Membrane lipid binding molecules for the isolation of bona fide extracellular vesicle types and associated biomarkers in liquid biopsy

Cancer exacts a heavy socioeconomic cost. Earlier detection and treatment are likely to mitigate this cost. Unfortunately, conventional tissue biopsy, the gold standard in cancer diagnosis cannot fulfill the goal of earlier detection. While liquid biopsy is a promising alternative to tissue biopsy, it has its challenges and limitations. A major challenge is the isolation of bona fide lipid membrane vesicles from biological fluids. In this review, we presented a new perspective of isolating different types of extracellular vesicles (EVs) by their affinity for membrane lipid binding ligands for liquid biopsy. EVs are lipid membrane particles naturally released by almost all cells and are found in almost all biological fluids suitable for liquid biopsy. They carry materials from the secreting cells that could affect the biology of the recipient cells and could thus inform on the state and progress of the disease. However, isolating bona fide EVs is a technical challenge as biological fluids have a complex composition and contain particles or aggregates that are physically similar to EVs. Here we review the use of membrane lipid-binding ligands to isolate different bona fide EV subtypes, and to circumvent the problem of co-isolating physically similar non-EV complexes in current EV isolation protocols. We will discuss the advantages of this technique and its potential for accelerated biomarker discovery and validation through examples of pre-clinical studies. We propose that isolating EV subtypes is a technically viable and robust strategy to overcome the current bottleneck of isolating EVs for liquid biopsy.

Structural characterization of SARS-CoV-2 dimeric ORF9b reveals potential fold-switching trigger mechanism

The constant emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)variants indicates the evolution and adaptation of the virus.Enhanced innate immune evasion through increased expression of viral antagonist proteins,including ORF9b,contributes to the improved transmission of the Alpha variant;hence,more attention should be paid to these viral proteins.ORF9b is an accessory protein that suppresses innate immunity via a monomer conformation by binding to Tom70.Here,we solved the dimeric structure of SARS-CoV-2 ORF9b with a long hydrophobic tunnel containing a lipid molecule that is crucial for the dimeric conformation and determined the specific lipid ligands as monoglycerides by conducting a liquid chromatography with tandem mass spectrometry analysis,suggesting an important role in the viral life cycle.Notably,a long intertwined loop accessible for host factor binding was observed in the structure.Eight phosphorylated residues in ORF9b were identified,and residues S50 and S53 were found to contribute to the stabilization of dimeric ORF9b.Additionally,we proposed a model of multifunctional ORF9b with a distinct conformation,suggesting that ORF9b is a fold-switching protein,while both lipids and phosphorylation contribute to the switching.Specifically,the ORF9b monomer interacts with Tom70 to suppress the innate immune response,whereas the ORF9b dimer binds to the membrane involving mature virion assembly.Our results provide a better understanding of the multiple functions of ORF9b.