Tcf7l2 plays pleiotropic roles in the control of glucose homeostasis, pancreas morphology, vascularization and regeneration

Type 2 diabetes (T2D) is a disease characterized by impaired insulin secretion. The Wnt signaling transcription factor Tcf7l2 is to date the T2D-associated gene with the largest effect on disease susceptibility. However, the mechanisms by which TCF7L2 variants affect insulin release from \u3b2-cells are not yet fully understood. By taking advantage of a tcf7l2 zebrafish mutant line, we first show that these animals are characterized by hyperglycemia and impaired islet development. Moreover, we demonstrate that the zebrafish tcf7l2 gene is highly expressed in the exocrine pancreas, suggesting potential bystander effects on \u3b2-cell growth, differentiation and regeneration. Finally, we describe a peculiar vascular phenotype in tcf7l2 mutant larvae, characterized by significant reduction in the average number and diameter of pancreatic islet capillaries. Overall, the zebrafish Tcf7l2 mutant, characterized by hyperglycemia, pancreatic and vascular defects, and reduced regeneration proves to be a suitable model to study the mechanism of action and the pleiotropic effects of Tcf7l2, the most relevant T2D GWAS hit in human populations

A Smad3 transgenic reporter reveals TGF-beta control of zebrafish spinal cord development

TGF-beta (TGFβ) family mediated Smad signaling is involved in mesoderm and endoderm specification, left-right asymmetry formation and neural tube development. The TGFβ1/2/3 and Activin/Nodal signal transduction cascades culminate with activation of SMAD2 and/or SMAD3 transcription factors and their overactivation are involved in different pathologies with an inflammatory and/or uncontrolled cell proliferation basis, such as cancer and fibrosis. We have developed a transgenic zebrafish reporter line responsive to Smad3 activity. Through chemical, genetic and molecular approaches we have seen that this transgenic line consistently reproduces in vivo Smad3-mediated TGFβ signaling. Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. In the CNS, Smad3 reporter activity is particularly high in the subpallium, tegumentum, cerebellar plate, medulla oblongata and the retina proliferative zone. In the spinal cord, the reporter is activated at the ventricular zone, where neuronal progenitor cells are located. Colocalization methods show in vivo that TGFβ signaling is particularly active in neuroD+ precursors. Using neuronal transgenic lines, we observed that TGFβ chemical inhibition leads to a decrease of differentiating cells and an increase of proliferation. Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. EdU proliferation assay and pH3 staining confirmed that Smad3 is mainly active in post-mitotic, non-proliferating cells. In summary, we demonstrate that the Smad3 reporter line allows us to follow in vivo Smad3 transcriptional activity and that Smad3, by controlling neural differentiation, promotes the progenitor to precursor switch allowing neural progenitors to exit cell cycle and differentiate

The Effect of Curcumin on Idiopathic Parkinson Disease: A Clinical and Skin Biopsy Study

There are currently no standardized therapies for Parkinson disease (PD). Curcumin shows anti-amyloidogenic properties in vitro and may be a promising treatment for PD. We evaluated the effects of curcumin supplementation on clinical scales and misfolded, phosphorylated α-synuclein (p-syn) accumulation in skin biopsies in 19 PD patients who received curcumin supplementation for 12 months and 14 PD patients to treated with curcumin. The patients underwent autonomic (COMPASS-31), motor (MDS-UPDRS and H&Y) and nonmotor (NMSS) questionnaires and skin biopsies to evaluate clinical involvement and p-syn load in skin nerves at the beginning and the end of study. Curcumin and curcuminoid levels were assayed in plasma and CSF. Supplemented patients showed detectable CSF curcuminoid levels that were lower than those in plasma. They showed a decrease of COMPASS-31 and NMSS scores, and a slight p-syn load decrease versus untreated patients who displayed a worsening of these parameters despite increased levodopa doses. Multiple regression models showed a significant effect of curcumin supplementation in decreasing the worsening of the clinical parameters and p-syn load at after curcumin treatment. These data suggest that curcumin can cross the blood-brain barrier, that it is effective in ameliorating clinical parameters and that it shows a tendency to decrease skin p-syn accumulation in PD patients

A Randomized Trial of Pharmacogenetic Warfarin Dosing in Naive Patients with Non-Valvular Atrial Fibrillation

Genotype-guided warfarin dosing have been proposed to improve patient's management. This study is aimed to determine whether a CYP2C9- VKORC1- CYP4F2-based pharmacogenetic algorithm is superior to a standard, clinically adopted, pharmacodynamic method. Two-hundred naive patients with non-valvular atrial fibrillation were randomized to trial arms and 180 completed the study. No significant differences were found in the number of out-of-range INRs (INR3.0) (p = 0.79) and in the mean percentage of time spent in the therapeutic range (TTR) after 19 days in the pharmacogenetic (51.9%) and in the control arm (53.2%, p = 0.71). The percentage of time spent at INR>4.0 was significantly lower in the pharmacogenetic (0.7%) than in the control arm (1.8%) (p = 0.02). Genotype-guided warfarin dosing is not superior in overall anticoagulation control when compared to accurate clinical standard of care

Pathways reporter analysis

Oral contribution: Pathways reporter analysi

Zebrafish pancreas development

An accurate understanding of the molecular events governing pancreas development can have an impact on clinical medicine related to diabetes, obesity and pancreatic cancer, diseases with a high impact in public health. Until 1996, the main animal models in which pancreas formation and differentiation could be studied were mouse and, for some instances related to early development, chicken and Xenopus. Zebrafish has penetrated this field very rapidly offering a new model of investigation; by joining functional genomics, genetics and in vivo whole mount visualization, Danio rerio has allowed large scale and fine multidimensional analysis of gene functions during pancreas formation and differentiation

Estimation of Real-Time Red Blood Cell Velocity in Conjunctival Vessels using a Modified Dynamic-Time-Warping Approach

We developed a tool to automatically analyse video sequences of conjunctival vessels, digitally imaged with high enough magnification to resolve movement of the blood within the vessel. After registering each frame of the sequence in order to compensate the movement of the patients or of the imaging instrumentation, we automatically tracked all vessels. From each vessel and from each frame we extract a one-dimensional signal representing the longitudinal variation of gray level along the vessel that is related to the presence of red blood cells. Then we estimate the local shift of the signals of a vessel between different frames, using a modified dynamic-time-warping approach. We test the algorithm first on simulated vessels, where the mean cell velocity is known, and on real video sequences. We show the effectivity of our method both regarding the estimation error and comparing it with a simpler cross-correlation approach, showing the possibility to design and develop a system to non-invasively quantify the blood velocity in the conjunctival vessels

Dynamic Time Warping and Cross Correlation for the estimation of cell movements in conjunctival capillaries: a comparison through simulation

We wish to assess the performance of an automatic analysis of video sequences of conjunctival vessels, digitally imaged with high enough magnification to resolve movement of the blood within the vessel. With a previously developed algorithm, from each vessel and from each frame we extract a one dimensional signal representing the longitudinal variation of gray level along the vessel, which is related to the presence of red blood cells. Then we estimate the local shift of the signals of a vessel between different frames, using a modified dynamic-time-warping approach. Since manual tracking of cells on a large batch of real video is unfeasible, we assess the performance of the algorithm on set of simulated vessels, where the mean cell velocity is known. By this means, we are also able to vary the mean blood cells velocity and the frequency of their velocity variation in time, so as to study the error variation with regard to these variables. We show the effectiveness of our method comparing it with a cross correlation approach. Moreover, along with the estimation error and robustness toward changes in the mean cells velocity, we show that at variance with the cross correlation method, the proposed algorithm is able to provide estimates on instantaneous velocity with an acceptable error, even if suffering from an overestimation bias that increases with the cells mean velocity

af9 Regulates gata2 Expression During Early Hemangioblast Specification and Vascular Pattern Formation In Zehrafish

Background: AF9 is a transcription factor that plays an essential role in hematopoiesis and embryonic development. The alteration of AF9 is principally associated in acute myeloid leukemia as fusion partner of human MLL (mixed-lineage leukemia) gene rearrangements. Zebrafish is an excellent model organism to study embryonic development and hematopoiesis. We have previously shown that zebrafish af9 is expressed within the intermediate cell mass (ICM), a site of primitive hematopoiesis in zebrafish. Here we study the loss of af9 in zebrafish development to further understand how af9 modulates early hematopoietic and embryonic development. Methods and results: Two morpholino antisense oligos (MOs), designed to block translation and inhibit pre-mRNA splicing of af9, were co-injected in embryos at 1-2 cell stage. To control for off-target effects, two morpholino mismatch oligos were designed and co-injected. Efficacy of MOs was demonstrated by Western blot analysis and RT-PCR in controls and MO-injected embryos (morphants). In vivo monitoring of both morphants and control embryos was carried out by microscopy. Effects of af9 depletion on vasculature and erythropoiesis were evaluated in Tg(fli1:eGFP) and Tg(gata1:DsRed) transgenic lines, respectively. Whole-mount in situ hybridization of known hematopoietic markers was used to decipher the developmental time-points in which af9 regulates blood development. Following injection of two MOs at 1-2 cell stage, we compared the morphological features of the morphants with control embryos at about 24 hours post-fertilization (hpf). The af9 morphants showed small head and eyes, disruption of tail development and pronounced swelling in the posterior ICM. Circulating blood cells were reduced from 26 hpf to later stages of development. At 48 hpf the heart was enlarged, showed a paucity of blood-cells and pericardial edema. Decreased number of blood cells in morphant embryos was further confirmed by o-dianisidine staining at 48 hpf and 72 hpf and in living af9-knockdown gata1:DsRed transgenic animals, suggesting that the differentiation of erythroblasts remains insufficient or impaired. Concordant with this observation, we examined the expression of specific markers for early hematopoiesis (scl, lmo2 and gata2) and primitive erythropoiesis (gata1, hbbe, and band3) using whole-mount in situ hybridization (WISH). At the 5-somite stage, the early hematopoietic precursor marker gata2 was markedly increased while scl and lmo2 remained unaffected in af9 morphants. Interestingly, by 24 hpf gata2 was found to be specifically over-expressed in ICM while no change was observed for scl and lmo2 markers. Besides, the erythroid progenitors and mature erythrocyte markers gata1, band3 and hbbe displayed nearly normal expression. To further confirm the role af9 in early hematopoiesis, we examined its expression in moonshine, a mutant zebrafish with defects in erythroid maturation due to deficiency of tif1γ, a key regulator of hematopoietic gene expression. WISH analysis in moonshine showed loss of af9 expression in the ICM at 24 hpf, suggesting that af9 functions genetically downstream of tif1γ in normal erythroid cell development. To determine the effect of af9 on endothelial and vascular development, we performed knockdown of af9 in fli1:eGFP transgenic line. By 24 hpf, these morphants showed significant increase of fluorescence intensity in the posterior ICM and a clear perturbation in the inter-segmental vessels (ISV) of the trunk at 30 hpf, indicating that af9 is required for early steps in hemangioblast specification and vascular pattern formation in zebrafish. Conclusion: af9 regulates gata2 expression during early hemangioblast specification and vascular pattern formation in zebrafish. af9 may also be involved in caudal segment morphogenesis. Taken together, these data provide the initial framework of a pathway that can be used to further integrate the molecular events regulating hemangioblast differentiation