Hypoxia-inducible factors in postnatal mouse molar dental pulp development: insights into expression patterns, localisation and metabolic pathways.

Hypoxia is relevant to several physiological and pathological processes and this also applies for the tooth. The adaptive response to lowering oxygen concentration is mediated by hypoxia-inducible factors (HIFs). Since HIFs were shown to participate in the promotion of angiogenesis, stem cell survival, odontoblast differentiation and dentin formation, they may play a beneficial role in the tooth reparative processes. Although some data were generated in vitro, little is known about the in vivo context of HIFs in tooth development. In order to contribute to this field, the mouse mandibular first molar was used as a model.The expression and in situ localisation of HIFs were examined at postnatal (P) days P0, P7, P14, using RT-PCR and immunostaining. The expression pattern of a broad spectrum of hypoxia-related genes was monitored by customised PCR Arrays. Metabolic aspects were evaluated by determination of the lactate level and mRNA expression of the mitochondrial marker Nd1.The results show constant high mRNA expression of Hif1a, increasing expression of Hif2a, and very low expression of Hif3a during early postnatal molar development. In the examined period the localisation of HIFs in the nuclei of odontoblasts and the subodontoblastic layer identified their presence during odontoblastic differentiation. Additionally, the lower lactate level and higher expression of mitochondrial Nd1 in advanced development points to decreasing glycolysis during differentiation. Postnatal nuclear localisation of HIFs indicates a hypoxic state in specific areas of dental pulp as oxygen demands depend on physiological events such as crown and root dentin mineralization.

Comparison of Metabolic Profiles of Fruits of Arctium lappa, Arctium minus, and Arctium tomentosum.

Metabolites of the edible and medicinal plant Arctium have been shown to possess beneficial activities. The phytochemical profile of Arctium lappa is well-explored and its fruits are known to contain mainly lignans, fatty acids, and sterols. But the fruits of other Arctium species have not been thoroughly investigated. Therefore, this study compares the metabolic profiles of the fruits of A. lappa, Arctium tomentosum, and Arctium minus. Targeted metabolomics led to the putative identification of 53 metabolites in the fruit extracts, the majority of these being lignans and fatty acids. Quantification of the major lignans showed that the year of collection had a significant effect on the lignan content. Furthermore, A. lappa fruits contained lesser amounts of arctigenin but greater amounts of arctigenin glycoside than A. minus fruits. Regarding the profile of fatty acids, A. minus fruits differed from the others in the presence of linolelaidic acid.

DiaPASEF proteotype analysis indicates changes in cell growth and metabolic switch induced by caspase-9 inhibition in chondrogenic cells.

Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase-9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase-9 inhibitor-treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC-MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC-MS system revealed 7849 protein groups (Q-value <0.01), a parallel analysis of iTRAQ-2DLC-MS3 and conventional DIA-MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down-/up-regulated protein groups after caspase-9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non-apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.

Health Beneficial Properties of Grapevine Seed Extract and Its Influence on Selected Biochemical Markers in the Blood, Liver and Kidneys of Rattus norvegicus.

Cadmium (Cd) is a heavy metal that occurs in all areas of the environment, including the food chain. In the body, it causes oxidative stress by producing free radicals that are harmful to the cells. Grape seed extract (GSE) contains a wide range of biologically active components that help to neutralize the adverse effects of free radicals. In this study, the effects of GSE prepared form semi-resistant grapevine cultivar Cerason, which is rich in phenolics, on biochemical markers of brown rats exposed to the effects of cadmium were monitored. GSE increased the plasma antioxidant activity and, in the kidneys and the liver, Cd content was significantly lowered by GSE co-administration. Accordingly, the increase in creatinine content and alanine aminotransferase activity and the decrease of catalase and superoxide dismutase activities caused by cadmium were slowed down by GSE co-administration. The results of this work reveal that grape seed extract offers a protective effect against the intake of heavy metals into the organism.

In sacco evaluation of ruminal degradability of isoflavones from full-fat soybean and extracted soybean meal-A pilot study.

The aim of the study was to determine the ruminal degradability of dry matter (DM), daidzein, genistein, glycitein and total isoflavones in ground full-fat soybean (GFFS) and solvent-extracted soybean meal (SSBM) using the in sacco method. The experiment was carried out in three replications on ruminally cannulated sheep that were fed twice a day with a diet consisted of hay and supplemental mixture (6:4, DM basis). The nylon bags with 2 g feed samples ground to 2 mm were incubated in the rumen for 0, 2, 4, 8, 16 and 24 h. The effective degradability (ED) of DM, daidzein, genistein, glycitein and total isoflavones was calculated at outflow rate of 0.06 h. The ED of DM in GFFS was 77.8% and was higher than in SSBM being 71.8% (p < 0.001). The ED of daidzein (96.8%) and genistein (93.6%) was higher for SSBM compared with GFFS (93.9% and 92.8%, p < 0.001 and p = 0.003, respectively) while ED of glycitein was lower for SSBM than for GFFS (75.5 and 81.7%, respectively, p < 0.001). All isoflavones in the incubations were extensively degraded in the rumen, and regardless of dietary source, they were almost completely degraded after 16 h of incubation. Further, the disappearance patterns, that is the functions describing the time courses of the analyte disappearance, were assessed. The disappearance patterns of daidzein, genistein, glycitein and total isoflavones were similar and showed greater disappearance of mentioned isoflavones from SSBM compared to GFFS (p < 0.001 for daidzein, genistein and total isoflavones and p = 0.002 for glycitein). The study provides knowledge on the effect of processing on degradability of isoflavones in rumen that can be used to clarify the interrelationship between isoflavones and rumen microbiota.

Origin of Wine Lignans.

Plant lignans possess several properties beneficial for human health and therefore, increasing their contents in foods and beverages is desirable. One of the lignan sources in human diet is wine. To elucidate the origin of lignans contained in wine, LC-MS was used to analyze resinol-related lignans in must, seeds, stems, and wine prepared using stainless steel tanks, oak barrels, and Qvevri (clay vessel). White wines aged in stainless steel tanks contained significantly lower amounts of lignan aglycones (20-60 µg/L) than red and Qvevri wines (300-500 µg/L). Generally, white wines aged in stainless steel tanks contained only low amounts of isolariciresinol and matairesinol. Qvevri wines and red wine aged in stainless steel tank contained up to five lignan compounds and in wine aged in oak barrel, six different lignans were identified. Consistently, only low concentration of isolariciresinol has been found in must, whereas more lignan compounds have been found in grape seeds (isolariciresinol, secoisolariciresinol, and pinoresinol) and stems (isolariciresinol and syringaresinol). Consequently, we conclude that lignan content in wine can be increased by maturation in contact with grape berries, seeds, or stems or with wood.

Production of Defense Phenolics in Tomato Leaves of Different Age.

Phenolics play an essential role in the defense reaction of crop plants against pathogens. However, the intensity of their production induced by infection may differ during the life of a plant. Here, we identified age-related differences in phenolic biosynthesis in the pathosystem Solanum lycopersicum cv. Amateur and Pseudomonas syringae pv. tomato DC3000. We analyzed concentrations of total phenolics, phenolic profiles, and concentrations of selected phenolic acids. The influence of bacterial infection, together with leaf and plant age, was assessed. The changes in concentrations of caffeic acid, 4-hydroxybenzoic acid, and salicylic acid glucoside caused by infection were found to be influenced by age. In concrete, the increases in the concentrations of these metabolites were all evident only in young plants.

In vitro metabolism of red clover isoflavones in rumen fluid.

The degradation of red clover isoflavones was studied in vitro using a rumen fluid buffer system. Various amounts of red clover extract (5-75 mg) together with hay or concentrate-rich diet were added to 40 ml of rumen fluid obtained from non-lactating and lactating dairy cows, respectively, and incubated for 0, 3, 6, 12 or 24 hr. Following incubation, concentrations of daidzein, genistein, formononetin, biochanin A and equol were determined in the samples. After 3 hr of incubation, isoflavone metabolism and equol production could be observed. The results obtained indicate that hay diet provides better conditions for isoflavone metabolism, as concentrations of daidzein, formononetin and biochanin A were higher in incubations based on the concentrate-rich diet and the production of equol was higher in incubations based on the hay diet. Furthermore, in incubations with higher amounts of added clover extract, a decrease in equol production was observed. Further studies are needed to clarify the role of adaptation of rumen microflora on isoflavone degradation kinetics and to clarify the interrelationship between various dietary factors, rumen microbiota and isoflavones. The knowledge of isoflavone metabolism kinetics in dependence on studied factors will be useful for the optimization of feeding dose.

Isoflavones.

Phytoestrogens are naturally occurring nonsteroidal phenolic plant compounds that, due to their molecular structure and size, resemble vertebrate steroids estrogens. This review is focused on plant flavonoids isoflavones, which are ranked among the most estrogenic compounds. The main dietary sources of isoflavones for humans are soybean and soybean products, which contain mainly daidzein and genistein. When they are consumed, they exert estrogenic and/or antiestrogenic effects. Isoflavones are considered chemoprotective and can be used as an alternative therapy for a wide range of hormonal disorders, including several cancer types, namely breast cancer and prostate cancer, cardiovascular diseases, osteoporosis, or menopausal symptoms. On the other hand, isoflavones may also be considered endocrine disruptors with possible negative influences on the state of health in a certain part of the population or on the environment. This review deals with isoflavone classification, structure, and occurrence, with their metabolism, biological, and health effects in humans and animals, and with their utilization and potential risks.

Determination of in vitro isoflavone degradation in rumen fluid.

The aim of this study was to determine the degradation of dietary isoflavones in rumen fluid under 2 feeding regimens. The experiments were performed in vitro using a rumen fluid buffer system. The rumen fluid was taken from cows fed either a hay diet or a concentrate-rich diet (the diet consisted of 34.6% maize silage, 17.6% haylage, 12.8% alfalfa hay, and 35.0% supplemental mixture on a dry matter basis). As a source of isoflavones, 40% soybean extract (Biomedica, Prague, Czech Republic) at levels of 5, 25, 50, and 75 mg per 40 mL of rumen fluid was used. Samples of soybean extract were incubated in triplicate at 39°C for 0, 3.0, 6.0, 12.0, and 24.0 h in incubation solution. The metabolism of daidzein and genistein was faster under concentrate-rich diet conditions. In general, production of equol started after 3 to 6 h of incubation and reached the highest rate after approximately 12 h of incubation regardless of the type of diet or concentration of extract. In most of the experiments, production of equol continued after 24 h of incubation. Generally, equol production was greater under the hay diet conditions. Furthermore, experiments with higher amounts of added soybean extract revealed possible inhibitory effects of high levels of isoflavones on the rumen microflora.

Changes in the fatty acid profiles and health indexes of bovine colostrum during the first days of lactation and their impact on human health.

Our objective was to analyze the changes in fatty acid (FA) profiles of bovine colostrum and immature milk during the first four days of lactation and assess their potential impact on human health. Colostrum and immature milk samples were collected from Czech Fleckvieh cows during their first to third lactation and the FA profiles were analyzed using multidimensional gas chromatography with a vacuum ultraviolet detector (GC×GC-VUV). The colostrum of primiparous cows contained lower levels of medium-chain and saturated fatty acids, and higher levels of mono- and unsaturated fatty acids compared to that of multiparous cows. The atherogenic and thrombogenicity indexes, as well as the hypocholesterolemic-to-hypercholesterolemic fatty acid ratio, were more favourable in primiparous cows. This makes colostrum fat an attractive product for human nutrition. To obtain the maximum health benefits, we recommend collecting and processing the colostrum of primiparous cows and immature milk at the end of the milk transition separately.

Markers of dental pulp stem cells in in vivo developmental context.

Teeth and their associated tissues contain several populations of mesenchymal stem cells, one of which is represented by dental pulp stem cells (DPSCs). These cells have mainly been characterised in vitro and numerous positive and negati ve markers for these cells have been suggested. To investigate the presence and localization of these molecules during development, forming dental pulp was examined using the mouse first mandibular molar as a model. The stages corresponding to postnatal (P) days 0, 7, 14, and 21 were investigated. The expression was monitored using customised PCR Arrays. Additionally, in situ localization of the key trio of markers (Cd73, Cd90, Cd105 coded by genes Nt5e, Thy1, Eng) was performed at prenatal and postnatal stages using immunohistochemistry. The expression panel of 24 genes assigned as in vitro markers of DPSCs or mesenchymal stem cells (MSCs) revealed their developmental dynamics during formation of dental pulp mesenchyme. Among the positive markers, Vcam1, Fgf2, Nes were identified as increasing and Cd44, Cd59b, Mcam, Alcam as decreasing between perinatal vs. postnatal stages towards adulthood. Within the panel of negative DPSC markers, Cd14, Itgb2, Ptprc displayed increased and Cd24a decreased levels at later stages of pulp formation. Within the key trio of markers, Nt5e did not show any significant expression difference within the investigated period. Thy1 displayed a strong decrease between P0 and P7 while Eng increased between these stages. In situ localization of Cd73, Cd90 and Cd105 showed them overlap in differentiated odontoblasts and in the sub-odontoblastic layer that is speculated to host odontoblast progenitors. The highly prevalent expression of particularly Cd73 and Cd90 opens the question of potential multiple functions of these molecules. The results from this study add to the in vitro based knowledge by showing dynamics in the expression of DPSC/MSC markers during dental pulp formation in an in vivo context and thus with respect to the natural environment important for commitment of stem cells.

Gene expression and metabolic activity of Streptococcus mutans during exposure to dietary carbohydrates glucose, sucrose, lactose, and xylitol.

Recent RNA sequencing studies have given us a deeper insight into the cariogenic impact of carbohydrate sources in the bacterium Streptococcus mutans, the principal microbial agent in dental caries etiopathogenesis. The process of dental caries development is facilitated by the ability of this bacterium to ferment some carbohydrates into organic acids contributing to a pH decrease in the oral cavity and the demineralization of the hard tissues of the tooth. Furthermore, in dental caries progression, biofilm formation, which starts and ends with free planktonic cells, plays an important role and has several unique properties called virulence factors. The most cariogenic carbohydrate is sucrose, an easily metabolizable source of energy that induces the acidification and synthesis of glucans, forming typical bacterial cell clumps. We used multifaceted methodological approaches to compare the transcriptomic and metabolomic profiles of S. mutans growing in planktonic culture on preferred and nonpreferred carbohydrates and in fasting conditions. Streptococcus mutans in a planktonic culture with lactose produced the same pH drop as glucose and sucrose. By contrast, xylitol and lactose showed high effectiveness in regulating intracellular polysaccharide metabolism, cell wall structure, and overall virulence involved in the initial phase of biofilm formation and structure but with an opposite pattern compared with sucrose and glucose. Our results confirmed the recent findings that xylitol and lactose play a vital role in biofilm structure. However, they do not reduce its formation, which is related to the creation of a cariogenic environment.

L-Aspartate and L-Glutamine Inhibit Beta-Aminobutyric Acid-Induced Resistance in Tomatoes.

Plant diseases caused by pathogens lead to economic and agricultural losses, while plant resistance is defined by robustness and timing of defence response. Exposure to microbial-associated molecular patterns or specific chemical compounds can promote plants into a primed state with more robust defence responses. ß-aminobutyric acid (BABA) is an endogenous stress metabolite that induces resistance, thereby protecting various plants' diverse stresses by induction of non-canonical activity after binding into aspartyl-tRNA synthetase (AspRS). In this study, by integrating BABA-induced changes in selected metabolites and transcript data, we describe the molecular processes involved in BABA-induced resistance (BABA-IR) in tomatoes. BABA significantly restricted the growth of the pathogens P. syringae pv. tomato DC3000 and was related to the accumulation of transcripts for pathogenesis-related proteins and jasmonic acid signalling but not salicylic acid signalling in Arabidopsis. The resistance was considerably reduced by applying amino acids L-Asp and L-Gln when L-Gln prevents general amino acid inhibition in plants. Analysis of amino acid changes suggests that BABA-IR inhibition by L-Asp is due to its rapid metabolisation to L-Gln and not its competition with BABA for the aspartyl-tRNA synthetase (AspRS) binding site. Our results showed differences between the effect of BABA on tomatoes and other model plants. They highlighted the importance of comparative studies between plants of agronomic interest subjected to treatment with BABA.

Feedborne Mycotoxins Beauvericin and Enniatins and Livestock Animals.

Mycotoxins are secondary metabolites produced by several species of fungi, including the Fusarium, Aspergillus, and Penicillium species. Currently, more than 300 structurally diverse mycotoxins are known, including a group called minor mycotoxins, namely enniatins, beauvericin, and fusaproliferin. Beauvericin and enniatins possess a variety of biological activities. Their antimicrobial, antibiotic, or ionoforic activities have been proven and according to various bioassays, they are believed to be toxic. They are mainly found in cereal grains and their products, but they have also been detected in forage feedstuff. Mycotoxins in feedstuffs of livestock animals are of dual concern. First one relates to the safety of animal-derived food. Based on the available data, the carry-over of minor mycotoxins from feed to edible animal tissues is possible. The second concern relates to detrimental effects of mycotoxins on animal health and performance. This review aims to summarize current knowledge on the relation of minor mycotoxins to livestock animals.

Production of Bovine Equol-Enriched Milk: A Review.

Milk and dairy products are important sources of nutrients in the human diet because they contain a number of essential substances and other biologically active components. Many of these substances can be modified, and thus offer opportunities to use milk and dairy products as functional food. Isoflavones are particularly important in human nutrition due to their diverse pharmacological and antioxidant properties. The clinical effectiveness of isoflavone-rich products is believed to be dependent on their ability to metabolize daidzein to equol, which may directly exert cancer preventive effects. However, only approximately 30-40% of humans are able to produce equol, while animals, in general, produce equol. Equol is the predominant product of bacterial metabolism of isoflavones and can be found in various amounts in some food of animal origin, especially in milk. Therefore, milk and dairy products can be considered to be sources of equol for humans who are not able to produce this metabolite. When the content of isoflavones in milk is to be modified, two groups of factors should be considered, i.e., dietary factors that include the source of isoflavones and the processing effects on feedstuffs and animal factors that include the intake of isoflavones, ruminal and postruminal changes, and the health and physiological status of animals. The approximate content of isoflavones in milk can be predicted using carry-over rates for different dietary sources or using a formula that describes the relationship between equol concentration in milk and formononetin intake. Processing and storage can affect the content and profile of isoflavones in milk and dairy products.

Osteogenic impact of pro-apoptotic caspase inhibitors in MC3T3-E1 cells.

Caspases are proteases traditionally associated with inflammation and cell death. Recently, they have also been shown to modulate cell proliferation and differentiation. The aim of the current research was to search for osteogenic molecules affected by caspase inhibition and to specify the individual caspases critical for these effects with a focus on proapoptotic caspases: caspase-2, -3, -6, -7, -8 and -9. Along with osteocalcin (Ocn), general caspase inhibition significantly decreased the expression of the Phex gene in differentiated MC3T3-E1 cells. The inhibition of individual caspases indicated that caspase-8 is a major contributor to the modification of Ocn and Phex expression. Caspase-2 and-6 had effects on Ocn and caspase-6 had an effect on Phex. These data confirm and expand the current knowledge about the nonapoptotic roles of caspases and the effect of their pharmacological inhibition on the osteogenic potential of osteoblastic cells.

Changes in equol and major soybean isoflavone contents during processing and storage of yogurts made from control or isoflavone-enriched bovine milk determined using LC-MS (TOF) analysis.

The effect of supplementing a basal diet for dairy cows with "Soybean extract 40" (Biomedica, Prague, Czech Republic), containing 40% soybean isoflavones, on the contents of daidzein, glycitein, genistein, and equol in milk as well as fresh and mature yogurts was estimated. To determine the contents of these isoflavonoids, an efficient analytical LC-MS (TOF) technique was used. The "Soybean extract 40" used in our study contained an especially high proportion of daidzein (307gkg-1). In both milk and yogurt samples, the amounts of daidzein and its metabolite equol were significantly higher in samples obtained from cows that received the isoflavone extract-supplemented diet than from those that received the basal diet, as the precursor daidzein contributed to the increased equol concentrations. Fermentation caused significant changes in the daidzein and glycitein concentrations. With maturation, the concentrations of daidzein and equol were unaffected, while the glycitein concentration decreased significantly.

Soybean-Derived Isoflavone Determination in Rumen Fluid and Milk by LC-MS-(TOF).

Soybean-derived isoflavones belong to the family of biologically active phytoestrogens. The purpose of this study was to develop a sensitive method, which permits quantification of the soybean isoflavonoids and equol in bovine rumen fluid and milk using LC-MS-(TOF). The samples of rumen fluid and milk were obtained from 12 lactating dairy cows ingesting 7,500-9,500 mg of total isoflavones daily. The validation of the developed method showed the limits of quantification to be in the range of 0.9-5.0 ng/mL. The precision was determined as relative standard deviation, which was lower than 25% in all cases. The recoveries of the most isoflavonoids were satisfactory. Lower recoveries of daidzin and glycitin can be solved by adding an internal standard. The presented method will be useful for kinetic studies of isoflavone metabolism in ruminants due to simultaneous quantification of free aglycones and glycosides in the rumen fluid.

Effects of Isoflavone-Enriched Feed on the Rumen Microbiota in Dairy Cows.

In this study, we compared the effects of two diets containing different isoflavone concentrations on the isoflavone transfer from feed into milk and on the rumen microbiota in lactating dairy cows. The on-farm experiment was conducted on twelve lactating Czech Fleckvieh x Holstein cows divided into two groups, each with similar mean milk yield. Twice daily, cows were individually fed a diet based on maize silage, meadow hay and supplemental mixture. Control group (CTRL) received the basal diet while the experimental group (EXP) received the basal diet supplemented with 40% soybean isoflavone extract. The average daily isoflavone intake in the EXP group (16 g/day) was twice as high as that in the CTRL group (8.4 g/day, P<0.001). Total isoflavone concentrations in milk from the CTRL and EXP groups were 96.89 and 276.07 µg/L, respectively (P<0.001). Equol concentrations in milk increased from 77.78 µg/L in the CTRL group to 186.30 µg/L in the EXP group (P<0.001). The V3-4 region of bacterial 16S rRNA genes was used for metagenomic analysis of the rumen microbiome. The experimental cows exhibited fewer OTUs at a distance level of 0.03 compared to control cows (P<0.05) and reduced microbial richness compared to control cows based on the calculated Inverse Simpson and Shannon indices. Non-metric multidimensional scaling analysis showed that the major contributor to separation between the experimental and control groups were changes in the representation of bacteria belonging to the phyla Bacteroidetes, Proteobacteria, Firmicutes, and Planctomycetes. Surprisingly, a statistically significant positive correlation was found only between isoflavones and the phyla Burkholderiales (r = 0.65, P<0.05) and unclassified Betaproteobacteria (r = 0.58, P<0.05). Previous mouse and human studies of isoflavone effects on the composition of gastrointestinal microbial populations generally report similar findings.