In Vitro and In Vivo Effects of Ulvan Polysaccharides from Ulva rigida.

One of the main bioactive compounds of interest from the Ulva species is the sulfated polysaccharide ulvan, which has recently attracted attention for its anticancer properties. This study investigated the cytotoxic activity of ulvan polysaccharides obtained from Ulva rigida in the following scenarios: (i) in vitro against healthy and carcinogenic cell lines (1064sk (human fibroblasts), HACAT (immortalized human keratinocytes), U-937 (a human leukemia cell line), G-361 (a human malignant melanoma), and HCT-116 (a colon cancer cell line)) and (ii) in vivo against zebrafish embryos. Ulvan exhibited cytotoxic effects on the three human cancer cell lines tested. However, only HCT-116 demonstrated sufficient sensitivity to this ulvan to make it relevant as a potential anticancer treatment, presenting an LC50 of 0.1 mg mL-1. The in vivo assay on the zebrafish embryos showed a linear relationship between the polysaccharide concentration and growth retardation at 7.8 hpf mL mg-1, with an LC50 of about 5.2 mg mL-1 at 48 hpf. At concentrations near the LC50, toxic effects, such as pericardial edema or chorion lysis, could be found in the experimental larvae. Our in vitro study supports the potential use of polysaccharides extracted from U. rigida as candidates for treating human colon cancer. However, the in vivo assay on zebrafish indicated that the potential use of ulvan as a promising, safe compound should be limited to specific concentrations below 0.001 mg mL-1 since it revealed side effects on the embryonic growth rate and osmolar balance.

Zebrafish Models for Human Skeletal Disorders.

In 2019, the Nosology Committee of the International Skeletal Dysplasia Society provided an updated version of the Nosology and Classification of Genetic Skeletal Disorders. This is a reference list of recognized diseases in humans and their causal genes published to help clinician diagnosis and scientific research advances. Complementary to mammalian models, zebrafish has emerged as an interesting species to evaluate chemical treatments against these human skeletal disorders. Due to its versatility and the low cost of experiments, more than 80 models are currently available. In this article, we review the state-of-art of this "aquarium to bedside" approach describing the models according to the list provided by the Nosology Committee. With this, we intend to stimulate research in the appropriate direction to efficiently meet the actual needs of clinicians under the scope of the Nosology Committee.

Synthesis, characterization and biological evaluation of octyltrimethylammonium tetrathiotungstate.

Octyltrimethylammonium tetrathiotungstate salt (ATT-C8) was synthesized and its ability to chelate copper was evaluated. The biological and toxic aspects were evaluated by in vitro and in vivo assays, using bovine aorta endothelial cells (BAEC) and zebrafish (Danio rerio) embryos. The obtained results suggest that ATT-C8 has better biocompatibility, showing a significantly lower lethal concentration 50 (LC50) value in comparison to ammonium tetrathiotungstate (ATT). Zebrafish embryos assay results indicate that both tetrathiotungstate salts at the studied concentrations increase the hatching time. Even more, an in vivo assay showed that synthesized materials behave as copper antagonists and have the ability to inhibit its toxicological effects. Also, both materials were found to be active for the in vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The characterization of the materials was carried out using the following spectroscopic techniques: Ultraviolet-Visible (UV-Vis), Fourier Transform Infrared (FTIR) and proton nuclear magnetic resonance (1H-NRM).

Fasentin diminishes endothelial cell proliferation, differentiation and invasion in a glucose metabolism-independent manner.

The synthetic compound fasentin has been described as a modulator of GLUT-1 and GLUT-4 transporters, thus inhibiting glucose uptake in some cancer cells. Endothelial glucose metabolism has been recently connected to angiogenesis and it is now an emerging topic in scientific research. Indeed, certain compounds with a known effect on glucose metabolism have also been shown to inhibit angiogenesis. In this work we tested the capability of fasentin to modulate angiogenesis in vitro and in vivo. We show that fasentin inhibited tube formation in endothelial cells by a mechanism that involves a negative effect on endothelial cell proliferation and invasion, without affecting other steps related to the angiogenic process. However, fasentin barely decreased glucose uptake in human dermal microvascular endothelial cells and the GLUT-1 inhibitor STF-31 failed to inhibit tube formation in these cells. Therefore, this modulatory capacity on endothelial cells function exerted by fasentin is most likely independent of a modulation of glucose metabolism. Taken together, our results show a novel biological activity of fasentin, which could be evaluated for its utility in cancer and other angiogenesis-dependent diseases.

Widening control of fin inter-rays in zebrafish and inferences about actinopterygian fins.

The amputation of a teleost fin rapidly triggers an intricate maze of hierarchically regulated signalling processes which ultimately reconstruct the diverse tissues of the appendage. Whereas the generation of the fin pattern along the proximodistal axis brings with it several well-known developmental regulators, the mechanisms by which the fin widens along its dorsoventral axis remain poorly understood. Utilizing the zebrafish as an experimental model of fin regeneration and studying more than 1000 actinopterygian species, we hypothesized a connection between specific inter-ray regulatory mechanisms and the morphological variability of inter-ray membranes found in nature. To tackle these issues, both cellular and molecular approaches have been adopted and our results suggest the existence of two distinguishable inter-ray areas in the zebrafish caudal fin, a marginal and a central region. The present work associates the activity of the cell membrane potassium channel kcnk5b, the fibroblast growth factor receptor 1 and the sonic hedgehog pathway to the control of several cell functions involved in inter-ray wound healing or dorsoventral regeneration of the zebrafish caudal fin. This ray-dependent regulation controls cell migration, cell-type patterning and gene expression. The possibility that modifications of these mechanisms are responsible for phenotypic variations found in euteleostean species, is discussed.

Fishing anti(lymph)angiogenic drugs with zebrafish.

Zebrafish, an amenable small teleost fish with a complex mammal-like circulatory system, is being increasingly used for drug screening and toxicity studies. It combines the biological complexity of in vivo models with a higher-throughput screening capability compared with other available animal models. Externally growing, transparent embryos, displaying well-defined blood and lymphatic vessels, allow the inexpensive, rapid, and automatable evaluation of drug candidates that are able to inhibit neovascularisation. Here, we briefly review zebrafish as a model for the screening of anti(lymph)angiogenic drugs, with emphasis on the advantages and limitations of the different zebrafish-based in vivo assays.

Copper-containing mesoporous bioactive glass promotes angiogenesis in an in vivo zebrafish model.

The osteogenic and angiogenic responses of organisms to the ionic products of degradation of bioactive glasses (BGs) are being intensively investigated. The promotion of angiogenesis by copper (Cu) has been known for more than three decades. This element can be incorporated to delivery carriers, such as BGs, and the materials used in biological assays. In this work, Cu-containing mesoporous bioactive glass (MBG) in the SiO2-CaO-P2O5 compositional system was prepared incorporating 5% mol Cu (MBG-5Cu) by replacement of the corresponding amount of Ca. The biological effects of the ionic products of MBG biodegradation were evaluated on a well-known endothelial cell line, the bovine aorta endothelial cells (BAEC), as well as in an in vivo zebrafish (Danio rerio) embryo assay. The results suggest that ionic products of both MBG (Cu free) and MBG-5Cu materials promote angiogenesis. In vitro cell cultures show that the ionic dissolution products of these materials are not toxic and promote BAEC viability and migration. In addition, the in vivo assay indicates that both exposition and microinjection of zebrafish embryos with Cu free MBG material increase vessel number and thickness of the subintestinal venous plexus (SIVP), whereas assays using MBG-5Cu enhance this effect. STATEMENT OF SIGNIFICANCE: Mesoporous bioactive glasses (MBGs) with high specific surface area, well-ordered pores, large pore volumes and controllable amount of ions are interesting to develop controlled drug delivery systems for bone tissue regeneration. Copper (Cu) incorporation to the basic SiO2-CaO-P2O5 composition has attracted high interest due to its multifunctional biological properties. Promotion of angiogenesis is one of these properties, which can be integrated to the biomaterial with lower cost and higher stability when compared with growth factors. This work reports the synthesis and characterization of Cu-containing MBG evaluating its angiogenic properties in the subintestinal vessel zebrafish assay. This transgenic in vivo assay is merging as an alternative model providing short-time consuming protocols and facilities during pro-angiogenic drug screenings. The report shows that the ionic products of this MBG material delivered to the zebrafish incubation media significantly enhance angiogenesis in comparison with control groups. Besides, results indicate Cu ions may exhibit a synergic effect with Si, Ca, and P ions in angiogenesis stimulation both in vitro and in vivo. To our knowledge, this is the first time that zebrafish in vivo assays are used to evaluate angiogenic activity of ionic dissolution products from MBG materials.

Regenerative Endodontic Procedures: A Perspective from Stem Cell Niche Biology.

INTRODUCTION: Endodontics uses cell therapy strategies to treat pulpal and periapical diseases. During these therapies, surgeons aim to reconstruct the natural microenvironments that regulate the activity of dental stem cells. METHODS: We searched for more than 400 articles in PubMed using key words from regenerative endodontics and dental stem cell biology. In 268 articles, we reviewed what factors may influence histologic results after preclinical dental treatments that use regenerative endodontic procedures after pulpectomy. RESULTS: Several factors, such as the origin of stem cells, the biomimicry of scaffolds used, and the size of lesions, are considered to influence the histologic appearance of the regenerated pulp-dentin complex after treatments. Information is accumulating on transcription factors that generate the pulp-dentin complex and survival/trophic factors that would benefit niche recovery and histologic results. CONCLUSIONS: In this article, we discuss the noninterchangeability of stem cells, the influence of dentin-entrapped molecule release on pulp regeneration and survival of stem cells, and the need of positional markers to assess treatments histologically. The ex vivo amplification of appropriate dental stem cells, the search for scaffolds storing the molecular diversity entrapped in the dentin, and the use of positional transcription factors as histologic markers are necessary to improve future preclinical experiments.

Toluquinol, a marine fungus metabolite, is a new angiosuppresor that interferes with the Akt pathway.

Toluquinol, a methylhydroquinone produced by a marine fungus, was selected in the course of a blind screening for new potential inhibitors of angiogenesis. In the present study we provide the first evidence that toluquinol is a new anti-angiogenic-compound. In a variety of experimental systems, representing the sequential events of the angiogenic process, toluquinol treatment of activated endothelial cells resulted in strong inhibitory effect. Toluquinol inhibited the growth of endothelial and tumor cells in culture in the micromolar range. Our results indicate that the observed growth inhibitory effect could be due, at least in part, to an induction of apoptosis. Toluquinol induced endothelial cell death is mediated via apoptosis after a cell cycle block and caspase activation. Capillary tube formation on Matrigel and migratory, invasive and proteolytic capabilities of endothelial cells were inhibited by addition of toluquinol at subtoxic concentrations. Inhibition of the mentioned essential steps of in vitro angiogenesis agrees with the observed inhibition of the in vivo angiogenesis, substantiated by using the chick chorioallatoic membrane assay and confirmed by the murine Matrigel plug, the zebrafish embryo neovascularization and the zebrafish caudal fin regeneration assays. Data here shown altogether indicate that toluquinol has antiangiogenic effects both in vitro and in vivo that are exerted partly by suppression of the VEGF and FGF-induced Akt activation of endothelial cells. These effects are carried out at lower concentrations to those required for other inhibitors of angiogenesis, what makes toluquinol a promising drug candidate for further evaluation in the treatment of cancer and other angiogenesis-related pathologies.

Dimethylfumarate inhibits angiogenesis in vitro and in vivo: a possible role for its antipsoriatic effect?

The fumaric acid esters (FAEs) have been used for the oral treatment of psoriasis for some 50 years. Given that a persistent and maintained angiogenesis is associated with several cutaneous diseases, including psoriasis, we sought in our study to gain further insight into their mechanism of action by investigating whether FAEs are able to interfere with angiogenesis mechanisms. Our results demonstrate that dimethylfumarate (DMF) inhibits certain functions of endothelial cells, namely, differentiation, proliferation, and migration. This activity was not exhibited by similar concentrations of monomethylfumarate or fumaric acid. Our data indicate that DMF inhibits the growth of transformed and nontransformed cells in a dose-dependent manner. The growth-inhibitory effect exerted by this compound on proliferating endothelial cells could be due, at least in part, to an induction of apoptosis. Inhibition by DMF of the mentioned essential steps of in vitro angiogenesis is consistent with the observed inhibition of in vivo angiogenesis, substantiated using chick chorioallantoic membrane and live fluorescent zebrafish embryo neovascularization assays. The antiangiogenic activity of DMF may contribute to the antipsoriatic, antitumoral, and antimetastatic activities of this compound and suggests its potential in the treatment of angiogenesis-related malignancies.

The stem cell niche should be a key issue for cell therapy in regenerative medicine.

Recent advances in stem cell research have highlighted the role played by such cells and their environment (the stem cell niche) in tissue renewal and homeostasis. The control and regulation of stem cells and their niche are remaining challenges for cell therapy and regenerative medicine on several tissues and organs. These advances are important for both, the basic knowledge of stem cell regulation, and their practical translational applications into clinical medicine. This article is primarily concerned with the mesenchymal stem cells (MSCs) and it reviews the current aspects of their own niche. We discuss on the need for a deeper understanding of the identity of this cell type and its microenvironment in order to improve the effectiveness of any cell therapy for regenerative medicine. Ex vivo reproduction of the conditions of the natural stem cell niche, when necessary, would provide success to tissue engineering. The first challenge of regenerative medicine is to find cells able to replace and/or repair the lost function of tissues and organs by disease or aging and the trophic and immunomodulatory effects recently found for MSCs open up for new opportunities. If MSCs are pericytes, as it has been proposed, perhaps it may explain the ubiquity of these cells and their possible role in miscellaneous repairs throughout the body opening for new chances for extensive tissue repair.

Freeze substitution followed by low melting point wax embedding preserves histomorphology and allows protein and mRNA localization techniques.

Fixation and embedding are major steps in tissue preservation for histological analysis. However, conventional fixatives like aldehyde-based solutions usually mask tissular epitopes preventing their immunolocalization. Alternative fixation methods used to avoid this drawback, such as cryopreservation, alcohol- or zinc salts-based fixatives do not efficiently preserve tissue and cell morphology. Likewise, paraffin and resin embedding, commonly used for thin sectioning, frequently damage epitopes due to the clearing agents and high temperatures needed along the embedding procedure. Alternatives like cryosectioning avoid the embedding steps but yield sections of poorer quality and are not suitable for all kinds of samples. To overcome these handicaps, we have developed a method that preserves histoarchitecture as well as tissue antigenic properties. This method, which we have named CryoWax, involves freeze substitution of the samples in isopentane and methanol, followed by embedding in low melting point polyester wax. CryoWax has proven efficient in obtaining thin sections of embryos and adult tissues from different species, including amphioxus, zebrafish, and mouse. CryoWax sections displayed optimal preservation of tissue morphology and were successfully immunostained for fixation- and temperature-sensitive antigens. Furthermore, CryoWax has been tested for in situ hybridization application, obtaining positive results.

Dermoskeleton morphogenesis in zebrafish fins.

Zebrafish fins have a proximal skeleton of endochondral bones and a distal skeleton of dermal bones. Recent experimental and genetic studies are discovering mechanisms to control fin skeleton morphogenesis. Whereas the endochondral skeleton has been extensively studied, the formation of the dermal skeleton requires further revision. The shape of the dermal skeleton of the fin is generated in its distal growing margin and along a proximal growing domain. In these positions, dermoskeletal fin morphogenesis can be explained by intertissue interactions and the function of several genetic pathways. These pathways regulate patterning, size, and cell differentiation along three axes. Finally, a common genetic control of late development, regeneration, and tissue homeostasis of the fin dermoskeleton is currently being analyzed. These pathways may be responsible for the similar shape obtained after each morphogenetic process. This provides an interesting conceptual framework for future studies on this topic.

The teaching of Developmental Biology in Spain: future challenges.

Developmental biology and/or embryology prospectuses in Spanish universities are reviewed. From old masterly classes to virtual teaching, a variety of methods are being used nowadays to teach these subjects. In a country like Spain, adapting to the European Higher Education Space, old and new teaching methods are educational alternatives in a university model which respects both tradition and modernity. In this report, consensus guidelines, concluded from this general survey, are suggested for teachers to ensure future progress.

Zebrafish fins as a model system for skeletal human studies.

Recent studies on the morphogenesis of the fins of Danio rerio (zebrafish) during development and regeneration suggest that a number of inductive signals involved in the process are similar to some of those that affect bone and cartilage differentiation in mammals and humans. Akimenko et al. (2002) has shown that bone morphogenetic protein-2b (BMP2b) is involved in the induction of dermal bone differentiation during fin regeneration. Many other groups have also shown that molecules from the transforming growth factor-beta superfamily (TGFb), including BMP2, are effective in promoting chondrogenesis and osteogenesis in vivo in higher vertebrates, including humans. In the present study, we review the state of the art of this topic by a comparative analysis of skeletal tissue development, regeneration and renewal processes in tetrapods, and fin regeneration in fishes. A general conclusion of this study states that lepidotrichia is a special skeletal tissue different to cartilage, bone, enamel, or dentine in fishes, according to its extracellular matrix (ECM) composition. However, the empirical analysis of inducing signals of skeletal tissues in fishes and tetrapods suggests that lepidotrichia is different to any responding features with main skeletal tissues. A number of new inductive molecules are arising from fin development and regeneration studies that might establish an empirical basis for further molecular approaches to mammal skeletal tissues differentiation. Despite the tissue dissimilarity, this empirical evidence might finally lead to clinical applications to skeletal disorders in humans.

Differential regulation of msx genes in the development of the gonopodium, an intromittent organ, and of the "sword," a sexually selected trait of swordtail fishes (Xiphophorus).

The possession of a conspicuous extension of colored ventral rays of the caudal fin in male fish of swordtails (genus Xiphophorus) is a prominent example for a trait that evolved by sexual selection. To understand the evolutionary history of this so-called sword molecularly, it is of interest to unravel the developmental pathways responsible for extended growth of sword rays during development of swordtail males. We isolated two msx genes and showed that they are differentially regulated during sword outgrowth. During sword growth in juvenile males, as well as during testosterone-induced sword development and fin ray regeneration in the sword after amputation, expression of msxC is markedly up-regulated in the sword forming fin rays. In contrast, msxE/1 is not differentially expressed in ventral and dorsal male fin rays, suggesting a link between the development of male secondary sexual characters in fins and up-regulation of msxC expression. In addition, we showed that msx gene expression patterns differ significantly between Xiphophorus and zebrafish. We also included in our study the gonopodium, a testosterone-dependent anal fin modification that serves as a fertilization organ in males of live-bearing fishes. Our finding that increased levels of msxC expression are associated with the testosterone-induced outgrowth of the gonopodium might suggest either that at least parts of the signaling pathways that pattern the evolutionary older gonopodium have been coopted to evolve a sexually selected innovation such as the sword or that increased msxC expression may be inherent to the growth process of long fin rays in general.

Old questions, new tools, and some answers to the mystery of fin regeneration.

Pluridisciplinary approaches led to the notion that fin regeneration is an intricate phenomenon involving epithelial-mesenchymal and reciprocal exchanges throughout the process as well as interactions between ray and interray tissue. The establishment of a blastema after fin amputation is the first event leading to the reconstruction of the missing part of the fin. Here, we review our knowledge on the origin of the blastema, its formation and growth, and of the mechanisms that control differentiation and patterning of the regenerate. Our current understanding results from studies of fin regeneration performed in various teleost fish over the past century. We also report the recent breakthroughs that have been made in the past decade with the arrival of a new model, the zebrafish, Danio rerio, which now offers the possibility to combine cytologic, molecular, and genetic analyses and open new perspectives in this field.

Function of trans-acting genes of theachaete-scute complex in sensory organ patterning in the mesonotum ofDrosophila.

Cell-cell interactions play a fundamental role in the differentiation of nervous elements in constant patterns, both during embryogenesis and imaginal development. In this paper we analyse the role of genes of theachaete-scute andEnhancer of split complexes, plus the genesextramacrochaetae, Notch, Delta, andHairless in the patterning of sensory elements in the mesonotum ofDrosophila. The phenotypes of different alleles of these genes, including lethals in genetic mosaics, reveal their participation in two processes, the singling out from epidermal cells of sensory organ mother cells and their subsequent differentiation. Studies of allelic combinations of different genes lead to a model of the genetic interactions involved in the processes of pattern formation. In this model, theachaete-scute complex plays a central role, determining sensory organ mother cells and preventing neighbouring cells from following the same developmental pathway.

Genetic and developmental analyses of chaetae pattern formation in Drosophila tergites.

The role of the achaete-scute complex and extramacrochaetae, Notch, Delta, Enhancer of split and Hairless genes in chaeta patterning in Drosophila tergites was studied in genetic mosaics and in mutant combinations. The mutant phenotypes of different alleles of each gene can be ordered in characteristic topographical seriations. These seriations are related to the pattern of proliferation of histoblasts and the time of singularization of sensory organ mother cells from surrounding epidermal cells. Genetic mosaics of lethal alleles show that these genes are fundamentally involved in this singularization and subsequent differentiation. The study of mutant combinations of alleles of these genes reveals specific relationships of epistasis and synergism between them. The results suggest that spatial and temporal variations in achaete-scute complex functional products in cells, modulated by the activity of other genes involved in signal transduction, define the patterned differentiation of sensory organs in tergites.