Nanopore sequencing in human genetic studies: application to structural variant detection

Background: Nanopore sequencing is a recent technology which allows direct real-time sequencing of DNA or RNA molecules and production of read lengths as long as the size of the original fragments. It is based on the application of an ionic current to move DNA strands through an array of nanopores embedded in an electrically-resistant membrane. An ASIC chip placed below this membrane is responsible for recording signal changes in the nanopore, which are then translated into a specific base sequence. Although major improvements have been made in the genomic short-read sequencing technologies, structural variants (SV) detection still stands as a challenge. The characteristics of nanopore sequencing make it well suited for whole genome sequencing and as a preferential tool to identify SV, namely those associated with tumours. In this work we present a complete workflow to detect SV in tumour samples using nanopore sequencing. Methods: We used a test tumour sample to prepare DNA libraries using the rapid sequencing kit (Oxford Nanopore Technologies-ONT) and sequenced those in a single R9.4 flow cell on the MinION device (ONT). We implemented a pipeline for SV detection using multiple bioinformatics tools: MinKNOW software (ONT) (run set up and data acquisition), Albacore pipeline (ONT) (basecalling), LAST aligner (mapping) and Picky (SV detection and analysis). Results: The use of the rapid sequencing kit allowed a fast preparation of sequencing libraries when compared with standard library preparation procedures. The MinION generated a total of 2.34 Gb of DNA. The highest number of reads were obtained during the first 8-10h of the sequencing run. Overall, 87.8% of reads had a quality (Q) value >7 (quality threshold) and 54.2% had a Q value >10. The longest read obtained had 74369 bases and the mean read length was of 4508 bases. All reads with a length >50 kb had Q values >= 7. A total of 3470 SV were identified, including deletions, duplications, translocations, insertions and inversions. Conclusions: Long-read sequencing technology is becoming an important tool as it poses itself as a powerful complementary method that suppresses the inherent limitations of other technologies. Nanopore sequencing is a fast and sensitive approach of great potential for human genomics research and for use in a clinical perspective, namely in the detection of SV in the human genome. A critical issue in nanopore sequencing is DNA quality and integrity because longer size reads make it easier to detect large SV.This work was supported by Toxomics (Centre for Toxicogenomics and Human Health), Nova Medical School and is a result of the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

Standardization of the Solar Array Design for Small Satellites

Our approach: Choose your configuration, get all the information you need and place your order Track and trace the progress of the manufacturing process Plug the solar array to your spacecraft and get ready for launc

Cyclodextrins in pulmonary drug delivery

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia.A veiculação de fármacos via pulmonar é relevante devido ao seu elevado índice terapêutico, maior seletividade, menor dose administrada e efeitos adversos sistémicos reduzidos em comparação com outras vias de administração. Os principais desafios por esta via de administração são os mecanismos de depuração e a administração eficiente de fármacos pouco solúveis em água. As ciclodextrinas são oligossacáridos cíclicos não redutores que se têm demonstrado promissores portadores de fármacos para diversas indicações, como cancro do pulmão, fibrose pulmonar, entre outras. As ciclodextrinas naturais e os seus derivados são utilizadas em formulações administradas via oral, bucal, retal, dérmica, ocular, parentérica e, menos frequentemente, via pulmonar. As principais vantagens destas moléculas como transportadoras de fármacos são o aumento da solubilidade e estabilidade do fármaco, assim como a diminuição da toxicidade local e sistémica. Neste trabalho, foi realizada uma revisão da investigação feita sobre as características dos complexos fármaco-ciclodextrina para libertação pulmonar. As características físicas e químicas de diversas nanopartículas contendo fármaco-ciclodextrina revelaram que as ciclodextrinas diminuíram o tamanho e aumentaram a estabilidade das nanopartículas. Os resultados sobre o desempenho in vitro e in vivo de formulações contendo fármaco-ciclodextrinas revelaram uma libertação in vivo eficaz e biodisponibilidade sistémica com baixa toxicidade. A maioria das formulações selecionadas para veiculação pulmonar contêm ciclodextrinas destinadas à libertação de extratos e produtos químicos e foram encontrados apenas alguns estudos com ciclodextrinas para libertação de proteínas. Foi encontrado apenas um medicamento com ciclodextrinas aprovado para libertação pulmonar – Aerodiol – com estradiol como substância ativa e metil-ß-ciclodextrina como um dos excipientes da formulação. Até onde sabemos, não há nenhum medicamento em ensaios clínicos para aplicação pulmonar que contenha ciclodextrinas como excipiente, apesar de ter sido encontrado um estudo piloto não registado, realizado no Chipre. Em suma, com este trabalho, focado nos avanços mais recentes do desenvolvimento de formulações contendo ciclodextrinas, é possível concluir que há ainda um longo caminho a percorrer no desenvolvimento de medicamentos para administração pulmonar com ciclodextrinas.Pulmonary drug delivery is relevant due to its high therapeutic ratio, increased selectivity, lower administrated dose, and reduced systemic side effects when compared with other administration routes. The major challenges for this administration route are the clearance mechanisms and the efficient delivery of poorly water-soluble drugs. Cyclodextrins are cyclic non reducing oligosaccharides that have been shown as promising carriers of drugs for several indications such as lung cancer, pulmonary fibrosis, among others. Natural cyclodextrins and its derivatives have been used in formulations administered by oral, buccal, rectal, dermal, ocular, parental routes, and less frequently used for pulmonary delivery. The main advantages of these molecules as drug carriers are drug solubility, stability increase, and decrease of local and systemic toxicity. In this work, a review of the research that have been done about characteristics of drug-cyclodextrin complexes for pulmonary delivery, was performed. Physicochemical characteristics of several drug-cyclodextrin nanoparticles revealed that these molecules decreased the size and increased the stability of the nanoparticles. Results on the in vitro and in vivo performance of drug-cyclodextrin formulations unveiled an effective in vivo delivery and systemic bioavailability with low toxicity. The majority of screened formulations, for pulmonary delivery, containing cyclodextrins aimed at delivering extracts and chemicals and only few studies with cyclodextrins for protein delivery were found. Only one approved medicine was found with cyclodextrins for pulmonary delivery - Aerodiol - with estradiol as the active substance and a formulation containing methyl-ß-cyclodextrin as one of the excipients. To our best knowledge, no registered clinical trial testing drugs for pulmonary delivery uses cyclodextrins as carriers or excipients, even though an unregistered pilot study carried out in Cyprus was found. Overall, with this work, focused on the most recent advances on the development of formulations containing cyclodextrins, it is possible to conclude that there is still a long way to go in the development of new medicines for pulmonary delivery using cyclodextrins

ARP Case Report nº 13: What is your diagnosis?

Presentation of the CaseA 73-year-old woman who has been consulting the gynaecologist regarding persistent pelvic pain since last month. No other complaints including systemic symptoms or metrorrhagia.The ultrasound study showed a large pelvic tumour mass and it was not possible to determine the organ of origin. Values of Ca-125 are normal.Pelvic MRI was requested for additional characterization of the pelvic mass and to determine the organ of origin

Caso Clínico ARP Nº13: Torção com Enfarte de Mioma Pediculado

A 73-year-old woman consulted the gynecologist for persistent pelvic pain for the past month. No other complaints including systemic symptoms or metrorrhagia. The ultrasound study showed a large pelvic tumour mass. It was not possible to determine the organ of origin. Values of Ca- 125 were normal.Pelvic MRI was requested for additional characterization of the pelvic mass and to determine the organ of origin.Magnetic resonance imaging revealed a well-defined solid tumour lesion located in the midline independent of the ovaries and bladder but contacting the uterine fundus by a small pedicle. The mass reveals blood inside and there was no enhancement after IV contrast. The findings suggest the diagnosis of torsion on pedunculated subserous uterine myoma with infarction.Mulher de 73 anos que recorre à médica ginecologista por dor pélvica persistente desde o último mês. Sem outras queixas nomeadamente sintomas sistémicos ou metrorragias. O estudo ecográfico realizado mostra volumosa massa tumoral pélvica não sendo possível determinar o órgão de origem. Os valores de Ca-125 são normais. Foi pedida RM pélvica para caracterização adicional da massa pélvica e determinar órgão de origem. A RM magnética revelou lesão tumoral sólida, bem delimitada, localizada na linha média independente dos ovários e da bexiga mas que contacta com o fundus uterino por um pequeno pedículo. A massa revela sangue no seu interior e aquando do contraste não exibe qualquer captação de contraste. O diagnóstico avançado e confirmado na cirurgia foi de mioma fundico pediculado complicado com torção e enfarte

NGS Panels applied to Hereditary Cancer Syndromes

Cancer is among the leading causes of morbidity and mortality worldwide (Okur et al, 2017). Germline pathogenic variants for monogenic, highly penetrant cancer susceptibility genes are observed in 5%–10% of all cancers (Lu et al, 2014). Hereditary cancers due to monogenic causes are characterized by earlier age of onset, other associated cancers, and often a family history of specific cancers. From the clinical perspective, it is important to recognize the affected individuals to provide them the best clinical management (Hennessy et al, 2010; Ledermann et al, 2014; Pennington et al, 2014) and to identify at-risk family members who will benefit from predictive genetic testing and enhanced surveillance, including early detection and/or risk reduction measures (Kurian et al, 2010; Okur et al, 2017). Germline variants identified in major cancer susceptibility genes associated with hereditary breast or ovarian cancer (HBOC) or hereditary colorectal cancer (HCRC), also account for 5-10% of the patients with these cancers. In the last years, new susceptibility genes, with different penetrance degrees, have been identified. Variants in any of those genes are rare and classical methodologies (e.g. Sanger sequencing - SS) are time consuming and expensive. Next-generation sequencing (NGS) has several advantages compared to SS, including the simultaneous analysis of many samples and sequencing of a large set of genes, higher sensitivity (down to 1% vs 15-20% in SS), lower cost and faster turnaround time, reasons that make NGS the best approach for molecular diagnosis. It is possible nowadays to choose between whole-genome sequencing (WGS), whole-exome sequencing (WES) and NGS limited to a set of genes (NGS-Panel). In cases where a suspected genetic disease or condition has been identified, targeted sequencing of specific genes or genomic regions is preferred (Grada et al, 2013). For that reason, we use NGS-Panel approach using TruSight Cancer (Illumina) to sequence DNA extracted from blood samples of patients with personal and/or familiar history of cancer. This hereditary cancer gene panel sequences 94 genes associated with both common (e.g., breast, colorectal) and rare hereditary cancers and allows the creation of virtual gene panels according to each phenotype or disease under study. NGS workflow analysis (Figure 1) includes five steps: quality assessment of raw data, read alignment to a reference genome, variant identification/calling, variant annotation and data visualization (Pabinger et al, 2013). The establishment of the most appropriate bioinformatics pipeline is crucial in order to achieve the best results. NGS data allows the identification of several types of variants like single nucleotide variants (SNVs), small insertions/deletions, inversions and also copy number variants (CNVs).FCT - UID/BIM/0009/2016info:eu-repo/semantics/publishedVersio

Mães e pais em reclusão: Diferenças e comunalidades no contacto com descendentes menores

Dissertação de mestrado integrado em Psicologia (área de especialização em Psicologia da Justiça)O aumento da população prisional, a nível mundial, traduziu-se num inequívoco acréscimo do número de pais e mães encarcerados. Comprovada a importância do contacto entre progenitores recluídos e seus filhos/as para o ajustamento psicológico de ambos, e dada a escassez de investigações nacionais a este nível, o presente estudo visou avaliar, primordialmente, a frequência de contacto estabelecida entre detentos e seus filhos menores, a qualidade da relação entre estes e o sentido de competência parental dos primeiros. A presença de diferenças entre os sexos, ao nível dos aspetos avaliados, foi igualmente aferida. Desta forma, administrou-se um questionário, constituído para os efeitos deste estudo, a 200 reclusos/as. Os resultados sugerem algumas diferenças entre homens e mulheres, bem como aspetos semelhantes para ambos os sexos. No que concerne às diferenças, talvez essas sejam o reflexo da sociedade atual em que a mãe, apesar de tudo, continua a ter o papel de cuidadora emocional e o pai, mais distante emocionalmente, terá encargos relacionados com a subsistência financeira da família.The increase in worldwide population in prison has resulted in an unequivocal growth of the number of incarcerated parents. Given the importance of the contact between incarcerated parents and their children to the psychological adjustment of both of them, and in face of the scarcity of national studies at this level, the present study aimed to assess the frequency of the contact established between prisoners and their minor children, the quality of the relationship between them, and the sense of parental competence of the incarcerated mothers and fathers. This study also assessed the presence of differences between sexes regarding the studied issues. A questionnaire composed for the purposes of this study was administered to 200 inmates. Results suggest some differences between men and women, as well as similar aspects for both sexes. Those differences are, probably, the reflection of today's society in which the mother continues to interpret the role of the emotional caregiver and the father, more emotionally distant, has occupations related to the financial maintenance of the family

A oxigenoterapia hiperbárica nas lesões neuro-músculoesqueléticas: um estudo retrospetivo

Projeto de Graduação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Licenciada em FisioterapiaObjetivo: Efetuar um levantamento de dados, através de uma análise retrospetiva, sobre a eficácia da Oxigenoterapia Hiperbárica (OTH) na reabilitação de sequelas neuro-músculoesqueléticas provocadas pela Doença de Descompressão (DD) e verificar quantos e que casos precisaram da fisioterapia. Metodologia: A amostra foi constituída por 140 indivíduos, com uma média de idades de 35,31 ± 9,49 anos, que tenham recebido tratamento de OTH em Portugal continental, nomeadamente nos serviços de medicina hiperbárica do Hospital Pedro Hispano (Matosinhos) e no centro médico subaquático de hiperbárica do Hospital das Forças Armadas (Lisboa) com o diagnóstico de DD. Resultados: Desta amostra, 129 (92,1%) dos indivíduos eram do género masculino e apenas 11 (7,9%) do género feminino. Do total, 111 (79,3%) pertenciam ao Hospital das Forças Armadas e 29 (20,7%) ao Hospital Pedro Hispano. A DD tipo 1 teve como resultado 60 (42%) de prevalência e o tipo 2 de 74 (52,9%) em 134 indivíduos, havendo 6 (4,3%) omissos. Apenas os diagnosticados com DD tipo 2, com comprometimento medular, 6 (8,1%) indivíduos realizaram fisioterapia. Conclusão: A OTH é o tratamento gold-standard para a DD e que a necessidade da fisioterapia, como tratamento complementar, revelou-se importante na DD tipo 2 com comprometimento medular.Objective: To conduct a data collection, through a retrospective analysis, on the efficacy of Hyperbaric Oxygen Therapy (HBO) in the rehabilitation of neuro-musculoskeletal sequelae caused by Decompression Disease (DD) and to verify how many and which cases required physiotherapy. Methodology: The sample consisted of 140 individuals, with a mean age of 35.31 ± 9.49 years, who had received HBO treatment in mainland Portugal, namely in the hyperbaric medicine services of Pedro Hispano Hospital (Matosinhos) and in the underwater hyperbaric medical center of the Armed Forces Hospital (Lisbon) with the diagnosis of DD. Results: Of this sample, 129 (92.1%) of the individuals were male and only 11 (7.9%) were female. Of the total, 111 (79.3%) belonged to the Armed Forces Hospital and 29 (20.7%) to the Pedro Hispano Hospital. Type 1 DD was prevalent in 60 (42%) and type 2 in 74 (52.9%) of 134 individuals, with 6 (4.3%) without information. Only 6 (8.1%) individuals underwent physiotherapy diagnosed with type 2 DD, all with spinal cord involvement. Conclusion: HBO is the gold standard treatment for DD and the need for physiotherapy, as a complementary treatment, has been shown to be important in type 2 DD with spinal cord involvement.N/

Efeitos de ortóteses e de exercícios aeróbios na capacidade física e na função pulmonar de adolescentes com escoliose idiopática: revisão sistemática

Projeto de Graduação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Licenciada em FisioterapiaObjetivo: Averiguar os efeitos da utilização de ortóteses e de exercícios aeróbios na capacidade física e na função pulmonar de adolescentes com Escoliose Idiopática (EI), de forma a compreender se estas duas diferentes abordagens de tratamento se podem complementar. Metodologia: Pesquisa computorizada nas bases de dados B-on e Pubmed, para identificar estudos clínicos que avaliassem a utilização de ortóteses e de exercícios aeróbios em adolescentes com EI. Resultados: Nesta revisão foram incluídos 9 estudos envolvendo 221 adolescentes. Dos estudos incluídos que referiam estes tipos de intervenção, 5 eram sobre a utilização de ortóteses e 4 sobre a utilização de exercícios aeróbios. Conclusões: As ortóteses parecem ter efeitos negativos e os exercícios aeróbios efeitos positivos, nos volumes pulmonares. Quanto à capacidade física os dados são inconclusivos. A sistematização do conhecimento nesta área beneficiaria mais se houvesse mais estudos clínicos sobre o assunto. Objective: To determine the effects of bracing and aerobic exercises on physical capacity and pulmonary function in adolescent idiopathic scoliosis (AIS), in order to understand if this two different approaches could complement. Methods: Research on computerized databases on B-On and PubMed to identify clinic trials that evaluated the effects of bracing and aerobic exercises in AIS. Results: This review included 9 studies involving 221 adolescents. Of the studies included, 5 were about bracing and 4 were about aerobic exercises. Conclusions: Bracing seems to have negative effects and aerobic exercises positive effects on pulmonary volumes. Regarding physical capacity data, they were inconclusive. The systematization of knowledge in this area would benefit more if there were more clinical studies on the subject