Free Volatile Compounds of Veronica austriaca ssp. jacquinii (Baumg.) Eb. Fisch. and Their Biological Activity

The composition of free volatile compounds of essential oils (EO) and hydrosols (Hy) from four different localities of the species Veronica austriaca ssp. jacquinii (Baumg.) Eb. Fisch. were analyzed by gas chromatography coupled with mass spectrometry. In the EOs, the most abundant compounds identified were hexahydrofarnesyl acetone (23.34–52.56%), hexadecanoic acid (palmitic acid, 26.71–58.91%) and octadecanol acetate (0–6.24%). The hydrosols were characterized by high abundance of methyl eugenol (23.35–57.93%), trans-p-mentha-1(7),8-dien-2-ol (5.24–7.69%) and thymol (3.48–9.45%). Glandular trichomes were analyzed using SEM (Scanning Electron Microscopy), as they are the sites of synthesis of free volatile compounds. We have detected glandular trichomes, consisting of a one stalk cell and two elliptically shaped head cells, and non-glandular (unbranched, bi-cellular to multicellular) trichomes on stems, leaves and the sepals. Data for volatile compounds from EOs and hydrosols were analyzed using Principal Component Analyses (PCA) to demonstrate variations in the composition of the volatile compounds identified. Isolated samples of EO and hydrosols were analyzed for their antioxidant activity using two methods, DPPH (2,2-diphenyl-1-picrylhydrazyl) and ORAC (Oxygen Radical Absorbance Capacity). The essential oils showed higher antioxidant activity than the hydrosols in ORAC method, but lower activity by the DPPH method. The isolates were also tested for their antiproliferative activity on different types of cancer cells and also on two lines of healthy cells, and the results showed that the extracts were not toxic to the cell lines tested. Total polyphenols, total tannins, total flavonoids and total phenolic acids were also analyzed and determined spectrophotometrically. The free volatile compounds of Veronica austriaca ssp. jacquinii can be considered as a safe natural product.

Molecular Phylogeny and Taxonomy of the Genus Spumella (Chrysophyceae) Based on Morphological and Molecular Evidence

The genus Spumella, established by Cienkowsky in 1870, is characterized by omnivory, two (rarely three) flagella, a short stick-like structure beneath the flagella, a threadlike stalk, cell division via constriction and cyst formation. Since the first phylogenetic study of Spumella-like flagellates, their paraphyly has consistently been shown, with separation into several genera. More recently, Spumella was carefully investigated using molecular and morphological data to propose seven new species. Classification of this genus and knowledge of its species diversity remain limited because Spumella-like flagellates are extremely difficult to identify based on limited morphological characters. To understand the phylogeny and taxonomy of Spumella, we analyzed molecular and morphological data from 47 strains, including 18 strains isolated from Korean ponds or swamps. Nuclear SSU, ITS and LSU rDNA data were used for maximum likelihood and Bayesian analyses. The molecular data divided the strains into 15 clades, including seven new lineages, each with unique molecular signatures for nuclear SSU rRNA from the E23-2 to E23-5 domains, the spacer between the E23-8 and E23-9 domains of the V4 region and domain 29 of the V5 region. Our results revealed increased species diversity in Spumella. In contrast to the molecular phylogeny results, the taxa showed very similar cell morphologies, suggesting morphological convergence into simple nanoflagellates to enable heterotrophy. Three new species produced stomatocysts in culture. Aspects of stomatocyst morphology, including collar structure, surface ornamentation, and cyst shape, were very useful in differentiating the three species. The general ultrastructure of Spumella bureschii strain Baekdongje012018B8 and S. benthica strain Hwarim032418A5 showed the typical chrysophyte form for the leucoplast, a vestigial chloroplast surrounded by four envelope membranes, supporting the hypothesis that Spumella evolved from a phototroph to a heterotroph via the loss of its photosynthetic ability. Seven new species are proposed: S. benthica, S. communis, S. longicolla, S. oblata, S. rotundata, S. similis, and S. sinechrysos.

Micromorphological Traits of Balcanic Micromeria and Closely Related Clinopodium Species (Lamiaceae)

A study of the trichomes types and distribution and pollen morphology was carried out in nine Micromeria taxa (M. cristata ssp. cristata, M. cristata ssp. kosaninii, M. croatica, M. graeca ssp. graeca, M. graeca ssp. fruticulosa, M. juliana, M. kerneri, M. longipedunculata and M. microphylla) and five closely related Clinopodium species (C. dalmaticum, C. frivaldszkyanum, C. pulegium, C. serpyllifolium and C. thymifolium) from the Lamiaceae family of the Balkan Peninsula. By scanning electron microscope, non-glandular trichomes, peltate and capitate trichomes were observed on the calyx, leaves and stem of the studied species. Two subtypes of capitate trichomes were observed in Micromeria species: subtype 1 (consisting of a basal epidermal cell and an elliptically shaped head cell) and subtype 2 (consisting of a basal epidermal cell, two to three stalk cells and a round head cell). In Clinopodium species, three types of capitate trichomes were observed: subtype 1, subtype 3 (consisting of a basal epidermal cell, a short peduncle cell, and a single round head cell), and subtype 4 (consisting of a basal epidermal cell, a stalk cell, and an elongated head cell). These results support the recent transfer of Micromeria species from the section Pseudomelissa to the genus Clinopodium.

Ultrastructural Alterations in Cells of Sunflower Linear Glandular Trichomes during Maturation

Sunflower and related taxa are known to possess a characteristic type of multicellular uniseriate trichome which produces sesquiterpenes and flavonoids of yet unknown function for this plant. Contrary to the metabolic profile, the cytological development and ultrastructural rearrangements during the biosynthetic activity of the trichome have not been studied in detail so far. Light, fluorescence and transmission electron microscopy were employed to investigate the functional structure of different trichome cells and their subcellular compartmentation in the pre-secretory, secretory and post-secretory phase. It was shown that the trichome was composed of four cell types, forming the trichome basis with a basal and a stalk cell, a variable number (mostly from five to eight) of barrel-shaped glandular cells and the tip consisting of a dome-shaped apical cell. Metabolic activity started at the trichome tip sometimes accompanied by the formation of small subcuticular cavities at the apical cell. Subsequently, metabolic activity progressed downwards in the upper glandular cells. Cells involved in the secretory process showed disintegration of the subcellular compartments and lost vitality in parallel to deposition of fluorescent and brownish metabolites. The subcuticular cavities usually collapsed in the early secretory stage, whereas the colored depositions remained in cells of senescent hairs.

The endosomal RIN2/Rab5C machinery prevents VEGFR2 degradation to control gene expression and tip cell identity during angiogenesis

Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic ‘tip’ and ‘stalk’ cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis.

The Role of Heparan Sulfate and Neuropilin 2 in VEGFA Signaling in Human Endothelial Tip Cells and Non-Tip Cells during Angiogenesis In Vitro

During angiogenesis, vascular endothelial growth factor A (VEGFA) regulates endothelial cell (EC) survival, tip cell formation, and stalk cell proliferation via VEGF receptor 2 (VEGFR2). VEGFR2 can interact with VEGFR2 co-receptors such as heparan sulfate proteoglycans (HSPGs) and neuropilin 2 (NRP2), but the exact roles of these co-receptors, or of sulfatase 2 (SULF2), an enzyme that removes sulfate groups from HSPGs and inhibits HSPG-mediated uptake of very low density lipoprotein (VLDL), in angiogenesis and tip cell biology are unknown. In the present study, we investigated whether the modulation of binding of VEGFA to VEGFR2 by knockdown of SULF2 or NRP2 affects sprouting angiogenesis, tip cell formation, proliferation of non-tip cells, and EC survival, or uptake of VLDL. To this end, we employed VEGFA splice variant 121, which lacks an HSPG binding domain, and VEGFA splice variant 165, which does have this domain, in in vitro models of angiogenic tip cells and vascular sprouting. We conclude that VEGFA165 and VEGFA121 have similar inducing effects on tip cells and sprouting in vitro, and that the binding of VEGFA165 to HSPGs in the extracellular matrix does not seem to play a role, as knockdown of SULF2 did not alter these effects. Co-binding of NRP2 appears to regulate VEGFA–VEGFR2-induced sprout initiation, but not tip cell formation. Finally, as the addition of VLDL increased sprout formation but not tip cell formation, and as VLDL uptake was limited to non-tip cells, our findings suggest that VLDL plays a role in sprout formation by providing biomass for stalk cell proliferation.

Evolution of Multicellular Complexity in The Dictyostelid Social Amoebas

Multicellularity evolved repeatedly in the history of life, but how it unfolded varies greatly between different lineages. Dictyostelid social amoebas offer a good system to study the evolution of multicellular complexity, with a well-resolved phylogeny and molecular genetic tools being available. We compare the life cycles of the Dictyostelids with closely related amoebozoans to show that complex life cycles were already present in the unicellular common ancestor of Dictyostelids. We propose frost resistance as an early driver of multicellular evolution in Dictyostelids and show that the cell signalling pathways for differentiating spore and stalk cells evolved from that for encystation. The stalk cell differentiation program was further modified, possibly through gene duplication, to evolve a new cell type, cup cells, in Group 4 Dictyostelids. Studies in various multicellular organisms, including Dictyostelids, volvocine algae, and metazoans, suggest as a common principle in the evolution of multicellular complexity that unicellular regulatory programs for adapting to environmental change serve as “proto-cell types” for subsequent evolution of multicellular organisms. Later, new cell types could further evolve by duplicating and diversifying the “proto-cell type” gene regulatory networks.

Dynamic Endothelial Stalk Cell–Matrix Interactions Regulate Angiogenic Sprout Diameter

Angiogenesis is a complex, multicellular process that involves bidirectional interactions between extracellular matrix (ECM) and collectively invading endothelial cell (EC) sprouts that extend the microvasculature during development, wound healing, and disease processes. While many aspects of angiogenesis have been well studied, the relationship between endothelial sprout morphology and subsequent neovessel function remains relatively unknown. Here, we investigated how various soluble and physical matrix cues that regulate endothelial sprouting speed and proliferation correspond to changes in sprout morphology, namely, sprout stalk diameter. We found that sprout stalk cells utilize a combination of cytoskeletal forces and proteolysis to physically compact and degrade the surrounding matrix, thus creating sufficient space in three-dimensional (3D) ECM for lateral expansion. As increasing sprout diameter precedes lumenization to generate perfusable neovessels, this work highlights how dynamic endothelial stalk cell–ECM interactions promote the generation of functional neovessels during sprouting angiogenesis to provide insight into the design of vascularized, implantable biomaterials.

Evolution of Multicellular Complexity in the Dictyostelid Social Amoebas

Multicellularity evolved repeatedly in the history of life, but how it unfolded varies greatly between different lineages. Dictyostelid social amoebas offer a good system to study the evolution of multicellular complexity, with a well-resolved phylogeny and molecular genetic tools being available. We compare the life cycles of the Dictyostelids with closely related amoebozoans to show that complex life cycles were already present in the unicellular common ancestor of Dictyostelids. We propose frost resistance as an early driver of multicellular evolution in Dictyostelids and show that the cell signalling pathways for differentiating spore and stalk cells evolved from that for encystation. The stalk cell differentiation program was further modified, possibly through gene duplication, to evolve a new cell type, cup cells, in Group 4 Dictyostelids. Studies in various multicellular organisms including Dictyostelids, volvocine algae, and metazoans suggest as a common principle in the evolution of multicellular complexity that unicellular regulatory programs for adapting to environmental change serve as “proto-cell types” for subsequent evolution of multicellular organisms. Later, new cell types could further evolve by duplicating and diversifying the “proto-cell type” gene regulatory networks.

Endemic Veronica saturejoides Vis. ssp. saturejoides–Chemical Composition and Antioxidant Activity of Free Volatile Compounds

Chemical profile and antioxidant activity of the species Veronica saturejoides Vis. ssp. saturejoides (Plantaginaceae)—which is endemic to Croatia, Bosnia and Herzegovina and Montenegro —were investigated. Volatile compounds produced by glandular trichomes (composed of one stalk cell and two elliptically formed head cells according to scanning electron microscope investigation) were isolated from the plants collected in two locations. Additionally, as a part of specialized metabolites, total polyphenols, total tannins, total flavonoids and total phenolic acids were determined spectrophotometrically. In the lipophilic volatile fractions-essential oils, the most abundant compounds identified were hexahydrofarnesyl acetone, caryophyllene oxide and hexadecanoic acid. In total, the class of oxygenated sesquiterpenes and the group of fatty aldehydes, acids and alcoholic compounds dominated in the essential oils. In the hydrophilic volatile fractions-hydrosols, the most abundant compounds identified were trans-p-mentha-1(7),8-dien-2-ol, allo-aromadendrene and (E)-caryophyllene. A group of oxygenated monoterpenes and the sesquiterpene hydrocarbons dominated in the hydrosols. Antioxidant activity of essential oils and hydrosols was tested with two methods: 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC). Essential oils showed higher antioxidant activity than hydrosols and showed similar antioxidant activity to Rosmarinus officinalis essential oil. Obtained results demonstrate that this genus is a potential source of volatiles with antioxidant activity.