Bee venom genotoxicity on Saccharomyces cerevisiae cells - The role of mitochondria and YAP1 transcription factor.

The present work aims to clarify the genotype differences of a model organism Saccharomyces cerevisiae in response to bee venom. The study evaluated various endpoints including cell survival, induction of physiologically active superoxide anions, mitotic gene conversion, mitotic crossing-over, reverse mutations, DNA double-strand breaks, and Ty1 retrotransposition. The role of the intact mitochondria and the YAP1 transcription factor was also evaluated. Our results indicate a genotype-specific response. The first experimental evidence has been provided that bee venom induces physiologically active superoxide anions and DNA double-strand breaks in S. cerevisiae. The lack of oxidative phosphorylation due to disrupted or missing mitochondrial DNA reduces but not diminishes the cytotoxicity of bee venom. The possible modes of action could be considered direct damage to membranes (cytotoxic effect) and indirect damage to DNA through oxidative stress (genotoxic effect). YAP1 transcription factor was not found to be directly involved in cell defense against bee venom treatment.

A Snapshot Picture of the Fungal Composition of Bee Bread in Four Locations in Bulgaria, Differing in Anthropogenic Influence.

Information about the fungal composition of bee bread, and the fermentation processes to which the fungi contribute significantly, is rather scarce or fragmentary. In this study, we performed an NGS-based metagenomics snapshot picture study of the fungal composition of bee bread in four locations in Bulgaria during the most active honeybee foraging period at the end of June 2020. The sampling locations were chosen to differ significantly in climatic conditions, landscape, and anthropogenic pressure, and the Illumina 2 × 250 paired-end reads platform was used for amplicon metagenomics study of the ITS2 region. We found that some of the already reported canonical beneficial core fungal species were present within the studied samples. However, some fungal genera such as Monilinia, Sclerotinia, Golovinomyces, Toxicocladosporium, Pseudopithomyces, Podosphaera and Septoriella were reported for the first time among the dominant genera for a honeybee related product. Anthropogenic pressure negatively influences the fungal composition of the bee bread in two different ways-urban/industrial pressure affects the presence of pathogenic species, while agricultural pressure is reflected in a decrease of the ratio of the beneficial fungi.

Phytochemical characterization and biological activity of apricot kernels' extract in yeast-cell based tests and hepatocellular and colorectal carcinoma cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Bitter apricot kernels' extract contains a broad spectrum of biologically active substances with a lot of attention to amygdalin - cyanogenic glycoside. The extract has been used in the pharmaceutical industry for years as an ingredient of different pharmaceuticals with anti-inflammatory, antimicrobial, or regenerative properties. In traditional medicine, the bitter apricot kernels are known as a remedy for respiratory disorders and skin diseases. The apricot kernels and amygdalin are often prescribed by practitioners for the prevention and treatment of various medical conditions, including colorectal cancer. THE PRESENT STUDY AIMS: to evaluate the phytochemical composition and the potential antimutagenic, antirecombinogenic, and antitumor effect of apricot kernels' extract at very low concentrations in yeast cell-based tests and mammalian hepatocellular and colon carcinoma cell lines. MATERIALS AND METHODS: Phytochemical analysis was performed by LC-MS profiling. Reverse-phase HPLC and UV detection were applied for the determination of amygdalin quantity in the extract. Biological activity was evaluated by Zimmermann's mutagenicity and Ty1 retrotransposition test. Cytotoxic/antiproliferative activity of apricot kernels' extract was performed on four types of cell lines - HepG2, HT-29, BALB/3T3, clone A31, and BJ using the standard MTT-dye reduction assay. RESULTS: Data revealed the presence of more than 1000 compounds and 4 cyanogenic glycosides among them - Amygdalin, Deidaclin, Linamarin and Prulaurasin. The Amygdalin concentration was measured to be 57.8 µg/ml. All extract concentrations demonstrated a strong antigenotoxic, antirecombinogenic, antimutagenic, and anticarcinogenic effect in the yeast cell-based tests. High selectivity of the extract action is established among different mammalian cell lines. Normal cell line BJ is found to be resistant to the extract action. HepG2 was found to be the most sensitive to apricot kernels' action. CONCLUSION: The present study provides the first phytochemical analysis of Bulgarian bitter apricot kernels. Three new cyanogenic glycosides were reported. Evidence is obtained that the apricot kernels' extract at low concentrations is not able to induce some of the events related to the initial steps of tumorigenesis. Additionally, a high selectivity of the extract action is established among different cell lines. The most sensitive cell line was found to be HepG2.

WGS-based characterization of the potentially beneficial Enterococcus faecium EFD from a beehive.

Nowadays, due to their potential application as probiotics for humans or animals, many beneficial lactic acid bacteria have been isolated from different natural environments. These include members of the genus Enterococcus - quite specific due to their ambiguous nature, varying from pathogens to probiotics. In our work we present a whole-genome sequencing (WGS)-based approach for assessing the potential of bacteriocin-producing Enterococcus isolates from beehives to serve as natural preserving agents against bacterial infections associated with honeybees. Potential Enterococcus spp. isolates from pollen granules were tested with the well diffusion assay for bacteriocin activity against Paenibacillus larvae, the causative agent of the American foulbrood disease (AFB). Two of them gave positive results and were determined at species level by 16S rRNA genes sequencing. They were then subjected to WGS using the Illumina HiSeq platform. The resulting raw data reads were processed and further analyzed by using only freely available web-based tools (the Shovill pipeline, QUAST, BAGEL4, ResFinder, VirulenceFinder and PlasmidFinder). The analysis revealed that both of them represent clonally identical isolates of the same strain. This specific strain was named Enterococcus faecium EFD, and was genotyped by the MLST-2.0 Server. Five bacteriocin genes were found in the assembled genome, providing a possible explanation for the antimicrobial properties of the isolate. The protein nature of the inhibitory agent/s was confirmed by treatment with proteinase K. No resistance determinants for clinically important antibiotics and functional virulence factor genes were detected. The bioinformatic analyses of the draft genome sequence suggest that E. faecium EFD is not pathogenic.The observation that E. faecium EFD was present within more than one of the beehives in the apiary proposes the idea that E. faecium EFD is there as a part of the normal beehive microbiota. This finding, in combination with its antibacterial activity against P. larvae, highlights this novel isolate as a potential natural preserving agent against AFB. Furthermore, the WGS-based approach reported here proved to be very cost- and time- efficient, for screening the applicability of new pro- and prebiotic Enterococcus strains as beehive protection agents.

New cell-based assay indicates dependence of antioxidant biological activity on the origin of reactive oxygen species.

The mobility of the Ty1 transposon in Saccharomyces cerevisiae was found to vary proportionally with the level of ROS generated in cells, which provides the possibility to determine antioxidant activity by changes in a cellular process instead of using chemical reactions. The study of propolis, royal jelly, and honey with the newly developed Ty1antiROS test reveals an inverse exponential dependence of antioxidant activity on increased concentrations. This dependence can be transformed to proportional by changing the source of ROS: instead of cell-produced to applied as hydrogen peroxide. The different test responses are not due to excess of added hydrogen peroxide, as evidenced by the exponential dependence found by usage of yap1Δ tester cells accumulating cell-generated ROS. Results indicate that the activity of antioxidants to oxidative radicals depends on the origin of ROS, and this activity is elevated for cell-generated ROS compared to ROS added as reagents in the assay.

The positive response of Ty1 retrotransposition test to carcinogens is due to increased levels of reactive oxygen species generated by the genotoxins.

In previous laboratory and environmental studies, the Ty1 short-term test showed positive responses (i.e. induced mobility of the Ty1 retrotransposon) to carcinogenic genotoxins. Here, we provide evidence for a causal relationship between increased level of reactive oxygen species and induction the mobility of the Ty1 retrotransposon. Results obtained in concentration and time-dependent experiments after treatment, the tester cells with carcinogenic genotoxins [benzo(a)pyrene, benzo(a)anthracene, ethylmethanesulfonate, formamide], free bile acids (chenodeoxycholic, lithocholic acids) and metals (arsenic, hexavelant chromium, lead) showed a simultaneous increase in both cellular level of the superoxide anions and Ty1 retrotransposition rates. Treatment with the noncarcinogenic genotoxins [benzo(e)pyrene, benzo(b)anthracen, anthracene], conjugated bile acids (taurodeoxycholic, glycodeoxycholic acids) and metals (zinc, trivalent chromium) did not change significantly superoxide anions level and Ty1 retrotransposition rate. The induction by carcinogens of the Ty1 mobility seems to depend on the accumulation of superoxide anions, since the addition of the scavenger N-acetylcysteine resulted in loss of both increased amount of superoxide anions and induced Ty1 retrotransposition. Increased hydrogen peroxide levels are also involved in the induction of Ty1 retrotransposition rates in response to treatment with carcinogenic genotoxins, as evidenced by disruption of YAP1 gene in the tester cells. It is concluded that the carcinogen-induced high level of reactive oxygen species play a primary and key role in determination the selective response of Ty1 test to carcinogenic genotoxins.