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

Transcriptome Analysis Reveals Dynamic Cultivar-Dependent Patterns of Gene Expression in Potato Spindle Tuber Viroid-Infected Pepper

Potato spindle tuber viroid (PSTVd) infects various plants. PSTVd pathogenesis is associated with interference with the cellular metabolism and defense signaling pathways via direct interaction with host factors or via the transcriptional or post-transcriptional modulation of gene expression. To better understand host defense mechanisms to PSTVd infection, we analyzed the gene expression in two pepper cultivars, Capsicum annuum Kurtovska kapia (KK) and Djulunska shipka (DS), which exhibit mild symptoms of PSTVd infection. Deep sequencing-based transcriptome analysis revealed differential gene expression upon infection, with some genes displaying contrasting expression patterns in KK and DS plants. More genes were downregulated in DS plants upon infection than in KK plants, which could underlie the more severe symptoms seen in DS plants. Gene ontology enrichment analysis revealed that most of the downregulated differentially expressed genes in both cultivars were enriched in the gene ontology term photosynthesis. The genes upregulated in DS plants fell in the biological process of gene ontology term defense response. We validated the expression of six overlapping differentially expressed genes that are involved in photosynthesis, plant hormone signaling, and defense pathways by quantitative polymerase chain reaction. The observed differences in the responses of the two cultivars to PSTVd infection expand the understanding of the fine-tuning of plant gene expression that is needed to overcome the infection

Mirgalaxy: Galaxy‐based framework for interactive analysis of microrna and isomir sequencing data

Tools for microRNA (miR) sequencing data analyses are broadly used in biomedical research. However, the complexity of computational approaches still remains a challenge for biologists with scarce experience in data analytics and bioinformatics. Here, we present miRGalaxy, a Galaxy‐based framework for comprehensive analysis of miRs and their sequence variants—miR isoforms (isomiRs). Though isomiRs are commonly reported in deep‐sequencing experiments, their detailed structure complexity and specific differential expression (DE) remain not fully examined by the majority of the available analysis tools. miRGalaxy encompasses biologist‐user‐friendly tools and workflows dedicated to the analysis of the isomiR‐ome and its complex behavior in various biological samples. miRGalaxy is developed as a modular, accessible, redistributable, shareable, and user‐friendly framework for scientists working with small RNA (sRNA)‐seq data. Due to its modular workflow, advanced users can customize the steps and tools for their needs. In addition, the framework provides an analysis report where the significant output results are summarized in charts and visualizations. miRGalaxy can be accessed via preconfigured Docker image flavor and a Toolshed installation if the user already has a running Galaxy instance. Over the last decade, studies on the expression of miRs and isomiRs in normal and deregulated tissues have led to the discovery of their potential as diagnostic biomarkers. The detection of miRs in biofluids further expanded the exploration of the miR repertoire as a source of liquid biopsy biomarkers. Here we show the miRGalaxy framework application for in‐depth analysis of the sRNA‐seq data from two different biofluids, milk and plasma, to identify, annotate, and discover specific differentially expressed miRs and isomiRs

Isomirs–hidden soldiers in the mirna regulatory army, and how to find them?

Numerous studies on microRNAs (miRNA) in cancer and other diseases have been ac-companied by diverse computational approaches and experimental methods to predict and validate miRNA biological and clinical significance as easily accessible disease biomarkers. In recent years, the application of the next-generation deep sequencing for the analysis and discovery of novel RNA biomarkers has clearly shown an expanding repertoire of diverse sequence variants of mature miRNAs, or isomiRs, resulting from alternative post-transcriptional processing events, and affected by (patho)physiological changes, population origin, individual’s gender, and age. Here, we provide an in-depth overview of currently available bioinformatics approaches for the detection and visualization of both mature miRNA and cognate isomiR sequences. An attempt has been made to present in a systematic way the advantages and downsides of in silico approaches in terms of their sensitivity and accuracy performance, as well as used methods, workflows, and processing steps, and end output dataset overlapping issues. The focus is given to the challenges and pitfalls of isomiR expression analysis. Specifically, we address the availability of tools enabling research without extensive bioinformatics background to explore this fascinating corner of the small RNAome universe that may facilitate the discovery of new and more reliable disease biomarkers

The Dynamics of miR-449a/c Expression during Uterine Cycles Are Associated with Endometrial Development

The human endometrium is a highly dynamic tissue. Increasing evidence has shown that microRNAs (miRs) play essential roles in human endometrium development. Our previous assay, based on small RNA-sequencing (sRNA-seq) indicated the complexity and dynamics of numerous sequence variants of miRs (isomiRs) that can act together to control genes of functional relevance to the receptive endometrium (RE). Here, we used a greater average depth of sRNA-seq to detect poorly expressed small RNAs. The sequencing data confirmed the up-regulation of miR-449c and uncovered other members of the miR-449 family up-regulated in RE—among them miR-449a, as well as several isoforms of both miR-449a and miR-449c, while the third family member, miR-449b, was not identified. Stem-looped RT-qPCR analysis of miR expression at four-time points of the endometrial cycle verified the increased expression of the miR-449a/c family members in RE, among which the 5′ isoform of miR-449c–miR-449c.1 was the most strongly up-regulated. Moreover, we found in a case study that the expression of miR-449c.1 and its precursor correlated with the histological assessment of the endometrial phase and patient age. We believe this study will promote the clinical investigation and application of the miR-449 family in the diagnosis and prognosis of human reproductive diseases

The Multiverse of Plant Small RNAs: How Can We Explore It?

Plant small RNAs (sRNAs) are a heterogeneous group of noncoding RNAs with a length of 20–24 nucleotides that are widely studied due to their importance as major regulators in various biological processes. sRNAs are divided into two main classes—microRNAs (miRNAs) and small interfering RNAs (siRNAs)—which differ in their biogenesis and functional pathways. Their identification and enrichment with new structural variants would not be possible without the use of various high-throughput sequencing (NGS) techniques, allowing for the detection of the total population of sRNAs in plants. Classifying sRNAs and predicting their functional role based on such high-performance datasets is a nontrivial bioinformatics task, as plants can generate millions of sRNAs from a variety of biosynthetic pathways. Over the years, many computing tools have been developed to meet this challenge. Here, we review more than 35 tools developed specifically for plant sRNAs over the past few years and explore some of their basic algorithms for performing tasks related to predicting, identifying, categorizing, and quantifying individual sRNAs in plant samples, as well as visualizing the results of these analyzes. We believe that this review will be practical for biologists who want to analyze their plant sRNA datasets but are overwhelmed by the number of tools available, thus answering the basic question of how to choose the right one for a particular study

Transposon-associated polymorphisms of stress-responsive gene promoters in selected accessions of Arabidopsis thaliana

Genetic diversity caused by transposable element movement can play an important role in plant adaptation to local environments. Regarding genes, transposon-induced alleles were mostly related to gene bodies and a few of them to promoter regions. In this study, promoter regions of 9 stress-related genes were searched for transposable element insertions in 12 natural accessions of Arabidopsis thaliana. The promoter screening was performed via PCR amplification with primers designed to flank transposable element insertions in the promoter regions of the reference accession Col-0. Transposable element-associated insertion/deletion (indel) polymorphisms were identified in 7 of the 12 promoter loci across studied accessions that can be developed further as molecular markers. The transposable element absence in the promoter regions of orthologous genes in A. lyrata indicated that the insertion of these transposable elements in A. thaliana lineage had occurred after its divergence from A. lyrata. Sequence analysis of the promoter regions of CML41 (Calmodulin-like protein 41) and CHAP (chaperone protein dnaJ-related) confirmed the indel polymorphic sites in four accessions - Col-0, Wassilewskija, Shahdara, and Pirin. The observed indel polymorphism of the CHAP promoter region was associated with specific gene expression profiles in the different accessions grown at a normal and elevated temperature in a plant growth chamber. The collected data can be a starting point for gene expression profiling studies under conditions resembling the natural habitats of accessions