miR-494-3p overexpression promotes megakaryocytopoiesis in primary myelofibrosis hematopoietic stem/progenitor cells by targeting SOCS6

Primary myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by hematopoietic stem cell-derived clonal myeloproliferation, involving especially the megakaryocyte lineage. To better characterize how the altered expression of microRNAs might contribute to PMF pathogenesis, we have previously performed the integrative analysis of gene and microRNA expression profiles of PMF hematopoietic stem/progenitor cells (HSPCs), which allowed us to identify miR- 494-3p as the upregulated microRNA predicted to target the highest number of downregulated mRNAs. To elucidate the role of miR-494-3p in hematopoietic differentiation, in the present study we demonstrated that miR-494-3p enforced expression in normal HSPCs promotes megakaryocytopoiesis. Gene expression profiling upon miR-494-3p overexpression allowed the identification of genes commonly downregulated both after microRNA overexpression and in PMF CD34+ cells. Among them, suppressor of cytokine signaling 6 (SOCS6) was confirmed to be a miR-494-3p target by luciferase assay. Western blot analysis showed reduced level of SOCS6 protein as well as STAT3 activation in miR-494-3p overexpressing cells. Furthermore, transient inhibition of SOCS6 expression in HSPCs demonstrated that SOCS6 silencing stimulates megakaryocytopoiesis, mimicking the phenotypic effects observed upon miR-494-3p overexpression. Finally, to disclose the contribution of miR-494-3p upregulation to PMF pathogenesis, we performed inhibition experiments in PMF HSPCs, which showed that miR-494-3p silencing led to SOCS6 upregulation and impaired megakaryocyte differentiation. Taken together, our results describe for the first time the role of miR-494- 3p during normal HSPC differentiation and suggest that its increased expression, and the subsequent downregulation of its target SOCS6, might contribute to the megakaryocyte hyperplasia commonly observed in PMF patients

Calreticulin Ins5 and Del52 mutations impair unfolded protein and oxidative stress responses in K562 cells expressing CALR mutants

Somatic mutations of calreticulin (CALR) have been described in approximately 60-80% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In this study, we showed that CALR mutants impair the ability to respond to the ER stress and reduce the activation of the pro-apoptotic pathway of the unfolded protein response (UPR). Moreover, our data demonstrated that CALR mutations induce increased sensitivity to oxidative stress, leading to increase oxidative DNA damage. We finally demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR. Altogether, our data identify novel mechanisms collaborating with MPL activation in CALR-mediated cellular transformation. CALR mutants negatively impact on the capability of cells to respond to oxidative stress leading to genomic instability and on the ability to react to ER stress, causing resistance to UPR-induced apoptosis

the SMARTPARKS Project

UID/SOC/04647/2013Small Islands face particular challenges in their sustainable development, and therefore require specific tailored approaches for planning and management. Unsurprisingly, protected areas have a special role in the conservation of biodiversity and natural resources crucial to the sustainability of such territories. How should the management and planning system of protected areas in small islands be, therefore, structured and operated so it can face the threats and challenges falling upon the already fragile and vulnerable insular ecosystems? This is the central question of SMARTPARKS Project. The core objective of the project consists of the conceptual development of a planning and management system for protected areas that can be integrated with the territorial management instruments in force, and that takes into consideration the specificities of insular ecosystems, correcting or perfecting the insufficiencies or flaws already pointed out to traditional planning systems of protected areas. This paper presents the SMARTPARKS Project, its rationale and main outcomes. Taking Pico Island Natural Park (Azores, Portugal) as its case study, the SMARTPARKS Project has adopted the ecosystem approach and the conciliation of conservation objectives with human needs and activities. Throughout its five tasks several studies were developed, and contributed to the functional analysis (developed during the last task) of each protected area constituting the Island Natural Park, in terms of their conservation and development values. This innovative application allows not only an integrated assessment of the protected areas but also a sustained monitoring. PT | RESUMOAs pequenas ilhas oceânicas enfrentam desafios particulares com vista ao seu desenvolvimento sustentável, necessitando consequentemente de abordagens técnicas de base científica específicas no desenvolvimento das suas estratégias de planeamento e gestão territoriais. É inquestionável a relevância do papel das áreas protegidas quer na conservação da biodiversidade e dos recursos naturais, quer na sustentabilidade dos territórios por elas abrangidos. De que modo deve ser então definido o modelo de planeamento e gestão de áreas protegidas em pequenas ilhas oceânicas para poder fazer face a todas as ameaças e desafios com que se deparam estes frágeis e vulneráveis ecossistemas costeiros? Esta é a questão principal pela qual se rege o projecto SMARTPARKS. O objectivo nuclear deste projecto consiste no desenvolvimento conceptual de um sistema integrado de planeamento e gestão de áreas protegidas que possa integrar, complementar e fortalecer os instrumentos de gestão territorial vigentes, e que tenha em consideração as particularidades e especificidades destes ecossistemas insulares, corrigindo ou minimizando as falhas e insuficiências já identificadas das ferramentas e técnicas tradicionais de planeamento territorial de áreas protegidas. Este artigo apresenta o projecto SMARTPARKS, o seu contexto, a sua abordagem conceptual e os seus principais resultados. O Parque Natural de Ilha do Pico (Arquipélago dos Açores, Portugal) constitui o caso de estudo deste projecto, estando o desenvolvimento conceptual do SMARTPARKS especialmente focado numa sinergia definida pela abordagem ecossistémica e pela sua tentativa de conciliação com os objectivos de conservação com todas as necessidades e actividades humanas de cariz sócio-económico e cultural desenvolvidas no território abrangido. Ao longo do seu desenvolvimento metodológico dividido em 5 grandes tarefas, vários estudos específicos foram realizados, contribuindo nomeadamente para a análise funcional (em termos de valores para a conservação e desenvolvimento) que foi feita para cada área protegida que compõe o Parque Natural de Ilha do Pico. Esta abordagem metodológica inovadora permite não só uma avaliação integrada das áreas protegidas como também a sua monitorização sustentável.publishersversionpublishe

Mutations impairing GSK3-mediated MAF phosphorylation cause cataract, deafness, intellectual disability, seizures, and a down syndrome-like facies

Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing developmen

Calreticulin affects hematopoietic stem/progenitor cell fate by impacting erythroid and megakaryocytic differentiation

Calreticulin (CALR) is a chaperone protein that localizes primarily to the endoplasmic reticulum (ER) lumen where it is responsible for the control of proper folding of neo-synthesized glycoproteins and for the retention of calcium. Recently, mutations affecting exon 9 of the CALR gene have been described in approximately 40% of patients with myeloproliferative neoplasms (MPNs). Although the role of mutated CALR in the development of MPNs has begun to be clarified, there are still no data available on the function of wild-type (WT) CALR during physiological hematopoiesis. In order to shed light on the role of WT CALR during normal hematopoiesis, we performed gene silencing and overexpression experiments in Hematopoietic Stem Progenitor Cells (HSPCs). Our results showed that CALR overexpression is able to affect physiological hematopoiesis by enhancing both erythroid and megakaryocytic (MK) differentiation. In agreement with overexpression data, CALR silencing caused a significant decrease in both erythroid and MK differentiation of human HSPCs. Gene expression profiling (GEP) analysis showed that CALR is able to affect the expression of several genes involved in HSPCs differentiation towards both the erythroid and MK lineages. Moreover, GEP data also highlighted the modulation of several genes involved in ER stress response, unfolded protein response (UPR), DNA repair and of several genes already described to play a role in MPN development, such as pro-inflammatory cytokines and hematological neoplasms-related markers. Altogether, our data unraveled a new and unexpected role for CALR in the regulation of normal hematopoietic differentiation. Moreover, by showing the impact of CALR on the expression of genes involved in several biological processes already described in cellular transformation, our data strongly suggest a more complex role for CALR in MPN development that goes beyond the activation of the THPO receptor and involves ER stress response, UPR and DNA repair

Variability in genes regulating vitamin D metabolism is associated with vitamin D levels in type 2 diabetes

Mortality rate is increased in type 2 diabetes (T2D). Low vitamin D levels are associated with increased mortality risk in T2D. In the general population, genetic variants affecting vitamin D metabolism (DHCR7 rs12785878, CYP2R1 rs10741657, GC rs4588) have been associated with serum vitamin D. We studied the association of these variants with serum vitamin D in 2163 patients with T2D from the "Sapienza University Mortality and Morbidity Event Rate (SUMMER) study in diabetes". Measurements of serum vitamin D were centralised. Genotypes were obtained by Eco™ Real-Time PCR. Data were adjusted for gender, age, BMI, HbA1c, T2D therapy and sampling season. DHCR7 rs12785878 (p = 1 x 10-4) and GC rs4588 (p = 1 x 10-6) but not CYP2R1 rs10741657 (p = 0.31) were significantly associated with vitamin D levels. One unit of a weighted genotype risk score (GRS) was strongly associated with vitamin D levels (p = 1.1 x 10-11) and insufficiency (<30 ng/ml) (OR, 95%CI = 1.28, 1.16-1.41, p = 1.1 x 10-7). In conclusion, DHCR7 rs12785878 and GC rs4588, but not CYP2R1 rs10741657, are significantly associated with vitamin D levels. When the 3 variants were considered together as GRS, a strong association with vitamin D levels and vitamin D insufficiency was observed, thus providing robust evidence that genes involved in vitamin D metabolism modulate serum vitamin D in T2D

Role of TGF-\u3b21/miR-382-5p/SOD2 axis in the induction of oxidative stress in CD34+ cells from primary myelofibrosis

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by an excessive production of pro-inflammatory cytokines resulting in chronic inflammation and genomic instability. Besides the driver mutations in JAK2, MPL, and CALR genes, the deregulation of miRNA expression may also contribute to the pathogenesis of PMF. To this end, we recently reported the upregulation of miR-382-5p in PMF CD34+ cells. In order to unveil the mechanistic details of the role of miR-382-5p in pathogenesis of PMF, we performed gene expression profiling of CD34+ cells overexpressing miR-382-5p. Among the downregulated genes, we identified superoxide dismutase 2 (SOD2), which is a predicted target of miR-382-5p. Subsequently, we confirmed miR-382-5p/SOD2 interaction by luciferase assay and we showed that miR-382-5p overexpression in CD34+ cells causes the decrease in SOD2 activity leading to reactive oxygen species (ROS) accumulation and oxidative DNA damage. In addition, our data indicate that inhibition of miR-382-5p in PMF CD34+ cells restores SOD2 function, induces ROS disposal, and reduces DNA oxidation. Since the pro-inflammatory cytokine transforming growth factor-\u3b21 (TGF-\u3b21) is a key player in PMF pathogenesis, we further investigated the effect of TGF-\u3b21 on ROS and miR-382-5p levels. Our data showed that TGF-\u3b21 treatment enhances miR-382-5p expression and reduces SOD2 activity leading to ROS accumulation. Finally, inhibition of TGF-\u3b21 signaling in PMF CD34+ cells by galunisertib significantly reduced miR-382-5p expression and ROS accumulation and restored SOD2 activity. As a whole, this study reports that TGF-\u3b21/miR-382-5p/SOD2 axis deregulation in PMF cells is linked to ROS overproduction that may contribute to enhanced oxidative stress and inflammation. Our results suggest that galunisertib may represent an effective drug reducing abnormal oxidative stress induced by TGF-\u3b21 in PMF patients. Database linking: GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103464

Adjuvant FOLFOX-4 in patients with radically resected gastric cancer: tolerability, and prognostic factors.

Purpose: To evaluate the toxicity and efficacy of the FOLFOX-4 regimen as adjuvant chemotherapy in patients with gastric cancer after radical surgery. Methods: Fifty-four patients (1 stage Ib, 6 stage II, 22 stage IIIa, 14 stage IIIb and 11 stage IV) received 8-12 cycles of FOLFOX-4 (oxaliplatin 85 mg/m2, day 1; leucovorin 100 mg/m2 i.v. days 1 and 2; 5fluorouracil 400 mg /m2 i.v. bolus days 1 and 2, and 600 mg /m2 in 22 hours i.v. continuous infusion days 1 and 2; every 14 days). Toxicity was recorded at each cycle according to the National Cancer Institute Common Toxicity Criteria. Disease-free (DFS) and overall survival (OS) were calculated according to the Kaplan-Meier method. Results: Thirty-eight patients (70.4%) completed the prescribed number of cycles of chemotherapy. The toxicity was mild. Grade 3-4 neutropenia occurred in 57% of patients, thrombocytopenia and anemia in 2% of cases. Peripheral neuropathy was experienced by 46% of patients (grade 4 in 2% of cases). Five patients experienced grade 3 gastrointestinal toxicity. After a median follow up of 33.1 months, 17 patients relapsed and 17 deceased. The mean observed DFS and OS were 49.7 (range 40.7-58.8) and 57.9 (range 49.6-66.2) months, respectively. At univariate analysis, females and patients who had received < 8 cycles of chemotherapy had a significantly worse probability of DFS and OS. The Cox model showed gender to be independent factors affecting DFS. Conclusions: Adjuvant FOLFOX-4 is feasible and well tolerated in patients radically resected for gastric cancer. Receiving < 4 months of adjuvant FOLFOX-4 could be detrimental for prognosis