Susceptibility of Cu(I) transport ATPases to sodium dodecyl sulfate. Relevance of the composition of the micellar phase.

Sodium dodecyl sulfate (SDS) is a well-known protein denaturing agent. A less known property of this detergent is that it can activate or inactivate some enzymes at sub-denaturing concentrations. In this work we explore the effect of SDS on the ATPase activity of a hyper-thermophilic and a mesophilic Cu(I) ATPases reconstituted in mixed micelles of phospholipids and a non-denaturing detergent. An iterative procedure was used to evaluate the partition of SDS between the aqueous and the micellar phases, allowing to determine the composition of micelles prepared from phospholipid/detergent mixtures. The incubation of enzymes with SDS in the presence of different amounts of phospholipids reveals that higher SDS concentrations are required to obtain the same degree of inactivation when the initial concentration of phospholipids is increased. Remarkably, we found that, if represented as a function of the mole fraction of SDS in the micelle, the degree of inactivation obtained at different amounts of amphiphiles converges to a single inactivation curve. To interpret this result, we propose a simple model involving active and inactive enzyme molecules in equilibrium. This model allowed us to estimate the Gibbs free energy change for the inactivation process and its derivative with respect to the mole fraction of SDS in the micellar phase, the latter being a measure of the susceptibility of the enzyme to SDS. Our results showed that the inactivation free energy changes are similar for both proteins. Conversely, susceptibility to SDS is significantly lower for the hyperthermophilic ATPase, suggesting an inverse relation between thermophilicity and susceptibility to SDS.

Direct Oral FXa Inhibitors Binding to Human Serum Albumin: Spectroscopic, Calorimetric, and Computational Studies.

Direct FXa inhibitors are an important class of bioactive molecules (rivaroxaban, apixaban, edoxaban, and betrixaban) applied for thromboprophylaxis in diverse cardiovascular pathologies. The interaction of active compounds with human serum albumin (HSA), the most abundant protein in blood plasma, is a key research area and provides crucial information about drugs' pharmacokinetics and pharmacodynamic properties. This research focuses on the study of the interactions between HSA and four commercially available direct oral FXa inhibitors, applying methodologies including steady-state and time-resolved fluorescence, isothermal titration calorimetry (ITC), and molecular dynamics. The HSA complexation of FXa inhibitors was found to occur via static quenching, and the complex formation in the ground states affects the fluorescence of HSA, with a moderate binding constant of 104 M-1. However, the ITC studies reported significantly different binding constants (103 M-1) compared with the results obtained through spectrophotometric methods. The suspected binding mode is supported by molecular dynamics simulations, where the predominant interactions were hydrogen bonds and hydrophobic interactions (mainly π-π stacking interactions between the phenyl ring of FXa inhibitors and the indole moiety of Trp214). Finally, the possible implications of the obtained results regarding pathologies such as hypoalbuminemia are briefly discussed.

A standardized method for genus Colletotrichum characterization by isothermal microcalorimetry using thermokinetic parameters.

A new standardized method, using isothermal microcalorimetry (IMC), was established to determine thermokinetic parameters from heat flow curves and to demonstrate the reproducibility and repeatability of the parameters of five Colletotrichum species on different days. Measurements on IMC were made at different periods and by two operators. Repeatability and reproducibility (R&R) measurement system analysis was performed on the technique used to measure the heat flow of Colletotrichum strains. The results showed that the %GageR&R was found to be within the acceptable ranges of a measurement system. Also, the parameters obtained from the curves were subjected to a combination of Principal Component Analysis (PCA) and Clustering, the data showed that the total heat (Ht) and maximum growth rate (µmax) are probably the most specific distinguishing characteristic of the strains evaluated in this study. This study demonstrates, for the first time, the usefulness of IMC in obtaining heat flow curves and thermokinetic parameters, providing repeatable and reproducible measurements over a period and under controlled conditions, for future identifications of phytopathogenic fungi.

Thermodynamics of Aggregation between the Cationic Surfactant and Polymer Based on Biodegradable Poly(vinyl Alcohol).

The formation of aggregates between carboxylated (PVCOOH) or neutral hydrolyzed (PVOH) poly(vinyl alcohol) and hexadecylpyridinium chloride (C16PyCl) was examined by conductimetry, turbidimetry, and isothermal titration calorimetry (ITC) in the presence of different NaCl concentrations. The interaction between the polymers and C16PyCl in pure water showed a critical aggregation concentration (cac = 0.8 mmol L-1) only for the neutral polymer. PVCOOH interacted with the surfactant through electrostatic attraction, forming macroscopic aggregates. Integral enthalpy changes for aggregate formation (ΔHagg) obtained from ITC curves varied from -0.61 (for the PVOH system in pure water) to -4.14 kJ mol-1 (for PVOH in the presence of 10.0 mmol L-1 NaCl), indicating that the formation of the aggregates was enthalpically favored. However, hydrophobic interactions drove the process for low surfactant concentration for both polymers. Saturation concentrations (C2) obtained from conductimetry were smaller than those from ITC, revealing that the binding of C16PyCl on the chain of the polymers at higher surfactant concentrations shows the same electric properties as that of free micelles on the solution. Increase of the ionic strength favored the aggregation and decreased the complexity of the ITC curves, suggesting that the reorganization of the surfactant monomers on the polymeric chain with the increase in their concentration was suppressed.

Membrane Fusion Biophysical Analysis of Fusogenic Liposomes.

Liposomes represent important drug carrier vehicles in biological systems. A fusogenic liposomal system composed of equimolar mixtures of the cationic lipid DOTAP and the phospholipid DOPE showed high fusion and delivery efficiencies with cells and lipid vesicles. However, aspects of the thermodynamics involving the interaction of these fusogenic liposomes and biomimetic systems remain unclear. Here, we investigate the fusion of this system with large unilamellar vesicles (LUVs) composed of the zwitterionic lipid POPC and increasing fractions of the anionic lipid POPG and up to 30 mol % cholesterol. The focus here is to concomitantly follow changes in size, zeta-potential, and enthalpy binding upon membrane interaction and fusion. Isothermal titration calorimetry (ITC) data showed that membrane fusion in our system is an exothermic process in the absence of cholesterol, suggesting that electrostatic attraction is the driving force for fusion. An endothermic component appeared and eventually dominated the titration at 30 mol % cholesterol, which we propose is caused by membrane fluidification when cholesterol is diluted upon fusion. The inflection points of the ITC data occurred around 0.5-0.7 POPG/DOTAP for all systems, the same stoichiometry for which zeta-potential and dynamic light scattering measurements showed an increase in size coupled with charge neutralization of the system, which is consistent with the fact that fusion in our system is charge-mediated. Microscopy observations of the final mixtures revealed the presence of giant vesicles, which is a clear indication of fusion, coexisting with intermediate-sized objects that could be the result of both fusion and/or aggregation. The results show that the fusion efficiency of the DOTAP:DOPE fusogenic system is modulated by the charge and membrane packing of the acceptor membrane and explain why the system fuses very efficiently with cells.

High-Speed and High-Temperature Calorimetric Solid-State Thermal Mass Flow Sensor for Aerospace Application: A Sensitivity Analysis.

A high-speed and high-temperature calorimetric solid-state thermal mass flow sensor (TMFS) design was proposed and its sensitivity to temperature and airflow speed were numerically assessed. The sensor operates at 573.15 Kelvin (300 °C), measuring speeds up to 265 m/s, and is customized to be a transducer for an aircraft Air Data System (ADS). The aim was to enhance the system reliability against ice accretion on pitot tubes' pressure intakes, which causes the system to be inoperative and the aircraft to lose protections that ensure its safe operation. In this paper, the authors assess how the distance between heater and thermal sensors affects the overall TMFS sensitivity and how it can benefit from the inclusion of a thermal barrier between these elements. The results show that, by increasing the distance between the heater and temperature sensors from 0.1 to 0.6 mm, the sensitivity to temperature variation is improved by up to 80%, and that to airspeed variation is improved by up to 100%. In addition, adding a thermal barrier made of Parylene-N improves it even further, by nearly 6 times, for both temperature and air speed variations.

Preparação e caracterização de nanocarreadores de beta-lactose à base de pectina e lisozima / Preparation and characterization of beta-lactose nanocarriers based on pectin and lysozyme

O objetivo deste trabalho foi preparar e caracterizar nanocarreadores via auto-organização a partir da pectina de citros e lisozima para o encapsulamento da ß-lactose. Foram estudadas três condições de interação entre os biopolímeros variando a razão molar pectina/lisozima (3:1, 2:1, 1:1, 1:2 e 1:3), o pH e o tempo de aquecimento. A confirmação da interação foi determinada por espectroscopia no infravermelho por transformada de Fourier (FTIR) e por calorimetria de varredura diferencial (DSC). Os espectros de infravermelho evidenciaram que ligações de hidrogênio foram as principais forças envolvidas na formação dos nanocarreadores e sugeriram a ausência de ß-lactose livre na superfície das nanopartículas. Os termogramas evidenciaram que as nanopartículas formadas na presença de ß-lactose têm maior estabilidade térmica do que as nanopartículas sem ß-lactose. Para ambas as formulações estudadas, na presença e na ausência de ß-lactose, a formação das nanopartículas ocorreu entre os valores de pKa e ponto isoelétrico (pI) da pectina e lisozima, respectivamente, sendo a melhor razão de interação pectina/lisozima 1:2, em pH 10, a 80 ºC por 30 min. As nanopartículas foram formadas via auto-organização e todos as partículas apresentaram distribuição de tamanho homogênea, formato esférico, diâmetro inferior a 100 nm e carga superficial negativa. A morfologia e o tamanho das partículas pouco alteraram com a incorporação da -lactose. A eficiência de encapsulação (EE) da ß-lactose foi superior a 96% para as concentrações estudadas. Ensaios preliminares in vitro, em células epiteliais de câncer de cólon (HCT-116), evidenciaram que as nanopartículas formadas são capazes de adentrar no meio intracelular, possivelmente, por via endocitose

This work aimed to prepare and characterize nanocarriers via self-assembly using citrus pectin and lysozyme for ß-lactose encapsulation. Three interaction conditions between the biopolymers were studied, varying the pectin/lysozyme molar ratio (3:1, 2:1, 1:1, 1:2 and 1:3), pH and heating time. Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) determined the interaction's confirmation. The infrared spectra showed that hydrogen bonds were the main forces involved in the formation of nanocarriers and suggested the absence of free ß-lactose on the surface of the nanoparticles. The thermograms showed that nanoparticles formed in the presence of ß-lactose have greater thermal stability than nanoparticles without ß-lactose. For both formulations studied, in the presence and absence of lactose, the formation of nanoparticles occurred between the pKa and isoelectric point (pI) values of pectin and lysozyme, respectively, with the best pectin/lysozyme interaction molar ratio 1:2, at pH 10, at 80 °C for 30 min. Nanoparticles were formed via self-assembly, and all particles presented homogeneous size distribution, spherical shape, diameter less than 100 nm, and negative surface charge. The morphology and size of the particles changed little with the incorporation of ß-lactose. The encapsulation efficiency (EE) of ß-lactose was higher than 96% for the concentrations studied. Preliminary in vitro assays in colon cancer epithelial cells (HCT-116) showed that the nanoparticles formed are capable of entering the intracellular medium, possibly via endocytosis

Structural and calorimetric studies reveal specific determinants for the binding of a high-affinity NLS to mammalian importin-alpha.

The classical nuclear import pathway is mediated by importin (Impα and Impß), which recognizes the cargo protein by its nuclear localization sequence (NLS). NLSs have been extensively studied resulting in different proposed consensus; however, recent studies showed that exceptions may occur. This mechanism may be also dependent on specific characteristics of different Impα. Aiming to better understand the importance of specific residues from consensus and adjacent regions of NLSs, we studied different mutations of a high-affinity NLS complexed to Impα by crystallography and calorimetry. We showed that although the consensus sequence allows Lys or Arg residues at the second residue of a monopartite sequence, the presence of Arg is very important to its binding in major and minor sites of Impα. Mutations in the N or C-terminus (position P1 or P6) of the NLS drastically reduces their affinity to the receptor, which is corroborated by the loss of hydrogen bonds and hydrophobic interactions. Surprisingly, a mutation in the far N-terminus of the NLS led to an increase in the affinity for both binding sites, corroborated by the structure with an additional hydrogen bond. The binding of NLSs to the human variant Impα1 revealed that these are similar to those found in structures presented here. For human variant Impα3, the bindings are only relevant for the major site. This study increases understanding of specific issues sparsely addressed in previous studies that are important to the task of predicting NLSs, which will be relevant in the eventual design of synthetic NLSs.

Membrane interactions of Ocellatins. Where do antimicrobial gaps stem from?

The antimicrobial peptides Ocellatin-LB1, -LB2 and -F1, isolated from frogs, are identical from residue 1 to 22, which correspond to the -LB1 sequence, whereas -LB2 carries an extra N and -F1 additional NKL residues at their C-termini. Despite the similar sequences, previous investigations showed different spectra of activities and biophysical investigations indicated a direct correlation between both membrane-disruptive properties and activities, i.e., ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. This study presents experimental evidence as well as results from theoretical studies that contribute to a deeper understanding on how these peptides exert their antimicrobial activities and how small differences in the amino acid composition and their secondary structure can be correlated to these activity gaps. Solid-state NMR experiments allied to the simulation of anisotropic NMR parameters allowed the determination of the membrane topologies of these ocellatins. Interestingly, the extra Asn residue at the Ocellatin-LB2 C-terminus results in increased topological flexibility, which is mainly related to wobbling of the helix main axis as noticed by molecular dynamics simulations. Binding kinetics and thermodynamics of the interactions have also been assessed by Surface Plasmon Resonance and Isothermal Titration Calorimetry. Therefore, these investigations allowed to understand in atomic detail the relationships between peptide structure and membrane topology, which are in tune within the series -F1 > > -LB1 ≥ -LB2, as well as how peptide dynamics can affect membrane topology, insertion and binding.

Epimers l- and d-Phenylseptin: How the relative stereochemistry affects the peptide-membrane interactions.

In recent decades, several epimers of peptides containing d-amino acids have been identified in antimicrobial sequences, a feature which has been associated with post-translational modification. Generally, d-isomers present similar or inferior antimicrobial activity, only surpassing their epimers in resistance to peptidases. The naturally occurring l-Phenylseptin (l-Phes) and d-Phenylseptin (d-Phes) peptides (FFFDTLKNLAGKVIGALT-nh2) were reported with d-epimer showing higher activity against Staphylococcus aureus and Xanthomonas axonopodis in comparison with the l-epimer. In this study, we combine structural (CD, solution NMR), orientational (solid-state NMR) and biophysical (ITC, DSC and DLS) studies to understand the role of the d-phenylalanine in the increase of the antimicrobial activity. Although both peptides are structurally similar in the helical region ranging from D4 to the C-terminus, significant structural differences were observed near the peptides' N-termini (which encompasses the FFF motif). Specific aromatic interactions involving the phenylalanine side chains of d-Phes is responsible to maintaining the F1-F3 residues on the hydrophobic face of the peptide, increasing its amphipathicity when compared to the l-epimer. The higher capability of d-Phes to exert an efficient anchoring in the hydrophobic core of the phospholipid bilayer indicates a pivotal role of the N-terminus in enhancing the interaction between the d-peptide and the membrane interface in relation to its epimer.

MagC is a NplC/P60-like member of the α-2-macroglobulin Mag complex of Pseudomonas aeruginosa that interacts with peptidoglycan.

Bacterial α-2 macroglobulins (A2Ms) structurally resemble the large spectrum protease inhibitors of the eukaryotic immune system. In Pseudomonas aeruginosa, MagD acts as an A2M and is expressed within a six-gene operon encoding the MagA-F proteins. In this work, we employ isothermal calorimetry (ITC), analytical ultracentrifugation (AUC), and X-ray crystallography to investigate the function of MagC and show that MagC associates with the macroglobulin complex and with the peptidoglycan (PG). However, the catalytic residues of MagC display an inactive conformation that could suggest that it binds to PG but does not degrade it. We hypothesize that MagC could serve as an anchor between the MagD macroglobulin and the PG and could provide stabilization and/or regulation for the entire complex.

Avaliação do desempenho de oleogéis estruturados com ceras vegetais como base lipídica reduzida em ácidos graxos saturados aplicados em cream cheeses / Performance evaluation of structured oleogels with vegetable waxes as a reduced lipid base in saturated fatty acids applied in cream cheeses

Diante das exigências crescentes das agências regulatórias do mundo todo quanto à redução/eliminação de ácidos graxos trans nos alimentos industrializados, bem como da conscientização do consumidor sobre a relação entre alimentação e saúde, o desenvolvimento de alternativas mais saudáveis aos óleos parcialmente hidrogenados e a outras fontes lipídicas com alto grau de saturaçã o se faz necessário. O oleogel, um sistema composto por um óleo preso em uma rede tridimensional formada por um agente estruturante, se apresenta como uma solução promissora. Dentre os diversos agentes estruturantes, as ceras vegetais se destacam por sua excelente capacidade de gelificação de óleos. Contudo, apresentam uma desvantagem sob o aspecto sensorial, pois podem conferir cerosidade e sabor residual desagradável aos alimentos. Com o objetivo de viabilizar o uso das ceras como agentes estruturantes em oleogéis face ao seu excelente desempenho tecnológico, este projeto propõe o estudo e a aplicação de oleogéis à base de óleo de soja (SBO) estruturado com ceras de farelo de arroz (RBW) a 2 e 4 % (m/m) ou carnaúba (CBW) a 3 e 6% (m/m), isoladamente. As matérias-primas foram caracterizadas e o comportamento de gelificação de cada cera foi avaliado por análises de textura por penetração de cone, estabilidade à perda de óleo por centrifugação, energia coesiva por parâmetro de solubilidade de Hansen (HSP) e comportamento de cristalização e fusão por calorimetria exploratória diferencial (DSC). Os resultados mostraram que ambas as ceras são capazes de formar oleogéis estruturalmente estáveis, contudo, o oleogel com 2% de RBW apresentou maior firmeza a 20 °C (190,4 gf/cm2) do que o oleogel com 6% de CBW a 5 °C (186,1 gf/cm2). Ao final de 5 dias, a capacidade de retenção de óleo do oleogel preparado com RBW foi de 100% às concentrações de 2 e 4% (m/m), contra 61 e 99,3% do oleogel elaborado com CBW às concentrações de 3 e 6% (m/m), respectivamente. Esses resultados podem ser explicados pela diferença entre as energias coesivas, ou seja, do grau de interação molecular entre o solvente e o soluto de cada oleogel. De acordo com os resultados de distância, que prevê se o gel formado será forte, fraco ou se não haverá formação de gel, o soluto CBW apresentou menor interação com o óleo (3,3 MPa1/2) do que o soluto RBW (3,7 MPa1/2). Os oleogéis foram aplicados como ingredientes em diferentes formulações de cream cheese, que foram analisados quanto a diferentes parâmetros de textura e esses resultados foram comparados a uma referência comercial. Nenhuma das amostras produzidas obteve resultados de textura estatisticamente iguais aos do cream cheese comercial (CC), o que pode ser explicado pelas diferenças de formulação e processamento dos produtos. Face aos resultados para textura e estabilidade à perda de óleo dos oleogéis de RBW, este agente estruturante apresenta ria maior potencial de aplicação, porém o oleogel CBW6 obteve alta capacidade de retenção de óleo (99,3%) e quando aplicado na formulação de cream cheese (CCBW6) apresentou resultados de firmeza e espalhabilidade mais próximos da amostra de referência, feita com gordura do leite (CMF)

Given the growing demands of regulatory agencies around the world regarding the reduction/elimination of trans fatty acids in processed foods, as well as consumer awareness about the relationship between food and health, the development of healthier alternatives to partially hydrogenated oils and others lipid sources with a high degree of saturation are necessary. Oleogel, a system composed of an oil trapped in a three-dimensional network formed by a structuring agent, presents itself as a promising solution. Among the various structuring agents, vegetable waxes stand out for their excellent oil gelling capacity. However, they have a sensory disadvantage, as they can give waxy and unpleasant aftertaste to foods. Aiming at enabling the use of waxes as structuring agents in oleogels in view of their excellent technological performance, this study proposes the evaluation and application of oleogels based on soybean oil (SBO) structured with rice bran wax (RBW) at 2 and 4% (m/m) or carnauba (CBW) at 3 and 6% (m/m). The raw materials were characterized and the gelling behavior of each wax was evaluated by analysis of texture by cone penetration, stability to oil loss by centrifugation, cohesive energy by Hansen solubility parameter (HSP) and crystallization and melting behavior. by differential scanning calorimetry (DSC). The results showed that both waxes are able to form structurally stable oleogels, however, oleogel with 2% RBW showed greater firmness at 20 °C (190.4 gf/cm2) than oleogel with 6% CBW at 5° C (186.1 gf/cm2). At the end of 5 days, the oil retention capacity of oleogel prepared with RBW was 100% at concentrations of 2 and 4% (m/m), against 61 and 99.3% of oleogel prepared with CBW at concentrations of 3 and 6% (m/m), respectively. These results can be explained by the difference between the cohesive energies, that is, the degree of molecular interaction between the solvent and the solute of each oleogel. According to the distance results, which predicts if the formed gel will be strong, weak or if there will be no gel formation, the CBW solute showed less interaction with the oil (3.3 MPa1/2) than the RBW solute (3 ,7 MPa1/2). Oleogels were applied as ingredients in different cream cheese formulations, which were analyzed for different texture parameters and these results were compared to a commercial reference. None of the samples produced had texture results statistically equal to those of commercial cream cheese (CC), which can be explained by the differences in formulation and processing of the products. Given the results for texture and oil binding capacity of RBW oleogels, this structuring agent would present greater application potential, but CBW6 oleogel obtained high oil biding capacity (99.3%) and when applied in cream cheese formulation (CCBW6) showed firmness and spreadability results closer to the reference sample, made with milk fat (CMF)

Desenvolvimento de achocolatados em pó com adição de subprodutos de frutas, processados por spray dryer e com modificador reológico / Development of powdered chocolate added with fruit byproducts, processed by spray dryer and rheological modifier

O achocolatado em pó é um dos derivados do cacau com maior inserção econômica e cultural em diversos países. O objetivo deste estudo foi avaliar a adição de ingredientes diferenciados nesse tipo de produto, como modificadores reológicos e fruta, bem como à alteração no tipo de cacau utilizado, ocasionando mudanças sensoriais e nutricionais positivas ao produto. O fruto sugerido neste estudo foi o cupuaçu (Theoboroma grandiflorum), fruto típico da região Norte, que apresenta excelente qualidade nutricional. Foram desenvolvidas 7 formulações de achocolatado por método convencional após simples mistura (padrão, com cacau alcalino, com cacau orgânico, com polpa de cupuaçu, com amido pré-gelatinizado, com amido pré-gelatinizado + polpa de cupuaçu, com goma guar, com goma guar + polpa de cupuaçu) e 4 formulações processadas por spray dryer após a simples mistura (padrão, com polpa de cupuaçu, com amido pré-gelatinizado, com amido + polpa de cupuaçu). Todas as formulações foram avaliadas quanto à composição nutricional, calorimetria exploratória de varredura (DSC), análises físico-químicas, reológica, quantificação dos compostos fenólicos e avaliação da capacidade antioxidante por métodos in vitro. Em seguida foi realizada análise sensorial com as formulações: padrão, com polpa de cupuaçu e com amido + polpa de cupuaçu. O achocolatado padrão apresentou tempo de mistura de 38 min, o que foi utilizado como parâmetro para as demais formulações. Os achocolatados que continham polpa de cupuaçu apresentaram maior teor proteico (14,5 a 16,3 g/100g) quando comparados com o padrão (13,6 g/100g). Todos os achocolatados apresentaram umidade entre 1,2% e 3,7%, e atividade de água entre 0,13 e 0,57, considerados microbiologicamente estáveis, sendo bom para a vida útil do produto. Os achocolatados obtiveram tempo de molhabilidade entre 07:15 min e 15:06 min; solubilidade de 1,56 IR% a 7,44 IR%; tamanho de partícula variando entre 0,216 mm e 0,347 mm (partículas finas). O uso do spray dryer não teve impacto significativo nas características físicas das formulações, assim como a utilização dos diferentes tipos de cacau não afetou a composição nutricional e qualidade física dos achocolatados. Houve aumento (p< 0,05) para o tempo de molhabilidade e solubilidade do achocolatado com cacau orgânico em comparação com o padrão (13:30 e 9:33 min; 2,64 e 1,56 IR%, respectivamente). A transição vítrea variou entre 35,2 a 35,7 mW enquanto o ponto de carbonização ficou entre 237,4 a 243,6 mW, indicando que a adição dos agentes espessantes e/ou do cupuaçu não interferiu (p<0,05) na análise térmica dos achocolatados. Todos os achocolatados diluídos em leite apresentaram-se como pseudoplásticos, com aumento de viscosidade nas menores temperaturas, conforme esperado. O achocolatado com cacau orgânico apresentou o maior teor de compostos fenólicos (8,27 mg AG g-1) enquanto observou-se redução no conteúdo de fenólicos nos produtos processados por spray dryer. Os achocolatados apresentaram capacidade antioxidante entre 31,76 µMETrolox/g e 75,62 µMETrolox/g, pelo método do DPPH. A adição do cupuaçu levou ao aumento da capacidade de sequestro de radicais DPPH quando comprados com o padrão (p<0,05). Não foi observada diferença significativa pelo método FRAP. A avaliação sensorial obteve aceitação situada na região positiva da escala (5 a 7). Os achocolatados formulados apresentam formulações adequadas a sua comercialização, com agregação de valor nutricional e econômico

The chocolate powdered is a cocoa-derived with greater economic and cultural integration in several countries. The objective of this study was to evaluate the addition of different ingredients in this type of product, such as rheological modifiers and fruit, as well as the change in the type of cocoa used, causing positive sensory and nutritional changes to the product. The fruit suggested in this study was cupuassu (Theoboroma grandiflorum), a typical fruit from the northern region, which has excellent nutritional quality. Seven powdered chocolate formulations were developed by conventional method after simple mixing (standard, with alkaline cocoa, with organic cocoa, with cupuassu pulp, pre-gelatinized starch, pre-gelatinized starch + cupuassu pulp, guar gum, with guar gum + cupuassu pulp) and 4 formulations processed by spray dryer after simple mixing (standard, with cupuassu pulp, pre-gelatinized starch, starch + cupuassu pulp). All formulations were evaluated for nutritional composition, differential scanning calorimetry (DSC), physicochemical, rheological analyzes, quantification of phenolic compounds and antioxidant capacity evaluation by in vitro methods. Then, sensory analysis was performed with the formulations: standard, with cupuassu pulp and starch + cupuassu pulp. The standard powdered chocolate had a mixing time of 38 min, which was used as parameter for the other formulations. The powdered chocolate containing cupuassu pulp had higher protein content (14.5 to 16.3 g / 100g) when compared to the standard (13.6 g / 100g). All powdered chocolate presented humidity between 1.2% and 3.7%, and water activity between 0.13 and 0.57, considered microbiologically stable, wich is good for the shelf life of the product. The powdered chocolate obtained wettability time between 07:15 min and 15:06 min; solubility from 1.56 IR% to 7.44 IR%; particle size ranging from 0.216 mm to 0.347 mm (fine particles). The use of the spray dryer had no significant impact on the physical characteristics of the formulations, as well as the use of different types of cocoa did not affect the nutritional composition and physical quality of the powdered chocolate. There was an increase (p <0.05) for the time of wettability and solubility in chocolate powdered formulated with organic cocoa when compared to the standard (9:33 and 13:30 min; IR 2.64 and 1.56%, respectively). The glass transition ranged from 35.2 to 35.7 mW while the carbonization point ranged from 237.4 to 243.6 mW, indicating that the addition of thickening agents and / or cupuassu did not interfere (p <0.05) in the thermal analysis of powdered chocolate. All powdered chocolate when diluted in milk presented as pseudoplastics, with viscosity increase at lower temperatures, as expected. Chocolate powdered with organic cocoa presented the highest content of phenolic compounds (8.27 mg AG g-1) whereas there was a reduction in phenolic content in products processed by spray dryer. The powdered chocolates presented antioxidant capacity between 31.76 µMETrolox / g and 75.62 µMETrolox / g, by the DPPH method. The addition of cupuassu led to increased ability to sequester DPPH radicals when compared to the standard (p <0.05). No significant difference was observed by the FRAP method. Sensory evaluation was accepted in the positive region of the scale (5 to 7). The formulated powdered chocolates have appropriate formulations for marketing, with added nutritional and economic value

Naringenin-lactoferrin binding: Impact on naringenin bitterness and thermodynamic characterization of the complex.

Naringenin (NG) is a flavonoid with many bioactive properties, however, its bitterness limits its use in foods. It is known that complex formation with proteins can mask this undesirable sensory property. Therefore, a trained panel evaluated the effect of bovine lactoferrin (LF) on NG bitterness using time-intensity analysis. LF reduced the maximum bitterness intensity and overall bitterness perception for NG by 27% and 33%, respectively. Isothermal titration nanocalorimetry (ITC), molecular docking (DC), and molecular dynamics (MD) were used to characterize NG-LF binding. These techniques provided similar values of ΔG° for binding ( [Formula: see text]  = -33.42 kJ mol-1; [Formula: see text]  = -32.22 kJ mol-1; [Formula: see text]  = -31.84 kJ mol-1). ITC showed that the complex formation is primarily entropy driven and DC suggested that NG binds at a hydrophobic site in LF. Here are presented strategic tools for promoting NG incorporation in food and health products.

CALX-CBD1 Ca2+-Binding Cooperativity Studied by NMR Spectroscopy and ITC with Bayesian Statistics.

The Na+/Ca2+ exchanger of Drosophila melanogaster, CALX, is the main Ca2+-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na+/Ca2+ exchangers have two Ca2+ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologs, CALX is inhibited by Ca2+ binding to CALX-CBD1, whereas CALX-CBD2 does not bind Ca2+ at physiological concentrations. CALX-CBD1 consists of a ß-sandwich and displays four Ca2+-binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca2+ binding to CALX-CBD1. We observed that this domain binds Ca2+ in the slow exchange regime at the NMR chemical shift timescale. Ca2+ binding restricts the dynamics in the Ca2+-binding region. Experiments of 15N chemical exchange saturation transfer and 15N R2 dispersion allowed the determination of Ca2+ dissociation rates (∼30 s-1). NMR titration curves of residues in the Ca2+-binding region were sigmoidal because of the contribution of chemical exchange to transverse magnetization relaxation rates, R2. Hence, a novel, to our knowledge, approach to analyze NMR titration curves was proposed. Ca2+-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded nHill ∼2.9, near maximal cooperativity (nHill = 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2+ is approximately three orders of magnitude higher than that of binding a single Ca2+. Hence, four Ca2+ ions bind almost simultaneously to CALX-CBD1. Cooperative Ca2+ binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2+ concentration in sensory neuronal cells.

Complex coacervates of ß-lactoglobulin/sodium alginate for the microencapsulation of black pepper (Piper nigrum L.) essential oil: Simulated gastrointestinal conditions and modeling release kinetics.

This study evaluated the most appropriate conditions (pH and biopolymers ratio) for the formation of the complex between ß-lactoglobulin (ß-lg) and sodium alginate (NaAlg). Furthermore, we microencapsulated black pepper essential oil (EO) using these biopolymers and transglutaminase as a cross-linking agent, and stability during in vitro digestion and its release in food models were studied. A ratio of 17:1 (ß-lg/NaAlg) at a pH of 4.5 was the optimal condition for the formation of the complex. The encapsulation efficiency (85.01% ± 0.26) and chemical and morphological characteristics suggested that black pepper EO was microencapsulated using polymers and cross-linking agent naturals. The particle size demonstrated that the capsules produced were on micro scale. The black pepper EO microcapsules lost lower release in water, and the Rigger-Peppas model indicated that the Fickian diffusion mechanism occurred. The microcapsules demonstrated a low release of black pepper EO during oral and gastric digestion and a higher release in intestinal digestion. The black pepper EO after digestion presented high stability (84.8% ± 0.07), and bioaccessibility (31.16% ± 0.3). The results suggest that the black pepper EO was microencapsulated and, preserved in aqueous food model and during oral and gastric conditions tested in vitro.

A new methodology to determine the effect of the adsorbate-adsorbent interactions on the analgesic adsorption onto activated carbon using kinetic and calorimetry data.

This work proposes a new methodology to determine the adsorption mechanism for salicylic acid and paracetamol on activated carbon based on the physicochemical characteristics of adsorbent and adsorbates. The methodology is divided into two parts: the determination of adsorption kinetics (order and mechanism) and the study of the chemical interactions (adsorbate-adsorbent and solvent-adsorbent) using calorimetry tests. Then, the results obtained in both techniques were correlated with the amount of drug adsorbed. The adsorption kinetics of salicylic acid and paracetamol on activated carbons with different oxygen contents could be described with widely kinetic models such as intraparticle, pseudo-first-order, pseudo-second-order, Avrami, and Elovich models; different information about the adsorption mechanism are offered by each of them. The results indicated that the pseudo-first-order rate constant decreases with the molecular size of analgesics and the carboxylic acid groups on the adsorbent surface; the rate constant values are between 0.12 and 2.31 h-1. The adsorbed amount of analgesics and the adsorption rate are greatest on activated carbons with basic characteristics (QRAC 0.45 > 0.24 > 0.21 mmol g-1 for phenol, salicylic acid, and paracetamol, respectively). The enthalpy changes follow the same trend in all activated carbons; for RAC, the results were ΔHimmPHEN = - 33.4 J g-1, ΔHimmSA = - 35.9 J g-1, and ΔHimmPAR = - 45.4 J g-1. The analgesic diffusion rate in the boundary layer increases with the formation of adsorbate-adsorbent interactions (exothermic process).

Characterization of Schistosoma mansoni Dihydrofolate Reductase (DHFR).

Dihydrofolate reductase (DHFR) is an essential enzyme for nucleotide metabolism used to obtain energy and structural nucleic acids. Schistosoma mansoni has all the pathways for pyrimidine biosynthesis, which include the thymidylate cycle and, consequentially, the DHFR enzyme. Here, we describe the characterization of Schistosoma mansoni DHFR (SmDHFR) using isothermal titration calorimetry for the enzymatic activity and thermodynamic determination, also the folate analogs inhibition. Moreover, X-ray crystallography was used to determine the enzyme atomic model at 1.95 Å.

Probing protein adsorption onto polymer-stabilized silver nanocolloids towards a better understanding on the evolution and consequences of biomolecular coronas.

The use of noble metal nanoparticles in biomedical and biotechnological applications is nowadays well established. Particularly, silver nanoparticles (AgNPs) were proven to be effective for instance as a biocide agent. They also find applications in tumor therapies and sensing applications being encouraging tools for in-vivo imaging. In this framework, whenever they are in contact with living systems, they are rapidly coated by a protein corona thereby influencing a variety of biological events including cellular uptake, blood circulation lifetime, cytotoxicity and, ultimately, the therapeutic effect. Taking these considerations into account, we have explored the behavior of polymer-coated AgNPs in model protein environments focusing on the self-development of protein coronas. The polymers polyethyleneimine (PEI), polyvinylpyrrolidone (PVP) and poly(2-vinyl pyridine)-b-poly(ethylene oxide) (PEO-b-P2VP) were used as stabilizing agents. The chemical nature of the polymer capping remarkably influences the behavior of the hybrid nanomaterials in protein environments. The PEO-b-P2VP and PVP-stabilized AgNPs are essentially inert to the model proteins adsorption. On the other hand, the PEI-stabilized AgNPs interact strongly with bovine serum albumin (BSA). Nevertheless, the same silver colloids were evidenced to be stable in IgG and lysozyme environments. The BSA adsorption into the PEI-stabilized AgNPs is most probably driven by hydrogen bonding and van der Waals interactions as suggested by isothermal titration calorimetry data. The development of protein coronas around the AgNPs may have relevant implications in a variety of biological events. Therefore, further investigations are currently underway to evaluate the influence of its presence on the cytotoxicity, hemolytic effects and biocide properties of the produced hybrid nanomaterials.

Crystal structure of a C-type lysozyme from Litopenaeus vanamei exhibiting a high binding constant to its chitotriose inhibitor.

Although information about invertebrate lysozymes is scarce, these enzymes have been described as components of the innate immune system, functioning as antibacterial proteins. Here we describe the first thermodynamic and structural study of a new C-type lysozyme from a Pacific white shrimp Litopenaeus vannamei (LvL), which has shown high activity against both Gram (+) and Gram (-) bacteria including Vibrio sp. that is one of the most severe pathogens in penaeid shrimp aquaculture. Compared with hen egg-white lysozyme, its sequence harbors a seven-residue insertion from amino acid 97 to 103, and a nine-residue extension at the C-terminus only found in penaeid crustaceans, making this enzyme one of the longest lysozyme reported to date. LvL was crystallized in the presence and absence of chitotriose. The former crystallized as a monomer in space group P61 and the latter in P212121 with two monomers in the asymmetric unit. Since the enzyme crystallized at a pH where lysozyme activity is deficient, the ligand could not be observed in the P61 structure; therefore, we performed a docking simulation with chitotriose to compare with the hen egg lysozyme crystallized in the presence of the ligand. Remarkably, additional amino acids in LvL caused an increase in the length of α-helix H4 (residues 97-103) that is directly related to ligand recognition. The Ka for chitotriose (4.1 × 105 M-1), as determined by Isothermal Titration Calorimetry, was one order of magnitude higher than those for lysozymes from hen and duck eggs. Our results revealed new interactions of chitiotriose with residues in helix H4.