1,720,978 research outputs found
Cre/lox-controlled spatiotemporal perturbation of FGF signaling in zebrafish
Full text linkBACKGROUND Spatiotemporal perturbation of signaling pathways in vivo remains challenging and requires precise transgenic control of signaling effectors. Fibroblast growth factor (FGF) signaling guides multiple developmental processes, including body axis formation and cell fate patterning. In zebrafish, mutants and chemical perturbations affecting FGF signaling have uncovered key developmental processes; however, these approaches cause embryo-wide perturbations, rendering assessment of cell-autonomous vs. non-autonomous requirements for FGF signaling in individual processes difficult. RESULTS Here, we created the novel transgenic line fgfr1-dn-cargo, encoding dominant-negative Fgfr1a with fluorescent tag under combined Cre/lox and heatshock control to perturb FGF signaling spatiotemporally. Validating efficient perturbation of FGF signaling by fgfr1-dn-cargo primed with ubiquitous CreERT2, we established that primed, heatshock-induced fgfr1-dn-cargo behaves similarly to pulsed treatment with the FGFR inhibitor SU5402. Priming fgfr1-dn-cargo with CreERT2 in the lateral plate mesoderm triggered selective cardiac and pectoral fin phenotypes without drastic impact on overall embryo patterning. Harnessing lateral plate mesoderm-specific FGF inhibition, we recapitulated the cell-autonomous and temporal requirement for FGF signaling in pectoral fin outgrowth, as previously inferred from pan-embryonic FGF inhibition. CONCLUSIONS As a paradigm for rapid Cre/lox-mediated signaling perturbations, our results establish fgfr1-dn-cargo as a genetic tool to define the spatiotemporal requirements for FGF signaling in zebrafish. Developmental Dynamics 247:1146-1159, 2018. © 2018 Wiley Periodicals, Inc
Distribution of Brain-Derived Neurotrophic Factor in the Brain of the Small-Spotted Catshark Scyliorhinus canicula, and Evolution of Neurotrophins in Basal Vertebrates
Full text linkNeurotrophins (NTFs) are structurally related neurotrophic factors essential for differentiation, survival, neurite outgrowth, and the plasticity of neurons. Abnormalities associated with neurotrophin-signaling (NTF-signaling) were associated with neuropathies, neurodegenerative disorders, and age-associated cognitive decline. Among the neurotrophins, brain-derived neurotrophic factor (BDNF) has the highest expression and is expressed in mammals by specific cells throughout the brain, with particularly high expression in the hippocampus and cerebral cortex. Whole genome sequencing efforts showed that NTF signaling evolved before the evolution of Vertebrates; thus, the shared ancestor of Protostomes, Cyclostomes, and Deuterostomes must have possessed a single ortholog of neurotrophins. After the first round of whole genome duplication that occurred in the last common ancestor of Vertebrates, the presence of two neurotrophins in Agnatha was hypothesized, while the monophyletic group of cartilaginous fishes, or Chondrichthyans, was situated immediately after the second whole genome duplication round that occurred in the last common ancestor of Gnathostomes. Chondrichthyans represent the outgroup of all other living jawed vertebrates (Gnathostomes) and the sister group of Osteichthyans (comprehensive of Actinopterygians and Sarcopterygians). We were able to first identify the second neurotrophin in Agnatha. Secondly, we expanded our analysis to include the Chondrichthyans, with their strategic phylogenetic position as the most basal extant Gnathostome taxon. Results from the phylogenetic analysis confirmed the presence of four neurotrophins in the Chondrichthyans, namely the orthologs of the four mammalian neurotrophins BDNF, NGF, NT-3, and NT-4. We then proceeded to study the expression of BDNF in the adult brain of the Chondrichthyan Scyliorhinus canicula. Our results showed that BDNF is highly expressed in the S. canicula brain and that its expression is highest in the Telencephalon, while the Mesencephalic and Diencephalic areas showed expression of BDNF in isolated and well-defined cell groups. NGF was expressed at much lower levels that could be detected by PCR but not by in situ hybridization. Our results warrant further investigations in Chondrichthyans to characterize the putative ancestral function of neurotrophins in Vertebrates
Localization and Characterization of Major Neurogenic Niches in the Brain of the Lesser-Spotted Dogfish Scyliorhinus canicula
Full text linkAdult neurogenesis is defined as the ability of specialized cells in the postnatal brain to produce new functional neurons and to integrate them into the already-established neuronal network. This phenomenon is common in all vertebrates and has been found to be extremely relevant for numerous processes, such as long-term memory, learning, and anxiety responses, and it has been also found to be involved in neurodegenerative and psychiatric disorders. Adult neurogenesis has been studied extensively in many vertebrate models, from fish to human, and observed also in the more basal cartilaginous fish, such as the lesser-spotted dogfish, Scyliorhinus canicula, but a detailed description of neurogenic niches in this animal is, to date, limited to the telencephalic areas. With this article, we aim to extend the characterization of the neurogenic niches of S. canicula in other main areas of the brain: we analyzed via double immunofluorescence sections of telencephalon, optic tectum, and cerebellum with markers of proliferation (PCNA) and mitosis (pH3) in conjunction with glial cell (S100β) and stem cell (Msi1) markers, to identify the actively proliferating cells inside the neurogenic niches. We also labeled adult postmitotic neurons (NeuN) to exclude double labeling with actively proliferating cells (PCNA). Lastly, we observed the presence of the autofluorescent aging marker, lipofuscin, contained inside lysosomes in neurogenic areas
The zebrafish/tumor xenograft angiogenesis assay as a tool for screening anti-angiogenic miRNAs
Full text linkThe zebrafish/tumor xenograft angiogenesis assay is used to approach tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Here, we evaluated whether the assay could allow the identification of microRNAs having an anti-angiogenic potential. For that, we transfected DU-145 prostate cancer cells with four microRNAs (miR-125a, miR-320, miR-487b, miR-492) responsive to both anti- and pro-angiogenic stimuli applied to human umbilical vein endothelial cells. After transfection, DU-145 cells were injected close to the developing subintestinal vessels of transgenic Tg(Kdrl:eGFP)s843 zebrafish embryos that express green fluorescent protein under the control of Kdrl promoter. At 72 h post-fertilization, we observed that green fluorescent protein–positive neo-vessels infiltrated the graft of DU-145 transfected with miR-125a, miR-320, and miR-487b. Vice versa, neo-vessel formation and tumor cell infiltration were inhibited when DU-145 cells transfected with miR-492 were used. These results indicated that the zebrafish/tumor xenograft assay was adequate to identify microRNAs able to suppress the release of angiogenic growth factors by angiogenic tumor cells
Barley beta-glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment
No full textManganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-D-glucan (β-D-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-D-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo.
Long-term treatment of HUVECs with 3% w/v β-D-glucan significantly increased the level of MnSOD by 200±2% compared to control and by 50±4% compared to untreated H2O2-stressed cells. β-D-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24h-treatment with 3% w/v β-D-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O2-) and an increased survival under oxidative stress. In addition, β-D-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-D-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-D-glucan effects. In conclusion, 3% w/v β-D-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O2-, cell survival and angiogenic response to oxidative stress. The identification of dietary β-D-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodeling and heart failure
miR-29a and miR-30c negatively regulate DNMT 3a in cardiac ischemic tissues: implications for cardiac remodelling
Full text linkRecent evidences indicate that epigenetic changes play an important rolein the transcriptional reprogramming of gene expression that characterizescardiac hypertrophy and failure and may dictate response to therapy.Several data demonstrate that microRNAs (miRNAs) play critical rolesboth in normal cardiac function and under pathological conditions. Herewe assessed, in in vivo rat models of myocardial infarction (MI) andischemia-reperfusion (IR), the relationship between two miRNAs (miR-29aand miR-30c) and de novo methyltransferase (DNMT3a) which, alteringthe chromatin accessibility for transcription factors, deeply impacts geneexpression. We showed that the levels of members of miR-29 and miR30 families were down regulated in ischemic tissues whilst the proteinlevels of DNMT3a were increased, such a relation was not present inhealthy tissues. Furthermore, by an in vitro assay, we demonstrated thatboth miRNAs are able to down regulate DNMT3a by directly interactingwith DNMT3a 3’UTR and that miR-29a or miR-30c overexpression in thecardiac HL1 cell line causes decrease of DNMT3a enzyme both at themRNA and protein levels. Our data, besides confirming the down regulationof the miR-29a and miR-30c in infarcted tissues, envisage a cross-talkbetween microRNAs and chromatin modifying enzymes suggesting a newmechanism that might generate the alterations of DNA methylation oftenobserved in myocardial pathophysiology
Nerve Growth Factor Receptor (NGFR/p75NTR) of the Small-Spotted Catshark (Scyliorhinus canicula): Evolutionary Conservation and Brain Function
Full text linkThe p75NTR receptor, a member of the tumor necrosis factor (TNF) receptor superfamily, can participate in signaling pathways either by forming heteromeric complexes with other receptors, such as the Trk family (tropomyosin receptor kinases), or by functioning independently. p75NTR was investigated prevalently in the brain and retina of mammals, whereas almost nothing is known about its conservation among species. Here, we reconstructed the phylogenetic arb of p75NTR and described for the first time the p75NTR expression in the brain of the basal vertebrate Chondrichthyan Scyliorhinus canicula (S. canicula), uncovering the existing parallelism between ancient vertebrates and mammals. p75NTR functional conservation among vertebrates was further investigated by cloning the S. canicula nerve growth factor (NGF) and performing the canonical posterior commissure (PC)-12 differentiation assay, which results in standard neurite-like production. We then investigated the S. canicula p75NTR, which proves to be capable of complementing a specific clone of PC-12 lacking p75NTR (PC-12 p75NTR-/-). All together, our results highlighted the expression and functional conservation of p75NTR among vertebrates during the evolution
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
- …
