Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.

Isoprostanes are biologically active molecules, produced when reactive oxygen species mediate the peroxidation of membrane polyunsaturated fatty acids. Previous work has demonstrated that the isoprostane 8-iso-prostaglandin E(2) (PGE(2)) stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial anion secretion across the human airway epithelial cell line, Calu-3. Since isoprostanes predominantly achieve their effects via binding to prostanoid receptors, we hypothesized that this 8-iso-PGE(2) stimulation of CFTR activity was the result of the isoprostane binding to a prostanoid receptor. Using RT-PCR, immunoblotting, and immunofluorescence, we here demonstrate that Calu-3 cells express the EP(1-4) and FP receptors, and localize these proteins in polarized cell monolayers. Using iodide efflux as a marker for CFTR-mediated Cl(-) efflux, we investigate whether prostanoid receptor agonists elicit a functional response from Calu-3 cells. Application of the agonists PGE(2), misoprostol (EP(2), EP(3), and EP(4)) and PGE(1)-OH (EP(3) and EP(4)) stimulate iodide efflux; however, iloprost, butaprost, sulprostone, and fluoprostenol (agonists of the EP(1), EP(2), EP(3), and FP receptors, respectively) have no effect. The iodide efflux seen with 8-iso-PGE(2) is abolished by the EP(4) receptor antagonist AH23848, the CFTR inhibitor 172, and inhibition of PKA and the PI3K pathway. In conclusion, we demonstrate that although Calu-3 cells possess numerous prostanoid receptors, only the EP(4) subtype appears capable of eliciting a functional iodide efflux response, which is mediated via the EP(4) receptor. We propose that 8-iso-PGE(2), acting via EP(4) receptor, could play an important role in the CFTR-mediated response to oxidant stress, and which would be compromised in the CF airways.

Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of ‘-omics’ research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol© reagent is the ability to accumulate multi-parametric data (e.g., RNA and protein profiles) on the same limited EV sample while minimizing sample preparation and processing time.

PAX5 and EBF1 work synergistically to regulate genes that are involved in B lymphocyte differentiation. We used the KIS-1 diffuse large B cell lymphoma cell line, which is reported to have elevated levels of PAX5 expression, to investigate the mechanism of EBF1- and PAX5-regulated gene expression. We demonstrate the lack of expression of hallmark B cell genes, including CD19, CD79b, and EBF1, in the KIS-1 cell line. Upon restoration of EBF1 expression we observed activation of CD19, CD79b and other genes with critical roles in B cell differentiation. Mass spectrometry analyses of proteins co-immunoprecipitated with PAX5 in KIS-1 identified components of the MLL H3K4 methylation complex, which drives histone modifications associated with transcription activation. Immunoblotting showed a stronger association of this complex with PAX5 in the presence of EBF1. Silencing of KMT2A, the catalytic component of MLL, repressed the ability of exogenous EBF1 to activate transcription of both CD19 and CD79b in KIS-1 cells. We also find association of PAX5 with the MLL complex and decreased CD19 expression following silencing of KMT2A in other human B cell lines. These data support an important role for the MLL complex in PAX5-mediated transcription regulation.

Soxhlet (SE), microwave-assisted (MAE) and ultrasound-assisted (UAE) extraction were compared using ten extraction solvents for their efficiency to extract phenolic and flavonoid antioxidants from Eastern Canada propolis. Extracts were compared for total phenolic (TPC) and total flavonoid (TFC) content, and radical scavenging activities. Anti-inflammatory activity through inhibition of 5-lipoxygenase (5-LO) products biosynthesis in HEK293 cells was also evaluated. The results showed that SE extracts using polar solvents had the highest TPC and TFC. Extracts obtained with ethanol, methanol and acetone were effective free radical scavengers, and showed 5-LO inhibition similar to zileuton. UAE was an effective extraction method since the extracts obtained were comparable to those using SE and the MAE while being done at room temperature. With UAE, extracts of less polar solvents showed similar free radical scavenging and 5-LO inhibition to extracts of much more polar solvents such as methanol or ethanol. Reversed-phase liquid chromatography tandem mass spectrometry confirmed the presence of 21 natural compounds in the propolis extracts based on the comparison of intact mass, chromatographic retention time and fragmentation patterns derived from commercial analytical standards. The current study is the first of its kind to concurrently investigate solvent polarity as well as extraction techniques of propolis.