Genome-wide analysis of the repertoire of TRIM genes in sea urchins

The eukaryotic TRIM (TRIpartite Motif) super-family represents one of the largest classes of putative E3 ubiquitin ligases involved in several processes, including epigenetic control of development and disease. In the post-genomic era, new approaches allow genome-wide studies of gene family. In particular, we performed a comprehensive analysis of the TRIM repertoire in selected sea urchin species. By combining iterations of ab initio predictions and pairwise comparative methods, we first retrieved the full complement of TRIM genes in Strongylocentrotus purpuratus, whose full genome sequence was available. Interestingly, such a DNA sequence set includes not previously classified, echinoderm-specific, TRIM genes as well as multiple copies of known ones. We also retrieved a landscape of cDNA sequences from staged EST libraries, indicating that most of these genes are actively transcribed during development. Phylogenetic analysis of the deduced proteins, using set of TRIMs from various species, revealed a degree of genetic variation between species. Worth of mention, we predicted the occurrence of transposition events involving some of these genes, according with the documented rapid evolution of this family. Next, we adopted heuristic algorithms and post-processing steps to investigate the evolutionarily distant Paracentrotus lividus, Allocentrotus fragilis and Lytechinus variegatus genomes, whose sequencing projects are actually in progress. We assembled partial pools of TRIM genes and specifically associated them to EST-derived cDNA sequences. Such a collection of data should provide a framework for unravel gene regulatory networks involving TRIM genes from an evolutionary perspective. Indeed, in the Pl species, we have previously isolated and functionally characterized the cDNA sequence encoding the first echinoderm TRIM factor, Strim1. Here, we identified five strim1 genes, all sharing a intronless organization, and roughly located their cis-regulatory apparatus

The Compass-like Locus, Exclusive to the Ambulacrarians, Encodes a Chromatin Insulator Binding Protein in the Sea Urchin Embryo

Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer activity, and this, in turn, accounts for the different level of accumulation of early linker and nucleosomal transcripts

EFFECT OF Γ-AMINOBUTYRRIC ACID (GABA) EXPOSURE ON EMBRYOGENESIS OF PARACENTROTUS LIVIDUS AND IDENTIFICATION OF GABA-RECEPTOR GENES IN SEA URCHINS

Developmental processes are controlled by regulatory genes encoding for transcription factors and signaling molecules. Functional relationships between these genes are described by gene regulatory networks (GRN), models which allow integration of various levels of information. The sea urchin embryo is an experimental model system which offers many advantages for the analysis of GRN. Recently, the GRN that governs the biomineralization of the sea urchin embryonic skeleton has begun to be deciphered. Preliminary evidence suggest that the γ- aminobutyric acid (GABA) signaling pathway is involved in skeletal morphogenesis during development of the sea urchin. GABA is a molecule synthesized by nearly all organism, from bacteria to humans, and it acts through ionotropic and metabotropic receptors (GABAA-Rs and GABAB-Rs, respectively). We report that Paracentrotus lividus embryos exposed to GABA at concentrations ranging from 0.01 to 1.0 mM showed aberrations in axial patterning, with a dose dependent effect. Washout experiments allowed to determine that the period of sensitivity is restricted from the blastula to the gastrula stage. In order to identify GABA-R genes we performed a comprehensive in silico analysis in selected sea urchin species (P. lividus, Strongylocentrotus purpuratus, and Lytechinus variegatus), and in phylogenetically related organisms, such as the hemichordate Saccoglossus kowalevskii, the chordate Ciona intestinalis, and the nematode Caenorhabditis elegans. By combining iteration of ab initio predictions and pairwise comparative methods, we identified the orthologous genes encoding for GABAB1 and GABAB2, the two subunits which assemble GABAB-R, and we confirmed that all of these organisms possess a unique α/β GABAA-R gene pair clustered in the genome. Furthermore, we have observed that the reciprocal disposition of GABAA-R genes is also evolutionarily conserved. Interestingly, in adjacent position to these genes, we have identified an additional gene, which shows significant sequence similarity to a invertebrate-specific GABAA-R gene. Indeed, such a gene has been only identified in C. elegans, Drosophila melanogaster, and Nematostella vectensis. We also retrieved several cDNA sequences from staged EST databases of the three sea urchin species inspected, indicating that these genes are actively transcribed during development. Some selected cDNA plasmids were also isolated from P. lividus total RNA samples and fully sequenced. Hypothetical proteins were deduced and used for phylogenetic analysis, including a selection of vertebrate and invertebrate GABAA-R subunit sequences. The resulting phylogenetic tree strongly support the hypothesis that the sea urchins contain genes encoding for both canonical and invertebrate-specific GABAA-R subunits. Such a collection of data should provide a support to better understand the involvement of GABA-signalling pathway in the skeletal GR

Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo

In the indirect developing sea urchin embryo, the primary mesenchyme cells (PMCs) acquire most of the positional and temporal information from the overlying ectoderm for skeletal initiation and growth. In this study, we characterize the function of the novel gene strim1, which encodes a tripartite motif-containing (TRIM) protein, that adds to the list of genes constituting the epithelial-mesenchymal signaling network. We report that strim1 is expressed in ectoderm regions adjacent to the bilateral clusters of PMCs and that its misexpression leads to severe skeletal abnormalities. Reciprocally, knock down of strim1 function abrogates PMC positioning and blocks skeletogenesis. Blastomere transplantation experiments establish that the defects in PMC patterning, number and skeletal growth depend upon strim1 misexpression in ectoderm cells. Furthermore, clonal expression of strim1 into knocked down embryos locally restores skeletogenesis. We also provide evidence that the Otp and Pax2/5/8 regulators, as well as FGFA, but not VEGF, ligand act downstream to strim1 in ectoderm cells, and that strim1 triggers the expression of the PMC marker sm30, an ectoderm-signaling dependent gene. We conclude that the strim1 function elicits specific gene expression both in ectoderm cells and PMCs to guide the skeletal biomineralization during morphogenesis

Técnica do retalho coronalmente avançado com uso de enxertos de tecido conjuntivo ou de fibrina rica em plaquetas. Revisão sistemática integrativa

Introdução: Nos últimos anos, devido à sua elevada frequência, um dos desafios para os profissionais de saúde oral é o tratamento das recessões gengivais. Está condição pode ser de origem anatómica, fisiológica ou patológica, a identificação dos seus agentes etiológicos determina o sucesso do tratamento na gestão das recessões gengivais. O retalho coronalmente avançado é uma técnica muito comum para o tratamento das recessões gengivais. Objetivos: O objetivo deste trabalho é avaliar se o tecido conjuntivo e os enxertos de fibrina ricas em plaquetas melhoram os resultados clínicos na técnica de retalho coronalmente avançado. Material e métodos: Para realizar a nossa investigação foram utilizados vários websites científicos, tais como PubMed e ResearchGate, utilizando palavras-chave: “Coronally Advanced Flap” “Connective Tissue Graft”; “Platelet Rich Fibrin”; “Gingival Recessions” e abrangendo artigos entre 2012 e 2022 de idioma inglês. Resultados: Fizemos uma pesquisa, com um número total de 760 artigos. 18 artigos foram selecionados para a tabela comparativa e cada artigo selecionado foi analisado para mostrar todos os resultados de retalho coronalmente avançado com ou sem a utilização de enxertos de PRF e de tecido conjuntivo. Discussão: A prevalência da recessão gengival é elevada, afetando potencialmente mais de 50% da população em pelo menos um dente. Ao fim de realizar esta revisões sistematica foi feita uma comparação dos artigos, tentando estabelecer quais dos enxertos entre tecido conjuntivo e fibrina rica em plaquetas é mais vantajoso para a técnica do retalho coronalmente avançado. Conclusão: O retalho coronalmente avançado combinado com enxerto de tecido conjuntivo tem demonstrado melhores resultados clínicos do que a utilização de fibrina rica em plaquetas. A fibrina rica em plaquetas demonstrou ter melhores resultados na dor e no desconforto pós-operatório, mas para substituir o enxerto de tecido conjuntivo, precisamos de estudos com período de estudo a longo prazo.Introduction: In recent years, due to its high frequency, one of the challenges for oral health professionals is the treatment of gingival recessions. This condition may be of anatomical, physiological or pathological origin, and the identification of its etiologic agents determines the success of treatment in the management of gingival recessions. The coronally advanced flap is a very common technique for the treatment of gingival recessions. Objectives: The aim of this paper is to evaluate whether connective tissue and platelet-rich fibrin grafts improve clinical outcomes in coronally advanced flap technique. Material and methods: To carry out our investigation we used several scientific websites, such as PubMed and ResearchGate, using keywords: "Coronally Advanced Flap" "Connective Tissue Graft"; "Platelet Rich Fibrin"; "Gingival Recessions" and covering articles between 2012 and 2022 of English language. Results: We performed a search, with a total number of 760 articles. 18 articles were selected for the comparative table and each selected article was analysed to show all the results of coronally advanced flap with or without the use of PRF and connective tissue grafts. Discussion The prevalence of gingival recession is high, potentially affecting more than 50% of the population in at least one tooth. In order to perform this systematic review a comparison of articles was made, trying to establish which of the grafts between connective tissue and platelet-rich fibrin is more advantageous for the coronally advanced flap technique. Conclusion: Coronal advanced flap combined with connective tissue graft has shown better clinical results than the use of platelet-rich fibrin. Platelet-rich fibrin has been shown to have better results in postoperative pain and discomfort, but to replace connective tissue graft, we need studies with long-term study period

Impairing Otp homeodomain function in oral ectoderm cells affects skeletogenesis in sea urchin embryos

AbstractIn the sea urchin embryo skeletogenesis is the result of a complex series of molecular and cellular events that coordinate the morphogenetic process. Past and recent evidence strongly indicate that skeletal initiation and growth are strictly dependent on signals emanating from the oral ectodermal wall. As previously suggested, Orthopedia (Otp), a homeodomain-containing transcription factor specifically expressed in a small subset of oral ectoderm cells, might be implicated in this signalling pathway. In this study, we utilize three different strategies to address the issue of whether Otp is an upstream regulator of sketelogenesis. We describe the effects of microinjection of Otp morpholino-substituted antisense oligonucleotides and dominant-negative Otp-engrailed mRNA in Paracentrotus lividus embryos. We demonstrate that inhibition of Otp expression completely abolishes skeletal synthesis. By contrast, coinjection of Otp mRNA and the morpholino antisense oligonucleotide specifically rescues the skeletogenic program. In addition, localized ectodermal expression of the Otp-GFP fusion gene construct driven by the hatching enzyme promoter, induces ectopic and abnormal spiculogenesis. We further show that an indirect target of this homeoprotein is the skeletogenic specific gene SM30, whose expression is known to be under the strict control of the oral ectoderm territory. Based on these results, we conclude that Otp triggers the ectoderm-specific signal that promotes skeletogenesis

cis-Regulatory sequences driving the expression of the Hbox12 homeobox-containing gene in the presumptive aboral ectoderm territory of the Paracentrotus lividus sea urchin embryo.

Embryonic development is coordinated by networks of evolutionary conserved regulatory genes encoding transcription factors and components of cell signalling pathways. In the sea urchin embryo, a number of genes encoding transcription factors display territorial restricted expression. Among these, the zygotic Hbox12 homeobox gene is transiently transcribed in a limited number of cells of the animal-lateral half of the early Paracentrotus lividus embryo, whose descendants will constitute part of the ectoderm territory. To obtain insights on the regulation of Hbox12 expression, we have explored the cis-regulatory apparatus of the gene. In this paper, we show that the intergenic region of the tandem Hbox12 repeats drives GFP expression in the presumptive aboral ectoderm and that a 234 bp fragment, defined aboral ectoderm (AE) module, accounts for the restricted expression of the transgene. Within this module, a consensus sequence for a Sox factor and the binding of the Otx activator are both required for correct Hbox12 gene expression. Spatial restriction to the aboral ectoderm is achieved by a combination of different repressive sequence elements. Negative sequence elements necessary for repression in the endomesoderm map within the most upstream 60 bp region and nearby the Sox binding site. Strikingly, a Myb-like consensus is necessary for repression in the oral ectoderm, while down-regulation at the gastrula stage depends on a GA-rich region. These results suggest a role for Hbox12 in aboral ectoderm specification and represent our first attempt in the identification of the gene regulatory circuits involved in this process

Constitutive Promoter Occupancy by the MBF-1 Activator and Chromatin Modification of the Developmental Regulated Sea Urchin alpha-H2A Histone Gene

The tandemly repeated sea urchin α-histone genes are developmentally regulated. These genes are transcribed up to the early blastula stage and permanently silenced as the embryos approach gastrulation. As previously described, expression of the α-H2A gene depends on the binding of the MBF-1 activator to the 5′ enhancer, while down-regulation relies on the functional interaction between the 3′ sns 5 insulator and the GA repeats located upstream of the enhancer. As persistent MBF-1 binding and enhancer activity are detected in gastrula embryos, we have studied the molecular mechanisms that prevent the bound MBF-1 from trans-activating the H2A promoter at this stage of development. Here we used chromatin immunoprecipitation to demonstrate that MBF-1 occupies its site regardless of the transcriptional state of the H2A gene. In addition, we have mapped two nucleosomes specifically positioned on the enhancer and promoter regions of the repressed H2A gene. Interestingly, insertion of a 26 bp oligonucleotide between the enhancer and the TATA box, led to upregulation of the H2A gene at gastrula stage, possibly by changing the position of the TATA nucleosome. Finally, we found association of histone de-acetylase and de-acetylation and methylation of K9 of histone H3 on the promoter and insulator of the repressed H2A chromatin. These data argue for a role of a defined positioned nucleosome in the promoter and histone tail post-translational modifications, in the 3′ insulator and 5′ regulatory regions, in the repression of the α-H2A gene despite the presence of the MBF-1 activator bound to the enhance

Electrical conductivity of carbon nanofiber reinforced resins: potentiality of Tunneling Atomic Force Microscopy (TUNA) technique

Epoxy nanocomposites able to meet pressing industrial requirements in the field of structural material have been developed and characterized. Tunneling Atomic Force Microscopy (TUNA), which is able to detect ultra-low currents ranging from 80 fA to 120 pA, was used to correlate the local topography with electrical properties of tetraglycidyl methylene dianiline (TGMDA) epoxy nanocomposites at low concentration of carbon nanofibers (CNFs) ranging from 0.05% up to 2% by wt. The results show the unique capability of TUNA technique in identifying conductive pathways in CNF/resins even without modifying the morphology with usual treatments employed to create electrical contacts to the ground