Anticancer Activity of Encapsulated Pearl Millet Polyphenol-Rich Extract against Proliferating and Non-Proliferating Breast Cancer Cells In Vitro.

Plant-derived polyphenols are bioactive compounds with potential health-promoting properties including antioxidant, anti-inflammatory, and anticancer activity. However, their beneficial effects and biomedical applications may be limited due to their low bioavailability. In the present study, we have considered a microencapsulation-based drug delivery system to investigate the anticancer effects of polyphenol-rich (apigenin, caffeic acid, and luteolin) fractions, extracted from a cereal crop pearl millet (Pennisetum glaucum), using three phenotypically different cellular models of breast cancer in vitro, namely triple negative HCC1806, ER-positive HCC1428, and HER2-positive AU565 cells. Encapsulated polyphenolic extract induced apoptotic cell death in breast cancer cells with different receptor status, whereas it was ineffective against non-tumorigenic MCF10F cells. Encapsulated polyphenolic extract was also found to be cytotoxic against drug-resistant doxorubicin-induced senescent breast cancer cells that were accompanied by increased levels of apoptotic and necrotic markers, cell cycle inhibitor p21 and proinflammatory cytokine IL8. Furthermore, diverse responses to the stimulation with encapsulated polyphenolic extract in senescent breast cancer cells were observed, as in the encapsulated polyphenolic extract-treated non-proliferating AU565 cells, the autophagic pathway, here cytotoxic autophagy, was also induced, as judged by elevated levels of beclin-1 and LC3b. We show for the first time the anti-breast cancer activity of encapsulated polyphenolic extract of pearl millet and postulate that microencapsulation may be a useful approach for potentiating the anticancer effects of phytochemicals with limited bioavailability.

Application of Green Algal Planktochlorella nurekis Biomasses to Modulate Growth of Selected Microbial Species.

As microalgae are producers of proteins, lipids, polysaccharides, pigments, vitamins and unique secondary metabolites, microalgal biotechnology has gained attention in recent decades. Microalgae can be used for biomass production and to obtain biotechnologically important products. Here, we present the application of a method of producing a natural, biologically active composite obtained from unicellular microalgae of the genus Planktochlorella sp. as a modulator of the growth of microorganisms that can be used in the cosmetics and pharmaceutical industries by exploiting the phenomenon of photo-reprogramming of metabolism. The combination of red and blue light allows the collection of biomass with unique biochemical profiles, especially fatty acid composition (Patent Application P.429620). The ethanolic and water extracts of algae biomass inhibited the growth of a number of pathogenic bacteria, namely Enterococcus faecalis, Staphylococcus aureus PCM 458, Streptococcus pyogenes PCM 2318, Pseudomonas aeruginosa, Escherichia coli PCM 2209 and Candida albicans ATCC 14053. The algal biocomposite obtained according to our procedure can be used also as a prebiotic supplement. The presented technology may allow the limitation of the use of antibiotics and environmentally harmful chemicals commonly used in preparations against Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli or Candida spp.

Synthetic Pesticides Used in Agricultural Production Promote Genetic Instability and Metabolic Variability in Candida spp.

The effects of triazole fungicide Tango® (epoxiconazole) and two neonicotinoid insecticide formulations Mospilan® (acetamiprid) and Calypso® (thiacloprid) were investigated in Candida albicans and three non-albicans species Candida pulcherrima, Candida glabrata and Candida tropicalis to assess the range of morphological, metabolic and genetic changes after their exposure to pesticides. Moreover, the bioavailability of pesticides, which gives us information about their metabolization was assessed using gas chromatography-mass spectrophotometry (GC-MS). The tested pesticides caused differences between the cells of the same species in the studied populations in response to ROS accumulation, the level of DNA damage, changes in fatty acids (FAs) and phospholipid profiles, change in the percentage of unsaturated to saturated FAs or the ability to biofilm. In addition, for the first time, the effect of tested neonicotinoid insecticides on the change of metabolic profile of colony cells during aging was demonstrated. Our data suggest that widely used pesticides, including insecticides, may increase cellular diversity in the Candida species population-known as clonal heterogeneity-and thus play an important role in acquiring resistance to antifungal agents.

Long-Term Adaption to High Osmotic Stress as a Tool for Improving Enological Characteristics in Industrial Wine Yeast.

Industrial wine yeasts owe their adaptability in constantly changing environments to a long evolutionary history that combines naturally occurring evolutionary events with human-enforced domestication. Among the many stressors associated with winemaking processes that have potentially detrimental impacts on yeast viability, growth, and fermentation performance are hyperosmolarity, high glucose concentrations at the beginning of fermentation, followed by the depletion of nutrients at the end of this process. Therefore, in this study, we subjected three widely used industrial wine yeasts to adaptive laboratory evolution under potassium chloride (KCl)-induced osmotic stress. At the end of the evolutionary experiment, we evaluated the tolerance to high osmotic stress of the evolved strains. All of the analyzed strains improved their fitness under high osmotic stress without worsening their economic characteristics, such as growth rate and viability. The evolved derivatives of two strains also gained the ability to accumulate glycogen, a readily mobilized storage form of glucose conferring enhanced viability and vitality of cells during prolonged nutrient deprivation. Moreover, laboratory-scale fermentation in grape juice showed that some of the KCl-evolved strains significantly enhanced glycerol synthesis and production of resveratrol-enriched wines, which in turn greatly improved the wine sensory profile. Altogether, these findings showed that long-term adaptations to osmotic stress can be an attractive approach to develop industrial yeasts.

A Non-Vector Approach to Increase Lipid Levels in the Microalga Planktochlorella nurekis.

Microalgae are freshwater and marine unicellular photosynthetic organisms that utilize sunlight to produce biomass. Due to fast microalgal growth rate and their unique biochemical profiles and potential applications in food and renewable energy industries, the interest in microalgal research is rapidly increasing. Biochemical and genetic engineering have been considered to improve microalgal biomass production but these manipulations also limited microalgal growth. The aim of the study was the biochemical characterization of recently identified microalgal strain Planktochlorella nurekis with elevated cell size and DNA levels compared to wild type strain that was achieved by a safe non-vector approach, namely co-treatment with colchicine and cytochalasin B (CC). A slight increase in growth rate was observed in twelve clones of CC-treated cells. For biochemical profiling, several parameters were considered, namely the content of proteins, amino acids, lipids, fatty acids, ß-glucans, chlorophylls, carotenoids, B vitamins and ash. CC-treated cells were characterized by elevated levels of lipids compared to unmodified cells. Moreover, the ratio of carotenoids to chlorophyll a and total antioxidant capacity were slightly increased in CC-treated cells. We suggest that Planktochlorella nurekis with modified DNA levels and improved lipid content can be considered to be used as a dietary supplement and biofuel feedstock.

The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation.

Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic and physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage.

Changes in DNA methylation patterns and repetitive sequences in blood lymphocytes of aged horses.

It is known that aged organisms have modified epigenomes. Epigenetic modifications, such as changes in global and locus-specific DNA methylation, and histone modifications are suspected to play an important role in cancer development and aging. In the present study, with the well-established horse aging model, we showed the global loss of DNA methylation in blood lymphocytes during juvenile-to-aged period. Additionally, we tested a pattern of DNA methylation of ribosomal DNA and selected genes such as IGF2 and found no significant changes during development and aging. We asked if genetic components such as polymorphisms within DNA methyltransferase genes, DNMT1, DNMT3a, and DNMT3b, may contribute to observed changes in global DNA methylation status. The analysis of seven intragenic polymorphisms did not reveal any significant association with changes in global DNA methylation. Telomere shortage and a loss of pericentromeric heterochromatin during juvenile-to-aged period were also observed. Transcriptional rDNA activity, assessed as the number and size of nucleolar organizer regions, reflecting physiological state of the cell, and mitotic index were decreased with increasing horse donor age. Moreover, changes during juvenile-to-aged period and adult-to-aged period were compared and discussed. Taken together, changes in global DNA methylation status originating in development and affecting the stability of repetitive sequences may be associated with previously reported genomic instability during horse aging.

Cadmium-induced changes in genomic DNA-methylation status increase aneuploidy events in a pig Robertsonian translocation model.

Although cadmium is a well-established human carcinogen, the mechanisms by which it induces cancer are poorly understood. It is suggested that cadmium-mediated carcinogenesis may include the modulation of gene expression and signal-transduction pathways, interference with antioxidant enzymes, inhibition of DNA repair and DNA methylation, and induction of apoptosis. Nevertheless, no predominant mechanism playing a role in metal-induced carcinogenesis has been reported. In the present study, we used a pig Robertsonian translocation model, which is a cross between a wild boar and domestic pig resulting in Robertsonian translocation (37,XX,der15;17 or 37,XY,der15;17), to determine the role of cadmium sulfate in the modulation of genomic DNA-methylation status and the induction of aneuploidy. We found a cadmium-mediated increase in aneuploidy within chromosome group A and C, but not within chromosome group D containing the translocated chromosome der15,17 which indicates that translocated chromosome is not more prone to chromosomal aberrations than are other chromosomes. We suggest that cadmium-induced aneuploidy (up to 5-µM concentration) may be mediated by global DNA hypermethylation as monitored with HPLC and 5-mdC immunostaining. In addition, the cyto- and genotoxic potential of cadmium was evaluated. Cadmium sulfate was able to induce apoptosis, inhibit cell-proliferative status and expression of nucleolar organizer regions (NORs), and increase oxidative DNA damage (8-oxoG content).

Age-related changes in genomic stability of horses.

Recently, the old horse has been proposed as a model to study telomere-dependent senescence, immunosenescence and inflamm-aging. In the present paper, we used 80 Hucul and Anglo-Arabian horses divided into 3 age groups (juvenile, adult, old) to evaluate age-dependent changes at the genomic and DNA level and in cell proliferative potential. The level of positive TUNEL cells (both apoptotic and with DNA fragmentation), oxidative DNA damage (8-oxoG immunostaining), sister chromatid exchange and bleomycin-induced chromatid breaks were significantly increased in the combined old group compared to the combined adult group. We observed a negative correlation between micronuclei formation and age, which may be associated with damaged cells undergoing apoptosis, rather than expressing micronuclei. We were unable to show any significant changes in the nuclear division index value, which reflects the proliferative status of the viable cell fraction during aging. Here, we show that breed-independent and age-associated changes in genomic stability may contribute, at least in part, to the aging process in the horse.

PRINS detection of 18S rDNA in pig, red fox and Chinese raccoon dog, and centromere DNA in horse.

The fluorescence in situ hybridization (FISH) technique is widely used in animal cytogenetics. Contrary to FISH procedure, primed in situ DNA synthesis (PRINS) does not require the DNA probe preparation (design, synthesis, gel purification of PCR products and labeling). The PRINS method with primers used as 'DNA probes' is both PCR-sensitive and allows for chromosomal localization of DNA sequences. Here, we show the application of PRINS reaction with one unlabeled oligonucleotide pair to identify 18S rDNA loci in three different animal species: domestic pig (Sus scrofa), red fox (Vulpes vulpes) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides). We present the data of indirect labeling with the digoxigenin-PRINS using two different pairs of primers complementary to centromeric region of horse (Equus caballus) chromosomes. Our new PRINS application may be considered as a useful tool for chromosome investigation in the field of domestic and wild animal genetics and evolution.

Redox status of equine seminal plasma reflects the pattern and magnitude of DNA damage in sperm cells.

Antioxidant status of seminal plasma from 23 stallions was evaluated. We found a negative correlation between total antioxidant capacity (ABTS(•+) decolorization assay) and thiol content of seminal plasma, and sperm DNA damage (8-oxoG immunostaining, TUNEL reaction, comet assay). Low seminal redox status was the strongest correlated with 8-oxoG level which may indicate that seminal total antioxidant capacity influences mainly the formation of single strand DNA breaks in sperm cells. Since inter-individual differences in seminal antioxidant status were reported, we postulated that the redox status of seminal plasma may be an additional important parameter, both with sperm quantitative and morphological analysis, for evaluation of equine semen quality.

Evaluation of the cyto- and genotoxic activity of yerba mate (Ilex paraguariensis) in human lymphocytes in vitro.

Despite its antioxidant capacity and well-known health benefits, yerba mate tea (Ilex paraguariensis) has been shown to possess some genotoxic and mutagenic activities and to increase incidence of some types of cancer. The aim of this study was to estimate the cyto- and genotoxicity of mate tea in human peripheral lymphocytes in vitro. We found that yerba mate extract induced a concentration-dependent, statistically significant increase in the level of apoptotic and necrotic cells and a decrease in the nuclear division index (NDI). Mate-exposed lymphocytes had a reduced transcriptional rDNA activity, which may be due to the stress conditions, and showed an elevated production of micronuclei. The FISH technique revealed the appearance of an acrocentric signal in mate-induced micronuclei, which suggests that under these conditions yerba mate extract may display aneugenic activity. Since caffeine is one of the most abundant compounds found in the dry mass of mate, we conducted additional experiments with caffeine alone. We showed that caffeine used at the same concentrations manifests a more potent cyto- and genotoxic effect that may account, at least in part, for the disadvantageous effects observed for yerba mate extract.

Plate ethanol-screening assay for selection of the Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability for xylose alcoholic fermentation.

A new method for the selection of Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability to ferment xylose to ethanol was developed. The method is based on the ability of P. stipitis and H. polymorpha colonies to grow and produce ethanol on agar plates with xylose as the sole carbon and energy source. Secreted ethanol, in contrast to xylose, supports growth of cells of the indicator xylose-negative strains (the wild-type strain of Saccharomyces cerevisiae or Deltaxyl1 mutant of H. polymorpha) mixed with agar medium. The size of the tester culture-growth zone around xylose-grown colonies appeared to be dependent on the amount of secreted ethanol. Mutants with altered (decreased or elevated) ethanol production in xylose medium have been isolated using this method. The mutants exhibited pleiotropic alterations in enzymatic activities of the intermediary xylose metabolism.