Nociceptor neurons suppress alveolar macrophage-induced Siglec-F neutrophil-mediated inflammation to protect against pulmonary fibrosis.

Pulmonary fibrosis results from persistent and pathological tissue repair, which is therapeutically challenging to attenuate and often fatal. The immune processes involved in fibrosis remain ill defined. Using a bleomycin-induced lung fibrosis murine model, we discovered that vagal TRPV1 nociceptors are protective.

Confirmatory Testing for Primary Aldosteronism : A Study of Diagnostic Test Accuracy.

Background: Confirmatory testing to verify the diagnosis of primary aldosteronism (PA) in patients who have an abnormal screening result is of uncertain benefit.

Objective: To perform a blinded assessment of the seated saline suppression test (SSST).

Design: Diagnostic test accuracy study.

Big data determination and validation of reference range for 24-h urine cortisol by liquid chromatography-mass spectrometry.

Objective: Twenty-four-hour urine-free cortisol (UFC) is a first-line test for Cushing syndrome (CS). A new mass spectrometry assay for UFC requires a validated, relevant reference range appropriate to a screening population.

Design: Combined retrospective and prospective cohort study in a government health system and tertiary endocrinology clinic, Canada.

Discordance between adrenal vein sampling and imaging: the role of unilateral cortisol excess.

Objective: Adrenal vein sampling (AVS) is considered the gold-standard for identifying unilateral primary aldosteronism (PA), but is sensitive to any asymmetry in cortisol production. Subclinical autonomous cortisol production likely contributes to discordance between cross-sectional imaging and AVS.

Design And Methods: Retrospective chart review was performed of patients in Calgary, Alberta who (1) had a diagnosis of PA with clear adrenal mass, (2) had discordant AVS and cross-sectional imaging, and (3) underwent dexamethasone-suppressed NP59-iodocholesterol adrenal scintigraphy (n = 25).

Advancing Newborn Screening in Washington State: A Novel Multiplexed LC-MS/MS Proteomic Assay for Wilson Disease and Inborn Errors of Immunity.

For many genetic disorders, there are no specific metabolic biomarkers nor analytical methods suitable for newborn population screening, even where highly effective preemptive treatments are available. The direct measurement of signature peptides as a surrogate marker for the protein in dried blood spots (DBSs) has been shown to successfully identify patients with Wilson Disease (WD) and three life-threatening inborn errors of immunity, X-linked agammaglobulinemia (XLA), Wiskott-Aldrich syndrome (WAS), and adenosine deaminase deficiency (ADAD). A novel proteomic-based multiplex assay to detect these four conditions from DBS using high-throughput LC-MS/MS was developed and validated.

Targeted proteomic approach for quantification of collagen type I and type III in formalin-fixed paraffin-embedded tissue.

Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity.

Distribution of videos demonstrating best practices in preventing hemolysis is associated with reduced hemolysis among nurse-collected specimens in hospitals.

Background: Minimizing hemolysis during phlebotomy ensures accurate chemistry results and reduces test cancellations and specimen recollections. We developed videos demonstrating best practices to reduce hemolysis and tested whether distribution to clinical nurse educators (CNEs) for provision to nursing staff affected the degree of specimen hemolysis in hospital inpatient units and outpatient clinics.

Methods: Videos of common blood collections demonstrating best practices to reduce hemolysis were filmed and then distributed via email link to all hospital-based CNEs in Calgary, Alberta, Canada.

Agreement of LC-MS assays for IGF-1 traceable to NIST and WHO standards permits harmonization of reference intervals between laboratories.

Objectives: In this study, we aimed to determine the feasibility of transferring IGF-1 reference intervals between two liquid chromatography-mass spectrometry assays with distinct assay formats and calibration traceability.

Design And Methods: To adopt a reference interval (RI) for our new assay we have conducted RI transference and verification studies according to the CLSI EP28-A3c and EP9c guidelines. Specifically, the analytical agreement between the assays was evaluated using the linear model and the appropriateness of the linear model for RI transference was assessed using Deming regression, correlation coefficients, Q-Q plot, difference plot and studentized residues for the LC-MS/MS against DiaSorin LiaisonXL IGF-1 immunoassay and the liquid chromatography-high resolution mass spectrometry (LC-MS/HRMS) IGF-1 assay.

Biological variation in clozapine and metabolite reporting during therapeutic drug monitoring.

Background: Clozapine (CLO) is an atypical antipsychotic used in management of treatment-resistant schizophrenia. Adverse drug reactions are caused by both CLO and its primary metabolite, norclozapine (NCLO). We defined the biological variability of CLO, NCLO, and the CLO to NCLO ratio (CNR) as well as assess the impact of reporting CLO and NCLO routinely.

Novel Approach to Establishing an Aldosterone: Renin Ratio Cutoff for Primary Aldosteronism.

Direct renin concentration is replacing plasma renin activity in many laboratories for the investigation of primary aldosteronism, which may have a significant impact on the resulting aldosterone:renin ratios. We sought to develop a population-based approach to establishing an aldosterone:renin ratio cutoff when transitioning between assays. A population-based study was performed in Calgary, Alberta, Canada of 4301 individuals who received testing from January 2012 to November 2015.

Fasting time and vitamin B12 levels in a community-based population.

Objectives: Vitamin B12, also known as cobalamin (Cbl), is an essential vitamin that manifests with numerous severe but non-specific symptoms in cases of deficiency. Assessing Cbl status often requires fasting, although this requirement is not standard between institutions. This study evaluated the impact of fasting on Cbl levels in a large community-based cohort in an effort to promote standardization of Cbl testing between sites.

One-step extraction and quantitation of toxic alcohols and ethylene glycol in plasma by capillary gas chromatography (GC) with flame ionization detection (FID).

Objectives: Clinical analysis of volatile alcohols (i.e. methanol, ethanol, isopropanol, and metabolite acetone) and ethylene glycol (EG) generally employs separate gas chromatography (GC) methods for analysis.

Resolubilization of precipitated intact membrane proteins with cold formic acid for analysis by mass spectrometry.

Protein precipitation in organic solvent is an effective strategy to deplete sodium dodecyl sulfate (SDS) ahead of MS analysis. Here we evaluate the recovery of membrane and water-soluble proteins through precipitation with chloroform/methanol/water or with acetone (80%). With each solvent system, membrane protein recovery was greater than 90%, which was generally higher than that of cytosolic proteins.

Reprint of "GELFrEE fractionation combined with mass spectrometry for proteome analysis of secreted toxins from Enteropathogenic Escherichia coli (EPEC)".

Enteropathogenic Escherichia coli, or EPEC, is a human pathogen associated with gastroenteritis and diarrheal disease whose pathogenicity is related to the secretion of effector proteins (exotoxins). Determining exotoxin expression level is of considerable interest to those studying toxin function and pathological phenotypes. Mass spectrometry (MS) provides an ideal platform for detection and quantification of proteins from complex mixtures.

Proteomic analysis of rat proximal tubule cells following stretch-induced apoptosis in an in vitro model of kidney obstruction.

Unlabelled: Urinary tract obstruction (UTO) is a commonly noted disorder on prenatal ultrasound that has the potential to lead to permanent loss of renal function. To study the molecular processes of the disease, an in vitro model has been developed which involves mechanical stretch of proximal tubule cells grown on flexible plates which mimics the physiological conditions during UTO. This study employs a one dimensional SDS-PAGE fractionation procedure, followed by in-gel digest and LC-MS/MS analysis in a semi-quantitative experiment using spectral counting to relatively quantify changes in protein expression following the established model of UTO.

Dual LC-MS platform for high-throughput proteome analysis.

We describe a dual-column interface for parallel chromatography to improve throughput during LC-MS experimentation. The system employs a high-voltage switch to operate two capillary column/nanospray emitters fixed at the MS orifice. Sequentially loading one column while operating the second nearly doubles the LC-MS duty cycle.

Proteomic Workflows for Biomarker Identification Using Mass Spectrometry - Technical and Statistical Considerations during Initial Discovery.

Identification of biomarkers capable of differentiating between pathophysiological states of an individual is a laudable goal in the field of proteomics. Protein biomarker discovery generally employs high throughput sample characterization by mass spectrometry (MS), being capable of identifying and quantifying thousands of proteins per sample. While MS-based technologies have rapidly matured, the identification of truly informative biomarkers remains elusive, with only a handful of clinically applicable tests stemming from proteomic workflows.

GELFrEE fractionation combined with mass spectrometry for proteome analysis of secreted toxins from Enteropathogenic Escherichia coli (EPEC).

Enteropathogenic Escherichia coli, or EPEC, is a human pathogen associated with gastroenteritis and diarrheal disease whose pathogenicity is related to the secretion of effector proteins (exotoxins). Determining exotoxin expression level is of considerable interest to those studying toxin function and pathological phenotypes. Mass spectrometry (MS) provides an ideal platform for detection and quantification of proteins from complex mixtures.

A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC).

As an alternative to direct UV absorbance measurements, estimation of total protein concentration is typically conducted through colorimetric reagent assays. However, for protein-limited applications, the proportion of the sample sacrificed to the assay becomes increasingly significant. This work demonstrates a method for quantitation of protein samples with high recovery.

The htpAB operon of Legionella pneumophila cannot be deleted in the presence of the groE chaperonin operon of Escherichia coli.

HtpB, the chaperonin of the intracellular bacterial pathogen Legionella pneumophila , displays several virulence-related functions in vitro. To confirm HtpB's role in vivo, host infections with an htpB deletion mutant would be required. However, we previously reported that the htpAB operon (encoding co-chaperonin and chaperonin) is essential.