Biological Properties of Bee Bread Collected from Apiaries Located across Greece.

Bee bread is the only fermented product of the beehive. It constitutes the main source of proteins, lipids, vitamins, and macro- and microelements in honeybee nutrition and it exerts antioxidant and antimicrobial properties, though research on these aspects has been limited so far. In this study 18 samples of Greek bee bread, two of which were monofloral, were collected during different seasons from diverse locations such as Crete and Mount Athos and were tested for their bioactivity. Samples were analyzed for their antibacterial properties, antioxidant activity, total phenolic content (TPC), and total flavonoid content (TFC). The antimicrobial activity of each sample was tested against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Salmonella typhimurium. Our data demonstrate that all samples exert inhibitory and most of them bactericidal activity against at least two pathogens. Furthermore, all samples exert significant antioxidant activity, where the monofloral Castanea Sativa sample demonstrated superior antioxidant activity. Nevertheless, the antioxidant and antimicrobial activity were not strongly correlated. Furthermore, machine learning methods demonstrated that the palynological composition of the samples is a good predictor of their TPC and ABTS activity. This is the first study that focuses on the biological properties of Greek bee bread and demonstrates that bee bread can be considered a functional food and a possible source of novel antimicrobial compounds.

Transcriptomic Analysis of Pseudomonas aeruginosa Response to Pine Honey via RNA Sequencing Indicates Multiple Mechanisms of Antibacterial Activity.

Pine honey is a unique type of honeydew honey produced exclusively in Eastern Mediterranean countries like Greece and Turkey. Although the antioxidant and anti-inflammatory properties of pine honey are well documented, few studies have investigated so far its antibacterial activity. This study investigates the antibacterial effects of pine honey against P. aeruginosa PA14 at the molecular level using a global transcriptome approach via RNA-sequencing. Pine honey treatment was applied at sub-inhibitory concentration and short exposure time (0.5× of minimum inhibitory concentration -MIC- for 45 min). Pine honey induced the differential expression (>two-fold change and p ≤ 0.05) of 463 genes, with 274 of them being down-regulated and 189 being up-regulated. Gene ontology (GO) analysis revealed that pine honey affected a wide range of biological processes (BP). The most affected down-regulated BP GO terms were oxidation-reduction process, transmembrane transport, proteolysis, signal transduction, biosynthetic process, phenazine biosynthetic process, bacterial chemotaxis, and antibiotic biosynthetic process. The up-regulated BP terms, affected by pine honey treatment, were those related to the regulation of DNA-templated transcription, siderophore transport, and phosphorylation. Pathway analysis revealed that pine honey treatment significantly affected two-component regulatory systems, ABC transporter systems, quorum sensing, bacterial chemotaxis, biofilm formation and SOS response. These data collectively indicate that multiple mechanisms of action are implicated in antibacterial activity exerted by pine honey against P. aeruginosa.

Biofunctional Feed Supplemented With By-products of Olive Oil Production Improves Tissue Antioxidant Profile of Lambs.

BACKGROUND/AIM: Olive mill wastewater (OMW) is a byproduct of olive oil production. The aim of the study was to estimate the redox profile of lambs' vital organs after consumption of an OMW-supplemented feed. MATERIALS AND METHODS: Twenty-four lambs received breast milk until day 15. Then, they were divided in two groups: control and OMW, n=12 each. The control group received standard ration, while the OMW group received OMW enriched feed along with mother's milk until day 42 and animals (n=6 per group) were sacrificed. The remaining 12 received the feeds until day 70 and sacrificed. Tissue samples were collected at day 42 and 70 and specific redox biomarkers were assessed. RESULTS: Overall, the OMW feed improved tissue redox profile by affecting the glutathione S-transferase (GST) activity and γ-glutamate-cysteine ligase (γ-GCL) expression in all tested tissues. Superoxide dismutase (SOD) activity was not affected. CONCLUSION: The polyphenol-rich byproduct reinforced lamb redox profile and may putatively improve their wellness and productivity.

Polyphenolic Composition of Rosa canina, Rosa sempervivens and Pyrocantha coccinea Extracts and Assessment of Their Antioxidant Activity in Human Endothelial Cells.

The aim of the present study was the investigation of the antioxidant activity of plant extracts from Rosa canina, Rosa sempervivens and Pyrocantha coccinea. The results showed that the bioactive compounds found at higher concentrations were in the R. canina extract: hyperoside, astragalin, rutin, (+)-catechin and (-)-epicatechin; in the R. sempervirens extract: quinic acid, (+)-catechin, (-)-epicatechin, astragalin and hyperoside; and in the P. coccinea extract: hyperoside, rutin, (-)-epicatechin, (+)-catechin, astragalin, vanillin, syringic acid and chlorogenic acid. The total polyphenolic content was 290.00, 267.67 and 226.93 mg Gallic Acid Equivalent (GAE)/g dw, and the total flavonoid content 118.56, 65.78 and 99.16 mg Catechin Equivalent (CE)/g dw for R. caninna, R. sempervirens and P. coccinea extracts, respectively. The extracts exhibited radical scavenging activity in DPPH and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)•⁺ assays and protection from ROO•-induced DNA damage in the following potency order: R. canina > R. sempervirens > P. coccinea. Finally, treatment with R. canina and P. coccinea extract significantly increased the levels of the antioxidant molecule glutathione, while R. canina extract significantly decreased Reactive Oxygen Species (ROS) in endothelial cells. The results herein indicated that the R. canina extract in particular may be used for developing food supplements or biofunctional foods for the prevention of oxidative stress-induced pathological conditions of endothelium.

Effects of Dietary Grape Pomace Supplementation on Performance, Carcass Traits and Meat Quality of Lambs.

BACKGROUND/AIM: A previous study revealed that the inclusion of grape pomace (GP) in the diet for growing lambs had beneficial effects on the redox status and fecal microbiota. Herein, we investigated the effect of GP inclusion on performance, carcass traits and fatty acid composition of meat. MATERIALS AND METHODS: In the experimental trial of 55 days, lambs were fed with standard or diet supplemented with GP. Performance, carcass traits and fatty acid profile of quadriceps muscle were assessed. RESULTS: GP inclusion in the diet improved growth performance, since the average daily gain was significantly increased by 2-fold in GP group. Regarding the fatty acid composition of meat, GP inclusion significantly increased the content of long chain n-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid, and reduced the n-6/n-3 ratio compared to the control group. CONCLUSION: GP supplementation in lamb diet may improve performance and may have beneficial effects on meat quality.

Novel Feed Including Olive Oil Mill Wastewater Bioactive Compounds Enhanced the Redox Status of Lambs.

BACKGROUND/AIM: The aim of the present study was to investigate the antioxidant effects of a feed supplemented with polyphenolic additives from olive mill wastewater (OMW) on lambs. MATERIALS AND METHODS: Lambs received breast milk until the postnatal period, and then they were divided into two groups and received control and OMW feed for 55 days. Redox biomarkers were measured in blood and tissues at days 15, 42 and 70 after feeding. RESULTS: Feed supplemented with OMW reduced thiobarbituric acid reactive species and protein carbonyls and increased total antioxidant capacity, glutathione and catalase activity in both blood and tissues. CONCLUSION: The administration of OMW-containing feed reinforced the antioxidant defense of lambs, which may improve their wellbeing and productivity. Additionally, this exploitation of OMW may solve problems of environmental pollution in areas with olive oil industries.

Enhancement of Antioxidant Mechanisms and Reduction of Oxidative Stress in Chickens after the Administration of Drinking Water Enriched with Polyphenolic Powder from Olive Mill Waste Waters.

The aim of the study was to examine the effects of a polyphenolic powder from olive mill wastewater (OMWW) administered through drinking water, on chickens' redox status. Thus, 75 chickens were divided into three groups. Group A was given just drinking water, while groups B and C were given drinking water containing 20 and 50 µg/ml of polyphenols, respectively, for 45 days. The antioxidant effects of the polyphenolic powder were assessed by measuring oxidative stress biomarkers in blood after 25 and 45 days of treatment. These markers were total antioxidant capacity (TAC), protein carbonyls (CARB), thiobarbituric acid reactive species (TBARS) and superoxide dismutase activity (SOD) in plasma, and glutathione (GSH) and catalase activity in erythrocytes. The results showed that CARB and TBARS were decreased significantly in groups B and C, and SOD decreased in group B compared to that in group A. TAC was increased significantly in group C and GSH was increased in group B, while catalase activity was increased in groups B and C compared to that in group A. In conclusion, this is the first study showing that supplementation of chickens with polyphenols from OMWW through drinking water enhanced their antioxidant mechanisms and reduced oxidative stress-induced damage.

Tissue specific effects of feeds supplemented with grape pomace or olive oil mill wastewater on detoxification enzymes in sheep.

The aim of the present study was to investigate the effects of livestock feed supplemented with grape pomace (GP) or olive oil mill wastewater (OMW) byproducts on the enzymatic activity and protein expression of antioxidants enzymes, in liver and spleen tissue of sheep. Thus, 36 male sheep of Chios breed were divided into 3 homogeneous groups, control group (n = 12), GP group (n = 12) and OMW group (n = 12), receiving standard or experimental feed. Liver and spleen tissues were collected at 42 and 70 days post-birth. The enzymatic activity of superoxide dismutase (SOD) and glutathione-s-transferase (GST) and also the protein expression of γ-synthase glutamyl custeine (γ-GCS) were determined in these tissues. The results showed GP group exhibited increased enzymatic activity of GST and protein expression of γ-GCS in liver compared to control group. In GP group's spleen, GST activity was increased compared to control but γ-GCS expression was not affected. In OMW group's liver, GST activity was increased and γ-GCS expression was reduced compared to control. In OMW group's spleen, GST activity was increased but GCS expression was not affected. SOD activity was not affected in both tissues either in GP or OMW group.

Novel feed including bioactive compounds from winery wastes improved broilers' redox status in blood and tissues of vital organs.

Currently, there is a great interest in the production of animal feed with antioxidant activity. The aim of this study was to examine the potential antioxidant effects of a feed supplemented with grape pomace (GP), a winery by-product with high environmental load, in chickens. Broilers of 15 days post birth were separated into two groups fed either with standard diet or with diet supplemented with GP for 35 days. Blood and tissues collections were performed after feeding for 15 and 35 days with the experimental diet (i.e. at 30 and 50 days post birth). Free radical toxicity markers, namely thiobarbituric acid reactive substances, protein carbonyls, total antioxidant capacity, reduced glutathione, catalase activity and rate of H2O2 decomposition were determined in blood and tissues of vital organs. The results indicated that feed supplemented with GP decreased oxidative stress-induced toxic effects and improved chickens' redox status, and so it may also improve their wellness and productivity. On the other hand, this exploitation of GP may solve problems of environmental pollution in areas with wineries.

Validation and optimization of a predictive model for radiation pneumonitis in patients with lung cancer.

The aim of the current retrospective study was to validate a predictive model for radiation pneumonitis (STRIPE) in an independent dataset and to investigate whether the addition of other potential risk factors could strengthen the accuracy of the model. Consecutive patients with non-small cell lung carcinoma (NSCLC; n=71) treated with definitive concurrent chemotherapy and radiotherapy were retrospectively assessed for radiation pneumonitis (RP). The results identified that 16 (23%) patients developed grade ≥2 RP. Furthermore, STRIPE score (intermediate vs. low risk) was independently associated with the development of RP [odds ratio (OR), 3.72; 95% confidence interval (CI), 1.00-13.89], whereas current smoking status was found to be protective against RP (OR, 0.09; 95% CI, 0.01-0.78). Similar discriminatory power of the STRIPE score was observed as in the original study. The addition of smoking status strengthened the model's discriminatory ability to predict RP. Thus, the addition of smoking status as a risk factor may strengthen the accuracy of the model for predicting RP in patients with NSCLC.

Antioxidant Effects of Sheep Whey Protein on Endothelial Cells.

Excessive production of reactive oxygen species (ROS) may cause endothelial dysfunction and consequently vascular disease. In the present study, the possible protective effects of sheep whey protein (SWP) from tert-butyl hydroperoxide- (tBHP-) induced oxidative stress in endothelial cells (EA.hy926) were assessed using oxidative stress biomarkers. These oxidative stress biomarkers were glutathione (GSH) and ROS levels determined by flow cytometry. Moreover, thiobarbituric acid-reactive substances (TBARS), protein carbonyls (CARB), and oxidized glutathione (GSSG) were determined spectrophotometrically. The results showed that SWP at 0.78, 1.56, 3.12, and 6.24 mg of protein mL(-1) increased GSH up to 141%, while it decreased GSSG to 46.7%, ROS to 58.5%, TBARS to 52.5%, and CARB to 49.0%. In conclusion, the present study demonstrated for the first time that SWP protected endothelial cells from oxidative stress. Thus, SWP may be used for developing food supplements or biofunctional foods to attenuate vascular disturbances associated with oxidative stress.

Assessment of eccentric exercise-induced oxidative stress using oxidation-reduction potential markers.

The aim of the present study was to investigate the use of static (sORP) and capacity ORP (cORP) oxidation-reduction potential markers as measured by the RedoxSYS Diagnostic System in plasma, for assessing eccentric exercise-induced oxidative stress. Nineteen volunteers performed eccentric exercise with the knee extensors. Blood was collected before, immediately after exercise, and 24, 48, and 72 h after exercise. Moreover, common redox biomarkers were measured, which were protein carbonyls, thiobarbituric acid-reactive substances, total antioxidant capacity in plasma, and catalase activity and glutathione levels in erythrocytes. When the participants were examined as one group, there were not significant differences in any marker after exercise. However, in 11 participants there was a high increase in cORP after exercise, while in 8 participants there was a high decrease. Thus, the participants were divided in low cORP group exhibiting significant decrease in cORP after exercise and in high cORP group exhibiting significant increase. Moreover, only in the low cORP group there was a significant increase in lipid peroxidation after exercise suggesting induction of oxidative stress. The results suggested that high decreases in cORP values after exercise may indicate induction of oxidative stress by eccentric exercise, while high increases in cORP values after exercise may indicate no existence of oxidative stress.

How can bioinformatics and toxicogenomics assist the next generation of research on physical exercise and athletic performance.

The past 2-3 decades have seen an explosion in analytical areas related to "omic" technologies. These advancements have reached a point where their application can be and are being used as a part of exercise physiology and sport performance research. Such advancements have drastically enabled researchers to analyze extremely large groups of data that can provide amounts of information never before made available. Although these "omic" technologies offer exciting possibilities, the analytical costs and time required to complete the statistical approaches are substantial. The areas of exercise physiology and sport performance continue to witness an exponential growth of published studies using any combination of these techniques. Because more investigators within these traditionally applied science disciplines use these approaches, the need for efficient, thoughtful, and accurate extraction of information from electronic databases is paramount. As before, these disciplines can learn much from other disciplines who have already developed software and technologies to rapidly enhance the quality of results received when searching for key information. In addition, further development and interest in areas such as toxicogenomics could aid in the development and identification of more accurate testing programs for illicit drugs, performance enhancing drugs abused in sport, and better therapeutic outcomes from prescribed drug use. This review is intended to offer a discussion related to how bioinformatics approaches may assist the new generation of "omic" research in areas related to exercise physiology and toxicogenomics. Consequently, more focus will be placed on popular tools that are already available for analyzing such complex data and highlighting additional strategies and considerations that can further aid in developing new tools and data management approaches to assist future research in this field. It is our contention that introducing more scientists to how this type of work can complement existing experimental approaches within exercise physiology and sport performance will foster additional discussion and stimulate new research in these areas.

Application of a new oxidation-reduction potential assessment method in strenuous exercise-induced oxidative stress.

OBJECTIVES: The aim of the study was to test a novel method for assessing oxidative stress, the RedoxSYS™ diagnostic system, a holistic, fast, minimally invasive, and requiring small sample volume method, that measures two parameters, the static (sORP) and the capacity (cORP) oxidation-reduction potential. METHODS: The redox status of 14 athletes participating in a mountain marathon race was assessed. Redox status in blood obtained 1 day before the race and immediately after the race was assessed using the RedoxSYS diagnostic system as well as conventional oxidative stress markers such as glutathione levels (GSH), catalase activity (CAT), thiobarbituric acid reactive substances (TBARS), protein carbonyls (CARB), and total antioxidant activity. RESULTS: The results showed that after the race, the sORP was increased significantly by 7% indicating oxidative stress induction, while cORP was decreased by 14.6% but not significantly. Moreover, the conventional oxidative stress markers GSH and CAT were decreased significantly by 13.1 and 23.4%, respectively, while TBARS and CARB were increased significantly by 26.1 and 15.6%, respectively, after the race indicating oxidative stress induction. DISCUSSION: The present study demonstrated for the first time that the RedoxSYS diagnostic system can be used for evaluating the exercise-induced oxidative stress in athletes.

R gene expression changes related to Cercospora hydrangeae L.

Nursery growing as well as common landscape hydrangeas are all susceptible to leaf spot fungus Cercospora hydrangeae. Warm and rainy weather causes the fungal spores to germinate quickly and spread over the plant leaves forming small purple or brown spots. Although Hydrangea plants are not killed by leaf spot, it detracts from the value of plants through the reduction of flowering and plant vigor. The aim of our study was to isolate, characterize and investigate the expression profile of Hydrangea macrophylla resistance (R) gene transcripts under C. hydrangeae fungus infection and examine their evolutionary relationships by phylogenetic analysis. R-genes are thought to be one of the components of the genetic resistance in plants and most of them encode nucleotide binding site-leucine rich repeat (NBS-LRR) proteins. A cDNA-NBS strategy was carried out using as template cDNAs isolated from control and infected plant leaves. The cDNA-NBS profiling gave an excellent bands reproducibility. Twenty new transcripts corresponding to NBS-LRR proteins were identified only in infected plants. The extent of positivity between the aminoacid sequences at NBS region varied from 45 to 90 %, which indicates the diversity among the RGAs. The results of this paper will provide a genomic framework for the further isolation of candidate disease resistance NBS-encoding genes in Hortensia, and contribute to the understanding of the evolutionary mode of NBS-encoding genes in Hydrangeaceae crops.

DGGE analysis of buffalo manure eubacteria for hydrogen production: effect of pH, temperature and pretreatments.

Buffalo dung is a low-cost substrate with plenty of carbohydrates, an optimal carbon/nitrogen ratio, and a rich microbial flora, and could become a valuable source of biogas. Therefore, in the present study we compared the type and amount of specific eubacteria to the different configurations of pH, temperature and thermal pretreatment after fermentation in batch reactors in order to understand the suitability of buffalo manure for hydrogen production. The phylogenetic structure of the microbial community in fermentation samples was studied using denaturing gradient gel electrophoresis to generate fingerprints of 16S rRNA genes. The sequences analysis revealed abundance of the phyla Bacteroidetes and Firmicutes, and in particular of the order Clostridiales. Very active hydrogen producing bacteria belonging to Clostridium cellulosi species were identified demonstrating the suitability of this substrate to produce hydrogen. Moreover, a large fraction of 16S-rDNA amplicons could not be assigned to lower taxonomic ranks, demonstrating that numerous microorganisms involved in anaerobic fermentation in digesters or bioreactors are still unclassified or unknown.

cDNA cloning and differential expression patterns of ascorbate peroxidase during post-harvest in Brassica rapa L.

Ascorbate is an antioxidant and a cofactor of many dioxygenases in plant and animal cell metabolism. A well-recognized enzyme consuming ascorbate is ascorbate peroxidase (APX), which catalyses the reduction of hydrogen peroxide to water with the simultaneous oxidation of ascorbate with a high specificity. The isolation and characterisation of new Apx cDNAs, could provide new insights about the physiological roles and regulation of these enzymes. In this work chloroplastic (Br-chlApx) and cytosolic (Br-cApx) isoform transcripts were isolated by RT-PCR in Brassica rapa and expression changes were analysed by semi-quantitative RT-PCR performed in different tissues (layer, stalk and florets) at different days (0, 4 and 14 day). The result showed that BrApx isoforms were differentially expressed and the Br-chlApx, in particular in the layer, had the highest expression level and remained unchanged also after 14 day after harvest. In addition, expression changes were compared with total BrAPX activity and the results showed that the activity decreased in all tissues at 14 day after harvest, independently of transcripts. Finally, additional solutes as the substrate of APX ascorbate and its oxidized form, dehydroascorbate, as well as α-tocopherol, the major vitamin E compound that prevents the propagation of lipid peroxidation in thylakoid membranes, were followed. The changes in the BrApx expression, BrAPX activity and metabolites can provide further evidence of the close relationships that exist between antioxidants which compensate for each other and suggest that there are multiple sites of reciprocal control.

A PCR based SNPs marker for specific characterization of English walnut (Juglans regia L.) cultivars.

English walnut (Juglans regia L.) is the most economically important species from all the 21 species belonging to the genus Juglans and is an important and healthy food as well as base material for timber industry. The aim of this study was to develop a simple technique for specific characterization of English walnut using DNA method. The first and second internal transcribed spacers (ITS1 and ITS2) as well as the intervening 5.8S coding region of the rRNA gene for 18 cultivars of J. regia L. isolated from different geographic origins were characterized. The size of the spacers sequences ranged from 257 to 263 bases for ITS1 and from 217 to 219 bases for ITS2. Variation of GC contents has also been observed and scored as 55-56.7 and 57.1-58.9% for ITS1 and ITS2, respectively. This data exhibited the presence of polymorphism among cultivars. Alignment of the ITS1-5.8S-ITS2 sequences from 18 walnut cultivars showed that there were 244 single nucleotide polymorphisms (SNPs) and 1 short insertion-deletion (indel) at 5' end ITS1. Amplification refractory mutation system strategy was successfully applied to the SNP markers of the ITS1 and ITS2 sequences for the fingerprinting analysis of 17 on 18 walnut cultivars. The prediction of ITS1 and ITS2 RNA secondary structure from each cultivar was improved by detecting key functional elements shared by all sequences in the alignments. Phylogenetic analysis of the ITS1-5.8S-ITS2 region clearly separated the isolated sequences into two clusters. The results showed that ITS1 and ITS2 region could be used to discriminate these walnut cultivars.

Polymorphism of a new Ty1-copia retrotransposon in durum wheat under salt and light stresses.

Long terminal repeat retrotransposons are the most abundant mobile elements in the plant genome and play an important role in the genome reorganization induced by environmental challenges. Their success depends on the ability of their promoters to respond to different signaling pathways that regulate plant adaptation to biotic and abiotic stresses. We have isolated a new Ty1-copia-like retrotransposon, named Ttd1a from the Triticum durum L. genome. To get insight into stress activation pathways in Ttd1a, we investigated the effect of salt and light stresses by RT-PCR and S-SAP profiling. We screened for Ttd1a insertion polymorphisms in plants grown to stress and showed that one new insertion was located near the resistance gene. Our analysis showed that the activation and mobilization of Ttd1a was controlled by salt and light stresses, which strengthened the hypothesis that stress mobilization of this element might play a role in the defense response to environmental stresses.

Reversible dilated cardiomyopathy due to subclinical hyperthyroidism.

We present a patient without primary heart disease in whom subclinical hyperthyroidism was accompanied by manifestations of dilated cardiomyopathy, as evaluated by echocardiography, coronary angiography, and radionuclide ventriculography. His condition was reversed 6 months after conventional treatment (furosemide, carvedilol, angiotensin-converting-enzyme inhibitor and thiamazole administration). This patient represents an exceptional case, as overt congestive heart failure with left ventricular dilatation and depressed ventricular ejection fraction is not a common finding in patients with hyperthyroidism, let alone patients with subclinical hyperthyroidism and no underlying heart disease.