Ten-eleven translocation proteins (TET1-3) are dioxygenases that oxidize 5-methyldeoxycytosine, thus taking part in passive and active demethylation. TETs have shown to be involved in immune cell ...development, affecting from self-renewal of stem cells and lineage commitment to terminal differentiation. In fact, dysfunction of TET proteins have been vastly associated with both myeloid and lymphoid leukemias. Recently, there has been accumulating evidence suggesting that TETs regulate immune cell function during innate and adaptive immune responses, thereby modulating inflammation. In this work, we pursue to review the current and recent evidence on the mechanistic aspects by which TETs regulate immune cell maturation and function. We will also discuss the complex interplay of TET expression and activity by several factors to modulate a multitude of inflammatory processes. Thus, modulating TET enzymes could be a novel pharmacological approach to target inflammation-related diseases and myeloid and lymphoid leukemias, when their activity is dysregulated.
Mutations in the enzyme isocitrate dehydrogenase 1 (IDH1) lead to metabolic alterations and a sustained formation of 2-hydroxyglutarate (2-HG). 2-HG is an oncometabolite as it inhibits the activity ...of α-ketoglutarate-dependent dioxygenases such as ten-eleven translocation (TET) enzymes. Inhibitors of mutant IDH enzymes, like ML309, are currently tested in order to lower the levels of 2-HG. Vitamin C (VC) is an inducer of TET enzymes. To test a new therapeutic avenue of synergistic effects, the anti-neoplastic activity of inhibition of mutant IDH1 via ML309 in the presence of VC was investigated in the colon cancer cell line HCT116 IDH1
R132H/+
(harbouring a mutated IDH1 allele) and the parental cells HCT116 IDH1
+/+
(wild type IDH1). Measurement of the oncometabolite indicated a 56-fold higher content of 2-HG in mutated cells compared to wild type cells. A significant reduction of 2-HG was observed in mutated cells after treatment with ML 309, whereas VC produced only minimally changes of the oncometabolite. However, combinatorial treatment with both, ML309 and VC, in mutated cells induced pronounced reduction of 2-HG leading to levels comparable to those in wild type cells. The decreased level of 2-HG in mutated cells after combinatorial treatment was accompanied by an enhanced global DNA hydroxymethylation and an increased gene expression of certain tumour suppressors. Moreover, mutated cells showed an increased percentage of apoptotic cells after treatment with non-cytotoxic concentrations of ML309 and VC. These results suggest that combinatorial therapy is of interest for further investigation to rescue TET activity and treatment of IDH1/2 mutated cancers.
The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but ...sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.
The protein fractions of cocoa have been implicated influencing both the bioactive potential and sensory properties of cocoa and cocoa products. The objective of the present review is to show the ...impact of different stages of cultivation and processing with regard to the changes induced in the protein fractions. Special focus has been laid on the major seed storage proteins throughout the different stages of processing. The study starts with classical introduction of the extraction and the characterization methods used, while addressing classification approaches of cocoa proteins evolved during the timeline. The changes in protein composition during ripening and maturation of cocoa seeds, together with the possible modifications during the post-harvest processing (fermentation, drying, and roasting), have been documented. Finally, the bioactive potential arising directly or indirectly from cocoa proteins has been elucidated. The "state of the art" suggests that exploration of other potentially bioactive components in cocoa needs to be undertaken, while considering the complexity of reaction products occurring during the roasting phase of the post-harvest processing. Finally, the utilization of partially processed cocoa beans (e.g., fermented, conciliatory thermal treatment) can be recommended, providing a large reservoir of bioactive potentials arising from the protein components that could be instrumented in functionalizing foods.
Wheat is one of the most consumed foods in the world and unfortunately causes allergic reactions which have important health effects. The α-amylase/trypsin inhibitors (ATIs) have been identified as ...potentially allergen components of wheat. Due to a lack of data on optimization of ATI extraction, a new wheat ATIs extraction approach combining solvent extraction and selective precipitation is proposed in this work. Two types of wheat cultivars (
L.),
and
were used and parameters such as solvent type, extraction time, temperature, stirring speed, salt type, salt concentration, buffer pH and centrifugation speed were analyzed using the Plackett-Burman design. Salt concentration, extraction time and pH appeared to have significant effects on the recovery of ATIs (
< 0.01). In both wheat cultivars,
and
, ammonium sulfate substantially reduced protein concentration and inhibition of amylase activity (IAA) compared to sodium chloride. The optimal conditions with desirability levels of 0.94 and 0.91 according to the Doehlert design were: salt concentrations of 1.67 and 1.22 M, extraction times of 53 and 118 min, and pHs of 7.1 and 7.9 for
and
, respectively. The corresponding responses were: protein concentrations of 0.31 and 0.35 mg and IAAs of 91.6 and 83.3%. Electrophoresis and MALDI-TOF/MS analysis showed that the extracted ATIs masses were between 10 and 20 kDa. Based on the initial LC-MS/MS analysis, up to 10 individual ATIs were identified in the extracted proteins under the optimal conditions. The positive implication of the present study lies in the quick assessment of their content in different varieties especially while considering their allergenic potential.
•Stems of Abrus precatorius were used for the first time to extract a beta-amylase.•Amylase was purified by three phase partitioning and optimized by Doehlert design.•HPLC of hydrolysis products ...allows to identify and characterize a beta-amylase.•Unidentified high molecular weight protease was detected.
The stems of Abrus precatorius were used to extract a beta-amylase enriched fraction. A three phase partitioning method and a Doehlert design with 3 variables (ratio of crude extract/t-butanol, the ammonium sulphate saturation and pH) were used. The data was fitted in a second-order polynomial model and the parameters were optimized to enrich beta-amylase. Experimental responses for the modulation were recovery of activity and the purification factor. The optimal conditions were: a ratio of crude extract/t-butanol of 0.87 (v/v), saturation in ammonium sulphate of 49.46% (w/v) and a pH of 5.2. An activity recovery of 156.2% and a purification factor of 10.17 were found. The enriched enzyme was identified as a beta-amylase and its molecular weight was 60.1kDa. Km and Vmax values were 79.37mg/ml and 5.13U/ml, respectively and the highest activity was registered at a temperature of 70°C and a pH between 6 and 6.5. A significant stabilization of the beta-amylase was observed up to 65°C.
Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous system (CNS) which is associated with lower life expectancy and disability. The experimental ...antigen-induced encephalomyelitis (EAE) in mice is a useful animal model of MS, which allows exploring the etiopathogenetic mechanisms and testing novel potential therapeutic drugs. A new therapeutic paradigm for the treatment of MS was introduced in 2010 through the sphingosine 1-phosphate (S1P) analogue fingolimod (FTY720, Gilenya®), which acts as a functional S1P1 antagonist on T lymphocytes to deplete these cells from the blood. In this study, we synthesized two novel structures, ST-1893 and ST-1894, which are derived from fingolimod and chemically feature a morpholine ring in the polar head group. These compounds showed a selective S1P1 activation profile and a sustained S1P1 internalization in cultures of S1P1-overexpressing Chinese hamster ovary (CHO)-K1 cells, consistent with a functional antagonism. In vivo, both compounds induced a profound lymphopenia in mice. Finally, these substances showed efficacy in the EAE model, where they reduced clinical symptoms of the disease, and, on the molecular level, they reduced the T-cell infiltration and several inflammatory mediators in the brain and spinal cord. In summary, these data suggest that S1P1-selective compounds may have an advantage over fingolimod and siponimod, not only in MS but also in other autoimmune diseases.
Sphingosine 1-phosphate (S1P) is an extensively studied signaling molecule that contributes to cell proliferation, survival, migration and other functions through binding to specific S1P receptors. ...The cycle of S1P1 internalization upon S1P binding and recycling to the cell surface when local S1P concentrations are low drives T cell trafficking. S1P1 modulators, such as fingolimod, disrupt this recycling by inducing persistent S1P1 internalization and receptor degradation, which results in blocked egress of T cells from the secondary lymphoid tissues. The approval of these compounds for the treatment of multiple sclerosis has placed the development of S1PR modulators in the focus of pharmacological research, mostly for autoimmune indications. Here, we report on a novel anellated bismorpholino derivative of oxy-fingolimod, named ST-2191, which exerts selective S1P1 agonist and functional antagonist potency. ST-2191 is also effective in reducing the lymphocyte number in mice, and this effect is not dependent on phosphorylation by sphingosine kinase 2 for activity. These data show that ST-2191 is a novel S1P1 modulator, but further experiments are needed to analyze the therapeutic impact of ST-2191 in animal models of autoimmune diseases.
Many biochemical processes are involved in regulating the consecutive transition of different phases of dormancy in sweet cherry buds. An evaluation based on a metabolic approach has, as yet, only ...been partly addressed. The aim of this work, therefore, was to determine which plant metabolites could serve as biomarkers for the different transitions in sweet cherry buds. The focus here was on those metabolites involved in oxidation-reduction processes during bud dormancy, as determined by targeted and untargeted mass spectrometry-based methods. The metabolites addressed included phenolic compounds, ascorbate/dehydroascorbate, reducing sugars, carotenoids and chlorophylls. The results demonstrate that the content of phenolic compounds decrease until the end of endodormancy. After a long period of constancy until the end of ecodormancy, a final phase of further decrease followed up to the phenophase open cluster. The main phenolic compounds were caffeoylquinic acids, coumaroylquinic acids and catechins, as well as quercetin and kaempferol derivatives. The data also support the protective role of ascorbate and glutathione in the para- and endodormancy phases. Consistent trends in the content of reducing sugars can be elucidated for the different phenophases of dormancy, too. The untargeted approach with principle component analysis (PCA) clearly differentiates the different timings of dormancy giving further valuable information.
Large quantities of biological waste are generated at various steps within the food production chain and a great utilization potential for this solid biological waste exists apart from the current ...main usage for the feedstuff sector. It remains unclear how the usage of biological waste as compost modulates plant metabolites. We investigated the effect of biological waste of the processing of coffee, aronia, and hop added to soil on the plant metabolite profile by means of liquid chromatography in pak choi sprouts. Here we demonstrate that the solid biological waste composts induced specific changes in the metabolite profiles and the changes are depending on the type of the organic residues and its concentration in soil. The targeted analysis of selected plant metabolites, associated with health beneficial properties of the Brassicaceae family, revealed increased concentrations of carotenoids (up to 3.2-fold) and decreased amounts of glucosinolates (up to 4.7-fold) as well as phenolic compounds (up to 1.5-fold).
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