Ultracentrifugation-Free Enrichment and Quantification of Small Extracellular Vesicles.

Cancer is a malignant tumor with the highest mortality of human diseases. The early diagnosis of cancer can greatly reduce its mortality. Ultracentrifugation is the most commonly employed technique to separate small extracellular vesicles (sEVs) due to their small size and rare abundance, but the low separation efficiency is a major concern. Herein, we proposed a DNAzyme-triggered assembly and disassembly system that converted single nano-sized sEVs into clusters that could be conveniently enriched by ordinary centrifugation and then be broken into single sEVs in the presence of magnesium ions. The simultaneous quantification of sEVs was realized by recording the increase in fluorescence upon nucleic acid cleavage, and a detection limit as low as 54 particles/µL was achieved. The whole analytical procedure could be completed in 1.5 h without the assistance of ultracentrifugation. Efficient enrichment and accurate quantification of sEVs are enabled through the proposed approach, broadening the potentials of sEVs in biological science, biomedical engineering, and personalized medicine.

High-Fidelity Determination and Tracing of Small Extracellular Vesicle Cargoes.

Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for elucidating the mechanisms involved in intercellular communication. However, high-fidelity determination of sEVs' molecular cargoes in situ has yet to be achieved due to the difficulty in transporting molecular probes into intact sEVs. Herein, a fLuorescent Intracellular-Guided Hairpin-Tetrahedron (fLIGHT) nanoprobe is described for direct visualization of sEV microRNAs in situ. Integrating the advantages of nondestructive sEV penetration via DNA origami and single-nucleotide discrimination as well as wash-free fluorescence readout using a hairpin probe, the proposed approach enables high-fidelity fluorescence visualization of sEVs' microRNA without RNA extraction or leakage, demonstrating the potential of on-site tracing of sEV cargoes. This strategy opens an avenue to establishing universal molecular detection and labeling platforms that can facilitate both sEV-derived fundamental biological studies and molecular diagnostics.

Comparative Proteomic Analysis of Coregulation of CIPK14 and WHIRLY1/3 Mediated Pale Yellowing of Leaves in Arabidopsis.

Pale yellowing of leaf variegation is observed in the mutant Arabidopsis lines Calcineurin B-Like-Interacting Protein Kinase14 (CIPK14) overexpression (oeCIPK14) and double-knockout WHIRLY1/WHIRLY3 (why1/3). Further, the relative distribution of WHIRLY1 (WHY1) protein between plastids and the nucleus is affected by the phosphorylation of WHY1 by CIPK14. To elucidate the coregulation of CIPK14 and WHIRLY1/WHIRLY3-mediated pale yellowing of leaves, a differential proteomic analysis was conducted between the oeCIPK14 variegated (oeCIPK14-var) line, why1/3 variegated (why1/3-var) line, and wild type (WT). More than 800 protein spots were resolved on each gel, and 67 differentially abundant proteins (DAPs) were identified by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF-MS). Of these 67 proteins, 34 DAPs were in the oeCIPK14-var line and 33 DAPs were in the why1/3-var line compared to the WT. Five overlapping proteins were differentially expressed in both the oeCIPK14-var and why1/3-var lines: ATP-dependent Clp protease proteolytic subunit-related protein 3 (ClpR3), Ribulose bisphosphate carboxylase large chain (RBCL), Beta-amylase 3 (BAM3), Ribosome-recycling factor (RRF), and Ribulose bisphosphate carboxylase small chain (RBCS). Bioinformatics analysis showed that most of the DAPs are involved in photosynthesis, defense and antioxidation pathways, protein metabolism, amino acid metabolism, energy metabolism, malate biosynthesis, lipid metabolism, and transcription. Thus, in the why1/3-var and oeCIPK14-var lines, there was a decrease in the photosystem parameters, including the content of chlorophyll, the photochemical efficiency of photosystem (PS II) (Fv/Fm), and electron transport rates (ETRs), but there was an increase in non-photochemical quenching (NPQ). Both mutants showed high sensitivity to intense light. Based on the annotation of the DAPs from both why1/3-var and oeCIPK14-var lines, we conclude that the CIPK14 phosphorylation-mediated WHY1 deficiency in plastids is related to the impairment of protein metabolism, leading to chloroplast dysfunction.

Universal platform for accurately damage-free mapping of sEVs cargo information.

SEVs (small extracellular vesicles) contents signatures appear to mirror pathological changes of diseases, and mapping sEVs contents profile is a promising approach for non-invasive diagnosis of the disease. Herein, we propose a universal system for accurately and damage-freely mapping of sEVs content profile using dual-recognition triggered CHA (catalytic hairpin assembly) and DNAzyme based signal amplification strategy. After immunoassay based capture of CD63 positive sEVs by anti-CD63 lgG coated on the surface of polystyrene plates, probes are incubated with fixed sEVs to penetrate sEVs membrane and act to sense sEVs contents. In detection step, integrated CHA and DNAzyme based strategy is initiated by released initiator from capture probe after recognizing targets, forming a dual circle signal recycling process, realizing signal amplification for high sensitivity. Given the attractive analytical features that i) a universal platform for indistinctive sEVs nucleic acids and protein molecules detection; ii) high sensitivity derived from dual circle signal recycling process; iii) enzyme-free characteristic of integrated CHA and DNAzyme minimizes the interference to sEVs biological activity; iv) mapping of sEVs contents profiles indicates a brand-new strategy for non-invasive diagnosis of the disease, the present approach shows great promise for analyzing additional different analytes in clinical and experimental researches.

Spatiotemporally Controllable MicroRNA Imaging in Living Cells via a Near-Infrared Light-Activated Nanoprobe.

In situ spatiotemporal microRNA (miRNA) imaging in mammal cells plays an essential role in illustrating its structures and biological functions. Herein, we proposed a near-infrared (NIR) light-activated nanoprobe for high-sensitive in situ controllable miRNA imaging in living cells. The NIR-activated nanoprobe employed an upconversion nanoparticle that acted as a NIR-to-UV transducer to trigger the following photocleavage toward a dumbbell DNA probe tethered on the surface of the nanoparticle. The structure change of the dumbbell probe then induced a catalytic hairpin assembly of target miRNAs, by which in situ readout of the amplified fluorescence signal was enabled. Additionally, both intracellular miRNA imaging and accurate quantification in live cells were realized without damaging the cell membranes. Compared with conventional in situ strategies, the proposed approach remarkedly increases imaging efficiency by eliminating those unfavored intercellular molecular imaging backgrounds. We assured that the proposed NIR-activated miRNA sensing strategy will add to the advancement for bioanalysis in living systems, which is of crucial importance in the diagnosis of various human diseases, especially cancers.