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Enhanced Fluorescent Protein Activity in Polymer Scaffold-Stabilized Phospholipid Nanoshells Using Neutral Redox Initiator Polymerization Conditions
摘要： Phospholipid nanoshells, for example, liposomes, provide a versatile enabling platform for the development of nanometer-sized biosensors and molecular delivery systems. Utilization of phospholipid nanoshells is limited by the inherent instability in complex biological environments, where the phospholipid nanoshell may disassemble and degrade, thus releasing the contents and destroying sensor function. Polymer sca?old stabilization (PSS), wherein the phospholipid nanoshells are prepared by partitioning reactive monomers into the lipid bilayer lamella followed by radical polymerization, has emerged to increase phospholipid nanoshell stability. In this work, we investigated the e?ects of three di?erent radical initiator conditions to fabricate stable PSS-phospholipid nanoshells yet retain the activity of encapsulated model ?uorescent sensor proteins. To identify nondestructive initiation conditions, UV photoinitiation, neutral redox initiation, and thermal initiation were investigated as a function of PSS-phospholipid nanoshell stabilization and ?uorescence emission intensity of enhanced green ?uorescent protein (eGFP) and tandem dimer Tomato (td-Tomato). All three initiator approaches yielded comparably stable PSS-phospholipid nanoshells, although slight variations in PSS-phospholipid nanoshell size were observed, ranging from ca. 140 nm for unstabilized phospholipid nanoshells to 300?500 nm for PSS-phospholipid nanoshells. Fluorescence emission intensity of encapsulated eGFP was completely attenuated under thermal initiation (0% vs control), moderately attenuated under UV photoinitiation (40 ± 4% vs control), and una?ected by neutral redox initiation (97 ± 3% vs control). Fluorescence emission intensity of encapsulated td-Tomato was signi?cantly attenuated under thermal initiation (13 ± 3% vs control), moderately attenuated UV photoinitiation (64 ± 5% vs control), and una?ected by neutral redox initiation (98% ± 4% vs control). Therefore, the neutral redox initiation method provides a signi?cant advancement toward the preparation of protein-functionalized PSS-phospholipid nanoshells. These results should help to guide future applications and designs of biosensor platforms using PSS-phospholipid nanoshells and other polymer systems employing protein transducers.
关键词： Phospholipid nanoshells,td-Tomato,Neutral redox initiation,UV photoinitiation,Radical initiator conditions,eGFP,Polymer sca?old stabilization,Thermal initiation,Fluorescent sensor proteins
更新于2025-09-09 09:28:46
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Identification of Heat-Induced Proteomes in Tomato Microspores Using LCM- Proteomics Analysis
摘要： Pollen development is highly susceptible to heat stress (HS) and the production of inviable pollen causes reduction in seed- and fruit-set in plants. This study was carried out to identify HS-induced pollen proteins and the associated biological processes in tomato (Solanum lycopersicum). Tomato ‘Micro-Tom’ plants were incubated under 32°C//22°C (day/night, 12/12 h) for two weeks for heat treatment, and the non-treated control plants were incubated for the same time period at 25°C /22°C. Flower buds of 5 mm in length were confirmed to contain the heat sensitive uninucleate microspores. Pollen cells were harvested using laser capture microdissection (LCM) and protein was extracted using a one-step method under high pressure and vacuum. Approximately 60,000 LCM-harvested microspore cells yielded about 18-20 μg proteins. The tandem mass tags (TMT) proteomics analysis identified a total of 6018 proteins, 4784 proteins were quantified, 37 proteins were identified as HS up-regulated significantly changed proteins (SCPs), and 83 proteins as HS down (dn)-regulated SCPs. Further analysis using the plant MetGenMap system showed that the HS up-regulated SCPs were enriched in the heat acclimation, pollen wall formation, protein folding/refolding gene ontology (GO) biological processes, and the HS dn-regulated SCPs were placed in the carbohydrate catabolism and de-novo protein biosynthesis GO terms. Biological processes such as mitosis, resistance to oxidative stresses, and carbohydrate and lipid metabolic processes contain both the HS up-, and dn-regulated SCPs. These results indicate that the LCM-TMT proteomics workflow is highly efficient in the identification of HS-induced pollen proteomes. These HS induced SCPs will be used for exploring heat tolerance of tomato pollens. The proteomics data are available via ProteomeXchange with identifier PXD010218.
关键词： Tomato,Protein functional classification,Heat stress,LCM-TMT-proteomics,Pollen,Viability,Microspores
更新于2025-09-09 09:28:46