Numerous studies on microRNAs (miRNA) in cancer and other diseases have been accompanied 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.

Embryo implantation depends on endometrial receptivity (ER). To achieve ER, the preparation of the uterine lining requires controlled priming by ovarian hormones and the expression of numerous genes in the endometrial tissue. microRNAs (miRs) have emerged as critical genetic regulators of ER in fertility and of the diseases that are associated with infertility. With the rapid development of next-generation sequencing technologies, it has become clear that miR genes can produce canonical miRs and variants—isomiRs. Here, we describe miR/isomiR expression dynamics across the four time points of natural chorionic gonadotropin (hCG)-administered cycles. Sequencing of the small RNAs (sRNA-seq) revealed that the most significant expression changes during the transition from the pre-receptive to the receptive phase occurred in the isomiR families of miR-125a, miR-125b, miR-10a, miR-10b, miR-449c, miR-92a, miR-92b, and miR-99a. Pairing the analysis of the differentially expressed (DE) miRs/isomiRs and their predicted DE mRNA targets uncovered 280 negatively correlating pairs. In the receptive endometrium, the 5′3′-isomiRs of miR-449c, which were among the most highly up-regulated isomiRs, showed a negative correlation with their target, transcription factor (TF) MYCN, which was down-regulated. Joint analysis of the miR/isomiR and TF expression identified several regulatory interactions. Based on these data, a regulatory TF-miR/isomiR gene-target circuit including let7g-5p and miR-345; the isomiR families of miR-10a, miR-10b, miR-92a, and miR-449c; and MYCN and TWIST1 was proposed to play a key role in the establishment of ER. Our work uncovers the complexity and dynamics of the endometrial isomiRs that can act cooperatively with miRs to control the functionally important genes that are critical to ER. Further studies of miR/isomiR expression patterns that are paired with those of their target mRNAs may provide a more in-depth picture of the endometrial pathologies that are associated with implantation failure.

Lactiplantibacillus plantarum stands out as a remarkably diverse species of lactic acid bacteria, occupying a myriad of ecological niches. Particularly noteworthy is its presence in human breast milk, which can serve as a reservoir of probiotic bacteria, contributing significantly to the establishment and constitution of infant gut microbiota. In light of this, our study attempted to conduct an initial investigation encompassing both genomic and phenotypic aspects of the L. plantarum PU3 strain, that holds potential as a probiotic agent. By employing the cutting-edge third-generation Nanopore sequencing technology, L. plantarum PU3 revealed a circular chromosome of 3,180,940 bp and nine plasmids of various lengths. The L. plantarum PU3 genome has a total of 2962 protein-coding and non-coding genes. Our in-depth investigations revealed more than 150 probiotic gene markers that unfold the genetic determinants for acid tolerance, bile resistance, adhesion, and oxidative and osmotic stress. The in vivo analysis showed the strain’s proficiency in utilizing various carbohydrates as growth substrates, complementing the in silico analysis of the genes involved in metabolic pathways. Notably, the strain demonstrated a pronounced affinity for D-sorbitol, D-mannitol, and D-Gluconic acid, among other carbohydrate sources. The in vitro experimental verification of acid, osmotic and bile tolerance validated the robustness of the strain in challenging environments. Encouragingly, no virulence factors were detected in the genome of PU3, suggesting its safety profile. In search of beneficial properties, we found potential bacteriocin biosynthesis clusters, suggesting its capability for antimicrobial activity. The characteristics exhibited by L. plantarum PU3 pave the way for promising strain potential, warranting further investigations to unlock its full capacity and contributions to probiotic and therapeutic avenues.

Microbial communities drive the biogeochemical cycles in wetlands and provide a number of ecosystem services. They underpin soil function, and are easily impacted by anthropogenic pressure. This study examined the bacterial microbiome in the natural wetland of Zlato Pole and the protected, periodically flooded rice paddies in the Maritsa River Basin using a metagenomic approach, based on high-throughput sequencing. Alpha-diversity analysis showed a significant variation between the three study sites for Chao1 and ACE (abundance based coverage estimator) richness estimators. A positive correlation was established with pH, with highest values detected for the rice paddies and the lowest, for the Zlato Pole sediments. The obtained sequences were assigned into 37 known bacterial phyla with over 97% bacterial sequences classified within only nine of them. The bacterial communities in rice paddies sediments were more evenly distributed, whereas the Zlato Pole sediment was the most biased. The consortiums in the rice paddies were dominated by Proteobacteria, followed by Actinobacteria and Acidobacteria. The bacterial assemblages in those sites could not be distinguished by analysis of similarity. The Zlato Pole sediment community held an isolated position, where Acidobacteria was replaced by Firmicutes and Bacteroidetes and the microbiome showed an extremely high abundance of Gammaproteobacteria and Bacilli. The dominance of Gammaproteobacteria and the presence of Deinococcus-Thermus phylum, along lower nutrient concentration and the absence of correlation with the environmental parameters, is evidence of constant anthropogenic pressure around the area. The obtained results could be applied in the plans for sustainable management of the protected wetlands.

Lactobacillus is a key genus of probiotics commonly utilized for the treatment of oral infections The primary aim of our research was to investigate the probiotic potential of the newly isolated Levilactobacillus brevis DPL5 strain from human breast milk, focusing on its ability to combat biofilm-forming pathogens such as Staphylococcus aureus. Employing in vitro approaches, we demonstrate L. brevis DPL5′s ability to endure at pH 3 with survival rates above 30%, and withstand the osmotic stress often found during industrial processes like fermentation and freeze drying, retaining over 90% viability. The lyophilized cell-free supernatant of L. brevis DPL5 had a significant antagonistic effect against biofilm-producing nasal strains of Staphylococcus aureus, and it completely eradicated biofilms at subinhibitory concentrations of 20 mg·mL⁻¹. Higher concentrations of 69 mg·mL⁻¹ were found to have a 99% bactericidal effect, based on the conducted probability analysis, indicating the production of bactericidal bioactive extracellular compounds capable of disrupting the biofilm formation of pathogens like S. aureus. Furthermore, genome-wide sequencing and analysis of L. brevis DPL5 with cutting-edge Nanopore technology has uncovered over 50 genes linked to probiotic activity, supporting its ability to adapt and thrive in the harsh gut environment. The genome also contains multiple biosynthetic gene clusters such as lanthipeptide class IV, Type III polyketide synthase (T3PKS), and ribosomally synthesized, and post-translationally modified peptides (RiPP-like compounds), all of which are associated with antibacterial properties. Our study paves the way for the further exploration of DPL5, setting the stage for innovative, nature-inspired solutions to combat stubborn bacterial infections.

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.