Određivanje mikrobne populacije kiselog tijesta iz Bugarske metagenomskim metodama pomoću triju komercijalnih alata za ekstrakciju DNA

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.Pozadina istraživanja. Kiselo tijesto predstavlja spontano oblikovan složeni ekosustav različitih bakterija mliječno-kiselog vrenja i kvasaca koji stvaranjem specifičnih metabolita određuju kakvoću pekarskih proizvoda. Za kreiranje i kontroliranu pripremu kiselog tijesta sa željenim nutritivnim značajkama, neophodno je razumjeti raznolikost mliječno-kiselih bakterija. Eksperimentalni pristup. Sekvenciranjem nove generacije V1-V3 hipervarijabilnih regija 16S rRNA gena ispitali smo mikrobnu zajednicu kiselog tijesta proizvedenog od cjelovitih žitarica vrste Triticum monococcum, porijeklom iz jugozapadne Bugarske. Odabir metode ekstrakcije DNA je ključni korak za dobivanje točnih rezultata sekvenciranja, budući da ona može bitno utjecati na ispitanu mikrobnu zajednicu. Stoga smo odabrali tri komercijalna alata za izolaciju DNA i ispitali njihov učinak na bakterijsku raznolikost. Rezultati i zaključci. Pomoću sva tri alata izdvojen je bakterijski DNA materijal koji je zadovoljio kontrolu kvalitete, te je uspješno sekvenciran na platformi Illumina MiSeq. Rezultati dobiveni različitim DNA protokolima pokazali su razlike u profilima mikrobnih zajednica. Alfa-indeksi raznolikosti (ACE, Chao1, Shannon, i Simpson) također su bili različiti za svaku skupinu. Usprkos tome, opažena je izražena dominacija bakterija koljena Firmicutes, razreda Bacilli, reda Lactobacillales, i to najviše porodice Lactobacillaceae, roda Lactobacillus (relativna zastupljenost od 63,11-82,28 %) i porodice Leuconostocaceae, roda Weissella (relativna zastupljenost od 3,67–36,31 %). Dvije dominantne vrste koje su identificirane u sva tri DNA izolata bile su Lactiplantibacillus plantarum, čija je relativna zastupljenost bila 16,15-31,24 % i Levilactobacillus brevis, čija je relativna zastupljenost bila 6,21-16,29 %. Novina i znanstveni doprinos. Dobiveni rezultati daju uvid u taksonomski sastav bakterijske populacije kiselog tijesta porijeklom iz Bugarske. Imajući na umu da je kiselo tijesto zahtjevna podloga za izolaciju DNA, te da ne postoji standardizirani protokol za njegovu obradu, svrha je ove pilot studije bila dati mali doprinos uspostavljanju i validaciji budućih protokola, koji će omogućiti preciznu evaluaciju specifične mikrobne populacije kiselih tijesta

16S-rRNA-Based metagenomic profiling of the bacterial communities in traditional Bulgarian sourdoughs

Sourdoughs (SDs) are spontaneously formed microbial ecosystems composed of various species of lactic acid bacteria (LAB) and acid-tolerant yeasts in food matrices of cereal flours mixed with water. To date, more than 90 LAB species have been isolated, significantly impacting the organoleptic characteristics, shelf life, and health properties of bakery products. To learn more about the unique bacterial communities involved in creating regional Bulgarian sourdoughs, we examined the metacommunities of five sourdoughs produced by spontaneous fermentation and maintained by backslopping in bakeries from three geographic locations. The 16S rRNA gene amplicon sequencing showed that the former genus Lactobacillus was predominant in the studied sourdoughs (51.0–78.9%). Weissella (0.9–42.8%), Herbaspirillum (1.6–3.8%), Serratia (0.1–11.7%), Pediococcus (0.2–7.5%), Bacteroides (0.1–1.3%), and Sphingomonas (0.1–0.5%) were also found in all 5 samples. Genera Leuconostoc, Enterococcus, Bacillus, and Asaia were sample-specific. It is interesting to note that the genus Weissella was more abundant in wholegrain samples. The greatest diversity at the species level was found in the former genus Lactobacillus, presented in the sourdough samples with 13 species. The UPGMA cluster analysis clearly demonstrated similarity in species’ relative abundance between samples from the same location. In addition, we can conclude that the presence of two main clusters—one including samples from mountainous places (the cities of Smolyan and Bansko) and the other including samples from the city of Ruse (the banks of the Danube River)—may indicate the impact of climate and geographic location (e.g., terrain, elevation, land use, and nearby water bodies and their streams) on the abundance of microbiome taxa. As the bacterial population is crucial for bread standardization, we expect the local bakery sector to be interested in the relationship between process variables and their effect on bacterial dynamics described in this research study.info:eu-repo/semantics/publishedVersio

Aloe-emodin triggers ROS and Ca 2+ production and decreases the levels of mitochondrial membrane potential of human brain capillary endothelial cells

The aim of this work was to investigate the mechanisms of cytotoxicity of phyto-hydroxyanthraquinone aloe-emodin (AE) on human brain microvascular endothelial cell line hCMEC/D3 and to assess the cellular response in the early stage of treatment in order to extend the knowledge of AE’s anti-angiogenic properties. The immortalized human brain capillary endothelial cells hCMEC/D3 were treated with a series of AE concentrations (5 - 200 μM) for a period of 24 hours. The cell viability was determined by MTS assay. The cellular adenosine triphosphate (ATP) levels were evaluated by CellTiter-Glo® luminescent assay. The intracellular reactive oxygen species (ROS) were determined by 2’,7’-dichlorofluorescein (CM- H2DCFDA) fluorescence assay. The mitochondrial membrane potential (MMP) was assessed using tetramethylrhodamine methyl ester (TMRM) staining, while Fluo-4 was used to measure 2 the intracellular free Ca 2+ concentrations inside living cells analysed by High Content Analysis using the Arrayscan VTI 740. Twenty-four- hour treatment of hCMEC/D3 cells with AE, in concentrations between 50 and 200 µM, decreased the cell viability as well as the intracellular ATP levels in a dose- dependent manner. Increased ROS production and disruption of the mitochondrial membrane potential have also been detected. Notably, AE at a concentration greater than 5 µM dramatically increased intracellular calcium levels. Our results collectively indicate that AE inhibits proliferation of human brain microvascular cells via a mechanism involving ROS generation, disruption of Ca 2+ homeostasis and mitochondrial damage

Molecular pathogenesis of spontaneous abortions - Whole genome copy number analysis and expression of angiogenic factors.

OBJECTIVE To study two major molecular alterations in spontaneous abortions (SA) with unexplained etiology - fetal genomic anomalies and the endometrial expression of main angiogenic factors VEGFA/VEGFR2 and chemokines SDF-1/CXCR4. MATERIALS AND METHODS Whole genome copy number analysis by arrayCGH or Next Generation Sequencing (NGS) was applied for detection of fetal genomic imbalances. The abortive decidua of SA without fetal aneuploidies was further investigated for expression levels of the abovementioned factors using real time PCR analysis. A total of 30 abortive materials were collected from spontaneous abortions after exclusion of known predisposing factors. RESULTS In 21 of 30 spontaneous abortions (70%), genomic anomalies were discovered by whole genome copy number analysis. Numerical anomalies were detected in 90% of aberrant cases, and in 10% - structural aberrations were revealed. An increased expression for essential factors of angiogenesis was identified in spontaneous abortions' tissues - 3.44 times for VEGFA and 10.29 times for VEGFR2. We found an average of 14 times increase in the expression levels of SDF-1 and 3.21 times for its receptor CXCR4. CONCLUSION We could suggest the occurrence of increased angiogenesis in SA without fetal aneuploidies, compared to the control tissues, which could lead to increased oxidative stress and fetal loss