Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

Melanoma is the deadliest form of skin cancer, and its incidence has alarmingly increased in the last few decades, creating a need for novel treatment approaches. Thus, we evaluated the combinatorial effect of doxorubicin (DOX) and hyperthermia on A375 and MNT-1 human melanoma cell lines. Cells were treated with DOX for 24, 48, and 72 h and their viabilities were assessed. The effect of DOX IC10 and IC20 (combined at 43 °C for 30, 60, and 120 min) on cell viability was further analyzed. Interference on cell cycle dynamics, reactive oxygen species (ROS) production, and apoptosis upon treatment (with 30 min at 43 °C and DOX at the IC20 for 48 h) were analyzed by flow cytometry. Combined treatment significantly decreased cell viability, but not in all tested conditions, suggesting that the effect depends on the drug concentration and heat treatment duration. Combined treatment also mediated a G2/M phase arrest in both cell lines, as well as increasing ROS levels. Additionally, it induced early apoptosis in MNT-1 cells, while in A375 cells this effect was similar to the one caused by hyperthermia alone. These findings demonstrate that hyperthermia enhances DOX effect through cell cycle arrest, oxidative stress, and apoptotic cell death.     

Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear

Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s⁻¹). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A₂ (TxA₂), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.     

Teriflunomide Preserves Neuronal Activity and Protects Mitochondria in Brain Slices Exposed to Oxidative Stress

Teriflunomide (TFN) limits relapses in relapsing–remitting multiple sclerosis (RRMS) by reducing lymphocytic proliferation through the inhibition of the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) and the subsequent modulation of de novo pyrimidine synthesis. Alterations of mitochondrial function as a consequence of oxidative stress have been reported during neuroinflammation. Previously, we showed that TFN prevents alterations of mitochondrial motility caused by oxidative stress in peripheral axons. Here, we aimed to validate TFN effects on mitochondria and neuronal activity in hippocampal brain slices, in which cellular distribution and synaptic circuits are largely preserved. TFN effects on metabolism and neuronal activity were investigated by assessing oxygen partial pressure and local field potential in acute slices. Additionally, we imaged mitochondria in brain slices from the transgenic Thy1-CFP/COX8A)S2Lich/J (mitoCFP) mice using two-photon microscopy. Although TFN could not prevent oxidative stress-related depletion of ATP, it preserved oxygen consumption and neuronal activity in CNS tissue during oxidative stress. Furthermore, TFN prevented mitochondrial shortening and fragmentation of puncta-shaped and network mitochondria during oxidative stress. Regarding motility, TFN accentuated the decrease in mitochondrial displacement and increase in speed observed during oxidative stress. Importantly, these effects were not associated with neuronal viability and did not lead to axonal damage. In conclusion, during conditions of oxidative stress, TFN preserves the functionality of neurons and prevents morphological and motility alterations of mitochondria.     

Comparative performance of carbon nanotube and nanoclay on thermal properties and flammability behavior of amorphous polyamide/SEBS blend

Multiwall carbon nanotubes (CNT) or montmorillonite clay (MMT-30B) were added to a poly(hexamethylene isophthalamide-co-terephthalamine) (an amorphous polyamide - aPA) and styrene-ethylene/butylene-styrene graphitized with maleic anhydride (SEBS) blend, in different concentrations, in order to investigate the morphology, thermal properties and flammability behavior. Different nanoparticle localizations in the phase blend were observed through transmission electronic microscopy. CNT nanoparticles are localized in SEBS phase, and MMT-30B nanoparticles in aPA phase. No significant changes were observed on transition temperatures and thermal stability with both nanoparticle additions. However, a slight increase on storage modulus for clay nanocomposites and a slight reduction for carbon nanotube nanocomposites were observed, due to their different phase localizations. Regarding flammability, CNT nanocomposites showed better performance as a flame retardant when compared to samples with MMT-30B. Although the MMT-30B nanocomposites could not be classified according to the UL-94 criteria, no dripped flaming particles were observed, due to the a char barrier formation on the polymer surface. The CNT nanocomposites were classified according to the UL-94 criteria as V-2. The CNT's selective localization on the SEBS phase decreases its heat-release rate, but no interconnected network structure was formed in the matrix to suppress the dripping flaming particles.     

Validation of an LC-MS/MS method for malachite green (MG), leucomalachite green (LMG), crystal violet (CV) and leucocrystal violet (LCV) residues in fish and shrimp

A quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous analyses of malachite green (MG), crystal violet (CV) and its major metabolites, leucomalachite green (LMG) and leucocrystal violet (LCV) residues in fish and shrimp samples has been validated. Fish and shrimp samples were extracted with citrate buffer/acetonitrile, and the extracts were purified on strong cation-exchange (SCX) solid-phase extraction (SPE) cartridge. After conversion of LMG into MG using a post column oxidation reactor containing lead (IV) oxide (PbO(2)), the effluents were analysed. Residues were analysed using positive-ion electrospray ionisation (ESI). Identification and quantification of analytes were based on the ion transitions monitored by multiple reaction monitoring (MRM). Validation of the method was carried out in accordance with the Decision 2002/657/EC, which establishes criteria and procedures for the validation of methods. The following parameters were determined: decision limit (CCα), detection capability (CCβ), linearity, accuracy, precision, selectivity, specificity and matrix effect. The decision limits (CCα) for MG, LMG, CV and LCV were 0.164, 0.161, 0.248 and 0.860 μg kg(-1). The respective detection capabilities (CCβ) were 0.222, 0.218, 0.355 and 1.162 μg kg(-1). Typical recoveries (intermediate precision) in shrimp, for MG, CV, LMG and LCV for 2.0 μg kg(-1) level fortified samples using the optimised procedure were in the range 69%, 97%, 80.3% and 71.8%, respectively. The findings demonstrate the suitability of the method to detect simultaneously MG, CV and its metabolite (LMG and LCV) in fish and shrimp.     

Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, β4-galactosidase BgaD-B from Bacillus circulans, β4-N-acetylgalactosaminidase from Talaromyces flavus, and β3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods.     

Use of microbial transglutaminase and sodium alginate in the preparation of restructured fish models using cold gelation: Effect of frozen storage

Changes in physicochemical properties of raw-appearing restructured models made from hake (Merluccius capensis) muscle, using cold gelation technology by addition of sodium alginate and microbial transglutaminase (MTGase), were studied during frozen storage at ? 15 °C.Among the more interesting results, addition of MTGase produced more protein aggregation in models processed by muscle homogenization and thus the protein network that formed in the gel was better than in samples with added sodium alginate. All samples presented enhanced changes in mechanical properties such as gel strength in the course of frozen storage. The protein network that formed was better organized as shown by the ultrastructure analysis and by protein aggregation data from the beginning of storage, and as a result Water Binding Capacity was greater in restructured models processed by muscle homogenization. Some of the models studied presented lipid oxidation during frozen storage, slightly more in the ones with MTGase added.Industrial RelevanceThe industrial relevance of the present work is to establish bases for using fish fillet trimmings and minced muscle to prepare restructured products that can be commercialized in a raw state and that could also support frozen storage in order to prolong the self-life in the market. The models studied here are not final products but the base with which to prepare them. One possibility of them is to process this kind of products with technologies normally used in whole fish muscle such as smoked fish fillets, carpaccios or marinated products. In addition, these raw restructured products can be commercialized to be cooked as a normal fresh fish fillet or pieces of fillet in a variety of preparations, and also products that contain a high amount of salt. Samples elaborated by binding larger muscle fragments imitate well the original fibrousness of the muscle so that it could be used to elaborate products similar to hamburgers or others that will be fried in batter.     

Strain-specific polyketide synthase genes of Aspergillus niger

In silico comparison of 34 putative pks genes in Aspergillus niger strain CBS 513.88 versus A. niger strain ATCC 1015 genome revealed significant nucleotide identity (>95% covering a minimum of 99% of the gene sequence) for 31 of these genes (approximately 91%). A. niger CBS 513.88 harbors three putative pks genes (An01g01130, An11g05940, and An15g07920), for which nucleotide identity was not found in A. niger ATCC 1015. To compare the results of the in silico analysis with the in vivo situation, experimental data were obtained for a large number of A. niger strains obtained from different substrates and geographical regions. Three putative pks genes that were found to be variable between the two A. niger strains using bioinformatics tools were in fact strain-specific genes based on experimental data. The PCR amplification signals for the An01g01130, An11g05940, and An15g07920 pks genes were detected in only 97%, 71%, and 26% of the strains, respectively. Southern blot analyses confirmed the PCR data. Because one of the strain-specific pks genes (An15g07920) is located in a putative ochratoxin cluster, we focused our investigation on that region. We assessed the ochratoxin production capability of the 119 A. niger strains and found a positive association between the presence of this pks gene and the capability of the respective strain to produce ochratoxin.     

A Rapid and Eco-Friendly Method for Determination of Hydrolysable Tannins and Its Application to Honey Samples

This paper describes the development of a rapid and clean method for the determination of hydrolysable tannins in honey samples, focusing on an easy and green sample pretreatment and low generation of waste. The proposed method is based on the reaction between KIO₃ and hydrolysable tannin compounds in acetate buffer medium (pH 4.75), with formation of a colored product measured at 525 nm. The analytical conditions were optimized using experimental design (central composite design). The linear range obtained was 40.0–740 mg L^?1 (r?=?0.998), and the detection (LOD) and quantification (LOQ) limits were 12.2 and 40.8 mg L^?1, respectively. Acetate buffer was used to ensure formation of the colored product obtained by reaction between the chromogenic reagent and the analyte. The method showed good results when applied to honey samples, with recoveries in the range of 78.3–96.1 %, and the use of diffuse reflectance spectroscopy coupled with spot test analysis provided low reagent consumption and minimized waste generation. The results obtained with the proposed method were confirmed by a spectrophotometric method.     

Wild Prunus Fruit Species as a Rich Source of Bioactive Compounds

Sugars, organic acids, carotenoids, tocopherols, chlorophylls, and phenolic compounds were quantified in fruit of 4 wild growing Prunus species (wild cherry, bird cherry, blackthorn, and mahaleb cherry) using HPLC‐DAD‐MSn. In wild Prunus, the major sugars were glucose and fructose, whereas malic and citric acids dominated among organic acids. The most abundant classes of phenolic compounds in the analyzed fruit species were anthocyanins, flavonols, derivatives of cinnamic acids, and flavanols. Two major groups of anthocyanins measured in Prunus fruits were cyanidin‐3‐rutinoside and cyanidin‐3‐glucoside. Flavonols were represented by 19 derivatives of quercetin, 10 derivatives of kaempferol, and 2 derivatives of isorhamnetin. The highest total flavonol content was measured in mahaleb cherry and bird cherry, followed by blackthorn and wild cherry fruit. Total phenolic content varied from 2373 (wild cherry) to 11053 mg GAE per kg (bird cherry) and ferric reducing antioxidant power antioxidant activity from 7.26 to 31.54 mM trolox equivalents per kg fruits.