Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor in Trypanosoma cruzi

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa

Materiais de construção e materiais líticos nas práticas funerárias neolíticas da serra da Boa Viagem (Centro-Oeste de Portugal). O caso do monumento megalítico do Cabeço dos Moinhos, Figueira da Foz

Este trabalho contribui para o conhecimento do Neolítico do Centro-Oeste português. O caso de estudo foi o monumento megalítico do Cabeço dos Moinhos, edificado na serra da Boa Viajem, no que é hoje a freguesia da Brenha, concelho da Figueira da Foz. Para além de ser óbvio que esta região se encontrava, durante o Neolítico, no limiar de diferentes mundos culturais, o meridional e o setentrional, a partir das matérias usadas na construção do monumento e das matérias usadas no fabrico de oferendas líticas e de objetos de adorno, teceram-se algumas considerações relacionadas com as práticas funerárias, os modos de vida e de vivenciar o mundo por parte das comunidades neolíticas que aqui ergueram estes monumentos. Em relação às oferendas líticas notou-se a ausência de traços de uso o que indicia que foram fabricadas apenas para ritos funerários. Além do simbolismo intrínseco às formas, as diferentes matérias em que foram construídos (locais, regionais e inter-regionais) indiciam a importância da interligação física e simbólica das comunidades locais com lugares próximos e remotos, o que não será alheio a um modo de vida com grande abertura ao mundo exterior e, talvez, pouco sedentarizado . O mesmo se poderá interpretar em termos arquitetónicos pois, se a construção sobre um afloramento calcário e em local culminante poderá revelar assimilação das propriedades inerentes ao local e à matéria aí existente, a reunião de matérias de diferentes proveniências na câmara/corredor implicará uma interligação física e mental entre os diferentes lugares vivenciados pelos seus construtores, ou seja, entre lugares de habitat, de subsistência e de veneração dos espíritos dos antepassados.This paper contributes to the understanding of the Neolithic burial practices in Western Central Portugal. It relies on the reviewing of Santos Rocha texts, which describes research carried out in the megalithic monument of Cabeço dos Moinhos (Brenha, Figueira da Foz), fieldwork and the study of bone remains and “offerings” that were found in the monument. Cabeço dos Moinhos tomb was built on a limestone outcrop located in a hill placed on the ridgeline of Serra da Boa Viagem. From this hill there is a wide visibility to the sandy coastal plain (to the north) and to the Baixo Mondego area (to the south). Under a mound of "yellowish soil without mixture" a polygonal chamber with corridor oriented east was built. The standing stones were made of greyish white limestone (local) and of whitish, greyish, yellowish and reddish sandstone. The last two are sourced from slopes located more than 250m south of the tomb. Santos Rocha (1949: 14) found some "skeletons lying in gravel beds" and rare burnt bones. The dead were buried together with pottery, lithic and bone objects. Some of these indicate a reutilization of the monument during the Chalcolithic. The absence of use-wear in the majority of the lithics suggests that they were produced only for the burial practices. In addition to the symbolism intrinsic to morphologies, the use of different “raw” materials suggests the importance of the reunion of their own properties at the time of the burial, setting a symbolic and physical interconnection between local communities and near and remote places. The same reasoning can be applied to the architecture of the monument: if its construction on a limestone outcrop which is a culminating spot suggests the assimilation of the properties associated to the place and to the matter that exists there, the presence of “raw” materials from different sources among the standing stones of the chamber and the corridor imply a mental and physical interconnection between different places experienced by the builders. The visualized landscape must also have been significant to the people who built and attended the Cabeço dos Moinhos megalithic monument.info:eu-repo/semantics/publishedVersio

Water use and soil water balance of Mediterranean tree crops assessed with the SIMDualKc model in orchards of southern Portugal

Orchards consist of complex agricultural systems, with a variety of characteristics (planting density, tree height, training system, canopy cover, irrigation method, interrow management) influencing crop evapotranspiration (ETc). Thus, irrigation water management requires finding crop coefficients (Kc) that represent the characteristics of local orchards, evidencing the need for site specific data. The main objective of this study was to derive the Kc of almond, olive, citrus, and pomegranate orchards in Alentejo, southern Portugal, wherein they became dominant over the last decade. Monitoring was carried out in nine orchards, which management decisions were performed by the farmers. The ETc was estimated from the soil water balance computed for each orchard using the FAO56 dual-Kc approach with the SIMDualKc model. The model successfully simulated the soil water contents measured in the various fields along two growing seasons, with root mean square error values lower than 0.005 m3 m− 3 and modeling efficiencies from 0.363 to 0.782. The estimated basal crop coefficients (Kcb) for the initial, mid- and end-seasons were respectively 0.22, 0.58, and 0.50 for almond; 0.32–0.33, 0.35–0.36, and 0.33–0.34 for olive; 0.40, 0.40–41, and 0.40–0.41 for citrus; and 0.24, 0.60, and 0.52 for pomegranate. Small variations in olive and citrus Kcb values were found to be related to differences in the fraction of the ground covered by trees’ canopies and tree height. The single Kc values, which included the component relative to soil evaporation, were also estimated. Furthermore, evaluation of the soil water balance in the nine case studies showed salinity effects in one almond orchard, mild irrigation water deficits in olive systems, and large nonconsumptive water use in citrus and pomegranate orchards. These results evidence the need for better management of orchards irrigation water in the region, and the current study provides for reliable information on the Kc of tree crops to support improving the management of local orchard systems and the preservation of soil and water resources. Aimed at these resources and the sustainability of their use, simulated alternative irrigation schedules were performed, which identified possible water savings of 20 mm in case of olives, up to 855 mm for citrus.info:eu-repo/semantics/publishedVersio

Water use, soil water balance and soil salinization risks of Mediterranean tree orchards in southern Portugal under current climate variability: Issues for salinity control and irrigation management

Secondary salinization has long been reported in the Roxo irrigation district (RID), southern Portugal, due to the use of saline-prone irrigation water and the existence of poorly structured soils. This study assessed the soil water and salt budgets in nine commercial orchards located in the RID using the multiple ion chemistry module available in the HYDRUS-1D model during the 2019 and 2020 growing seasons. The studied crops were almond, olive, citrus (orange, mandarin, and clementine), and pomegranate. The model successfully simulated soil water contents measured in the different fields but there was a clear underestimation of the electrical conductivity of the soil saturation paste extract (ECe) in some locations, while simulations of the sodium adsorption ratio (SAR) were generally acceptable. Modeling errors were mostly associated with missing information on fertigation events rather than related to the effects of irrigation water quality. The water and salt balances were also computed for the 1979–2020 period. Considering the probability of non-exceedance of salt accumulation during this period, the risk of salinity build-up was high to very high for the very dry years in most fields, except in the citrus sites. The factors influencing the salt accumulation were the irrigation strategy, the seasonal irrigation and rainfall depths, the duration of the crop growth period, the rainfall distribution in the late and non-growing stages, the soil drainage conditions, and the irrigation water quality. For the current climate conditions and irrigation water quality, the risk of soil salinity levels affecting crop development and yields was found to be minor. This means that, despite salts tended to accumulate in the rootzone over a season, under current conditions the salinity stress did not reach harmful levels for plants. Only in two of the study sites, there was a need to promote salt leaching. Hence, this study shows that soil salinization risks in the study area are low but, for given locations during drier seasons, there is a need for tailored irrigation solutions aimed at the conservation of soil and water resources.info:eu-repo/semantics/publishedVersio

Synthesis of pure stereoisomers of benzo[b]thienyldehydrophenylalanines by Suzuki cross-coupling : preliminary studies of antimicrobial activity

Several benzo[b]thienyldehydrophenylalanines were synthesized from pure stereoisomers of the methyl ester of N-(tert-butoxycarbonyl)-beta-bromodehydrophenylalanine as an extension of our previously reported method for the synthesis of dehydrotryptophan analogues to dehydrophenylalanine derivatives. The latter were obtained in high yields by N-deprotection and bromination of N,N-bis-(tert-butoxycarbonyl)-(Z)-dehydrophenylalanine using TFA and NBS. This was carried out in two steps or in a one pot procedure resulting in different E/Z ratios. These compounds were coupled under Suzuki cross-coupling conditions [Pd(PPh3)4, Na2CO3, DME/H2O] with several boronic benzo[b]thienyl acids in good to high yields maintaining the stereochemistry of the starting materials. The best yields were obtained when the boronic acid was in position 7 of the benzo[b]thiophene and with the E isomer of the brominated dehydrophenylalanine. In some cases it was possible to increase the lower yields by changing the Pd source to PdCl2(PPh)2. A model dipeptide was prepared coupling a benzo[b]thienyldehydrophenylalanine with the methyl ester of alanine. Preliminary antimicrobial studies were performed with both isomers of one of the beta,beta-diaryldehydroalanines obtained. The results show that the compounds are selective and very active (very low MICs) against Gram positive bacteria (B. cereus and B. subtilis) the Z-isomer being more active. The compounds are also active against Candida albicans presenting similar MICs.Fundação para a Ciência e Tecnologia - POCTI/1999/QUI/32689, SFRH/BD/4709/2001

High yielding synthesis of N-ethyl dehydroamino acids

Recently we reported the use of a sequence of alkylation and dehydration methodologies to obtain N-ethyl-α, β-dehydroamino acid derivatives. The application of this N-alkylation procedure to several methyl esters of β, β-dibromo and β--bromo, β-substituted dehydroamino acids protected with standard amine protecting groups was subsequently reported. The corresponding N-ethyl, β-bromo dehydroamino acid derivatives were obtained in fair to high yields and some were used as substrates in Suzuki cross coupling reactions to give N-ethyl, β, β-disubstituted dehydroalanine derivatives. Herein, we further explore N-ethylation of β-halo dehydroamino acid derivatives using triethyloxonium tetrafluoroborate as alkylating agent but substituting N,N-diisopropylethylamine for potassium tert-butoxide as auxiliary base. In these conditions, for all β-halo dehydroamino acid derivatives, reactions were complete and the N-ethylated derivative could be isolated in high yield. This method was also applied for N-ethylation of non-halogenated dehydroamino acids. Again, with all compounds the reactions were complete and the N-ethyl dehydroamino acid derivatives could be isolated in high yields. Some of these N-ethyl dehydroamino acid methyl ester derivatives were converted in high yields to their corresponding acids and coupled to an amino acid methyl ester to give N-ethyl dehydrodipeptide derivatives in good yields. Thus, this method constitutes a general procedure for high yielding synthesis of N-ethylated dehydroamino acids, which can be further applied in peptide synthesis.Foundation for Science and Technology (FCT)-Portugal and Fundo Europeu de Desenvolvimento Regional (FEDER) for financial support to Chemistry Centre of University of Minho. The NMR spectrometer Bruker Avance II+ 400 is part of the National NMR Network and was purchased in the framework of the National Program for Scientific Re-equipment; contract REDE/1517/RMN/2005, with funds from POCI 2010, FEDER and FCT

Actividades de educação ambiental realizadas pelo CCPA no decorrer da XIII Expedição Científica do Departamento de Biologia – Flores e Corvo 2007

XIII Expedição Científica do Departamento de Biologia - Flores e Corvo 2007.Tendo em conta a crescente importância da educação ambiental para a construção da cidadania, força motriz de um desenvolvimento sustentável, o Centro de Conservação e Protecção do Ambiente (CCPA) promoveu a realização de diversas actividades de cariz teórico-prático sobre questões ambientais e de valorização do património biológico, junto dos utentes da Ecoteca das Flores e dos alunos da Escola Básica Integrada das Colmeias e da Escola EB 2.3/S de Melgaço, que participaram na XIII Expedição Científica do Departamento de Biologia às ilhas das Flores e Corvo

Evaluation of drying and storage conditions on nutritional and sensory properties of dried galega kale (Brassica oleracea L. var. Acephala)

This work evaluated the effect of the air-drying temperature and vacuum packaging on quality and nutritional compounds of dehydrated galega kale, previously steam blanched, during 5 months of storage. When compared with the vacuum packaged kale, the packaging without vacuum yielded improved nutritional features, in general. This approach combined with a drying temperature of 40°C resulted in retention percentages of 62, 38, 92, and 48% for vitamin C, total phenolic content, total antioxidant capacity, and chlorophylls, respectively. The acceptance of the dried product by the consumer was assessed through a focus group. The participants classified the product as a practical and convenient alternative to cook healthier dishes. The appearance of dried galega kale was described as being fragile and being with nice smell, natural colour, and flavour similar to the fresh product. In conclusion, the herein presented product was addressed as an innovation with multiple possible applications in several recipes.info:eu-repo/semantics/publishedVersio