Identification and molecular characterization of bone-related micrornas: functional implications

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014MicroRNAs (miRNAs) are a conserved class of small RNAs providing a post-transcriptional mechanism for fine-tuning of intricate physiological and pathological cellular processes, such as those affecting development. Skeletogenesis however, was so far poorly investigated and mainly focused on mammalian models, with a general lack of knowledge concerning other vertebrates. We aimed at the identification of bone-related miRNAs and their characterization from an evolutionary perspective, using fish (mostly zebrafish) as model, in comparison to mammalian systems. First, we focused on miR-223, a miRNA that was associated with bone remodelling. We demonstrated that miR-223 genomic organization/context and primary/secondary structures are largely maintained between human and zebrafish. As in mammals, miR-223 expression in zebrafish was highly correlated with hematopoietic events and osteoclastogenesis. Finally, miR-223 targets identified in mammals were also predicted in zebrafish, supporting a functional conservation of this miRNA. In a second set of experiments, we studied the biological role of miR-29a, a bone-related miRNA that was fairly investigated in mammals, but with no mineralogenic effects yet demonstrated. We took advantage of our fish bone-derived systems to explore miR-29a mineralogenic effects through gain-of-function experiments. We demonstrated a strong stimulation of this process through a mechanism probably involving the canonical Wnt signalling. Once more, through bioinformatics analysis, patterns of expression and target prediction/validation, we provided evidences for miR-29 conservation throughout evolution. Finally, we explored miR-214 putative roles on skeleton formation in vertebrates. Although our initial hypothesis of miR-214 involvement in osteogenesis was recently demonstrated by Wang et al. (2013), we proceeded with our investigation and finally showed that miR-214 is also associated with chondrogenesis. Overexpression of miR-214 in ATDC5 cells mitigated differentiation and down-regulated Mgp and Osteocalcin, probably by targeting Atf4. This work provides novel evidence that some miRNAs have conserved functions across vertebrates and, probably, conserved regulatory mechanisms of action.Nos últimos anos, assistiu-se a uma marcante expansão na área da biologia molecular, devendo-se isto principalmente à descoberta de pequenas moléculas de RNA não codante e ao seu modo peculiar de intervir na regulação genética. Dentro deste grupo de moléculas, os microRNAs (miRNAs) são, definitivamente, a classe melhor compreendida, o que se comprova pelo crescimento exponencial do número de trabalhos publicados desde a sua descoberta. Os miRNAs, na sua forma matura, são RNAs com aproximadamente 22 nucleótidos (nt), altamente conservados em vertebrados e que asseguram um controlo apertado de vários processos celulares através de uma regulação pós-transcricional. Esta regulação ocorre através da ligação específica do miRNA à 3’UTR do RNA mensageiro (mRNA). Neste mecanismo, destaca-se o envolvimento do complexo RISC (RNA-induced silencing complex; associado ao miRNA), a complementaridade da denominada região “seed” (extremidade 5’ do miRNA) ao mRNA, e o consequente bloqueio da tradução ou degradação do mRNA. Desta forma, cada miRNA pode regular centenas de genes transcritos, e de facto, hoje em dia pensa-se que a maioria dos genes humanos são controlados por miRNAs. Assim, os miRNAs são considerados não só importantes reguladores de múltiplos processos biológicos, incluindo desenvolvimento, diferenciação e apoptose celular, mas também responsáveis por vários processos patológicos, como o cancro, onde se observou que inúmeros miRNAs têm a sua expressão desregulada. Assim, a caracterização dos miRNAs (a vários níveis) é fundamental para a compreensão das suas funções, permitindo alargar também o conhecimento dos processos biológicos e patológicos onde estão envolvidos. Apesar do conhecimento sobre miRNAs ter aumentado francamente nos últimos anos, o papel dos miRNAs na formação e homeostasia do osso ainda está pouco caracterizado, e a maioria dos estudos tem abordado principalmente esta forma de regulação em mamíferos, havendo assim uma lacuna de conhecimento na regulação destes processos noutros vertebrados. Neste sentido, este trabalho focou-se na identificação de miRNAs potencialmente envolvidos na regulação do osso e na sua caracterização numa perspectiva evolutiva, usando o peixe (essencialmente o peixe-zebra) como modelo, e em comparação com mamíferos. Numa primeira abordagem, focámos a nossa investigação no estudo do miR-223, um miRNA anteriormente associado à diferenciação celular da linhagem hematopoiética e ao processo de remodelação óssea. Neste estudo, demonstramos que a organização e contexto genómicos do miR-223 estão preservados em vertebrados, verificando-se uma conservação das estruturas primária e secundária do pre-miR-223 em 46 espécies. Este estudo mostra ainda que a expressão deste miRNA se correlaciona com determinadas fases do desenvolvimento do peixe-zebra onde a hematopoiese e a osteoclastogénese são eventos predominantes. Além disso, este estudo mostra que o miR-223 apresenta uma expressão elevada no principal órgão hematopoético de peixes e ratinhos adultos (rim anterior e medula óssea, respectivamente), sugerindo que a função hematopoiética também se encontra conservada. Por último, através de análise bioinformática demonstrámos que a regulação de genes alvo do miR-223 em mamíferos também deverá estar mantida em peixe-zebra. Na secção seguinte estudámos o papel biológico do miR-29a, cujo efeito osteogénico em mamíferos se encontra bem caracterizado, mas sem nenhum fenótipo mineralogénico ainda associado. Neste estudo utilizámos uma linha celular derivada do osso de peixe previamente desenvolvida no nosso laboratório e com capacidade de mineralização in vitro. A fim de explorar os efeitos mineralogénicos do miR-29a foram realizadas experiências de ganho de função. O aumento dos níveis endógenos deste miRNA resultaram num incremento da mineralização da matriz extra-celular, o que provavelmente terá sido devido a uma aceleração da diferenciação celular pelo potenciamento da via de sinalização Wnt, tal como evidenciado pela acumulação de um dos seus principais componentes, a -catenina. Além disso, foi demonstrada a conservação da função deste miRNA através de estudos baseados em homologia de sequências, análise de sintenia, padrão de expressão tecidular e na manutenção da regulação do SPARC, um alvo previamente descrito em mamíferos. Reforçou-se assim a ideia de que o miR-29a é um regulador crucial na diferenciação de osteoblastos, induzindo um aumento da mineralização em sistemas in vitro. Finalmente, explorámos a hipótese do miR-214 ser regulador da formação do esqueleto/osso, em vertebrados. Apesar da nossa primeira hipótese, que consistia no envolvimento do miR-214 na osteogénese, ter sido entretanto demonstrada através do trabalho realizado por Wang et al. (2013), continuámos com este estudo, tentando demonstrar um potencial envolvimento deste miRNA na condrogénese, um processo essencial na formação do esqueleto de vertebrados. Através do padrão de expressão espacial e temporal do miR-214 durante o desenvolvimento do peixe-zebra, verificou-se uma clara associação com estruturas cartilagíneas. Adicionalmente, demonstrámos que a região reguladora (promotor) do transcrito primário deste miRNA se encontra conservada em oito vertebrados, assim como os locais de ligação de factores de transcrição (associados à condrogénese e/ou osteogénese) identificados. De acordo com a análise funcional deste promotor, concluiu-se que esta região reguladora (quer de peixe-zebra quer de humano) é activada e regulada de forma semelhante em condrócitos e osteoblastos. Por último, verificou-se que a sobreexpressão do miR-214 nas células ATDC5, um modelo in vitro para a condrogénese, atenua a diferenciação condrocítica, possivelmente através da regulação do gene Atf4. O decréscimo simultâneo de dois marcadores ósseos, a Mgp e a osteocalcina, aquando da sobreexpressão deste miRNA sugere que a mineralização dos condrócitos poderá estar comprometida nesta condição. Assim, propomos que o miR-214 desempenha um papel fundamental na formação do esqueleto de vertebrados, não apenas pela regulação da osteogénese, mas também pelo controlo da condrogénese, promovendo assim a normal e equilibrada formação de estruturas ósseas e cartilagíneas. No seu conjunto, estes estudos evidenciam uma conservação na função e mecanismos de regulação de muitos dos miRNAs identificados em vertebrados. Este conhecimento é bastante importante, por exemplo para a investigação de tratamento de patologias, uma vez que permite a utilização de modelos alternativos no rastreio de potenciais alvos terapêuticos, com particular destaque para as vias reguladas por miRNAs. Nesta perspectiva, em doenças como por exemplo a osteoporose, onde se verifica uma perda de massa óssea, terapias que estimulem a acção de miRNAs que promovam a osteoblastogénese ou que inibam a osteoclástogénese, são atractivas e com grande potencial na estimulação da formação óssea ou na redução da reabsorção óssea excessiva, respectivamente.Fundação para a Ciência e Tecnologia (SFRH/BD/38607/2007),pelo financiamento da bolsa de doutoramento, Fundação Calouste Gulbenkian, através do programa ‘‘Na Fronteira das Ciências da Vida’’ pelo co-financiamento deste trabalho

Evidences for a new role of miR-214 in chondrogenesis

miR-214 is known to play a role in mammalian skeletal development through inhibition of osteogenesis and stimulation of osteoclastogenesis, but data regarding other vertebrates, as well as a possible role in chondrogenesis, remain unknown. Here, we show that miR-214 expression is detected in bone and cartilage of zebrafish skeleton, and is downregulated during murine ATDC5 chondrocyte differentiation. Additionally, we observed a conservation of the transcriptional regulation of miR-214 primary transcript Dnm3os in vertebrates, being regulated by Ets1 in ATDC5 chondrogenic cells. Moreover, overexpression of miR-214 in vitro and in vivo mitigated chondrocyte differentiation probably by targeting activating transcription factor 4 (Atf4). Indeed, miR-214 overexpression in vivo hampered cranial cartilage formation of zebrafish and coincided with downregulation of atf4 and of the key chondrogenic players sox9 and col2a1. We show that miR-214 overexpression exerts a negative role in chondrogenesis by impacting on chondrocyte differentiation possibly through conserved mechanisms.Calouste Gulbenkian Foundation (program "Na Fronteira das Ciencias da Vida"); FCT [UID/Multi/04326/2013, PEst-C/MAR/LA0015/2011, SFRH/BD/38607/2007, SFRH/BPD/45034/2008, SFRH/BPD/111289/2015]; European Commission (ERDF-COMPETE) [PEst-C/MAR/LA0015/2011]info:eu-repo/semantics/publishedVersio

DNA Methylation of PI3K/AKT pathway-related genes predicts outcome in patients with pancreatic cancer: a comprehensive bioinformatics-based study

Pancreatic cancer (PCA) is one of the most lethal malignancies worldwide with a 5-year survival rate of 9%. Despite the advances in the field, the need for an earlier detection and effective therapies is paramount. PCA high heterogeneity suggests that epigenetic alterations play a key role in tumour development. However, only few epigenetic biomarkers or therapeutic targets have been identified so far. Here we explored the potential of distinct DNA methylation signatures as biomarkers for early detection and prognosis of PCA. PI3K/AKT-related genes differentially expressed in PCA were identified using the Pancreatic Expression Database (n = 153). Methylation data from PCA patients was obtained from The Cancer Genome Atlas (n = 183), crossed with clinical data to evaluate the biomarker potential of the epigenetic signatures identified and validated in independent cohorts. The majority of selected genes presented higher expression and hypomethylation in tumour tissue. The methylation signatures of specific genes in the PI3K/AKT pathway could distinguish normal from malignant tissue at initial disease stages with AUC > 0.8, revealing their potential as PCA diagnostic tools. ITGA4, SFN, ITGA2, and PIK3R1 methylation levels could be independent prognostic indicators of patients’ survival. Methylation status of SFN and PIK3R1 were also associated with disease recurrence. Our study reveals that the methylation levels of PIK3/AKT genes involved in PCA could be used to diagnose and predict patients’ clinical outcome with high sensitivity and specificity. These results provide new evidence of the potential of epigenetic alterations as biomarkers for disease screening and management and highlight possible therapeutic targets.This work was supported by the FCT Research Center Grant UID/BIM/04773/2013 CBMR 1334, Câmara Municipal de Loulé, by the Liga Portuguesa Contra o Cancro/Portuguese League against Cancer through the Grant LPCC-PT Foundation 2016 to V.P.R. and by the Spanish Ministry of Science, Innovation and Universities through Grant RTI2018-094629-B-I00 to W.L.info:eu-repo/semantics/publishedVersio

A Human Stem Cell-Derived Neurosensory-Epithelial Circuitry on a Chip to Model Herpes Simplex Virus Reactivation

Both emerging viruses and well-known viral pathogens endowed with neurotropism can either directly impair neuronal functions or induce physio-pathological changes by diffusing from the periphery through neurosensory-epithelial connections. However, developing a reliable and reproducible in vitro system modeling the connectivity between the different human sensory neurons and peripheral tissues is still a challenge and precludes the deepest comprehension of viral latency and reactivation at the cellular and molecular levels. This study shows a stable topographic neurosensory-epithelial connection on a chip using human stem cell-derived dorsal root ganglia (DRG) organoids. Bulk and single-cell transcriptomics showed that different combinations of key receptors for herpes simplex virus 1 (HSV-1) are expressed by each sensory neuronal cell type. This neuronal-epithelial circuitry enabled a detailed analysis of HSV infectivity, faithfully modeling its dynamics and cell type specificity. The reconstitution of an organized connectivity between human sensory neurons and keratinocytes into microfluidic chips provides a powerful in vitro platform for modeling viral latency and reactivation of human viral pathogens

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Gla portein localization and histological characterization of bone stuctures in relevant aquaculture fish

Osteocalcin (Oc) and Matrix Gia Protein (MGP) are vitamin K-dependente proteins known for their Ca2" binding capacity. Considering the problem of skeletal malformations in Mediterranean fish aquaculture, we investigated a possible correlation between vertebral deformities and changes of Oc and Mgp accumulation sites. ln this work we proposed to characterize bony and cartilaginous tissues from important fish for aquaculture, using molecular tools. The sites of gene expression and accumulation of osteocalcin and Mgp were investigated throughout Northern blot analysis and immunohistochemistry, following the cloning of those cDNAs not yet available, together with histological analysis. ln Scophthalmus maximus, oc mRNA was detected in vertebrae and branchial arches while mgp mRNA was highly expressed in branchial arches, followed by vertebrae, kidney and heart. ln S. maximus and Diplodus sargus, Mgp accumulated in chondrocytes from cartilages, in the vertebral mineralizing fronts and in notochord cells. Mgp was also found in scales of D. sargus. Ocaccumulated mainly in bone tissues, like the ceratobranchial bone and vertebral bone matrix. ln cultured Sparus aurata, Mgp accumulated mainly in vertebrae growth zones and in notochord cells while Oc was immunodetected in bone matrix of vertebrae and vertebral arches, both in non-deformed and in deformed vertebrae. We also identified Oc in the non-calcified notochord cells of deformed vertebrae, suggesting that abnormal skeletal development resulted in modifications on sites of osteocalcin expression and/or accumulation. Accordingly, histological analysis of normal and deformed vertebrae revealed calcification in blood vessel walls and pathological formation of chondroid bone, in the affected area of deformed vertebrae.Osteocalcina (Oo) e Proteína Gla da Matriz (MGP2 são proteínas dependentes da vitamina K, com capacidade para ligarem Ca2+. Considerando o problema do desenvolvimento de malformações ósseas nos peixes de aquacultura no Mediterrâneo, investigou-se a existência de uma possível correlação entre a presença de deformações esqueléticas e alterações nos locais de acumulação da osteocalcina e Mgp. Neste trabalho, caracterizámos tecidos ósseos e cartilaginosos de peixes importantes em aquacultura por análise histológica e, determinámos locais de expressão e acumulação da osteocalcina e Mgp por hibridação tipo Northern e immunohistoquímica. Em Scophthalmus maximus, detectou-se expressão do ARNm da oc na vértebra e arcos branquiais. O ARNm da mgp teve maior expressão nos arcos branquiais, seguidos da vértebra, rim e coração. Em S. maximus e Diplodus sargus, observou-se acumulação de Mgp nos condrócitos de cartilagens, nas zonas de mineralização das vértebras e nas células da notocorda. A Mgp foi também detectada nas escamas de D. sargus. A acumulação de Oc foi detectada em tecidos ósseos, como o osso ceratobranquial e matriz óssea das vértebras. Em Sparus aurata, detectou-se Mgp nas zonas de crescimento das vértebras e nas células da notocorda, enquanto que a Oc foi detectada na matriz óssea de vértebras e arcos vertebrais, em vértebras deformadas e não deformadas. No entanto, identificou-se acumulação de Oc nas células não calcificadas da notocorda das vértebras deformadas, sugerindo que o desenvolvimento esquelético anormal levou a alterações da expressão e/ou acumulação desta proteína. Análises histológicas de vértebras normais e deformadas demonstraram calcificação nos vasos sanguíneos e formação patológica de osso condróide, na zona da vértebra afectada pela deformação

Vertebral deformities and local accumulation of gla proteins in sparus aurata

Development of skeletal deformities is a frequent problem in Mediterranean fish aquaculture. Investigation of proteins involved in tissue calcification may help us uncover a possible correlation between the appearance of skeletal deformities and a defect in the site of expression and/or accumulation of a particular gene or protein. The purpose of this study was to investigate if the presence of skeletal deformities was related with any changes in the sites of osteocalcin (Oc or Bgp – Bone Gla Protein) and matrix Gla protein (Mgp) accumulation using immunohistochemistry methods

Evidence for the conservation of miR-223 in zebrafish (Danio rerio): implications for function

MicroRNAs (miRNAs) are an abundant and conserved class of small RNAs, which play important regulatory functions by interacting with the 3' untranslated region (UTR) of target mRNAs. Through this mechanism, miR-223 was shown to regulate genes involved in mammalian haematopoiesis, both in physiological and pathological contexts. MiR-223 is essential for normal myelopoiesis in mammals, promoting granulocyte, osteoclast and megakaryocyte differentiation and suppressing erythropoiesis. However, there is a general lack of knowledge regarding miR-223 function in other vertebrates, which could help to clarify its role in other processes, such as development. In this work, we explored the functional conservation of miR-223 using zebrafish as a model. We show that miR-223 gene structure and genomic context have been maintained between human and zebrafish. In addition, we identified 22 novel sequences of miR-223 precursor and demonstrate that it contains domains highly conserved among vertebrates, suggesting function preservation throughout evolution. Furthermore, collected evidences show that miR-223 expression is highly correlated with haematopoietic events and osteoclastogenesis throughout zebrafish development. In adults, expression of miR-223 in zebrafish tissues mimics the distribution in mice, with high levels found in the major fish haematopoietic organ, the head kidney. These results suggest a conservation of miR-223 role in haematopoiesis, and osteoclastogenesis between zebrafish and human. Accordingly, validated targets of miR-223 in mammalian models were investigated and defined as putative targets in zebrafish, by in silico and gene expression analysis. Our data compiles critical evidence showing that miR-223, a highly conserved miRNA, appears to have kept similar regulatory functions throughout evolution.This work was supported by a prize granted by the Calouste Gulbenkian Foundation (program “Na Fronteira das Ciências da Vida - 2009”; to D.M.T.), by the European Regional Development Fund (ERDF) through COMPETE Program and by national funds through the FCT — Foundation for Science and Technology, under the project “PEst-C/MAR/LA0015/ 2011” and by Helse SørØst, Norway. V.P.R. and D.M.T. were the recipients of doctoral (SFRH/BD/38607/ 2007) and post-doctoral (SFRH/BPD/45034/2008) fellowships respectively, fromthe Portuguese Foundation for Science and Technology (FCT)info:eu-repo/semantics/publishedVersio