Stability of human salivary extracellular vesicles containing dipeptidyl peptidase IV under simulated gastrointestinal tract conditions.

BACKGROUND: Extracellular vesicles (EVs) have been isolated from various sources, including primary and cultured cell lines and body fluids. Previous studies, including those conducted in our laboratory, have reported the stability of EVs under various storage conditions. METHODS: EVs from human whole saliva were separated via size-exclusion chromatography. To simulate the effects of gastric or intestinal fluids on the stability of EVs, pepsin or pancreatin was added to the samples. Additionally, to determine the effect of bile acids, sodium cholate was added. The samples were then subjected to western blotting, dynamic light scattering, and transmission electron microscopy analyses. In addition, the activity of dipeptidyl peptidase (DPP) IV retained in the samples was examined to monitor the stability of EVs. RESULTS: Under acidic conditions, with pepsin mimicking the milieu of the stomach, the EVs remained stable. However, they partially lost their membrane integrity in the presence of pancreatin and sodium cholate, indicating that they may be destabilized after passing through the duodenum. Although several associated proteins, such as mucin 5B and CD9 were degraded, DPP IV was stable, and its activity was retained under the simulated gastrointestinal conditions. CONCLUSION: Our data indicate that although EVs can pass through the stomach without undergoing significant damage, they may be disrupted in the intestine to release their contents. The consistent delivery of active components such as DPP IV from EVs into the intestine might play a role in the efficient modulation of homeostasis of the signal transduction pathways occurring in the gastrointestinal tract.

A redescription of morphologically similar species from the genus Euglena: E. laciniata, E. sanguinea, E. sociabilis, and E. splendens(1).

Euglena sanguinea (Ehrenberg 1831) was one of the first green euglenoid species described in the literature. At first, the species aroused the interest of researchers mainly due to the blood-red color of its cells, which, as it later turned out, is not a constant feature. Complicated chloroplast morphology, labeled by Pringsheim as the "peculiar chromatophore system", made the correct identification of the species difficult, which is the reason why, throughout the 20th century, new species resembling E. sanguinea were continually being named due to a lack of suitable diagnostic features to distinguish E. sanguinea. Interest in E. sanguinea has returned in recent years, following findings that the species can produce ichthyotoxins. This was followed by the need to classify E. sanguinea correctly, which was achieved through the verification of morphological and molecular data for all species similar to E. sanguinea. As the result of the analysis, the number of species sharing some morphological similarities with E. sanguinea could be reduced from 12, as described in the literature, to four, with established epitypes and updated diagnostic descriptions. The most important diagnostic features included: the presence of mucocysts (i.e., whether they were visible before and/or after staining), the number of chloroplasts, the size of the double-sheathed pyrenoids, and the presence of the large paramylon grain in the vicinity of the stigma. Moreover, sequence analysis revealed the presence of unusually long SSU rDNA sequences in E. sanguinea. Previously, SSU rDNA sequences of such length were known to be present in primary osmotrophic euglenoids.

TAXONOMIC REVISIONS OF MORPHOLOGICALLY SIMILAR SPECIES FROM TWO EUGLENOID GENERA: EUGLENA (E. GRANULATA AND E. VELATA) AND EUGLENARIA (EU. ANABAENA, EU. CAUDATA, AND EU. CLAVATA)(1).

The establishment of epitypes (together with the emended diagnoses) for three species of Euglenaria Karnkowska, E. W. Linton et Kwiatowski [Eu. anabaena (Mainx) Karnkowska et E. W. Linton; Eu. caudata (Hübner) Karnkowska et E. W. Linton; and Eu. clavata (Skuja) Karnkowska et E. W. Linton] and two species of Euglena Ehrenberg [E. granulata (Klebs) Schmitz and E. velata Klebs] was achieved due to literature studies, verification of morphological diagnostic features (cell size, cell shape, number of chloroplasts, the presence of mucocysts), as well as molecular characters (SSU rDNA). Now all these species are easy to identify and distinguish, despite their high morphological similarity, that is, spindle-shaped (or cylindrically spindle-shaped) cells and parietal, lobed chloroplasts with a single pyrenoid, accompanied by bilateral paramylon caps located on both sides of the chloroplast. E. granulata is the only species in this group that has spherical mucocysts. E. velata is distinguished by the largest cells (90-150 µm) and has the highest number of chloroplasts (>30). Eu. anabaena has the fewest chloroplasts (usually 3-6), and its cells are always (whether the organism is swimming or not) spindle-shaped or cylindrically spindle-shaped, in contrast to the cells of Eu. clavata, which are club-shaped (clavate) while swimming and only after stopping change to resemble the shape of a spindle or a cylindrical spindle; Eu. clavata has numerous chloroplasts (15-20). Eu. caudata is characterized by asymmetrical spindle-shaped (fusiform) cells, that is, with an elongated rear section and a shorter front section; the number of chloroplasts normally ranges from 7 to 15.

A novel phylogenetic clade of picocyanobacteria from the Mazurian lakes (Poland) reflects the early ontogeny of glacial lakes.

The community of picocyanobacteria inhabiting the Great Mazurian Lakes system (comprising lakes ranging from mesotrophic to hypertrophic) is dominated by phycoerythrin-rich cells, which outnumber phycocyanin-rich cells, even in hypertrophic lakes. The genetic diversity and phylogeny of 43 strains of picocyanobacteria isolated from four Mazurian lakes were studied by analyzing the nucleotide sequences of the 16S rRNA gene and cpcBA-IGS operon. Phylogenetic analyses assigned some of the strains to several previously described clusters (Groups A, B, C, E and I) and revealed the existence of a novel clade, Group M (Mazurian), which exhibited a low level of similarity to the other clusters. Both phycocyanin and phycoerythrin picocyanobacteria were assigned to this clade based on an analysis of the 16S rRNA gene. The cpcBA sequence analysis assigned only phycocyanin strains to Group M, whereas the phycoerythrin strains from the M ribogroup were assigned to Groups B and E. We hypothesize that Group M originally contained only phycocyanin picocyanobacteria. The phycoerythrin found in strains belonging to ribogroup M seems to have been acquired through horizontal gene transfer as an adaptation to the changing environment early in the ontogeny of these glacial lakes.

THE SPECIES EUGLENA DESES (EUGLENACEAE) REVISITED: NEW MORPHOLOGICAL AND MOLECULAR DATA1.

For this study, we have examined the literature and the morphological diversity, as well as analyzed the nuclear SSU rDNA sequences of two very common and cosmopolitan species formerly known as Euglena deses Ehrenb. and Euglena intermedia (G. A. Klebs) F. Schmitz. Our studies have shown that there is evidence for distinguishing only one species (E. deses). Here, we define new diagnostic features for E. deses, namely, periplast ornamentation (the presence of small papillae-discovered for the first time in this species) and the lateral location of the anterior canal opening, from which the flagellum emerges. We also designate the epitype and emend the diagnosis for E. deses.

Composition of picocyanobacteria community in the Great Mazurian Lakes: isolation of phycoerythrin-rich and phycocyanin-rich ecotypes from the system--comparison of two methods.

The study showed that the picocyanobacteria community of the Great Mazurian Lakes system (GML) was dominated by phycoerythrin-rich (PE) ecotypes and demonstrated a gradual decrease of the ratio between PE and phycocyanin-rich (PC) ecotypes. The Great Mazurian Lakes offer better conditions for the PE ecotype than for the PC one, despite the considerably high trophic status, probably thanks to low turbidity and attenuation of light in the water column. The successful isolation of PE and PC picocyanobacteria was achieved by two methods: the classic plate method and a modified flow-cytometry method. The modified flow-cytometry method proved to be superior: being more selective for PE picocyanobacteria as well as less time consuming and less laborious. The modifications introduced to the method, such us concentration of cyanobacterial cells by centrifugation to the density required by the flow cytometer, did not hinder the isolation while allowing to skip an intermediate phase of enrichment cultures that had been formerly proposed. The first phylogenetic analyses based on cpcBA operon and 16S rRNA gene demonstrated that picocyanobacteria isolates from GML could, with a high bootstrap support, be grouped into five and four clusters, respectively. Based on a cpcBA-IGS analysis and IGS length the study suggests that at least one of the clusters is new and has not been previously described.

Reconstructing euglenoid evolutionary relationships using three genes: nuclear SSU and LSU, and chloroplast SSU rDNA sequences and the description of Euglenaria gen. nov. (Euglenophyta).

Using Maximum Likelihood and Bayesian analyses of three genes, nuclear SSU (nSSU) and LSU (nLSU) rDNA, and chloroplast SSU (cpSSU) rDNA, the relationships among 82 plastid-containing strains of euglenophytes were clarified. The resulting tree split into two major clades: clade one contained Euglena, Trachelomonas, Strombomonas, Colacium, Monomorphina, Cryptoglena and Euglenaria; clade two contained Lepocinclis, Phacus and Discoplastis. The majority of the members of Euglena were contained in clade A, but seven members were outside of this clade. Euglena limnophila grouped with, and was thus transferred to Phacus. Euglena proxima was a single taxon at the base of clade one and is unassociated with any subclade. Five members of Euglena grouped together within clade one and were transferred into the newly erected genus Euglenaria. The monophyly of the remaining genera was supported by Bayesian and Maximum Likelihood analyses. Combining datasets resolved the relationships among ten genera of photosynthetic euglenoids.

PHYLOGENY AND SYSTEMATICS OF EUGLENA (EUGLENACEAE) SPECIES WITH AXIAL, STELLATE CHLOROPLASTS BASED ON MORPHOLOGICAL AND MOLECULAR DATA-NEW TAXA, EMENDED DIAGNOSES, AND EPITYPIFICATIONS(1).

Morphological and molecular studies, as well as original literature reexamination, necessitate establishment of five Euglena species with a single axial, stellate chloroplast [Euglena viridis (O. F. Müller) Ehrenberg 1830, Euglena pseudoviridis Chadefaud 1937, Euglena stellata Mainx 1926, Euglena pseudostellata sp. nov., and Euglena cantabrica Pringsheim 1956], three species with two chloroplasts (Euglena geniculata Dujardin ex Schmitz 1884, Euglena chadefaudii Bourrelly 1951, and Euglena pseudochadefaudii sp. nov.), and one species with three chloroplasts (Euglena tristella Chu 1946). The primary morphological features, allowing distinction of the considered species are the presence and the shape of mucocysts, as well as the number of chloroplasts. Spherical mucocysts occur in E. cantabrica and E. geniculata, while spindle-shaped mucocysts are present in E. stellata, E. pseudostellata, E. chadefaudii, E. pseudochadefaudii, and E. tristella. No mucocysts are observed in E. viridis and E. pseudoviridis. Two new species (E. pseudochadefaudii sp. nov. and E. pseudostellata sp. nov.) differ from the respective species, E. chadefaudii and E. stellata, only at the molecular level. Molecular signatures and characteristic sequences are designated for nine distinguished species. Emended diagnoses for all and delimitation of epitypes for seven species (except E. viridis and E. tristella) are proposed.