Chromatograms and Mass Spectra of High-Mannose and Paucimannose -Glycans for Rapid Isomeric Identifications.

-Linked glycosylation is one of the most essential post-translational modifications of proteins. However, -glycan structural determination remains challenging because of the small differences in structures between isomers. In this study, we constructed a database containing collision-induced dissociation MS mass spectra and chromatograms of high-performance liquid chromatography for the rapid identification of high-mannose and paucimannose -glycan isomers.

Identification of the High Mannose -Glycan Isomers Undescribed by Conventional Multicellular Eukaryotic Biosynthetic Pathways.

-linked glycosylation is one of the most important post-translational modifications of proteins. Current knowledge of multicellular eukaryote -glycan biosynthesis suggests high mannose -glycans are generated in the endoplasmic reticulum and Golgi apparatus through conserved biosynthetic pathways. According to conventional biosynthetic pathways, four ManGlcNAc isomers, three ManGlcNAc isomers, and one ManGlcNAc isomer are generated during this process.

Membrane Protein Modification Modulates Big and Small Extracellular Vesicle Biodistribution and Tumorigenic Potential in Breast Cancers In Vivo.

Extracellular vesicles (EVs) are released by cells to mediate intercellular communication under pathological and physiological conditions. While small EVs (sEVs; <100-200 nm, exosomes) are intensely investigated, the properties and functions of medium and large EVs (big EVs (bEVs); >200 nm, microvesicles) are less well explored. Here, bEVs and sEVs are identified as distinct EV populations, and it is determined that bEVs are released in a greater bEV:sEV ratio in the aggressive human triple-negative breast cancer (TNBC) subtype.

Multiplexed bioluminescence-mediated tracking of DNA double-strand break repairs in vitro and in vivo.

The dynamics of DNA double-strand break (DSB) repairs including homology-directed repair and nonhomologous end joining play an important role in diseases and therapies. However, investigating DSB repair is typically a low-throughput and cross-sectional process, requiring disruption of cells and organisms for subsequent nuclease-, sequencing- or reporter-based assays. In this protocol, we provide instructions for establishing a bioluminescent repair reporter system using engineered Gaussia and Vargula luciferases for noninvasive tracking of homology-directed repair and nonhomologous end joining, respectively, induced by SceI meganuclease, SpCas9 or SpCas9 D10A nickase-mediated editing.

Isolation and recovery of extracellular vesicles using optically-induced dielectrophoresis on an integrated microfluidic platform.

Cell-released, membrane-encapsulated extracellular vesicles (EVs) serve as a means of intercellular communication by delivering bioactive cargos including proteins, nucleic acids and lipids. EVs have been widely used for a variety of biomedical applications such as biomarkers for disease diagnosis and drug delivery vehicles for therapy. Herein, this study reports a novel method for label-free, contact-free isolation and recovery of EVs via optically-induced dielectrophoresis (ODEP) on a pneumatically-driven microfluidic platform with minimal human intervention.

Multiresolution Imaging Using Bioluminescence Resonance Energy Transfer Identifies Distinct Biodistribution Profiles of Extracellular Vesicles and Exomeres with Redirected Tropism.

Extracellular particles (EPs) including extracellular vesicles (EVs) and exomeres play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. Therefore, a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, is created to enable pan-EP labeling ranging from exomeres (<50 nm) to small (<200 nm) and medium and large (>200 nm) EVs.

A multiplexed bioluminescent reporter for sensitive and non-invasive tracking of DNA double strand break repair dynamics in vitro and in vivo.

Tracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively.

Sonogenetic Modulation of Cellular Activities Using an Engineered Auditory-Sensing Protein.

Biomolecules that respond to different external stimuli enable the remote control of genetically modified cells. We report herein a sonogenetic approach that can manipulate target cell activities by focused ultrasound stimulation. This system requires an ultrasound-responsive protein derived from an engineered auditory-sensing protein prestin.

Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles.

Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm).

Delivery of nitric oxide with a nanocarrier promotes tumour vessel normalization and potentiates anti-cancer therapies.

Abnormal tumour vasculature has a significant impact on tumour progression and response to therapy. Nitric oxide (NO) regulates angiogenesis and maintains vascular homeostasis and, thus, can be delivered to normalize tumour vasculature. However, a NO-delivery system with a prolonged half-life and a sustained release mechanism is currently lacking.

Methods for Systematic Identification of Membrane Proteins for Specific Capture of Cancer-Derived Extracellular Vesicles.

Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas.

Imaging extracellular vesicles: current and emerging methods.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells. They range from 30 nm to several micrometers in diameter, and ferry biological cargos such as proteins, lipids, RNAs and DNAs for local and distant intercellular communications. EVs have since been found to play a role in development, as well as in diseases including cancers.

Proteomic Analysis of Extracellular Vesicles for Cancer Diagnostics.

Extracellular vesicles (EVs) including exosomes and microvesicles are lipid bilayer-encapsulated nanoparticles released by cells, ranging from 40 nm to several microns in diameter. Biological cargoes including proteins, RNAs, and DNAs can be ferried by EVs to neighboring and distant cells via biofluids, serving as a means of cell-to-cell communication under normal and pathological conditions, especially cancers. On the other hand, EVs have been investigated as a novel "information capsule" for early disease detection and monitoring via liquid biopsy.

Tracking Extracellular Vesicles Delivery and RNA Translation Using Multiplexed Reporters.

Elucidating extracellular vesicle (EV; e.g., exosomes, microvesicles) delivery and translation of its RNA cargo with an accurate spatiotemporal resolution is critical in helping understand EV's role under normal and pathological conditions.

Role of exosomes/microvesicles in the nervous system and use in emerging therapies.

Extracellular membrane vesicles (EMVs) are nanometer sized vesicles, including exosomes and microvesicles capable of transferring DNAs, mRNAs, microRNAs, non-coding RNAs, proteins, and lipids among cells without direct cell-to-cell contact, thereby representing a novel form of intercellular communication. Many cells in the nervous system have been shown to release EMVs, implicating their active roles in development, function, and pathologies of this system. While substantial progress has been made in understanding the biogenesis, biophysical properties, and involvement of EMVs in diseases, relatively less information is known about their biological function in the normal nervous system.