Trypanosoma brucei and Trypanosoma cruzi DNA mismatch repair proteins act differently in the response to DNA damage caused by oxidative stress

MSH2, associated with MSH3 or MSH6, is a central component of the eukaryotic DNA Mismatch Repair (MMR) pathway responsible for the recognition and correction of base mismatches that occur during DNA replication and recombination. Previous studies have shown that MSH2 plays an additional DNA repair role in response to oxidative damage in Trypanosoma cruzi and Trypanosoma brucei. By performing co-immunoprecipitation followed by mass spectrometry with parasites expressing tagged proteins, we confirmed that the parasites’ MSH2 forms complexes with MSH3 and MSH6. To investigate the involvement of these two other MMR components in the oxidative stress response, we generated knockout mutants of MSH6 and MSH3 in T. brucei bloodstream forms and MSH6 mutants in T. cruzi epimastigotes. Differently from the phenotype observed with T. cruzi MSH2 knockout epimastigotes, loss of one or two alleles of T. cruzi msh6 resulted in increased susceptibility to H2O2 exposure, besides impaired MMR. In contrast, T. brucei msh6 or msh3 null mutants displayed increased tolerance to MNNG treatment, indicating that MMR is affected, but no difference in the response to H2O2 treatment when compared to wild type cells. Taken together, our results suggest that, while T. cruzi MSH6 and MSH2 are involved with the oxidative stress response in addition to their role as components of the MMR, the DNA repair pathway that deals with oxidative stress damage operates differently in T. brucei

Valorization of passion fruit stalk by the preparation of cellulose nanofibers and immobilization of trypsin

Fibers from passion fruit stalks were first recovered with a pulping and bleaching process. Two mechanical treatments were further applied to the fibers, homogenization (with and without ultrasound), and blender application. The effect of those treatments on fibers was evaluated. The chemical composition of the different stages of fibers undergoing treatment were measured according to TAPPI standards and were also analyzed by SEM, FTIR, and XRD. Trypsin was immobilized by adsorption and by covalent binding. The biocatalyst support-trypsin was evaluated in terms of immobilization yield, retention, and enzymatic activity. The experimental results demonstrated that the final cellulose concentration in the fibers was 44 % higher than that in the raw stalks. The cellulose nanofibers obtained by homogenization presented a size distribution between 20200 nm, and the application of ultrasound did not show a significant effect on size (between 50 to 300 nm). Trypsin immobilized using glycidol presented an immobilization yield of 67 % and presented higher retention and enzymatic activity (1.17±0.05 U/mg protein and 44.0±2.0 %, respectively). These results show that passion fruit stalks can be successfully used as a source of cellulose nanofibers and also can be used as carriers for the immobilization of trypsin.The authors acknowledge the financial support of Universidad Nacional de Colombia (Hermes code project 34573), COLCIENCIAS call 727-2015 National Doctorates. FONTAGRO (ATN/RF-16111-RG Productividad y Competitividad Frutícola Andina), and Erasmus+Programme Key Action 1. This work was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UID/BIO/04469/2020 unit and by the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Phenomenological viability of orbifold models with three Higgs families

We discuss the phenomenological viability of string multi-Higgs doublet models, namely a scenario of heterotic $Z_3$ orbifolds with two Wilson lines, which naturally predicts three supersymmetric families of matter and Higgs fields. We study the orbifold parameter space, and discuss the compatibility of the predicted Yukawa couplings with current experimental data. We address the implications of tree-level flavour changing neutral processes in constraining the Higgs sector of the model, finding that viable scenarios can be obtained for a reasonably light Higgs spectrum. We also take into account the tree-level contributions to indirect CP violation, showing that the experimental value of $\epsilon_K$ can be accommodated in the present framework.Comment: 31 pages, 12 figures. Comments and references added. Final version to be published in JHE

Combination of coagulation–flocculation–decantation and ozonation processes for winery wastewater treatment

This research assessed a novel treatment process of winery wastewater, through the application of a chemical-based process aiming to decrease the high organic carbon content, which represents a difficulty for wastewater treatment plants and a public health problem. Firstly, a coagulation–flocculation–decantation process (CFD process) was optimized by a simplex lattice design. Afterwards, the efficiency of a UV-C/ferrous iron/ozone system was assessed for organic carbon removal in winery wastewater. This system was applied alone and in combination with the CFD process (as a pre- and post-treatment). The coagulation–flocculation–decantation process, with a mixture of 0.48 g/L potassium caseinate and 0.52 g/L bentonite at pH 4.0, achieved 98.3, 97.6, and 87.8% removals of turbidity, total suspended solids, and total polyphenols, respectively. For the ozonation process, the required pH and ferrous iron concentration (Fe2+) were crucial variables in treatment optimization. With the application of the best operational conditions (pH = 4.0, [Fe2+] = 1.0 mM), the UV-C/ferrous iron/ozone system achieved 63.2% total organic carbon (TOC) removal and an energy consumption of 1843 kWh∙m−3∙order−1. The combination of CFD and ozonation processes increased the TOC removal to 66.1 and 65.5%, respectively, for the ozone/ferrous iron/UV-C/CFD and CFD/ozone/ferrous iron/UV-C systems. In addition, the germination index of several seeds was assessed and excellent values (>80%) were observed, which revealed the reduction in phytotoxicity. In conclusion, the combination of CFD and UV-C/ferrous iron/ozone processes is efficient for WW treatment.NORTE 2020 | Ref. NORTE-01-0145-FEDER-000041Fundação para a Ciência e a Tecnologia | Ref. UIDB/00616/2020Fundação para a Ciência e a Tecnologia | Ref. FCT-IF/00802/201

Idade e crescimento do boca-negra, Helicolenus dactylopterus dactylopterus (Delaroche, 1809) dos Açores

A idade e o crescimento de boca-negra, Helicolenus dactylopterus dactylopterus (Delaroche, 1809), foram estudadas pela observação dos otólitos (sagittae) esquerdos inteiros (n = 401) obtidos de exemplares (14-47 cm de comprimento total) capturados em águas Açoreanas. Enumeraram-se os anéis opacos observados na face anti-sulcal dos otólitos. Os intervalos de idades foram 3-14 anos para machos e 3-12 anos para fêmeas. Estimaram-se e compararam-se os parâmetros da equação de crescimento de von Bertalanffy, entre sexos e entre métodos (leitura directa de otólitos, retrocálculo e análise de distribuições de frequências de comprimentos). Não se verificaram diferenças importantes. Os resultados são diferentes da literatura publicada para a região. Discutem-se as causas e implicações dos resultados obtidos.ABSTRACT: Bluemouth, Helicolenus dactylopterus dactylopterus (Delaroche, 1809), age and growth were studied by whole-view examination of left sagittae (n = 401) obtained from specimens (14-47 cm in total length) caught off the Azores. Opaque rings observed on the anti-sulcal surface of sagittae were enumerated as age estimates. Ages ranged from 3 to 14 years in males and 3 to 12 years in females. The von Bertalanffy growth equation was fitted to average length at age data, and compared between sexes and methods (direct examination of otoliths, backcalculation and length-frequency analysis). No important differences in growth between sexes were found. Results are different from published literature for the region. The causes and implications of the results are discussed

Purification of plasmids using aqueous two-phase systems with amino affinity ligands

The increasing development and future application of molecular therapies such as gene therapy and DNA vaccination is expected to have a great impact in health care. However for their wide application large amounts of plasmid DNA (pDNA) are required with a stringent clearance of impurities. This prompted the development of new, efficient and cost-effective large-scale processes for the production and purification of pDNA. Most of the purification processes described are based on chromatography but dispite their high resolution, frequently they are difficult to scale-up, have low capacity and present low yields. In order to overcome these disadvantages other methodologies are also being developed. Aqueous two-phase systems (ATPS) are one of the most promising approaches for pDNA purification given their several advantages like easy scale-up, high capacity and the possibility of continuous operation. Despite their great potential ATPS have low selectivity, which limits the purification outcome. The addition of certain molecules with affinity for the target molecules (pDNA in this case) may increase their selectivity. In this work it was studied the possibility of using amino ligands for the affinity purification of pDNA from bacterial alkaline lysates. Two free amino acids, lysine and arginine, their respective Polyethylene glycol (PEG) conjugates, PEG-lysine and PEGarginine, and PEGamine were tested. The system used was composed of 16,2% (w/w) PEG 600 and 17,4% (w/w) dextran 100 (DEX) and it was evaluate the ability of each ligand to steer the pDNA to the phase where less impurities are accumulated (PEG rich phase). The results show that free amino acids did not have any effect on pDNA partitioning but the PEG conjugates were able to steer the pDNA to the PEG phase, at low concentrations. With the addition of 0,2% of PEG-lysine, or 0,5% of PEG-arginine or 4% of PEG-amine in relation to the total PEG, all the pDNA is recovered in the PEG phase. However it presents some RNA contamination, that could be removed by re-extracting with a new phase containing 30% of ammonium sulphate (NH4)2S04. The purified pDNA is obtained in the bottom phase of this new system with no measurable presence of RNA or proteins

Modulation of the Redox Potential and Electron/Proton Transfer Mechanisms in the Outer Membrane Cytochrome OmcF From Geobacter sulfurreducens

PD/00193/2012 UID/FIS/00068/2019 PTDC/BBBBQB/3554/2014 PTDC/BIA-BQM/31981/2017 PD/BD/114445/2016 UID/Multi/04378/2019 ROTEIRO/0031/2013 -PINFRA/22161/2016The monoheme outer membrane cytochrome F (OmcF) from Geobacter sulfurreducens plays an important role in Fe(III) reduction and electric current production. The electrochemical characterization of this cytochrome has shown that its redox potential is modulated by the solution pH (redox-Bohr effect) endowing the protein with the necessary properties to couple electron and proton transfer in the physiological range. The analysis of the OmcF structures in the reduced and oxidized states showed that with the exception of the side chain of histidine 47 (His47), all other residues with protonatable side chains are distant from the heme iron and, therefore, are unlikely to affect the redox potential of the protein. The protonatable site at the imidazole ring of His47 is in the close proximity to the heme and, therefore, this residue was suggested as the redox-Bohr center. In the present work, we tested this hypothesis by replacing the His47 with non-protonatable residues (isoleucine – OmcFH47I and phenylalanine – OmcFH47F). The structure of the mutant OmcFH47I was determined by X-ray crystallography to 1.13 Å resolution and showed only minimal changes at the site of the mutation. Both mutants were 15N-labeled and their overall folding was confirmed to be the same as the wild-type by NMR spectroscopy. The pH dependence of the redox potential of the mutants was measured by cyclic voltammetry. Compared to the wild-type protein, the magnitude of the redox-Bohr effect in the mutants was smaller, but not fully abolished, confirming the role of His47 on the pH modulation of OmcF’s redox potential. However, the pH effect on the heme substituents’ NMR chemical shifts suggested that the heme propionate P13 also contributes to the overall redox-Bohr effect in OmcF. In physiological terms, the contribution of two independent acid–base centers to the observed redox-Bohr effect confers OmcF a higher versatility to environmental changes by coupling electron/proton transfer within a wider pH range.publishersversionpublishe

The role of adjuvants in therapeutic protection against paracoccidioidomycosis after immunization with the P10 peptide

Paracoccidioidomycosis (PCM), a common chronic mycosis in Latin America, is a granulomatous systemic disease caused by the thermo-dimorphic fungus Paracoccidioides brasiliensis. The glycoprotein gp43 is the main antigen target of P. brasiliensis and a 15-mer internal peptide (QTLIAIHTLAIRYAN), known as P10, defines a major CD4+-specific T cell epitope. Previous results have indicated that, besides having a preventive role in conventional immunizations prior to challenge with the fungus, protective anti-fungal effects can be induced in P. brasiliensis-infected mice treated with P10 administered with complete Freund’s adjuvant (CFA). The peptide elicits an IFN-γ-dependent Th1 immune response and is the main candidate for effective immunotherapy of patients with PCM, as an adjunctive approach to conventional chemotherapy. In the present study we tested the therapeutic effects of P10 combined with different adjuvants [aluminum hydroxide, CFA, flagellin, and the cationic lipid dioctadecyl-dimethylammonium bromide (DODAB)] in BALB/c mice previously infected with the P. brasiliensis Pb18 strain. Significant reductions in the number of colony forming units of the fungus were detected in lungs of mice immunized with P10 associated with the different adjuvants 52 days after infection. Mice treated with DODAB and P10, followed by mice treated with P10 and flagellin, showed the most prominent effects as demonstrated by the lowest numbers of viable yeast cells as well as reductions in granuloma formation and fibrosis. Concomitantly, secretion of IFN-γ and TNF-α, in contrast to interleukin (IL)-4 and IL-10, was enhanced in the lungs of mice immunized with P10 in combination with the tested adjuvants, with the best results observed in mice treated with P10 and DODAB. In conclusion, the present results demonstrate that the co-administration of the synthetic P10 peptide with several adjuvants, particularly DODAB, have significant therapeutic effects in experimental PCM

Smart monitoring of constructed wetlands to improve efficiency and water quality

The Smart monitoring of constructed wetlands to improve efficiency and water quality (SmarterCW) project aims to monitor biological wastewater treatment processes by gathering continuous data from remote water and environmental sensors. The acquired data can be processed and analysed through data science tools to better understand the complex and coupled phenomena underneath wastewater treatment, as well as, to monitor and optimize the system performance. The results will improve the efficiency and control of nature-based wastewater treatment technologies. The methodology comprises the following tasks and activities: Implementation of a set of electrochemical sensors in the input and output flow streams of pilot-scale constructed wetlands; Acquisition of water quality parameters such as pH, electrical conductivity, temperature, and ionic compounds; Acquisition of environmental parameters, such as temperature and humidity; Application of data analysis tools to design and optimize conceptual models to correlate pollutants removal with operative parameters in green technologies for wastewater treatment. This methodology was applied to a patent-protected pilot-scale modular constructed wetland in which filling media consists of a mixture of solid waste. The system is complemented by a high-level IoT communication layer structure to support remote real-time water and environmental monitoring, system performance, and data dissemination. The project contributes to: Water and Environment through the efficient management and use of water resources and waste reduction, management, treatment, and valorisation; Materials and raw-materials through efficient, secure, and sustainable use of resources; and Environmental Education promoting environmental awareness and best environmental practices through the dissemination of scientific data and results using Information and Communication Technologies (ICT) tools and IoT platforms. The project also contributes to giving response to Societal Challenges, such as Environment protection, sustainable management of natural resources, water, biodiversity, and ecosystems; Enabling the transition to a green society and economy through eco-innovation.info:eu-repo/semantics/submittedVersio