Escherichia coli expression and purification of four antimicrobial peptides fused to a family 3 carbohydrate-binding module (CBM) from Clostridium thermocellum

Antimicrobial peptides (AMPs) are molecules that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. Several hundreds of AMPs have been identified and characterized. These molecules are presently gaining increasing importance, as a consequence of their remarkable resistance to microorganism adaptation. Carbohydrate-binding modules (CBMs) are non-catalytic domains that anchor glycoside hydrolases into complex carbohydrates. Clostridium thermocellum produces a multi-enzyme complex of cellulases and hemicellulases, termed the cellulosome, which is organized by the scaffoldin protein CipA. Binding of the cellulosome to the plant cell wall results from the action of CipA family 3 CBM (CBM3), which presents a high affinity for crystalline cellulose. Here CipA family 3 CBM was fused to four different AMPs using recombinant DNA technology and the fusion recombinant proteins were expressed at high levels in Escherichia coli cells. CBM3 does not present antibacterial activity and does not bind to the bacterial surface. However, the four recombinant proteins retained the ability to bind cellulose, suggesting that CBM3 is a good candidate polypeptide to direct the binding of AMPs into cellulosic supports. A comprehensive characterization of the antimicrobial activity of the recombinant fusion proteins is currently under evaluation.Instituto de Tecnologia Química e Biológica,Fundação para a Ciência e a Tecnologia (FCT

Dextrin nanoparticles as a protein delivery system: the interleukin-10 case study

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Analysis of DNA damage and repair in saccharomyces cerevisiae using the comet assay in the characterization of antigenotoxicity of plant extracts and phytochemicals

In this work we used the model organism Saccharomyces cerevisiae to characterise the biological activity and the mechanism of action of phytochemicals. One of the goals is to use mutant strains affected in basic mechanisms of oxidative stress response and DNA repair in order to uncover the molecular targets of phytochemicals. We have assessed DNA damage and repair using the comet assay, evaluated as “comet tail length”, which displayed a dose-response relationship with different DNA-damaging agents1. Subsequently, we used this system to assess the antigenotoxic properties of a leaf extract from Ginkgo biloba (GBE). Typical experiments involved incubation of yeast cells, or spheroplasts, with GBE before and during oxidative shock with hydrogen peroxide. Our results obtained with the comet assay show that DNA damage was significantly decreased upon GBE treatment in a dose-dependent manner. In addition, DNA repair kinetics was significantly improved in cells incubated with GBE. However, in the mutant strain affected in CDC9, encoding a DNA ligase involved in the mechanisms of nucleotide excision repair and base excision repair, oxidative DNA damage repair kinetics was unchanged with GBE, suggesting that the activity of this extract involves one of these mechanisms, or both. Hydrogen peroxide-induced cell cycle arrest in G2 was abolished when cells were incubated with GBE after oxidative shock, suggesting that the improved repair kinetics allows progression of the cell cycle and/or GBE can have a direct effect on its regulation. As expected, GBE treatment improved survival of yeast cells when challenged with oxidative shock with H2O2 and intracellular oxidation was considerably decreased upon pre-treatment with GBE as revealed by flow cytometry. Reference(s) 1. Azevedo F., Marques, F., Fokt, H., Oliveira, R. and Johansson, B. (2011) Measuring oxidative DNA damage and DNA repair using the yeast comet assay. Yeast, 28, 55-6

Changes in leaf functional traits with leaf age: when do leaves decrease their photosynthetic capacity in Amazonian trees?

Most leaf functional trait studies in the Amazon basin do not consider ontogenetic variations (leaf age), which may influence ecosystem productivity throughout the year. When leaf age is taken into account, it is generally considered discontinuous, and leaves are classified into age categories based on qualitative observations. Here, we quantified age-dependent changes in leaf functional traits such as the maximum carboxylation rate of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) (Vcmax), stomatal control (Cgs%), leaf dry mass per area and leaf macronutrient concentrations for nine naturally growing Amazon tropical trees with variable phenological strategies. Leaf ages were assessed by monthly censuses of branch-level leaf demography; we also performed leaf trait measurements accounting for leaf chronological age based on days elapsed since the first inclusion in the leaf demography, not predetermined age classes. At the tree community scale, a nonlinear relationship between Vcmax and leaf age existed: young, developing leaves showed the lowest mean photosynthetic capacity, increasing to a maximum at 45 days and then decreasing gradually with age in both continuous and categorical age group analyses. Maturation times among species and phenological habits differed substantially, from 8 ± 30 to 238 ± 30 days, and the rate of decline of Vcmax varied from −0.003 to −0.065 μmol CO2 m−2 s−1 day−1. Stomatal control increased significantly in young leaves but remained constant after peaking. Mass-based phosphorus and potassium concentrations displayed negative relationships with leaf age, whereas nitrogen did not vary temporally. Differences in life strategies, leaf nutrient concentrations and phenological types, not the leaf age effect alone, may thus be important factors for understanding observed photosynthesis seasonality in Amazonian forests. Furthermore, assigning leaf age categories in diverse tree communities may not be the recommended method for studying carbon uptake seasonality in the Amazon, since the relationship between Vcmax and leaf age could not be confirmed for all trees

Engolo and Capoeira. From Ethnic to Diasporic Combat Games in the Southern Atlantic

This article provides a re-examination of the main Afrocentric narrative of capoeira origins, the engolo or ‘Zebra Dance’, in light of historical primary sources and new ethnographic evidence gathered during fieldwork in south-west Angola. By examining engolo’s bodily techniques, its socio-historical context and cultural meanings, the piece emphasises its insertion into a pastoral lifestyle and highlights the relatively narrow ethnic character of the practice in Angola. This analysis and the comparison with capoeira helps us to develop certain hypotheses about the formation, migration, and re-invention of diasporic combat games between southern Angola and coastal Brazil, and more broadly, to increase our understanding of how African cultures spread across the southern Atlantic

Direct evidence for phosphorus limitation on Amazon forest productivity

The productivity of rainforests growing on highly weathered tropical soils is expected to be limited by phosphorus availability1. Yet, controlled fertilization experiments have been unable to demonstrate a dominant role for phosphorus in controlling tropical forest net primary productivity. Recent syntheses have demonstrated that responses to nitrogen addition are as large as to phosphorus2, and adaptations to low phosphorus availability appear to enable net primary productivity to be maintained across major soil phosphorus gradients3. Thus, the extent to which phosphorus availability limits tropical forest productivity is highly uncertain. The majority of the Amazonia, however, is characterized by soils that are more depleted in phosphorus than those in which most tropical fertilization experiments have taken place2. Thus, we established a phosphorus, nitrogen and base cation addition experiment in an old growth Amazon rainforest, with a low soil phosphorus content that is representative of approximately 60% of the Amazon basin. Here we show that net primary productivity increased exclusively with phosphorus addition. After 2 years, strong responses were observed in fine root (+29%) and canopy productivity (+19%), but not stem growth. The direct evidence of phosphorus limitation of net primary productivity suggests that phosphorus availability may restrict Amazon forest responses to CO2 fertilization4, with major implications for future carbon sequestration and forest resilience to climate change.The authors acknowledge funding from the UK Natural Environment Research Council (NERC), grant number NE/L007223/1. This is publication 850 in the technical series of the BDFFP. C.A.Q. acknowledges the grants from Brazilian National Council for Scientific and Technological Development (CNPq) CNPq/LBA 68/2013, CNPq/MCTI/FNDCT no. 18/2021 and his productivity grant. C.A.Q., H.F.V.C., F.D.S., I.A., L.F.L., E.O.M. and S.G. acknowledge the AmazonFACE programme for financial support in cooperation with Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Institute of Amazonian Research as part of the grants CAPES-INPA/88887.154643/2017-00 and 88881.154644/2017-01. T.F.D. acknowledges funds from FundacAo de Amparo a Pesquisa do Estado de SAo Paulo (FAPESP), grant 2015/50488-5, and the Partnership for Enhanced Engagement in Research (PEER) programme grant AID-OAA-A-11-00012. L.E.O.C.A. thanks CNPq (314416/2020-0)