The Lacticaseibacillus paracasei species is comprised by nomadic bacteria inhabiting a wide variety of ecological niches, from fermented foodstuffs to host-associated microenvironments. Lc. paracasei SP5 is a novel strain, originally isolated from kefir grains that presents desirable probiotic and biotechnological attributes. In this study, we applied genomic tools to further characterize the probiotic and biotechnological potential of the strain. Firstly, whole genome sequencing and assembly, were performed to construct the chromosome map of the strain and determine its genomic stability. Lc. paracasei SP5 carriers several insertion sequences, however, no plasmids or mobile elements were detected. Furthermore, phylogenomic and comparative genomic analyses were utilized to study the nomadic attributes of the strain, and more specifically, its metabolic capacity and ability to withstand environmental stresses imposed during food processing and passage through the gastrointestinal (GI) tract. More specifically, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-active enzyme (CAZymes) analyses provided evidence for the ability of the stain to utilize an array of carbohydrates as growth substrates. Consequently, genes for heat, cold, osmotic shock, acidic pH, and bile salt tolerance were annotated. Importantly bioinformatic analysis showed that the novel strain does not harbor acquired antimicrobial resistance genes nor virulence factors, in agreement with previous experimental data. Putative bacteriocin biosynthesis clusters were identified using BAGEL4, suggesting its potential antimicrobial activity. Concerning microbe-host interactions, adhesins, moonlighting proteins, exopolysaccharide (EPS) biosynthesis genes and pilins mediating the adhesive phenotype were, also, pinpointed in the genome of Lc. paracasei SP5. Validation of this phenotype was performed by employing a microbiological method and confocal microscopy. Conclusively, Lc. paracasei SP5 harbors genes necessary for the manifestation of the probiotic character and application in the food industry. Upcoming studies will focus on the mechanisms of action of the novel strain at multiple levels.

Biomedical research requires both in vitro and in vivo studies in order to explore disease processes or drug interactions. Foundational investigations have been performed at the cellular level using two-dimensional cultures as the gold-standard method since the early 20th century. However, three-dimensional (3D) cultures have emerged as a new tool for tissue modeling over the last few years, bridging the gap between in vitro and animal model studies. Cancer has been a worldwide challenge for the biomedical community due to its high morbidity and mortality rates. Various methods have been developed to produce multicellular tumor spheroids (MCTSs), including scaffold-free and scaffold-based structures, which usually depend on the demands of the cells used and the related biological question. MCTSs are increasingly utilized in studies involving cancer cell metabolism and cell cycle defects. These studies produce massive amounts of data, which demand elaborate and complex tools for thorough analysis. In this review, we discuss the advantages and disadvantages of several up-to-date methods used to construct MCTSs. In addition, we also present advanced methods for analyzing MCTS features. As MCTSs more closely mimic the in vivo tumor environment, compared to 2D monolayers, they can evolve to be an appealing model for in vitro tumor biology studies.

IntroductionLactobacilli are avid producers of antimicrobial compounds responsible for their adaptation and survival in microbe-rich matrices. The bactericidal or bacteriostatic ability of lactic acid bacteria (LAB) can be exploited for the identification of novel antimicrobial compounds to be incorporated in functional foodstuffs or pharmaceutical supplements. In this study, the antimicrobial and antibiofilm properties of Lactiplantibacillus pentosus L33, Lactiplantibacillus plantarum L125 and Lacticaseibacillus paracasei SP5, previously isolated form fermented products, were examined, against clinical isolates of Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Enteritidis and Escherichia coli.MethodsThe ability of viable cells to inhibit pathogen colonization on HT-29 cell monolayers, as well as their co-aggregation capacity, were examined utilizing the competitive exclusion assay. The antimicrobial activity of cell-free culture supernatants (CFCS) was determined against planktonic cells and biofilms, using microbiological assays, confocal microscopy, and gene expression analysis of biofilm formation-related genes. Furthermore, in vitro analysis was supplemented with in silico prediction of bacteriocin clusters and of other loci involved in antimicrobial activity.ResultsThe three lactobacilli were able to limit the viability of planktonic cells of S. aureus and E. coli in suspension. Greater inhibition of biofilm formation was recorded after co-incubation of S. enterica with the CFCS of Lc. paracasei SP5. Predictions based on sequence revealed the ability of strains to produce single or two-peptide Class II bacteriocins, presenting sequence and structural conservation with functional bacteriocins.DiscussionThe efficiency of the potentially probiotic bacteria to elicit antimicrobial effects presented a strain- and pathogen-specific pattern. Future studies, utilizing multi-omic approaches, will focus on the structural and functional characterization of molecules involved in the recorded phenotypes.