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Administration of plasma therapy may contribute to viral control and survival of COVID-19 patients receiving B-cell-depleting agents that impair humoral immunity. However, little is known on the impact of anti-CD20 pre-exposition on the kinetics of SARS-CoV-2-specific antibodies. Here, we evaluated the relationship between anti-spike immunoglobulin G (IgG) kinetics and the clinical status or intra-host viral evolution after plasma therapy in 36 eligible hospitalized COVID-19 patients, pre-exposed or not to B-cell-depleting treatments. The majority of anti-CD20 pre-exposed patients (14/17) showed progressive declines of anti-spike IgG titres following plasma therapy, contrasting with the 4/19 patients who had not received B-cell-depleting agents (p = 0.0006). Patients with antibody decay also depicted prolonged clinical symptoms according to the World Health Organization (WHO) severity classification (p = 0.0267) and SARS-CoV-2 viral loads (p = 0.0032) before complete virus clearance. Moreover, they had higher mutation rates than patients able to mount an endogenous humoral response (p = 0.015), including three patients with one to four spike mutations, potentially associated with immune escape. No relevant differences were observed between patients treated with plasma from convalescent and/or mRNA-vaccinated donors. Our study emphasizes the need for an individualized clinical care and follow-up in the management of COVID-19 patients with B-cell lymphopenia.

BackgroundAdministration of plasma therapy may contribute to viral control and survival of COVID-19 patients receiving B-cell depleting agents that hinder the endogenous humoral response. However, little is known on the impact of anti-CD20 pre-exposition and the use of different sources of plasma (convalescent versus vaccinated) on the kinetics of SARS-CoV-2-specific antibodies and viral evolution after plasma therapy.MethodsEligible COVID-19 patients (n = 36), half of them after anti-CD20 targeted therapy, were treated with therapeutic plasma from convalescent (n = 17) or mRNA-vaccinated (n = 19) donors. Each plasma-transfused patient was thoroughly monitored over time by anti-S IgG quantification and whole-genome SARS-CoV-2 sequencing.ResultsThe majority of anti-CD20 pre-exposed patients (15/18) showed progressive declines of anti-S protein IgG titers following plasma therapy, indicating that they mostly relied on the passive transfer of anti-SARS-CoV-2 antibodies. Such antibody kinetics correlated with prolonged infection before virus clearance, contrasting with the endogenous humoral response predominantly present in patients who had not received B-cell depleting agents (15/18). No relevant differences were observed between patients treated with plasma from convalescent and/or vaccinated donors. Finally, 4/30 genotyped patients showed increased intra-host viral evolution and 3/30 included 1 to 4 spike mutations, potentially associated to immune escape.ConclusionsConvalescent and/or vaccinated plasma therapy may provide anti-SARS-CoV-2 antibodies and clinical benefit to B-cell depleted COVID-19 patients. Only a limited number of patients acquired viral mutations prior to clinical recovery, yet our study further emphasizes the need for long-term surveillance for intra-host variant evolution, to guide best therapeutic strategies.

In the Mount-Lebanon, the agricultural territory of Aaqoura is significant despite high elevations reaching 2100 m. In the 1960’s, apple orchards were subsidized by a state-owned service of planning: the Green Plan, but the increasing urbanization in Lebanon requires nowadays a solid knowledge of the remaining and not yet developed lands for agricultural extension. The challenge of this study is dual: environmental and socio-economic. Two models of potential site identification and location of new cultivable lands and new hill lakes have been developed in this study. These models are based on pedologic, topographic, geologic criteria and simultaneously integrate socio-economic surveys with farmers in order to assess the convenience of such new infrastructures and to strengthen its relevance in this region. The results of these models lead to two maps that locate sites with high potential for the implementation of new infrastructure, but its analysis must be completed through survey’s results. After field verifications of these maps, 14% of the area is still available for new agricultural lands and only 4% for hill lakes on account of steep slopes, permeability of soil, and farmers stressed out economic challenges, such as the lack of profitable markets, which undermines the local agricultural growth. However, farmers are interested in new hill lakes for the purpose of satisfying orchard high water needs. This integrated approach of rural development, appeared as an interesting tool for planning office such as the Green Plan as much as for farmers since they are the first users of these infrastructures.

Convalescent plasma to treat coronavirus disease 2019 (COVID-19) is currently the focus of numerous clinical trials worldwide, but the criteria of treatment efficacy remain largely unknown. Here, we describe a severely immunosuppressed patient following rituximab and chemotherapy for chronic lymphoid leukemia, who became infected by SARS-CoV-2. His prolonged viral disease was successfully cured after four cycles of convalescent plasma. Its immunomodulatory properties led to the rapid improvement of inflammation, pneumonia and blood cell counts, already after the first cycle. Importantly, the cumulative increase in anti-SARS-CoV-2 neutralizing antibodies following each plasma transfusion was associated to progressive viral clearance, resulting in clinical recovery from infection. Our data provide insight into the different modes of action of plasma components. Understanding the underlying mechanisms will help to optimize the treatment of COVID-19 patients.

This paper is a first attempt to compute the total water needs of an agricultural plain with remote sensing and ground data in Iran. The cropping areas were mapped with Sentinels-2 images, based on NDVI profiles classification. This model was validated and 85% of the areas were correctly classified. Second, the crop water needs were computed using PYSEBAL and Landsat-8 images. Crop evapotranspiration (ETseason) and Irrigation Requirements (IRseason) were calculated for each crop and then validated by comparing IR collected in the field from farmers with computed IRPYSEBAL on 5 plots. IRPYSEBAL underestimated the reality with an average of 10% while the overestimation average was 17%. The second validation was the comparison of Daily ET from FAO-56 method and Daily ET PYSEBAL showed a RMSE of 0.67 mm/day and MAE of 0.52 mm/day, which assesses the accuracy of PYSEBAL. ETseason varies according to weather parameters in the plain and IRseason, according to different irrigation practices. The most water demanding crops were identified: rice (IR: 1427 mm) and corn (669). The total water balance of Marvdasht was negative in 2018 with 0.2859 km3 of extracted groundwater for irrigation for only 0.098 km3 of available water for aquifers recharge.

In this work, we have considered a new multimodality imaging for macroscopy based on Second Harmonic Generation (SHG) method to monitor invasivelessly the matrix collagen. As the triple helicoidally structure of collagen molecules appearing as not centrosymetric, very organized and spatially oriented, collagen fibrils give rise to a very strong SHG signal and can be imaged without any exogenous dye. To integrate a multidimensional scale with a large field of view (non-sliced samples), we have adapted and validated an instrumental coupling between a two photon excitation laser and a macroscope to collect cartography of SHG signal. We introduced an index (F-SHG) based on decay time response measured by TCSPC for respectively Fluorescence (F) and Second Harmonic Generation (SHG) values. For various sample where protein collagen is the major component of extracellular matrix (vessel, skin, carotide vessel, rat femoral head cartilage, mouse tumor, human wharton’s jelly and rat tendon) or not (nacre), we compared the index distribution obtained with MacroSHG. In this work, we showed for the first time that multiscale large field imaging (Macroscopy) combined to Multimodality approaches (SHG-TCSPC) could be an innovative and non-invasive technique to detect and identify some biological interest molecules (collagen) in biomedical topics.

For this study, we have considered a new large field of view imaging dedicated to matrix collagen (no stained samples). To integrate a multidimensional scale (non-sliced samples), a femtosecond oscillator (two photon excitation laser) has been coupled with a large field optical setup to collect SHG signal. We introduced an index (F-SHG) based on decay time response measured by TCSPC for, respectively, Fluorescence (F) and Second Harmonic Generation (SHG) values. For samples where protein collagen is the major component of extracellular matrix (skin) or not (nacre), we compared the index distribution (from 2 to 12) obtained with large field optical setup. In this work, we showed for the first time that multiscale large field imaging combined to multimodality approaches (SHG-TCSPC) could be an innovative and non invasive technique to detect and identify some biological interest molecules (collagen) in biomedical topics.

L'inondation de 1859 marque la dernière grande crue d'inondation de l'Isère. La tranquillité de l'Isère au cours du XXe siècle est-elle définitive, est-elle liée à une modification climatique? Pour répondre à ces questions, l'étude vise dans un premier temps à cerner les particularités climatiques de ces événements hydrologiques. Dans un second temps, afin d'évaluer le caractère plus ou moins exceptionnel de ces conditions climatiques, les valeurs annuelles maximales des précipitations cumulées, d'une journée à six journées consécutives, ont été ajustées à la loi de Gumbel. La démarche a ensuite été reconduite avec les précipitations journalières couplées à une hausse des températures. Il apparaît d'une manière assez nette que ces crues font suite à des conditions climatiques véritablement exceptionnelles; un épisode pluvieux durable qui se combine avec une phase de redoux. Aussi, l'absence de ces grandes crues au cours XXe siècle n'est pas étonnante sur un plan statistique, et il ne faudrait peut-être pas y voir la marque d'un changement climatique régional ou planétaire., The great 1859 flood at Grenoble in the southeastern France was the last water-level rise of such magnitude for the Isère River, and Grenoble has not been flooded again since that time. However, one can legitimately wonder whether the calm behavior of the Isère during the 20th century was something definitive or whether it was related to climate change. The present study aimed at answering this question, firstly by determining the climatic characteristics of the hydrological episodes in question, while, secondly, using Gumbel's Law to adjust the maximum annual values of accumulated precipitation over a period lasting from one to six consecutive days, in order to assess how exceptional such climatic conditions actually are. The procedure was then repeated by linking daily precipitation to rises in temperature. It is clearly shown that flood episodes follow truly exceptional climatic conditions. Thus, the absence of such severe floods during the 20th century is not statistically surprising, and perhaps should not be seen as a sign of regional or planetary climate change.

To mask the antigenic sites of cells for cell therapies, especially for blood transfusion, we investigated the hemocompatibility of two poly(2-(dimethylamino)ethyl methacrylate-co-poly(ethyleneglycol) compared with that of the homopolymer without PEG. Our strategy relies on the potential ability of these copolymers to self-assemble at the erythrocyte surface. The cationic sequence of the copolymer should be able to interact with the glycocalyx by ionic interaction. The other sequence, based on a polyethyleneglycol moiety, should prevent both nonspecific interactions and specific recognition of the biological surface. The hemocompatibility of these copolymers was assessed by analyzing alterations in human erythrocyte membrane viscoelasticity, morphology, granularity, and aggregation. Their properties to mask ABO system and three erythrocyte glycophorin sites were investigated. No alterations in the erythrocyte morphology were observed by confocal microscopy. On the other hand, a partial masking of different specific glycophorin sites leads to future optimization of the macromolecular structures of these functionalized copolymers.

This study investigates erythromycin toxicity toward activated sludge as a function of exposure time and antibiotic concentration. Batch experiments were conducted and microscopic techniques ranging from bright-field microscopy to epifluorescence and confocal laser scanning microscopy (CLSM), combined with a fluorescent viability indicator ( BacLight ® Bacterial Viability Kit, Molecular Probes), allowed us to study erythromycin time-kill activity. The erythromycin toxicity was observed at lower concentration when exposure time increased. A 4 μg/L erythromycin concentration was toxic to heterotrophic bacteria on a 5-day time exposure, and a 5 mg/L concentration inhibited nitrification. These findings are in agreement with the microscopic studies, which showed a latency time before the lower antibiotic concentrations began to kill bacteria. Microscope slide wells were used as micro-reactors in which erythromycin concentration ranged from 0.1 to 1 mg/L. After 45 min there were 94% (SD 3.8) of living bacteria in control micro-reactors, 67% (SD 3.1) in micro-reactors that contained 0.1 mg/L erythromycin and 37% (SD 18.6) in micro-reactors that contained 1 mg/L erythromycin. CLSM allowed visualization of isolated stained cells in the three-dimensional structure of damaged flocs.

International audience; Osteoarthritis is a degenerative pathology leading to degradation of the extracellular matrix (ECM). Similar effects can be visualized when applying mechanical or biochemical constraints on cartilaginous tissue. Here, we characterized modification of the ECM appearing under mechanical compression and/or biochemical action (hypoxia environment, nitric oxide and collagenase action). In recent decades, multiphoton microscopy has proved its interest for observing living, thick and opaque biological tissues. Thus, the main components of the cartilaginous ECM can be observed without fluorescent labeling. In particular, the collagen network emits strong second harmonic generation (SHG) signal which could be collected at half of the excitation wavelength. Combining autofluorescence and SHG signal detection enables to obtain complementary structural information. Here, we proved that multiphoton microscopy represents an appropriate tool for ex vitro cartilage imaging. First, we showed that SHG signal specifically comes from collagen (collagenase digestion). Further, we verified that the use of an appropriate band-pass filter enables to reject the autofluorescence from the ECM. Once this specificity was shown, we followed modification of the cartilage ECM submitted to mechanical or biochemical constraints (compression, enzymatic digestion). By performing textural analysis of SHG images (Haralick's method), we showed the restructuration of the collagen network according to constraints.

This study presents the measurement of streamflows by constant dilution of a yellow-orange food colorant (E110). Field measurements are carried out in the Doria River, a mountain torrent in the Savoie pre-Alps, France. A limnimetric station installed 12 years ago is periodically calibrated by mechanical and chemical measurements. Moreover, simultaneous mechanical and chemical methods allow the statistical validation of the proposed technique. The main advantages of this method are the lack of toxicity, the absence of visible river colouring, comparable to that of aquatic organic matter, and the low detection limit. This method allows high flow measurement under suitable conditions.

International audience; Gap junctions play an important role in vital functions, including the regulation of cell growth and cell differentiation. Connexins 43 (Cx43) are the most widely expressed gap junction proteins. Cellular localization of phosphorylated Cx43 has been implicated in the capacity of gap junctional intercellular communication (GJIC). To follow the functionality of GJIC of different cell types, in monolayer cultures, characterized by different patterns of phosphorylated Cx43, we used a fluorescence recovery after photobleaching (FRAP) technique, and compared two tracers, 5(6)-carboxyfluorescein diacetate (CFDA) and calcein acetoxymethylester (AM). The GJIC capacity was quantified by estimating fluorescence redistribution parameters. The functionality of GJIC was in relation with the staining localization of phosphorylated Cx43 to the cell-cell contact areas, corresponding to gap junctions between contacting cells. GJIC involvement in fluorescence restitution after photobleaching was checked by a gap junction channel inhibition assay. We demonstrated that the choice of the dye did not significantly influence the fluorescence recovery percentages despite a cell line-dependent CFDA release, whereas it had an important impact on fluorescence kinetic profiles. This study reinforces the interest of the gap-FRAP approach to quantify modifications in the functionality of gap junctions and, above all, argues about the limits of CFDA for 3-D future approaches.

Several strategies have been proposed to improve the efficiency of photosensitizers used in photodynamic therapy (PDT). In this context, the synthesis of mono- (1) and di-glucosylated (2) porphyrins, and mono-glucosylated chlorin (3) was performed. HT29 human adenocarcinoma cells were significantly more sensitive to asymmetric and less hydrophobic glucosylated photosensitizers-mediated PDT (1, 3), compared to tetraphenylporphyrin (TPP). The lowest photosensitivity observed for TPP was consistent with the lowest uptake. Moreover, the most pronounced photodynamic activity measured for 3 was in relation with the improvement of cellular uptake, the singlet oxygen quantum yield and the high extinction coefficient value at 650 nm compared to porphyrins. Cellular localization analysis showed that 1 and 3 accumulated mainly inside the endoplasmic reticulum.

International audience; The seeding of endothelial cells (ECs) on biomaterial surfaces became a major challenge, allowing to improve the non-thrombogenic properties of these surfaces. Recently, the use of polyelectrolyte films has been suggested as a new versatile technique of surface modification aimed at tissue engineering. In this study, we evaluate the adhesion properties of ECs on two types of polyelectrolyte films ending either by poly(D-lysine) (PDL), or poly(allylamine hydrochloride) (PAH), and compared them to data obtained on PDL or PAH monolayers, glass and fibronectin (Fn)-coated glass. ECs seeded on polyelectrolyte films showed a good morphology, allowing ECs to resist physiological shear stress better compared to ECs seeded on glass or Fn. The expression of beta1 integrins was slightly lower on polyelectrolyte films than on control surfaces. However, the phosphorylation of focal adhesion kinase, involved in the transduction of adhesion signal, was not modified on PAH ending films compared to control surfaces; whereas it became lower on PDL ending films. Finally, PAH ending films improve strongly ECs adhesion without disturbing the adhesion mechanism, necessary for the development of a new endothelium. These types of films or similar build-ups could thus be used in the future as a way to modify surfaces for vascular tissue engineering.

Optimization of suspended matter-fluxes quantification of an Alpine river: the Isère at Grenoble (France). This study seeks to determine suspended-matter flux of the Isère at Grenoble, one of the most important rivers of the northern French Alps. Since 1994, and especially 1996, systematic samplings of left bank make it possible to follow in one point the variations of the suspended particles and matter (SPM) of this river. But from a single measurement, sometimes not very representative of the whole observable concentrations within the section, it remains still difficult to estimate precisely the SPM flux. The results of this study show in particular the successive appearance of several turbidity gradients, whose occurrence is explained mainly by the hydrological and hydro-sedimentary dynamics of this river. The estimate of these gradients thus makes it possible to extrapolate specific measurements more easily and consequently to daily define the quantities of suspended matter exported by the Isère. Since 1996, the results show a very high flow specific of Isère, at Grenoble, variable between 124 and more than 460 t km-2 yr-1., Cette étude cherche à cerner les flux de matière en suspension de l'Isère, l'un des plus important cours d'eau des Alpes du Nord, à Grenoble. Depuis 1994, et surtout 1996, des prélèvements systématiques en rive gauche permettent de suivre en un point les variations de la matière en suspension de ce cours d'eau. Mais à partir d'une seule mesure, parfois peu représentative de l'ensemble des concentrations observables au sein de la section, il reste encore difficile d'estimer précisément les flux de MES. Les résultats de cette étude montrent notamment l'apparition successive de plusieurs gradients de turbidité, dont l'occurrence s'explique en grande partie par la dynamique hydrologique et hydrosédimentaire de ce cours d'eau, L'estimation de ces gradients permet ainsi d'extrapoler plus aisément les mesures ponctuelles et, dès lors, de définir quotidiennement les quantités de matières en suspension exportées par l'Isère. Depuis 1996, les résultats montrent un flux spécifique de l'Isère à Grenoble très élevé, variant entre 124 et plus de 460 t km-2 an-1.

ration de la taille de Grenoble de cerner le mieux possible ces deux événements exceptionnels estimés notamment à partir des travaux de M. Pardé. Par ailleurs, ce travail permet de compléter les informations sur les débits de crue des événements anciens de l'Isère, pour lesquelles parfois seule la hauteur de la ligne d'eau était connue., Dumas Dominique. Les deux crues mémorables de l'Isère à Grenoble (1651 et 1859). Analyse des estimations de M. Pardé. In: Revue de géographie alpine, tome 92, n°3, 2004. Mélanges 2004. pp. 27-38.

Articular cartilage is an hydrated tissue that withstands and distributes mechanical stresses. The chondrocytes respond to mechanical signals by regulating their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). The objective of this work was to compare, under mechanical stress, the ECMs synthesized by rat chondrocytes seeded onto biosystems based on alginate (Alg), hyaluronic acid (HA) and a HA amphiphilic derivative. The mechanical stress simulates the traumatisms resulting from accidental shocks or intensive physical exercise by knocking the biosystems together. The investigation of ECMs neosynthesized by chondrocytes was carried out according to various criteria: proliferation, proteoglycans synthesis activity, expression of type I and type II collagens and the expression of α5/β1 integrin. The results obtained for the stress applied on neosynthesized matrixes of 3, 10, 17 and 24 days evidenced a high proliferation and proteoglycans synthesis activity for cells submitted to a knocking process. For all biosystems, the neosynthesized matrix contained an important level of collagen, which was in part of type II, whatever the biosystems. Finally, the chemical modification of HA by long hydrophobic alkyl chains, affords an amphiphilic derivative with viscoelastic properties perfectly mimicking those of matricial environment of chondrocytes. This study showed that the HA amphiphilic derivative induced biological effects similar to those of parent HA containing no hydrophobic modifications.

Study of the osmotic resistance to hypotonic medium of platelets has often been suggested as a global test to assess the viability of these cells in transfusion or to study modification during haematological pathologies. A number of authors have analysed the behaviour of platelets in hypotonic media by a variety of methods (cell count, determinations of substances released, morphology, etc.), but most studies are currently based on the so-called “Hypotonic Shock Response” test (HSR). In this study, the authors describe a new automated and reproducible apparatus, called fragilimeter, using slow dialysis to assess platelet osmotic resistance. The variations in light transmission through a platelet suspension according to ionic strength are linked to the change in cellular volume and lysis and characterise the osmotic behaviour of the cells. The results revealed the good reproducibility and sensibility of the technique. This apparatus allows also the realisation of the “HSR” test.

- This article presents the Sargon qanat providing drinkable water to Kyanabad village wich is located at about 10 kilometres SW of Shiraz. The study was conducted at a local scale on three differents perspectives. Construction modalities, hydrological characteristics of the Sargon qanat and the fourty-year systems evolution are presented. Morphological measurements of the qanat and environmental observations allow to raise new hypothesis on the construction of these local systems found in the fold zone of the Zagros. The description of the hydrological characteristics of the Sargon qanat shows the efficiency of these traditional management schemes. The semi-arid climate of this region is the main cause of these qanat and of their long duration. However the comparison at three different dates of these management schemes around Kyanabad village show very well their sudden disparition during the last decades., Résumé. - Cet article présente le qanat de Sargon alimentant en eau potable le village de Kyanabad, situé à environ 10 km au SW de Shiraz. L'étude a été menée à une échelle très locale et sous trois angles différents. Ainsi sont présentées tour à tour les modalités de la construction, les caractéristiques hydrologiques du qanat de Sargon et l'évolution depuis quarante ans de ces systèmes autour du village de Kyanabad. Des mesures morphométriques précises du qanat et des observations environnementales permettent de dégager des hypothèses sur la construction de ces systèmes traditionnels situés dans la zone plissée du Zagros. La description des caractéristiques hydrologiques du qanat de Sargon montre l'efficacité de ces aménagements. Le climat semi-aride de la région reste la cause première de la création de ces qanat ainsi que celle de leur pérennité. Cependant, la comparaison à trois dates différentes de ces aménagements autour du village de Kyanabad illustre bien leur disparition brutale au cours de ces dernières décennies., Dumas Dominique, Mietton Michel. Structure et fonctionnement du qanat de Sargon (Shiraz-lran) (Structure and hydrological behavior of the Sargon qanat (Shiraz-lran)). In: Bulletin de l'Association de géographes français, 75e année, 1998-2 ( juin). Milieux arides et sociétés anciennes. Géographie et télécommunications, sous la direction de Brigitte Coque, Pierre Gentelle, Henry Bakis et Bernard Dezert. pp. 170-178.

This work studied the effect of cholesteryl hemisuccinate incorporation on membrane fluidity and on the kinetics of oxygen diffusion at different depths in the erythrocyte membrane. Cholesterol concentration in the membrane was expressed as the cholesterol–protein ratio (C/Pt). The membrane fluidity, as assessed by a fluorescence polarization method with diphenylhexatriene and 1-(4-trimethylamino)-6-phenylhexa-1,3,5-triene, decreased as the C/Pt ratio increased. Time-resolved fluorescence spectroscopy of pyrene dodecanoic acid (PDA) under an increasing C/Pt ratio in the erythrocyte membrane revealed enhanced oxygen diffusion in the middle of the membrane bilayer (in which PDA was incorporated), which was not the case with pyrene butyric acid (PBA) incorporated in the internal part of the membrane surface. It has generally been accepted that increased membrane fluidity reduces the physical barrier to oxygen permeation. Such conflicting observations on oxygen permeation in the rigidified erythrocyte membrane could be due to variations in oxygen solubility (preferential partitioning) in different polarity microdomains (cholesterol and phospholipid partitions).

Gap junctional intercellular communication has been shown to be involved in the carcinogenesis process. Gap-FRAP (Fluorescence Recovery After Photobleaching) technique could be used to estimate gap junctions functionality and their potential involvement for distinguish normal and cancer cells. We developed a sensitive microspectrofluorimetric method to characterize the kinetics of fluorescence recovery after photobleaching. We used a positive control cell line (fibroblast), a negative control (FaDu) and an intermediate (KB) head and neck cancer cell lines. Our results indicate that using our approach, the fluorescence kinetic profiles remain comparable with the method widely used by confocal microscopy.

International audience; Chondrocalcin is among the most highly synthesized polypeptides in cartilage. This protein is released from its parent molecule, type II pro-collagen, after secretion by chondrocytes. A participation of extracellular, isolated chondrocalcin in mineralization was proposed more than 25 years ago, but never demonstrated. Here, exogenous chondrocalcin was found to trigger MMP13 secretion and cartilage destruction ex vivo in human cartilage explants and did so by modulating the expression of interleukin-1 beta in primary chondrocyte cultures in vitro. Chondrocalcin was found internalized by chondrocytes. Uptake was found mediated by a single 18-mer peptide of chondrocalcin, which does not exhibit homology to any known cell-penetrating peptide. The isolated peptide, when artificially linked as a tetramer, inhibited gene expression regulation by chondrocalcin, suggesting a functional link between uptake and gene expression regulation. At the same time, the tetrameric peptide potentiated chondrocalcin uptake by chondrocytes, suggesting a cooperative mechanism of entry. The corresponding peptide from type I pro-collagen supported identical cell-penetration, suggesting that this property may be conserved among C-propeptides of fibrillar pro-collagens. Structural modeling localized this peptide to the tips of procollagen C-propeptide trimers. Our findings shed light on unexpected function and mechanism of action of these highly expressed proteins from vertebrates.

International audience; Nacre (or mother of pearl) can facilitate bone cell differentiation and ă can speed up their mineralization. Here we report on the capability of ă nacre to induce differentiation of human bone marrow mesenchymal stem ă cells (hBM-MSCs) and the production of extracellular matrix. hBM-MSCs ă were encapsulated in an alginate hydrogel containing different ă concentrations of powdered nacre and cultured in the same environment ă until Day 28. Analysis of osteogenic gene expression, histochemistry, ă second harmonic generation (SHG) microscopy, and Raman scattering ă spectroscopy were used to characterize the synthesis of the ă extracellular matrix. In the presence of nacre powder, a significant ă increase in matrix synthesis from D21 in comparison with pure alginate ă was observed. Histochemistry revealed the formation of a new tissue ă composed of collagen fibers in the presence of nacre (immunostaining and ă SHG), and hydroxyapatite crystals (Raman) in the alginate beads. These ă results suggest that nacre is efficient in hBM-MSCs differentiation, ă extracellular matrix production and mineralization in alginate 3D ă biomaterials. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part ă A: 101A: 3211-3218, 2013.

Crosslinking of actin filaments into bundles is essential for the assembly and stabilization of specific cytoskeletal structures. However, relatively little is known about the molecular mechanisms underlying actin bundle formation. The two LIM-domain-containing proteins define a novel and evolutionarily conserved family of actin-bundling proteins whose actin-binding and -crosslinking activities primarily rely on their LIM domains. Using TIRF microscopy, we describe real-time formation of actin bundles induced by tobacco NtWLIM1 in vitro. We show that NtWLIM1 binds to single filaments and subsequently promotes their interaction and zippering into tight bundles of mixed polarity. NtWLIM1-induced bundles grew by both elongation of internal filaments and addition of preformed fragments at their extremities. Importantly, these data are highly consistent with the modes of bundle formation and growth observed in transgenic Arabidopsis plants expressing a GFP-fused Arabidopsis AtWLIM1 protein. Using two complementary live cell imaging approaches, a close relationship between NtWLIM1 subcellular localization and self-association was established. Indeed, both BiFC and FLIM-FRET data revealed that, although unstable NtWLIM1 complexes can sporadically form in the cytosol, stable complexes concentrate along the actin cytoskeleton. Remarkably, disruption of the actin cytoskeleton significantly impaired self-association of NtWLIM1. In addition, biochemical analyses support the idea that F-actin facilitates the switch of purified recombinant NtWLIM1 from a monomeric to a di- or oligomeric state. On the basis of our data, we propose a model in which actin binding promotes the formation and stabilization of NtWLIM1 complexes, which in turn might drive the crosslinking of actin filaments.

Multi-scale characterization of structures and mechanical behavior of biological tissues are of huge importance in order to evaluate the quality of a biological tissue and/or to provide bio-inspired scaffold for functional tissue engineering. Indeed, the more information on main biological tissue structures we get, the more relevant we will be to design new functional prostheses for regenerative medicine or to accurately evaluate tissues. From this perspective, we have investigated the structures and their mechanical properties from nanoscopic to macroscopic scale of fresh ex-vivo white New-Zealand rabbit Achilles tendon using second harmonic generation (SHG) microscopy, atomic force microscopy (AFM) and tensile tests to provide a “simple” model whose parameters are relevant of its micro or nano structure. Thus, collagen fiber's crimping was identified then measured from SHG images as a plane sine wave with 28.4±5.8 μm of amplitude and 141±41 μm of wavelength. Young's moduli of fibrils (3.0 GPa) and amorphous phases (223 MPa) were obtained using TH-AFM. From these investigations, a non-linear Zener model linking a statistical Weibull's distribution of taut fibers under traction to crimp fibers were developed. This model showed that for small strain (

Integrin αv functions as a receptor for adhesion proteins and is expressed at high density in vascular smooth muscle cells (VSMC)1,2,3,4,5 whose phenotypic modulation plays a crucial role in arterial ageing and atherosclerosis.6,7. Our aim was to define the arterial phenotype in mice conditionally inactivated for the integrin αv subunit in VSMC8,9,10(αv SMKO) and its role in angiotensin II (AngII)-induced arterial fibrosis. Transgenic mice αv SMKO and their control littermates (WT) were treated with two doses of AngII, low (0.3 mg/kg/day) and high (1.5 mg/kg/day), for 4 weeks. At baseline, blood pressure was lower in αvSMKO compared to WT mice. Carotid distensibility was increased in αv SMKO mice (13.3±0.7 vs 10.3±0.6 mmHg−1.10−3). With low dose AngII isobaric distensibility remained higher in αvSMKOmice (12.4±1.2 vs 10.7±1.0 mmHg−1.10−3).With high dose AngII the increase in collagen content in carotid media was lower in αvSMKOthan in WT (19 vs 35%) for a similar increase in blood pressure (30 mmHg) and arterial wall hypertrophy. Collagen immunostaining and fluorescence measurements (multiphoton microscopy second harmonic generation) confirmed that high dose AngII induced lower increases in collagen content in αvSMKO mice versus WT (8.9±1.7 vs 14.2±1.4 greyscale mean/pixel). The combination of similar arterial wall hypertrophy with less fibrosis in mutant mice explains an increased distensibility in response to AngII. The αv subunit regulates AngII-induced arterial fibrosis as determined by collagen staining, immunostaining and fluorescence. Pharmacological targeting of vascular αv integrin may have clinical applications in the treatment of patients with fibrosis associated with hypertension and atherosclerosis.

Tendons and ligaments are complex multi-scale collageneous structures playing a fundamental role in mouvement. Even if these tissues are extensively studied in the past decades, modeling their non-linear viscoelastic properties is still a tough challenge. In order to reveal the relationship between the multi-scale structures and the macroscopic mechanical properties, we used atomic force microscopy (AFM) and second harmonic generation (SHG) microscopy to study unstreateched microtome slices of rabbit Achilles tendons, and an Adamel Lomargy DY.22 tensile test machine to study the dynamic properties of these tissues. Based on our data, a Zener model was used to describe the dynamic loading and unloading cycles.Copyright © 2012 by ASME

Since the Industrial Revolution, a substantial part of the land in mid-altitude mountain areas has been abandoned to woodland. The forest intercepts and retains a certain percentage of rainfall on its foliage, thereby reducing the amount of water actually reaching the ground. The gradual transformation of the landscape over recent decades has therefore had an impact on the transfer of atmospheric water into a form that can be used as a resource. The present study, conducted at the scale of the Chartreuse massif (Pre-Alps, France), examines annual precipitation, the role of the forest in intercepting this precipitation, and changes in the extent of the forest cover. The impact of the extension of the forest cover from the middle of the 19th century on the amount of water lost through runoff and on groundwater reserves can thus be evaluated. The study shows that since the middle of the 19th century the extension of the forest cover in the Chartreuse massif has increased water losses through interception by almost 100 mm per year. It also reveals that over this same period water losses have not been compensated for by an increase in precipitation. In short, the hydrological balance has been considerably modified, with the amount of water available for runoff and groundwater reserves being less than that generally calculated simply from meteorological data records.

The potential interest of DPH-PC was checked with a macrophagic cell line (P388D1). The uptake of DPH-PC was associated with a rapid increase in both fluorescence intensity and a slow decrease in anisotropy values. A flow cytometry comparative study with DPH revealed in both cases the existence of two cell subpopulations with different labeling levels. The analysis of fluorescence decay of DPH-PC showed two components. The fractional intensity of the main component (9.7 ns) is higher than 92%. The Lorentzian distribution of the main lifetime presents an important homogeneity. The observation that an increase in temperature induced a decrease in steady state anisotropy values but did not affect the lifetime suggests that the anisotropy variations effectively reflect modifications in the cohesion of probe micro-surroundings. A transmembrane diffusional phenomenon of a fraction of fluorescent phospholipids (205) was suggested by a study with a nonpermeant membrane quencher. The transmembrane diffusion was confirmed by extraction of the phospholipid analog with fatty acid free BSA. The use of inhibitors of endogenous phospholipase A2 showed a progressive hydrolysis of the fluorescent phospholipid. Nevertheless, the hydrolysis can be neglected in the case of short term interactions with cells (

Hybridoma cell growth and monoclonal antibody production were investigated with a laboratory-made system in which cells were grown in dialysis tubing (MW cut-off 25 kD). The dialysis system contained 10 ml of cell suspension and was immersed in 200 ml of culture medium which when replaced or was at 4-day intervals. With this system, monoclonal antibody concentrations similar to those observed in ascites (concentrations in the order of one gram per liter) were obtained. With no medium replacement, the antibody production was 3.3 g/l and the cell productivity 3.2 x 10(-8) micrograms of IgM produced per cell in one minute. With medium replacement the antibody production was higher, 4.4 g/l but the cell productivity was lower, 1.49 x 10(-8) micrograms per cell in one minute. Cells cultivated in non-optimized conditions were better producers than cells growing in a good environment.

International audience; We propose an innovative invasiveless technique in the field of nonlinear optical imaging to facilitate monitoring of cell/scaffold combinations for tissue repair. By using a near infrared (NIR) femtosecond excitation, we were able to introduce a new index based on decay time response for fluorescence (F) and Second Harmonic Generation (SHG) obtained with Time Correlated Single Photon Counting (TCSPC) microscopy to monitor structural information on the state of the matrix collagen. Some human Mesenchymal Stem Cells (hMSCs) seeded in 3D scaffolds were tested with different culture times (from D7 to D56) to analyze the effect of Tumor Growth Factor beta 1 (TGF-β1) on type-2 collagen expression in the matrix. After 14 days in the presence of TGF-β1, our results showed an increase in the expression of type-2 collagen synthesized by hMSCs, and a change in collagen conformation, as an indication of its ability to be detected as a harmonophore by TCSPC-SHG without the need for an exogenous probe.

The prevalence of bladder cancer is very high, due to its high recurrence rate in superficial bladder cancer (30 to 85%), which is the staging of approximately 80% of the patients at first diagnosis. Risk of recurrence and progression is associated with grade, stage, presence of concomitant carcinoma in situ, size and number of lesions, as well as time to first recurrence. Recurrences can be partly attributed to new occurrences but also to residual tumors after resection. Incomplete tumor removal has been observed in 30 to 50% of TUR's, especially when dealing with T1 or poorly visible malignant or pre-malignant disease1. Fluorescence guided resection with 5 amino levulinic acid (ALA) or its hexyl ester derivative (Hexvix, has now unequivocally been demonstrated to increase detection rate and a growing number of studies indicate this has a positive impact on recurrence and progression ratesImplantation of viable tumor cells, dispersed during resection, is a third factor influencing bladder cancer recurrence. The aim of early intravesical therapy is to interfere with cell viability and thus reduce implantation risks.

As a comparatively non-destructive imaging technique into living specimens, fluorescence microscopy has a number of strong advantages over alternative imaging modalities (X-ray, MRI, CT-scan, arthro-scan, etc..). The limited analysis in thick tissue has given rise to the development of other techniques, multiphoton excitation microscopy in particular. A need for increased sensitivity and resolution has been driving the development of new sophisticated fluorescence techniques based on microscopies to study: the tissue microstructure in situ (CLSM, SHG) on deeper thick sections of tissue (multiphoton), molecular diffusion (FRAP, FCS) with fluorescent protein variants and molecular interaction (spectral, FRET, FLIM). In this paper, we have considered developments based on near infrared (NIR) femtosecond excitation in the imaging of articular tissue and discussed the technical limitations and perspectives.

Cartilage degenerative diseases like osteoarthritis affect the organization of the biological extracellular matrix (ECM) surrounding chondrocytes. This ECM is mainly composed by collagen giving rise to a strong Second Harmonic Generation (SHG) Signal, due to its high non linear susceptibility. Mechanical stress leads to perturbation of the collagen network comparable to modification occurring in disease. To be sure that SHG signal comes specifically from the collagen network, the enzymatical action of Collagenase was followed. We clearly noted the decrease of the collagen specific signal according to incubation time due to enzymatic degradation. To characterize structural modification on the arrangement of collagen fibers in the ECM, we used image analysis based on co-occurrence matrix (Haralick). Textural features give information like homogeneity ('Angular Second Moment') or size of textural elements ('Inverse Difference Moment', 'Correlation'). Samples submitted to compression are characterized by higher 'Correlation', associated with a decrease of 'IDM' and 'ASM'. Those evolutions suggest the presence of long linear structures, an effect of packing of collagen fibrils and the apparition of nodes where the density of collagen is important versus areas showing a lack of molecules. Collagen I, II and VI are biomarkers characterising disease states since its presence is increased in pathological cartilage (osteoarthritis). Fluorescence Lifetime Imaging Microscopy (FLIM) associated to Spectral and SHG analysis confirmed the presence of Collagen I and II in the extracellular and Collagen VI in the pericellular matrix of chondrocytes. SHG, FLIM and Spectral Imaging combined with multiphoton excitation enable tissue imaging at deep penetration. We pointed out a local modification of the ECM of cartilage without any labelling (SHG) under mechanical stress. Thus the association of all these techniques represents a potential diagnosis tool for disorganization of collagen.

Proper management of Chartreuse Mountains water resources requires precise knowledge of the rainfall reaching ground level. Foliage partially intercepts and absorbs rainfall, thus reducing the amount which actually reaches the soil. Runoff and underground water is consequently available in lower quantities than what would be expected simply from examining rainfall records. The influence of the Chartreuse range’s dense forest cover on rainfall is not yet fully understood. In this study, water loss due to forest interception was assessed and defined. It was estimated as the equivalent of 300 mm per annum, or 16 % of overall annual precipitation falling on the range. This is a significant amount and shows the importance of including the forest rainfall interception factor in climatological studies of water resources, especially in thickly forested upland areas.

Several works have recently shown that Mesenchymal Stem Cells (MSC) collected from bone marrow can differentiate in vitro into cartilage cells under the effects of transforming growth factors or critical transcription factors [1]. The transforming growth factor TGF-β is a family of multifunctional cytokines controlling cell growth, differentiation, and extra cellular matrix deposition. The biological effects of TGF-β are mediated by type I (TBR-I) and II (TBR-II) receptors. In this work we presented a preliminary study to characterize the localization and mobility of TBR-II using mesenchymal stem cells derived from bone marrow by Fluorescence Correlation Spectroscopy (FCS). This sensitive technique is mainly used for biological applications for measuring dynamic processes (number density, interaction fractions and molecular dynamics) in a fluorescent signal on the nanosecond to second time scales [2]. To apply this analytical method to living cells, some steps of standardization have to be optimized in the case of indirect immunolabeling.

International audience; The present study addresses the impact of different aggregation states of meta-tetra(hydroxyphenyl)chlorin (mTHPC) on the photoinactivation of cells. Measurements of the photophysical properties of mTHPC in MCF-7 cells showed progressive sensitizer aggregation with increasing incubation time. Reconstructed absorption spectra of intracellular mTHPC showed a significant decrease in the molar extinction coefficient and broadening of the Soret band at 24 h incubation compared to 3 h. Intracellular photobleaching of mTHPC slowed down, and the profile changed from mono- to bi-exponential upon incubation. Fluorescence lifetime imaging (FLIM) measurements revealed a substantial decrease in the lifetime of mTHPC fluorescence at 24 h compared to 3 h. In addition, the intracellular localization of mTHPC as observed by fluorescence microscopy changed from a diffuse homogeneous fluorescence pattern at short incubation times to a punctiform pattern at 24 h. The efficiency of photodynamic therapy (PDT) assessed by a clonogenic assay was three times greater at 24 h. However, when the survival curves were replotted as a function of the number of absorbed photons, the efficiency was 1.8 times greater at 3 h than at 24 h. The loss of photosensitizing efficiency at higher mTHPC concentrations was attributed to self-quenching of the triplet states of the sensitizers.

The aim of this work was to study synthetic polycation effects on erythrocyte agglutination mediated by anti-glycophorin using image digital analysis. Polycations are oligomers or polymers of natural or synthetic origin, which bear a great number of positive charges at pH 7.4. Several of these polycations are nowadays used in clinic for human and veterinary purposes. New applications of polycations to the development of new drug delivery systems are investigated, in order to promote the drug absorption through the gastro-intestinal and blood brain barriers. However, up to now, there are no clear relationships between macromolecular features of polycations (molecular weight, mean charge density, charge repartition, etc.) and their interactions with blood elements (which bear superficial negative charges). The interaction on the red blood cell membrane with synthetic polycations having well-controlled macromolecular features and functionalized with pendent polyethylene glycol segments was investigated. The alterations over stationary and dynamic viscoelastic properties of erythrocyte membranes were analyzed through laser diffractometry. Image digital analysis was used to study erythrocyte agglutination mediated by anti-glycophorin. Results show different reactivities of the polycations on the erythrocyte membrane. These findings could provide more information about the mechanisms of polycation interaction on erythrocyte membranes. We consider that this work could provide useful tools to understand and improve the haemocompatibility of polycations and enlarge their potential in clinic.

The gap-FRAP approach can be suggested to study modifications in functionality of gap junctions. Our results indicate that the degree of gap junctional intercellular communication could be estimated by this technique in vitro.

Several reports show that cancerous cells are linked to early decrease in gap-junction number and functionality diminution. This precancerous phenomenon may be accessed by different fluorescence techniques and particularly by gap-FRAP technique (gap-junction fluorescence recovery after photobleaching). Measurements at cell or tissue scale allowed by this method lead to consider its potential interest in endoscopic technics applied to early cancer detection. Experiments were performed on HT-29 (human colon adenocarcinoma), MCF-7 (human breast cancer cell) and CCD-1137Sk (human Fibroblasts) in the presence of 5.6CFDA. Dye was bleached by laser light (488nm) during few seconds depending on the region of interest (one or fewer cells). Fluorescence recovery kinetic after photobleaching was measured by imaging and spectral analysis with a confocal laser scanning microscope as reference technique. Then, a microspectrofluorimeter was used in order to evaluate the faisability on a fiber optics based system offering measurement condition close to the tissue clinical endoscopy conditions.Preliminary results obtained on the various cells lines show significant differences in kinetics for normal and cancerous cells. We have shown that CCD-1137Sk line cells possess functional communicating junctions, contrary to the carcinogenic HT-29 and MCF-7 cells. Results obtained by microspectrofluorimetry are related to confocal microscopes ones confirming the feasibility in endoscopy.

molecular transport processes on a larger scale, through a fluorescence inhibition method. The system Boxcar EG&G (4100) performs the lifetimmeasurement by scanning the temporal gate along the fluorescence decay. Op tical sections oadhesive molecules (ICAM-1) labelling were obtained with a 3D-scanning microscopy (CSPI-Scanalytic’s, EPR