Falsely Elevated Vancomycin Concentrations in a Patient Not Receiving Vancomycin.

Therapeutic drug monitoring (TDM) of vancomycin is commonly performed using immunoassays. This case describes falsely elevated vancomycin serum concentrations, possibly secondary to endogenous protein interference. Vancomycin was prescribed for a patient with a suspected septic knee. A blood sample for TDM was inadvertently collected before the first dose. The reported concentration was 36.1 mg/L using the Roche Modular P analyzer and remained high over the next 48 hours and 8 months later in the absence of vancomycin therapy. Vancomycin was undetectable in the patient sample by liquid chromatography-tandem mass spectrometry. The sample was subsequently investigated for endogenous protein interference. The responsible interference was removed by polyethylene glycol precipitation and heat inactivation. Four alternative immunoassays with varying test principles measured vancomycin concentrations ranging from undetectable to 108 mg/L. A glucose-6-phosphate dehydrogenase detection method was common to the two immunoassays exhibiting the greatest interference. To our knowledge, this is the first report of falsely elevated vancomycin concentrations on the Roche Modular P analyzer. Immunoassays are generally robust in facilitating TDM but are susceptible to cross-reactivity. Assay interference should be considered and laboratory professionals contacted when vancomycin levels do not correlate with clinical expectations.

Comparison of Fecal Calprotectin Methods for Predicting Relapse of Pediatric Inflammatory Bowel Disease.

Background. Pediatric inflammatory bowel disease (IBD) is on the rise worldwide. Endoscopies are necessary for IBD assessment but are invasive, expensive, and inconvenient. Recently, fecal calprotectin (FCal) was proposed as a noninvasive and specific marker of gut inflammation. We evaluated the analytical performance of three FCal assays and their clinical performance in predicting relapse in pediatric IBD. Methods. This study used 40 pediatric IBD and 40 random non-IBD patients' fecal samples. Two automated ELISAs (Bühlmann and PhiCal® Calprotectin-EIA) and an EliA (Phadia 250 EliA-Calprotectin) were used to evaluate the analytical performance. The clinical performance was assessed by PhiCal Calprotectin-EIA, EliA-Calprotectin, and Bühlmann immunochromatographic point-of-care test (POCT). Results. All assays displayed acceptable analytical performance below and above the medical decision cut-off [imprecision (CV < 10% intra-assay; <15% interassay); linearity (overall mean % deviation < 16.5%)]. The agreement with PhiCal Calprotectin-EIA was 100% and 78.6% for Bühlmann (95% CI, 87.5-100; Kappa: 1) and EliA-Calprotectin (95% CI, 60.5-89.8; Kappa: 0.32), respectively, and 63.6% between Bühlmann and EliA-Calprotectin (95% CI, 46.6-77.8; Kappa: 0.16). All assays evaluated had similar clinical performance [AUC: 0.84 (EliA-Calprotectin); 0.83 (POCT and PhiCal Calprotectin-EIA)]. Conclusion. FCal levels determined using the same method and assay together with clinical history would be a noninvasive and useful tool in monitoring pediatric IBD.

Systematic protein-protein interaction mapping for clinically relevant human GPCRs.

G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.

A genome scale overexpression screen to reveal drug activity in human cells.

Target identification is a critical step in the lengthy and expensive process of drug development. Here, we describe a genome-wide screening platform that uses systematic overexpression of pooled human ORFs to understand drug mode-of-action and resistance mechanisms. We first calibrated our screen with the well-characterized drug methotrexate. We then identified new genes involved in the bioactivity of diverse drugs including antineoplastic agents and biologically active molecules. Finally, we focused on the transcription factor RHOXF2 whose overexpression conferred resistance to DNA damaging agents. This approach represents an orthogonal method for functional screening and, to our knowledge, has never been reported before.

CHIP-MYTH: a novel interactive proteomics method for the assessment of agonist-dependent interactions of the human ß2-adrenergic receptor.

G-protein coupled receptors (GPCRs) are involved in a variety of disease processes and comprise major drug targets. However, the complexity of integral membrane proteins such as GPCRs makes the identification of their interacting partners and subsequent drug development challenging. A comprehensive understanding of GPCR protein interaction networks is needed to design effective therapeutic strategies to inhibit these drug targets. Here, we developed a novel split-ubiquitin membrane yeast two-hybrid (MYTH) technology called CHIP-MYTH, which allows the unbiased characterization of interaction partners of full-length GPCRs in a drug-dependent manner. This was achieved by coupling DNA microarray technology to the MYTH approach, which allows a quantitative evaluation of interacting partners of a given integral membrane protein in the presence or absence of drug. As a proof of principle, we applied the CHIP-MYTH approach to the human ß2-adrenergic receptor (ß2AR), a target of interest in the treatment of asthma, chronic obstructive pulmonary disease (COPD), neurological disease, cardiovascular disease, and obesity. A CHIP-MYTH screen was performed in the presence or absence of salmeterol, a long-acting ß2AR-agonist. Our results suggest that ß2AR activation with salmeterol can induce the dissociation of heterotrimeric G-proteins, Gαßγ, into Gα and Gßγ subunits, which in turn activates downstream signaling cascades. Using CHIP-MYTH, we confirmed previously known and identified novel ß2AR interactors involved in GPCR-mediated signaling cascades. Several of these interactions were confirmed in mammalian cells using LUminescence-based Mammalian IntERactome (LUMIER) and co-immunoprecipitation assays. In summary, the CHIP-MYTH approach is ideal for conducting comprehensive protein-protein interactions (PPI) screenings of full-length GPCRs in the presence or absence of drugs, thus providing a valuable tool to further our understanding of GPCR-mediated signaling.

Aerosolized liposomal Amphotericin B: a potential prophylaxis of invasive pulmonary aspergillosis in immunocompromised patients.

BACKGROUND: Aerosolized liposomal Amphotericin B may reduce the incidence of invasive pulmonary Aspergillosis in adults with chemotherapy-induced prolonged neutropenia with less nephrotoxicity. The breath-actuated AeroEclipse® BAN nebulizer is very efficient and minimizes environmental drug contamination since no aerosol is produced, unless the patient is inspiring through the device. Our aim is to develop an appropriate delivery system suitable for children that does not disrupt the liposomes due to the shear forces in nebulization. METHODS: This is an in vitro experimental study in vitro. Six ml of 4 mg/ml liposomal Amphotericin B solution (AmBisome®; Astellas Pharma Inc., Markham, Ontario, CA) was nebulized with the breath-actuated nebulizer (AeroEclipse®; Trudell Medical International, Canada) and captured by the glass liquid impinger. Sodium dodecyl sulfate was used as detergent to disrupt the liposomes in control samples. Gel filtration, electron microscopy, and high performance liquid chromatography (HPLC) were used to compare the size and shape of the liposomes, and amount of the drug before and after nebulization. The aerosol particle size was obtained by the laser diffraction. RESULTS: After nebulization, 97.5% of amphotericin B was captured by the liquid impinger and detected by HPLC. Gel filtration and electron microscopy demonstrated that the drug remained in its liposomal configuration after nebulization. The mass median diameter (MMD) was 3.7 µm and 66% of aerosol particles were less than 5 µm in diameter. CONCLUSIONS: We demonstrated that liposomal Amphotericin B can be nebulized successfully without disrupting the liposomes and minimize drug loss by using the breath-actuated nebulizer.

MOR is not enough: identification of novel mu-opioid receptor interacting proteins using traditional and modified membrane yeast two-hybrid screens.

The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.

Miniature short hairpin RNA screens to characterize antiproliferative drugs.

The application of new proteomics and genomics technologies support a view in which few drugs act solely by inhibiting a single cellular target. Indeed, drug activity is modulated by complex, often incompletely understood cellular mechanisms. Therefore, efforts to decipher mode of action through genetic perturbation such as RNAi typically yields "hits" that fall into several categories. Of particular interest to the present study, we aimed to characterize secondary activities of drugs on cells. Inhibiting a known target can result in clinically relevant synthetic phenotypes. In one scenario, drug perturbation could, for example, improperly activate a protein that normally inhibits a particular kinase. In other cases, additional, lower affinity targets can be inhibited as in the example of inhibition of c-Kit observed in Bcr-Abl-positive cells treated with Gleevec. Drug transport and metabolism also play an important role in the way any chemicals act within the cells. Finally, RNAi per se can also affect cell fitness by more general off-target effects, e.g., via the modulation of apoptosis or DNA damage repair. Regardless of the root cause of these unwanted effects, understanding the scope of a drug's activity and polypharmacology is essential for better understanding its mechanism(s) of action, and such information can guide development of improved therapies. We describe a rapid, cost-effective approach to characterize primary and secondary effects of small-molecules by using small-scale libraries of virally integrated short hairpin RNAs. We demonstrate this principle using a "minipool" composed of shRNAs that target the genes encoding the reported protein targets of approved drugs. Among the 28 known reported drug-target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug-target interactions based on drug-induced synthetic lethality. We provide a detailed protocol for performing such screens and for analyzing the data. This cost-effective approach to mammalian knockdown screens, combined with the increasing maturation of RNAi technology will expand the accessibility of similar approaches in academic settings.

Regulation of CD133 by HDAC6 promotes ß-catenin signaling to suppress cancer cell differentiation.

The pentaspan membrane glycoprotein CD133 marks lineage-specific cancer progenitor cells and is associated with poor prognosis in a number of tumor types. Despite its utility as a cancer progenitor cell marker, CD133 protein regulation and molecular function remain poorly understood. We find that the deacetylase HDAC6 physically associates with CD133 to negatively regulate CD133 trafficking down the endosomal-lysosomal pathway for degradation. We further demonstrate that CD133, HDAC6, and the central molecule of the canonical Wnt signaling pathway, ß-catenin, can physically associate as a ternary complex. This association stabilizes ß-catenin via HDAC6 deacetylase activity, which leads to activation of ß-catenin signaling targets. Downregulation of either CD133 or HDAC6 results in increased ß-catenin acetylation and degradation, which correlates with decreased proliferation in vitro and tumor xenograft growth in vivo. Given that CD133 marks progenitor cells in a wide range of cancers, targeting CD133 may be a means to treat multiple cancer types.

Barcode sequencing for understanding drug-gene interactions.

With the advent of next-generation sequencing (NGS) technology, methods previously developed for microarrays have been adapted for use by NGS. Here we describe in detail a protocol for Barcode analysis by sequencing (Bar-seq) to assess pooled competitive growth of individually barcoded yeast deletion mutants. This protocol has been optimized on two sequencing platforms: Illumina's Genome Analyzer IIx/HiSeq2000 and Life Technologies SOLiD3/5500. In addition, we provide guidelines for assessment of human knockdown cells using short-hairpin RNAs (shRNA) and an Illumina sequencing readout.

Detecting interactions with membrane proteins using a membrane two-hybrid assay in yeast.

The biological function of proteins may be predicted by identification of their interacting partners, and one of the major goals of the postgenomic era is the mapping of protein interaction networks. Membrane proteins are of particular interest because of their role in disease and because of their prevalence as major pharmaceutical targets. Unfortunately, because of their hydrophobic nature, they have long been difficult to study in a high-throughput format. A powerful technology recently developed to facilitate the characterization of membrane protein interactions is the membrane yeast two-hybrid (MYTH) assay. MYTH adapts the principle of split ubiquitin for use as a potent in vivo sensor of protein-protein interactions, allowing large-scale screening for interactors of full-length membrane proteins, from a range of organisms, using Saccharomyces cerevisiae as a host. In this article, we describe a protocol for MYTH bait generation, validation and library screening. The entire MYTH procedure can generally be completed in 4-6 weeks.