Human colostrum in vitro protein digestion: peptidomics by liquid chromatography-Orbitrap-high-resolution MS and prospection for bioactive peptides via bioinformatics.

Breast milk is known to contain bioactive peptides that are released during digestion, being a major source of bioactive peptides to the new-born, some of which act against invading pathogens. However, the formation of bioactive peptides during digestion of human colostrum remains largely uninvestigated. This study aimed to investigate the formation of peptides during simulated digestion of human colostrum from adult women and to prospect antimicrobial peptides. For this purpose, we used high-resolution MS to monitor the release of peptides during in vitro digestion. Bioinformatics was used for the prospection of antimicrobial activity of peptides. During simulated digestion (oral, gastric and duodenal phases), 2318 peptide sequences derived from 112 precursor proteins were identified. At the end of simulated digestion, casein-derived peptide sequences were the most frequently observed. Among precursors, some proteins were seen for the first time in this study. The resulting peptides were rich in proline, glutamine, valine and leucine residues, providing characteristic traits of antimicrobial peptides. From bioinformatics analysis, seven peptides showed potentially high antimicrobial activity towards bacteria, viruses and fungi, from which the latter was the most prominent predicted activity. Antimicrobial peptides released during digestion may provide a defence platform with controlled release for the new-born.

Data reconciliation connected to guard bands to set specification limits related to risk assessment for radiopharmaceutical activity.

Radiopharmaceuticals have been used to diagnose several diseases, particularly because the procedure is non-invasive. However, it is important that the correct amount of radiopharmaceutical is used to avoid inaccurate diagnostic results and suboptimal therapeutic outcomes. The amount of the radiopharmaceutical is measured when produced (by the supplier) and a second time (by the receiver), before it's use. When measured at the receiver, the result is corrected for its normal radioactivity decay. Even then, it is possible that both measurements should be considered nominal different or even statistically different when compared through various statistical tools. This research combines two innovative techniques in the field of clinical metrology. The first technique is data reconciliation, which not only enhances measurement accuracy but also reduces measurement uncertainty. The second technique involves using uncertainty information to establish specification limits for compliance assessments. In this way, our proposal aimed to minimize the risk of making incorrect decisions regarding the conformity of the concentration of radiopharmaceutical activity, that is, rejecting an item or batch that is within specification or accepting an item or batch that is outside of specification. A spreadsheet, based on these metrology fundamentals, is available to help the user with the calculations, presenting numerical and graphical results for some common radioisotopes. Reliable specification limits can be calculated and used to determine if the radiopharmaceutical is in accordance with its proposed application.

An NMR-Based Chemometric Strategy to Identify Leishmania donovani Nucleoside Hydrolase Inhibitors from the Brazilian Tree Ormosia arborea.

Nucleoside hydrolases are a strategic target for the development of drugs to treat leishmaniasis, a neglected disease that affects 700 thousand to one million people annually. The present study aimed to identify Leishmania donovani nucleoside hydrolase (LdNH) inhibitors from the leaves of Ormosia arborea, a tree endemic to Brazilian ecosystems, through a strategy based on 1H NMR analyses and chemometrics. The aqueous EtOH extract of O. arborea leaves inhibited LdNH activity by 95%. The extract was fractionated in triplicate (13 in each step, making a total of 39 fractions). Partial least squares discriminant analysis (PLS-DA) was used to correlate the 1H NMR spectra of the fractions with their LdNH inhibitory activity and thus to identify the spectral regions associated with the bioactivity. The strategy aimed at isolating the probable bioactive substances and led to two new A-type proanthocyanidins, linked to a p-coumaroyl unit (1 and 2), which appeared as noncompetitive inhibitors of LdNH (IC50: 28.2 ± 3.0 µM and 25.6 ± 4.1 µM, respectively). This study confirms the usefulness of the NMR-based chemometric methods to accelerate the discovery of drugs from natural products.

High-intensity interval training followed by postexercise cold-water immersion does not alter angiogenic circulating cells, but increases circulating endothelial cells.

High-intensity interval training (HIIT) induces vascular adaptations that might be attenuated by postexercise cold-water immersion (CWI). Circulating angiogenic cells (CAC) participate in the vascular adaptations and circulating endothelial cells (CEC) indicate endothelial damage. CAC and CEC are involved in vascular adaptation. Therefore, the aim of the study was to investigate postexercise CWI during HIIT on CAC and CEC and on muscle angiogenesis-related molecules. Seventeen male subjects performed 13 HIIT sessions followed by 15 min of passive recovery (n = 9) or CWI at 10 °C (n = 8). HIIT comprised cycling (8-12 bouts, 90%-110% peak power). The first and the thirteenth sessions were similar (8 bouts at 90% of peak power). Venous blood was drawn before exercise (baseline) and after the recovery strategy (postrecovery) in the first (pretraining) and in the thirteenth (post-training) sessions. For CAC and CEC identification lymphocyte surface markers (CD133, CD34, and VEGFR2) were used. Vastus lateralis muscle biopsies were performed pre- and post-training for protein (p-eNOSser1177) and gene (VEGF and HIF-1) expression analysis related to angiogenesis. CAC was not affected by HIIT or postexercise CWI. Postexercise CWI increased acute and baseline CEC number. Angiogenic protein and genes were not differently modulated by post-CWI. HIIT followed by either recovery strategy did not alter CAC number. Postexercise CWI increased a marker of endothelial damage both acutely and chronically, suggesting that this postexercise recovery strategy might cause endothelial damage. Novelty HIIT followed by CWI did not alter CAC. HIIT followed by CWI increased CEC. Postexercise CWI might cause endothelial damage.

High-intensity interval training reduces monocyte activation in obese adults.

Alterations in the distribution and activation of monocyte subsets are frequently observed in individuals with obesity and their participation in the pathological complications of obesity is proposed. High-intensity interval training (HIIT) can be a time-efficient alternative to counteract the inflammatory outcomes of obesity, but so far, its effects on monocytes in obesity has not been fully explored. In this study, we investigated whether 8 weeks of HIIT can modify the distribution and activation of the three monocyte subsets (classical, intermediate and non-classical monocytes) in individuals with obesity. Our data show that individuals with obesity have a higher percentage of non-classical monocytes compared to control, lean individuals, and consequently an imbalance among the CD16+ monocyte subsets. Also, the expression of HLA-DR by intermediate monocytes is higher in insulin-resistant obese individuals, which indicates monocyte activation in obesity. After 8 weeks of HIIT, the percentage of non-classical monocytes was reduced in individuals with obesity, restoring the balance among the CD16+ monocytes. Also, the expression of HLA-DR by intermediate monocytes in insulin-resistant obese subjects was lower after HIIT. Both findings indicate that monocyte activation in individuals with obesity was reduced by HIIT. These modifications were observed in the absence of changes in weight and body composition, although they were accompanied by the improvement in the metabolic status (reduced insulin levels). Our findings indicate that HIIT can be considered a time-efficient strategy to manage obesity-related monocyte alterations and strengthen the immunomodulatory potential of HIIT.

Optimization of Aqueous Extraction from Kalanchoe pinnata Leaves to Obtain the Highest Content of an Anti-inflammatory Flavonoid using a Response Surface Model.

INTRODUCTION: The medicinal plant Kalanchoe pinnata is a phenolic-rich species used worldwide. The reports on its pharmacological uses have increased by 70% in the last 10 years. The leaves of this plant are the main source of an unusual quercetin-diglycosyl flavonoid (QAR, quercetin arabinopyranosyl rhamnopyranoside), which can be easily extracted using water. QAR possess a strong in vivo anti-inflammatory activity. OBJECTIVE: To optimize the aqueous extraction of QAR from K. pinnata leaves using a three-level full factorial design. MATERIAL AND METHODS: After a previous screening design, time (x1 ) and temperature (x2 ) were chosen as the two independent variables for optimization. Freeze-dried leaves were extracted with water (20% w/v), at 30°C, 40°C or 50°C for 5, 18 or 30 min. QAR content (determined by HPLC-DAD) and yield of extracts were analyzed. The optimized extracts were also evaluated for cytotoxicity. RESULTS: The optimal heating times for extract yield and QAR content were similar in two-dimensional (2D) surface responses (between 12.8 and 30 min), but their optimal extraction temperatures were ranged between 40°C and 50°C for QAR content and 30°C and 38°C for extract yield. A compromise region for both parameters was at the mean points that were 40°C for the extraction temperature and 18 min for the total time. CONCLUSION: The optimized process is faster and spends less energy than the previous one (water; 30 min at 55°C); therefore is greener and more attractive for industrial purposes. This is the first report of extraction optimization of this bioactive flavonoid. Copyright © 2018 John Wiley & Sons, Ltd.

Post-exercise cold water immersion does not alter high intensity interval training-induced exercise performance and Hsp72 responses, but enhances mitochondrial markers.

This study aims to evaluate the effect of regular post-exercise cold water immersion (CWI) on intramuscular markers of cellular stress response and signaling molecules related to mitochondria biogenesis and exercise performance after 4 weeks of high intensity interval training (HIIT). Seventeen healthy subjects were allocated into two groups: control (CON, n = 9) or CWI (n = 8). Each HIIT session consisted of 8-12 cycling exercise stimuli (90-110 % of peak power) for 60 s followed by 75 s of active recovery three times per week, for 4 weeks (12 HIIT sessions). After each HIIT session, the CWI had their lower limbs immersed in cold water (10 °C) for 15 min and the CON recovered at room temperature. Exercise performance was evaluated before and after HIIT by a 15-km cycling time trial. Vastus lateralis biopsies were obtained pre and 72 h post training. Samples were analyzed for heat shock protein 72 kDa (Hsp72), adenosine monophosphate-activated protein kinase (AMPK), and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) assessed by western blot. In addition, the mRNA expression of heat shock factor-1 (HSF-1), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), nuclear respiratory factor 1 and 2 (NRF1 and 2), mitochondrial transcription factor A (Tfam), calcium calmodulin-dependent protein kinase 2 (CaMK2) and enzymes citrate synthase (CS), carnitine palmitoyltransferase I (CPT1), and pyruvate dehydrogenase kinase (PDK4) were assessed by real-time PCR. Time to complete the 15-km cycling time trial was reduced with training (p < 0.001), but was not different between groups (p = 0.33). The Hsp72 (p = 0.01), p38 MAPK, and AMPK (p = 0.04) contents increased with training, but were not different between groups (p > 0.05). No differences were observed with training or condition for mRNA expression of PGC-1α (p = 0.31), CPT1 (p = 0.14), CS (p = 0.44), and NRF-2 (p = 0.82). However, HFS-1 (p = 0.007), PDK4 (p = 0.03), and Tfam (p = 0.03) mRNA were higher in CWI. NRF-1 decrease in both groups after training (p = 0.006). CaMK2 decreased with HIIT (p = 0.003) but it was not affected by CWI (p = 0.99). Cold water immersion does not alter HIIT-induced Hsp72, AMPK, p38 MAPK, and exercise performance but was able to increase some markers of cellular stress response and signaling molecules related to mitochondria biogenesis.

Phenolic compounds of Brazilian beers from different types and styles and application of chemometrics for modeling antioxidant capacity.

In the present study we aimed at investigating, for the first time, phenolic compounds in Brazilian beers of different types and styles. We also aimed at applying chemometrics for modeling beer's antioxidant capacity as a function of their physicochemical attributes (density, refractive index, bitterness and ethanol content). Samples (n=29) were analyzed by PCA originating five groups, especially according to ethanol contents and bitterness. In general, Group V (alcoholic beers with very high bitterness) presented higher refractive index, bitterness, ethanol and phenolics contents than Groups I (non-alcoholic beers) and II (alcoholic beers with low bitterness). Brazilian beers phenolics profile was distinct from that of European beers, with high contents of gallic acid (0.5-14.7 mg/L) and low contents of ferulic acid (0.2-1.8 mg/L). Using PLS, beer's antioxidant capacity measured by FRAP assay could be predicted with acceptable precision by data of ethanol content and density, bitterness and refractive index values.

The effect of different water immersion temperatures on post-exercise parasympathetic reactivation.

PURPOSE: We evaluated the effect of different water immersion (WI) temperatures on post-exercise cardiac parasympathetic reactivation. METHODS: Eight young, physically active men participated in four experimental conditions composed of resting (REST), exercise session (resistance and endurance exercises), post-exercise recovery strategies, including 15 min of WI at 15°C (CWI), 28°C (TWI), 38°C (HWI) or control (CTRL, seated at room temperature), followed by passive resting. The following indices were assessed before and during WI, 30 min post-WI and 4 hours post-exercise: mean R-R (mR-R), the natural logarithm (ln) of the square root of the mean of the sum of the squares of differences between adjacent normal R-R (ln rMSSD) and the ln of instantaneous beat-to-beat variability (ln SD1). RESULTS: The results showed that during WI mRR was reduced for CTRL, TWI and HWI versus REST, and ln rMSSD and ln SD1 were reduced for TWI and HWI versus REST. During post-WI, mRR, ln rMSSD and ln SD1 were reduced for HWI versus REST, and mRR values for CWI were higher versus CTRL. Four hours post exercise, mRR was reduced for HWI versus REST, although no difference was observed among conditions. CONCLUSIONS: We conclude that CWI accelerates, while HWI blunts post-exercise parasympathetic reactivation, but these recovery strategies are short-lasting and not evident 4 hours after the exercise session.

Validation of impregnation process for homeopathic globules by spectrophotometric-UV method

We compared the impregnation techniques for globules according to the Manual of Technical Norms for Homeopathic Pharmacies (MNTFH) of the Association of Homeopathic Pharmacists (ABFH), Brazilian Homeopathic Pharmacopoeia (FHB) and variations of these techniques. The variables were evaluated in this procedure, three different sizes of globules (N o . 3, 5 and 7), the hydroalcoholic solution of 70% (v/v) Minoxidil 2% (w/v) was used to impregnate the globules in concentrations of 2, 3, 4, 5% (v/w) and the impregnation at 10 %(v/w) was used hydroalcoholic solutions at 70, 80 and 90% (v/v), and four impregnation techniques various ( A -glass, B -paper, C -cup and D -FHB). As the results of content uniformity did not demonstrate a normal distribution, the one way ANOVA and a nonparametric statistical model were used for evaluation. Considering the average, the standard deviation (SD), the individual variance of each group and the principal components analysis graphs (PCA), it was observed that the “A” impregnation of globules technique, with 5% (v/w) of the impregnation concentrations and the No.5 globule presented the best uniformity of dose. As to the drying, there was a need to use a heat source.

Compararam-se as técnicas de impregnação para glóbulos segundo o Manual de Normas Técnicas para Farmácias Homeopáticas (MNTFH) da Associação Brasileira de Farmacêuticos Homeopatas (ABFH), Farmacopeia Homeopática Brasileira (FHB) e variações destas técnicas. As variáveis avaliadas neste processo foram: três tamanhos diferentes de glóbulos (n.º 3, 5 e 7); a solução hidroalcóolica a 70% (v/v) de minoxidil a 2% (p/v) foi utilizada para impregnar os glóbulos nas concentrações de 2, 3, 4, 5% (v/p) e na impregnação a 10% (v/p) utilizaram-se as soluções hidroalcóolicas a 70, 80 e 90% (v/v); e quatro técnicas de impregnação diferentes ( A -vidro, B -papel, C -copo e D -FHB). A impregnação foi validada através da uniformidade de dose por conteúdo, sendo o minoxidil a substância quantificada. Como os resultados da uniformidade de dose por conteúdo não demonstraram distribuição normal, utilizaram-se o One way ANOVA e um modelo estatístico não paramétrico para sua avaliação. Considerando-se a média, o desvio padrão (DP), a variância individual de cada grupo e os gráficos de análise de componentes principais (ACP), observou-se que a impregnação que utilizou o glóbulo nº5, a concentração para impregnação de 5% (v/p), graduação alcoólica de 70% (v/v) e técnica “A” apresentou a melhor uniformidade de dose. Quanto à secagem, verificou-se a necessidade do uso de uma fonte de calor.

Production of Bacillus sphaericus entomopathogenic biomass using brewery residues.

The use of brewery residues--yeast and trub--has been evaluated aiming to minimize the costs of the industrial production of Bacillus sphaericus-based bioinsecticide. Both brewery residues promoted growth and sporulation of the three B. sphaericus strains that were isolated from Brazilian soils (S1, S2, and S20). However, distinct growth and sporulation behaviors were observed in relation to the different nutritional conditions and strain used. The maximum sporulation percentage was obtained through the cultivation of S20 strain in brewery residual yeast. In general, the entomopathogenic biomasses produced showed good results for toxicity to Culex larvae. The minimum values of larvae population (LC50) were observed for the S20 strain grown on yeast brewery residue-containing media. After fermentation, a considerable decrease in the organic material of alternative media was verified, although the residual values were still higher than that considered appropriate for effluent discharge.

Production of Bacillus sphaericus entomopathogenic biomass using brewery residues.

The use of brewery residues--yeast and trub--has been evaluated aiming to minimize the costs of the industrial production of Bacillus sphaericus-based bioinsecticide. Both brewery residues promoted growth and sporulation of the three B. sphaericus strains that were isolated from Brazilian soils (S1, S2, and S20). However, distinct growth and sporulation behaviors were observed in relation to the different nutritional conditions and strain used. The maximum sporulation percentage was obtained through the cultivation of S20 strain in brewery residual yeast. In general, the entomopathogenic biomasses produced showed good results for toxicity to Culex larvae. The minimum values of larvae population (LC50) were observed for the S20 strain grown on yeast brewery residue-containing media. After fermentation, a considerable decrease in the organic material of alternative media was verified, although the residual values were still higher than that considered appropriate for effluent discharge.