Avian neural crest cell migration is diversely regulated by the two major hyaluronan-binding proteoglycans PG-M/versican and aggrecan

It has been proposed that hyaluronan-binding proteoglycans play an important role as guiding cues during neural crest (NC) cell migration, but their precise function has not been elucidated. In this study, we examine the distribution, structure and putative role of the two major hyaluronan-binding proteoglycans, PG-M/versicans and aggrecan, during the course of avian NC development. PG-M/versicans V0 and V1 are shown to be the prevalent isoforms at initial and advanced phases of NC cell movement, whereas the V2 and V3 transcripts are first detected following gangliogenesis. During NC cell dispersion, mRNAs for PG-M/versicans V0/V1 are transcribed by tissues lining the NC migratory pathways, as well as by tissues delimiting nonpermissive areas. Immunohistochemistry confirm the deposition of the macromolecules in these regions and highlight regional differences in the density of these proteoglycans. PG-M/versicans assembled within the sclerotome rearrange from an initially uniform distribution to a preferentially caudal localization, both at the mRNA and protein level. This reorganization is a direct consequence of the metameric NC cell migration through the rostral portion of the somites. As suggested by previous in situ hybridizations, aggrecan shows a virtually opposite distribution to PG-M/versicans being confined to the perinotochordal ECM and extending dorsolaterally in a segmentally organized manner eventually to the entire spinal cord at axial levels interspacing the ganglia. PG-M/versicans purified from the NC migratory routes are highly polydispersed, have an apparent M(r) of 1,200-2,000 kDa, are primarily substituted with chondroitin-6-sulfates and, upon chondroitinase ABC digestion, are found to be composed of core proteins with apparent M(r)of 360–530, 000. TEM/rotary shadowing analysis of the isolated PG-M/versicans confirmed that they exhibit the characteristic bi-globular shape, have core proteins with sizes predicted for the V0/V1 isoforms and carry relatively few extended glycosaminoglycan chains. Orthotopical implantation of PG-M/versicans immobilized onto transplantable micromembranes tend to ‘attract’ moving cells toward them, whereas similar implantations of a notochordal type-aggrecan retain both single and cohorts of moving NC cells in close proximity of the implant and thereby perturb their spatiotemporal migratory pattern. NC cells fail to migrate through three-dimensional collagen type I-aggrecan substrata in vitro, but locomote in a haptotactic manner through collagen type I-PG-M/versican V0 substrata via engagement of HNK-1 antigen-bearing cell surface components. The present data suggest that PG-M/versicans and notochordal aggrecan exert divergent guiding functions during NC cell dispersion, which are mediated by both their core proteins and glycosaminoglycan side chains and may involve ‘haptotactic-like’ motility phenomena. Whereas aggrecan defines strictly impenetrable embryonic areas, PG-M/versicans are central components of the NC migratory pathways favoring the directed movement of the cells

Characterisation of gene expression profiles of yeast cells expressing BRCA1 missense variants

Germline mutations in breast cancer susceptibility gene 1 (BRCA1) confer high risk of developing breast and ovarian cancers. Even though most BRCA1 cancer-predisposing mutations produce a non-functional truncated protein, 5-10% of them cause single amino acid substitutions. This second type of mutations represents a useful tool for examining BRCA1 molecular functions. Human BRCA1 inhibits cell proliferation in transformed Saccharomyces cerevisiae cells and this effect is abolished by disease-associated mutations in the BRCT domain. Moreover, BRCA1 mutations located both inside and outside the BRCT domain may induce an increase in the homologous recombination frequency in yeast cells. Here we present a microarray analysis of gene expression induced in yeast cells transformed with five BRCA1 missense variants, in comparison with gene expression induced by wildtype BRCA1. Data analysis was performed by grouping the BRCA1 variants into three sets: Recombination (R)-set (Y179C and S1164I), Recombination and Proliferation (RP)-set(I1766S and M1775R) and Proliferation (P)-set (A1789T), according to their effects on yeast cell phenotype. We found 470, 740 and 1136 differentially expressed genes in R-, P- and RP-set, respectively. Our results point to some molecular mechanisms critical for the control of cell proliferation and of genome integrity providing support to a possible pathogenic role of the analysed mutations. They also confirm that yeast, despite the absence of a BRCA1 homologue, represents a valid model system to examine BRCA1 molecular functions, as the molecular pathways activated by BRCA1 variants are conserved in humans

Efficacy of conventional versus innovative therapies for treating skin wounds in veterinary medicine

open16siINTRODUCTION: The skin is the largest organ of mammals. The loss of skin integrity may induce important dysfunctions or even death. For superficial wounds, the endogenous healing mechanisms in combination with traditional wound care are sufficient to achieve functional repair. In contrast, in larger wounds, like third and fourth degree burns, chronic wound or deep ulcers it is difficult to obtain the restitutio ad integrum and fibrosis and/or scar tissue develops1,2. The aim of this study was to verify the efficacy of conventional and innovative topic treatments on skin regeneration, induced experimentally in sheep. To achieve this goal different types of investigations (clinical, molecular, histological, immunohistochemical) were performed. METHODS: Six skin lesions (4x4cm) were surgically created on the back of six healthy adult sheep; every single wound was destined, in a randomized way, to one of the following treatments: Acemannan gel, Manuka Honey, hyaluronic acid, Plasma3 (ionized gas), allogeneic mesenchymal stem cells isolated from peripheral blood (PB-MSCs). The sixth wound was the placebo. Biopsies were collected with a surgical punch (0,6x0,6 cm) at time T0, T15 and T40 days. Lesions were clinically evaluated considering the presence and color of wound fluid, the state of hydration, the wound surface/surroundings and other parameters. Histological examinations considered crust formation, re-epithelization and epidermal thickness, dermis edema, extension of granulation tissue, acute and chronic inflammation. Immunohistochemistry for evaluation of inflammation, vascularization and cell proliferation was performed using CD3, CD20, MHCII, von Willebrand factor (vWF) and KI67 antibodies. Furthermore, Real time-PCR investigated genes as V ascular endothelial growth factors (VEGF), Transforming growth factor beta 1(TGFβ1), Vimentin (VIM), Collagen 1α1 (Col1α1) and hair Keratin (hKER). RESULTS: Clinically, the lesions treated with plasma healed more rapidly respect to other treatments and a reduced bacterial load was observed. At T7 wounds treated with stem cells and plasma were less macerated than lesions treated with other therapies. At T15 the wounds treated with hyaluronic acid showed a normal state of hydration while lesions treated with Manuka Honey exhibited a normal hydration from the third week only (Acemannan gel at fourth week). From the second week onwards all wounds did not show presence of fluid and exhibited a dry and clean secondary layer. All lesions, excluded wounds treated with acemannan gel, presented a red (hyaluronic acid and plasma) and dark red (Manuka Honey, PB-MSCs) granulation tissue starting from the first week. Molecular analysis showed a correspondence between clinical and molecular/histologic results. For instance, VEGF mRNA expression confirms angiogenetic events observed at histological level while TGF-β, CD3 and CD20 mRNA/protein expression indicated the presence/absence of inflammation in the used treatments. DISCUSSION & CONCLUSIONS: Innovative therapies led to surprising results regarding regeneration of mammalian skin. Indeed, on the basis of clinical analysis, wounds treated with plasma and MSC healed more rapidly. Further examinations are ongoing in order to elucidate possible mechanisms explaining these differences. REFERENCES: 1S.Y. Broeckx, S. Maes, T. Martinello, et al (2014) Equine epidermis: a source of epithelial-like stem/progenitor cells with in vitro and in vivo regenerative capacities Stem Cells Dev, pp 1134-48. 2J.H. Spaas, C. Gomiero, S.Y. Broeckx, et al (2016) Wound healing markers after autologous and allogeneic epithelial-like stem cell treatment Cytotherapy 2016 (in press). 3E. Martines, M. Zuin, R. Cavazzana, et al. (2009) A novel plasma source for sterilization of living tissues, New J. Phys. 11, 115014.openPatruno, MARCO VINCENZO; Gomiero, Chiara; Martinello, Tiziana; Perazzi, Anna; Gemignani, F; DE BENEDICTIS, GIULIA MARIA; Ferro, Silvia; Zuin, M; Martines, E; Cordaro, Luigi; Brun, Paola; Maccatrozzo, Lisa; Broeckx, Sy; Spaas, Jh; Chiers, K; Iacopetti, IlariaPatruno, MARCO VINCENZO; Gomiero, Chiara; Martinello, Tiziana; Perazzi, Anna; Gemignani, F; DE BENEDICTIS, GIULIA MARIA; Ferro, Silvia; Zuin, M; Martines, E; Cordaro, Luigi; Brun, Paola; Maccatrozzo, Lisa; Broeckx, Sy; Spaas, Jh; Chiers, K; Iacopetti, Ilari

Stem cells and neural signalling: the case of neoblast recruitment and plasticity in low dose X-ray treated planarians

Planarians (Platyhelminthes) possess an abundant population of adult stem cells, the neoblasts, capable to give rise to both somatic and germ cells. Although neoblasts share similar morphological features, several pieces of evidence suggest that they constitute a heterogeneous population of cells with distinct ultrastructural and molecular features. We found that in planarians treated with low X-ray doses (5 Gy), only a few neoblasts survive. Among these cells, those located close to the nervous system activate an intense proliferation program and migrate to reconstitute the whole complex neoblast population. This phenomenon is inhibited by the substance P receptor antagonist spantide, and accompanied by the up-regulation of a number of genes implicated in neuronal signalling and plasticity, suggesting that signals of neural origin modulate neoblast proliferation and/or migration. Here, we review these findings and the literature available on the influence of the nervous system on stem cell activity, both in planarians and vertebrates, and we propose 5 Gy-treated planarians as a unique model system to study the influence of neural signalling on stem cell biolog

IDENTIFICATION OF GENES EXPRESSED IN DIFFERENTIATED CELLS INVOLVED IN STEM CELL REPOPULATION FOLLOWING LOW-DOSE XRAY TREATMENT IN PLANARIAN

A major challenge in stem cell research is the comprehension of the extrinsic control exerted in vivo by the niche. Despite the significant advances derived from the successful application of molecular, cellular, and genomic approaches in planarians, the nature of extrinsic signals regulating stem cell biology remains to be understood. With the aim to identify genes, preferentially enriched in differentiated cells, whose expression is involved in the regulation of stem cells, we took advantage from the stem cell repopulation process that follows low-dose X-ray treatment in planarians. Due to its awfulness, our idea is that this process might represent a context in which the release of signaling molecules by differentiated tissues is highly emphasized. We searched for differentially expressed tags in a DGE library obtained from low-dose treated animals sacrificed between 4 and 7 days after irradiation compared to a DGE library from control animals and we selected some upregulated genes on the basis of their fold change. Expression analysis of the selected genes in intact un-irradiated planarians by WISH experiments revealed that these genes were preferentially expressed in differentiated tissues such as the gut and in the cephalic ganglia and their expression strongly increased in low-dose irradiated animals respect to not-irradiated animals. Genetic silencing of some of them by RNA interference impaired the stem cell repopulation, suggesting a tight Extrinsic control of stem cell activity

Stem cells and regeneration, the case of planarians

Planarians are free-living flatworms, capable of regenerating any missing body part. This remarkable regenerative capacity is due to the presence of an abundant population of stem cells, the neoblasts, dispersed throughout the planarian body. Neoblasts are extremely heterogeneous in terms of X-ray sensitivity, expression profiles and differentiation fates, and include pluripotent clonogenic stem cells, the c-neoblasts. Planarians represent a suitable model system for studying stem cells in their physiological environment, and for the identification of both cell autonomous and extrinsic regulators of stem cell fate. In the last years we identified several neoblast-specific genes, most of which are involved in epigenetic modifications and post-translational regulation. Silencing of some of them by RNA interference induces lethality, inability to regenerate and early differentiation failure. Recently, we also focused our attention on the biosynthetic pathway of polyamines, as putative extrinsic factors involved in stem-cell/differentiated tissue crosstalk. We identified 6 planarian homologues of the ornithine decarboxylase (ODC) gene, the rate-limiting enzyme in polyamine synthesis: four of them are expressed in neoblast late progeny, and functionally involved in stem cell commitment towards the epidermal lineage

Starvation resistance in planarians: multiple strategies to get a thrifty phenotype

: Starvation resistance is a life-saving mechanism for many organisms facing food availability fluctuation in the natural environment. Different strategies have been episodically identified for some model organisms, the first of which was the ability to suppress metabolic rate. Among the identified strategies, the ability of planarians to shrink their body under fasting conditions and revert the process after feeding (the growth-degrowth process) represents a fascinating mechanism to face long periods of fasting. The growth-degrowth process is strictly related to the capability of planarians to continuously maintain tissue homeostasis and body proportions even in challenging conditions, thanks to the presence of a population of pluripotent stem cells. Here, we take advantage of several previous studies describing the growth-degrowth process and of recent progress in the understanding of planarian homeostasis mechanisms, to identify tissue-selective transcriptional downregulation as a driving strategy for the development of a thrifty phenotype, and the p53 transcription factor as a player in adjusting tissue homeostasis in accordance with food availability

Pharmacokinetics of gallium nitrate after oral administration in adult horses \u2013 pilot study

Gallium (Ga), a metal in group IIIA of the periodic table, has shown a remarkable activity against bone resorption and could therefore possibly prove useful in the treatment of certain diseases in sport horses, for example navicular disease. The aim of this study was to gain more information concerning the kinetics of Ga after oral administration of gallium nitrate (GaN) in adult horses. Six horses received a single dose of 10 mg\u2044 kg of GaN mixed with the food ration. Absorption was slow (Tmax = 10 \ub1 3 h, T\ubdabs = 2 \ub1 0.8 h), and a Cmax of 26 \ub1 11 lg \u2044 L was achieved. Excretion followed a one-phase elimination model, with a long half-life (T\ubdel = 52 \ub1 14 h). By means of a mathematical model, we estimated that the plasmatic levels should reach 93 lg \u2044 L (1.33 lM) at steady state, following the repeated daily administration of 10 mg\u2044 kg of GaN. A three times lower concentration has been demonstrated as effective in inhibiting the osteolytic activity of osteoclasts in vitro. The results of this study suggest that the administration of oral GaN at a rate of 10 mg\u2044 kg per day may be considered for future clinical studies

Cell quiescence in planarian stem cells, interplay between p53 and nutritional stimuli

Cell quiescence appeared early in evolution as an adaptive response to adverse conditions (i.e. nutrient depletion). In metazoans, quiescence has been involved in additional processes like tissue homeostasis, which is made possible by the presence of adult stem cells (ASCs). Cell cycle control machinery is a common hub for quiescence entrance, and evidence indicates a role for p53 in establishing the quiescent state of undamaged cells. Mechanisms responsible for waking up quiescent cells remain elusive, and nutritional stimulus, as a legacy of its original role, still appears to be a player in quiescence exit. Planarians, rich in ASCs, represent a suitable system in which we characterized a quiescent population of ASCs, the dorsal midline cord (DMC) cells, exhibiting unique transcriptional features and maintained quiescent by p53 and awakened upon feeding. The function of DMC cells is puzzling and we speculate that DMC cells, despite retaining ancient properties, might represent a functional drift in which quiescence has been recruited to provide evolutionary advantages