Preparative laser capture microdissection and single-pot cell wall material preparation: a novel method for tissue-specific analysis.

International audienceIn adaptation to their function the walls of plant cell display tissue-specific variations of composition according to their developmental stage, cell type and stress of various origin. It is therefore important to obtain a precise analytical data describing the cell wall composition with respect to these different factors. In the present work, laser capture microdissection (LCM) was used for isolating different tissues from the stem of Urtica dioica L. at a semi-preparative scale. The technique was associated for the first time to a one-pot sequential cell wall preparation and hydrolysis for the carbohydrate analysis of each cell type. The results demonstrate that the combination of LCM and micro-analytical methods can provide individual cell type composition and should improve our knowledge of the biochemical diversity of cell walls in plants. This approach will be of potential interest for the understanding of the effects of stress or genetic engineering on the composition of the cell walls

The rate of fibrinolysis is increased by free retraction of human gingival fibroblast populated fibrin lattices.

We previously demonstrated that human gingival fibroblasts (HGF), but not their dermal counterparts, when seeded in retracting fibrin lattices induced intense fibrinolysis that was observed at the earliest stages of contraction and led to complete matrix degradation by day 7 of culture. Our aim was to examine the influence of mechanical forces in such fibrinolytic processes. HGF were seeded in retracting (R) e.g. free floating or non retracting (NR) e.g. anchored fibrin lattices (FL). Cultures were analysed from day 1-12 by phase contrast microscopy and scanning electron microscopy (s.e.m.). Levels of fibrin degradation products (FDP) and tissue plasminogen activator (tPA) accumulating in culture media were quantified by ELISA. Urokinase (uPA) and gelatinase A (MMP2) were identified by zymographic techniques. At the s.e.m. level, vacuolization around some HGF was noticed at the earliest stages of culture for RFL and complete degradation of lattices occurred at day 7. Formation of lysed matrix cavity was far less intense in NRFL even after 12 days of culture. FDP amounts at day 4 of culture were equal to 79 +/- 14 and 8.5 +/- 0.6 micrograms/10(5) cells for RFL and NRFL, respectively; tPA levels were equal to 5.8 +/- 0.6 (RFL) and 2.1 +/- 0.3 ng/10(5) cells (NRFL) and differences were still evident at day 7. The kinetics of tPA production were identical in either retracting fibrin or collagen lattices. On the contrary, uPA and proMMP2 productions were similar in RFL and NRFL. Isometric forces, but not the matrix support, were responsible for accelerated tPA production and fibrinolysis in HGF populated lattices

Ensuring the safety and security of frozen lung cancer tissue collections through the encapsulation of dried DNA

International audienceCollected specimens for research purposes may or may not be made available depending on their scarcity and/or on the project needs. Their protection against degradation or in the event of an incident is pivotal. Duplication and storage on a different site is the best way to assure their sustainability. The conservation of samples at room temperature (RT) by duplication can facilitate their protection. We describe a security system for the collection of non-small cell lung cancers (NSCLC) stored in the biobank of the Nice Hospital Center, France, by duplication and conservation of lyophilized (dried), encapsulated DNA kept at RT. Therefore, three frozen tissue collections from non-smoking, early stage and sarcomatoid carcinoma NSCLC patients were selected for this study. DNA was extracted, lyophilized and encapsulated at RT under anoxic conditions using the DNAshell technology. In total, 1974 samples from 987 patients were encapsulated. Six and two capsules from each sample were stored in the biobanks of the Nice and Grenoble (France) Hospitals, respectively. In conclusion, DNA maintained at RT allows for the conservation, duplication and durability of collections of interest stored in biobanks. This is a low-cost and safe technology that requires a limited amount of space and has a low environmental impact. © 2018 by the authors. Licensee MDPI, Basel, Switzerland

Mieux comprendre le rôle de l’exposition précoce à la pollution de l’air sur la santé via une approche originale combinant une cohorte épidémiologique (SEPAGES) et une étude toxicologique

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Deciphering the Impact of Early-Life Exposures to Highly Variable Environmental Factors on Foetal and Child Health : Design of SEPAGES Couple-Child Cohort

In humans, studies based on Developmental Origins of Health and Disease (DOHaD) concept and targeting short half-lived chemicals, including many endocrine disruptors, generally assessed exposures from spot biospecimens. Effects of early-life exposure to atmospheric pollutants were reported, based on outdoor air pollution levels. For both exposure families, exposure misclassification is expected from these designs: for non-persistent chemicals, because a spot biospecimen is unlikely to capture exposure over windows longer than a few days; for air pollutants, because indoor levels are ignored. We developed a couple-child cohort relying on deep phenotyping and extended personal exposure assessment aiming to better characterize the effects of components of the exposome, including air pollutants and non-persistent endocrine disruptors, on child health and development. Pregnant women were included in SEPAGES couple-child cohort (Grenoble area) from 2014 to 2017. Maternal and children exposure to air pollutants was repeatedly assessed by personal monitors. DNA, RNA, serum, plasma, placenta, cord blood, meconium, child and mother stools, living cells, milk, hair and repeated urine samples were collected. A total of 484 pregnant women were recruited, with excellent compliance to the repeated urine sampling protocol (median, 43 urine samples per woman during pregnancy). The main health outcomes are child respiratory health using early objective measures, growth and neurodevelopment. Compared to former studies, the accuracy of assessment of non-persistent exposures is expected to be strongly improved in this new type of birth cohort tailored for the exposome concept, with deep phenotyping and extended exposure characterization. By targeting weaknesses in exposure assessment of the current approaches of cohorts on effects of early life environmental exposures with strong temporal variations, and relying on a rich biobank to provide insight on the underlying biological pathways whereby exposures affect health, this design is expected to provide deeper understanding of the interplay between the Exposome and child development and health