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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.

Aujourd'hui, le fleuve Betsiboka alimente une vaste plaine agricole située au NW de Madagascar. Ce fleuve est pourtant très mal connu sur un plan hydrologique. Sur la base de mesures des écoulements entre 1958 et 1971, cette étude cherche dans un premier temps à reconstituer les débits contemporains de ce fleuve, puis dans un second temps à cerner comment les écoulements peuvent évoluer au cours du XXIème siècle. Les résultats de cette étude montrent qu'une baisse des débits semble se dessiner à la fin de ce siècle. Elle diminuerait la ressource en eau apportée par le fleuve, selon les scénarii climatiques retenus, de 2.4 à 2.7 km3 d'eau par an. Cette diminution de la ressource serait par ailleurs plus marquée sur la période de décembre à février, durant justement la période de repiquage du riz qui implique un apport en eau élevé.

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.

The year 2021 has seen many breakthroughs in general internal medicine, despite the ongoing COVID-19 pandemic, with multiple implications in our daily clinical practice. From shorter antibiotic treatment duration in community-acquired pneumonia, to new indications for colchicine treatment, without forgetting better targets of hemoglobin for transfusion, questioning of the interest of high dose vitamin D substitution when preventing falls in older patients and finally disappointing hopes for new indications of albumin substitution in cirrhosis, the literature is full of new evidence. Each year, the chief residents of the internal medicine ward in Lausanne university hospital (CHUV) in Switzerland meet up to share their readings: here is a selection of ten articles, chosen, summarized, and commented for you.L’année 2021, malgré la pandémie de Covid-19, a vu de nombreux progrès en médecine interne générale, avec de multiples implications pour notre pratique quotidienne. D’une durée diminuée d’antibiothérapie pour le traitement de la pneumonie communautaire à de nouvelles indications au traitement de colchicine, en passant par des précisions sur les cibles de transfusion érythrocytaire, ainsi qu’une remise en question de l’intérêt de la vitamine D à haute dose dans la prévention des chutes chez la personne âgée, et pour finir des espoirs déçus de nouvelle indication à la substitution d’albumine dans la cirrhose, les nouveautés abondent dans la littérature. Chaque année, les cheffes et chefs de clinique du Service de médecine interne du CHUV se réunissent pour partager leurs lectures : voici une sélection de dix articles choisis, revus et commentés pour vous.

Due to their very poor prognosis and a fatal outcome, secondary brain tumors are one of the biggest challenges in oncology today. From the point of view of the early diagnosis of these brain micro- and macro-tumors, the sensitivity and specificity of the diagnostic tools constitute an obstacle. Molecular imaging, such as Positron Emission Tomography (PET), is a promising technique but remains limited in the search for cerebral localizations, given the commercially available radiotracers. Indeed, the [18F]FDG PET remains constrained by the physiological fixation of the cerebral cortex, which hinders the visualization of cerebral metastases. Tumor angiogenesis is recognized as a crucial phenomenon in the progression of malignant tumors and is correlated with overexpression of the neuropilin-1 (NRP-1) receptor. Here, we describe the synthesis and the photophysical properties of the new gallium-68 radiolabeled peptide to target NRP-1. The KDKPPR peptide was coupled with gallium-68 anchored into a bifunctional NODAGA chelating agent, as well as Cy5 for fluorescence detection. The Cy5 absorbance spectra did not change, whereas the molar extinction coefficient (ε) decreased drastically. An enhancement of the fluorescence quantum yield (φF) could be observed due to the better water solubility of Cy5. [68Ga]Ga-NODAGA-K(Cy5)DKPPR was radiosynthesized efficiently, presented hydrophilic properties (log D = −1.86), and had high in vitro stability (>120 min). The molecular affinity and the cytotoxicity of this new chelated radiotracer were evaluated in vitro on endothelial cells (HUVEC) and MDA-MB-231 cancer cells (hormone-independent and triple-negative line) and in vivo on a brain model of metastasis in a nude rat using the MDA-MB-231 cell line. No in vitro toxicity has been observed. The in vivo preliminary experiments showed promising results, with a high contrast between the healthy brain and metastatic foci for [68Ga]Ga-NODAGA-K(Cy5)DKPPR.

Albumin has multiple physiological functions such as maintenance of oncotic pressure, binding of plasmatic molecule, immunomodulation and endothelial stabilization. Considering potential negative effects of hypoalbuminemia, the question of albumin substitution arises. For cirrhotic patients, indications for substitution are well established with a scientific consensus for spontaneous bacterial peritonitis, paracentesis-induced circulatory dysfunction and hepatorenal syndrome. However, in the case of hepatic encephalopathy or of hypoalbuminemia in non-cirrhotic patients, the scientific consensus is absent, and the results of clinical studies differ. In this article, we present the different indications of albumin substitution by examining the most recent studies.L’albumine exerce de multiples fonctions physiologiques, notamment : maintien de la pression oncotique, liaison à d’autres molécules plasmatiques, immunomodulation et stabilisation endothéliale. Face aux potentiels effets délétères de l’hypoalbuminémie, la question d’une substitution se pose. Chez le patient cirrhotique, les indications à la substitution sont bien définies avec un consensus scientifique pour la péritonite bactérienne spontanée, la dysfonction circulatoire postparacentèse de grand volume et le syndrome hépatorénal. Néanmoins, en cas d’encéphalopathie hépatique ou d’hypoalbuminémie chez le patient non cirrhotique, le consensus est absent et les résultats des études divergent. Dans cet article, nous présentons les différentes indications à sa substitution, en mettant l’accent sur les études récentes en la matière.

Given the lack of in situ hydroclimatic measurements and networks in Madagascar, the GRACE (2003–2016) spatial gravimetry data, combined with other satellite data such as CHIRPS rainfall estimates or fire monitoring using GFED products, make it possible to establish an interannual assessment of certain climatic and environmental covariations at the northwest scale of the country. The results show a negative trend in continental rainfall and water content, especially after 2007, but also a time lag in the linear variations and trends of the Water Equivalent Height as well as the number of detected fires (variable indirectly measuring the pressure of deforestation by slash and burn agriculture).

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.

2020 has continued to bring important progress in all areas of internal medicine, impacting our daily practice. From a shift to oral stepdown antibiotics in infectious endocarditis, to new indications for apixaban, SLGT-2 inhibitors and macrolide antibiotics, passing by the catheter-based renal denervation in hypertension, early paracentesis in cirrhosis and new diagnostic criteria in pulmonary embolism, internal medicine journals are full of novelties. Every year, the chief residents of internal medicine at the Swiss University Hospital of Lausanne meet up to share their readings: here is their selection of eleven articles, summarized and commented for you.L’année 2020 a vu de notables progrès dans tous les domaines de la médecine interne, avec un impact important sur notre pratique quotidienne. Du passage à une antibiothérapie orale dans l’endocardite infectieuse, aux nouvelles indications pour l’apixaban, les inhibiteurs du SGLT2 (sodium-glucose co-transporteur de type 2) et les macrolides en passant par la dénervation rénale dans l’hypertension artérielle, la ponction d’ascite et les changements de critères diagnostiques de l’embolie pulmonaire, les nouveautés abondent dans la littérature. Chaque année, les chefs de clinique du Service de médecine interne du CHUV se réunissent pour partager leurs lectures : voici une sélection de onze articles choisis, revus et commentés pour vous.

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.

International audience; In this work, we studied the nature of the molecular bonds involved in the red blood cell aggregation process by the coherent anti-Stokes Raman spectroscopy technique. Images were acquired with a commercial Leica TCS SP8 CARS confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) temporally and spatially overlapping the pulses of two sources in the focal plane of the microscope. A pump wavelength of 810 nm to 817 nm was used for the CARS mode simultaneously with the Stokes beam at 1064 nm to excite the vibratory resonance of the symmetric hydrocarbon bonds in the lipids and that of the bonds in amino acids of the proteins. The Raman shift was also observed at the 1200 cm−1 range to study possible variations in the sialic acid on the cell membrane produced by concentrations of dextran 500 in the suspension medium. Curves of lifetime emission distribution were obtained for untreated erythrocytes and treated erythrocytes with dextran 500, particularly at a pump wavelength of 904 nm.

Based on remotely sensed imagery and socioeconomic data, this research analyzes the reasons why farmers choose one crop over another in the Bekaa Valley in Lebanon. This study mapped the area of the cultivated crop in 2017 with Sentinel-2 images. An accurate and new method was developed to extract the field boundaries from the evolution of the normalized difference vegetation index (NDVI) profile throughout the season. We collected 386 GPS locations for fields that are used for crop cultivation, from which the NDVI profile was extracted. The 386 reference fields were separated into two groups: reference locations and test locations. The Euclidean distance (ED) was calculated between these two groups, and the classification was strongly correlated to the known crop type in the field (overall accuracy: 90%). Our study area cultivated wheat (32%), spring potatoes (25%), spring vegetables (27%), orchards (11%), vineyards (7%), and alfalfa (&lt, 1%). Socioeconomic surveys showed that farmers favored these crops over others on account of their profitability. Nonetheless, the surveys highlighted a paradox: despite the lack of a political frame for agriculture in Lebanon, farmers&rsquo, crop choices strongly depend on a few existing policies.

Annulus fibrosus (AF) impairment is associated with reherniation, discogenic pain, and disc degeneration after surgical partial discectomy. Due to a limited intrinsic healing capacity, defects in the AF persist over time and it is hence necessary to adopt an appropriate strategy to close and repair the damaged AF. In this study, a cell-free biodegradable scaffold made of polycaprolactone (PCL), electrospun, aligned microfibers exhibited high levels of cell colonization, alignment, and AF-like extracellular matrix deposition when evaluated in an explant culture model. The biomimetic multilayer fibrous scaffold was then assessed in an ovine model of AF impairment. After 4 weeks, no dislocation of the implants was detected, and only one sample out of six showed a partial delamination. Histological and immunohistochemical analyses revealed integration of the implant with the surrounding tissue as well as homogeneously aligned collagen fiber organization within each lamella compared to the disorganized and scarcer fibrous tissue in a randomly organized control fibrous scaffold. In conclusion, this biomimetic electrospun implant exhibited promising properties in terms of AF defect closure, with AF-like neotissue formation that fully integrated with the surrounding ovine tissue. Accepted version

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.

International audience

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.

Significance A concept called reactive compatibilizer-tracer is proposed. The latter bears reactive groups capable of reacting with its counterpart on forming a copolymer for in situ compatibilization, and fluorescent labels allowing determining very small amounts of the in situ formed compatibilizer and formation of micelles when it occurs. Owing to this concept, it is shown that a reactive compatibilizer may be very efficient at the beginning of a reactive blending process and may suddenly become completely inefficient, resulting in an abrupt and drastic increase in size of the dispersed phase domains. © 2015 American Institute of Chemical Engineers AIChE J, 2015

In accordance with their mission and as part of their long-term observation work, Ecrins National Park (NP) is making important contributions to the study and moni-toring of phenomena related to climate change. Its work takes information collected at different meteorological stations and supplements it with analyses of vegetation changes through aerial and satellite images, measuring and monitoring of glaciers, and interdisciplinary programmes focused on high-altitude lakes, mountain pastures and indicator species. Through its mission and long-term observations, Ecrins NP (http://www.ecrins-parcnational.fr/) contributes to the environmental surveillance of many ecosystems. Working within many different networks and in partnership with various research teams, the park participates in the development of a better understanding of the important changes taking place.

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.

International audience; Osteoarthritis (OA) is the most common cause of joint chronic pain and involves the entire joints. Subchondral osteoarthritic osteoblasts present a mineralization defect and, to date, only a few molecules (Vitamin D3 and Bone Morphogenetic Protein2) could improve the mineralization potential of this cell type. In this context, we have tested for the first time the effect of nacre extract on the mineralization capacity of osteoblasts from OA patients. Nacre extract is known to contain osteogenic molecules which have demonstrated their activities notably on the MC3T3 pre-osteoblastic cell line. For this goal, molecules were extracted from nacre (ESM, Ethanol Soluble Matrix) and tested on osteoblasts of the subchondral bone from OA patients undergoing total knee replacement and on MC3T3 cells for comparison. We chose to investigate the mineralization with Alizarin Red staining and with the study of extracellular matrix (ECM) structure and composition. In a complementary way the structure of the ECM secreted during the mineralization phase was investigated using second harmonic generation (SHG). Nacre extract was able to induce the early presence (after 7 days) of precipitated calcium in cells. Raman spectroscopy and electron microscopy showed the presence of nanograins of an early crystalline form of calcium phosphate in OA osteoblasts ECM and hydroxyapatite in MC3T3 ECM. SHG collagen fibers signal was present in both cell types but lower for OA osteoblasts. In conclusion, nacre extract was able to rapidly restore the mineralization capacity of osteoarthritis osteoblasts, therefore confirming the potential of nacre as a source of osteogenic compounds. (C) 2015 Elsevier Inc. All rights reserved.

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; The present study investigates the relationship between the subcellular localisation of Foscan and intrinsic apoptotic pathway post Foscan-based photodynamic therapy (PDT). With this purpose, mammary carcinoma MCF-7 cells were incubated with Foscan for 3 or 24 h and then subjected to equitoxic light doses. Fluorescence microscopy revealed very good Foscan co-localization to endoplasmic reticulum (ER) and Golgi apparatus after 3 h incubation with MCF-7 cells. Progressive increase in incubation time shows leakage of Foscan from Golgi apparatus. Twenty-four hours incubation yielded a fluence-dependent enhanced induction of the ER-resident glucose-regulated protein 78 (Bip/GRP78), along with a weak mitochondrial damage, thus underscoring the ER as the main site of photodamage after prolonged incubation. Analysis of events implicated in apoptotic pathway after 24 h incubation demonstrated photodamage to Bcl-2 protein in total cellular extract, but not in the mitochondrial fraction. We further determined an increase in caspases-7 and -6 activation, which was strongly related to the expression of GRP78. The above findings demonstrate that Foscan localisation in ER improves the photoactivation of the caspase-7 apoptotic pathway, which is poorly related to mitochondrial damage.

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.

International audience; There is currently great interest in the development of efficient and specific carrier delivery platforms for systemic photodynamic therapy. Therefore, we aimed to develop covalent conjugates between the photosensitizer chlorin e6 (Ce6) and PAMAM G4.5 dendrimers. Singlet oxygen generation (SOG) efficiency and fluorescence emission were moderately affected by the covalent binding of the Ce6 to the dendrimer. Compared to free Ce6, PAMAM anchored Ce6 displays a much higher photodynamic effect, which is ascribable to better internalization in a tumor cell model. Intracellular fate and internalization pathway of our different compounds were investigated using specific inhibition conditions and confocal fluorescence microscopy. Free Ce6 was shown to enter the cells by a simple diffusion mechanism, while G4.5-Ce6-PEG internalization was dependent on the caveolae pathway, whereas G4.5-Ce6 was subjected to the clathrin-mediated endocytosis pathway. Subcellular localization of PAMAM anchored Ce6, PEGylated or not, was very similar suggesting that the nanoparticles behave similarly in the cells. As a conclusion, we have demonstrated that PEGylated G4.5 PAMAM-Ce6 dendrimers may offer effective biocompatible nanoparticles for improved photodynamic treatment in a preclinical tumor model.

National audience

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.