Identification of Bulgarian Sourdough Microbiota by Metagenomic Approach Using Three Commercially Available DNA Extraction Protocols.

Research background: Sourdough is a spontaneously formed, complex microbial ecosystem of various lactic acid bacteria (LAB) and yeast which, by producing specific metabolites, determines the quality of the baked products. In order to design and control the sourdough with preferred nutritional characteristics, it is crucial that the LAB diversity of the product of interest be elucidated. Experimental approach: Using the opportunities of next-generation sequencing (NGS) of the V1-V3 hypervariable gene region of 16S rRNA, we studied the microbial ecosystem of a whole grain sourdough made of Triticum monococcum, originating from Southwestern Bulgaria. Since the DNA extraction method is considered crucial for the accuracy of the sequencing results, as it can introduce significant differences in the examined microbiota, we used three different commercial kits for DNA isolation and analyzed their impact on the observed bacterial diversity. Results and conclusions: All three DNA extraction kits provided bacterial DNA which passed quality control and was successfully sequenced on Illumina MiSeq platform. The results received from the different DNA protocols showed variations in the microbial profiles. Alpha diversity indices (ACE, Chao1, Shannon, and Simpson) were also different among the three groups of results. Nevertheless, a strong dominance of phylum Firmicutes, class Bacilli, order Lactobacillales, represented mostly by family Lactobacillaceae, genus Lactobacillus (relative abundance of 63.11-82.28%) and family Leuconostocaceae, genus Weissella (relative abundance of 3.67-36.31%) was observed. Lactiplantibacillus plantarum and Levilactobacillus brevis with relative abundance of 16.15-31.24% and 6.21-16.29% respectively, were the two dominant species identified in all three DNA isolates. Novelty and scientific contribution: The presented results give insight into the taxonomic composition of bacterial community of a specific Bulgarian sourdough. Having in mind that the sourdough is a difficult matrix for DNA isolation on the one hand, and that there is no standardized DNA extraction protocol for this matrix on the other hand, this pilot study aims to give a small contribution to the future establishment and validation of such a protocol, which will allow accurate assessment of the specific microbiota of sourdough samples.

Photobiomodulation with 590 nm Wavelength Delays the Telomere Shortening and Replicative Senescence of Human Dermal Fibroblasts In Vitro.

Background: Cellular senescence is one of the major factors contributing to the aging process. Photobiomodulation (PBM) is known to trigger an array of cellular responses, but there are no data on how it affects the process of cellular senescence. In this study, we analyze the effect of PBM on the cellular senescence and telomere dynamics. Methods: Human dermal fibroblasts were irradiated by a panel of light-emitting diodes with 590 nm and dose 30 J/cm2 accumulated over 1200 sec repeated in 4-day cycle within 40 days. After the last cycle of PBM treatment, the difference in number of senescent cells between PBM treated groups end nontreated control groups was measured by senescent sensitive ß-galactosidase assay, and the difference in average telomere length between the experimental end control groups was analyzed using relative human telomere length quantitative Polymerase Chain Reaction (qPCR) assay. Results: After 10 cycles of irradiation, the percentage of senescent cells in PBM-treated cultures was 19.7% ± 4.5%, p < 0.05 smaller than the percentage of senescent cells in the control group, and their relative telomere length was 1.19 ± 0.09-fold, p < 0.05 greater than nontreated controls. Conclusions: Our study demonstrates for the first time that PBM with appropriate parameters can delay the attrition of the telomeres and the entry of cells into senescence, suggesting a potential involvement of telomerase reactivation. A hypothetical mechanism for this light-induced antiaging effect is discussed.

The mammalian INO80 chromatin remodeling complex is required for replication stress recovery.

A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart.

The inhibitor of histone deacetylases sodium butyrate enhances the cytotoxicity of mitomycin C.

The use of histone deacetylase inhibitors has been proposed as a promising approach to increase the cell killing effect of DNA damage-inducing drugs in chemotherapy. However, the molecular mechanism of their action remains understudied. In the present article, we have assessed the effect of the histone deacetylase inhibitor sodium butyrate on the DNA damage response induced by the crosslinking agent mitomycin C. Sodium butyrate increased mitomycin C cytotoxicity, but did not impair the repair pathways required to remove mitomycin C-induced lesions as neither the rate of nucleotide excision repair nor the homologous recombination repair rate were diminished. Sodium butyrate treatment abrogated the S-phase cell-cycle checkpoint in mitomycin C-treated cells and induced the G(2)-M checkpoint. However, sodium butyrate treatment alone resulted in accumulation of reactive oxygen species, double-strand breaks in DNA, and apoptosis. These results imply that the accumulation of reactive oxygen species-mediated increase in DNA lesion burden may be the major mechanism by which sodium butyrate enhances the cytotoxicity of mitomycin C.