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Plant cells secrete membrane-enclosed micrometer- and nanometer-sized vesicles that, similarly to the extracellular vesicles (EVs) released by mammalian or bacterial cells, carry a complex molecular cargo of proteins, nucleic acids, lipids, and primary and secondary metabolites. While it is technically complicated to isolate EVs from whole plants or their tissues, in vitro plant cell cultures provide excellent model systems for their study. Plant EVs have been isolated from the conditioned culture media of plant cell, pollen, hairy root, and protoplast cultures, and recent studies have gathered important structural and biological data that provide a framework to decipher their physiological roles and unveil previously unacknowledged links to their diverse biological functions. The primary function of plant EVs seems to be in the secretion that underlies cell growth and morphogenesis, cell wall composition, and cell-cell communication processes. Besides their physiological functions, plant EVs may participate in defence mechanisms against different plant pathogens, including fungi, viruses, and bacteria. Whereas edible and medicinal-plant-derived nanovesicles isolated from homogenised plant materials ex vivo are widely studied and exploited, today, plant EV research is still in its infancy. This review, for the first time, highlights the different in vitro sources that have been used to isolate plant EVs, together with the structural and biological studies that investigate the molecular cargo, and pinpoints the possible role of plant EVs as mediators in plant-pathogen interactions, which may contribute to opening up new scenarios for agricultural applications, biotechnology, and innovative strategies for plant disease management.

Cell culture-conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM-derived EVs (CCM-EV). The CCM-EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell-specific recommendations may need to be established for non-mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM-EV research., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Plant-derived nanovesicles (PDNVs) have become attractive alternatives to mammalian cell-derived extracellular vesicles (EVs) both as therapeutic approaches and drug-delivery vehicles. In this study, we isolated tomato fruit-derived NVs and separated them by the iodixanol density gradient ultracentrifugation (DGUC) into twelve fractions. Three visible bands were observed at densities 1.064 ± 0.007 g/mL, 1.103 ± 0.006 g/mL and 1.122 ± 0.012 g/mL. Crude tomato PDNVs and DGUC fractions were characterized by particle size-distribution, concentration, lipid and protein contents as well as protein composition using mass spectrometry-based proteomics. Cytotoxicity and anti-inflammatory activity of the DGUC fractions associated to these bands were assessed in the lipopolysaccharide (LPS)-stimulated human monocytic THP-1 cell culture. The middle and the low-density visible DGUC fractions of tomato PDNVs showed a significant reduction in LPS-induced inflammatory IL-1β cytokine mRNA production. Functional analysis of proteins identified in these fractions reveals the presence of 14-3-3 proteins, endoplasmic reticulum luminal binding proteins and GTP binding proteins associated to gene ontology (GO) term GO:0050794 and the regulation of several cellular processes including inflammation. The most abundant middle-density DGUC fraction was loaded with curcumin using direct loading, sonication and extrusion methods and anti-inflammatory activity was compared. The highest entrapment efficiency and drug loading capacity was obtained by direct loading. Curcumin loaded by sonication increased the basal anti-inflammatory activity of tomato PDNVs.

Small particles in natural sources are a subject of interest for their potential role in intercellular, inter-organism, and inter-species interactions, but their harvesting and assessment present a challenge due to their small size and transient identity. We applied a recently developed interferometric light microscopy (ILM) to assess the number density and hydrodynamic radius (R h ) of isolated small cellular particles (SCPs) from blood preparations (plasma and washed erythrocytes) (B), spruce needle homogenate (S), suspension of flagellae of microalgae Tetraselmis chuii (T), conditioned culture media of microalgae Phaeodactylum tricornutum (P), and liposomes (L). The aliquots were also assessed by flow cytometry (FCM), dynamic light scattering (DLS), ultraviolet-visible spectrometry (UV-vis), and imaging by cryogenic transmission electron microscopy (cryo-TEM). In R h , ILM showed agreement with DLS within the measurement error in 10 out of 13 samples and was the only method used here that yielded particle density. Cryo-TEM revealed that representative SCPs from Tetraselmis chuii flagella (T) did not have a globular shape, so the interpretation by R h of the batch methods was biased. Cryo-TEM showed the presence of thin filaments in isolates from Phaeodactylum tricornutum conditioned culture media (P), which provides an explanation for the considerably larger R h obtained by batch methods than the sizes of particles observed by cryo-TEM images. ILM proved convenient for assessment of number density and R h of SCPs in blood preparations (e.g., plasma); therefore, its use in population and clinical studies is indicated.

(1) Background: Extracellular vesicles (EVs) are considered to be efficient nanocarriers for improved drug delivery and can be derived from mammalian or plant cells. Cucumber-derived EVs are not yet described in the literature. Therefore, the aim of this study was to produce and characterize cucumber-derived EVs and to investigate their suitability to improve the dermal penetration efficacy of a lipophilic active ingredient (AI) surrogate. (2) Methods: The EVs were obtained by classical EVs isolation methods and by high pressure homogenization (HPH). They were characterized regarding their physico-chemical and biopharmaceutical properties. (3) Results: Utilization of classical isolation and purification methods for EVs resulted in cucumber-derived EVs. Their dermal penetration efficacy for the AI surrogate was 2-fold higher when compared to a classical formulation and enabled a pronounced transdermal penetration into the viable dermis. HPH resulted in submicron sized particles composed of a mixture of disrupted plant cells. A successful isolation of pure EVs from this mixture was not possible with classical EVs isolation methods. The presence of EVs was, therefore, proven indirectly. For this, the lipophilic drug surrogate was admixed to the cucumber juice either prior to or after HPH. Admixing of the drug surrogate to the cucumber prior to the HPH resulted in a 1.5-fold increase in the dermal penetration efficacy, whereas the addition of the AI surrogate to the cucumber after HPH was not able to improve the penetration efficacy. (4) Conclusions: Results, therefore, indicate that HPH causes the formation of EVs in which AI can be incorporated. The formation of plant EVs by HPH was also indicated by zeta potential analysis.

Background: Nanometer-sized membrane-surrounded vesicles from different parts of plants including fruits are gaining increasing attention due to their anti-inflammatory and anticancer effects demonstrated by in vitro and in vivo studies, and as nanovectors for molecular delivery of exogenous substances. These nanomaterials are very complex and contain a diverse arsenal of bioactive molecules, such as nucleic acids, proteins, and lipids. Our knowledge about the transport of allergens in vesicles isolated from plant food is limited today. Methods: Here, to investigate the allergenicity of strawberry-derived microvesicles (MVs), nanovesicles (NVs), and subpopulations of NV, we have set up a multidisciplinary approach. The strategy combines proteomics-based protein identification, immunological investigations, bioinformatics, and data mining to gain biological insights useful to evaluate the presence of potential allergens and the immunoglobulin E (IgE) inhibitory activity of vesicle preparations. Results: Immunological test showed that several proteins of strawberry-derived vesicles compete for IgE binding with allergens spotted on the FABER biochip. This includes the known strawberry allergens Fra a 1, Fra a 3, and Fra a 4, and also other IgE-binding proteins not yet described as allergens in this food, such as gibberellin-regulated proteins, 2S albumin, pectate lyase, and trypsin inhibitors. Proteomics identified homologous sequences of the three strawberry allergens and their isoforms in total protein extract (TPE) but only Fra a 1 and Fra a 4 in the vesicle samples. Label-free quantitative proteomic analysis revealed no significant enrichment of these proteins in strawberry vesicles with respect to TPE. Conclusion: Immunological tests and bioinformatics analysis of proteomics data sets revealed that MVs and NVs isolated from strawberries can carry functional allergens their isoforms as well as proteins potentially allergenic based on their structural features. This should be considered when these new nanomaterials are used for human nutraceutical or biomedical applications., Competing Interests: AM as, an ADL stakeholder, develops, manufactures, and distributes the FABER test as a diagnostic service. IG as an employee of ADL contributed to the development, manufacture, and performance of the FABER test. MAC receives funding from ADL., (Copyright © 2021 Stanly, Kim, Antonucci, Fiume, Guescini, Kim, Ciardiello, Giangrieco, Mari and Pocsfalvi.)

Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato ( Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC-MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry-based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 10 9 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii , using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production., Competing Interests: The authors report no conflict of interest., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Background: Alfalfa (Medicago sativa L) is one of the most planted crops worldwide primarily used to feed animals. The use of alfalfa in human diet as sprouts, infusions and nutritional supplements is rapidly gaining popularity. Despite this, allergenicity assessment of this novel plant food is largely lacking., Results: Here, leaf protein extract of alfalfa was studied using a combined proteomics, Immunoglobulin E (IgE)-binding inhibition assay and in silico approach to find potential allergens. We have identified and annotated 129 proteins using in-gel digestion proteomics and Blast2Go suit. A search against COMPARE database, using the identified proteins as query sequences, revealed high similarity with several allergenic proteins. The Single Point Highest Inhibition Achievable assay (SPHIAa) performed on the multiplex FABER ® allergy testing system confirmed the in silico results and showed some additional potential allergens. This approach allowed the detection of proteins in alfalfa leaves cross-reacting with plant allergens from three different allergen families such as lipid transfer, thaumatin-like and Bet v 1-like protein families. In addition, the absence of structural determinants cross-reacting with seed storage allergenic proteins and with animal allergens was recorded., Conclusion: This study reports for the first time potential allergenic proteins in alfalfa. The results suggest that this plant food can be safely introduced, as a protein-rich supplement, in the diet of patients allergic to animal food allergens. Allergic patients towards certain plant food allergens need to be careful about consuming alfalfa because they might have allergic symptoms. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Tiny membrane-enclosed cellular fragments that can mediate interactions between cells and organisms have recently become a subject of increasing attention. In this work the mechanism of formation of cell membrane nanovesicles (CNVs) was studied experimentally and theoretically. CNVs were isolated by centrifugation and washing of blood cells and observed by optical microscopy and scanning electron microscopy. The shape of the biological membrane in the budding process, as observed in phospholipid vesicles, in erythrocytes and in CNVs, was described by an unifying model. Taking the mean curvature h and the curvature deviator d of the membrane surface as the relevant parameters, the shape and the distribution of membrane constituents were determined theoretically by minimization of membrane free energy. Considering these results and previous results on vesiculation of red blood cells it was interpreted that the budding processes may lead to formation of different types of CNVs as regards the compartment (exo/endovesicles), shape (spherical/tubular/torocytic) and composition (enriched/depleted in particular kinds of molecules). It was concluded that the specificity of pinched off nanovesicles derives from the shape of the membrane constituents and not primarily from their chemical identity, which explains evidences on great heterogeneity of isolated extracellular vesicles with respect to composition., Competing Interests: The authors have declared that no competing interests exist.

Fruit juice is one of the most easily accessible resources for the isolation of plant-derived vesicles. Here we found that micro- and nano-sized vesicles (MVs and NVs) from four Citrus species, C. sinensis , C. limon , C. paradisi and C. aurantium , specifically inhibit the proliferation of lung, skin and breast cancer cells, with no substantial effect on the growth of non-cancer cells. Cellular and molecular analyses demonstrate that grapefruit-derived vesicles cause cell cycle arrest at G2/M checkpoint associated with a reduced cyclins B1 and B2 expression levels and the upregulation of cell cycle inhibitor p21. Further data suggest the inhibition of Akt and ERK signalling, reduced intercellular cell adhesion molecule-1 and cathepsins expressions, and the presence of cleaved PARP-1, all associated with the observed changes at the cellular level. Gas chromatography-mass spectrometry-based metabolomics reveals distinct metabolite profiles for the juice and vesicle fractions. NVs exhibit a high relative amount of amino acids and organic acids whereas MVs and fruit juice are characterized by a high percentage of sugars and sugar derivatives. Grapefruit-derived NVs are in particular rich in alpha-hydroxy acids and leucine/isoleucine, myo-inositol and doconexent, while quininic acid was detected in MVs. Our findings reveal the metabolite signatures of grapefruit-derived vesicles and substantiate their potential use in new anticancer strategies.

Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell and inter-kingdom communication. Despite their relevant biological implications, the existence and role of plant EVs released into the environment has been unexplored. Herein, we purified round-shaped small vesicles (EVs) by differential ultracentrifugation of a sampling solution containing root exudates of hydroponically grown tomato plants. Biophysical analyses, by means of dynamic light scattering, microfluidic resistive pulse sensing and scanning electron microscopy, showed that the size of root-released EVs range in the nanometric scale (50-100 nm). Shot-gun proteomics of tomato EVs identified 179 unique proteins, several of which are known to be involved in plant-microbe interactions. In addition, the application of root-released EVs induced a significant inhibition of spore germination and of germination tube development of the plant pathogens Fusarium oxysporum , Botrytis cinerea and Alternaria alternata. Interestingly, these EVs contain several proteins involved in plant defense, suggesting that they could be new components of the plant innate immune system.

Micro- and nano-sized vesicles (MVs and NVs, respectively) from edible plant resources are gaining increasing interest as green, sustainable, and biocompatible materials for the development of next-generation delivery vectors. The isolation of vesicles from complex plant matrix is a significant challenge considering the trade-off between yield and purity. Here, we used differential ultracentrifugation (dUC) for the bulk production of MVs and NVs from tomato ( Solanum lycopersicum L.) fruit and analyzed their physical and morphological characteristics and biocargo profiles. The protein and phospholipid cargo shared considerable similarities between MVs and NVs. Phosphatidic acid was the most abundant phospholipid identified in NVs and MVs. The bulk vesicle isolates were further purified using sucrose density gradient ultracentrifugation (gUC) or size-exclusion chromatography (SEC). We showed that SEC using gravity column efficiently removed co-purifying matrix components including proteins and small molecular species. dUC/SEC yielded a high yield of purified vesicles in terms of number of particles (2.6 × 10 15 particles) and protein quantities (6.9 ± 1.5 mg) per kilogram of tomato. dUC/gUC method separated two vesicle populations on the basis of buoyant density. Proteomics and in silico studies of the SEC-purified MVs and NVs support the presence of different intra- and extracellular vesicles with highly abundant lipoxygenase (LOX), ATPases, and heat shock proteins (HSPs), as well as a set of proteins that overlaps with that previously reported in tomato chromoplast.

Essential oils (EOs) obtained from aromatic plants are widely used worldwide, especially in cosmetic and food products due to their aroma and biological properties and health benefits. Some EOs have significant antimicrobial and antioxidant activities, and thus could effectively increase the shelf lives of foodstuff and beverages. In this study, fourteen essential oils (clove, eucalyptus, fennel, lavender, oregano, palmarosa, pepper, star anise, tea tree, turmeric, Chinese yin yang, Japanese yin yang, and ylang ylang) from different medicinal plant families were screened by gas-chromatography-mass spectrometry (GC-MS) for their different chemical profiles and bioassays were performed to assess their antifungal and antioxidant activities. The results obtained were assessed by principal component analysis (PCA). PCA distinguished six groups characterized by different terpene chemotypes. Amongst the EOs studied, the clove EO showed the strongest antioxidant activity characterized by an EC 50 of 0.36 µL/mL. The oregano EO had the greatest antiyeast activity characterized by a minimal inhibitory concentration of 10 µL/mL. In conclusion, clove and oregano EOs are strong antifungal and antioxidant agents, respectively, with great potential in the food industry to avoid spoilage and to increase shelf life.

Background: The outbreak of severe acute respiratory syndrome β-coronavirus 2 (SARS-CoV-2) has the potential to become a long-lasting global health crisis. The number of people infected with the novel coronavirus has surpassed 22 million globally, resulting in over 700,000 deaths with more than 15 million people having recovered (https://covid19.who.int). Enormous efforts are underway for rapid vaccine and treatment developments. Amongst the many ways of tackling the novel coronavirus disease 2019 (COVID-19) pandemic, extracellular vesicles (EVs) are emerging., Summary: EVs are lipid bilayer-enclosed structures secreted from all types of cells, including those lining the respiratory tract. They have established roles in lung immunity and are involved in the pathogenesis of various lung diseases, including viral infection. In this review, we point out the roles and possible contribution of EVs in viral infections, as well as ongoing EV-based approaches for the treatment of COVID-19, including clinical trials. Key Messages: EVs share structural similarities to viruses and recent findings demonstrate that viruses exploit EVs for cellular exit and EVs exploit viral entry mechanisms for cargo delivery. Moreover, EV-virus interplay could be exploited for future antiviral drug and vaccine development. EV-based therapies, especially the mesenchymal stem cell-derived EVs, are being intensively studied for the treatment of COVID-19., (© 2020 The Author(s) Published by S. Karger AG, Basel.)

The cellular vesicle is a fluid-filled structure separated from the surrounding environment by a biological membrane. Here, we isolated nanovesicles (NVs) from the juice of clementines using a discontinuous density gradient ultracentrifugation method. To gain information about the protein content of vesicles, mass spectrometry-based organelle proteomics and bioinformatics were applied to the exosome-like vesicle fraction isolated in the 1 mol/L sucrose/D 2 O cushion. Analysis of 1018 identified proteins revealed a highly complex mixture of different intra, extracellular and artificially-formed vesicle populations. In particular, clathrin-coated vesicles were significantly expressed in this sample. Membrane transporters are significantly represented in clementines nanovesicles. We have found 162 proteins associated with the transport Gene Ontology term (GO: 0006810) which includes; 71 transmembrane transport related, 53 vesicle mediated and 50 intracellular transporters. Platellin-3 like carrier protein containing a Sec14 domain is known to have a role in plant-virus interaction and that is one of the most abundant proteins in our dataset. The presence of transmembrane transporters like ATPases, aquaporins, ATP Binding Cassette (ABC) transporters and tetraspanins, regulators of protein trafficking suggests that nanovesicles of clementines can actively interact with their environment in a controlled way.

Potential applications of extracellular vesicles (EVs) are attracting increasing interest in the fields of medicine, cosmetics, and nutrition. However, the manufacturing of EVs is currently characterized by low yields. This limitation severely hampers progress in research at the laboratory and clinical scales, as well as the realization of successful and cost-effective EV-based products. Moreover, the high level of heterogeneity of EVs further complicates reproducible manufacturing on a large scale. In this review, possible directions toward the scalable production of EVs are discussed. In particular, two strategies are considered: i) the optimization of upstream unit operations and ii) the exploitation of well-established and mature technologies already in use in other industrial bioprocesses., (© 2019 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Indole-3-acetic acid (IAA) is the main auxin acting as a phytohormone in many plant developmental processes. The ability to synthesize IAA is widely associated with plant growth-promoting rhizobacteria (PGPR). Several studies have been published on the potential application of PGPR to improve plant growth through the enhancement of their main metabolic processes. In this study, the IAA-overproducing Ensifer meliloti strain RD64 and its parental strain 1021 were used to inoculate Medicago sativa plants. After verifying that the endogenous biosynthesis of IAA did not lead to genomic changes during the initial phases of the symbiotic process, we analyzed whether the overproduction of bacterial IAA inside root nodules influenced, in a coordinated manner, the activity of the nitrogen-fixing apparatus and the photosynthetic function, which are the two processes playing a key role in legume plant growth and productivity. Higher nitrogen-fixing activity and a greater amount of total nitrogen (N), carbon (C), Rubisco, nitrogen-rich amino acids, soluble sugars, and organic acids were measured for RD64-nodulated plants compared to the plants nodulated by the wild-type strain 1021. Furthermore, the RD64-nodulated plants showed a biomass increase over time, with the highest increment (more than 60%) being reached at six weeks after infection. Our findings show that the RD64-nodulated plants need more substrate derived from photosynthesis to generate the ATP required for their increased nitrogenase activity. This high carbohydrate demand further stimulates the photosynthetic function with the production of molecules that can be used to promote plant growth. We thus speculate that the use of PGPR able to stimulate both C and N metabolism with a balanced C/N ratio represents an efficient strategy to obtain substantial gains in plant productivity.

Qualitative and quantitative data obtained on micro and nanovesicle enriched fractions isolated from four citrus species, C. sinensis , C. limon , C. paradisi and C. aurantium are presented. It includes physiochemical characterization by transmission electron microscopy (TEM) and dynamic laser scattering (DLS); and molecular characterization of the biocargo of citrus vesicles by quantitative label-free proteomics. Vesicular transport related proteins of C. sinensis were predicted by (i) finding orthologues based on previously described vesicular transport proteins and (ii) GO term enrichment analysis. Based on the protein content different types of intra and intercellular vesicles were dissected and the distribution of different Enzyme classes (ECs) were determined. This data article is related to "Protein biocargo of citrus fruit-derived vesicles reveals heterogeneous transport and extracellular vesicle populations" (Pocsfalvi et al., 2018).

Cell-derived vesicles are membrane-enclosed organelles that transport material inside and outside the cell. Plant-derived vesicles are receiving more and more attention due to their potential as nanovectors for the delivery of biologically active substances. Here, we studied the heterogeneity and protein biocargo in citrus fruit juice sac cell-derived vesicles populations. Micro- and nano-sized vesicle fractions were isolated from four citrus species, C. sinensis, C. limon, C. paradisi and C. aurantium, characterized using physicochemical methods and protein cargos were compared using label-free quantitative shotgun proteomics. In each sample approximately 600-800 proteins were identified. Orthologues of most of the top-ranking proteins have previously been reported in extracellular vesicles of mammalian origin. High expression levels of patellin-3-like, clathrin heavy chain, heat shock proteins, 14-3-3 protein, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase 6 were measured in all samples while aquaporin was highly expressed only in the nanovesicle fractions. Bioinformatics revealed more than hundred protein orthologues potentially implicated in vesicular trafficking. In particular, the presence of CCV, COPI and COPII coat proteins indicates the presence of heterogeneous populations of intracellular transport vesicles. Moreover, a high number of different enzymes including hydrolases and oxidoreductases are ubiquities in citrus fruit sac cell-derived vesicles., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Introduction: Extracellular vesicles are emerging sources of biomarkers for modern preventive and precision medicine. Extracellular vesicles in body fluids offer a unique opportunity for integrative biomarker approaches due to their complex biocargo that includes proteins, lipids, nucleic acids and metabolites. Mass spectrometry-based proteomics data suggest that a significant portion of human proteins are sorted into extracellular vesicles and amenable for biomarker discovery schemes. Areas covered: this review focuses on key aspects of isolation, quality control and subsequent analysis of blood plasma- and conditioned medium-derived extracellular vesicle proteins, and summarizes the current state-of-the-art in the field. Furthermore, it provides introduction and guidelines for mass spectrometry-based proteomic analysis of extracellular vesicles. Expert commentary: Comparison of newly developed isolation and purification techniques with classical ultracentrifugation-based approaches are highly recommended. It is also essential to use multiple analytical approaches to characterize the isolated extracellular vesicles prior to characterization of their biocargo. Rigor in data reproducibility, critical data analysis, awareness of potential pitfalls, standardization and benchmarking are required for extracellular vesicle research to fulfil the current expectation that these subcellular structures can become a valid source of next generation biomarkers.

Chemo-enzymatic synthesis of oligosaccharides exploits the diversity of glycosidases and their ability to promote transglycosylation reactions in parallel with hydrolysis. Methods to increase the transglycosylation/hydrolysis ratio include site-directed mutagenesis and medium modification. The former approach was successful in several cases and has provided the best synthetic yields with glycosynthases-mutants at the catalytic nucleophile position that promote transglycosylation with high efficiency, but do not hydrolyze the oligosaccharide products. Several glycosidases have proven recalcitrant to this conversion, thus alternative methods to increase the transglycosylation/hydrolysis ratio by mutation would be very useful. Here we show that a mutant of a β-galactosidase from Alicyclobacillus acidocaldarius in an invariant residue in the active site of the enzymes of this family (glutamic acid 361) carries out efficient transglycosylation reactions on different acceptors only in the presence of external ions with yields up to 177-fold higher than that of the wild type. This is the first case in which sodium azide and sodium formate in combination with site-directed mutagenesis have been used to introduce transglycosylation activity into a glycosidase. These observations will hopefully guide further efforts to generate useful synthases., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

An extracellular halophilic alpha-amylase (AmyA) was produced by the haloarchaeon Haloterrigena turkmenica grown in medium enriched with 0.2% (w/v) starch. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) analyses showed a major band at 66.0kDa and a peak of 54.0kDa, respectively. Analysis of tryptic fragments of the protein present in the major SDS-PAGE band by nano-LC-ESI-MS/MS led to identification of the alpha-amylase catalytic region, encoded by the htur2110 gene, as the protein possessing the described activity. Optimal values for activity were 55°C, pH 8.5 and 2M NaCl, and high thermostability was showed at 55°C and 3M NaCl. AmyA activity was enhanced by Triton X-100 and was not influenced by n-hexane and chloroform. Starch hydrolysis produced different oligomers with maltose as the smallest end-product. The efficiency of AmyA in degrading starch contained in agronomic residues was tested in grape cane chosen as model substrate. Preliminary results showed that starch was degraded making the enzyme a potential candidate for utilization of agro-industrial waste in fuel and chemicals production. AmyA is one of the few investigated amylases produced by haloarchaea, and the first alpha-amylase described among microorganisms belonging to the genus Haloterrigena., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.