Systems epidemiology to devise new interventions for multi-host tuberculosis

Animal tuberculosis (TB) is an infectious disease of livestock and wildlife mainly caused by Mycobacterium bovis and, in a minor extent, Mycobacterium caprae. In Portugal, animal TB is maintained in a multi-host system involving livestock and wild ungulates. The understanding of the processes driving transmission at this interface is key to inform control. In this work, M. caprae isolates (n=55) from Portugal were characterized by spoligotyping and MIRU-VNTR, supporting the clonal structure, co-infection and in vivo microevolution of this ecotype. M. bovis (n=948) from cattle, red deer and wild boar from TB hotspot regions were genotyped. Bayesian inference identified five ancestral populations and associated the most probable ancient M. bovis subpopulation with cattle and Beja, providing clues on the epidemics origin. A multinomial spatiotemporal probability model identified two significant TB clusters: one that persisted in 2004-2010 with Barrancos (Beja) at the centre, highlighting a significant higher risk associated to cattle; a second cluster, predominant in 2012-2016, holding the county Rosmaninhal (Castelo Branco) at the centre, for which wild boar contributed the most in relative risk. Whole-genome sequences (WGS) of 44 representative M. bovis distinguished five genetic clades and supported sustained transmission and multiple introductions in this multi-host system. Exploratory evolutionary analysis gave further support to pathogen transition between different hosts. Comparative genomics applied to M. bovis (n=70) representing the global clonal complex diversity predicted an open pan-genome and showed diversification of discrete subpopulations through core and accessory genomes. Consistent non-synonymous SNPs illustrated clade-specific virulence landscapes correlating with disease severity. Positive selection and weaker effects of recombination compared with mutation were evidenced as predominant evolutionary forces. Altogether, our results provide novel evidence on the population structure and evolution of M. caprae and M. bovis, delivering insights that could be used to inform adaptive TB control choices in different hosts and regions

The open pan-genome architecture and virulence landscape of Mycobacterium bovis

Animal tuberculosis (TB) is an emergent disease caused by Mycobacterium bovis, one of the animal-adapted ecotypes of the Mycobacterium tuberculosis complex (MTC). In this work, whole-genome comparative analyses of 70 M. bovis were performed to gain insights into the pan-genome architecture. The comparison across M. bovis predicted genome composition enabled clustering into the core- and accessory-genome components, with 2736 CDS for the former, while the accessory moiety included 3897 CDS, of which 2656 are restricted to one/two genomes only. These analyses predicted an open pan-genome architecture, with an average of 32 CDS added by each genome and show the diversification of discrete M. bovis subpopulations supported by both core- and accessory-genome components. The functional annotation of the pan-genome classified each CDS into one or several COG (Clusters of Orthologous Groups) categories, revealing ‘transcription’ (total average CDSs, n=258), ‘lipid metabolism and transport’ (n=242), ‘energy production and conversion’ (n=214) and ‘unknown function’ (n=876) as the most represented. The closer analysis of polymorphisms in virulence-related genes in a restrict group of M. bovis from a multi-host system enabled the identification of clade-monomorphic non-synonymous SNPs, illustrating clade-specific virulence landscapes and correlating with disease severity. This first comparative pan-genome study of a diverse collection of M. bovis encompassing all clonal complexes indicates a high percentage of accessory genes and denotes an open, dynamic non-conservative pan-genome structure, with high evolutionary potential, defying the canons of MTC biology. Furthermore, it shows that M. bovis can shape its virulence repertoire, either by acquisition and loss of genes or by SNP-based diversification, likely towards host immune evasion, adaptation and persistence.info:eu-repo/semantics/publishedVersio

Genome-wide estimation of recombination, mutation and positive selection enlightens diversification drivers of Mycobacterium bovis

Genome sequencing has reinvigorated the infectious disease research feld, shedding light on disease epidemiology, pathogenesis, host–pathogen interactions and also evolutionary processes exerted upon pathogens. Mycobacterium tuberculosis complex (MTBC), enclosing M. bovis as one of its animal-adapted members causing tuberculosis (TB) in terrestrial mammals, is a paradigmatic model of bacterial evolution. As other MTBC members, M. bovis is postulated as a strictly clonal, slowly evolving pathogen, with apparently no signs of recombination or horizontal gene transfer. In this work, we applied comparative genomics to a whole genome sequence (WGS) dataset composed by 70 M. bovis from diferent lineages (European and African) to gain insights into the evolutionary forces that shape genetic diversifcation in M. bovis. Three distinct approaches were used to estimate signs of recombination. Globally, a small number of recombinant events was identifed and confrmed by two independent methods with solid support. Still, recombination reveals a weaker efect on M. bovis diversity compared with mutation (overall r/m= 0.037). The diferential r/m average values obtained across the clonal complexes of M. bovis in our dataset are consistent with the general notion that the extent of recombination may vary widely among lineages assigned to the same taxonomical species. Based on this work, recombination in M. bovis cannot be excluded and should thus be a topic of further efort in future comparative genomics studies for which WGS of large datasets from diferent epidemiological scenarios across the world is crucial. A smaller M. bovis dataset (n= 42) from a multi-host TB endemic scenario was then subjected to additional analyses, with the identifcation of more than 1,800 sites wherein at least one strain showed a single nucleotide polymorphism (SNP). The majority (87.1%) was located in coding regions, with the global ratio of non-synonymous upon synonymous alterations (dN/dS) exceeding 1.5, suggesting that positive selection is an important evolutionary force exerted upon M. bovis. A higher percentage of SNPs was detected in genes enriched into “lipid metabolism”, “cell wall and cell processes” and “intermediary metabolism and respiration” functional categories, revealing their underlying importance in M. bovis biology and evolution. A closer look on genes prone to horizontal gene transfer in the MTBC ancestor and included in the 3R (DNA repair, replication and recombination) system revealed a global average negative value for Taijima’s D neutrality test, suggesting that past selective sweeps and population expansion after a recent bottleneck remain as major evolutionary drivers of the obligatory pathogen M. bovis in its struggle with the host.info:eu-repo/semantics/publishedVersio

Extractability and mobility of mercury from agricultural soils surrounding industrial and mining contaminated areas

This study focussed on a comparison of the extractability of mercury in soils with two different contamination sources (a chlor-alkali plant and mining activities) and on the evaluation of the influence of specific soil properties on the behaviour of the contaminant. The method applied here did not target the identification of individual species, but instead provided information concerning the mobility of mercury species in soil. Mercury fractions were classified as mobile, semi-mobile and non-mobile. The fractionation study revealed that in all samples mercury was mainly present in the semi-mobile phase (between 63 and 97%). The highest mercury mobility (2.7 mg kg-1) was found in soils from the industrial area. Mining soils exhibited higher percentage of non-mobile mercury, up to 35%, due to their elevated sulfur content. Results of factor analysis indicate that the presence of mercury in the mobile phase could be related to manganese and aluminum soil contents. A positive relation between mercury in the semi-mobile fraction and the aluminium content was also observed. By contrary, organic matter and sulfur contents contributed to mercury retention in the soil matrix reducing the mobility of the metal. Despite known limitations of sequential extraction procedures, the methodology applied in this study for the fractionation of mercury in contaminated soil samples provided relevant information on mercury's relative mobility

Multi-target optimization of solid phase microextraction to analyse key flavour compounds in wort and beer

Despite the literature comprises numerous studies dealing with the analysis of wort and beer flavour-related compounds by HS-SPME followed by GC-MS quantification, no generalized consensus exists regarding the optimal conditions for the extraction procedure. The complex chemistry nature of these matrices, the number of analytes, as well as the number and interactions among parameters affecting the extraction performance, requires the adoption of optimal experimental design protocols. This aspect is often overlooked and often not properly addressed in practice. Therefore, in the present work, the optimal conditions under which a range of wort and beer analytes can be extracted and quantified were analysed. The optimal extraction conditions were presented at two levels of aggregation: global (untargeted) and key-flavour analysis. Experimental data was generated by Definitive-Screening-Design, followed by model development and optimization. Both approaches were compared and critically analysed. For vicinal-diketones group, a complete validation study for the optimal conditions is presented.publishe

Immunohistochemistry in the diagnosis of membranous glomerulonephritis: a comparative study of chromogen + counterstaining combination

A glomerulonefrite membranosa faz parte das doenças glomerulares que provocam glomerulonefrite crônica, apresentando-se como uma das causas da doença renal terminal. As técnicas de imunofluorescência são o gold standard no estudo imunológico desta patologia em biópsia renal, através da deteção de imunocomplexos (e.g. IgG e C3) e do seu padrão de distribuição granular característico. No entanto, a imunofluorescência não permite uma contextualização histológica e os fluorocromos utilizados possuem um reduzido tempo de atividade, ao contrário das técnicas imunoenzimáticas que utilizam cromogénios coloridos precipitados que permitem a obtenção de uma marcação permanente e a sua contextualização histológica por via da utilização de eficientes colorações de contraste. Com a finalidade de contribuir para a qualidade do diagnóstico da glomerulonefrite membranosa, em biópsias renais, procurou-se, com esta pesquisa, identificar uma técnica imunoenzimática, através da conjugação entre diferentes cromogênios e colorações de contraste, que permita a deteção de depósitos de IgG e C3, com padrão granular. Foram constituídos diferentes binômios cromogênio + coloração, com os cromogênios 3,3›- Diaminobenzidine Tetrahydrochloride e 3-Amino-9-ethylcarbazole e as colorações Periodic Acid Schiff, Periodic Acid Methenamine Silver e Hematoxilina. Foram utilizadas 72 secções de tecido provenientes de seis de casos de biópsias renais com diagnóstico de glomerulonefrite membranosa, fixados em formalina a 10% e incluídos em parafina. A recolha de dados foi realizada por observação microscópica com preenchimento de uma grelha de classificação dos parâmetros: preservação da morfologia, intensidade da marcação específica, quantidade relativa de estruturas marcadas, marcação inespecífica/fundo, contraste e padrão da marcação, que permitiu a classificação dos binómios estudados num score quantitativo de 0-100 pontos. O binômio que apresentou melhores resultados foi 3-Amino-9-ethylcarbazole + Hematoxilina (score 71,81) e o binômio 3,3›- Diaminobenzidine Tetrahydrochloride+Periodic Acid Methenamine Silver (score 7,81), apresentou os piores resultados. O resultado do teste Kruskal-Wallis indica-nos a presença de diferenças estatísticas entre os binómios em estudo (p=0,000). A Hematoxilina pode ser considerada a coloração mais eficaz, pois cumpriu a sua função de auxiliar e facilitar a observação do tipo de padrão com os dois cromogênios utilizados. O cromogênio 3-Amino-9-ethylcarbazole apresentou resultados semelhantes aos produzidos pelo 3,3›-Diaminobenzidine Tetrahydrochloride, no entanto, permitiu identificar em todos os casos o padrão granular de imunomarcação, ao contrário do que aconteceu com este último.ABSTRACT - Membranous glomerulonephritis is one of the glomerular diseases that induce chronic glomerulonephritis and one of the causes of terminal renal disease. Immunofluorescence techniques are the gold standard in the immunologic study of this disease in renal biopsy by the detection of the immunocomplexs and their granular specific pattern. However, immunofluorescence does not allow a histological contextualization and the fluorochromes that are used have a reduced timeline, contrary to immunoenzimatic techniques that use colorful chromogens which permit to obtain a permanent stain and a histological contextualization, with the help of efficient counterstaining. In order to contribute to the quality of diagnosis of membranous glomeurlonephritis in renal biopsies, we tried with this research to identify a combination of different chromogens and counterstainings that allows the detection of IgG and C3 deposits with granular pattern. So we establish different chromogen + counterstaing combination, with 3,3’- Diaminobenzidine Tetrahydrochloride and 3-Amino-9-ethylcarbazole as chromogens and Periodic Acid Schiff, Periodic Acid Methenamine Silver and Hematoxilin as counterstainings. We used 72 sections of 6 renal biopsies with membranous glomerulonephritis, formalin-fixed and paraffin-embedded. Data collection was performed by completing an assessment grid with parameters: preservation of morphology, intensity of specific staining, relative amount of labeled structures, nonspecific staining / background contrast and pattern of staining, which allowed the assignment of a 0-100 points quantitative score. 3-Amino-9-ethylcarbazole+Hematoxilin was the combination that accomplish the higher score (71.81) and 3,3’- Diaminobenzidine Tetrahydrochloride+ Periodic Acid Methenamine Silver the worst (7.81). Kruskal-Wallis test show us that exist statistical difference between the combinations in study (p=0.000). Hematoxilin was the most versatile counterstaining, because it help and facilitate the observation of the pattern produced by the two chromogen used. The chromogen 3-Amino-9-ethylcarbazole presented similar results to 3,3’-Diaminobenzidine Tetrahydrochloride, but, with the first one, all cases were evaluated as granular pattern unlike what happen with the last one

Modulating intermolecular interactions and rheological properties in silk fibroin using ultrasound frequencies

info:eu-repo/semantics/draf

Self-control feeding in children

Introduction: Overweight and childhood obesity are a public health problem. Therefore, it is fundamental to intervene in the factors at the behavioural level that can lead to changes. Objectives: To evaluate children’s food self-control and to relate it to gender, the phratry, nutritional status and food preferences. To verify the influence of the nutritionist (about food literacy and the nutritional education) in the children’s self-control and dietary preferences. Methods: The study was carried out in a sample of 66 children, aged between 4 and 6 years old, from four kindergarten in the district of Leiria. The criteria used for the analysis was: food self-control, food preferences, nutritional status, food literacy of the head of education and the effect of food education. The statistical treatment of data was performed using SPSS ß version 21 software. Results: In the analysed sample, mainly female (53.0%), 57.6% of the children were not the first children and had normal nutritional status (77.3%); 19.7% were overweight and obese and 3.0% were thinner. The majority of the children (54.5%) presented food self- control. The caregivers showed good knowledge in the scope of food, with an average score of 82.0%. There was a positive significant correlation between the educator and the first choice of the children. Regarding the children’s choices secondary to the nutritionist’s intervention, no significant differences were found. However, although not statistically significant it is clinically relevant, because 25 of the 66 participating children improved their choices. Conclusions: In the preschool stage, the nutritionist intervention can be highly beneficial. Educating for healthy habits and lifestyles, increasing awareness for food good practices and strengthening children self-control might contribute positively to the struggle against childhood obesity.info:eu-repo/semantics/publishedVersio