Identification of optimal conditions for human placental explant culture and extracellular vesicle release.

Extracellular vesicles (EVs) can mediate intercellular communication, including signaling between the placenta and maternal tissues. Human placental explant culture is a versatile model system to investigate placental function. We performed systematic studies in different tissue culture media types and oxygen tensions to identify a defined serum-free culture condition that supports high trophoblast viability and metabolism, as well as the release of similar populations of EVs, compared to traditional undefined conditions that contain media additives potentially contaminated with exogenous EVs.

Serpin-loaded extracellular vesicles promote tissue repair in a mouse model of impaired wound healing.

Chronic metabolic diseases such as diabetes are characterized by delayed wound healing and a dysregulation of the inflammatory phase of wound repair. Our study focuses on changes in the payload of extracellular vesicles (EVs) communicating between immune cells and stromal cells in the wound bed, which regulate the rate of wound closure. Adoptive transfer of EVs from genetically defined mouse models are used here to demonstrate a functional and molecular basis for differences in the pro-reparative biological activity of diabetic (db/db) vs.

Cancer-cell-secreted miR-122 suppresses O-GlcNAcylation to promote skeletal muscle proteolysis.

A decline in skeletal muscle mass and low muscular strength are prognostic factors in advanced human cancers. Here we found that breast cancer suppressed O-linked N-acetylglucosamine (O-GlcNAc) protein modification in muscle through extracellular-vesicle-encapsulated miR-122, which targets O-GlcNAc transferase (OGT). Mechanistically, O-GlcNAcylation of ryanodine receptor 1 (RYR1) competed with NEK10-mediated phosphorylation and increased K48-linked ubiquitination and proteasomal degradation; the miR-122-mediated decrease in OGT resulted in increased RYR1 abundance.

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens.

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets-the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays.

Methamphetamine use alters human plasma extracellular vesicles and their microRNA cargo: An exploratory study.

Methamphetamine (MA) is the largest drug threat across the globe, with health effects including neurotoxicity and cardiovascular disease. Recent studies have begun to link microRNAs (miRNAs) to the processes related to MA use and addiction. Our studies are the first to analyse plasma EVs and their miRNA cargo in humans actively using MA (MA-ACT) and control participants (CTL).

MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments.

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization.

1,300 Days and Counting: A Risk Model Approach to Preventing Retained Foreign Objects (RFOs).

Background: A retained foreign object (RFO) is a devastating surgical complication that typically results in additional surgeries, increased length of stay, and risk of infections and is potentially fatal. Memorial Sloan Kettering Cancer Center (MSKCC) convened a multidisciplinary task force to undertake an improvement initiative to reduce the frequency of RFO incidents.

Methods: A needs assessment was undertaken using focus group interviews, review of past RFOs, and operating room (OR) observations, and a comprehensive intervention plan was initiated.

Analysis of Individual Extracellular Vesicles by Flow Cytometry.

Extracellular vesicles (EVs) are released by cells and can be found in cell culture supernatants and biofluids. EVs carry proteins, nucleic acids, and other cellular components and can deliver these to nearby or distant cells, making EVs of interest as both disease biomarkers and therapeutic targets. EVs in biofluids are heterogeneous, coming from different cell types and from different sources with the cell, which limits the usefulness of bulk EV analysis methods that report the average features of all EVs present.

Analysis of extracellular RNA in cerebrospinal fluid.

We examined the extracellular vesicle (EV) and RNA composition of pooled normal cerebrospinal fluid (CSF) samples and CSF from five major neurological disorders: Alzheimer's disease (AD), Parkinson's disease (PD), low-grade glioma (LGG), glioblastoma multiforme (GBM), and subarachnoid haemorrhage (SAH), representing neurodegenerative disease, cancer, and severe acute brain injury. We evaluated: (I) size and quantity of EVs by nanoparticle tracking analysis (NTA) and vesicle flow cytometry (VFC), (II) RNA yield and purity using four RNA isolation kits, (III) replication of RNA yields within and between laboratories, and (IV) composition of total and EV RNAs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNASeq). The CSF contained ~106 EVs/μL by NTA and VFC.

Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF).

Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM).

High sensitivity flow cytometry of membrane vesicles.

Extracellular vesicles (EVs) are attracting attention as vehicles for inter-cellular signaling that may have value as diagnostic or therapeutic targets. EVs are released by many cell types and by different mechanisms, resulting in phenotypic heterogeneity that makes them a challenge to study. Flow cytometry is a popular tool for characterizing heterogeneous mixtures of particles such as cell types within blood, but the small size of EVs makes them difficult to measure using conventional flow cytometry.

Multiplexed TEM Specimen Preparation and Analysis of Plasmonic Nanoparticles.

We describe a system for rapidly screening hundreds of nanoparticle samples using transmission electron microscopy (TEM). The system uses a liquid handling robot to place up to 96 individual samples onto a single standard TEM grid at separate locations. The grid is then transferred into the TEM and automated software is used to acquire multiscale images of each sample.

Optimization of SERS tag intensity, binding footprint, and emittance.

Nanoparticle surface enhanced Raman scattering (SERS) tags have attracted interest as labels for use in a variety of applications, including biomolecular assays. An obstacle to progress in this area is a lack of standardized approaches to compare the brightness of different SERS tags within and between laboratories. Here we present an approach based on binding of SERS tags to beads with known binding capacities that allows evaluation of the average intensity, the relative binding footprint of particles in a SERS tag preparation, and the size-normalized intensity or emittance.

Visible and near infrared fluorescence spectral flow cytometry.

There is a long standing interest in measuring complete emission spectra from individual cells in flow cytometry. We have developed flow cytometry instruments and analysis approaches to enable this to be done routinely and robustly. Our spectral flow cytometers use a holographic grating to disperse light from single cells onto a CCD for high speed, wavelength-resolved detection.

A lactate dehydrogenase ELISA-based assay for the in vitro determination of Plasmodium berghei sensitivity to anti-malarial drugs.

Background: Plasmodium berghei rodent malaria is a well-known model for the investigation of anti-malarial drug efficacy in vivo. However, the availability of drug in vitro assays in P. berghei is reduced when compared with the spectrum of techniques existing for Plasmodium falciparum.

Single cell analysis using surface enhanced Raman scattering (SERS) tags.

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing.

Chapter 3. High-throughput protein purification for x-ray crystallography and NMR.

In structural biology, the most critical issue is the availability of high-quality samples. "Structural-biology-grade" proteins must be generated in a quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance. The additional challenge for structural genomics is the need for high numbers of proteins at low cost where protein targets quite often have low sequence similarities, unknown properties and are poorly characterized.