The ESCRT-III machinery participates in the production of extracellular vesicles and protein export during Plasmodium falciparum infection.

Infection with Plasmodium falciparum enhances extracellular vesicle (EV) production in parasitized red blood cells (pRBCs), an important mechanism for parasite-to-parasite communication during the asexual intraerythrocytic life cycle. The endosomal sorting complex required for transport (ESCRT), and in particular the ESCRT-III sub-complex, participates in the formation of EVs in higher eukaryotes. However, RBCs have lost the majority of their organelles through the maturation process, including an important reduction in their vesicular network. Therefore, the mechanism of EV production in P. falciparum-infected RBCs remains to be elucidated. Here we demonstrate that P. falciparum possesses a functional ESCRT-III machinery activated by an alternative recruitment pathway involving the action of PfBro1 and PfVps32/PfVps60 proteins. Additionally, multivesicular body formation and membrane shedding, both reported mechanisms of EV production, were reconstituted in the membrane model of giant unilamellar vesicles using the purified recombinant proteins. Moreover, the presence of PfVps32, PfVps60 and PfBro1 in EVs purified from a pRBC culture was confirmed by super-resolution microscopy and dot blot assays. Finally, disruption of the PfVps60 gene led to a reduction in the number of the produced EVs in the KO strain and affected the distribution of other ESCRT-III components. Overall, our results increase the knowledge on the underlying molecular mechanisms during malaria pathogenesis and demonstrate that ESCRT-III P. falciparum proteins participate in EV production.

Magainin 2 and PGLa in bacterial membrane mimics III: Membrane fusion and disruption.

We previously speculated that the synergistically enhanced antimicrobial activity of Magainin 2 and PGLa is related to membrane adhesion, fusion, and further membrane remodeling. Here we combined computer simulations with time-resolved in vitro fluorescence microscopy, cryoelectron microscopy, and small-angle X-ray scattering to interrogate such morphological and topological changes of vesicles at nanoscopic and microscopic length scales in real time. Coarse-grained simulations revealed formation of an elongated and bent fusion zone between vesicles in the presence of equimolar peptide mixtures. Vesicle adhesion and fusion were observed to occur within a few seconds by cryoelectron microscopy and corroborated by small-angle X-ray scattering measurements. The latter experiments indicated continued and time-extended structural remodeling for individual peptides or chemically linked peptide heterodimers but with different kinetics. Fluorescence microscopy further captured peptide-dependent adhesion, fusion, and occasional bursting of giant unilamellar vesicles a few seconds after peptide addition. The synergistic interactions between the peptides shorten the time response of vesicles and enhance membrane fusogenic and disruption properties of the equimolar mixture compared with the individual peptides.

Metabolite Profiling of Gardenia jasminoides Ellis In Vitro Cultures with Different Levels of Differentiation.

Gardenia jasminoides Ellis is an aromatic and medicinal plant of high economic value. Much research has focused on the phytochemistry and biological activities of Gardenia fruit extracts; however, the potential of the Gardenia plant in vitro cultures used as mass production systems of valuable secondary metabolites has been understudied. This paper presents data on metabolite profiling (GC/MS and HPLC), antioxidant activities (DPPH, TEAC, FRAP, and CUPRAC), and SSR profiles of G. jasminoides plant leaves and in vitro cultures with different levels of differentiation (shoots, callus, and cell suspension). The data show strong correlations (r = 0.9777 to r = 0.9908) between antioxidant activity and the concentrations of chlorogenic acid, salicylic acid, rutin, and hesperidin. Eleven co-dominant microsatellite simple sequence repeats (SSRs) markers were used to evaluate genetic variations (average PIC = 0.738 ± 0.153). All of the investigated Gardenia in vitro cultures showed high genetic variabilities (average Na = 5.636 ± 2.157, average Ne = 3.0 ± 1.095). This is the first report on a study on metabolite profiles, antioxidant activities, and genetic variations of G. jasminoides in vitro cultures with different levels of differentiation.

Fluctuation spectroscopy of giant unilamellar vesicles using confocal and phase contrast microscopy.

A widely used method to measure the bending rigidity of bilayer membranes is fluctuation spectroscopy, which analyses the thermally-driven membrane undulations of giant unilamellar vesicles recorded with either phase-contrast or confocal microscopy. Here, we analyze the fluctuations of the same vesicle using both techniques and obtain consistent values for the bending modulus. We discuss the factors that may lead to discrepancies.

Recent Progress in Amaryllidaceae Biotechnology.

Plants belonging to the monocotyledonous Amaryllidaceae family include about 1100 species divided among 75 genera. They are well known as medicinal and ornamental plants, producing pharmaceutically important alkaloids, the most intensively investigated of which are galanthamine and lycorine. Amaryllidaceae alkaloids possess various biological activities, the most important one being their anti-acetylcholinesterase activity, used for the treatment of Alzheimer's disease. Due to increased demand for Amaryllidaceae alkaloids (mainly galanthamine) and the limited availability of plant sources, in vitro culture technology has attracted the attention of researchers as a prospective alternative for their sustainable production. Plant in vitro systems have been extensively used for continuous, sustainable, and economically viable production of bioactive plant secondary metabolites. Over the past two decades, a significant success has been demonstrated in the development of in vitro systems synthesizing Amaryllidaceae alkaloids. The present review discusses the state of the art of in vitro Amaryllidaceae alkaloids production, summarizing recently documented plant in vitro systems producing them, as well as the authors' point of view on the development of biotechnological production processes with a focus on the future prospects of in vitro culture technology for the commercial production of these valuable alkaloids.

Chemical Composition, In Vitro Antioxidant Potential, and Antimicrobial Activities of Essential Oils and Hydrosols from Native American Muscadine Grapes.

Essential oils and hydrosols of two cultivars of muscadine grapes (Muscadinia rotundifolia (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 Bacillus cereus, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. However, the essential oils from the flowers of both cultivars showed moderate antifungal activities against Candida albicans, 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.

Sage in vitro cultures: a promising tool for the production of bioactive terpenes and phenolic substances.

Extracts of Salvia species are used in traditional medicine to treat various diseases. The economic importance of this genus has increased in recent years due to evidence that some of its secondary metabolites have valuable pharmaceutical and nutraceutical properties.The bioactivity of sage extracts is mainly due to their content of terpenes and polyphenols. The increasing demand for sage products combined with environmental, ecological and climatic limitations on the production of sage metabolites from field-grown plants have led to extensive investigations into biotechnological approaches for the production of Salvia phytochemicals. The purpose of this review is to evaluate recent progress in investigations of sage in vitro systems as tools for producing important terpenoids and polyphenols and in development of methods for manipulating regulatory processes to enhance secondary metabolite production in such systems.

Plant In Vitro Culture Factories for Pentacyclic Triterpenoid Production.

Pentacyclic triterpenoids are a diverse subclass of naturally occurring terpenes with various biological activities and applications. These compounds are broadly distributed in natural plant resources, but their low abundance and the slow growth cycle of plants pose challenges to their extraction and production. The biosynthesis of pentacyclic triterpenoids occurs through two main pathways, the mevalonic acid (MVA) pathway and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which involve several enzymes and modifications. Plant in vitro cultures, including elicited and hairy root cultures, have emerged as an effective and sustainable system for pentacyclic triterpenoid production, circumventing the limitations associated with natural plant resources. Bioreactor systems and controlling key parameters, such as media composition, temperature, light quality, and elicitor treatments, have been optimized to enhance the production and characterization of specific pentacyclic triterpenoids. These systems offer a promising bioprocessing tool for producing pentacyclic triterpenoids characterized by a low carbon footprint and a sustainable source of these compounds for various industrial applications.

Exploring the Biosafety Potential of Haberlea rhodopensis Friv. In Vitro Culture Total Ethanol Extract: A Comprehensive Assessment of Genotoxicity, Mitotoxicity, and Cytotoxicity for Therapeutic Applications.

The escalating elderly population worldwide has prompted a surge of interest in longevity medicine. Its goal is to interfere with the speed of ageing by slowing it down or even reversing its accompanying effects. As a field, it is rapidly growing and spreading into different branches. One of these is the use of nutraceuticals as anti-ageing drugs. This field is gaining massive popularity nowadays, as people are shifting towards a more natural approach to life and seeking to use natural products as a source of medicine. The present article focuses on the cellular effect of Haberlea rhodopensis Friv. in vitro culture total ethanol extract (HRT), produced by a sustainable biotechnological approach. The extract showed a similar phytochemical profile to plant leaf extract and was rich in primary bioactive ingredients-caffeoyl phenylethanoid glycosides, myconoside, and paucifloside. This study examined the biosafety potential, cytotoxicity, genotoxicity, and mitochondrial activity of the extract using in vitro cultures. The results showed high cell survival rates and minimal cytotoxic effects on Lep3 cells, with no induction of reactive oxygen species nor genotoxicity. Additionally, the extract positively influenced mitochondrial activity, indicating potential benefits for cellular health. The results are promising and show the beneficial effect of HRT without the observation of any adverse effects, which sets the foundation for its further testing and potential therapeutic applications.

Stepwise remodeling and subcompartment formation in individual vesicles by three ESCRT-III proteins.

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.

Photomanipulation of Minimal Synthetic Cells: Area Increase, Softening, and Interleaflet Coupling of Membrane Models Doped with Azobenzene-Lipid Photoswitches.

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.

Phenolic Profile, Antioxidant and DNA-Protective Capacity, and Microscopic Characters of Ailanthus altissima Aerial Substances.

Invasive species as sources of natural components are of increasing interest for scientific research. This is the case of Ailanthus altissima, 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 A. altissima 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 A. altissima 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 A. altissima could be a natural source of DNA protection and antioxidants.

Femtoliter Injection of ESCRT-III Proteins into Adhered Giant Unilamellar Vesicles.

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.

Zwitterionic Dendrimersomes: A Closer Xenobiotic Mimic of Cell Membranes.

Building functional mimics of cell membranes is an important task toward the development of synthetic cells. So far, lipid and amphiphilic block copolymers are the most widely used amphiphiles with the bilayers by the former lacking stability while membranes by the latter are typically characterized by very slow dynamics. Herein, a new type of Janus dendrimer containing a zwitterionic phosphocholine hydrophilic headgroup (JDPC ) and a 3,5-substituted dihydrobenzoate-based hydrophobic dendron is introduced. JDPC self-assembles in water into zwitterionic dendrimersomes (z-DSs) that faithfully recapitulate the cell membrane in thickness, flexibility, and fluidity, while being resilient to harsh conditions and displaying faster pore closing dynamics in the event of membrane rupture. This enables the fabrication of hybrid DSs with components of natural membranes, including pore-forming peptides, structure-directing lipids, and glycans to create raft-like domains or onion vesicles. Moreover, z-DSs can be used to create active synthetic cells with life-like features that mimic vesicle fusion and motility as well as environmental sensing. Despite their fully synthetic nature, z-DSs are minimal cell mimics that can integrate and interact with living matter with the programmability to imitate life-like features and beyond.

Two-phase temporary immersion system for Agrobacterium rhizogenes genetic transformation of sage (Salvia tomentosa Mill.).

Hairy root cultures of Salvia tomentosa were initiated by transformation with Agrobacterium rhizogenes. To prevent necrosis in the explants and to protect young hairy roots, Amberlite XAD-4 resin, in combination with a temporary immersion cultivation system, was applied. HPLC analyzes showed that the resin adsorbed more than 93% of the released phenolic acids and 100% of the released flavonoids. The decreased content of the released phenolics significantly reduced their destructive effects on the plant tissues, prevented, and speeded up the appearance of hairy roots.

ESCRT-III induces phase separation in model membranes prior to budding and causes invagination of the liquid-ordered phase.

Membrane fission triggered by the endosomal sorting complex required for transport (ESCRT) is an important process observed in several pathogenic and non-pathogenic cellular events. From a synthetic-biology viewpoint, ESCRT proteins represent an interesting machinery for the construction of cell mimetic sub-compartments produced by fission. Since their discovery, the studies on ESCRT-III-mediated action, have mainly focused on protein dynamics, ignoring the role of lipid organization and membrane phase state. Recently, it has been suggested that membrane buds formed by the action of ESCRT-III are generated from transient microdomains in endosomal membranes. However, the interplay between membrane domain formation and ESCRT remodeling pathways has not been investigated. Here, giant unilamellar vesicles made of ternary lipid mixtures, either homogeneous in phase or exhibiting liquid-ordered/liquid-disordered phase coexistence, were employed as a model membrane system. These vesicles were incubated with purified recombinant ESCRT-III proteins from the parasite Entamoeba histolytica. In homogeneous membranes, we observe that EhVps32 can trigger domain formation while EhVps20 preferentially co-localizes in the liquid disordered phase. The addition of EhVps24 appears to induce the formation of intraluminal vesicles produced from the liquid-ordered phase. In phase separated membranes, the intraluminal vesicles are also generated from the liquid-ordered phase and presumably emerge from the phase boundary region. Our findings reinforce the hypothesis that ESCRT-mediated remodeling depends on the membrane phase state. Furthermore, the obtained results point to a potential synthetic biology approach for establishing eukaryotic mimics of artificial cells with microcompartments of specific membrane composition, which can also differ from that of the mother vesicle.

Recent applications of plant cell culture technology in cosmetics and foods.

Plants have been used as the main source of phytochemicals with nutritional, medicinal, cultural and cosmetic applications since times immemorial. Nowadays, achieving sustainable development, global climate change, restricted access to fresh water, limited food supply and growing energy demands are among the critical global challenges faced by humanity. Plant cell culture technology has the potential to address some of these challenges by providing effective tools for sustainable supply of phyto-ingredients with reduced energy, carbon and water footprints. The main aim of this review is to discuss the recent trends in the development of plant cell culture technologies for production of plant-derived substances with application in food products and cosmetic formulations. The specific technological steps and requirements for the final products are discussed in the light of the advances in cultivation technologies used for growing differentiated and undifferentiated plant in vitro systems. Future prospects and existing challenges of the commercialization of plant cell culture-derived products have been outlined through the prism of the authors' point of view. We expect this review will encourage scientists, policymakers and business enterprises to join efforts for speeding-up the mass commercialization and popularization of plant cell culture technology as an eco-friendly alternative method for sustainable production of plant-derived additives with application in food and cosmetic products.

Antioxidant activity and phenolic content of betalain extracts from intact plants and hairy root cultures of the red beetroot Beta vulgaris cv. Detroit dark red.

Betalains are water-soluble plant pigments that are widely used as food colorants, and have a wide range of desirable biological activities, including antioxidant, anti-inflammatory, hepatoprotective, anti-cancer properties. They can be produced from various plants, notably beetroot, but betalain products obtained in this way also have some undesirable properties and are difficult to standardize. A potentially attractive alternative is to use hairy root cultures. In the study reported here, we found that betalain extracts obtained from hairy root cultures of the red beetroot B. vulgaris cv. Detroit Dark Red also had higher antioxidant activity than extracts obtained from mature beetroots: six-fold higher 2,2-dyphenyl-1-picrylhydrazyl radical scavenging ability (90.7% inhibition, EC(50) = 0.11 mg, vs 14.2% inhibition, EC(50) = 0.70 mg) and 3.28-fold higher oxygen radical absorbance capacity (4,100 microM TE/g dry extract, vs 1,250 microM TE/g dry extract). The high antioxidant activity of the hairy root extracts was associated with increased concentrations (more than 20-fold) of total phenolic concomitant compounds, which may have synergistic effects with betalains. The presence of 4-hydroxybenzoic acid, caffeic acid, catechin hydrate, and epicatechin were detected in both types of extract, but at different concentrations. Rutin was only present at high concentration (1.096 mg.g(-1) dry extract) in betalain extracts from the hairy root cultures, whereas chlorogenic acid was only detected at measurable concentrations in extracts from intact plants.

Optimized nutrient medium for galanthamine production in Leucojum aestivum L. in vitro shoot system.

The common effect of NH4+, NO3-, KH2PO4 and sucrose on the biosynthesis of galanthamine by a Leucojum aestivum shoot culture was studied. Polynominal regression models were elaborated for the description of the galanthamine biosynthesis as a consequence of variation of the investigated variables (NH4+ between 0.20 and 0.54 g/L; NO3- between 1.44 and 3.44 g/L; KH2PO4 between 0.10 and 0.24 g/L, and sucrose between 30.00 and 60.00 g/L). Optimization procedures allowed us to establish the optimal concentrations of the investigated variables and to propose the modified MS nutrient medium, with 4.50 g/L KNO3, 0.89 g/L NH4NO3, 1.25 g/L (NH4)2SO4, 0.10 g/L KH2PO4 and 60 g/L sucrose, for the galanthamine production by a Leucojum aestivum shoot culture. The proposed modified MS medium provided considerable increase of both the production yield and the relative content of the target alkaloid in the alkaloid mixture.

In sito galanthamine extraction during the cultivation of Leucojum aestivum L. shoot culture in two-phase bubble column cultivation system.

Two-phase bioreactor cultivation system was developed and applied for in sito recovery of extracellular galanthamine during the cultivation of Leucojum aestivum L. shoot culture in a modified column bioreactor system. The inclusion of an external circulation column with adsorbent resin Amberlite XAD-4 as a second phase, on the 21st day of the beginning of cultivation resulted in 1.25 folds increase in biomass accumulation and maximal amounts of accumulated galanthamine of 6 mg/L (3.1 mg/L intracellular and 2.9 mg/L extracellular). It was demonstrated that the inclusion of a second phase at the cultivation of the L. aestivum shoot culture in a bubble column bioreactor with internal sections redirected the alkaloid metabolism to galanthamine synthesis and inhibits the synthesis of hemanthamine and lycorine type alkaloids. Our research demonstrated that the application of the two-phase cultivation systems could be an important tool to increase the yields of valuable secondary metabolites in plant tissue culture-based bioprocess.