Profiling of adrenal corticosteroids in blood and local tissues of mice during chronic stress.

Stress increases plasma concentrations of corticosteroids, however, their tissue levels are unclear. Using a repeated social defeat paradigm, we examined the impact of chronic stress on tissue levels of corticosterone (CORT), progesterone (PROG), 11-deoxycorticosterone (11DOC) and 11-dehydrocorticosterone (11DHC) and on gut microbiota, which may reshape the stress response. Male BALB/c mice, liquid chromatography-tandem mass spectrometry and 16S RNA gene sequencing were used to screen steroid levels and fecal microbiome, respectively. Stress induced greater increase of CORT in the brain, liver, and kidney than in the colon and lymphoid organs, whereas 11DHC was the highest in the colon, liver and kidney and much lower in the brain and lymphoid organs. The CORT/11DHC ratio in plasma was similar to the brain but much lower in other organs. Stress also altered tissue levels of PROG and 11DOC and the PROG/11DOC ratio was much higher in lymphoid organs that in plasma and other organs. Stress impacted the ß- but not the α-diversity of the gut microbiota and LEfSe analysis revealed several biomarkers associated with stress treatment. Our data indicate that social defeat stress modulates gut microbiota diversity and induces tissue-dependent changes in local levels of corticosteroids, which often do not reflect their systemic levels.

The Adapted POM Analysis of Avenanthramides In Silico.

POM analysis and related approaches are significant tools based on calculating various physico-chemical properties and predicting biological activity, ADME parameters, and toxicity of a molecule. These methods are used to evaluate a molecule's potential to become a drug candidate. Avenanthramides (AVNs) are promising secondary metabolites specific to Avena spp. (oat). They comprise the amides of anthranilic acid linked to various polyphenolic acids with or without post-condensation molecule transformation. These natural compounds have been reported to exert numerous biological effects, including antioxidant, anti-inflammatory, hepatoprotective, antiatherogenic, and antiproliferative properties. To date, almost 50 various AVNs have been identified. We performed a modified POM analysis of 42 AVNs using MOLINSPIRATION, SWISSADME, and OSIRIS software. The evaluation of primary in silico parameters revealed significant differences among individual AVNs, highlighting the most promising candidates. These preliminary results may help coordinate and initiate other research projects focused on particular AVNs, especially those with predicted bioactivity, low toxicity, optimal ADME parameters, and promising perspectives.

Nutritional Evaluation of Quinoa Genetic Resources Growing in the Climatic Conditions of Central Europe.

Quinoa displays huge genetic variability and adaptability to distinct climatic conditions. Quinoa seeds are a good source of nutrients; however, the overall nutritional composition and nutrient content is influenced by numerous factors. This study focused on the nutritional and morphologic evaluation of various quinoa genotypes grown in the Czech Republic. Significant differences between years were observed for morphological traits (plant height, inflorescence length, weight of thousand seeds). The weather conditions in the year 2018 were favorable for all the morphological traits. The protein content of quinoa accessions ranged between 13.44 and 20.01% and it was positively correlated to mauritianin. Total phenolic content varied greatly from year to year, while the antioxidant activity remained relatively stable. The most abundant phenolic compounds were the flavonoids miquelianin, rutin, and isoquercetin. Isoquercetin, quercetin, and N-feruoloyl octopamine showed the highest stability under variable weather conditions in the analyzed years. A total of six compounds were detected and quantified in quinoa for the first time. Most varieties performed well under Central European conditions and can be considered a good source of nutrients and bioactive compounds. These data can be used as a source of information for plant breeders aiming to improve the quality traits of quinoa.

Specific Avenin Cross-Reactivity with G12 Antibody in a Wide Range of Current Oat Cultivars.

Current clinical studies confirm that the consumption of oats for people suffering from celiac disease is safe. Some studies have confirmed different levels of immunoreactive gluten epitopes of oats in different cultivars, while others explain these differences due to contamination with gluten-rich species or as random cross-reactivity ELISA of homologous oat epitopes with anti-wheat gliadin antibodies. The aim of our two-year study was therefore to map cross-reactive oat epitopes in a set of 132 oat cultivars using a G12-based ELISA kit. The results were focused on the varietal and annual level of cross-reactivity (interference) of avenin epitopes with the G12 antibody on the identification of potential cultivars with significantly different interferences and assessing the degree of risk of possible false-contamination with external gluten. Although repeated evaluations confirmed high year-to-year variability (RSD ≥ 30%) in approximately 2/3 of the cultivars, the content of interfering avenin epitopes with G12 did not exceed the considered safe limit (20 mg·kg-1) for celiacs. At the same time, not only annual but, above all, significant cultivar dependences in the interference of avenins to the G12 antibody were demonstrated. Genetic dependence was further confirmed in connection with the proven avenin polymorphism as well as immunoblotting with the identification of interfering peptides with the G12 antibody in the 25 and 30 kDa regions. It was the occurrence of two bands around 30 kDa that predominantly occurred in oat cultivars with a relatively higher content of cross-reactive avenins (12-16 mg·kg-1). Due to the fact that the contents of interfering avenins ranged in several cultivars even over 16 mg·kg-1, the choice of a suitable oat cultivar may be crucial for gluten-free food producers, as it reduces the risk of a possible false-response of the commercial ELISA kits when checking the real-gluten contamination.

Avenanthramides: Unique Bioactive Substances of Oat Grain in the Context of Cultivar, Cropping System, Weather Conditions and Other Grain Parameters.

Our study was focused on the evaluation of the content of a wider spectrum of eight avenanthramides (AVNs) as unique components of oat grain under the effects of four selected factors (cultivar, locality, cropping system, and year). The weather effects on changes in the AVN content and their relationship to other important parameters of oat grain were further evaluated in more detail. A sensitive UHPLC system coupled with a QExactive Orbitrap mass spectrometer was used for AVN quantification. AVNs confirmed a high variability (RDS = 72.7-113.5%), which was dominantly influenced by the locality and year factors. While most AVN types confirmed mutually high correlations (r = 0.7-0.9), their correlations with the other 10 grain parameters were lower (r ≤ 0.48). Their significant correlations (0.27-0.46) with ß-D-glucan could be used in perspective in breeding programs for the synergetic increase of both parameters. PCA analysis and Spearman correlations based on individual cultivars confirmed a significant effect of June and July precipitation on the increase of Σ AVNs. However, the results also indicated that higher precipitation can generate favorable conditions for related factors, such as preharvest sprouting evoking a direct increase of AVNs synthesis in oat grain.

Breeding Buckwheat for Nutritional Quality in the Czech Republic.

Buckwheat is a nutritionally valuable crop, an alternative to common cereals also usable in gluten-free diets. The selection of buckwheat genotypes suitable for further breeding requires the characterization and evaluation of genetic resources. The main objective of this work was to evaluate selected phenotypic and morphological traits using international buckwheat descriptors, including total phenolic content and antioxidant activity, on a unique set of 136 common buckwheat accessions grown in 2019-2020 under Czech Republic conditions. In addition, UHPLC-ESI- MS/MS was used to analyze a wide spectrum of 20 phenolic compounds in buckwheat seeds, including four flavanols, three phenolic acids, seven flavonols, four flavones, and two flavanones. Significant differences among years and genotypes were observed for morphological traits (plant height and 1000-seed weight) and antioxidant activity, as well as levels of observed chemical compounds. Antioxidant activity, crude protein content, plant height and rutin content were characterized by higher mean values in 2020 than in 2019 and vice versa for total polyphenol content and 1000-seed weight. Crude protein content was the most stable across years, while total polyphenol content and rutin content varied greatly from year to year. The most abundant phenolic compounds were rutin, hyperoside, epicatechin, catechin, vitexin, isovitexin, orientin and isoorientin. Protein content was negatively correlated with plant height, catechin and epicatechin content. On the other hand, AA and TPC were positively correlated with rutin, hyperoside and chlorogenic acid. Five accessions showed high stability of the evaluated traits under changing conditions within both years of observation. These materials can be used in breeding programmes aimed at improving buckwheat genotypes with emphasis on quality traits.

The Gut Microbiota Affects Corticosterone Production in the Murine Small Intestine.

Glucocorticoids (GCs) are hormones that are released in response to stressors and exhibit many activities, including immunomodulatory and anti-inflammatory activities. They are primarily synthesized in the adrenal gland but are also produced in peripheral tissues via regeneration of adrenal 11-oxo metabolites or by de novo synthesis from cholesterol. The present study investigated the influence of the microbiota on de novo steroidogenesis and regeneration of corticosterone in the intestine of germ-free (GF) and specific pathogen-free mice challenged with a physical stressor (anti-CD3 antibody i.p. injection). In the small intestine, acute immune stress resulted in increased mRNA levels of the proinflammatory cytokines IL1ß, IL6 and Tnfα and genes involved in de novo steroidogenesis (Stard3 and Cyp11a1), as well as in regeneration of active GCs from their 11-oxo metabolites (Hsd11b1). GF mice showed a generally reduced transcriptional response to immune stress, which was accompanied by decreased intestinal corticosterone production and reduced expression of the GC-sensitive marker Fkbp5. In contrast, the interaction between stress and the microbiota was not detected at the level of plasma corticosterone or the transcriptional response of adrenal steroidogenic enzymes. The results indicate a differential immune stress-induced intestinal response to proinflammatory stimuli and local corticosterone production driven by the gut microbiota.

Proteomic analysis of protein composition of rat hippocampus exposed to morphine for 10 days; comparison with animals after 20 days of morphine withdrawal.

Opioid addiction is recognized as a chronic relapsing brain disease resulting from repeated exposure to opioid drugs. Cellular and molecular mechanisms underlying the ability of organism to return back to the physiological norm after cessation of drug supply are not fully understood. The aim of this work was to extend our previous studies of morphine-induced alteration of rat forebrain cortex protein composition to the hippocampus. Rats were exposed to morphine for 10 days and sacrificed 24 h (groups +M10 and -M10) or 20 days after the last dose of morphine (groups +M10/-M20 and -M10/-M20). The six altered proteins (≥2-fold) were identified in group (+M10) when compared with group (-M10) by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). The number of differentially expressed proteins was increased to thirteen after 20 days of the drug withdrawal. Noticeably, the altered level of α-synuclein, ß-synuclein, α-enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also determined in both (±M10) and (±M10/-M20) samples of hippocampus. Immunoblot analysis of 2D gels by specific antibodies oriented against α/ß-synucleins and GAPDH confirmed the data obtained by 2D-DIGE analysis. Label-free quantification identified nineteen differentially expressed proteins in group (+M10) when compared with group (-M10). After 20 days of morphine withdrawal (±M10/-M20), the number of altered proteins was increased to twenty. We conclude that the morphine-induced alteration of protein composition in rat hippocampus after cessation of drug supply proceeds in a different manner when compared with the forebrain cortex. In forebrain cortex, the total number of altered proteins was decreased after 20 days without morphine, whilst in hippocampus, it was increased.

Comprehensive analysis of oat avenanthramides using hybrid quadrupole-Orbitrap mass spectrometry: Possible detection of new compounds.

RATIONALE: Avenanthramides (AVNs) are constituents unique to oats and have many outstanding health benefits. AVNs are antioxidants and possess anti-inflammatory, antifungal and antibacterial activity. The number of known AVNs increased recently because of the latest developments in high-resolution tandem mass spectrometry (HRMS/MS) techniques. METHODS: Oat seed extract from 10 oat cultivars was analysed using ultra-high-performance liquid chromatography (UHPLC) and Q Exactive hybrid quadrupole-Orbitrap mass spectrometry (HRMS/MS) with positive heated electrospray ionization. RESULTS: Thirty-five AVNs were identified and characterized in seed extracts, and the structures of 10 novel AVNs were tentatively elucidated, among which were AVNs bearing a cinamoyl or sinapoyl moiety. These AVNs are reported in oats for the first time. The method was validated using AVN standards (AVNs 2c, 2f and 2p), with limits of detection and quantitation at low picomole levels. Recovery of AVN standards varied from 83% to 106%, and relative standard deviations ranged from 2% to 9%. The total AVNs in the selected oat varieties ranged from 36.0 to 302.5 µg/g (dry weight), with AVN 2c, AVN 2f and AVN 2p representing approximately 65%-70% of that total. CONCLUSIONS: Our comprehensive method for detecting the full avenanthramide spectrum can contribute to better understanding the chemical and biological properties of individual AVNs for utilization in developing new oat cultivars and novel functional foods.

Proteomic analysis of dentin-enamel junction and adjacent protein-containing enamel matrix layer of healthy human molar teeth.

The dentin-enamel junction (DEJ) is the border where two different mineralized structures - enamel and dentin - meet. The protein-rich DEJ, together with the inner enamel region of mature teeth, is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the DEJ and the adjacent enamel organic matrix (EOM). We studied proteomes of the DEJ and EOM of healthy human molars and compared them with dentin and enamel proteomes from the same teeth. These tissues were cut out of tooth sections by laser capture microdissection, proteins were extracted and cleaved by trypsin, then processed by liquid chromatography coupled to tandem mass spectrometry to analyze the proteome profiles of these tissues. This study identified 46 proteins in DEJ and EOM. The proteins identified have a variety of functions, including calcium ion-binding, formation of extracellular matrix, formation of cytoskeleton, cytoskeletal protein binding, cell adhesion, and transport. Collagens were identified as the most dominant proteins. Tissue-specific proteins, such as ameloblastin and amelogenin, were also detected. Our findings reveal new insight into proteomics of DEJ and EOM, highly mineralized tissues that are obviously difficult to analyze.

Proteomic analysis of human tooth pulp proteomes - Comparison of caries-resistant and caries-susceptible persons.

UNLABELLED: Most people in the world suffer from dental caries, >90% of adults experience caries on enamel and root surfaces during their life. However, the overall roles of all factors in the development of dental caries still remain unclear and are worthy of recent investigation. In this study we used a proteomic 2D-DIGE approach in connection with MS/MS to investigate the different protein abundances in the tooth pulp of human third molars obtained from caries-resistant and caries-susceptible people. Statistical analysis of the two protein maps obtained on large gel (17cm length) and mini gel (7cm length) followed by nLC-MS/MS analysis enabled the identification of 16 significantly changed spots with unique protein identifications corresponding to 12 non-redundant proteins. Seven proteins exhibited higher and four proteins exhibited lower expression in the caries-resistant samples compared to the caries-susceptible samples. Additionally, one protein (alpha-1-antitrypsin) exhibited both expressions (up and down). Most of the differentially expressed proteins were associated with protein metabolism, energy production, cytoskeletal organization and transport. These differentially expressed proteins are likely involved in the natural resistance or susceptibility of humans to the development of dental caries and suggest that the resistance mechanism is multifactorial. BIOLOGICAL SIGNIFICANCE: Dental caries are not a serious and life-threatening disease, but their healing requires many remedies and takes up a lot of time. Moreover, neglecting the problem may lead to tooth loss, which can strongly reduce the quality of life. Therefore the identifying effective and safe oral medicine and determining the causes of caries-resistance were viewed as the main aims of this study. Our work aims to elucidate the mechanism of natural human resistance to the development of dental caries by studying the proteomes of tooth pulp isolated from patients who displayed different prevalences of tooth caries. This study is the first protein tooth pulp comparison of sound teeth obtained from caries-resistant versus caries-susceptible people.

Proteomic analysis of human tooth pulp: proteomics of human tooth.

INTRODUCTION: The unique pulp-dentin complex demonstrates strong regenerative potential, which enables it to respond to disease and traumatic injury. Identifying the proteins of the pulp-dentin complex is crucial to understanding the mechanisms of regeneration, tissue calcification, defense processes, and the reparation of dentin by dental pulp. The lack of knowledge of these proteins limits the development of more efficient therapies. METHODS: The proteomic profile of human tooth pulp was investigated and compared with the proteome of human dentin and blood. The samples of tooth pulp were obtained from 5 sound permanent human third molars of 5 adults (n = 5). The extracted proteins were separated by 2-dimensional gel electrophoresis, analyzed by nano-liquid chromatography tandem mass spectrometry, and identified by correlating mass spectra to the proteomic databases. RESULTS: A total of 342 proteins were identified with high confidence, and 2 proteins were detected for the first time in an actual human sample. The identified tooth pulp proteins have a variety of functions: structural, catalytic, transporter, protease activity, immune response, and many others. In a comparison with dentin and blood plasma, 140 (pulp/dentin) shared proteins were identified, 37 of which were not observed in plasma. It can be suggested that they might participate in the unique pulp-dentin complex. CONCLUSIONS: This proteomic investigation of human tooth pulp, together with the previously published study of human dentin, is one of the most comprehensive proteome lists of human teeth to date.

Biosynthesis of colabomycin E, a new manumycin-family metabolite, involves an unusual chain-length factor.

Colabomycin E is a new member of the manumycin-type metabolites produced by the strain Streptomyces aureus SOK1/5-04 and identified by genetic screening from a library of streptomycete strains. The structures of colabomycin E and accompanying congeners were resolved. The entire biosynthetic gene cluster was cloned and expressed in Streptomyces lividans. Bioinformatic analysis and mutagenic studies identified components of the biosynthetic pathway that are involved in the formation of both polyketide chains. Recombinant polyketide synthases (PKSs) assembled from the components of colabomycin E and asukamycin biosynthetic routes catalyzing the biosynthesis of "lower" carbon chains were constructed and expressed in S. aureus SOK1/5-04 ΔcolC11-14 deletion mutant. Analysis of the metabolites produced by recombinant strains provided evidence that in both biosynthetic pathways the length of the lower carbon chain is controlled by an unusual chain-length factor supporting biosynthesis either of a triketide in asukamycin or of a tetraketide in colabomycin E. Biological activity assays indicated that colabomycin E significantly inhibited IL-1ß release from THP-1 cells and might thus potentially act as an anti-inflammatory agent.

Serine protease from midgut of Bombus terrestris males.

A serine protease was isolated from midguts of the bumblebee male Bombus terrestris by a combination of precipitation procedures with column chromatography. The purified enzyme exhibited two bands with molecular masses of 25 and 26 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These bands showed a proteolytic activity in zymography assay. Midgut enzymes showed optimum proteolytic activity at pH 9 and 35°C using N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-alanine 4-nitroanilide as a substrate. The Michaelis constant (Km) and maximum reaction rate (Vmax) were 0.55±0.042 mM and 0.714±0.056 µmol p-nitroalanine produced min(-1) mg protein(-1) , respectively. Inhibition was affected by trypsin inhibitor, but not by phenylmethylsulfonyl fluoride and N-tosyl-L-phenylalanine chloromethyl ketone, which indicated the trypsin-like but not chymotrypsin-like specificity. The identity of the serine protease was confirmed by nanoliquid-tandem mass spectrometry. Eleven unique peptides of the B. terrestris serine protease were found. It shows high homology to a previously reported B. ignitus serine protease covering more than 65% of the protein amino acid sequence.

Comprehensive proteomic analysis of human dentin.

Proteomic analysis of the human body is a significant recent scientific endeavour. In this study, we investigated the proteomic profile of human dentin using modern analytical and mass spectrometric techniques. Five healthy permanent human molars from five adults were cut, pulverized, denaturated with guanidine buffer, and demineralized with EDTA buffer. The extracted proteins were analysed by gel electrophoresis (SDS-PAGE and two-dimensional gel electrophoresis), digested with trypsin, and separated by liquid chromatography/high-resolution tandem mass spectrometry. We identified 289 proteins with high confidence, 90 of which had not been previously detected in human dentin. Nine (currently hypothetical) proteins were identified for the first time in an actual human sample. The proteins have a variety of functions, including calcium-ion binding, formation of the extracellular matrix, formation of the cytoskeleton, cytoskeletal protein binding, immune response, and transport. In conclusion, this is the first use of two-dimensional electrophoresis for investigating human dentin.

Synthesis and characterization of styrene oxide adducts with cysteine, histidine, and lysine in human globin.

Styrene 7,8-oxide (SO), a reactive metabolic intermediate of the industrial chemical styrene, binds covalently at nucleophilic amino acid residues of blood proteins in vivo and in vitro. In this study, SO adducts with cysteine, lysine, and histidine were synthesized, characterized, and then used as authentic standards to assign and quantitate the SO adducts in globin incubated with SO. S-(2-Hydroxy-1-phenylethyl)cysteine and S-(2-hydroxy-2-phenylethyl)cysteine were prepared by direct alkylation of cysteine with (R)-SO or (S)-SO. To prepare the SO adducts with lysine and histidine, Nalpha-Boc-protected amino acids were alkylated with (R)-SO or (S)-SO followed by deprotection of the Boc group to obtain Nepsilon-(2-hydroxy-1-phenylethyl)lysine and Nepsilon-(2-hydroxy-2-phenylethyl)lysine as well as Npi-(2-hydroxy-1-phenylethyl)histidine, Npi-(2-hydroxy-2-phenylethyl)histidine, Ntau-(2-hydroxy-1-phenylethyl)histidine, and Ntau-(2-hydroxy-2-phenylethyl)histidine. The individual regioisomers were isolated from their mixtures by semipreparative HPLC, and their structure was assigned using NMR techniques. The SO-modified globin, isolated from human hemoglobin incubated in vitro with racemic SO at a molar ratio SO/globin of 100:1 or 10:1, was digested with pronase and subjected to LC/MS and GC/MS analysis. All known regioisomers of the SO adducts were detected, with S-(2-hydroxy-1-phenylethyl)cysteine, Nepsilon-(2-hydroxy-1-phenylethyl)lysine, and Ntau-(2-hydroxy-2-phenylethyl)histidine being the most abundant in the modified globin. Deuterated analogues of the SO adducts were employed as internal standards. The SO-amino acid adducts described here appear to be suitable biomarkers for long-term exposures to styrene or SO.