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This study was designed to investigate chemical and antioxidant properties, as well as the antimicrobial and antibiofilm behaviour of Cinnamomum cassia essential oil (CCEO). MALDI-TOF MS Biotyper mass spectrometry was applied to evaluate the biofilms of Stenotrophonomonas maltophilia and Bacillus subtilis, while the antibiofilm ability of CCEO was assessed on wooden and glass surfaces. The antimicrobial activity by disc diffusion method, microdilution method, and vapour phase for two biofilm-producing bacteria and three Penicillium spp. were used. Antimicrobial and antibiofilm properties were assessed using the agar microdilution protocol. The vapour phase of Penicillium citrinum, P. crustosum, P. expansum, S. maltophilia, and B. subtilis on bread, carrot, potato, sweet potato, and apple in situ was studied. Specific molecular variations related to the biofilm formation and genetic analogies were evaluated with MSP spectra dendrograms of S. maltophilia and B. subtilis profiles were grown on different days. The results of disc diffusion and broth diffusion methods showed that CCEO was strongly effective against all tested microorganisms and the vapour phase method was effective and active against all Penicillium spp., but not strongly effective against bacteria in food preservation of food matrices.

Inter primer binding site (iPBS) polymorphism was investigated for common ivy (Hedera helix, L.) to obtain the knowledge on genetic diversity in this species. Actually, a very limited information exists about application of DNA markers in Hedera helix. Natural and planted European populations of ivy were analysed using an iPBS marker 5′ACCTGGCGTGCCA3′ with a total number of 238 fragments generated. Of these, 86% were polymorphic. There were determined certain attributes of this marker such as the diversity index (DI) and polymorphism information content (PIC). The value of the diversity index was 0.79 and the polymorphic information index was 0.78. The proportion of polymorphisms of the individual amplified loci ranged from 0.32% to 6.98%. Cluster analysis was performed to determine the relationships among the European ivy populations where the distribution in the dendrogram under the habitat specifity was found for the used iPBS marker. We concluded that iPBS was very efficient in analysing the genetic diversity in Hedera helix, L. and that this marker can serve as a suitable tool to find genomically specific fingerprints relevant to the factors influencing the distribution of genetic variation.

Ginkgo biloba L. is characterized by its high level of resistance to climatic conditions, diseases, and pests. In Slovakia, there is a rich collection of genetic resources of ginkgo consisting of 288 trees growing in 103 locations and providing valuable biological material for scientific research. There have been documented 45 trees of ginkgo older than 100 years (ranging from 112 to 242 years of age). Their dendrometrical parameters were recorded. For genomic imprinting, three types of microRNA-based markers were selected; highly conserved gb-miR160, moderately conserved gb-miR482 and the species-specific gb-miR75. The most efficient one can be considered the marker gb-miR482 with its genotype-unique miRNA profiles probably related to this marker functioning in the defence mechanisms of the ginkgo species. Unique miRNA loci were recorded in genomes of young ginkgo trees. We found that, by selecting the appropriate microRNA-based markers, it is possible to characterize the ginkgo genome in the context of microclimatic conditions.

Molecular markers ISSR were used to analyse γ-irradiated mutants of amaranth Ficha cultivar and K-433 hybrid; tested six microsatellite - primed PCR markers. First analysis of gamma-irradiated amaranth mutant lines was done using the ISSR. In the study, primers that distinguish the mutant lines from control plant were reported as well as primer with the differentiation ability for all analysed mutant lines. The very specific changes in the mutant lines' non-coding regions based on inter microsatellite length polymorphism were analysed. Mutant lines of the Ficha cultivar (C15, C26, C27, C82, C236) shared a genetic dissimilarity of 0.16 and their ISSR profiles were more similar to the Ficha than those of K-433 hybrid mutant lines. The K-433 mutant lines (D54, D279, D282) shared genetic dissimilarity of 0.22 but are more distinct to their control plant as a whole, as those of the Ficha mutant lines. Different ISSR fingerprints patters of the mutant lines when compared to the Ficha cultivar and K-433 hybrid ISSR specific profiles may be part of a consequence of the complex response of the intergenic space of mutant lines to the gamma-radiance. [ABSTRACT FROM AUTHOR]

Physiological and molecular marker-based changes were studied in the tissues of two-year-old Pyrus pyraster (L.) Burgsd. seedlings under salt treatment. For 60 days, 5 mL of 100 mM NaCl solution was applied to each plant per day to a cumulative volume of 300 mL in the substrate. In response to osmotic stress, the seedlings increased their water use efficiency (WUE) on day 20 of regular NaCl application and maintained a stable net photosynthetic rate (A n ) per unit area. Under conditions of increasing salinity, the young plants maintained a balanced water regime of the leaf tissues (Ψ wl ). The seedlings invested mass to their root growth (R/S), retained a substantial portion (72%) of Na + ions in the roots, and protected their leaves against intoxication and damage. A significant decrease in the leaf gas exchange parameters (g s , E, A n ) was manifested on day 60 of the experiment when the cumulative NaCl intake was 300 mL per plant. The variability in the reactions of the seedlings to salinity is related to the use of open-pollinated progeny (54 genotypes) in the experiment. Lus-miR168 showed tissue- and genotype-specific genome responses to the applied stress. Polymorphic miRNA-based loci were mostly detected in the root samples on the 20th and 35th days of the experiment. The cumulative effect of the salt treatment was reflected in the predominance of polymorphic loci in the leaves. We can confirm that miRNA-based markers represent a sensitive detection tool for plant stress response on an individual level. The screening and selection of the optimal type of miRNA for this type of research is crucial. The cytochrome P450-Based Analog (PBA) techniques were unable to detect polymorphism among the control and treated seedlings, except for the primer pair CYP2BF+R, where, in the roots of the stressed plant, insertions in the amplicons were obtained. The expression ratios of cytochrome P450 in the salt-stressed plants were higher in the roots in the case of 20/100 mL and in the leaves with higher doses. The observed physiological and molecular responses to salinity reflect the potential of P. pyraster seedlings in adaptation to osmotic and ionic stress.

Melissa officinalis L., a well-known herb with diverse industrial and ethnopharmacological properties. Although, there has been a significant lack in the breeding attempts of this invaluable herb. This study aimed to enhance the agronomical traits of M. officinalis through in vitro polyploidization. Nodal segments were micropropagated and subjected to oryzalin treatment at concentrations of 20, 40, and 60 mM for 24 and 48 hours. Flow cytometry, chromosome counting, and stomatal characteristics were employed to confirm the ploidy level of the surviving plants. The survival rate of the treated explants decreased exponentially with increasing oryzalin concentration and duration. The highest polyploid induction rate (8%) was achieved with 40 mM oryzalin treatment for 24 hours. The induced tetraploid plants exhibited vigorous growth, characterized by longer shoots, larger leaves, and a higher leaf count. Chlorophyll content and fluorescence parameters elucidated disparities in photosynthetic performance between diploid and tetraploid genotypes. Tetraploid plants demonstrated a 75% increase in average essential oil yield, attributed to the significantly larger size of peltate trichomes. Analysis of essential oil composition in diploid and tetraploid plants indicated the presence of three major components: geranial, neral, and citronellal. While citronellal remained consistent, geranial and neral increased by 11.06% and 9.49%, respectively, in the tetraploid population. This effective methodology, utilizing oryzalin as an anti-mitotic agent for polyploid induction in M. officinalis , resulted in a polyploid genotype with superior morpho-physiological traits. The polyploid lemon balm generated through this method has the potential to meet commercial demands and contribute significantly to the improvement of lemon balm cultivation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bharati, Gupta, Novy, Severová, Šrédl, Žiarovská and Fernández-Cusimamani.)

Cadmium (Cd) is a heavy metal that can cause damage to living organisms at different levels. Even at low concentrations, Cd can be toxic to plants, causing harm at multiple levels. As they are unable to move away from areas contaminated by Cd, plants have developed various defence mechanisms to protect themselves. Hyperaccumulators, which can accumulate and detoxify heavy metals more efficiently, are highly valued by scientists studying plant accumulation and detoxification mechanisms, as they provide a promising source of genes for developing plants suitable for phytoremediation techniques. So far, several genes have been identified as being upregulated when plants are exposed to Cd. These genes include genes encoding transcription factors such as iron-regulated transporter-like protein (ZIP), natural resistance associated macrophage protein (NRAMP) gene family, genes encoding phytochelatin synthases (PCs), superoxide dismutase (SOD) genes, heavy metal ATPase (HMA), cation diffusion facilitator gene family (CDF), Cd resistance gene family (PCR), ATP-binding cassette transporter gene family (ABC), the precursor 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and precursor 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) multigene family are also influenced. Thanks to advances in omics sciences and transcriptome analysis, we are gaining more insights into the genes involved in Cd stress response. Recent studies have also shown that Cd can affect the expression of genes related to antioxidant enzymes, hormonal pathways, and energy metabolism.

This study was designed to describe bacterial profiles of ejaculates collected following a long and short ejaculatory abstinence set in the context of changes in the conventional, oxidative, and immunological characteristics of semen. Two specimens were collected in succession from normozoospermic men ( n = 51) following 2 days and 2 h, respectively. Semen samples were processed and analyzed according to the World Health Organization (WHO) 2021 guidelines. Afterwards, sperm DNA fragmentation, mitochondrial function, levels of reactive oxygen species (ROS), total antioxidant capacity, and oxidative damage to sperm lipids and proteins were evaluated in each specimen. Selected cytokine levels were quantified using the ELISA method. Bacterial identification by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that samples collected following two days of abstinence presented with a higher bacterial load and diversity, and a greater prevalence of potentially uropathogenic bacteria including Escherichia coli , Staphylococcus aureus and Enterococcus faecalis . Only staphylococci and Escherichia coli remained present in specimens obtained after 2 h of abstinence. Whilst all samples accomplished the criteria set by WHO, a significantly higher motility ( p < 0.05), membrane integrity ( p < 0.05), mitochondrial membrane potential ( p < 0.05), and DNA integrity ( p < 0.0001) were detected following 2 h of ejaculatory abstinence. On the other hand, significantly higher ROS levels ( p < 0.001), protein oxidation ( p < 0.001), and lipid peroxidation ( p < 0.01) accompanied by significantly higher concentrations of tumor necrosis factor alpha ( p < 0.05), interleukin-6 ( p < 0.01), and interferon gamma ( p < 0.05) were observed in specimens collected after two days of abstinence. It may be summarized that shorter ejaculatory abstinence does not compromise sperm quality in normozoospermic men, while it contributes to a decreased occurrence of bacteria in semen which is accompanied by a lower probability of damage to spermatozoa by ROS or pro-inflammatory cytokines.

This study aimed to characterize the bacterial profiles and their association with selected semen quality traits among two chicken breeds. Thirty Lohmann Brown and thirty ROSS 308 roosters were selected for semen quality estimation, including sperm motility, membrane and acrosome integrity, mitochondrial activity, and DNA fragmentation. The oxidative profile of the semen, including the production of reactive oxygen species (ROS), antioxidant capacity, protein, and lipid oxidation, were assessed as well. Moreover, the levels of pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukins 1 and 6 (IL-1, IL-6) and C-reactive protein, as well as the concentrations of selected antibacterial proteins (cathelicidin, β-defensin and lysozyme) in the seminal plasma were evaluated with the enzyme-linked immunosorbent assay. The prevailing bacterial genera identified by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were Citrobacter spp., Enterococcus spp., Escherichia spp. and Staphylococcus spp. While the bacterial load was significantly higher in the ROSS 308 line ( p < 0.05), a higher number of potentially uropathogenic bacteria was found in the Lohmann Brown roosters. Antimicrobial susceptibility tests revealed a substantial resistance of randomly selected bacterial strains, particularly to ampicillin, tetracycline, chloramphenicol, and tobramycin. Furthermore, Lohmann Brown ejaculates containing an increased proportion of Escherichia coli presented with significantly ( p < 0.05) elevated levels of TNF-α and IL-6, as well as ROS overproduction and lipid peroxidation. Inversely, significantly ( p < 0.05) higher levels of β-defensin and lysozyme were found in the semen collected from the ROSS 308 roosters, which was characterized by a higher quality in comparison to the Lohmann Brown roosters. In conclusion, we emphasize the criticality of bacteriospermia in the poultry industry and highlight the need to include a more complex microbiological screening of semen samples designated for artificial insemination.

Melissa officinalis L. is well known for its lemon-scented aroma and various pharmacological properties. Despite these valuable properties, the genes involved in the biosynthetic pathways in M. officinalis are not yet well-explored when compared to other members of the mint family. For that, gene expression studies using quantitative real-time PCR (qRT-PCR) are an excellent tool. Although qRT-PCR can provide accurate results, its accuracy is highly reliant on the expression and stability of the reference gene used for normalization. Hence, selecting a suitable experiment-specific reference gene is very crucial to obtain accurate results. However, to date, there are no reports for experiment-specific reference genes in M. officinalis . Therefore, in the current study, ten commonly used reference genes were assessed for their suitability as optimal reference genes in M. officinalis under various abiotic stress conditions and different plant organs. The candidate genes were ranked based on BestKeeper, comparative ΔCt, geNorm, NormFinder, and RefFinder. Based on the results, we recommend the combination of EF-1α and GAPDH as the best reference genes to normalize gene expression studies in M. officinalis . On the contrary, HLH71 was identified as the least-performing gene. Thereafter, the reliability of the optimal gene combination was assessed by evaluating the relative gene expression of the phenylalanine ammonia lyase ( PAL ) gene under two elicitor treatments (gibberellic acid and jasmonic acid). PAL is a crucial gene involved directly or indirectly in the production of various economically important secondary metabolites in plants. Suitable reference genes for each experimental condition are also discussed. The findings of the current study form a basis for current and future gene expression studies in M. officinalis and other related species.

The aim of the study was to analyse selected aspects of the natural variability of selected varieties of Vitis vinifera . Grapevine is one of the most popular and desirable crops in the world due to the great tradition of wine production, but grape extracts also have a wide range of pharmaceutical effects on the human body. It is important to identify different varieties for the conservation of genetic resources, but also for commercial and cultivation purposes. The variability of conserved DNA-derived polymorphism profiles, as well as microbial characteristics, were analysed in this study. Six different varieties of Vitis vinifera L. were used in the study: Cabernet Savignon, Chardonney, Welschriesling, Weisser Riesling, Gewurztramines and Gruner Veltliner. Genetic polymorphism was analysed by CDDP markers for WRKY genes. Polymorphic amplicon profiles were generated by all primer combinations used in the study. Gruner Veltliner and Welschriesling were the most similar, with a similarity value at 0.778. Microbiological quality of grape and antimicrobial activity against Gram-positive and Gram-negative bacteria and yeasts were analysed further. The plate diluting method for microbial quality and the disc diffusion method for antimicrobial activity were evaluated. The number of total count of bacteria ranged between 3.12 in Cabernet Sauvignon to 3.62 log cfu/g in Grűner Veltliner. The best antimicrobial activity showed Gewurztramines against Salmonella enterica , Yersinia enterocolitica , Pseudomonas aeroginosa , Staphylococcus aureus , Listeria monocytogenes , Candida albicans , Candida krusei , and Candida tropicalis . The best antimicrobial activity was found against Enterococcus faecalis in variety Weisser Riesling.

Since the molecular similarities and differences among physiological capacitation and cryocapacitation have not been studied in detail, this study was designed to assess the gene and protein expression levels of the Cation channel of sperm (CatSper) 1 and 2, sodium bicarbonate (Na+/HCO3−) cotransporter (NBC) and protein kinase A (PKA) in un-capacitated (control), in vitro capacitated (CAP) and cryopreserved (CRYO) bovine spermatozoa. All samples were subjected to motility evaluation using the computer assisted sperm analysis and chlortetracycline (CTC) assay for the assessment of the capacitation patterns. Furthermore, quantitative reverse transcription PCR (qRT-PCR) and Western blots were used to monitor the expression patterns of the selected capacitation markers. The results showed a significant reduction in the gene and protein expression levels of CatSper1 and 2 in the CRYO group when compared to the CAP group (p < 0.0001). In the case of NBC, the results were not significantly different or were inconclusive. While a non-significant down-regulation of PKA was found in the CRYO group, a significant reduction in the expression of the PKA protein was found in frozen-thawed spermatozoa in comparison to the CAP group (p < 0.05). In conclusion, we may hypothesize that while in vitro capacitated and cryopreserved spermatozoa exhibit CTC-patterns consistent with capacitation events, the molecular machinery underlying CTC-positivity may be different.

The nuclear reactor accident in Chernobyl, Ukraine, resulted in effects both locally and farther away. Most of the contaminated areas were the agricultural fields and forests. Experimental fields were established near Chernobyl-radioactively contaminated fields localized 5 km from Chernobyl Nuclear Power Plant as well as the remediated soil that is localized directly in the Chernobyl town. Two flax varieties growing under chronic exposition to ionizing radiation were used for this study-the local Ukrainian variety Kyivskyi and a commercial variety Bethune. The screening of the length polymorphism generated by transposable elements insertions were performed. All known types of common flax transposon, retrotransposons and iPBS approach were used. In the iPBS multiplex analyze, for the Kyivskyi variety, a unique addition was found in the seeds from the radioactive contaminated field and for the Bethune variety, a total of five amplicon additions were obtained and one deletion. For the TRIM Cassandra fingerprints, two amplicon additions were generated in the seeds from radioactive contaminated fields for the Bethune variety. In summary, the obtained data represent the genetic diversity between control and irradiated subgroups of flax seeds from Chernobyl area and the presence of activated transposable elements due to the irradiation stress.

This study focused on the identification of bacterial profiles of semen in normozoospermic men and their possible involvement in changes to the sperm structural integrity and functional activity. Furthermore, we studied possible fluctuations of selected cytokines, oxidative markers, and antibacterial proteins as a result of bacterial presence in the ejaculate. Sperm motility was assessed with computer-assisted sperm analysis, while sperm apoptosis, necrosis and acrosome integrity were examined with fluorescent methods. Reactive oxygen species (ROS) generation was quantified via luminometry, sperm DNA fragmentation was evaluated using the TUNEL protocol and chromatin-dispersion test, while the JC-1 assay was applied to evaluate the mitochondrial membrane potential. Cytokine levels were quantified with the biochip assay, whilst selected antibacterial proteins were quantified using the ELISA method. The predominant species identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were Staphylococcus hominis , Staphylococcus capitis and Micrococcus luteus . The results revealed that the sperm quality decreased proportionally to the increasing bacterial load and occurrence of conditionally pathogenic bacteria, including Enterococcus faecalis , Staphylococcus aureus and Escherichia coli . Antimicrobial susceptibility tests revealed a substantial resistance of randomly selected bacterial strains to ampicillin, vancomycin, tobramycin, and tetracycline. Furthermore, an increased bacterial quantity in semen was accompanied by elevated levels of pro-inflammatory cytokines, including interleukin-1, interleukin-2, interleukin-6, tumor necrosis factor alpha as well as ROS overproduction and lipid peroxidation of the sperm membranes. Our results suggest that semen quality may be notably affected by the bacterial quantity as well as quality. It seems that bacteriospermia may be associated with inflammatory processes, oxidative stress, sperm structural deterioration, and a subsequent risk for the development of subfertility, even in normozoospermic males.

Oyster mushroom ( Pleurotus ostreatus ) is still one of the most cultivated edible and medicinal mushrooms. Despite its frequent cultivation around the world, there is currently just a little information available on the variability of strains in terms of the content of β-glucans in them. This work presents an extensive study of 60 strains in terms of the content of α-glucans and β-glucans in their caps and stipes. The authenticity of the production strains based on an analysis of the variability of their genome by CDDP (Conserved DNA-derived polymorphism) markers was confirmed, whereas identical CDDP profiles were identified between samples 45, 89, 95, and 96. Genetic variability of the analyzed production strains showed a high polymorphism and effective discriminative power of the used marking technique. Medium positive correlations were found among the CDDP profiles and β-glucan content in the group of strains that generated the same CDDP profiles, and low negative correlation was found among these profiles in the group of low β-glucan content strains. For the determination of glucans content, Mushroom and Yeast analytical enzymatic kit (Megazyme, Bray, Co. Wicklow, Ireland) were used. The results clearly showed that the stipe contains on average 33% more β-glucans than the cap. The minimum detected β-glucan content in the stipe was in strain no. 72, specifically 22%, and the maximum in strain no. 43, specifically 56%, which after the conversion represents a difference of 155%. From the point of view of β-glucan content, the stated strain no. 43 appears to be very suitable for the commercial production of β-glucans under certain conditions.

Bacterial contamination of semen is an often overlooked, yet important, factor contributing to decreased sperm vitality. Understanding the impact of bacterial presence on sperm structural integrity and functional activity may assist the development of effective strategies to prevent, or manage, bacteriospermia in the breeding practice. The aim of this study was to describe the bacterial profiles of ram semen ( n = 35), and we also focused on the associations between bacteriospermia, sperm structure, and function, as well as oxidative and inflammatory characteristics of semen. For a better insight, the samples were divided into three groups, according to the breeds used in the study: native Wallachian (NW), improved Wallachian (IW), and Slovak dairy (SD) breeds. The results showed a significantly lower motility and membrane integrity in the NW group in comparison to the IW and SD groups, which was accompanied by a significantly higher concentration of leukocytes, increased reactive oxygen species (ROS) generation, and subsequent oxidative insults to the sperm lipids and proteins. Accordingly, the NW group presented with the highest bacterial load, in which Staphylococcus and Escherichia were the predominant representatives. The Pearson correlation analysis uncovered positive relationships amongst the bacterial load and leukocytospermia (r = 0.613), the extent of lipid peroxidation (r = 0.598), protein oxidation (r = 0.514), and DNA fragmentation (r = 0.638). Furthermore, positive correlations were found between the bacterial load and pro-inflammatory molecules, such as the C-reactive protein (r = 0.592), interleukin 1 (r = 0.709), and interleukin 6 (r = 0.474), indicating a possible involvement of the immune response in the process of bacteriospermia. Overall, our data indicate that ram semen quality may be equally affected by the bacterial load and diversity. Furthermore, we can assume that the presence of bacteria in ejaculates triggers inflammatory processes, causes ROS overproduction, and, thereby, contributes to alterations in the sperm structure, while at the same time compromising the fertilization ability of male gametes.

Although bacterial contamination of ejaculates may cause difficulties in cattle reproduction, standard protocols for a routine microbiological analysis of bovine semen are still missing. Understanding of the mechanisms of bacterial damage to spermatozoa may contribute to the prevention and management of bacteriospermia in the future. Therefore, this study was designed to investigate bacterial profiles of fresh bovine ejaculates (n = 30), while at the same time we focused on assessing the relationships between bacteriospermia and selected sperm quality parameters as well as an array of oxidative stress and inflammatory markers. The samples were divided into three quality groups according to the sperm motility: Excellent (EX) - over 90% > Good (GO) - between 89% and 80% > Moderate (MO) - under 80%. The results showed a significant increase in reactive oxygen species (ROS) generation in the GO group when compared to the EX group. In the MO group, a deterioration of almost all quality parameters was observed when compared to the EX group. In particular, sperm motility, mitochondrial membrane potential, ROS production and IL-6 concentration exhibited a significant decline. Pearson correlation analysis revealed positive associations among bacterial load and the presence of leukocytes in semen (r = 0.965), malondialdehyde concentration (r = 0.816) and DNA fragmentation (r = 0.784). MALDI-TOF MS Biotyper analysis showed a prevalence of the Staphylococcus genus. The quantification of bacterial colonies revealed a significantly increased (P < 0.01) bacterial load in the MO group when compared with the EX as well as the GO group. Overall, our results suggest that sperm quality may be affected by both, bacterial composition, and bacterial load. It appears that an increased presence of bacterial species triggers the immune response, causes oxidative stress, and thereby contributes to sperm structural alterations while diminishing their fertilization ability. Abbreviations: EX: Excellent; GO: Good; MO: Moderate; MOT: Motility; ROS: Reactive Oxygen Species; MMP: Mitochondrial Membrane Potential; IL-1: Interleukin 1; IL-6: Interleukin 6; IL-8: Interleukin 8; IL-12: Interleukin 12; CRP: C-reactive protein; DNA: Deoxyribonucleic acid; MALDI-TOF MS: Matrix-assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry; LPO: Lipid peroxidation; CFU: Colony-forming units MDA: Malondialdehyde; CASA: Computer-assisted Sperm Analysis; WS: Working solution; RIPA: Radio-immunoprecipitation assay; TBARS: Thiobarbituric Acid Reactive Substances; BHB: D-β-hydroxybutyrate.

Bacterial contamination of semen is an important factor connected to the health status of bulls that may significantly affect semen quality for artificial insemination. Moreover, some important bovine diseases may be transmitted through semen. Up to now, only a very limited number of complex studies describing the semen microbiome of bulls have been published, as many bacteria are hard to cultivate using traditional techniques. The 16S rRNA high-throughput sequencing strategy allows for the reliable identification of bacterial profiles of bovine semen together with the detection of noncultivable bacterial species. Fresh samples from Holstein Friesian breeding bulls ( n = 55) were examined for the natural variability in the present bacteria. Semen doses were selected randomly from Slovak Biological Services in Nitra, Slovak Republic. The most predominant phyla within the whole dataset were Firmicutes (31%), Proteobacteria (22%), Fusobacteria (18%), Actinobacteria (13%) and Bacteroidetes (12%). Samples of semen were divided into two separate clusters according to their microbiome compositions using a cording partition around a medoids analysis. Microbiomes of the first cluster (CL1) of samples ( n = 20) were based on Actinobacteria (CL1 average = 25%; CL = 28%) and Firmicutes (CL1 = 38%; CL2 = 27%), while the second cluster (CL2; n = 35) contained samples characterized by a high prevalence of Fusobacteria (CL1 = 4%; CL2 = 26%). Some important indicator microbial groups were differentially distributed between the clusters.

Staphylococcus spp. is not only a commensal bacteria but also a major human pathogen that causes a wide range of clinical infections. Recent evidence suggests that Staphylococcus has the ability to colonize the reproductive system and to affect its structure and functions. The objective of this study was to determine the chemical properties and antibacterial effects of select essential oils (EOs): Amyris balsamifera L., Boswellia carterii Birdw., Canarium luzonicum (Blume) A. Gray, Cinnamomum camphora (L.) J. Presl., Cinnamomum camphora var. linaloolifera Y. Fuita, Citrus x aurantium L., Gaultheria procumbens L., Litsea cubeba (Lour.) Pers., Melaleuca ericifolia Smith., Melaleuca leucadendra L., Pogostemon cablin (Blanco) Benth., Citrus limon (L.) Osbeck, Santalum album L., and Vetiveria zizanoides (L.) Roberty against 50 Staphylococcus spp. cultures isolated from human semen, specifically Staphylococcus aureus , S. capiti , S. epidermidis , S. haemoliticus , and S. hominis . The disc diffusion and broth microdilution methods were used to assess the antimicrobial potential and to determine the minimum inhibitory concentration (MIC) of the selected EOs. The best anti- Staphylococcus activities were found with both methods for the essential oils of C. luzonicum (Blume) A. Gray, A. balsamifera , C. camphora , and P. cabli .

The aim of study was to isolate and identify the gut bacteria of Apis mellifera and to evaluate antagonistic effect of the bacteriota against Paenibacillus larvae , which causes American foulbrood (AFB) in honeybees. The dilution plating method was used for the quantification of selected microbial groups from digestive tract of bees, with an emphasis on the bacteriota of the bees' intestines. Bacteria were identified using mass spectrometry (MALDI-TOF-MS Biotyper). Overall, five classes, 27 genera and 66 species of bacteria were identified. Genera Lactobacillus (10 species) and Bacillus (8 species) were the most abundant. Gram-negative bacteria were represented with 16 genera, whereas Gram-positive with 10 genera. Delftia acidovorans and Escherichia coli were the most abundant in the digestive tract of honey bee. Resistance to a selection of antimicrobials was assessed for the bacterial isolates from bee gut and confirmed against all antimicrobials included in the study, with the exception of cefepime. Lactobacillus spp., especially L. kunkeei , L. crispatus and L. acidophilus . showed the strongest antimicrobial activity against P. larvae , the causal pathogen of AFB. Antimicrobial activity of essential oils against isolated bacteria and two isolates of P. larvae were assessed. Application of a broad selection of plant essential oils indicated that Thymus vulgaris had the highest antimicrobial activity against P. larvae .

The main aim of the study was to investigate the chemical composition, antioxidant, antimicrobial, and antibiofilm activity of Citrus aurantium essential oil (CAEO). The biofilm profile of Stenotrophonomonas maltophilia and Bacillus subtilis were assessed using the mass spectrometry MALDI-TOF MS Biotyper and the antibiofilm activity of Citrus aurantium (CAEO) was studied on wood and glass surfaces. A semi-quantitative composition using a modified version was applied for the CAEO characterization. The antioxidant activity of CAEO was determined using the DPPH method. The antimicrobial activity was analyzed by disc diffusion for two biofilm producing bacteria, while the vapor phase was used for three penicillia. The antibiofilm activity was observed with the agar microdilution method. The molecular differences of biofilm formation on different days were analyzed, and the genetic similarity was studied with dendrograms constructed from MSP spectra to illustrate the grouping profiles of S. maltophilia and B. subtilis . A differentiated branch was obtained for early growth variants of S. maltophilia for planktonic cells and all experimental groups. The time span can be reported for the grouping pattern of B. subtilis preferentially when comparing to the media matrix, but without clear differences among variants. Furthermore, the minimum inhibitory doses of the CAEO were investigated against microscopic fungi. The results showed that CAEO was most active against Penicillium crustosum , in the vapor phase, on bread and carrot in situ.

Phytoremediation efficiency depends on the ability of plants to accumulate, translocate and resist high levels of metals without symptoms of toxicity. This study was conducted to evaluate the potential of grain amaranth for remediation of soils contaminated with Cd. Three grain amaranth varieties, "Pribina" ( A. cruentus ), "Zobor" ( A. hypochondriacus x A. hybridus ) and Plainsman ( A. hypochondriacus x A. hybridus ) were tested under different level of Cd (0, 5, 10 and 15 mg/L) in a hydroponic experimental treatment. All could be classified as Cd excluders or Cd-hypertolerant varieties able to grow and accumulate significant amounts of Cd from the hydroponic solution, preferentially in the roots. Under the highest level of Cd exposure, qRT-PCR expression analysis of five stress-related genes was examined in above- and below-ground biomass. The results show that the Cd concentration significantly increased the mRNA level of chitinase 5 ( Chit 5) in amaranth roots as the primary site of metal stress. The involvement of phytochelatin synthase ( PCS1 ) in Cd detoxification is suggested. Based on our findings, we can conclude that variety "Pribina" is the most Cd-tolerant among three tested and can be expected to be used in the phytomanagement of Cd loaded soils as an effective phytostabiliser., Competing Interests: The authors declare no conflict of interest.

The aim of this study was to characterize extracts from the leaves of Ginkgo biloba L. from selected Slovakian localities in terms of the content of bioactive constituents, antioxidants and their antimicrobial properties. The results indicated that the content of antioxidants was sample-specific, and this specificity was statistically significant. Ginkgo biloba L. from the locality of Košice had the best activity determined by the free radical scavenging activity (DPPH) (1.545 mg Trolox equivalent antioxidant capacity (TEAC)/g fresh matter (FM)) as well as the molybdenum-reducing antioxidant power (35.485 mg TEAC/g FM) methods. The highest content of total polyphenols (2.803 mg gallic acid equivalent (GAE)/g FM) and flavonoids (4.649 μg quercetin equivalent (QE)/g FM) was also detected in this sample. All samples of G. biloba leaf extracts showed significant antimicrobial activity against one or more of the examined bacterial species, and Staphylococcus aureus subsp. aureus CCM 2461 was found to be the most susceptible (minimal inhibition concentration MIC50 and MIC90 values of 64.2 and 72.2 µg/mL, respectively). Based on the results it was concluded that Ginkgo biloba L. extracts can be used as antimicrobial and antioxidant additives. Selected miRNA-based molecular markers were used to examine the environmental adaptability of Ginkgo biloba L. An almost-complete genotype clustering pattern based on locality was determined in the analysis that involved a species-specific gb-miR5261 marker. Morphologically specific exemplar, cv. Ohatsuki, was excluded., Competing Interests: The authors declare no conflict of interest.

The studies of plant bacterial endophytes, colonizing the plant tissues without any signs of diseases, are essential for understanding of ecological interactions. The aim of our study is to detect microbiological contamination and to assess the antimicrobial, antioxidant activity, total phenolic, carotenoid content, genome size, and ploidy of non-cultivated Rosa canina sampled from urban areas. Samples of Rosa canina fruits were collected in three locations in Slovakia. The highest total viable count and the Enterobacteriaceae count in fruits were 4.32 log CFU/g and 4.29 log CFU/g, respectively. Counts of the mesophilic anaerobic sporulating bacteria, Pseudomonas spp., and of the microscopic fungi and yeasts were 3.00, 2.15 log CFU/g, 3.65 log CFU/g, and 2.76 log CFU/g, respectively. Regarding the antimicrobial activity, Escherichia coli and Klebsiela oxytoca were the most sensitive species among the assayed microorganisms to the treatment with the ethanolic extracts of Rosa canina fruits. The fruits were rich in bioactive compounds, polyphenols, and carotenoids, that could be related to their antioxidant activity. Genome sizes of analyzed samples ranged from 2.3 to 2.96. DNA-based fingerprinting obtained by iPBS markers of the Rosa canina var. lapidicola Heinr. Braun., was characterized by some distinctive inserted loci. An interdisciplinary study was performed for the dog roses from different parts of Slovakia that resulted in deeper characterization of this species.

The aim of this study was to assess the chemical composition, antioxidant, antimicrobial and antibiofilm activity of the Coriandrum sativum essential oil. Changes in the biofilm profile of Stenotropomonas maltophilia and Bacillus subtilis were studied using MALDI-TOF MS Biotyper on glass and wooden surfaces. The molecular differences of biofilms in different days were observed as well. The major volatile compounds of the coriander essential oil in the present study were β -linalool 66.07%. Coriander essential oil radical scavenging activity was 51.05% of inhibition. Coriander essential oil expressed the strongest antibacterial activity against B. subtilis followed by S. maltophilia and Penicillium expansum. The strongest antibiofilm activity of the coriander essential oil was found against S. maltophilia. A clearly differentiated branch was obtained for early growth variants of S. maltophilia in case of planktonic cells and all experimental groups and time span can be reported for the grouping pattern of B. subtilis preferentially when comparing to the media matrix, but without clear differences among variants. The results indicate that coriander was effective against the tested Penicillium expansum in the vapor phase after 14 days with MID 50 367.19 and MID 90 445.92 µL/L of air.

Endophytic bacteria are an important part of different functions in plants that lead to plants' production characteristics as well as their stress response mechanisms. Endophytic bacterial diversity was analyzed in this study to describe 16S rRNA variability and changes in the leaves of drought-tolerant and drought-susceptible wheat when growth under in vitro conditions. A metagenomic analysis was applied and a pilot exploratory study was performed to prove this type of analysis as applicable to tracking endophytic bacterial diversity changes when a drought stress is applied to an in vitro culture of wheat. The study showed that the changes in the bacterial endophytes' variabilities associated preferentially with the drought stress varietal characteristics of the analyzed wheat instead of the applied stress conditions., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Ionizing radiation in environment comes from various natural and anthropogenic sources. The effect of radioactivity released after the CNPP (Chernobyl Nuclear Power Plant) on plant systems remains of great interest. Even now, more than three decades after the nuclear accident, the long-lived radionuclides represent a strong stress factor. Herein, the emphasis has been placed on analysis of genetic variability represented by activation of LTR (Long Terminal Repeat)-retrotransposons. Polymorphism in LTR-retrotransposon insertions has been investigated throughout the genome of two flax varieties, Kyivskyi and Bethune. For this purpose, two retrotransposon-based marker techniques, IRAP (Inter-Retrotransposon Amplified Polymorphism) and iPBS (inter-Primer Binding Site), have been employed. The hypothesis that chronic radioactive stress may induce mechanism of retransposition has been supported by the activation of FL9, FL11 and FL12 LTR-retrotransposons in flax seeds harvested from radioactive environment. Out of two retrotransposon-based approaches, IRAP appears to be more suitable for identification of LTR-retrotransposon polymorphism. Even though the LTR-retrotransposon polymorphism was identified, the results suggest the high level of plant adaptation in the radioactive Chernobyl area. However, it is not really surprising that plants developed an effective strategy to survive in radio-contaminated environment over the past 30 years.

The present study is focused on the characterization of yacon [Smallanthus sonchifolius (Poepp. et Endl.) H. Robinson] accessions from different geographic origins (Bolivia, Ecuador, and Peru) by iPBS markers and metabolomic fingerprinting. The results showed that the number of amplified polymorphic fragment levels ranged from 20 up to 27 with a level of polymorphism ranging from 80 to 100%. Five of the iPBS primers used in this study provided no specific banding pattern able to discriminate between the different yacon accessions. However, two iPBS primer pairs were able to separate Peru accessions from those of Ecuador and Bolivia. The UPLC-HRMS/MS-based metabolomic fingerprinting showed highly similar metabolomic fingerprints characterized by the accumulation of high quantities of sesquiterpene lactones and diterpenes, but no apparent geographic clustering. The present study demonstrates that yacon accessions from different geographical origins maintained ex situ (in the Czech Republic) present a rather low chemical and genetic diversity., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Effective and reliable methods of identification of Pseudomonas species are important for the characterization of microorganisms. Freshwater ecosystems are an important source of Pseudomonas species, including those pathogenic to fish and humans. The aim of the present study was to compare the identification conducted with MALDI-TOF MS Biotyper and 16S rDNA sequencing of fish-borne Pseudomonas spp. Altogether, 13 different Pseudomonas spp. were isolated from freshwater fish. Phylogenetic analysis showed a clear taxonomic placement only for 13 out of 15 Pseudomonas isolates. Accordance of identification method was found only in 6 out of 15 isolates. The human pathogenic Pseudomonas spp. were not found in our study, indicating that the fish could be considered as safe for consumption. The present study revealed a high discriminatory power of the mass spectra investigation and 16S rDNA gene sequencing technology for the identification of Pseudomonas spp. associated with freshwater fish., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Many food innovations rely on the introduction and use of new or modified proteins. New or modified food proteins may lead to major health risks due to their inherent potential to cause food allergy. Currently, the pre-market allergenicity assessment for new or modified food proteins and protein sources relies on methods for identifying allergenic hazards based on characteristics of known allergens. However, there is no general consensus on the allergenicity parameters to use and the criteria that should apply for the evaluation and decisions to be made. In this paper, we propose that the strategy for allergenicity risk assessment of new or modified food proteins and the methodologies applied should be governed by the risk management questions to be answered, reflected in the information needed by risk managers to enable their informed decision making. We generated an inventory of health outcome-related assessment parameters and criteria potentially important for risk management decision-making and we discuss the implications of selecting different optional criteria (e.g. cut-off values) for what could be accepted as safe with regards to the health outcomes in the (at risk) population. The impact of these various options on both method development and risk management practices was investigated., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A plant selection system based on the phosphomannose isomerase gene (pmi) as a selectable marker is often used to avoid selection using antibiotic resistance. Nevertheless, pmi gene is endogenous in several plant species and therefore difficult to use in such cases. Here we evaluated and compared Agrobacterium-mediated transformation of Linum usitatissimum breeding line AGT-952 (without endogenous pmi gene) and Nicotiana tabacum var. WSC-38 (with endogenous pmi gene). Transformation was evaluated for vectors bearing transgenes that have the potential to be involved in improved phytoremediation of contaminated environment. Tobacco regenerants selection resulted in 6.8% transformation efficiency when using a medium supplemented with 30 g/L mannose with stepwise decrease of the sucrose concentration. Similar transformation efficiency (5.3%) was achieved in transformation of flax. Relatively low selection efficiency was achieved (12.5% and 34.8%, respectively). The final detection of efficient pmi selection was conducted using PCR and the non-endogenous genes; pmi transgene for flax and todC2 transgene for tobacco plants.

The aim of the study was to determinate the level of expression of silver birch allergen Betv1 in pollen samples from different Ukraine areas by RT-qPCR SYBR Green assay. Protocol for quantifying the expression of Betv1 allergen was developed when testing of three housekeeping genes-cyclophylin, alpha-tubulin and transcription factor CBF1. Samples from urbanized area was analysed by real-time PCR when a sample from forest growth conditions was used as a calibrator. Real-time PCR based quantifying of Betv1 provides a useful method for rapid and sensitive analyses of this silver birch allergen. Our results show higher expression levels in samples from central parts of urbanized area as housing estates when compared to the samples from borders of the urbanized area.