Light can effectively interrogate biological systems in a reversible and physiologically compatible manner with high spatiotemporal precision. Understanding the biophysics of photo-induced processes ...in bio-systems is crucial for achieving relevant clinical applications. Employing membranes doped with the photolipid azobenzene-phosphatidylcholine (azo-PC), a holistic picture of light-triggered changes in membrane kinetics, morphology, and material properties obtained from correlative studies on cell-sized vesicles, Langmuir monolayers, supported lipid bilayers, and molecular dynamics simulations is provided. Light-induced membrane area increases as high as ≈25% and a ten-fold decrease in the membrane bending rigidity is observed upon trans-to-cis azo-PC isomerization associated with membrane leaflet coupling and molecular curvature changes. Vesicle electrodeformation measurements and atomic force microscopy reveal that trans azo-PC bilayers are thicker than palmitoyl-oleoyl phosphatidylcholine (POPC) bilayers but have higher specific membrane capacitance and dielectric constant suggesting an increased ability to store electric charges across the membrane. Lastly, incubating POPC vesicles with azo-PC solutions results in the insertion of azo-PC in the membrane enabling them to become photoresponsive. All these results demonstrate that light can be used to finely manipulate the shape, mechanical and electric properties of photolipid-doped minimal cell models, and liposomal drug carriers, thus, presenting a promising therapeutic alternative for the repair of cellular disorders.
The endosomal sorting complex required for transport (ESCRT) machinery mediates membrane fission reactions that exhibit a different topology from that observed in clathrin-coated vesicles. In all of ...the ESCRT-mediated events, the nascent vesicle buds away from the cytosol. However, ESCRT proteins are able to act upon membranes with different geometries. For instance, the formation of multivesicular bodies (MVBs) and the biogenesis of extracellular vesicles both require the participation of the ESCRT-III sub-complex, and they differ in their initial membrane geometry before budding starts: the protein complex acts either from outside the membrane organelle (causing inward budding) or from within (causing outward budding). Several studies have reconstituted the action of the ESCRT-III subunits in supported bilayers and cell-sized vesicles mimicking the geometry occurring during MVBs formation (in-bud), but extracellular vesicle budding (out-bud) mechanisms remain less explored, because of the outstanding difficulties encountered in encapsulation of functional ESCRT-III in vesicles. Here, we provide a different approach that allows the recreation of the out-bud formation, by combining giant unilamellar vesicles as a membrane model and a microinjection system. The vesicles are immobilized prior to injection via weak adhesion to the chamber coverslip, which also ensures preserving the membrane excess area required for budding. After protein injection, vesicles exhibit outward budding. The approach presented in this work can be used in the future to disentangle the mechanisms underlying ESCRT-III-mediated fission, recreating the geometry of extracellular bud production, which remains a challenge. Moreover, the microinjection methodology can be also adapted to interrogate the action of other cytosolic components on the encapsulating membranous organelle. Graphic abstract: Out-bud formation after ESCRT-III protein injection into GUVs.
Essential oils and hydrosols of two cultivars of muscadine grapes (
(Michx.) Small.) were obtained by hydro-distillation of flowers and berry skins. Twenty-three volatile compounds were identified in ...essential oils from the muscadine flowers, and twenty volatiles in their corresponding hydrosols. The composition of volatiles in berry skins differed significantly from that of the vine flowers. The antioxidant potential of investigated essential oils and hydrosols was evaluated using five in vitro assays: DPPH (2,2-diphenyl-1-picrylhydrazyl) method, TEAC (Trolox equivalent antioxidant capacity), FRAP (Ferric reducing antioxidant power), CUPRAC (cupric ion reducing antioxidant capacity), and NO (nitric oxide radical scavenging assay). The essential oils from the flowers of both cultivars showed the strongest antioxidant power, whereas the hydrosols were the significantly less active. All investigated essential oils showed very weak antibacterial activities against
,
,
, and
. However, the essential oils from the flowers of both cultivars showed moderate antifungal activities against
, which were stronger for the oil from "Carlos" (white muscadine cultivar). To the best of our knowledge, this is the first report on obtaining and characterizing essential oils and hydrosols from muscadine grapes. This study demonstrated the variations in aromatic compounds accumulated in flowers and mature berry skins of muscadine grapes, and evaluated their possible antioxidant and antimicrobial activities. The presented results will be the base for future research, focused on a better understanding of the molecular and regulatory mechanisms involved in aromatic compound biosynthesis and accumulation in muscadine grapes.
Biomembranes are constantly remodeled and in cells, these processes are controlled and modulated by an assortment of membrane proteins. Here, it is shown that such remodeling can also be induced by ...photoresponsive molecules. The morphological control of giant vesicles in the presence of a water‐soluble ortho‐tetrafluoroazobenzene photoswitch (F‐azo) is demonstrated and it is shown that the shape transformations are based on an increase in membrane area and generation of spontaneous curvature. The vesicles exhibit budding and the buds can be retracted by using light of a different wavelength. In the presence of F‐azo, the membrane area can increase by more than 5% as assessed from vesicle electrodeformation. To elucidate the underlying molecular mechanism and the partitioning of F‐azo in the membrane, molecular dynamics simulations are employed. Comparison with theoretically calculated shapes reveals that the budded shapes are governed by curvature elasticity, that the spontaneous curvature can be decomposed into a local and a nonlocal contribution, and that the local spontaneous curvature is about 1/(2.5 µm). The results show that exo‐ and endocytotic events can be controlled by light and that these photoinduced processes provide an attractive method to change membrane area and morphology.
Morphological control of giant vesicles exhibited in exo‐ and endocytotic events is established in the presence of an ortho‐tetrafluoroazobenzene photoswitch. The vesicles undergo reversible budding modulated by light of different wavelengths. The shape transformations result from membrane area increase and generation of spontaneous curvature. Molecular dynamics simulations elucidate the underlying molecular mechanism of the photoswitch action.
The coiled-coil forming peptides 'K' enriched in lysine and 'E' enriched in glutamic acid have been used as a minimal SNARE mimetic system for membrane fusion. Here we describe atomistic molecular ...dynamics simulations to characterize the interactions of these peptides with lipid bilayers for two different compositions. For neutral phosphatidylcholine (PC)/phosphatidylethanolamine (PE) bilayers the peptides experience a strong repulsive barrier against adsorption, also observed in potential of mean force (PMF) profiles calculated with umbrella sampling. For peptide K, a minimum of -12 k
T in the PMF provides an upper bound for the binding free energy whereas no stable membrane bound state could be observed for peptide E. In contrast, the electrostatic interactions with negatively charged phosphatidylglycerol (PG) lipids lead to fast adsorption of both peptides at the head-water interface. Experimental data using fluorescently labeled peptides confirm the stronger binding to PG containing bilayers. Lipid anchors have little effect on the peptide-bilayer interactions or peptide structure, when the peptide also binds to the bilayer in the absence of a lipid anchor. For peptide E, which does not bind to the PC bilayer without a lipid anchor, the presence of such an anchor strengthens the electrostatic interactions between the charged side chains and the zwitterionic head-groups and leads to a stabilization of the peptide's helical fold by the membrane.
Polyphenols are bioactive natural molecules biogenerated through secondary metabolic pathways. They are involved in different functions in the ecology, physiology, and biochemistry of plants such as ...chemical defense against predators and in plant-plant interferences. These compounds are known to have important biological activities related to human health such as antioxidant action, antiinflammatory and antimicrobial effects. The immense health benefits as well as use of many polyphenolic compounds as anti-infective agents against human pathogens have heightened the need for continuous supply of rare and expensive secondary metabolites. It has been demonstrated that the chemical structure of dietary polyphenols, such as the number and position of hydroxyl groups, can change their biological properties and bioavailability. This review focuses on prospects for, and success in metabolic engineering, including manipulation of structural regulatory genes to develop plants with tailor-made, optimized levels and composition of polyphenols.
The endosomal sorting complex required for transport (ESCRT) is a multi-protein machinery involved in several membrane remodeling processes. Different approaches have been used to resolve how ESCRT ...proteins scission membranes. However, the underlying mechanisms generating membrane deformations are still a matter of debate. Here, giant unilamellar vesicles, microfluidic technology, and micropipette aspiration are combined to continuously follow the ESCRT-III-mediated membrane remodeling on the single-vesicle level for the first time. With this approach, we identify different mechanisms by which a minimal set of three ESCRT-III proteins from Entamoeba histolytica reshape the membrane. These proteins modulate the membrane stiffness and spontaneous curvature to regulate bud size and generate intraluminal vesicles even in the absence of ATP. We demonstrate that the bud stability depends on the protein concentration and membrane tension. The approaches introduced here should open the road to diverse applications in synthetic biology for establishing artificial cells with several membrane compartments.
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•ESCRT-III proteins change membrane stiffness and spontaneous curvature•These properties modulate the size of buds and intraluminal vesicles•Bud stability depends on protein concentration and membrane tension•The ESCRT-III forms domain-like protein-rich patches on membranes
Biotechnology; Membranes; Cell biology; Biophysics
Cultivation of plant cells under controlled conditions is a highly effective and fast developing technology that recently was considered as a branch of cellular agriculture. Cellular agriculture is a ...multidisciplinary approach for sustainable and renewable production of agricultural goods and raw materials at cellular, rather than organismal, level. However, in contrast to the rapid advance in development of cultured meat and precision fermentation, the production of nutritional supplements from plant cells is still in its infancy. One of the limiting factors, striating commercialization of plant cells for food production, is the low yields of target bioactive metabolites. In this work, the changes in phenolics, anthocyanins and exopolysaccharides accumulation during cultivation of Aronia melanocarpa (Michx.) Elliott cell suspension cultures in darknessor under illumination (16 light and 8 dark) were investigated. The data showed that the highest contents of total phenolics (8.17 ± 0.39 mg GAE/g DW), total anthocyanins (0.011 ± 0.001 mg cyanidin-3-glucoside equivalents/g DW) and antioxidant activities (DPPH—21.36 ± 0.29 µM TE/g DW; TEAC—10.08 ± 0.07 µM TE/g DW; FRAP—34.85 ± 1.47 µM TE/g DW; and CUPRAC—126.74 ± 9.15 µM TE/g DW) were achieved when the cells were grown under illumination (16 light and 8 dark). In contrast, when the culture was grown indarkness, the highest amounts of accumulated dry biomass (8.68 ± 0.35 g/L) and exopolysaccharides production (2.10 ± 0.07 g/L) were reached. The results demonstrated that light can be used as an affordable and highly effective factor to control the production of valuable antioxidants by black chokeberry cell suspension culture.
The book Alkaloids - Alternatives in Synthesis, Modification, and Application collects several chapters written by distinguished scientists and recognized experts in their respective fields of ...research. The purpose of this book is to focus the attention of a broad range of students, researchers, and specialists on some innovative and highly perspective areas in alkaloid research. The book covers several topics, guiding the readers from the development of nonconventional biotechnologies for alternative production of valuable alkaloids, through the application of modern chemical methods of asymmetric synthesis for production of synthetic and semisynthetic alkaloid derivatives, medicinal application of alkaloids as anesthetics and pain-relief drugs, analytical techniques for alkaloid profiling and their application in chemotaxonomy, quality control and standardization of raw plant material, to the importance of the control and reduction of alkaloid contents during production of animal feedstuffs.
Invasive species as sources of natural components are of increasing interest for scientific research. This is the case of
, which belongs to the top 100 of the most dangerous invasive plant species ...in Europe, and which is the subject of the present study. The purpose of the research was to analyze the main phenolic compounds in the flowers, leaves, and stem bark of
and determine the DNA-protective and antioxidant potential of their ethanolic extracts. HPLC profiling revealed the presence of 6 flavonoids and 10 phenolic acids, of which 15 were found in flowers, 14 in leaves, and 11 in the stem bark. Rutin (5.68 mg/g dw in flowers), hesperidin (2.67 mg/g dw in leaves) and (+)-catechin (2.15 mg/g dw in stem bark) were the best-represented flavonoids. Rosmarinic (10.32 mg/g dw in leaves) and salicylic (6.19 mg/g dw in leaves) acids were predominant among phenolic acids. All plant extracts tested showed in vitro antioxidant activity (determined by DPPH, ABTS, FRAP, and CUPRAC assays) and DNA-protection capacity (assay with supercoiled plasmid DNA-pUC19). The highest antioxidant activity was recorded in the flower parts (in the range from 661 to 893 mmol TE/g dw), followed by the leaves. A DNA protective potential for
.
leaf and flower extracts has not been established to date. In addition, the main microscopic diagnostic features of studied plant substances were described, with data for the flower parts being reported for the first time. The present study proves that
could be a natural source of DNA protection and antioxidants.
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