellulose fibre with expected contrasted MFAs compared to flax. To estimate the MFA, we analysed the images collected that clearly show the microfibrils. The values found are in line with the literature data obtained with conventional techniques. However, new important details of the microfibrils orientation of elementary flax fibres and cotton trichomes are highlighted, such as inhomogeneities in a single flax fibre, leading to MFAs varying between 0 and 10 degrees along the fibre with an average value around 5 degrees. The results obtained give an important contribution to the knowledge of the plant fibre ultrastructure, giving some structural details never provided with measurements of fibre bundles.

Rrp44 is a conserved eukaryotic protein showing endonuclease and exoribonuclease activity that plays essential functions in RNA maturation and degradation. InTrypanosoma brucei, depletion of Rrp44 (TbRrp44) blocks processing of large subunit ribosomal RNA precursors, leading to disruption of ribosome synthesis and inhibition of cell proliferation. We used a combination of molecular and chemical markers to investigate the fate ofT. bruceicells upon knockdown of TbRrp44. In addition, synchrotron deep UV microscopy and cryo soft X-ray tomography were used to investigate cell morphology and ultra-structure modifications. Downregulation of TbRrp44 results in induction of autophagy, inactivation of mitochondria and expansion of acidic and lysosome-derived vacuoles in parallel with enlargement of cell size. Nuclei also increase in size without changes in DNA content. 3D tomographic reconstructions revealed extreme vacuolation of the cytoplasm of TbRrp44 knockdown cells and general alteration of organelles. Calcium-containing vesicles (acidocalcisomes) were identified by X-ray absorption near-edge structure spectra (XANES) of calcium L2,3edges on fully hydrated cells. The volumes of acidocalcisomes and lipid droplet were quantified from 3D reconstructions. Both were found in higher number and with larger volumes in TbRrp44 knockdown cells. These multiple defects indicate that a combination of signals, starting from nucleolar stress and activation of autophagy, converge to induce lysosome expansion. With time, the cytoplasm is taken up by lysosome-derived vacuoles, which may be one of the final stages leading to cell death triggered by TbRrp44 depletion. These studies provide the first evidence on the ultra-structure cell modifications caused by Rrp44 ribonuclease deficiency.Author summaryTrypanosoma bruceiis a parasitic protozoan belonging to the Kinetoplastidae Class, which displays distinct cellular, genomic and molecular features. These features include extra intervening sequences in the large subunit ribosomal RNA (rRNA) precursor that are not found in the homologous rRNA precursor of host cells. Genetic downregulation of theT. bruceiRrp44 (TbRrp44) ribonuclease shows that it is required for accurate excision of these intervening sequences to produce mature rRNA molecules. Here, we have used a multidisciplinary approach based on molecular and chemical markers, synchrotron deep UV microscopy and cryo soft X-ray tomography to show that downregulation of TbRrp44 leads to a series of cellular alterations that eventually result in cell death. Mitochondrial activity is particularly affected in parallel with induction of autophagy response, increase in size of the cell and of organelles such as acidocalcisomes and lipid droplets, and with a massive expansion of lysosome-derived vacuoles. The ultrastructural modifications that take place inT. bruceicells are nicely highlighted in the 3D reconstructions generated using cryo soft X-ray tomography images. This study provides new insights into the multiple cellular consequences of Rrp44 ribonuclease depletion inT. bruceiparasites.

Cryo soft X-ray tomography and deep-ultraviolet transmittance imaging reveal partitioning of neutral lipids in a lipid droplet core. Furthermore, deep-ultraviolet transmittance and tryptophan/tyrosine auto-fluorescence multimodal imaging on living cells were combined to obtain complementary information on organelle chemical contents., A lipid droplet (LD) core of a cell consists mainly of neutral lipids, tri­­acyl­glycerols and/or steryl esters (SEs). The structuration of these lipids inside the core is still under debate. Lipid segregation inside LDs has been observed but is sometimes suggested to be an artefact of LD isolation and chemical fixation. LD imaging in their native state and in unaltered cellular environments appears essential to overcome these possible technical pitfalls. Here, imaging techniques for ultrastructural study of native LDs in cellulo are provided and it is shown that LDs are organized structures. Cryo soft X-ray tomography and deep-ultraviolet (DUV) transmittance imaging are showing a partitioning of SEs at the periphery of the LD core. Furthermore, DUV transmittance and tryptophan/tyrosine auto-fluorescence imaging on living cells are combined to obtain complementary information on cell chemical contents. This multimodal approach paves the way for a new label-free organelle imaging technique in living cells.

Various studies have shown that food structure has an impact on digestion kinetics. We focus here on the effects of gastric and intestinal enzymes (in-vitro digestion) on two canola seed storage proteins, napin and cruciferin. To monitor structure effect we conducted experiments on gels of these proteins at different pHs, yielding different structures and elastic modulus. What is new is to get information on the mechanisms at the lowest scales, using imaging and radiation scattering at large facilities: Synchrotron fluorescence microscopy, X-Ray scattering, at SOLEIL synchrotron, and Small-Angle Neutron Scattering, at Laboratoire Leon Brillouin reactor. We can identify the mechanisms at each step and in two distinct scale ranges, observed simultaneously, the one of the individual protein scale and the one of the structure connectivity: • during gelation individual canola proteins are not deeply modified in comparison with their state in solution ; larger scale gel heterogeneity appears due to connectivity or aggregation • in the gastric step (up to 40 min): ○ at short scale (large q) we see that the proteins disintegration is much slowed down in gels than in solutions, particularly in the gastric phase; ○ at larger scales (low q), we see that the gel structure is also self-resistant to the action of the enzyme (pepsin). • in the intestinal step, such kinetics differences hold until major disintegration after no more than 15 min.

17 pages; International audience; Molluscs, the largest marine phylum, display extraordinary shell diversity and sophisticated biomineral architectures. However, mineral-associated biomolecules involved in biomineralization are still poorly characterised.We report the first comprehensive structural and biomolecular study of Spondylus gaederopus, a pectinoid bivalve with a peculiar shell texture. Used since prehistoric times, this is the best-known shell of Europe’s cultural heritage. We find that Spondylus microstructure is very poor in mineral-bound organics, which are mostly intercrystalline and concentrated at the interface between structural layers.Using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) we characterized several shell protein fractions, isolated following different bleaching treatments. Several peptides were identified as well as six shell proteins, which display features and domains typically found in biomineralized tissues, including the prevalence of intrinsically disordered regions. It is very likely that these sequences only partially represent the full proteome of Spondylus, considering the lack of genomics data for this genus and the fact that most of the reconstructed peptides do not match with any known shell proteins, representing consequently lineage-specific sequences.This work sheds light onto the shell matrix involved in the biomineralization in spondylids. Our proteomics data suggest that Spondylus has evolved a shell-forming toolkit, distinct from that of other better studied pectinoids – fine-tuned to produce shell structures with high mechanical properties, while limited in organic content. This study therefore represents an important milestone for future studies on biomineralized skeletons and provides the first reference dataset for forthcoming molecular studies of Spondylus archaeological artifacts.

International audience; Starch granules can be extruded to obtain a thermoplastic material. Thermoplastic starch (TPS) usually requires a significant break down of the starch granular organization to form a continuous polysaccharide matrix. In this work, we extrude potato starch with and without a plasticizer and store samples at high humidity to generate recrystallization. A multi-scale investigation of the microstructure is performed by combining different techniques: WAXS and solid-state NMR to describe macromolecule organization and Second Harmonic Generation (SHG) imaging to provide spatial information. Finally, the ability of the material to swell and remain sound in water is assessed. Glycerol-plasticized samples swell the least despite many granules with native-like structure embedded in the starch matrix. Glycerol limits the fragmentation and melting of the granules and crystallites during extrusion but also reduces the proportion of starch molecules in constrained conformations, enabling the formation of a polymer network that can sustain the penetration of water.

Pompe disease is a lysosomal storage disorder caused by acid-α-glucosidase (GAA) deficiency, leading to glycogen storage. The disease manifests as a fatal cardiomyopathy in infantile form. Enzyme replacement therapy (ERT) has recently prolonged the lifespan of these patients, revealing a new natural history. The neurologic phenotype and the persistence of selective muscular weakness in some patients could be attributed to the central nervous system (CNS) storage uncorrected by ERT. GAA-KO 6neo/6neo mice were treated with a single intrathecal administration of adeno-associated recombinant vector (AAV) mediated gene transfer of human GAA at 1 month and their neurologic, neuromuscular, and cardiac function was assessed for 1 year. We demonstrate a significant functional neurologic correction in treated animals from 4 months onward, a neuromuscular improvement from 9 months onward, and a correction of the hypertrophic cardiomyopathy at 12 months. The regions most affected by the disease i.e. the brainstem, spinal cord, and the left cardiac ventricular wall all show enzymatic, biochemical and histological correction. Muscle glycogen storage is not affected by the treatment, thus suggesting that the restoration of muscle functionality is directly related to the CNS correction. This unprecedented global and long-term CNS and cardiac cure offer new perspectives for the management of patients. Electronic supplementary material The online version of this article (doi:10.1186/s40478-017-0464-2) contains supplementary material, which is available to authorized users.

International audience; PB1-F2 is a non-structural protein of influenza A viruses (IAV) that modulates viral pathogenesis in a host-specific manner. In mammals, this protein has been shown to increase IAV virulence by delaying the early immune response and, eventually, exacerbating lung inflammation at the late stage of infection. PB1-F2 is a small protein, but displays very high sequence polymorphism and sequence length disparity depending on viral strain. These features result in strong variations in the cellular activity of PB1-F2. Studies have also reported that the effect of PB1-F2 is cell-type dependent. It has notably been shown that PB1-F2 can promote apoptosis in immune cells, but not in epithelial cells. This phenomenon appears to be partly related to the higher order structure of the protein, given that the presence of PB1-F2 β-aggregated structures in infected immune cells correlates with cell death induction. In this work, we evaluated, by synchrotron Fourier transform infrared spectromicroscopy, the impact of the transient expression of PB1-F2 on the biochemical composition of the human epithelial cell line HEK293T. Two PB1-F2 variants that are closely related to each other but derived from a strain with high [A/BrevigMission/1/1918 (H1N1)] or a low [A/WSN/1933 (H1N1)] virulence were studied here. Infrared spectra analysis revealed no specific enrichment of β-aggregated structures in PB1-F2-expressing cells. Nevertheless, this analysis suggested that there is a higher content of β-sheet secondary structures in the PB1-F2 from A/WSN/1933 than that from A/BrevigMission/1/1918. Our data also showed no change in membrane composition in the presence of PB1-F2, implying that PB1-F2 does not promote apoptosis in HEK293T cells. Finally, we found that the PB1-F2 from A/WSN/1933 interferes with adenosine triphosphate production, suggesting that this PB1-F2 variant may disturb the mitochondrial activity.

Ultraviolet (UV) synchrotron radiation circular dichroism (SRCD) spectroscopy has made an important contribution to the determination and understanding of the structure of bio-molecules. In this paper, we report an innovative approach that we term time-resolved SRCD (tr-SRCD), which overcomes the limitations of current broadband UV SRCD setups. This technique allows accessing ultrafast time scales (down to nanoseconds), previously measurable only by other methods, such as infrared (IR), nuclear magnetic resonance (NMR), fluorescence and absorbance spectroscopies and small angle X-ray scattering (SAXS). The tr-SRCD setup takes advantage of the natural polarisation of the synchrotron radiation emitted by a bending magnet to record broadband UV CD faster than any current SRCD setup, improving the acquisition speed from 10 mHz to 130 Hz and the accessible temporal resolution by several orders of magnitude. We illustrate the new approach by following the isomers concentration changes of an azopeptide after a photoisomerisation. This breakthrough in SRCD spectroscopy opens up a wide range of potential applications to the detailed characterisation of biological processes, such as protein folding, protein-ligand binding.

International audience; Ultraviolet (UV) synchrotron radiation circular dichroism (SRCD) spectroscopy has made an important contribution to the determination and understanding of the structure of bio-molecules. In this paper, we report an innovative approach that we term time-resolved SRCD (tr-SRCD), which overcomes the limitations of current broadband UV SRCD setups. This technique allows accessing ultrafast time scales (down to nanoseconds), previously measurable only by other methods, such as infrared (IR), nuclear magnetic resonance (NMR), fluorescence and absorbance spectroscopies and small angle X-ray scattering (SAXS). The tr-SRCD setup takes advantage of the natural polarisation of the synchrotron radiation emitted by a bending magnet to record broadband UV CD faster than any current SRCD setup, improving the acquisition speed from 10 mHz to 130 Hz and the accessible temporal resolution by several orders of magnitude. We illustrate the new approach by following the isomers concentration changes of an azopeptide after a photoisomerisation. This breakthrough in SRCD spectroscopy opens up a wide range of potential applications to the detailed characterisation of biological processes, such as protein folding, protein-ligand binding.

Among all the tumors of the central nervous system (CNS), glioma are the most deadly and the most malignant. Surgical resection is the standard therapeutic method to treat this type of brain cancer. But the diffusive character of these tumors create many problems for surgeons during the operation. In fact, these tumors migrate outside the tumor solid zone and invade the surrounding healthy tissues. These infiltrative tissues have the same visual appearance as healthy tissues, making it very difficult for surgeons to distinguish the healthy ones from the diffused ones. The surgeon, therefore, cannot properly remove the tumor margins increasing the recurrence risk of the tumor. To resolve this problem, our team has developed a multimodal two-photon fibered endomicroscope, compatible with the surgeon trocar, to better delimitate tumor boundaries by relying on the endogenous fluorescence of brain tissues. In this context, and in order to characterize the optical signature of glioma tumors, this study offers multimodal and multi-scaled optical measurements from healthy tissues to high grade glioma. We can interrogate tissue from deep ultra-violet to near infrared excitation by working with spectroscopy, fluorescence lifetime imaging, two-photon fluorescene imaging and Second Harmonic Generation (SHG) imaging. Optically derived ratios such as the Tryptophan/Collagen ratio, the optical redox ratio and the long lifetime intensity fraction, discriminated diseased tissue from its normal counterparts when fitted by Gaussian ellipsoids and choosing a threshold for each. Additionally two-photon fluorescence and SHG images were shown to display similar histological features as Hematoxylin-Eosin stained images.

Phenolic compounds in fruit are involved in responses to biotic and abiotic stresses and are responsible for organoleptic properties. To establish the distribution of these secondary metabolites at the tissue and sub-cellular scales, mapping of fluorescence in apple epidermis and outer cortex tissue in cryogenic condition was performed after deep-UV excitation at 275 nm. Douce Moen and Guillevic cider apple varieties were sampled and frozen after harvest, after 30 days at 4 °C and after 20 days at room temperature. Image analysis of fluorescence emission images acquired between 300 and 650 nm allowed the assignment of fluorescence signals to phenolic compound families based on reference molecules. Emission attributed to monomeric and/or condensed flavanol was localized in whole tissue with major fluorescence in the cuticle region. Hydroxycinnamic acids were found predominantly in the outer cortex and appeared in the cell wall. Fluorescent pigments were mostly found in the epidermis. The distribution of flavanols in the sub-cuticle and phenolic acids in the outer cortex distinguished apple varieties. Storage conditions had no impact on phenolic distribution. The proposed fluorescent imaging and analysis approach enables studies on phenolic distribution in relation to fruit development, biotic/abiotic stress resistance and quality.

International audience; Among Central Nervous System (CNS) tumors, diffuse glioma are the most infiltrating and malignant tumors. According to the World Health Organization (WHO), they are classified into different grades, referring to their pathological class and histological properties. To treat glioma tumors, many methods have been proposed, still the standard one remains the maximal safe total resection. During the operation, many difficulties obstruct the surgeon to identify the infiltrated areas, which contains diffuse tumors cells, around the solid area of the tumor, overlapping the healthy areas, and presenting the same visual appearances. If not totally removed, these infiltrating zones can increase the risk of recurrence and affects the survival rate of patient. To overcome this problem, we develop a multimodal two-photon endomicroscope, based on the endogenous fluorescence of brain tissues, to assist the surgeon during the surgery. The tool will provide him information on the infiltrated areas and their histological nature. In this paper, we tried to discriminate between metastasis, low grade and high grade glioma from healthy fresh tissues, presenting a multimodal study using deep ultraviolet, visible and near infrared excitation to acquire spectral measurements, Fluorescence Lifetime Imaging (FLIM) and Two-Photon Emission Fluorescence (TPEF) imaging. We compared also our TPEF and FLIM images to the histological images.

Pompe disease (glycogen storage disease type II) is a lysosomal storage disorder due to a mutation in the gene that encodes acid alpha-glucosidase (GAA). GAA deficiency causes the excessive storage of lysosomal glycogen in many cell types, leading to cell and, subsequently, tissue dysfunction. Cardiac, respiratory and skeletal muscles are the most severely affected. Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA, Myozyme®, Genzyme, Cambridge) is the only approved treatment for Pompe disease. A new therapeutic strategy was developed consisting of delivering adeno-associated virus serotype 9 (AAV9) and serotype 10 (AAV10) vectors expressing human GAA into cerebrospinal fluid of GAA-KO 6neo/6neo Pompe mice. The purpose of this work was to investigate synchrotron Fourier transform infrared (sFT-IR) and deep ultraviolet (sDUV) microspectroscopies to detect new biomarkers of the disease and the AAV gene therapy in the cardiac muscle, one of the most affected organs in Pompe disease. Multivariate statistics applied to sFT-IR spectra between 4000 cm–1 and 950 cm–1 highlighted the potential of sFT-IR to discriminate Pompe (-/-), Wild type and AAV-treated animals from C–H stretching vibrations of CH3, from C–O, C–N and C–C stretching vibrations of amide I, II, III bands and from specific IR signature of the glycogen. Investigations performed by sDUV microscopy showed a significant increase of the tryptophan autofluorescent signal in the right ventricle for the AAV9-treated Pompe mice. The high-resolution sDUV microspectroscopy experiments suggested a correlation between the tryptophan-rich area and the GAA-rich area. These unprecedented results demonstrate that high-resolution UV microspectroscopy can be a complementary innovative approach to monitor the chemical change in label-free cardiac muscle section. Moreover, this non-destructive technology can be applied to a small amount of tissue allowing therapeutic assessment from biopsy of human patients.

International audience; In their native state, milk lipids are present in the form of dispersed droplets called Milk Fat Globules (MFG, diameter 0.1-20 μm, average = 4 μm in bovine milk) [1,2]. The native MFG is constituted by a triglyceride core covered with an external trilayered membrane inherited from its secretory past. This membrane is mainly based on polar lipids (glycerophospholipids, sphingolipids and glycosphingolipids), proteins (25 % of total membrane with high proportion of glycoproteins and enzymes), neutral lipids and minor components. The major biological function of the MFG is to deliver energy to the mammal newborn, for that, the MFG has to be hydrolyzed in the gastro-intestinal tract by lipases, in successive steps. The lipases should interact with the MFG membrane and diffuse into the supramolecular object. The aim of this work was to determine the mechanisms involved in the enzymatic hydrolysis along the digestive process. We used millifluidic cells designed to trap MFG or model droplets and injected successively each lipase (gastric, pancreatic related 2, pancreatic triglyceride lipase) involved in the digestion process and thus mimicking different physiological stages (preterm infant, fullterm infant and adult). Thanks to the deep UV-microscope present at DISCO beamline, the autofluorescence of tryptophan (and tyrosine) of lipase aminoacids allowed protein observation without external labeling. The gastric and intestinal neonatal disintegration of milk fat globules (human or bovine) or other model systems (bovine anhydrous milk fat droplets stabilized by bovine milk membrane extracts or soy lecithin) was approached through transmission images recording. The analysis of the number of globules and their state of aggregation/coalescence during digestion (approached via diameter, size, mode and specific surface evolution against time) gave indication on the kinetics and mechanisms of lipolysis. Fluorescence (327-335 nm) image indicated lipase distribution. Gastric phase was mainly marked by the heterogeneous adsorption of the gastric lipase in the MFG membrane. Afterwards, heterogeneous adsorption of pancreatic triglyceride lipase could also be observed followed by the disappearance of the MFG core. The progression of lipases fronts was monitored and the kinetics of lipolysis characterized allowing the differentiation of substrate and of in vitro digestion models (full term infant, versus preterm infant versus adult). The microfluidics set up was a very interesting tool to follow sequentially the action of lipases, to save volume of purified lipase and to image simultaneously several positions. The high sensitivity of the DISCO beamline allowing the detection of digestive lipases auto-fluorescence, is a unique tool to unravel their mechanisms of adsorption onto complex substrate using free-label imaging and has generated useful data for infant formula optimization

Fibres from annual plants are a sustainable alternative to glass fibres in composite manufacturing. However, both synthetic fibres, such as carbon fibres, and cellulosic fibres exhibit heterogeneities along their lengths, which appear as localised morphological misorientations known as kink-bands. In plant fibres, kink-bands occur due to growing conditions under abiotic stress during the retting stage and the fibre extraction process. Many studies have been conducted to identify the origin of such kink-bands and their impact on the mechanical behaviour of composites, but their characteristics and fine ultrastructure remain unclear. The presence of cavities in transition zones was assessed by scanning electron microscopy (SEM) and atomic force microscopy (AFM) and confirmed by low-intensity second harmonic generation microscopy (SHG) signals, especially when large kink-bands were considered. Moreover, the transverse indentation modulus was obtained by AFM in peak force mode; no substantial differences were observed between the kink-band and defect-free regions, with average values ranging from 6.2–7.3 GPa. Additionally, important MFA changes were measured through SHG imaging, especially in large kink-bands with local misorientation up to 47°. Thus, this in-depth investigation of kink-band areas reveals ultrastructure heterogeneities and the presence of local defects.

Abiotic hydrocarbons and carboxylic acids are known to be formed on Earth, notably during the hydrothermal alteration of mantle rocks. Although the abiotic formation of amino acids has been predicted both from experimental studies and thermodynamic calculations, its occurrence has not been demonstrated in terrestrial settings. Here, using a multimodal approach that combines high-resolution imaging techniques, we obtain evidence for the occurrence of aromatic amino acids formed abiotically and subsequently preserved at depth beneath the Atlantis Massif (Mid-Atlantic Ridge). These aromatic amino acids may have been formed through Friedel–Crafts reactions catalysed by an iron-rich saponite clay during a late alteration stage of the massif serpentinites. Demonstrating the potential of fluid-rock interactions in the oceanic lithosphere to generate amino acids abiotically gives credence to the hydrothermal theory for the origin of life, and may shed light on ancient metabolisms and the functioning of the present-day deep biosphere. High-resolution imaging techniques show that aromatic amino acids such as tryptophan formed abiotically and were subsequently preserved at depth beneath the Atlantis Massif of the Mid-Atlantic Ridge, supporting the hydrothermal theory for the origin of life.

Finding traces of life or organic components of prebiotic interest in the rock record is an appealing goal for numerous fields in Earth and space sciences. However, this is often hampered by the scarceness and highly heterogeneous distribution of organic compounds within rocks. We assess here an innovative analytical strategy combining Synchrotron radiation-based Fourier-Transform Infrared microspectroscopy (S-FTIR) and multivariate analysis techniques to track and characterize organic compounds at the pore level in complex oceanic rocks. S-FTIR hyperspectral images are analysed individually or as multiple image combinations (multiset analysis) using Principal Component Analyses (PCA) and Multivariate Curve Resolution – Alternating Least Squares (MCR-ALS). This approach allows extracting simultaneously pure organic and mineral spectral signatures and determining their spatial distributions and relationships. MCR-ALS analysis provides resolved S-FTIR signatures of 8 pure mineral and organic components showing the close association at a micrometric scale of organic compounds and secondary clays formed during rock alteration and known to catalyse organic synthesis. These results highlights the potential of the serpentinizing oceanic lithosphere to generate and preserve organic compounds of abiotic origin, in favour of the hydrothermal theory for the origin of life.

Cryopreservation-related stresses in Lactobacillus delbrueckii SUBSP. Bulgaricus: Global and multi-scale study. 55. Annual Meeting of the Society for Cryobiology

Titanium dioxide (TiO2) is commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, a risk of intestinal barrier disruption, including dysbiosis of the gut microbiota, is increasingly suspected because of the presence of a nano-sized fraction in this additive. We hypothesized that food-grade E171 and Aeroxyde P25 (identical to the NM-105 OECD reference nanomaterial in the European Union Joint Research Centre) interact with both commensal intestinal bacteria and transient food-borne bacteria under non-UV-irradiated conditions. Based on differences in their physicochemical properties, we expect a difference in their respective effects. To test these hypotheses, we chose a panel of eight Gram-positive/Gram-negative bacterial strains, isolated from different biotopes and belonging to the species Escherichia coli, Lactobacillus rhamnosus, Lactococcus lactis (subsp. lactis and cremoris), Streptococcus thermophilus, and Lactobacillus sakei. Bacterial cells were exposed to food-grade E171 vs. P25 in vitro and the interactions were explored with innovative (nano)imaging methods. The ability of bacteria to trap TiO2 was demonstrated using synchrotron UV fluorescence imaging with single cell resolution. Subsequent alterations in the growth profiles were shown, notably for the transient food-borne L. lactis and the commensal intestinal E. coli in contact with food-grade TiO2. However, for both species, the reduction in cell cultivability remained moderate, and the morphological and ultrastructural damages, observed with electron microscopy, were restricted to a small number of cells. E. coli exposed to food-grade TiO2 showed some internalization of TiO2 (7% of cells), observed with high-resolution nano-secondary ion mass spectrometry (Nano-SIMS) chemical imaging. Taken together, these data show that E171 may be trapped by commensal and transient food-borne bacteria within the gut. In return, it may induce some physiological alterations in the most sensitive species, with a putative impact on gut microbiota composition and functioning, especially after chronic exposure.

International audience

Agricultural resources give us food but also potential sources of feedstocks for the chemical industry. As demand from the growing human population rises, the food industry and the chemical industry face similar problems of scaling operations while sourcing the largest possible amount of at least reasonable-quality raw materials. Food is composed of complex structures formed from molecular assemblies ( e.g. particles, fibres, crystals) whose properties depend in part on the molecular species present. In this context, investigations are needed to better understand raw material structure and structure transformation mechanisms in order to improve manufacturing processes and the properties of the final product ( e.g. food), which means dedicated methodologies need to be developed. This review presents case studies illustrating advanced technologies designed for characterizing biopolymers, supramolecular complexes, cell membranes, enzymatic degradation of food matrices and biopolymers, flavor release dynamics during eating, cerebral multisensory integration of food and eating behavior.