Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma

Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull‐down, and OptiPrep^TM density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep^TM density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.     

Synthesis of 7-oxabicyclo[2.2.1]hept-5-en-2-yl derivatives and their screening for antimicrobial and antioxidant properties

Novel 7-oxabicyclo[2.2.1]hept-5-en-2-yl derivatives have been synthesized using boron trifluoride diethyl etherate catalyzed Diels–Alder reaction. This method presents considerable synthetic advantages in terms of high atom economy, mild reaction condition and good yields. The synthesized compounds have been screened for their antibacterial and antioxidant activities.     

Proteomics Analysis of A33 Immunoaffinity-purified Exosomes Released from the Human Colon Tumor Cell Line LIM1215 Reveals a Tissue-specific Protein Signature

Exosomes are 40–100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly purified material. Here, we describe an immunoaffinity capture method using the colon epithelial cell-specific A33 antibody to purify colorectal cancer cell (LIM1215)-derived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which 112 proteins (28%) contained signal peptides and a significant enrichment of proteins containing coiled coil, RAS, and MIRO domains. A comparative protein profiling analysis of LIM1215-, murine mast cell-, and human urine-derived exosomes revealed a subset of proteins common to all exosomes such as endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, signaling, trafficking, and cytoskeletal proteins. A conspicuous finding of this comparative analysis was the presence of host cell-specific (LIM1215 exosome) proteins such as A33, cadherin-17, carcinoembryonic antigen, epithelial cell surface antigen (EpCAM), proliferating cell nuclear antigen, epidermal growth factor receptor, mucin 13, misshapen-like kinase 1, keratin 18, mitogen-activated protein kinase 4, claudins (1, 3, and 7), centrosomal protein 55 kDa, and ephrin-B1 and -B2. Furthermore, we report the presence of the enzyme phospholipid scramblase implicated in transbilayer lipid distribution membrane remodeling. The LIM1215-specific exosomal proteins identified in this study may provide insights into colon cancer biology and potential diagnostic biomarkers.Exosomes represent a distinct class of membrane nanovesicles (40–100-nm diameter) of endocytic origin that are released from diverse cell types under both normal and pathological conditions (1). Although initial studies focused on exosomes released from various cell types in vitro, exosomes have also been reported in diverse body fluids such as urine (2), amniotic fluid (3, 4), malignant ascites (5–7), bronchoalveolar lavage fluid (8), synovial fluid (9), platelets (10), breast milk (11), and blood (12). Exosomes are formed through the inward budding of late endosomal membranes that give rise to intraluminal vesicles (ILVs)1 within intracellular multivesicular bodies (MVBs). MVBs have a well known intermediary function in the degradation of either proteins internalized from the cell surface (e.g. cell surface receptors) or intracellular proteins sorted from the trans-Golgi network. Proteins destined for degradation are sorted, typically in a ubiquitin-dependent manner, into the ILVs of the nascent MVBs, which then fuse with pre-existing lysosomes (13). An alternate fate for MVBs involves their fusion with the plasma membrane and ensuing release of ILVs into the extracellular environment as exosomes. The biogenesis of exosomes has been linked to the protein complex ESCRT machinery, which is required for both formation of MVBs and the recruitment of their endosome-derived cargo proteins (14).Exosomes exhibit pleiotropic biological functions including immunomodulatory activity, mediation of cell-cell communication, and, possibly, the transport and propagation of infectious cargo such as prions and retroviruses (1, 15, 16). Despite these advances in our understanding of exosome function, the physiological significance of exosomes is still not fully understood. The observation that exosomes contains inactive RNA and microRNAs that can be transferred to another cell and be translated in the recipient suggest that exosomes may provide a novel vehicle for genetic exchange between cells (17). More recently, the finding of glioblastoma tumor cell-derived exosomes that contain mRNA mutant/variants and microRNAs characteristic of the glioma coupled with the finding of these microvesicles in serum of glioblastoma patients suggests that blood-based exosomes may provide important diagnostic information and aid in therapeutic decisions for cancer patients (18).The molecular composition of exosomes purified from the cell culture medium from various cell types and diverse body fluids has been analyzed by proteomics as well as fluorescence-activated cell sorting, Western blot analysis, and immunohistochemistry (1, 19). In addition to displaying a protein composition that reflects their endosomal origin, these proteome profiling studies also indicate a unique protein fingerprint that reflects their cellular origin as well as possible physiological role and targeting properties. However, interpretation of exosomal proteome profiles in a biological context also highlights a cautionary note, especially if exosomes are not highly purified. For example, retroviruses such as HIV particles that bud from the cell surface using the same endocytic pathway machinery as exosomes to egress from hematopoietic cells can be a confounding factor in biochemical and physiological analyses of exosomes. Furthermore, exosomes and HIV-1 particles have similar biophysical properties such as size (40–100 and 100 nm, respectively) and buoyant density (1.13–1.21 g/liter (20) and 1.13–1.21 g/liter (21), respectively) as well as molecular composition and their ability to activate immune cells. Although earlier studies describe exosomes carrying virion cargo (22–24), recent exosome purification strategies deploying immunoaffinity capture (25) or a combination of immunoaffinity capture and density gradient centrifugation (26) demonstrate that exosomes from hematopoietic cells can be purified free of virions like HIV-1.In-depth proteomics studies with large data sets that might contribute to the understanding of the biological function of exosomes are, to date, limited (2, 17). Moreover, strategies used to purify exosomes differ between laboratories (1) with little consensus concerning criteria of purity. Isolation strategies typically involve a combination of differential centrifugation, filtration, concentration, and flotation density gradient followed by characterization using electron microscopy, flow cytometry, and Western blotting (for a review, see Simpson et al. (1)). As a first step toward understanding the physiological role of exosomes in colon cancer biology, we describe here a robust strategy to isolate and characterize exosomes released from LIM1215 colorectal carcinoma cells (27) for the purpose of proteome analysis. This isolation strategy utilized the colon epithelial cell-specific A33 antibody (28–31) to immunoaffinity capture A33-containing exosomes using microbeads. Here, we report for the first time an in-depth proteomics analysis of A33-containing exosomes released from the LIM1215 colon carcinoma cell line. Using these data, we performed a comparative bioinformatics analysis with human urinary and mast cell-derived exosomes.     

Exosomes: Extracellular organelles important in intercellular communication

In addition to intracellular organelles, eukaryotic cells also contain extracellular organelles that are released, or shed, into the microenvironment. These membranous extracellular organelles include exosomes, shedding microvesicles (SMVs) and apoptotic blebs (ABs), many of which exhibit pleiotropic biological functions. Because extracellular organelle terminology is often confounding, with many preparations reported in the literature being mixtures of extracellular vesicles, there is a growing need to clarify nomenclature and to improve purification strategies in order to discriminate the biochemical and functional activities of these moieties. Exosomes are formed by the inward budding of multivesicular bodies (MVBs) and are released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane (PM). In this review we focus on various strategies for purifying exosomes and discuss their biophysical and biochemical properties. An update on proteomic analysis of exosomes from various cell types and body fluids is provided and host-cell specific proteomic signatures are also discussed. Because the ectodomain of ~ 42% of exosomal integral membrane proteins are also found in the secretome, these vesicles provide a potential source of serum-based membrane protein biomarkers that are reflective of the host cell. ExoCarta, an exosomal protein and RNA database (http://exocarta.ludwig.edu.au), is described.     

Growth promotion of groundnut by IAA producing rhizobacteria Bacillus licheniformis MML2501

B. licheniformis MML2501 which was isolated from groundnut rhizosphere soil showed increased populations on spermozphere colonisation and significantly increased the seed germination and other growth parameters in groundnut under in vitro conditions. B. licheniformis MML2501 did not solubilise phosphate but produced indole acetic acid (IAA), with a maximum of 23 μg/ml under optimised conditions such as pH 7.0, temperature 35°C, tryphtophan at a concentration of 16 mM and at 200 rpm shaken conditions. The production of IAA by B. licheniformis MML2501 was further confirmed by TLC and HPLC analyses, in which the R_f value and retention time of 0.66 and 4.1 min respectively, match with that of the authentic IAA. Seed treatment of B. licheniformis MML2501 in groundnut showed a significant increase in seed germination, other growth parameters and yield parameters under potted plant experiments.     

Tandem application of cationic colloidal silica and Triton X-114 for plasma membrane protein isolation and purification: towards developing an MDCK protein database

Plasma membrane (PM) proteins are attractive therapeutic targets because of their accessibility to drugs. Although genes encoding PM proteins represent 20-30% of eukaryotic genomes, a detailed characterisation of their encoded proteins is underrepresented, due, to their low copy number and the inherent difficulties in their isolation and purification as a consequence of their high hydrophobicity. We describe here a strategy that combines two orthogonal methods to isolate and purify PM proteins from Madin Darby canine kidney (MDCK) cells. In this two-step method, we first used cationic colloidal silica (CCS) to isolate adherent (Ad) and non-adherent (nAd) PM fractions, and then subjected each fraction to Triton X-114 (TX-114) phase partitioning to further enrich for hydrophobic proteins. While CCS alone identified 255/757 (34%) membrane proteins, CCS/TX-114 in combination yielded 453/745 (61%). Strikingly, of those proteins unique to CCS/TX-114, 277/393 (70%) had membrane annotation. Further characterisation of the CCS/TX-114 data set using Uniprot and transmembrane hidden Markov model revealed that 306/745 (41%) contained one or more transmembrane domains (TMDs), including proteins with 25 and 17 TMDs. Of the remaining proteins in the data set, 69/439 (16%) are known to contain lipid modifications. Of all membrane proteins identified, 93 had PM origin, including proteins that mediate cell adhesion, modulate transmembrane ion transport, and cell-cell communication. These studies reveal that the application of CCS to first isolate Ad and nAd PM fractions, followed by their detergent-phase TX-114 partitioning, to be a powerful method to isolate low-abundance PM proteins, and a useful adjunct for in-depth cell surface proteome analyses.     

Proteomic profiling of secretome and adherent plasma membranes from distinct mammary epithelial cell subpopulations

The stem cell niche comprises stem cells (SCs), stromal cells, soluble factors, extracellular matrix constituents and vascular networks. The ability to identify signals that regulate SC self-renewal and differentiation is confounded by the difficulty in isolating pure SC niche components in sufficient quantities to enable their biochemical characterisation. Here, we report the extracellular (secretome) and adherent plasma membrane proteomes of three distinct epithelial cell subpopulations isolated and immortalized from the mouse mammary gland--basal and mammary stem cell (basal/MaSC), luminal progenitor (LP) and mature luminal (ML) cell lines. GeLC-MS/MS-based proteomic profiling revealed a distinct switch in components modulating Wnt and ephrin signalling, and integrin-mediated interactions amongst the three cell subpopulations. For example, expression of ephrin B2, ephrin receptors A1, and A2, as well as integrins α2β1 and α6β4 were shown to be enriched in basal/MaSCs, relative to LP and ML cells. Conspicuously, Wnt10a was uniquely detected in basal/MaSCs, and may modulate the canonical Wnt signalling pathway to maintain basal/MaSC activity. By contrast, non-canonical Wnt signalling might be elevated in ML cells, as evidenced by the high expression levels of Wnt5a, Wnt5b, and the transmembrane tyrosine kinase Ror2.     

Why are hippocampal CA1 neurons vulnerable but motor cortex neurons resistant to transient ischemia?

It is well-known that heat-shock protein 70.1 (Hsp70.1), a major protein of the human Hsp70 family, plays cytoprotective roles by both its chaperone function and stabilization of lysosomal membranes. Recently, we found that calpain-mediated cleavage of carbonylated Hsp70.1 in the hippocampal cornu Ammonis1 (CA1) contributes to neuronal death after transient global ischemia. This study aims to elucidate the differential neuronal vulnerability between the motor cortex and CA1 sector against ischemia/reperfusion. Fluoro-Jade B staining and terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labeling analysis of the monkey brain undergoing 20min whole brain ischemia followed by reperfusion, showed that the motor cortex is significantly resistant to the ischemic insult compared with CA1. Up-regulation of Hsp70.1 but absence of its cleavage by calpain facilitated its binding with NF-κB p65/IκBα complex to minimize NF-κB p65 activation, which contributed to a neuroprotection in the motor cortex. In contrast, because activated μ-calpain cleaved carbonylated Hsp70.1 in CA1, the resultant Hsp70.1 dysfunction not only destabilized lysosomal membrane but also induced a sustained activation of NF-κB p65, both of which resulted in delayed neuronal death. We propose that the cascades underlying lysosomal stabilization and regulating NF-κB activation by Hsp70.1 may influence neuronal survival/death after the ischemia/reperfusion.     

Synthesis of quinoline derivatives for fluorescent imaging certain bacteria

Highly fluorescent quinoline derivatives were synthesized using Sc(OTf)₃ catalyzed imino Diels–Alder reaction. Both the aromatic and their analogous tetradehydroquinoline derivatives were explored for the detection of bacteria using fluorescent imaging studies. Surprisingly the aromatic quinoline derivatives show a remarkable fluorescent response that can be useful in the detection of both gram positive and gram negative bacteria even at a concentration in the range of 0.078 mM.     

Screening of marine actinomycetes isolated from the Bay of Bengal,India for antimicrobial activity and industrial enzymes

A total of 288 marine samples were collected from different locations of the Bay of Bengal starting from Pulicat lake to Kanyakumari, and 208 isolates of marine actinomycetes were isolated using starch casein agar medium. The growth pattern, mycelial coloration, production of exopolysaccharides and diffusible pigment and abundance of Streptomyces spp. were documented. Among marine actinomycetes, Streptomyces spp. were present in large proportion (88%). Among 208 marine actinomycetes, 111 isolates exhibited antimicrobial activity against human pathogens, and 151 showed antifungal activity against two plant pathogens. Among 208 isolates, 183, 157, 116, 72 and 68 isolates produced lipase, caseinase, gelatinase, cellulase and amylase, respectively. The results of diversity, antimicrobial activity and enzymes production have increased the scope of finding industrially important marine actinomycetes from the Bay of Bengal and these organisms could be vital sources for the discovery of industrially useful molecules/enzymes.