Validação e implementação do kit Quantifiler® Trio DNA Quantification em amostras forenses

Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015O perfil genético de cada indivíduo pode ser obtido através do estudo de vários marcadores polimórficos presentes no DNA nuclear, nomeadamente os STRs. Em Genética Forense, a quantidade de DNA pode variar consideravelmente entre diferentes amostras, factor que poderá comprometer a obtenção dos respetivos perfis genéticos. Deste modo, torna-se indispensável determinar a quantidade de DNA presente nas amostras forenses através de uma metodologia de quantificação, recorrendo a alguns kits comerciais. Esta etapa permite avaliar se uma amostra pode ou não seguir para amplificação por PCR e, em caso afirmativo, qual o kit de amplificação e volume de DNA que devem ser utilizados, garantindo, assim, o sucesso dos resultados. De todas as amostras analisadas no contexto forense, as degradadas constituem o maior desafio, devido à presença de DNA em quantidade e qualidade muitas vezes limitante. Para ultrapassar esse obstáculo, surgiu um novo kit de quantificação denominado Quantifiler® Trio DNA Quantification. Este kit apresenta características melhoradas comparativamente aos kits anteriores, possibilitando detetar quantidades de DNA mais reduzidas, bem como proporções de DNA masculino versus DNA feminino mais díspares em amostras de mistura. Como mais-valia, o kit permite avaliar o nível de degradação das amostras. Para esse efeito, deteta dois alvos localizados no DNA autossómico, um alvo de 80 pares de bases e um alvo de 214 pares de bases, sendo que a razão entre as concentrações destes dois alvos constitui o índice de degradação de cada amostra. No Serviço de Genética e Biologia Forenses - Delegação do Centro do Instituto Nacional de Medicina Legal e Ciências Forenses, I.P., procedeu-se à validação interna do kit Quantifiler® Trio, com base no Procedimento Geral de Validação de Ensaios, documento elaborado pelo serviço que define todos os passos e estudos a realizar durante este processo. Para testar o desempenho do kit foram estudados os parâmetros de precisão, limiares analíticos, especificidade e contaminação. A par disso, foi igualmente avaliada a influência dos resultados de quantificação obtidos com o kit na determinação dos perfis genéticos de amostras de mistura com diferentes proporções, assim como de amostras com diferentes índices de degradação, recorrendo ao kit de amplificação GlobalFiler®. Este estudo demonstrou uma grande precisão e sensibilidade do kit Quantifiler® Trio, associado à especificidade deste para o DNA humano. Por fim, a informação dada pelo índice de degradação revelou ser fundamental para prever o impacto que diferentes níveis de degradação poderão ter na obtenção de um perfil genético.The genetic profile of each individual can be obtained through the study of several polymorphic markers in the nuclear DNA, mainly the STRs. In Forensic Genetics, the amount of DNA can vary considerably among different samples, a factor which may interfere with the achievement of the respective genetic profiles. Thus, it is essential to determine the amount of DNA present in forensic samples by a quantification methodology, using commercial kits. This step provides guidelines on whether or not a sample is suitable for PCR amplification and, if possible, which amplification kit and DNA volume should be used, therefore ensuring the success of the results. In all samples analyzed in forensic context, degraded samples represent a major challenge, due to the presence of DNA in often compromised quantity and quality. To overcome this obstacle, a new quantification kit called Quantifiler® Trio DNA Quantification was developed. This kit has improved features when compared to previous kits used, making it possible to detect smaller amounts of DNA and proportions of male DNA versus female DNA more discrepants in mixture samples. In addition, the kit enables to evaluate the level of degradation of samples. For this purpose, it detects two targets located in the autosomal DNA, a target of 80 base pairs and another of 214 base pairs. The ratio between the concentrations of these two targets is referred to as the degradation index of each sample. In the Forensics Genetic and Biology Service - Central Delegation of the National Institute of Legal Medicine and Forensic Sciences, I.P., the internal validation of Quantifiler® Trio kit was carried out, based on the General Procedure Testing Validation, a document developed in the service which defines all the steps and studies to be conducted during this process. The kit performance was tested based on the study of precision parameters, analytical thresholds, specificity and contamination. Furthermore, it was also evaluated the influence of the quantification results obtained with the Quantifiler® Trio kit in determining genetic profiles of mixture samples with different ratios, as well as samples with different levels of degradation, using the GlobalFiler® PCR Amplification kit. This study demonstrated a high accuracy and sensitivity of the Quantifiler® Trio kit, along with its specificity for human DNA. Finally, the information provided by the degradation index was found to be essential to predict the impact that different levels of degradation may have on obtaining a genetic profile

Microsatellites’ mutation modeling through the analysis of the Y-chromosomal transmission: Results of a GHEP-ISFG collaborative study

The Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) organized a collaborative study on mutations of Y-chromosomal short tandem repeats (Y-STRs). New data from 2225 father-son duos and data from 44 previously published reports, corresponding to 25,729 duos, were collected and analyzed. Marker-specific mutation rates were estimated for 33 Y-STRs. Although highly dependent on the analyzed marker, mutations compatible with the gain or loss of a single repeat were 23.2 times more likely than those involving a greater number of repeats. Longer alleles (relatively to the modal one) showed to be nearly twice more mutable than the shorter ones. Within the subset of longer alleles, the loss of repeats showed to be nearly twice more likely than the gain. Conversely, shorter alleles showed a symmetrical trend, with repeat gains being twofold more frequent than reductions. A positive correlation between the paternal age and the mutation rate was observed, strengthening previous findings. The results of a machine learning approach, via logistic regression analyses, allowed the establishment of algebraic formulas for estimating the probability of mutation depending on paternal age and allele length for DYS389I, DYS393 and DYS627. Algebraic formulas could also be established considering only the allele length as predictor for DYS19, DYS389I, DYS389II-I, DYS390, DYS391, DYS393, DYS437, DYS439, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS533, DYS576, DYS626 and DYS627 loci. For the remaining Y-STRs, a lack of statistical significance was observed, probably as a consequence of the small effective size of the subsets available, a common difficulty in the modeling of rare events as is the case of mutations. The amount of data used in the different analyses varied widely, depending on how the data were reported in the publications analyzed. This shows a regrettable waste of produced data, due to inadequate communication of the results, supporting an urgent need of publication guidelines for mutation studies.info:eu-repo/semantics/publishedVersio

Autoimmune and autoinflammatory mechanisms in uveitis

The eye, as currently viewed, is neither immunologically ignorant nor sequestered from the systemic environment. The eye utilises distinct immunoregulatory mechanisms to preserve tissue and cellular function in the face of immune-mediated insult; clinically, inflammation following such an insult is termed uveitis. The intra-ocular inflammation in uveitis may be clinically obvious as a result of infection (e.g. toxoplasma, herpes), but in the main infection, if any, remains covert. We now recognise that healthy tissues including the retina have regulatory mechanisms imparted by control of myeloid cells through receptors (e.g. CD200R) and soluble inhibitory factors (e.g. alpha-MSH), regulation of the blood retinal barrier, and active immune surveillance. Once homoeostasis has been disrupted and inflammation ensues, the mechanisms to regulate inflammation, including T cell apoptosis, generation of Treg cells, and myeloid cell suppression in situ, are less successful. Why inflammation becomes persistent remains unknown, but extrapolating from animal models, possibilities include differential trafficking of T cells from the retina, residency of CD8(+) T cells, and alterations of myeloid cell phenotype and function. Translating lessons learned from animal models to humans has been helped by system biology approaches and informatics, which suggest that diseased animals and people share similar changes in T cell phenotypes and monocyte function to date. Together the data infer a possible cryptic infectious drive in uveitis that unlocks and drives persistent autoimmune responses, or promotes further innate immune responses. Thus there may be many mechanisms in common with those observed in autoinflammatory disorders

Pneumococcal lineages associated with serotype replacement and antibiotic resistance in childhood invasive pneumococcal disease in the post-PCV13 era: an international whole-genome sequencing study

Background: Invasive pneumococcal disease remains an important health priority owing to increasing disease incidence caused by pneumococci expressing non-vaccine serotypes. We previously defined 621 Global Pneumococcal Sequence Clusters (GPSCs) by analysing 20 027 pneumococcal isolates collected worldwide and from previously published genomic data. In this study, we aimed to investigate the pneumococcal lineages behind the predominant serotypes, the mechanism of serotype replacement in disease, as well as the major pneumococcal lineages contributing to invasive pneumococcal disease in the post-vaccine era and their antibiotic resistant traits. / Methods: We whole-genome sequenced 3233 invasive pneumococcal disease isolates from laboratory-based surveillance programmes in Hong Kong (n=78), Israel (n=701), Malawi (n=226), South Africa (n=1351), The Gambia (n=203), and the USA (n=674). The genomes represented pneumococci from before and after pneumococcal conjugate vaccine (PCV) introductions and were from children younger than 3 years. We identified predominant serotypes by prevalence and their major contributing lineages in each country, and assessed any serotype replacement by comparing the incidence rate between the pre-PCV and PCV periods for Israel, South Africa, and the USA. We defined the status of a lineage as vaccine-type GPSC (≥50% 13-valent PCV [PCV13] serotypes) or non-vaccine-type GPSC (>50% non-PCV13 serotypes) on the basis of its initial serotype composition detected in the earliest vaccine period to measure their individual contribution toward serotype replacement in each country. Major pneumococcal lineages in the PCV period were identified by pooled incidence rate using a random effects model. / Findings: The five most prevalent serotypes in the PCV13 period varied between countries, with only serotypes 5, 12F, 15B/C, 19A, 33F, and 35B/D common to two or more countries. The five most prevalent serotypes in the PCV13 period varied between countries, with only serotypes 5, 12F, 15B/C, 19A, 33F, and 35B/D common to two or more countries. These serotypes were associated with more than one lineage, except for serotype 5 (GPSC8). Serotype replacement was mainly mediated by expansion of non-vaccine serotypes within vaccine-type GPSCs and, to a lesser extent, by increases in non-vaccine-type GPSCs. A globally spreading lineage, GPSC3, expressing invasive serotypes 8 in South Africa and 33F in the USA and Israel, was the most common lineage causing non-vaccine serotype invasive pneumococcal disease in the PCV13 period. We observed that same prevalent non-vaccine serotypes could be associated with distinctive lineages in different countries, which exhibited dissimilar antibiotic resistance profiles. In non-vaccine serotype isolates, we detected significant increases in the prevalence of resistance to penicillin (52 [21%] of 249 vs 169 [29%] of 575, p=0·0016) and erythromycin (three [1%] of 249 vs 65 [11%] of 575, p=0·0031) in the PCV13 period compared with the pre-PCV period. / Interpretation: Globally spreading lineages expressing invasive serotypes have an important role in serotype replacement, and emerging non-vaccine serotypes associated with different pneumococcal lineages in different countries might be explained by local antibiotic-selective pressures. Continued genomic surveillance of the dynamics of the pneumococcal population with increased geographical representation in the post-vaccine period will generate further knowledge for optimising future vaccine design. / Funding: Bill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control

Covalent Label Transfer Between Peroxisomal Importomer Components Reveals Export-Driven Import Interactions

Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal Malate Dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes, owing to high degrees of protease susceptibility or resistance, respectively. Here, we present a means for analysis of in vitro import through a covalent biotin label transfer, and employ this method to the import of PEX5C. Label transfer demonstrates that PEX5C construct is monomeric in the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labelling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest, and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from 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

Enhanced hydrogen production from thermochemical processes

To alleviate the pressing problem of greenhouse gas emissions, the development and deployment of sustainable energy technologies is necessary. One potentially viable approach for replacing fossil fuels is the development of a H2 economy. Not only can H2 be used to produce heat and electricity, it is also utilised in ammonia synthesis and hydrocracking. H2 is traditionally generated from thermochemical processes such as steam reforming of hydrocarbons and the water-gas-shift (WGS) reaction. However, these processes suffer from low H2 yields owing to their reversible nature. Removing H2 with membranes and/or extracting CO2 with solid sorbents in situ can overcome these issues by shifting the component equilibrium towards enhanced H2 production via Le Chatelier's principle. This can potentially result in reduced energy consumption, smaller reactor sizes and, therefore, lower capital costs. In light of this, a significant amount of work has been conducted over the past few decades to refine these processes through the development of novel materials and complex models. Here, we critically review the most recent developments in these studies, identify possible research gaps, and offer recommendations for future research

Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine

[This corrects the article DOI: 10.1186/s13054-016-1208-6.]

Segurança do paciente no uso de medicamentos após a alta hospitalar: estudo exploratório1

No Brasil, são escassos os estudos sobre estratégias para a segurança do paciente no processo de uso de medicamentos após a alta hospitalar, o que dificulta o conhecimento sobre a atuação de hospitais brasileiros nessa área. Neste artigo, buscou-se compreender a dinâmica e os desafios do cuidado fornecido ao paciente pela equipe hospitalar, visando à segurança no processo de uso de medicamentos após a alta hospitalar. Realizou-se pesquisa exploratória por meio de entrevistas com médicos, enfermeiros, farmacêuticos e assistentes sociais do Hospital Universitário da Universidade de São Paulo. Foram pesquisadas as atividades de cuidado com a farmacoterapia durante e após a hospitalização, incluindo o acesso a medicamentos após alta, a existência de articulação do hospital com outros serviços de saúde, e barreiras para desenvolver essas atividades. A principal estratégia adotada é a orientação de alta, realizada de forma estruturada, principalmente para cuidadores de pacientes pediátricos. Em situações específicas, ocorre mobilização da equipe para viabilização do acesso a medicamentos prescritos na alta. Reconciliação medicamentosa está em fase de implantação, e visita domiciliar é realizada apenas para pacientes críticos com problemas de locomoção. As principais barreiras identificadas foram insuficiência de recursos humanos e falta de tecnologias de informação. Conclui-se que são desenvolvidas algumas estratégias, porém com limitações e sem articulação adequada com outros serviços de saúde para a continuidade do cuidado. Isto sugere a necessidade de concentração de esforços para transpor as barreiras identificadas, contribuindo para a segurança do paciente na interface entre hospital, atenção básica e domicílio