Extracellular vesicles (EVs) are heterogeneous lipid containers with a complex molecular cargo comprising several populations with unique roles in biological processes. These vesicles are closely associated with specific physiological features, which makes them invaluable in the detection and monitoring of various diseases. EVs play a key role in pathophysiological processes by actively triggering genetic or metabolic responses. However, the heterogeneity of their structure and composition hinders their application in medical diagnosis and therapies. This diversity makes it difficult to establish their exact physiological roles, and the functions and composition of different EV (sub)populations. Ensemble averaging approaches currently employed for EV characterization, such as western blotting or 'omics' technologies, tend to obscure rather than reveal these heterogeneities. Recent developments in single-vesicle analysis have made it possible to overcome these limitations and have facilitated the development of practical clinical applications. In this review, we discuss the benefits and challenges inherent to the current methods for the analysis of single vesicles and review the contribution of these approaches to the understanding of EV biology. We describe the contributions of these recent technological advances to the characterization and phenotyping of EVs, examination of the role of EVs in cell-to-cell communication pathways and the identification and validation of EVs as disease biomarkers. Finally, we discuss the potential of innovative single-vesicle imaging and analysis methodologies using microfluidic devices, which promise to deliver rapid and effective basic and practical applications for minimally invasive prognosis systems.

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Carotenoids are lipophilic plastidial isoprenoids highly valued as nutrients and natural pigments. A correct balance of chlorophylls and carotenoids is required for photosynthesis and therefore highly regulated, making carotenoid enrichment of green tissues challenging. Here we show that leaf carotenoid levels can be boosted through engineering their biosynthesis outside the chloroplast. Transient expression experiments in Nicotiana benthamiana leaves indicated that high extraplastidial production of carotenoids requires an enhanced supply of their isoprenoid precursors in the cytosol, which was achieved using a deregulated form of the main ratedetermining enzyme of the mevalonic acid (MVA) pathway. Constructs encoding bacterial enzymes were used to convert these MVA-derived precursors into carotenoid biosynthetic intermediates that do not normally accumulate in leaves, such as phytoene and lycopene. Cytosolic versions of these enzymes produced extraplastidial carotenoids at levels similar to those of total endogenous (i.e. chloroplast) carotenoids. Strategies to enhance the development of endomembrane structures and lipid bodies as potential extraplastidial carotenoid storage systems were not successful to further increase carotenoid contents. Phytoene was found to be more bioaccessible when accumulated outside plastids, whereas lycopene formed cytosolic crystalloids very similar to those found in the chromoplasts of ripe tomatoes. This extraplastidial production of phytoene and lycopene led to an increased antioxidant capacity of leaves. Finally, we demonstrate that our system can be adapted for the biofortification of leafy vegetables such as lettuce.

The improvement of culturing techniques to model the environment and physiological conditions surrounding tumors has also been applied to the study of extracellular vesicles (EVs) in cancer research. EVs role is not only limited to cell-to-cell communication in tumor physiology, they are also a promising source of biomarkers, and a tool to deliver drugs and induce antitumoral activity. In the present review, we have addressed the improvements achieved by using 3D culture models to evaluate the role of EVs in tumor progression and the potential applications of EVs in diagnostics and therapeutics. The most employed assays are gel-based spheroids, often utilized to examine the cell invasion rate and angiogenesis markers upon EVs treatment. To study EVs as drug carriers, a more complex multicellular cultures and organoids from cancer stem cell populations have been developed. Such strategies provide a closer response to in vivo physiology observed responses. They are also the best models to understand the complex interactions between different populations of cells and the extracellular matrix, in which tumor-derived EVs modify epithelial or mesenchymal cells to become protumor agents. Finally, the growth of cells in 3D bioreactor-like systems is appointed as the best approach to industrial EVs production, a necessary step toward clinical translation of EVs-based therapy.

14Formate can be directly produced from CO2 and renewable electricity, making it a promising microbial 15 feedstock for sustainable bioproduction. Cupriavidus necator is one of the few biotechnologically-relevant 16 hosts that can grow on formate, but it uses the inefficient Calvin cycle. Here, we redesign C. necator 17 metabolism for formate assimilation via the highly efficient synthetic reductive glycine pathway. First, we 18 demonstrate that the upper pathway segment supports glycine biosynthesis from formate. Next, we explore 19 the endogenous route for glycine assimilation and discover a wasteful oxidation-dependent pathway. By 20 integrating glycine biosynthesis and assimilation we are able to replace C. necator's Calvin cycle with the 21 synthetic pathway and achieve formatotrophic growth. We then engineer more efficient glycine metabolism 22 and use short-term evolution to optimize pathway activity, doubling the growth yield on formate and 23 quadrupling the growth rate. This study thus paves the way towards an ideal microbial platform for realizing 24 the formate bioeconomy. 25 26 27 28 Microbial biosynthesis offers an environmentally friendly alternative to fossil-based production. However, the 31 limited availability and questionable sustainability of microbial feedstocks hamper the expansion of 32 biotechnological production and the establishment of a circular carbon economy. The common substrates for 33 microbial bioproduction are plant-based sugars, the utilization of which competes with food supply and 34 necessitates vast land use that negatively impacts the environment. Moreover, alternative feedstocks, such 35 as lignocellulosic biomass, suffer from crucial drawbacks, such as difficult and expensive processing 1 . A 36 fundamental limitation of all photosynthesis-based resources is the low energy conversion efficiency 37 associated with this process, typically below 1% 2,3 . 38Electromicrobial production has gained attention as an alternative route towards sustainable biotechnology 4,5 . 39 This strategy is based on the use of two key feedstocks: CO2-free electricitye.g. from solar, wind, hydro -40 the production of which is rapidly growing, and CO2, a virtually unlimited carbon source, captured either from 41 point sources or directly from air. Some microbes can grow by receiving electrons directly from a cathode; 42 however, low current densities limit the economic viability of this approach 6,7 . A more feasible option is the 43 electrochemical production of small reduced compounds 6 that are subsequently fed to microbes and then 44 converted into value-added chemicals. Among the possible mediator compounds, hydrogen, carbon 45 monoxide, and formate can be produced at high efficiency and rate 8 . Whereas hydrogen and carbon monoxide 46 are gases of low solubility, formate is completely miscible and can be readily introduced to microbial cells 47 without mass transfer limitations and without major safety concerns 9 . Hence, establishing a "formate bio-48 economy" has been proposed as a ro...