Protein stability arises from a combination of factors which are often difficult to rationalise. Therefore its improvement is better addressed through directed evolution than by rational design ...approaches. In this study, five rounds of mutagenesis/recombination followed by high-throughput screening (≈10,000 clones) yielded the hit 1B6 showing a 300-fold higher half life at 50°C than that exhibited by the homodimeric wild type PpAzoR azoreductase from Pseudomonas putida MET94. The characterization using fluorescence, calorimetry and light scattering shows that 1B6 has a folded state slightly less stable than the wild type (with lower melting and optimal temperatures) but in contrast is more resistant to irreversible denaturation. The superior kinetic stability of 1B6 variant was therefore related to an increased resistance of the unfolded monomers to aggregation through the introduction of mutations that disturbed hydrophobic patches and increased the surface net charge of the protein. Variants 2A1 and 2A1-Y179H with increased thermodynamic stability (10 to 20°C higher melting temperature than wild type) were also examined showing the distinctive nature of mutations that lead to improved structural robustness: these occur in residues that are mostly involved in strengthening the solvent-exposed loops or the inter-dimer interactions of the folded state.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Serological assays are valuable tools to study SARS‐CoV‐2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. ...SARS‐CoV‐2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS‐CoV‐2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID‐19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293‐E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme‐linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in‐depth characterization of SARS‐CoV‐2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS‐CoV‐2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high‐quality SARS‐CoV‐2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in‐depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID‐19 serologic tests.
Spike and RBD were produced in human derived cells hosts. Shake flasks, stirred tank and wave bioreactors were compared. The impact of these different scalable bioprocessing approaches on Spike and RBD production yields and quality attributes was evaluated. An in‐depth characterization of SARS‐CoV‐2 Spike and RBD proteins was performed, namely the antigen s oligomeric state, glycosylation profiles and thermal stability during storage. The correlation of these quality attributes with ELISA performance is discussed.
The feasibility of bacterial cellulose (BC) as a novel substrate for retinal pigment epithelium (RPE) culture was evaluated. Thin (41.6 ± 2.2 μm of average thickness) and heat-dried BC substrates ...were surface-modified via acetylation and polysaccharide adsorption, using chitosan and carboxymethyl cellulose. All substrates were characterized according to their surface chemistry, wettability, energy, topography, and also regarding their permeability, dimensional stability, mechanical properties, and endotoxin content. Then, their ability to promote RPE cell adhesion and proliferation in vitro was assessed. All surface-modified BC substrates presented similar permeation coefficients with solutes of up to 300 kDa. Acetylation of BC decreased it’s swelling and the amount of endotoxins. Surface modification of BC greatly enhanced the adhesion and proliferation of RPE cells. All samples showed similar stress–strain behavior; BC and acetylated BC showed the highest elastic modulus, but the latter exhibited a slightly smaller tensile strength and elongation at break as compared to pristine BC. Although similar proliferation rates were observed among the modified substrates, the acetylated ones showed higher initial cell adhesion. This difference may be mainly due to the moderately hydrophilic surface obtained after acetylation.
Pesticides are the main tactics for pest control because they reduce the pest population very fast and their efficiency does not depend on abiotic factors. However, the indiscriminate use of these ...substances can speed up the development of resistant populations and causing environmental contamination. Therefore, alternative methods of pest control are sought, such as the use of botanical compounds. Nanoencapsulation of volatile compounds has been shown to be an important tool that can be used to overcome the lack of stability of these compounds. In this work, we describe the preparation and characterization of chitosan nanoparticles functionalized with β-cyclodextrin containing carvacrol and linalool. The toxicity and biological activity were evaluated. Decreases of toxicity were observed when the compounds were nanoencapsulated. The nanoparticles presented insecticidal activity against the species Helicoverpa armigera (corn earworm) and Tetranychus urticae (spider mite). In addition, repellent activity and reduction in oviposition were observed for the mites.
Asthma is characterized by chronic lung inflammation and airway hyperresponsiveness. Despite recent advances in the understanding of its pathophysiology, asthma remains a major public health problem ...and, at present, there are no effective interventions capable of reversing airway remodeling. Mesenchymal stromal cell (MSC)-based therapy mitigates lung inflammation in experimental allergic asthma; however, its ability to reduce airway remodeling is limited. We aimed to investigate whether pre-treatment with eicosapentaenoic acid (EPA) potentiates the therapeutic properties of MSCs in experimental allergic asthma. Seventy-two C57BL/6 mice were used. House dust mite (HDM) extract was intranasally administered to induce severe allergic asthma in mice. Unstimulated or EPA-stimulated MSCs were administered intratracheally 24 h after final HDM challenge. Lung mechanics, histology, protein levels of biomarkers, and cellularity in bronchoalveolar lavage fluid (BALF), thymus, lymph nodes, and bone marrow were analyzed. Furthermore, the effects of EPA on lipid body formation and secretion of resolvin-D
(RvD
), prostaglandin E
(PGE
), interleukin (IL)-10, and transforming growth factor (TGF)-β1 by MSCs were evaluated
. EPA-stimulated MSCs, compared to unstimulated MSCs, yielded greater therapeutic effects by further reducing bronchoconstriction, alveolar collapse, total cell counts (in BALF, bone marrow, and lymph nodes), and collagen fiber content in airways, while increasing IL-10 levels in BALF and M2 macrophage counts in lungs. In conclusion, EPA potentiated MSC-based therapy in experimental allergic asthma, leading to increased secretion of pro-resolution and anti-inflammatory mediators (RvD
, PGE
, IL-10, and TGF-β), modulation of macrophages toward an anti-inflammatory phenotype, and reduction in the remodeling process. Taken together, these modifications may explain the greater improvement in lung mechanics obtained. This may be a promising novel strategy to potentiate MSCs effects.
RESUMO Com o objetivo de prolongar o período de conservação e manter a qualidade do mamão UENF/ Caliman01, foram testadas diferentes concentrações de CaCl2 aplicadas por infiltração a vácuo. Os ...frutos foram separados em seis lotes, o controle(sem tratamento) e os tratamentos que receberam a aplicação de CaCl2 a 0%, 2%, 4%, 6% e 8% CaCl2(p/v) por imersão sob vácuo, por 3 minutos, a 50kPa de tensão, com posterior análises da perda de massafresca, firmeza do fruto, firmeza do mesocarpo, ângulo de cor hue, teores de sólidos solúveis (SS), acideztitulável (AT), ácido ascórbico, açúcares solúveis totais e razão SS/AT. Os tratamentos com cálcio não interferiram na perda de massa que, de modo geral, incrementou ao longo do tempo, porém os frutos permaneceram verdes e firmes por mais tempo quando tratados com CaCl2 6% e 8% (p/v). O uso do CaCl2 não interferiu nas características químicas do fruto, tais como sólidos solúveis, acidez titulável e razão SS/AT, porém diminuiu asíntese de ácido ascórbico e a degradação dos açúcares solúveis.
In this study, the biosurfactant production by an Aureobasidium thailandense LB01 was reported for the first time. Different agro-industrial by-products (corn steep liquor, sugarcane molasses, and ...olive oil mill wastewater) were evaluated as alternative low-cost substrates. The composition of the culture medium was optimized through response surface methodology. The highest biosurfactant production (139±16mg/L) was achieved using a culture medium containing yeast extract (2g/L); olive oil mill wastewater (1.5%, w/w); glucose (6g/L) and KH2PO4 (1g/L) after 48h of fermentation. The partially purified biosurfactant exhibited a critical micelle concentration of 550mg/L, reducing the surface tension of water up to 31.2mN/m. Its molecular structure was found to be similar to a lauric acid ester. The biosurfactant exhibited a better performance than the chemical surfactant sodium dodecyl sulfate (SDS) in oil dispersion assays, thus suggesting its potential application in bioremediation.
The treatment of dermatophytoses, the most common human fungal infections, requires new alternatives. The aim of this study was to determine the antidermatophytic activity of the aqueous Azorean ...Black Tea extract (ABT), together with an approach to the mechanisms of action. The phytochemical analysis of ABT extract was performed by HPLC. The dermatophytes susceptibility was assessed using a broth microdilution assay; potential synergies with terbinafine and griseofulvin were evaluated by the checkerboard assay. The mechanism of action was appraised by the quantification of the fungal cell wall chitin and β-1,3-glucan, and by membrane ergosterol. The presence of ultrastructural modifications was studied by Transmission Electron Microscopy (TEM). The ABT extract contained organic and phenolic acids, flavonoids, theaflavins and alkaloids. It showed an antidermatophytic effect, with MIC values of 250 µg/mL for
, 125 µg/mL for
and 500 µg/mL for
; at these concentrations, the extract was fungicidal. An additive effect of ABT in association to terbinafine on these three dermatophytes was observed. The ABT extract caused a significant reduction in β-1,3-glucan content, indicating the synthesis of this cell wall component as a possible target. The present study identifies the antidermatophytic activity of the ABT and highlights its potential to improve the effectiveness of conventional topical treatment currently used for the management of skin or mucosal fungal infections.
Semi-arid regions are known for erratic precipitation patterns with significant effects on the hydrological cycle and water resources availability. High temporal and spatial variation in ...precipitation causes large variability in runoff over short durations. Due to low soil water storage capacity, base flow is often missing and rivers fall dry for long periods. Because of its climatic characteristics, the semi-arid north-eastern region of Brazil is prone to droughts. To counter these, reservoirs were built to ensure water supply during dry months. This paper describes problems and solutions when calibrating and validating the eco-hydrological model SWIM for semi-arid regions on the example of the Pajeú watershed in north-eastern Brazil. The model was calibrated to river discharge data before the year 1983, with no or little effects of water management, applying a simple and an enhanced approach. Uncertainties result mainly from the meteorological data and observed river discharges. After model calibration water management was included in the simulations. Observed and simulated reservoir volumes and river discharges are compared. The calibrated and validated models were used to simulate the impacts of climate change on hydrological processes and water resources management using data of two representative concentration pathways (RCP) and five earth system models (ESM). The differences in changes in natural and managed mean discharges are negligible (< 5%) under RCP8.5 but notable (> 5%) under RCP2.6 for the ESM ensemble mean. In semi-arid catchments, the enhanced approach should be preferred, because in addition to discharge, a second variable, here evapotranspiration, is considered for model validation.
PDF
