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L'étude des régulations hormonales, reponsables du contrôle de grandes fonctions telles que le développement, la croissance et la reproduction, présente chez l'anguille un intérêt tout particulier du fait du cycle biologique original de ce poisson. Les objectifs d'une telle étude sont à la fois fondamentaux (connaissance de la biologie de l'espèce, notion d'espèce modèle, écophysiologie, évolution) et appliqués (caractérisation des populations, maîtrise du cycle biologique). Cependant, la complexité et la méconnaissance actuelle de ce cycle apportent des difficultés supplémentaires (absence de référence naturelle pour les caractéristiques physiologiques et environnementales des dernières étapes du cycle) qui s'ajoutent à celles concernant classiquement la mise en oeuvre d'une telle étude chez une espèce particulière (nécessité de la mise au point des outils spécifiques d'investigation), espèce de surcroît sauvage (hétérogénéité physiologique et génétique des animaux étudiés) et e n danger (limitation du nombre d'animaux expérimentaux). C'est la mise en oeuvre de tout un ensemble de méthodes d'investigation, allant de la mise au point des outils moléculaires (purifications d'hormones, obtention d'anticorps, dosages immunologiques, clonage, obtention de sondes...) à la réalisation d'expériences in vitro (cultures de cellules, incubation ou périfusion d'organes) et in vivo (ablations de glandes, traitements hormonaux, prélèvements sanguins...) juqu'aux études sur le terrain, qui permet une approche complète et intégrée de l'endocrinologie de ce poisson.

Abstract Background The formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised. Results Recent expressed sequence tag (EST) investigations of the mantle tissue from the tropical abalone (Haliotis asinina) provide an opportunity to further characterise the proteins in the shell by a proteomic approach. In this study, we have identified a total of 14 proteins from distinct calcified layers of the shell. Only two of these proteins have been previously characterised from abalone shells. Among the novel proteins are several glutamine- and methionine-rich motifs and hydrophobic glycine-, alanine- and acidic aspartate-rich domains. In addition, two of the new proteins contained Kunitz-like and WAP (whey acidic protein) protease inhibitor domains. Conclusion This is one of the first comprehensive proteomic study of a molluscan shell, and should provide a platform for further characterization of matrix protein functions and interactions.

Long-term modelization of cancer as it changes in the human body is a difficult goal, particularly when designing and testing new therapeutic strategies. This becomes even more difficult with metastasis modeling to show chemotherapeutic molecule delivery directly to tumoral cells. Advanced therapeutics, including oncolytic viruses, antibody-based and cell-based therapies are increasing. The question is, are screening tests also evolving? Next-generation therapeutics need equally advanced screening tests, which whilst difficult to achieve, are the goal of our work here, creating models of micro- and macrotumors using 3D bioprinting. We developed advanced colorectal cancer tumor processing techniques to provide options for cellular expansion, microtumor printing, and long-term models, which allow for the evaluation of the kinetics of penetration testing, therapeutic success, targeted therapies, and personalized medicine. We describe how we tested tumors from a primary colorectal patient and, applying 3D bioprinting, matured long-term models for oncolytic metastatic screening. Three-dimensional microtumors were kept alive for the longest time ever recorded in vitro, allowing longitudinal studies, screening of oncolytic viruses and realistic modelization of colorectal cancer. These 3D bioprinted models were maintained for around 6 months and were able to demonstrate the effective delivery of a product to the tumoral environment and represent a step forward in therapeutic screening.

Acne-prone skin is associated with dysbiosis involving Cutibacterium acnes ( C. acnes ) and Staphylococcus epidermidis ( S. epidermidis ) causing increased seborrhea in sebaceous glands (SG) and inflammation. Human primary sebocytes were cultivated using 1.10 6 UFC/mL C. acnes Type IA (facial acne, ATCC6919) and/or 1.10 5 UFC/mL S. epidermidis (unknown origin, ATCC12228) for 48 h in our SEB4GLN-optimized media without antibiotics. Bacteria and sebocytes were enumerated and assessed to determine their viability. Lipid production was imaged and quantified via Nile Red staining. SG with hair follicles were microdissected from healthy skin and cultured using 1.10 5 UFC/mL C. acnes Type 1A and/or 1.10 4 UFC/mL S. epidermidis (wild-type facial skin strain) through prior fixation and immunostaining for MC5R, C. acnes and nuclei (DAPI) via Z-stack confocal microscopy bioimaging (Leica SP5X & FIJI software, Version 2.9.0). C. acnes growth was not impacted when co-cultivated with sebocytes (2D) or SG (3D) models. Phylotype IA stimulated sebocyte lipid production, which had no impact on viability. The S. epidermidis reference strain overproliferated, inducing sebocyte mortality. For 3D SG model, culture conditions were optimized using a wild-type facial skin strain at a lower concentration, 1:10 ratio to C. acnes, reduced contact time, sequential inoculation and rinsing step. Bioimaging revealed strong C. acnes labeling in the active areas of the pilosebaceous unit. S. epidermidis formed biofilm, which was distributed across the SG via non-specific fluorescence imaging. We developed an innovative model of a sebaceous gland that mimics acne-prone skin with lipid overproduction and virulent phylotype IA C. acnes inoculation.

The tailoring of the skin microbiome is challenging and is a research hotspot in the pathogenesis of immune-mediated inflammatory skin diseases such as acne. Commonly encountered preservatives used as functional ingredients have an impact on the skin microbiota and are known to inhibit the survival of skin commensal bacteria. The selected species is Lactiplantibacillus plantarum , formulated with natural enhancers for topical use (SkinDuo TM ). Ex vivo human skin models were used as a test system to assess the strain viability which was then validated on healthy volunteers. SkinDuo TM showed increased viability over time for in vitro skin models and a stable viability of over 50% on healthy skin. The strain was tested on human primary sebocytes obtained from sebaceous gland rich areas of facial skin and inoculated with the most abundant bacteria from the skin microbiota. Results on human ex vivo sebaceous gland models with the virulent phylotype of Cutibacterium acnes and Staphylococcus epidermidis present a significant reduction in viability, lipid production, and anti-inflammatory markers. We have developed an innovative anti-acne serum with L. plantarum that mimics the over-production of lipids, anti-inflammatory properties, and improves acne-disease skin models. Based on these results, we suggest that SkinDuo TM may be introduced as an acne-mitigating agent.

Colorectal cancer (CRC) is the third most common malignancy and has recently moved up to the second leading cause of death among carcinomas. Prognosis, especially for advanced diseases or certain molecular subtypes of CRC, remains poor, which highlights the urgent need for better therapeutic strategies. However, currently, as little as 0.1% of all drugs make it from bench to bedside because of the inherently high false-positive and false-negative rates of current preclinical and clinical drug testing data. Therefore, the success of developing novel treatment agents lies in the introduction of improved preclinical disease models which resemble in vivo carcinomas closer, possess higher predictive properties, and offer opportunities for individualized therapies. Aiming to address these needs, we have established an affordable, flexible, and highly reproducible 3D bioprinted CRC model. The histological assessment of Caco-2 cells in 3D bioprints revealed the formation of glandular-like structures which show greater pathomorphological resemblance to tumors than monolayer cultures do. RNA expression profiles in 3D bioprinted cells were marked by upregulation of genes involved in cell adhesion, hypoxia, EGFR/KRAS signaling, and downregulation of cell cycle programs. Testing this 3D experimental platform with three of the most commonly used chemotherapeutics in CRC (5-fluoruracil, oxaliplatin, and irinotecan) revealed overall increased resistance compared to 2D cell cultures. Last, we demonstrate that our workflow can be successfully extended to primary CRC samples. Thereby, we describe a novel accessible platform for disease modeling and drug testing, which may present an innovative opportunity for personalized therapeutic screening., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sbirkov, Molander, Milet, Bodurov, Atanasov, Penkov, Belev, Forraz, McGuckin and Sarafian.)

Beige adipocyte differentiation within white adipose tissue, referred to as browning, is seen as a possible mechanism for increasing energy expenditure. The molecular regulation underlying the thermogenic browning process has not been entirely elucidated. Here, we identify the zinc finger transcription factor EGR1 as a negative regulator of the beige fat program. Loss of Egr1 in mice promotes browning in the absence of external stimulation and leads to an increase of Ucp1 expression, which encodes the key thermogenic mitochondrial uncoupling protein-1. Moreover, EGR1 is recruited to the proximal region of the Ucp1 promoter in subcutaneous inguinal white adipose tissue. Transcriptomic analysis of subcutaneous inguinal white adipose tissue in the absence of Egr1 identifies the molecular signature of white adipocyte browning downstream of Egr1 deletion and highlights a concomitant increase of beige differentiation marker and a decrease in extracellular matrix gene expression. Conversely, Egr1 overexpression in mesenchymal stem cells decreases beige adipocyte differentiation, while increasing extracellular matrix production. These results reveal a role for Egr1 in blocking energy expenditure via direct Ucp1 transcription repression and highlight Egr1 as a therapeutic target for counteracting obesity.

The molecular programme underlying tendon development has not been fully identified. Interactions with components of the musculoskeletal system are important for limb tendon formation. Limb tendons initiate their development independently of muscles; however, muscles are required for further tendon differentiation. We show that both FGF/ERK MAPK and TGFβ/SMAD2/3 signalling pathways are required and sufficient for SCX expression in chick undifferentiated limb cells, whereas the FGF/ERK MAPK pathway inhibits Scx expression in mouse undifferentiated limb mesodermal cells. During differentiation, muscle contraction is required to maintain SCX, TNMD and THBS2 expression in chick limbs. The activities of FGF/ERK MAPK and TGFβ/SMAD2/3 signalling pathways are decreased in tendons under immobilisation conditions. Application of FGF4 or TGFβ2 ligands prevents SCX downregulation in immobilised limbs. TGFβ2 but not FGF4 prevent TNMD and THBS2 downregulation under immobilisation conditions. We did not identify any intracellular crosstalk between both signalling pathways in their positive effect on SCX expression. Independently of each other, both FGF and TGFβ promote tendon commitment of limb mesodermal cells and act downstream of mechanical forces to regulate tendon differentiation during chick limb development., (© 2016. Published by The Company of Biologists Ltd.)

Members of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. We report here the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. We isolated 17 putative modifiers involved in various function or process: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation.

The tumor suppressor retinoblastoma protein (pRb) is inactivated in a wide variety of cancers. While its role during cell cycle is well characterized, little is known about its properties on apoptosis regulation and apoptosis-induced cell responses. pRb shorter forms that can modulate pRB apoptotic properties, resulting from cleavages at caspase specific sites are observed in several cellular contexts. A bioinformatics analysis showed that a putative caspase cleavage site (TELD) is found in the Drosophila homologue of pRb(RBF) at a position similar to the site generating the p76Rb form in mammals. Thus, we generated a punctual mutant form of RBF in which the aspartate of the TELD site is replaced by an alanine. This mutant form, RBFD253A, conserved the JNK-dependent pro-apoptotic properties of RBF but gained the ability of inducing overgrowth phenotypes in adult wings. We show that this overgrowth is a consequence of an abnormal proliferation in wing imaginal discs, which depends on the JNK pathway activation but not on wingless (wg) ectopic expression. These results show for the first time that the TELD site of RBF could be important to control the function of RBF in tissue homeostasis in vivo.

Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Defining which key factors control commitment of an embryonic lineage among a myriad of candidates is a longstanding challenge in developmental biology and an essential prerequisite for developing stem cell-based therapies. Commitment implies that the induced cells not only express early lineage markers but further undergo an autonomous differentiation into the lineage. The embryonic neural crest generates a highly diverse array of derivatives, including melanocytes, neurons, glia, cartilage, mesenchyme, and bone. A complex gene regulatory network has recently classified genes involved in the many steps of neural crest induction, specification, migration, and differentiation. However, which factor or combination of factors is sufficient to trigger full commitment of this multipotent lineage remains unknown. Here, we show that, in contrast to other potential combinations of candidate factors, coactivating transcription factors Pax3 and Zic1 not only initiate neural crest specification from various early embryonic lineages in Xenopus and chicken embryos but also trigger full neural crest determination. These two factors are sufficient to drive migration and differentiation of several neural crest derivatives in minimal culture conditions in vitro or ectopic locations in vivo. After transplantation, the induced cells migrate to and integrate into normal neural crest craniofacial target territories, indicating an efficient spatial recognition in vivo. Thus, Pax3 and Zic1 cooperate and execute a transcriptional switch sufficient to activate full multipotent neural crest development and differentiation.

Controling embryonic stem cell fate in vitro has been a major challenge in the past decade. Several protocols have been developed to obtain neural crest derivatives in culture, using more or less defined conditions. Here, we present various strategies used to date to obtain neural crest specification and the markers that can be used to identify human neural crest cells., (Copyright © 2012. Published by Elsevier Inc.)

The neural crest is a transient and multipotent cell population arising at the edge of the neural plate in vertebrates. Recent findings highlight that neural crest patterning is initiated during gastrulation, i.e. earlier than classically described, in a progenitor domain named the neural border. This chapter reviews the dynamic and complex molecular interactions underlying neural border formation and neural crest emergence., (Copyright © 2012. Published by Elsevier Inc.)

Background: The formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised., Results: Recent expressed sequence tag (EST) investigations of the mantle tissue from the tropical abalone (Haliotis asinina) provide an opportunity to further characterise the proteins in the shell by a proteomic approach. In this study, we have identified a total of 14 proteins from distinct calcified layers of the shell. Only two of these proteins have been previously characterised from abalone shells. Among the novel proteins are several glutamine- and methionine-rich motifs and hydrophobic glycine-, alanine- and acidic aspartate-rich domains. In addition, two of the new proteins contained Kunitz-like and WAP (whey acidic protein) protease inhibitor domains., Conclusion: This is one of the first comprehensive proteomic study of a molluscan shell, and should provide a platform for further characterization of matrix protein functions and interactions.

The retinoblastoma protein, pRb, plays important roles in many processes implicated in cell fate decisions, including cell cycle, differentiation and apoptosis. In cell cycle regulation, pRb interacts principally with the E2F transcription factor family members to inhibit the transcription of many genes controlling cell cycle progression. In this study, we focused on the role of pRb in apoptosis, which is much less clear than its role in cell cycle regulation. Indeed, pRb has been found to be either pro- or anti-apoptotic. To clarify how the proliferative status of the cells impacts the role of pRb in apoptosis, we used Drosophila to induce RBF (the pRb fly homologue) expression in different cellular and developmental contexts. We found that RBF expression induces apoptosis in different proliferative tissues in a caspase-dependent manner, whereas this effect was not observed in differentiated post-mitotic cells. Furthermore, RBF-induced apoptosis in proliferating cells was inhibited by co-expression of dE2F1, an antagonistic partner of RBF in cell cycle regulation. These results are in agreement with the view that the apoptotic properties of pRb are tightly linked to, and are probably a consequence of, an effect on cell cycle progression. Moreover, we show for the first time that RBF has a direct anti-apoptotic effect on Dmp53-induced cell death in post-mitotic cells only. Taken together, these data clearly show that RBF can exert a dual role in the control of apoptotic processes, and that its properties depend on the proliferative status of the cells.

This study evaluates the effect of the mother-of-pearl (nacre) organic matrix on mammalian osteoclast activity and on cathepsin K protease. Rabbit osteoclasts were cultured on bovine cortical bone slices in the presence of water-soluble molecules extracted from nacre of the pearl oyster Pinctada margaritifera. Osteoclast resorption activity was determined by quantification of the resorption surface area on bovine bone slices. Papain and cathepsin K, B and L inhibition tests were performed in the presence of the nacre water-soluble extracts. The active crude extract was fractionated by dialysis and reversed-phase high-performance liquid chromatography before electrospray mass spectrometry analysis of inhibitory fractions. The water-soluble molecules extracted from nacre decreased bone resorption without jeopardizing osteoclast survival. The hydrolytic activity of cysteine proteinases was reduced when the enzymes were incubated with the nacre water-soluble molecules. Trending towards characterization of the molecules involved, it appears that cathepsin K inhibitors remain in different nacre water-soluble organic matrix subfractions, composed of low molecular weight molecules. Mollusk shell nacre contains molecules capable of reducing osteoclast bone resorption activity by inhibiting cathepsin K, giving a new facet of the bioactivity of nacre as bone biomaterial.

Nacre organic matrix has been conventionally classified as both 'water-soluble' and 'water-insoluble', based on its solubility in aqueous solutions after decalcification with acid or EDTA. Some characteristics (aspartic acid-rich, silk-fibroin-like content) were specifically attributed to either one or the other. The comparative study on the technique of extraction (extraction with water alone vs. demineralization with EDTA) presented here, seems to reveal that this generally accepted classification may need to be reconsidered. Actually, the nondecalcified soluble organic matrix, extracted in ultra-pure water, displays many of the characteristics of what until now has been called 'insoluble matrix'. We present the results obtained on this extract and on a conventional EDTA-soluble matrix, with various characterization methods: fractionation by size-exclusion and anion-exchange HPLC, amino acid analysis, glycosaminoglycan and calcium quantification, SDS/PAGE and FTIR spectroscopy. We propose that the model for the interlamellar matrix sheets of nacre given by Nakahara [In: Biomineralization and Biological Metal Accumulation, Westbroek, P. & deJong, E.W., eds, (1983) pp. 225-230. Reidel, Dordrecht, Holland] and Weiner and Traub [Phil. Trans. R. Soc. Lond. B (1984) 304, 425-434] may no longer be valid. The most recent model, proposed by Levi-Kalisman et al. [J. Struct. Biol. (2001) 135, 8-17], seemed to be more in accordance with our findings.