Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein-extraction methods.

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.

CD24 induces changes to the surface receptors of B cell microvesicles with variable effects on their RNA and protein cargo.

The CD24 cell surface receptor promotes apoptosis in developing B cells, and we recently found that it induces B cells to release plasma membrane-derived, CD24-bearing microvesicles (MVs). Here we have performed a systematic characterization of B cell MVs released from WEHI-231 B lymphoma cells in response to CD24 stimulation. We found that B cells constitutively release MVs of approximately 120 nm, and that CD24 induces an increase in phosphatidylserine-positive MV release.

Quantitative proteomics to study a small molecule targeting the loss of von Hippel-Lindau in renal cell carcinomas.

Inactivation of the tumor suppressor gene, von Hippel-Lindau (VHL), is known to play an important role in the development of sporadic clear cell renal cell carcinomas (ccRCCs). Even if available targeted therapies for metastatic RCCs (mRCCs) have helped to improve progression-free survival rates, they have no durable clinical response. We have previously shown the feasibility of specifically targeting the loss of VHL with the identification of a small molecule, STF-62247.

PML nuclear bodies contribute to the basal expression of the mTOR inhibitor DDIT4.

The promyelocytic leukemia (PML) protein is an essential component of PML nuclear bodies (PML NBs) frequently lost in cancer. PML NBs coordinate chromosomal regions via modification of nuclear proteins that in turn may regulate genes in the vicinity of these bodies. However, few PML NB-associated genes have been identified.

Rapid isolation of extracellular vesicles from cell culture and biological fluids using a synthetic peptide with specific affinity for heat shock proteins.

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.

A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA.

Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence.

A transgenic zebrafish model expressing KIT-D816V recapitulates features of aggressive systemic mastocytosis.

Systemic mastocytosis (SM) is a rare myeloproliferative disease without curative therapy. Despite clinical variability, the majority of patients harbour a KIT-D816V mutation, but efforts to inhibit mutant KIT with tyrosine kinase inhibitors have been unsatisfactory, indicating a need for new preclinical approaches to identify alternative targets and novel therapies in this disease. Murine models to date have been limited and do not fully recapitulate the most aggressive forms of SM.

Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli.

Cell culture metabolomics: applications and future directions.

Metabolomics represents a global quantitative assessment of metabolites within a biological system. The metabolic analysis of cell cultures has many potential applications and advantages to currently used methods for cell line testing. Metabolite concentrations represent sensitive markers of both genomic and phenotypic changes.

N-(4-(6,7-Disubstituted-quinolin-4-yloxy)-3-fluorophenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides: a novel series of dual c-Met/VEGFR2 receptor tyrosine kinase inhibitors.

A series of N-(4-(6,7-disubstituted-quinolin-4-yloxy)-3-fluorophenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides targeting c-Met and VEGFR2 tyrosine kinases was designed and synthesized. The compounds were potent against these two enzymes with IC(50) values in the low nanomolar range in vitro, possessed favorable pharmacokinetic profiles and showed high efficacy in vivo in several human tumor xenograft models in mice.

NMR metabolic analysis of samples using fuzzy K-means clustering.

The global analysis of metabolites can be used to define the phenotypes of cells, tissues or organisms. Classifying groups of samples based on their metabolic profile is one of the main topics of metabolomics research. Crisp clustering methods assign each feature to one cluster, thereby omitting information about the multiplicity of sample subtypes.

Discovery of a novel and potent series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases.

A series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases is described. The compounds demonstrated potency with IC(50) values in the low nanomolar range in vitro while the lead compound also showed in vivo activity against various human tumor xenograft models in mice. Further exploration of this class of compounds is underway.

DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells.

Transcriptional silencing by CpG island methylation is a prevalent mechanism of tumor-suppressor gene suppression in cancers. Genetic experiments have defined the importance of the DNA methyltransferase Dnmt1 for the maintenance of methylation in mouse cells and its role in neoplasia. In human bladder cancer cells, selective depletion of DNMT1 with antisense inhibitors has been shown to induce demethylation and reactivation of the silenced tumor-suppressor gene CDKN2A.

An essential role for DNA methyltransferase DNMT3B in cancer cell survival.

Abnormal methylation and associated silencing of tumor suppressor genes is a common feature of many types of cancers. The observation of persistent methylation in human cancer cells lacking the maintenance methyltransferase DNMT1 suggests the involvement of other DNA methyltransferases in gene silencing in cancer. To test this hypothesis, we have evaluated methylation and gene expression in cancer cells specifically depleted of DNMT3A or DNMT3B, de novo methyltransferases that are expressed in adult tissues.

Comparison of inhibition of ovalbumin-induced bronchoconstriction in guinea pigs and in vitro inhibition of tumor necrosis factor-alpha formation with phosphodiesterase 4 (PDE4) selective inhibitors.

Phosphodiesterase 4 (PDE4) inhibitors elevate cyclic adenosine 5'-monophosphate (cAMP), and this elevation has been shown to inhibit inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Using TNF-alpha as a biomarker, we have developed transcription-based assays to examine inhibition of PDE4 activity in human and guinea pig whole blood. In vitro inhibition by PDE4 inhibitors was measured using quantitative PCR (qPCR) analysis of TNF-alpha mRNA levels in whole blood stimulated with lipopolysaccharide (LPS).