A membrane process to extract Citrus Lemon derived extracellular vesicles

Extra cellular vesicles are membrane bound and lipid based nano particles having the size range of 30 to 1000 nm released by a plethora of cells. Their prime function is cellular communication but in the recent studies, the potential of these vesicles to maintain physiological and pathological processes as well as their nano-sized constituents opened doors to its applications in therapeutics, and diagnostics of variety of diseases such as cancer. Their main constituents include lipids, proteins, and RNAs. They are categorized into subtypes such as exosomes, micro-vesicles and apoptotic bodies In recent studies, extracellular vesicles that are derived from plants are gaining high regard due to their variety of advantages such as safety, non-toxicity, and high availability which promotes large scale production. EVs are isolated from mammalian and plant cells using multitude of techniques such as Ultracentrifugation, SEC, Precipitation and so on. Due to the variety in the sources as well as shortcomings arising from the isolation method, a scalable and inexpensive EV isolation method is yet to be designed. This study focusses on isolation of EVs from citrus lemon juice through diafiltration. Lemon is a promising source due to its biological properties to act as antioxidant, anticancer, and anti-inflammatory agents. Lemon derived vesicles was proven to have several proteins analogous to mammalian vesicles. A diafiltration could be carried out for successful removal of impurities and it is a scalable, continuous technique with potentially lower process times. The concentration of purified product and impurities are analysed using Size Exclusion Chromatography in analytical mode. It is also considered imperative to compare the results from diafiltration with gold standard UC. BCA is proposed to evaluate total protein content and DLS for size measurements. Finally, the ideal mode of storage of EVs to protect its internals and its structure is analysed with storage tests.

Portofino Seaweed Garden: a local citizen science case study on Ericaria amentacea (C.Agardh) Molinari & Guiry, 2020 restoration

Ericaria amentacea is an endemic alga of the Mediterranean Sea that lives in the littoral rocky fringe. The species is sensitive to environmental changes, so it’s used to assess the water ecological quality. Nevertheless, E. amentacea is deeply impacted by coastal development which cause rapid regression despite data on its distribution and healthy status are still limited. Moreover, it’s little known by people outside the scientific community. In this context, Portofino Seaweed Garden was born, a conservation and citizen science project aim to involve marine outdoor enthusiasts in protecting and restoring E. amentacea, creating a submerged garden. Restoration measures have been encouraged by EU regulations. Here, using citizen science, 1) I evaluated the spatial variability of E. amentacea abundance along the central-eastern Ligurian coast, to evaluate its status and choose donor and restoration sites. 2) I carried out an E. amentacea restoration (with outplanting lab-cultured embryos on 50 clay tiles). 3) I assessed the community involvement and education of volunteers. Simple protocol was created to train them on the monitoring. Unprecedented E. amentacea reproductive mismatch affected the restoration performance, probably caused by marine heat wave that hit the Mediterranean in summer 2022. After fertile apices collection in Pontetto (GE) and during laboratory phase, gametes spawned on the discs didn’t settle as expected. Only 16 tiles showed juveniles and they were outplanted at Punta Castello (C zone of Portofino MPA). Unfortunately, they didn’t survive in the field due to an interplay of physical and biological factors. From citizen science point of view, the project demonstrated positive outcomes of collaborations between people and scientists by involving more than 100 participants. Citizen scientists became specialize in the protocol providing quality data for E. amentacea conservation. Current results suggest that outplanting should be further tested.