Dr Josh Farley

Wound infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are notoriously difficult to treat due to biofilm formation and multidrug resistance, necessitating the development of novel antimicrobial agents. To address this challenge, we designed and synthesized a series of resveratrol-antimicrobial peptide mimic conjugates using a molecular splicing strategy. Among them, lead compound III-5 exhibited a minimum inhibitory concentration (MIC) of 6.25 μg/mL against MRSA, which is an approximately 40-fold improvement in antimicrobial activity over the precursor resveratrol. Moreover, III-5 demonstrated low hemolytic activity, a low propensity to induce drug resistance, and favorable anti-inflammatory properties. The membrane-targeted III-5 effectively disrupted bacterial cell membrane integrity and significantly inhibited both biofilm formation and the eradication of preformed mature biofilms. Transcriptomic analysis indicated a membrane-targeted mechanism, interfering with lipoteichoic acid biosynthesis, cell wall organization, and two-component systems. Together, these membrane-targeted actions synergistically impair cell wall integrity and suppress biofilm formation. Furthermore, in a murine model of MRSA-infected wounds, treatment with III-5-loaded PVA-SA hydrogel achieved a 96% wound healing rate by day 14, significantly accelerating wound closure and reducing the bacterial burden. Collectively, these findings position resveratrol-antibacterial peptide mimic conjugates as a promising antimicrobial candidate for combating MRSA-associated wound infections and provide valuable insights for the development of novel antimicrobial agents.

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.

Extracellular vesicles (EVs) are small membranous vesicles secreted by cells into their surrounding extracellular environment. Similar to mammalian EVs, plant EVs have emerged as essential mediators of intercellular communication in plants that facilitate the transfer of biological material between cells. They also play essential roles in diverse physiological processes including stress responses, developmental regulation, and defense mechanisms against pathogens. In addition, plant EVs have demonstrated promising health benefits as well as potential therapeutic effects in mammalian health. Despite the plethora of potential applications using plant EVs, their isolation and characterization remains challenging. In contrast to mammalian EVs, which benefit from more standardized isolation protocols, methods for isolating plant EVs can vary depending on the starting material used, resulting in diverse levels of purity and composition. Additionally, the field suffers from the lack of plant EV markers. Nevertheless, three main EV subclasses have been described from leaf apoplasts: tetraspanin 8 positive (TET8), penetration‐1‐positive (PEN1), and EXPO vesicles derived from exocyst‐positive organelles (EXPO). Here, we present an optimized protocol for the isolation and enrichment of small EVs (sEVs; <200 nm) from the apoplastic fluid from Nicotiana benthamiana leaves by ultracentrifugation. We analyze the preparation through transmitted electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. We believe this method will establish a basic protocol for the isolation of EVs from N. benthamiana leaves, and we discuss technical considerations to be evaluated by each researcher working towards improving their plant sEV preparations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation and enrichment of small extracellular vesicles (sEVs) from the apoplastic fluid of Nicotiana benthamiana leaves

Mechanisms devoted to the secretion of proteins via extracellular vesicles (EVs) have been found in mammals, yeasts, and plants. Since they transport a number of leader-less proteins to the plasma membrane or the extracellular space, EVs are considered part of Unconventional protein secretion (UPS) routes. UPS involving EVs are a relatively new field in plants. Aside from their role in plant physiology and immunity, plant extracts containing EVs have also been shown to be beneficial for human health. Therefore, exploring the use of plant EVs in biomedicine and their potential as drug delivery tools is an exciting avenue. Here we give a summary of the state of knowledge on plant EVs, their crosstalk with mammalian systems and potential research routes that could lead to practical applications in therapeutic drug delivery.