Multivariate optimization results in an edible extract from Ilex paraguariensis unexplored residues with a high amount of phenolic compounds.

Ilex paraguariensis A. St. Hil. plants are used for the preparation of food and drinks which are widely consumed worldwide. During the harvest season of these plants, 2-5 ton hec-1 of agricultural residue is generated, which remains underutilized. Therefore, this study aimed to obtain an edible extract with high content of bioactive compounds and antimicrobial properties from the agricultural residue of I. paraguariensis for industrial use in food applications. The extraction conditions were optimized through a multivariate experimental design using ethanol:water. The extracted compounds were characterized by HPLC-ESY-QTOF-MS. In the optimal extraction conditions, 55 compounds were extracted, including 8 compounds that were not previously reported in I. paraguariensis. The method proved to be simple, fast, economical and environmentally friendly, with the use of green solvents. This optimization allowed for the extraction of 15.07 g of phenolic compounds per 100 g of residue. The extract showed high antioxidant activity and the capacity to inhibit Staphylococcus aureus. Results indicate that it is possible to obtain an edible extract with a high content of bioactive compounds, particularly phenolic compounds, from the I. paraguariensis residue, which has high prospects for the valorization of unexplored natural resources.

Inflammation markers in the saliva of infants born from Zika-infected mothers: exploring potential mechanisms of microcephaly during fetal development.

Zika virus (ZIKV) has emerged as one of the most medically relevant viral infections of the past decades; the devastating effects of this virus over the developing brain are a major matter of concern during pregnancy. Although the connection with congenital malformations are well documented, the mechanisms by which ZIKV reach the central nervous system (CNS) and the causes of impaired cortical growth in affected fetuses need to be better addressed. We performed a non-invasive, metabolomics-based screening of saliva from infants with congenital Zika syndrome (CZS), born from mothers that were infected with ZIKV during pregnancy. We were able to identify three biomarkers that suggest that this population suffered from an important inflammatory process; with the detection of mediators associated with glial activation, we propose that microcephaly is a product of immune response to the virus, as well as excitotoxicity mechanisms, which remain ongoing even after birth.

The presence of ochratoxin A does not influence Saccharomyces cerevisiae growth kinetics but leads to the formation of modified ochratoxins.

Yeasts are able to reduce the levels of ochratoxin A in fermentative processes; and, through their enzymatic complex, these micro-organisms are also capable of forming modified mycotoxins. These mycotoxins are often underreported, and may increase health risks after ingestion of contaminated food. In this sense, this study aims to evaluate whether the presence of ochratoxin A influences yeast growth kinetic parameters and to elucidate the formation of modified ochratoxin by Saccharomyces cerevisiae strains during fermentation. Three S. cerevisiae strains (12 M, 01 PP, 41 PP) were exposed to OTA at the concentrations of 10, 20 and 30 µg/L. The Baranyi model was fitted to the growth data (Log CFU/mL), and the identification of modified ochratoxins was performed through High Resolution Mass Spectrometry. The presence of ochratoxin A did not influence the growth of S. cerevisiae strains. Four pathways were proposed for the metabolization of OTA: dechlorination, hydrolysis, hydroxylation, and conjugation. Among the elected targets, the following were identified: ochratoxin α, ochratoxin ß, ochratoxin α methyl ester, ochratoxin B methyl ester, ethylamide ochratoxin A, ochratoxin C, hydroxy-ochratoxin A, hydroxy-ochratoxin A methyl ester, and ochratoxin A cellobiose ester. These derivatives formed from yeast metabolism may contribute to the occurrence of underreporting levels of total mycotoxin in fermented products.

Author Correction: A quantitative study on growth variability and production of ochratoxin A and its derivatives by A. carbonarius and A. niger in grape-based medium.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

A quantitative study on growth variability and production of ochratoxin A and its derivatives by A. carbonarius and A. niger in grape-based medium.

Aspergillus carbonarius and Aspergillus niger are the main responsible fungi for the accumulation of ochratoxin A (OTA) in wine grapes. Some strains are able to convert the parent mycotoxin into other compounds by means of hydrolysis and/or conjugation reactions through their defense mechanisms and enzymatic activity, leading to the formation of a modified mycotoxin. Thus, the variability of growth and metabolite production are inherent to the strain, occurring distinctively even when submitted to similar conditions. In this sense, this contribution aimed at determining the variability in multiplication and production of OTA by strains of A. carbonarius and A. niger isolated from grapes, as well as investigating the formation of modified mycotoxins. Strains were incubated in grape-based medium, and the diameter of the colonies measured daily. The determination of OTA was performed by high-performance liquid chromatography and the identification of modified mycotoxins was carried out using high-resolution mass spectrometry. Variabilities in terms of growth and OTA production were assessed across five different strains. Peak production of OTA was detected on day 15, and a decline on day 21 was observed, indicating that the observed reduction may be associated with the degradation or modification of the OTA over time by the fungus. Ethylamide ochratoxin A, a modified mycotoxin identified in this study, provides evidence that there may be underreporting of total mycotoxin levels in food, increasing uncertainty concerning health risks to the population.

Coenzyme Q10 or Creatine Counteract Pravastatin-Induced Liver Redox Changes in Hypercholesterolemic Mice.

Statins are the preferred therapy to treat hypercholesterolemia. Their main action consists of inhibiting the cholesterol biosynthesis pathway. Previous studies report mitochondrial oxidative stress and membrane permeability transition (MPT) of several experimental models submitted to diverse statins treatments. The aim of the present study was to investigate whether chronic treatment with the hydrophilic pravastatin induces hepatotoxicity in LDL receptor knockout mice (LDLr-/-), a model for human familial hypercholesterolemia. We evaluated respiration and reactive oxygen production rates, cyclosporine-A sensitive mitochondrial calcium release, antioxidant enzyme activities in liver mitochondria or homogenates obtained from LDLr-/- mice treated with pravastatin for 3 months. We observed that pravastatin induced higher H2O2 production rate (40%), decreased activity of aconitase (28%), a superoxide-sensitive Krebs cycle enzyme, and increased susceptibility to Ca2+-induced MPT (32%) in liver mitochondria. Among several antioxidant enzymes, only glucose-6-phosphate dehydrogenase (G6PD) activity was increased (44%) in the liver of treated mice. Reduced glutathione content and reduced to oxidized glutathione ratio were increased in livers of pravastatin treated mice (1.5- and 2-fold, respectively). The presence of oxidized lipid species were detected in pravastatin group but protein oxidation markers (carbonyl and SH- groups) were not altered. Diet supplementation with the antioxidants CoQ10 or creatine fully reversed all pravastatin effects (reduced H2O2 generation, susceptibility to MPT and normalized aconitase and G6PD activity). Taken together, these results suggest that 1- pravastatin induces liver mitochondrial redox imbalance that may explain the hepatic side effects reported in a small number of patients, and 2- the co-treatment with safe antioxidants neutralize these side effects.

A fast semi-quantitative screening for cocoa content in chocolates using MALDI-MSI.

Chocolate is a popular food bearing a number of different classifications that are differentiated by proportions of cocoa solids, milk and cocoa butter. Literature brings evidence that chocolates with a high percentage of cocoa solids contribute to good health maintenance due to the presence of phenolic compounds. On the other hand, it is known that the productive process, including pre-processing, may influence the level of these substances in the finished product. Thus, accurate strategies to measure the levels of this class of molecules that can be highly adaptable throughout the manufacturing process are important to ensure high-quality products. Mass spectrometry is an analytical tool of high sensitivity and specificity that is leading the research in food analysis towards new directions. By using mass spectrometry imaging in direct food analysis, this contribution developed an effective methodology for comparatively establishing the levels of catechin/epicatechin as phenolics content markers for cocoa content in a series of commercial chocolates from a single manufacturer, rendering a versatile tool that can be applied in fast screening of cocoa content in finished products and during manufacturing.

Corrigendum: Serum Metabolic Alterations upon Zika Infection.

[This corrects the article on p. 1954 in vol. 8, PMID: 29067015.].

Serum Metabolic Alterations upon Zika Infection.

Zika virus (ZIKV) infection has recently emerged as a major concern worldwide due to its strong association with nervous system malformation (microcephaly) of fetuses in pregnant women infected by the virus. Signs and symptoms of ZIKV infection are often mistaken with other common viral infections. Since transmission may occur through biological fluids exchange and coitus, in addition to mosquito bite, this condition is an important infectious disease. Thus, understanding the mechanism of viral infection has become an important research focus, as well as providing potential targets for assertive clinical diagnosis and quality screening for hemoderivatives. Within this context, the present work analyzed blood plasma from 79 subjects, divided as a control group and a ZIKV-infected group. Samples underwent direct-infusion mass spectrometry and statistical analysis, where eight markers related to the pathophysiological process of ZIKV infection were elected and characterized. Among these, Angiotensin (1-7) and Angiotensin I were upregulated under infection, showing an attempt to induce autophagy of the infected cells. However, this finding is concerning about hypertensive individuals under treatment with inhibitors of the Renin-Angiotensin System (RAS), which could reduce this response against the virus and exacerbate the symptoms of the infection. Moreover, one of the most abundant glycosphingolipids in the nervous tissue, Ganglioside GM2, was also elected in the present study as an infection biomarker. Considered an important pathogen receptor at membrane's outer layer, this finding represents the importance of gangliosides for ZIKV infection and its association with brain tropism. Furthermore, a series of phosphatidylinositols were also identified as biomarkers, implying a significant role of the PI3K-AKT-mTOR Pathway in this mechanism. Finally, these pathways may also be understood as potential targets to be considered in pharmacological intervention studies on ZIKV infection management.

Pravastatin Chronic Treatment Sensitizes Hypercholesterolemic Mice Muscle to Mitochondrial Permeability Transition: Protection by Creatine or Coenzyme Q10.

Statins are efficient cholesterol-lowering medicines utilized worldwide. However, 10% of patients suffer from adverse effects specially related to skeletal muscle function. Pro- or anti-oxidant effects of statins have been reported. Here we hypothesized that statins induce muscle mitochondrial oxidative stress leading to mitochondrial permeability transition (MPT) which may explain statin muscle toxicity. Thus, our aims were to investigate the effects of statin chronic treatment on muscle mitochondrial respiration rates, MPT and redox state indicators in the context of hypercholesterolemia. For this purpose, we studied muscle biopsies of the hypercholesterolemic LDL receptor knockout mice (LDLr-/-) treated with pravastatin during 3 months. Plantaris, but not soleus muscle of treated mice showed significant inhibition of respiration rates induced by ADP (-14%), oligomycin (-20%) or FCCP (-40%). Inhibitions of respiratory rates were sensitive to EGTA (Ca2+ chelator), cyclosporin A (MPT inhibitor), ruthenium red (inhibitor of mitochondria Ca2+ uptake) and coenzyme Q10 (antioxidant), indicating that pravastatin treatment favors Ca2+ induced MPT. Diet supplementation with creatine (antioxidant) also protected treated mice against pravastatin sensitization to Ca2+ induced MPT. Among several antioxidant enzymes analyzed, only catalase activity was increased by 30% in plantaris muscle of pravastatin treated mice. Oxidized lipids, but not proteins biomarkers were identified in treated LDLr-/- plantaris muscle. Taken together, the present results suggest that chronic pravastatin administration to a model of familial hypercholesterolemia promotes mitochondrial dysfunctions in plantaris muscle that can be counteracted by antioxidants administered either in vitro (CoQ10) or in vivo (creatine). Therefore, we propose that inhibition of muscle mitochondrial respiration by pravastatin leads to an oxidative stress that, in the presence of calcium, opens the permeability transition pore. This mitochondrial oxidative stress caused by statin treatment also signals for cellular antioxidant system responses such as catalase upregulation. These results suggest that the detrimental effects of statins on muscle mitochondria could be prevented by co-administration of a safe antioxidant such as creatine or CoQ10.

Analysis and characterisation of bovine oocyte and embryo biomarkers by matrix-assisted desorption ionisation mass spectrometry imaging.

In the field of 'single cell analysis', many classical strategies like immunofluorescence and electron microscopy are the primary techniques of choice. However, these methodologies are time consuming and do not permit direct identification of specific molecular classes, such as lipids. In the present study, a novel mass spectrometry-based analytical approach was applied to bovine oocytes and embryos. This new metabolomics-based application uses mass spectrometry imaging (MSI), efficient data processing and multivariate data analysis. Metabolic fingerprinting (MF) was applied to the analysis of unfertilised oocytes, 2-, 4- and 8-cell embryos and blastocysts. A semiquantitative strategy for sphingomyelin [SM (16:0)+Na](+) (m/z 725) and phosphatidylcholine [PC (32:0)+Na](+) (m/z 756) was developed, showing that lipid concentration was useful for selecting the best metabolic biomarkers. This study demonstrates that a combination of MF, MSI features and chemometric analysis can be applied to discriminate cell stages, characterising specific biomarkers and relating them to developmental pathways. This information furthers our understanding of fertilisation and preimplantation events during bovine embryo development.

Thermal degradation of sucralose: a combination of analytical methods to determine stability and chlorinated byproducts.

In the late years, much attention has been brought to the scientific community regarding the safety of sucralose and its industrial applications. Although it is the most used artificial sweetener in foods and pharmaceuticals, many questions still arise on its potential to form chlorinated byproducts in high temperatures, as demonstrated by several recent studies. In the present contribution, we use a combination of differential scanning calorimetry and thermogravimetric analysis coupled with infrared spectroscopy (DSC/TGA/IR), Hot-stage microscopy (HSM) and high-resolution mass spectrometry (HRMS) on samples submitted to water bath at mild temperatures to evaluate a broad spectrum of hazardous compounds formed in the degradation of this product. TGA/IR has revealed that there is effective decomposition in form of CO2 along with the formation of hydrogen chloride and other minor compounds. HSM results have provided accurate information, where the melting of the crystals was observed, followed by decomposition. Chlorinated derivatives, including polychlorinated aromatic hydrocarbons (PCAHs) were also confirmed by HRMS. These findings not only corroborate the suspected instability of sucralose to high temperatures, but also indicate that even exposed to mild conditions the formation of hazardous polychlorinated compounds is observed.

Novel R-(+)-limonene-based thiosemicarbazones and their antitumor activity against human tumor cell lines.

In an attempt to develop potent and selective antitumor agents, a series of novel thiosemicarbazones derived from a natural monoterpene R-(+)-limonene was synthesized and their antitumor activity was evaluated. Overall, the majority of tested compounds exhibited considerable inhibitory effects on the growth of a wide range of cancer cell lines. Almost all of tested thiosemicarbazones were especially sensitive to prostate cells (PC-3). Derivatives 5, 6, 8, 9, 10, 11 and 13 presented the most potent antitumor activity against PC-3 cells. These compounds showed lower value of GI50 (0.04-0.05 µM) than the reference drug paclitaxel, besides a high selectivity for the same cell line. The 4-fluorobenzaldehyde derivative 10 was the most selective compound for prostate cells, while 2-hydroxybenzaldehyde derivative 8 was the most active compound, with potent antitumor activity against all tested cell lines.

Rapid and simultaneous in situ assessment of aflatoxins and stilbenes using silica plate imprinting mass spectrometry imaging.

A fast and direct combination of techniques for simultaneous mycotoxin and phytoalexin identification in peanut skin and kernel is described. Silica Plate Imprinting Laser Desorption/Ionization Mass Spectrometry Imaging (SPILDI-MSI) is a powerful technique that exhibits great advantages, such as solvent-free and matrix-free characteristics, as well as no sample preparation or separation steps. It also permits accurate identification of mycotoxins and phytoalexins with unique fingerprint profiles in just a few seconds. Results are expressed as chemical images of the 4 identified types of aflatoxins (B1, B2, G1 and G2) and a stilbenoid (resveratrol). Also, SPILDI-MSI allows the comparison between the spatial distribution of aflatoxins and resveratrol found in kernel and skin. This novel application has proven to be useful for instantaneous qualitative assessment of aflatoxins and stilbenoids both in the peanut skin and kernel and offers precise tracking of fungal contamination in nuts and other foodstuffs.

Lipid characterization of embryo zones by silica plate laser desorption ionization mass spectrometry imaging (SP-LDI-MSI).

Lipid pathways play important biological roles in mammalian embryology, directing early developmental pathways to differentiation. Phospholipids and triglycerides, among others, are the main composing lipids of zona pellucida in several embryo species. Lipid analysis in embryos by mass spectrometry usually requires sample preparation and/or matrix application. This novel approach using silica plate laser desorption/ionization mass spectrometry imaging (SP-LDI-MSI) allows direct single-cell imaging and embryo region discrimination with no matrix coating. Its application is herein described for two- and eight-cell embryos. Lipid biomarkers for blastomere and intact zona pellucida are reported and corroborated by both fragmentation reactions (MS/MS) and images. Results obtained in this work are understood to be of great use for further developments on in vitro bovine fertilization. Since much of the processes can be monitored by characteristic biomarkers, it is now possible to precisely identify cell division errors during early embryo stages, as well as evaluate pre-implantation conditions.

Irradiated riboflavin diminishes the aggressiveness of melanoma in vitro and in vivo.

Melanoma is one of the most aggressive skin cancers due to its high capacity to metastasize. Treatment of metastatic melanomas is challenging for clinicians, as most therapeutic agents have failed to demonstrate improved survival. Thus, new candidates with antimetastatic activity are much needed. Riboavin (RF) is a component of the vitamin B complex and a potent photosensitizer. Previously, our group showed that the RF photoproducts (iRF) have potential as an antitumoral agent. Hence, we investigated the capacity of iRF on modulating melanoma B16F10 cells aggressiveness in vitro and in vivo. iRF decreases B16F10 cells survival by inhibiting mTOR as well as Src kinase. Moreover, melanoma cell migration was disrupted after treatment with iRF, mainly by inhibition of metalloproteinase (MMP) activity and expression, and by increasing TIMP expression. Interestingly, we observed that the Hedgehog (HH) pathway was inhibited by iRF. Two mediators of HH signaling, GLI1 and PTCH, were downregulated, while SUFU expression (an inhibitor of this cascade) was enhanced. Furthermore, inhibition of HH pathway signaling by cyclopamine and Gant 61 potentiated the antiproliferative action of RF. Accordingly, when a HH ligand was applied, the effect of iRF was almost completely abrogated. Our findings indicate that Hedgehog pathway is involved on the modulation of melanoma cell aggressiveness by iRF. Moreover, iRF treatment decreased pulmonary tumor formation in a murine experimental metastasis model. Research to clarify the molecular action of flavins, in vivo, is currently in progress. Taken together, the present data provides evidence that riboflavin photoproducts may provide potential candidates for improving the efficiency of melanoma treatment.