Extracellular vesicle-bound DNA in urine is indicative of kidney allograft injury.

Extracellular vesicle-bound DNA (evDNA) is an understudied extracellular vesicle (EV) cargo, particularly in cancer-unrelated research. Although evDNA has been detected in urine, little is known about its characteristics, localization, and biomarker potential for kidney pathologies. To address this, we enriched EVs from urine of well-characterized kidney transplant recipients undergoing allograft biopsy, characterized their evDNA and its association to allograft injury. The SEC-based method enriched pure EVs from urine of kidney transplant recipients, regardless of the allograft injury. Urinary evDNA represented up to 29.2 ± 8% (mean ± SD) of cell-free DNA (cfDNA) and correlated with cfDNA in several characteristics but was less fragmented (P < 0.001). Importantly, using DNase treatment and immunogold labelling TEM, we demonstrated that evDNA was bound to the surface of urinary EVs. Normalised evDNA yield (P = 0.042) and evDNA copy number (P = 0.027) significantly differed between patients with normal histology, rejection injury and non-rejection injury, the later groups having significantly larger uEVs (mean diameter, P = 0.045) and more DNA bound per uEV. ddDNA is detectable in uEV samples of kidney allograft recipients, but its quantity is highly variable. In a proof-of-principle study, several evDNA characteristics correlated with clinical and histological parameters (P = 0.040), supporting that the potential of evDNA as a biomarker for kidney allograft injury should be further investigated.

Optimization of isolation protocol and characterization of urinary extracellular vesicles as biomarkers of kidney allograft injury.

AIMS: Long-term kidney allograft survival requires a personalized approach to allograft injury recognition in a timely and reliable manner. Kidney biopsy is invasive and unsuitable for continuous function assessment. Alternatively, in urine, we find extracellular vesicles (uEVs), stable carriers of kidney pathology signals. Analysis of uEVs and their cargo could allow for more frequent and non-invasive assessment of allograft function. We aimed to optimize the uEVs isolation method applicable for kidney allograft injury biomarker studies. MATERIALS AND METHODS: To this end, we optimized several steps of size-exclusion chromatography (SEC)-based method for uEVs isolation from second morning urine of kidney allograft recipients. uEVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), western analysis, and quantitative PCR. RESULTS: According to TEM and NTA, SEC isolated high concentrations (8.64 × 108 EVs/mL of urine) of EVs that showed typical morphology and mean size (171 nm), but addition of EDTA and filtration step were needed to remove impurities. Additionally, typical EV proteins Hsc70, CD63, flotillin, tubulin, GAPDH, and miR hsa-let-7i were detected in isolated uEVs, further confirming their identity. CONCLUSION: Optimized method based on SEC was effective and adequate in isolating pure EVs from urine of kidney allograft recipients and could be used in future biomarker studies.

Texture of Hot-Air-Dried Persimmon (Diospyros kaki) Chips: Instrumental, Sensory, and Consumer Input for Product Development.

Persimmon (Diospyros kaki) is an underutilized tree fruit. Previous studies have shown the feasibility of making a hot-air-dried, chip-style product from persimmon. However, the texture of this type of product has not been explored or connected to consumer preference. Thus, for dried samples representing 37 cultivars, this study aimed to (1) predict trained sensory panel texture attributes from instrumental measurements, (2) predict consumer liking from instrumental measurements and sensory texture attributes, and (3) elucidate whether astringency type affects dried product texture. Partial least-squares regression models of fair-to-good quality predicted all measured sensory texture attributes (except Tooth Packing) from instrumental measurements. Modeling also identified that consumer preference is for a moist, smooth texture. Lastly, while astringency type has significant (p < 0.05) effects on several individual texture attributes, astringency type should not be used a priori to screen-in or -out persimmon cultivars for processing into a hot-air-dried product.

Association between histopathological changes and expression of selected microRNAs in skin of adult patients with IgA vasculitis.

AIMS: IgA vasculitis (IgAV) is a common small-vessel systemic vasculitisthat is histologically characterised by granulocyte infiltration and IgA deposition in vessel walls. Information on microRNA (miRNA) involvement inIgAVis limited. The aim of this study was to analyse the association between histopathological changes and expression profiles of 14 miRNAs in the affected skin of 70 adult patients with IgAV. METHODS AND RESULTS: miRNA expression analysis was performed by quantitative real-time polymerase chain reaction and evaluation of histopathological changes by light and immunofluorescence microscopy on formalin-fixed paraffin-embedded skin excision samples. In IgAV-affected skin, granulocyte infiltration was significantly associated with vessel fibrinoid necrosis. Of the analysed miRNAs, four showed two-fold increased expression (let-7d, let-7f, miR-21-5p, and miR-203-3p), five showed five-fold increased expression (let-7b, miR-17-5p, miR-155-5p, miR-423-5p, and miR-451a), and threeshowed 15-fold increased expression (let-7a, miR-21-3p, miR-223-3p), as compared with controls (all P < 0.001). miR-146a-5p and miR-148b-3p showed three-fold decreased expression (P = 0.981 and P < 0.001). The expression of miR-223-3p also showed a significant positive association with granulocyte infiltration and fibrinoid necrosis. CONCLUSIONS: Altered miRNA expression, especially of miRNA-223-3p, may be associated with the skin inflammatory state in IgAV. The majority of aberrantly expressed miRNAs in IgAV-affected skin are known to influence the nuclear factor-κB signalling pathway, which is crucial for activation of key proinflammatory genes, including those encoding tumour necrosis factor-α, interleukin (IL)-6, and IL-8. Furthermore, miR-146a-5p and miR-148b-3p, which are negative regulators of inflammatory gene expression, showed decreased expression and could contribute to the exaggerated inflammation. Further investigation of miRNA expression in the affected tissues could improve our knowledge of IgAV pathogenesis, and possibly help to identify novel biomarkers in body fluids.

Synthesis of descriptive sensory attributes and hedonic rankings of dried persimmon (Diospyros kaki sp.).

This work aimed to characterize the sensory attributes of hot air-dried persimmon (Diospyros kaki) chips, correlate these attributes with consumer hedonic information, and, by doing so, present recommendations for cultivars that are most suitable for hot-air drying. A trained sensory panel evaluated dried persimmon samples (representing 40 cultivars) for flavor, taste/aftertaste, and texture. In addition, in each of two tests conducted in different years, more than 100 consumers provided hedonic evaluations of 21 unique samples in a ranking task with a balanced incomplete block design. A partial least squares regression model correlating the mean hedonic ranking to the trained panel data was developed using the data from the first consumer panel. The predictions from the model were correlated with the second panel to verify the model. It was found that including taste, aftertaste, and texture data (but not specific flavor attribute data) produced a predictive model (Spearman's ρ=0.83). This indicates that flavor is likely secondary to taste and texture in dried persimmon chips. Using the validated predictive model, 6 of the 40 persimmon cultivars tested are recommended for a dried chip product; these cultivars are 'Fuyu', 'Lycopersicon', 'Maekawa Jiro', 'Nishimura Wase', 'Tishihtzu', and 'Yotsumizo'.

Vitamin D-fortified chitosan films from mushroom waste.

Brown mushroom (Agaricus bisporus) stalk bases from mushroom waste were treated with UV-B light to rapidly increase vitamin D2 content. Chitin was also recovered from this waste and converted into chitosan by N-deacetylation. FTIR spectra showed that the mushroom chitosan were similar to chitosan from animal sources. Chitosan films were prepared using high molecular weight (HW), low molecular weight (LW) and fungal chitosan. UV-B treated mushroom particles were also incorporated into fungal chitosan films. The fungal chitosan films showed similar density, porosity and water vapor barrier properties to the LW and HW chitosan films. However, fungal chitosan films were more hydrophobic and less flexible than the LW and HW chitosan films. Addition of mushroom particles did not significantly affect mechanical or water barrier properties of the fungal chitosan films.

Membrane-Filtered Olive Mill Wastewater: Quality Assessment of the Dried Phenolic-Rich Fraction.

A current trend in olive mill wastewater (OMWW) management is to not only decrease environmental pollution but also to extract and utilize valuable by-products. Therefore, the objectives of this study were to explore different techniques for drying a phenolic-rich membrane filtration fraction of OMWW and compare the techniques in terms of the dried product quality and feasibility of the process. The OMWW from 2 (3-phase and 2-phase) California mills was subjected to a 2-step membrane filtration process using a novel vibratory system. The reverse osmosis retentate (RO-R) is a phenolic-rich coproduct stream, and the reverse osmosis permeate is a near-pure water stream that could be recycled into the milling process. Spray-, freeze-, and infrared-drying were applied to obtain solid material from the RO-R. Drying of the RO-R was made possible only with addition of 10% maltodextrin as a carrier. The total soluble phenolics in dried RO-R were in the range 0.15 to 0.58 mg gallic acid equivalents/g of dry weight for 2-phase RO-R, and 1.38 to 2.17 mg gallic acid equivalents/g of dry weight for the 3-phase RO-R. Spray-dried RO-R from 3-phase OMWW showed remarkable antioxidant activity. Protocatechuic acid, tyrosol, vanillic acid, and p-coumaric acid were quantified in all dried RO-R, whereas 3-hydroxytyrosol was found in 3-phase dried RO-R. This combination of separation and drying technologies helps to add value and shelf-stability to an olive oil by-product and increase environmental sustainability of its production.

Comparative effect of thymol or its glucose conjugate, thymol-ß-D-glucopyranoside, on Campylobacter in avian gut contents.

Campylobacter jejuni is an important human food-borne pathogen that can contaminate meat and poultry during processing. Consequently, strategies are sought to reduce the carriage of C. jejuni in food animals before they arrive at the abattoir. Thymol is a natural product that reduces survivability of Campylobacter in vitro, but its rapid absorption from the proximal alimentary tract limits its bactericidal efficacy in vivo. Thymol-ß-D-glucopyranoside is more resistant to absorption than free thymol, but its administration to chickens has not been reported. In the present studies, 1 mM thymol-ß-D-glucopyranoside was shown to exhibit near equal anti-Campylobacter activity as 1 mM thymol when incubated anaerobically in avian crop or cecal contents in vitro, resulting in reductions of 1.10-2.32 log10 colony forming units mL(-1) in C. jejuni concentrations after 24 h incubation. In a follow-up live animal study, oral administration of thymol-ß-D-glucopyranoside, but not free thymol, significantly lowered (>10-fold) recovery of Campylobacter from the crop of market-aged broilers when compared to placebo-treated controls (n = 6 broilers/treatment). Neither thymol-ß-D-glucopyranoside nor thymol affected recovery of Campylobacter from cecal contents of the treated broilers. These results indicate that rapid absorption or passage of free thymol from the crop precluded its anti-Campylobacter activity at this site and throughout the entire gastrointestinal tract. Conversely, lower recovery of Campylobacter from the crop of birds treated with thymol-ß-D-glucopyranoside indicates this conjugate was retained and able to be hydrolyzed to biologically active free thymol at this site as intended, yet was not sufficiently protected to allow passage of efficacious amounts of the intact glycoside to the lower gut. Nevertheless, these results warrant further research to see if higher doses or encapsulation of thymol-ß-D-glucopyranoside or similar glycosides may yield an efficacious additive to reduce carriage of Campylobacter as well as other pathogens throughout the avian gut.

Comparison of anti-Campylobacter activity of free thymol and thymol-ß-D-glucopyranoside in absence or presence of ß-glycoside-hydrolysing gut bacteria.

Thymol is a natural product that exhibits antimicrobial activity in vitro but in vivo results indicate that absorption within the proximal alimentary tract precludes its delivery to the distal gut. Presently, the anti-Campylobacter activity of thymol was compared against that of thymol-ß-D-glucopyranoside, the latter being resistant to absorption. When treated with 1 mM thymol, Campylobacter coli and jejuni were reduced during pure or co-culture with a ß-glycoside-hydrolysing Parabacteroides distasonis. Thymol-ß-D-glucopyranoside treatment (1 mM) did not reduce C. coli and jejuni during pure culture but did during co-culture with P. distasonis or during mixed culture with porcine or bovine faecal microbes possessing ß-glycoside-hydrolysing activity. Fermentation acid production was reduced by thymol-ß-D-glucopyranoside treatment, indicating that fermentation was inhibited, which may limit its application to just before harvest. Results suggest that thymol-ß-D-glucopyranoside or similar ß-glycosides may be able to escape absorption within the proximal gut and become activated by bacterial ß-glycosidases in the distal gut.

Ex vivo absorption of thymol and thymol-ß-D-glucopyranoside in piglet everted jejunal segments.

Food-producing animals are reservoirs of Campylobacter, a leading bacterial cause of human foodborne illness. The natural product thymol can reduce the survivability of Campylobacter, but its rapid absorption in the proximal gastrointestinal tract may preclude its use as a feed additive to reduce intestinal colonization of these pathogens. This work examined the ex vivo absorption of thymol and thymol-ß-d-glucopyranoside in everted porcine jejunal segments, as the latter was hypothesized to be more resistant to absorption. A modified gas chromatography and extraction method was developed to determine 1.0-500 mg/L thymol. From 1 and 3 mM solutions, 0.293 ± 0.04 and 0.898 ± 0.212 mM thymol, respectively, p = 0.0347, were absorbed, and 0.125 ± 0.041 and 0.317 ± 0.143 mM thymol-ß-d-glucopyranoside, respectively, p = 0.0892, were absorbed. Results indicate that thymol-ß-d-glucopyranoside was absorbed 2.3 to 2.8 times less effectively than thymol, thus providing evidence that thymol-ß-d-glucopyranoside may potentially be used as a feed additive to transport thymol to the piglet lower gut.

Lipid oxidative changes in chitosan-oregano coated traditional dry fermented sausage Petrovská klobása.

The effect of a chitosan coating with added essential oil of oregano (Origanum vulgare) on lipid oxidation of dry fermented sausage (Petrovská klobása) was investigated. Fatty acid profile, aldehyde contents and sensory analysis of odor and flavor were determined after drying and during seven months of storage. Between coated and control sausage, a difference was observed after two months storage in fatty acid profiles (myristic, oleic and linoleic acids), but after seven months storage there was no difference. Decrease in polyunsaturated acid content was observed (from 17.25% to 15.70%), as well as an increase in total aldehydes (from 4.54 µg/g to 31.80 µg/g), due to lipid oxidation during storage. After seven months storage, the content of most aldehydes was significantly lower in coated sausage than in the control. Sensory characteristics of odor and flavor were better for coated sausage, after seven months of storage. Results suggest that chitosan-oregano coating can be successfully applied to protect dry fermented sausages from lipid oxidation.

Buckwheat (Fagopyrum esculentum Moench) grain and fractions: antioxidant compounds and activities.

UNLABELLED: Buckwheat (Fagopyrum esculentum Moench) is an alternative crop belonging to the Polygonaceae family. In comparison to antioxidant activity of frequently used cereals, buckwheat has been reported to possess higher antioxidant activity (AOA), mainly due to high rutin content. The objective of this work was to determine the main antioxidant compounds and AOA of buckwheat grain fractions (whole grain, hull, and groat). Buckwheat grain fractions were extracted with ethanol/water (80/20, v/v), followed by determination of total phenolic and flavonoid content. Quantification of phenolic compounds and tocopherols was performed by high-performance liquid chromatography (HPLC). The AOA was estimated by 2 direct electron spin resonance (ESR) and 4 indirect (spectrophotometric) tests. Significantly higher contents of total phenolics and total flavonoids were found in buckwheat hull than in whole grain and groat. Protocatechuic, syringic, and sinapic acid, rutin, and quercetin were found in all tested fractions, whereas vanilic acid was found in whole grain and hull. The content of total tocopherols in investigated samples ranged from 23.3 mmol/g for hull to 61.8 mmol/g for groat. Hull was superior in scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH(•)), hydroxyl ((•)OH), and superoxide anion (O(2)(•-)) radicals, reducing activity, AOA by ß-carotene bleaching method, and chelating activity on Fe(2+) as evidenced by its lower IC(50) value. Obtained results can broaden the utilization of buckwheat, especially a share of hull in whole grain flour production. PRACTICAL APPLICATION: Obtained results suggest possibility to supplement the whole grain buckwheat flour with hull, which leads toward better usage of by-products in buckwheat production, and enhancement of antioxidant potential of the final product.

Health-promoting phytochemicals of Italian common wheat varieties grown under low-input agricultural management.

BACKGROUND: The increasing interest in organic food products and environmental friendly practices has emphasised the importance of selecting crop varieties suitable for the low-input sector. Moreover, in recent years the relationship between diet and human health has gained much attention among consumers. The aim of this study was to comparatively evaluate the agronomic performance and the nutrient and phytochemical composition of old and modern Italian wheat genotypes grown under low-input management. RESULTS: Research highlighted that several old wheat genotypes were comparable to the modern ones in terms of agronomic performance and nutrient content. Genotype and environmental conditions (growing season), as well as their interaction, significantly affected the phytochemical composition of wheat grains for most of the analysed bioactive compounds. High variability was observed among the wheat genotypes for dietary fibre (154.7-183.3 g kg⁻¹), polyphenol (1.94-2.77 mg g⁻¹), tocopherol (9.1-21.2 mg kg⁻¹) and carotenoid (701.4-3243 µg kg⁻¹) content. CONCLUSION: The comparative study of old and modern wheat varieties highlighted that, under low-input conditions, ancient genotypes may equal modern ones in terms of agronomic traits and additionally provide nutraceutical value-added wheat grains. The most promising ancient varieties for the unique phytochemical profiles are Gentil rosso, Marzuolo d'aqui and Verna.

Antioxidant activity of medicinal plant extracts in cookies.

UNLABELLED: The antioxidant activity of ethanolic extracts of parsley, buckthorn, mint, caraway, and their mixture "Vitalplant" was evaluated, and the potential of "Vitalplant" mixture extract to retard the process of lipid oxidation in cookies was tested. The antioxidant activity was estimated by 2 direct (ESR) and 4 indirect (spectrophotometric) tests and correlated with the total phenolic and flavonoid content. The potential of "Vitalplant" mixture extract to retard the process of lipid oxidation in cookies was measured by thiobarbituric acid (TBA)-reactive-substances assay (TBARS) and DPPH˙ (1,1-diphenyl-2-picryl-hydrazyl) test. Significantly different (P < 0.05) amounts of total phenolics were found among extracts in the following order: mint > buckthorn > "Vitalplant" > parsley > caraway. Total flavonoid content varied from 0.510% (parsley) to 2.05% ("Vitalplant"). A statistically significant correlation was found between IC(50) values on DPPH˙ and total flavonoid content of the samples (r=- 0.94, P < 0.05) and between IC(50) values on DPPH˙ and IC(50) values on O(2) (·-) scavenging activity (r= 0.89, P < 0.05). A correlation between reducing activity and total phenolic content was found to be positive and statistically significant (r= 0.94, P < 0.05). "Vitalplant" mixture exhibited a relatively high antioxidant activity in most of the tests, which can be explained by synergistic effects of the ingredients of which it is composed. Finally, "Vitalplant" extract addition (2%, 4%, and 6%) improved antioxidant activity and oxidative stability of the cookies in dose-dependent manner. PRACTICAL APPLICATION: Supplementation of cookies with a mixture of Petroselini fructus, Frangulae cortex, Mentha piperitae folium, Carvi fructus can retard the process of lipid oxidation and elevate antioxidant activity of the final product.