Cell senescence, apoptosis and DNA damage cooperate in the remodeling processes accounting for heart morphogenesis

During embryonic development, organ morphogenesis requires major tissue rearrangements that are tightly regulated at the genetic level. A large number of studies performed in recent decades assigned a central role to programmed cell death for such morphogenetic tissue rearrangements that often sculpt the shape of embryonic organs. However, accumulating evidence indicates that far from being the only factor responsible for sculpting organ morphology, programmed cell death is accompanied by other tissue remodeling events that ensure the outcome of morphogenesis. In this regard, cell senescence has been recently associated with morphogenetic degenerative embryonic processes as an early tissue remodeling event in development of the limbs, kidney and inner ear. Here, we have explored cell senescence by monitoring ?-galactosidase activity during embryonic heart development where programmed cell death is believed to exert an important morphogenetic function. We report the occurrence of extensive cell senescence foci during heart morphogenesis. These foci overlap spatially and temporally with the areas of programmed cell death that are associated with remodeling of the outflow tract to build the roots of the great arteries and with the septation of cardiac cavities. qPCR analysis allowed us to identify a gene expression profile characteristic of the so-called senescence secretory associated phenotype in the remodeling outflow tract of the embryonic heart. In addition, we confirmed local upregulation of numerous tumor suppressor genes including p21, p53, p63, p73 and Btg2. Interestingly, the areas of cell senescence were also accompanied by intense lysosomal activation and non-apoptotic DNA damage revealed by ?H2AX immunolabeling. Considering the importance of sustained DNA damage as a triggering factor for cell senescence and apoptosis, we propose the coordinated contribution of DNA damage, senescence and apoptotic cell death to assure tissue remodeling in the developing vertebrate heart.Funding: Thanks are due to Montse Fernandez-Calderon, Sonia Perez-Mantecon and Susana Dawalibi for technical assistance. This work was supported by Grant BFU2017-84046-P from the Spanish Ministry of Science, Innovation and Universities. C S-F is a recipient of a FPI predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universitie

Cell Death in the Embryonic Developing Limb

In amniote vertebrates, the development of form and structure of the limb bud is accompanied by precise patterns of massive mesodermal cell death with morphological features of apoptosis. These areas of cell death appear to eliminate undifferentiated cells which are required only for a limited time period of limb development. Predictable skeletal and morphological anomalies of the limb occur when the pattern of cell death is modified in mutant species or under experimental conditions. Most evidence points to the occurrence of local triggering mechanisms to account for the establishment of the areas of cell death and the subsequent activation of cell death genes. Modifications of the extracellular matrix and diminution in the contribution of growth factors by neighbouring tissues appear as the most likely potential candidates for triggering the cell death program. Information on the genetical basis of cell death in the developing limb is very scarce. Among the increasing number of cell death genes identified in other cell death systems, such as p-53 and the ced-3/ICE and ced-9/bcl-2 gene families, only bcl-2 has been studied in detail during limb development and yet, the information obtained is contradictory. Bcl-2 is not expressed in the areas of cell death of the developing limb, but normal limbs develop in mice with disruption of the bcl-2 gene. Obviously, the clarification of the role of the cell death genes constitute a major task in future studies of cell death in the developing limb

Activin/TGFβ and BMP crosstalk determines digit chondrogenesis

AbstractThe progress zone (PZ) is a specialized area at the distal margin of the developing limb where mesodermal cells are kept in proliferation and undifferentiated, allowing limb outgrowth. At stages of digit morphogenesis the PZ cells can undergo two possible fates, either aggregate initiating chondrogenic differentiation to configure the digit blastemas, or to die by apoptosis if they are incorporated in the interdigital mesenchyme. While both processes are controlled by bone morphogenetic proteins (BMPs) the molecular basis for such contrasting differential behavior of the autopodial mesoderm remains unknown. Here we show that a well-defined crescent domain of high BMP activity located at the tip of the forming digits, which we termed the digit crescent (DC), directs incorporation and differentiation of the PZ mesenchymal cells into the digit aggregates. The presence of this domain does not correlate with an exclusive expression domain of BMP receptors and its abrogation by surgical approaches or by local application of BMP antagonists is followed by digit truncation and cell death. We further show that establishment of the DC is directed by Activin/TGFβ signaling, which inhibits Smad 6 and Bambi, two specific BMP antagonists expressed in the interdigits and progress zone mesoderm. The interaction between Activin/TGFβ and BMP pathways at the level of DC promotes the expression of the chondrogenic factor SOX9 accompanied by a local decrease in cell proliferation. Characteristically, the DC domain is asymmetric, it being extended towards the posterior interdigit. The presence of the DC is transitorily dependent of the adjacent posterior interdigit and its maintenance requires also the integrity of the AER

Horizontal travelling heater method growth of Hg1−xCdxTe with crucible rotation

A horizontal travelling heater method (THM) for growing cylindrical cyrstals from a partially filled solution zone has been investigated for the first time. By applying ampoule rotation, the whole cross section of the crystal is successively brought into contact with the liquid solution, which is effectively stirred by forced convection. This approach was used to grow single-crystalline Hg1−xCdxTe ingots from a Te-rich solution zone. The structural perfection and metallurgical homogeneity are equivalent to vertically-grown THM material

Activin/TGFβ and BMP crosstalk determines digit chondrogenesis

AbstractThe progress zone (PZ) is a specialized area at the distal margin of the developing limb where mesodermal cells are kept in proliferation and undifferentiated, allowing limb outgrowth. At stages of digit morphogenesis the PZ cells can undergo two possible fates, either aggregate initiating chondrogenic differentiation to configure the digit blastemas, or to die by apoptosis if they are incorporated in the interdigital mesenchyme. While both processes are controlled by bone morphogenetic proteins (BMPs) the molecular basis for such contrasting differential behavior of the autopodial mesoderm remains unknown. Here we show that a well-defined crescent domain of high BMP activity located at the tip of the forming digits, which we termed the digit crescent (DC), directs incorporation and differentiation of the PZ mesenchymal cells into the digit aggregates. The presence of this domain does not correlate with an exclusive expression domain of BMP receptors and its abrogation by surgical approaches or by local application of BMP antagonists is followed by digit truncation and cell death. We further show that establishment of the DC is directed by Activin/TGFβ signaling, which inhibits Smad 6 and Bambi, two specific BMP antagonists expressed in the interdigits and progress zone mesoderm. The interaction between Activin/TGFβ and BMP pathways at the level of DC promotes the expression of the chondrogenic factor SOX9 accompanied by a local decrease in cell proliferation. Characteristically, the DC domain is asymmetric, it being extended towards the posterior interdigit. The presence of the DC is transitorily dependent of the adjacent posterior interdigit and its maintenance requires also the integrity of the AER

Tgfβ2 and 3 are coexpressed with their extracellular regulator Ltbp1 in the early limb bud and modulate mesodermal outgrowth and BMP signaling in chicken embryos

<p>Abstract</p> <p>Background</p> <p>Transforming growth factor β proteins (Tgfβs) are secreted cytokines with well-defined functions in the differentiation of the musculoskeletal system of the developing limb. Here we have studied in chicken embryos, whether these cytokines are implicated in the development of the embryonic limb bud at stages preceding tissue differentiation.</p> <p>Results</p> <p>Immunohistochemical detection of phosphorylated Smad2 and Smad3 indicates that signaling by this pathway is active in the undifferentiated mesoderm and AER. Gene expression analysis shows that transcripts of <it>tgfβ2 </it>and <it>tgfβ3 </it>but not <it>tgfβ1 </it>are abundant in the growing undifferentiated limb mesoderm. Transcripts of <it>tgfβ2 </it>are also found in the AER, which is the signaling center responsible for limb outgrowth. Furthermore, we show that Latent Tgfβ Binding protein 1 (LTBP1), which is a key extracellular modulator of Tgfβ ligand bioavailability, is coexpressed with <it>Tgfβs </it>in the early limb bud. Administration of exogenous Tgfβs to limb buds growing in explant cultures provides evidence of these cytokines playing a role in the regulation of mesodermal limb proliferation. In addition, analysis of gene regulation in these experiments revealed that Tgfβ signaling has no effect on the expression of master genes of musculoskeletal tissue differentiation but negatively regulates the expression of the BMP-antagonist Gremlin.</p> <p>Conclusion</p> <p>We propose the occurrence of an interplay between Tgfβ and BMP signaling functionally associated with the regulation of early limb outgrowth by modulating limb mesenchymal cell proliferation.</p

Identification of the Otopetrin Domain, a conserved domain in vertebrate otopetrins and invertebrate otopetrin-like family members

<p>Abstract</p> <p>Background</p> <p><it>Otopetrin 1 (Otop1) </it>encodes a multi-transmembrane domain protein with no homology to known transporters, channels, exchangers, or receptors. Otop1 is necessary for the formation of otoconia and otoliths, calcium carbonate biominerals within the inner ear of mammals and teleost fish that are required for the detection of linear acceleration and gravity. Vertebrate <it>Otop1 </it>and its paralogues <it>Otop2 </it>and <it>Otop3 </it>define a new gene family with homology to the invertebrate Domain of Unknown Function 270 genes (<it>DUF270</it>; pfam03189).</p> <p>Results</p> <p>Multi-species comparison of the predicted primary sequences and predicted secondary structures of 62 vertebrate otopetrin, and arthropod and nematode DUF270 proteins, has established that the genes encoding these proteins constitute a single family that we renamed the Otopetrin Domain Protein (<it>ODP</it>) gene family. Signature features of ODP proteins are three "Otopetrin Domains" that are highly conserved between vertebrates, arthropods and nematodes, and a highly constrained predicted loop structure.</p> <p>Conclusion</p> <p>Our studies suggest a refined topologic model for ODP insertion into the lipid bilayer of 12 transmembrane domains, and highlight conserved amino-acid residues that will aid in the biochemical examination of ODP family function. The high degree of sequence and structural similarity of the ODP proteins may suggest a conserved role in the intracellular trafficking of calcium and the formation of biominerals.</p

Ion-doped brushite cements for bone regeneration

Decades of research in orthopaedics has culminated in the quest for formidable yet resorbable biomaterials using bioactive materials. Brushite cements most salient features embrace high biocompatibility, bioresorbability, osteoconductivity, self-setting characteristics, handling, and injectability properties. Such type of materials is also effectively applied as drug delivery systems. However, brushite cements possess limited mechanical strength and fast setting times. By means of incorporating bioactive ions, which are incredibly promising in directing cell fate when incorporated within biomaterials, it can yield biomaterials with superior mechanical properties. Therefore, it is a key to develop fine-tuned regenerative medicine therapeutics. A comprehensive overview of the current accomplishments of ion-doped brushite cements for bone tissue repair and regeneration is provided herein. The role of ionic substitution on the cements physicochemical properties, such as structural, setting time, hydration products, injectability, mechanical behaviour and ion release is discussed. Cell-material interactions, osteogenesis, angiogenesis, and antibacterial activity of the ion-doped cements, as well as its potential use as drug delivery carriers are also presented.This study was funded by the Portuguese Foundation for Science and Technology (FCT) and the German Academic Exchange Service (Deutscher Akademischer Austauschdienst, DAAD) for the transnational cooperation FCT/DAAD 2018-2019. The authors also thank the funds provided under the distinctions attributed to JMO (IF/01285/2015) and SP (CEECIND/03673/2017). Furthermore, funding by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG), Grant Nr. HU 2498/1-1; GB 1/22-1, and the Emerging Talents Initiative of the FAU is acknowledged

Testimony at court: a randomised controlled trial investigating the art and science of persuading witnesses and victims to attend trial

The presence of civilian witnesses and victims in court is central to the effective operation of the criminal justice system. However, there is evidence of significant non-attendance which can result in ineffective and cracked trials. To address this, West Midlands Police Witness Care Unit and the Behavioural Insights Team designed an intervention using behavioural insight principles consisting of (1) a new conversation guide for Witness Care Officers (WCOs); (2) a redesigned ‘Warning Letter’ confirming details of the proceedings; and (3) a new reminder call and SMS. The impact of the new approach was evaluated through a randomised controlled trial in which 36 WCOs were randomly assigned to either “business as usual” (control) or treatment. The evaluation used an intention-to-treat design with implementation guided and encouraged at several points. Subgroup analysis was undertaken to explore whether differential effects were seen for domestic violence cases or between those that were victims and witnesses. Results indicated that the treatment approach was directionally positive in all cases, but that the increase in attendance was not statistically significant. This is in line with findings of other similar research in this area