Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans

Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F. hepatica NEJ glycosylation and the first report of N-glycosylation of F. hepatica cathepsins. The significance of these findings for immunological studies and vaccine development is discussed.

Fasciola hepatica surface tegument: glycoproteins at the interface of parasite and host

Fasciola hepatica, commonly known as liver fluke, is a trematode which causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterisation of FhTeg glycosylation using lectin microarrays to characterise carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. While some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components which could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.

Timing ótimo para investir na proteção costeira na zona centro de Portugal

A presente dissertação insere-se no âmbito da gestão das zonas costeiras, que na atualidade enfrentam um grave problema de erosão. Este torna-se um problema social com especial importância devido à concentração populacional e atividade económica que se verifica nas regiões litorais. Neste contexto muitos têm sido os estudos físicos e económicos levados a cabo no sentido de encontrar soluções para lidar com este problema. O principal objetivo desta dissertação foi avaliar qual a importância que o timing em que é efetuado o investimento na proteção pode ter na defesa das zonas costeiras. Por outras palavras, perceber se existem ou não vantagens em adiar o investimento na proteção por um determinado período de tempo. Foi definida como área de estudo o trecho costeiro de 20 km compreendido entre a Praia da Barra (Aveiro) e a Praia de Mira (Coimbra). Através da utilização do modelo numérico Long-Term Configuration (LTC) foi simulada a evolução da linha de costa por um período de 80 anos, seguida de uma análise custo-benefício ambiental em que foi considerada a possibilidade de investimentos em estruturas de defesa (no caso desta dissertação esporões com 100m, 200m e 300m de comprimento) a serem efetuados após 0, 10, 20 ou 30 anos. Foram também contempladas restrições orçamentais, e ainda, efetuada uma análise de sensibilidade à taxa de desconto utilizada. Os resultados mostram que quanto mais cedo se iniciar proteção das zonas costeiras, mais benefícios económicos serão retirados desse investimento. Os cenários que se apresentaram como os mais vantajosos são aqueles em que o investimento é levado a cabo no Ano 0 e onde se opta por uma proteção integral ou quase integral da linha de costa. Ainda assim, em todos os timings de investimento analisados foi possível encontrar cenários economicamente viáveis e dos quais resultariam benefícios económicos. A análise com inclusão de restrições orçamentais mostra que a utilização destas deixa de fazer sentido à medida que o investimento é adiado, funcionando então o próprio retardar do investimento como uma medida de contenção orçamental. A análise de sensibilidade à taxa de desconto revelou que a opção por uma taxa de desconto de 4% acaba por não influenciar de maneira significativa os resultados obtidos.

Severe and Symptomatic Calcium Disorders Caused by Secondary Bone Disease in a Patient with a Thyroid Storm

A patient with Graves’ disease was admitted with a thyroid storm. She had severe hypercalcaemia caused by thyrotoxicosis. Treatment was complicated by vomiting and diarrhoea. With intravenous ondansetron, hydration and bisphosphonates, GI symptoms improved and oral thyreostatics could be started. This, combined with bisphosphonate administration, resulted in a mild hungry bone syndrome.

On estimating attenuation from the amplitude of the spectrally whitened ambient seismic field

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