Isolation Method and Characterization of Outer Membranes Vesicles of Helicobacter pylori Grown in a Chemically Defined Medium

Outer membrane vesicles (OMVs) are small vesicles constitutively shed by all Gram-negative bacterium, which have been proposed to play a role in Helicobacter pylori persistence and pathogenesis. The methods currently available for the isolation of H. pylori OMVs are diverse and time-consuming, raising the need for a protocol standardization, which was the main aim of this study. Here, we showed that the chemically defined F12 medium, supplemented with cholesterol, nutritionally supports bacterial growth and maintains H. pylori viability for at least 72 h. Additionally, we developed an abridged protocol for isolation of OMVs from these bacterial cultures, which comprises a low-speed centrifugation, supernatant filtration through a 0.45 µm pore, and two ultracentrifugations for OMVs’ recovery and washing. Using this approach, a good yield of highly pure bona fide OMVs was recovered from cultures of different H. pylori strains and in different periods of bacterial growth, as assessed by nanoparticle tracking analysis, transmission electron microscopy (TEM), and proteomic analyses, confirming the reliability of the protocol. Analysis of the proteome of OMVs isolated from H. pylori F12-cholesterol cultures at different time points of bacterial growth revealed differentially expressed proteins, including the vacuolating cytotoxin VacA. In conclusion, this work proposes a time- and cost-efficient protocol for the isolation of H. pylori OMVs from a chemically defined culture medium that is suitable for implementation in research and in the biopharmaceutical field.This article is a result of the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The i3S HEMS Scientific Platform is member of the national infrastructure PPBI – Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122). The i3S Proteomics Scientific Platform is funded by the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125). JM and ML had fellowships from FCT – Fundação para a Ciência e a Tecnologia (SFRH/BD/116965/2016 and SFRH/BDP/110065/2015). ML has a FCT RJEC Id 3762 contract

Integrated analysis of climate, soil, topography and vegetative growth in Iberian viticultural regions

The Iberian viticultural regions are convened according to the Denomination of Origin (DO) and present different climates, soils, topography and management practices. All these elements influence the vegetative growth of different varieties throughout the peninsula, and are tied to grape quality and wine type. In the current study, an integrated analysis of climate, soil, topography and vegetative growth was performed for the Iberian DO regions, using state-of-the-art datasets. For climatic assessment, a categorized index, accounting for phenological/thermal development, water availability and grape ripening conditions was computed. Soil textural classes were established to distinguish soil types. Elevation and aspect (orientation) were also taken into account, as the leading topographic elements. A spectral vegetation index was used to assess grapevine vegetative growth and an integrated analysis of all variables was performed. The results showed that the integrated climate-soil-topography influence on vine performance is evident. Most Iberian vineyards are grown in temperate dry climates with loamy soils, presenting low vegetative growth. Vineyards in temperate humid conditions tend to show higher vegetative growth. Conversely, in cooler/warmer climates, lower vigour vineyards prevail and other factors, such as soil type and precipitation acquire more important roles in driving vigour. Vines in prevailing loamy soils are grown over a wide climatic diversity, suggesting that precipitation is the primary factor influencing vigour. The present assessment of terroir characteristics allows direct comparison among wine regions and may have great value to viticulturists, particularly under a changing climate

Methotrexate-loaded lipid-core nanocapsules are highly effective in the control of inflammation in synovial cells and a chronic arthritis model

Antônio Luiz Boechat,1,2,* Catiúscia Padilha de Oliveira,3,* Andrea Monteiro Tarragô,2 Allyson Guimarães da Costa,2 Adriana Malheiro,1,2 Silvia Stanisçuaski Guterres,3 Adriana Raffin Pohlmann3,41Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Manaus, 2Programa de Pós-Graduação e Imunologia Básica e Aplicada, Universidade Federal do Amazonas, Manaus, 3Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, 4Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil*These authors contributed equally to this workBackground: Rheumatoid arthritis (RA) is the most common autoimmune disease in the word, affecting 1% of the population. Long-term prognosis in RA was greatly improved following the introduction of highly effective medications such as methotrexate (MTX). Despite the importance of this drug in RA, 8%–16% of patients must discontinue the treatment because of adverse effects. Last decade, we developed a promising new nanocarrier as a drug-delivery system, lipid-core nanocapsules.Objective: The aim of the investigation reported here was to evaluate if methotrexate-loaded lipid-core nanocapsules (MTX-LNC) reduce proinflammatory and T-cell-derived cytokines in activated mononuclear cells derived from RA patients and even in functional MTX-resistant conditions. We also aimed to find out if MTX-LNC would reduce inflammation in experimentally inflammatory arthritis at lower doses than MTX solution.Methods: Formulations were prepared by self-assembling methodology. The adjuvant arthritis was induced in Lewis rats (AIA) and the effect on edema formation, TNF-a levels, and interleukin-1 beta levels after treatment was evaluated. Mononuclear cells obtained from the synovial fluid of RA patients during articular infiltration procedures were treated with MTX solution and MTX-LNC. For in vitro experiments, the same dose of MTX was used in comparing MTX and MTX-LNC, while the dose of MTX in the MTX-LNC was 75% lower than the drug in solution in in vivo experiments.Results: Formulations presented nanometric and unimodal size distribution profiles, with D[4.3] of 175±17 nm and span of 1.6±0.2. Experimental results showed that MTX-LNC had the same effect as MTX on arthritis inhibition on day 28 of the experiment (P<0.0001); however, this effect was achieved earlier, on day 21 (P<0.0001), by MTX-LNC, and this formulation had reduced both TNF-α (P=0.001) and IL-1α (P=0.0002) serum levels by the last day of the experiment. Further, the MTX-LNC were more effective at reducing the cytokine production from mononuclear synovial cells than MTX.Conclusion: The MTX-LNC were better than the MTX solution at reducing proinflammatory cytokines and T-cell-derived cytokines such as interferon-gamma and interleukin-17A. This result, combined with the reduction in the dose required for therapy, shows that MTX-LNC are a very promising system for the treatment of RA.Keywords: drug delivery, drug targeting, arthritis, cytokines, TNF-α, IL-6, IL-1, IL-17A, IFN-&gamma

In situ atomic-scale observation of oxygen-driven core-shell formation in Pt3Co nanoparticles

The catalytic performance of core-shell platinum alloy nanoparticles is typically superior to that of pure platinum nanoparticles for the oxygen reduction reaction in fuel cell cathodes. Thorough understanding of core-shell formation is critical for atomic-scale design and control of the platinum shell, which is known to be the structural feature responsible for the enhancement. Here we reveal details of a counter-intuitive core-shell formation process in platinum-cobalt nanoparticles at elevated temperature under oxygen at atmospheric pressure, by using advanced in situ electron microscopy. Initial segregation of a thin platinum, rather than cobalt oxide, surface layer occurs concurrently with ordering of the intermetallic core, followed by the layer-by-layer growth of a platinum shell via Ostwald ripening during the oxygen annealing treatment. Calculations based on density functional theory demonstrate that this process follows an energetically favourable path. These findings are expected to be useful for the future design of structured platinum alloy nanocatalysts.Core-shell platinum alloy nanoparticles are promising catalysts for oxygen reduction, however a deeper understanding of core-shell formation is still required. Here the authors report oxygen-driven formation of core-shell Pt3Co nanoparticles, seen at the atomic scale with in situ electron microscopy at ambient pressure

Peroxisomes and their central role in metabolic interaction networks in humans

Peroxisomes catalyze a number of essential metabolic functions and impairments in any of these are usually associated with major clinical signs and symptoms. In contrast to mitochondria which are autonomous organelles that can catalyze the degradation of fatty acids, certain amino acids and other compounds all by themselves, peroxisomes are non-autonomous organelles which are highly dependent on the interaction with other organelles and compartments to fulfill their role in metabolism. This includes mitochondria, the endoplasmic reticulum, lysosomes, and the cytosol. In this paper we will discuss the central role of peroxisomes in different metabolic interaction networks in humans, including fatty acid oxidation, ether phospholipid biosynthesis, bile acid synthesis, fatty acid alpha-oxidation and glyoxylate metabolism