Early metabolic response after resistance exercise with blood flow restriction in well-trained men: a metabolomics approach.

The present study aimed to compare the early metabolic response between high-load resistance exercise (HL-RE) and low-load resistance exercise with blood flow restriction (LL-BFR). Nine young, well-trained men participated in a randomized crossover design in which each subject completed LL-BFR, HL-RE, or condition control (no exercise) with a 1-week interval between them. Blood samples were taken immediately before and 5 min after the exercise sessions. Nuclear magnetic resonance spectroscopy identified and quantified 48 metabolites, 6 of which presented significant changes among the exercise protocols. The HL-RE promoted a higher increase in pyruvate, lactate, and alanine compared with the LL-BFR and the control. HL-RE and LL-BFR promoted a higher increase in succinate compared with the control; however, there was no difference between HL-RE and LL-BFR. Also, while there was no difference in acetoacetate between HL-RE and LL-BFR, a greater decrease was observed in both compared with the control. Finally, LL-BFR promoted a greater decrease in choline compared with the control. In conclusion, this study provides by metabolomics a new insight in metabolic response between LL-BFR and HL-RE by demonstrating a distinct response to some metabolites that are not commonly analyzed.

Metabolic time-course response after resistance exercise: A metabolomics approach.

This study analysed the time course of the global metabolic acute response after resistance exercise (RE), with the use of proton nuclear magnetic resonance (1H NMR) spectroscopy. Ten young healthy males performed 4 sets of 10 repetitions at 70% of one-repetition maximum in the leg press and knee extension exercises and had the serum metabolome assessed at 5, 15, 30 and 60 min post-RE. Measurements were also performed 1 h earlier and immediately before the exercises, as an attempt to characterise each participant's serum metabolome at rest. One-way ANOVA was applied and the significance level was set at P ≤ 0.05. RE promoted an increase in 2-hydroxybutyrate, 2-oxoisocaproate, 3-hydroxyisobutyrate, alanine, hypoxanthine, lactate, pyruvate and succinate concentrations. However, isoleucine, leucine, lysine, ornithine and valine had their concentrations decreased post-RE compared with at rest. This is the first study to show significant changes in serum concentration of metabolites such as 2-oxoisocaproate, 2-hydroxybutyrate, 3-hydroxyisobutyrate, lysine, hypoxanthine and pyruvate post-RE, attesting metabolomics as an interesting approach to advance in the understanding of global RE-induced metabolic changes. Moreover, the present data could influence the time point of blood collection in the future studies that aims to investigate metabolism and exercise.

Metabolic Profiling and Classification of Propolis Samples from Southern Brazil: An NMR-Based Platform Coupled with Machine Learning.

The chemical composition of propolis is affected by environmental factors and harvest season, making it difficult to standardize its extracts for medicinal usage. By detecting a typical chemical profile associated with propolis from a specific production region or season, certain types of propolis may be used to obtain a specific pharmacological activity. In this study, propolis from three agroecological regions (plain, plateau, and highlands) from southern Brazil, collected over the four seasons of 2010, were investigated through a novel NMR-based metabolomics data analysis workflow. Chemometrics and machine learning algorithms (PLS-DA and RF), including methods to estimate variable importance in classification, were used in this study. The machine learning and feature selection methods permitted construction of models for propolis sample classification with high accuracy (>75%, reaching ∼90% in the best case), better discriminating samples regarding their collection seasons comparatively to the harvest regions. PLS-DA and RF allowed the identification of biomarkers for sample discrimination, expanding the set of discriminating features and adding relevant information for the identification of the class-determining metabolites. The NMR-based metabolomics analytical platform, coupled to bioinformatic tools, allowed characterization and classification of Brazilian propolis samples regarding the metabolite signature of important compounds, i.e., chemical fingerprint, harvest seasons, and production regions.

Catheter-based induction of renal ischemia/reperfusion in swine: description of an experimental model.

Several techniques to induce renal ischemia have been proposed: clamp, PVA particles, and catheter-balloon. We report the development of a controlled, single-insult model of unilateral renal ischemia/reperfusion (I/R) without contralateral nephrectomy, using a suitable model, the pig. This is a balloon-catheter-based model using a percutaneous, interventional radiology procedure. One angioplasty balloon-catheter was placed into the right renal artery and inflated for 120 min and reperfusion over 24 h. Serial serums were sampled from the inferior vena cava and urine was directly sampled from the bladder throughout the experiment, and both kidneys were excised after 24 h of reperfusion. Analyses of renal structure and function were performed by hematoxylin-eosin/periodic Acid-Schiff, serum creatinine (SCr), blood urea nitrogen (BUN), fractional excretion of ions, and glucose, SDS-PAGE analysis of urinary proteins, and serum neutrophil gelatinase-associated lipocalin (NGAL). Total nitrated protein was quantified to characterize oxidative stress. Acute tubular necrosis (ATN) was identified in every animal, but only two animals showed levels of SCr above 150% of baseline values. As expected, I/R increased SCr and BUN. Fractional sodium, potassium, chloride, and bicarbonate excretion were modulated during ischemia. Serum-nitrated proteins and NGAL had two profiles: decreased with ischemia and increased after reperfusion. This decline was associated with increased protein excretion during ischemia and early reperfusion. Altogether, these data show that the renal I/R model can be performed by percutaneous approach in the swine model. This is a suitable translational model to study new early renal ischemic biomarkers and pathophysiological mechanisms in renal ischemia.

Xanthan Gum Removal for 1H-NMR Analysis of the Intracellular Metabolome of the Bacteria Xanthomonas axonopodis pv. citri 306.

Xanthomonas is a genus of phytopathogenic bacteria, which produces a slimy, polysaccharide matrix known as xanthan gum, which involves, protects and helps the bacteria during host colonization. Although broadly used as a stabilizer and thickener in the cosmetic and food industries, xanthan gum can be a troubling artifact in molecular investigations due to its rheological properties. In particular, a cross-reaction between reference compounds and the xanthan gum could compromise metabolic quantification by NMR spectroscopy. Aiming at an efficient gum extraction protocol, for a 1H-NMR-based metabolic profiling study of Xanthomonas, we tested four different interventions on the broadly used methanol-chloroform extraction protocol for the intracellular metabolic contents observation. Lower limits for bacterial pellet volumes for extraction were also probed, and a strategy is illustrated with an initial analysis of X. citri's metabolism by 1H-NMR spectroscopy.

Functional diversification of cerato-platanins in Moniliophthora perniciosa as seen by differential expression and protein function specialization.

Cerato-platanins (CP) are small, cysteine-rich fungal-secreted proteins involved in the various stages of the host-fungus interaction process, acting as phytotoxins, elicitors, and allergens. We identified 12 CP genes (MpCP1 to MpCP12) in the genome of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao, and showed that they present distinct expression profiles throughout fungal development and infection. We determined the X-ray crystal structures of MpCP1, MpCP2, MpCP3, and MpCP5, representative of different branches of a phylogenetic tree and expressed at different stages of the disease. Structure-based biochemistry, in combination with nuclear magnetic resonance and mass spectrometry, allowed us to define specialized capabilities regarding self-assembling and the direct binding to chitin and N-acetylglucosamine (NAG) tetramers, a fungal cell wall building block, and to map a previously unknown binding region in MpCP5. Moreover, fibers of MpCP2 were shown to act as expansin and facilitate basidiospore germination whereas soluble MpCP5 blocked NAG6-induced defense response. The correlation between these roles, the fungus life cycle, and its tug-of-war interaction with cacao plants is discussed.

Structural characterization of the H-NS protein from Xylella fastidiosa and its interaction with DNA.

The nucleoid-associated protein H-NS is a major component of the bacterial nucleoid involved in DNA compaction and transcription regulation. The NMR solution structure of the Xylella fastidiosa H-NS C-terminal domain (residues 56-134) is presented here and consists of two beta-strands and two alpha helices, with one loop connecting the two beta-strands and a second loop connecting the second beta strand and the first helix. The amide (1)H and (15)N chemical shift signals for a sample of XfH-NS(56-134) were monitored in the course of a titration series with a 14-bp DNA duplex. Most of the residues involved in contacts to DNA are located around the first and second loops and in the first helix at a positively charged side of the protein surface. The overall structure of the Xylella H-NS C-terminal domain differ significantly from Escherichia coli and Salmonella enterica H-NS proteins, even though the DNA binding motif in loop 2 adopt similar conformation, as well as ß-strand 2 and loop 1. Interestingly, we have also found that the DNA binding site is expanded to include helix 1, which is not seen in the other structures.

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168.

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.