Andrewsbirch1041

In amyotrophic lateral sclerosis (ALS) motor neurons (MNs) undergo dying-back, where the distal axon degenerates before the soma. The hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS, but the mechanism of pathogenesis is largely unknown with both gain- and loss-of-function mechanisms being proposed. To better understand C9ORF72-ALS pathogenesis, we generated isogenic induced pluripotent stem cells. MNs with HRE in C9ORF72 showed decreased axonal trafficking compared with gene corrected MNs. However, knocking out C9ORF72 did not recapitulate these changes in MNs from healthy controls, suggesting a gain-of-function mechanism. In contrast, knocking out C9ORF72 in MNs with HRE exacerbated axonal trafficking defects and increased apoptosis as well as decreased levels of HSP70 and HSP40, and inhibition of HSPs exacerbated ALS phenotypes in MNs with HRE. Therefore, we propose that the HRE in C9ORF72 induces ALS pathogenesis via a combination of gain- and loss-of-function mechanisms. Trypanosoma are blood-borne parasites and are the causative agents of neglected tropical diseases (NTDs) affecting both humans and animals. These parasites mainly rely on glycolysis for their energy production within the mammalian host, which is why trypanosomal glycolytic enzymes have been pursued as interesting targets for the development of trypanocidal drugs. The structure-function relationships of pyruvate kinases (PYKs) from trypanosomatids (Trypanosoma and Leishmania) have been well-studied within this context. In this paper, we describe the structural and enzymatic characterization of PYK from T. congolense (TcoPYK), the main causative agent of Animal African Trypanosomosis (AAT), by employing a combination of enzymatic assays, thermal unfolding studies and X-ray crystallography. BACKGROUND Biotin is an interference in many streptavidin-biotin based immunoassays, causing falsely decreased results with sandwich immunoassays and falsely increased results with competitive immunoassays. It has been discussed that premixing streptavidin coated beads and biotinylated capturing molecules may prevent biotin interference. This study was designed to test whether such modification could mitigate biotin interference in two originally susceptible sandwich immunoassays. METHODS Roche C-peptide and human growth hormone (hGH) immunoassays utilize three reagent containers for streptavidin coated beads (M), biotinylated capturing antibody (R1) and ruthenylated antibody (R2). SMIP34 The reagents were modified by premixing reagent M and R1. Following incubation, the beads were placed back in the M-container and R1-supernatant back to R1-container. Patient specimens were selected, spiked with biotin to 1055 ng/mL, and measured by both the original, unmodified reagent and modified reagent on Roche cobas e411 analyzer. The biotin interference dose response curves were also compared using pooled patient specimen spiked with different concentrations of biotin. RESULTS For the original reagent, 1055 ng/mL of biotin decreased C- peptide results by 88% and hGH results by 97%. After reagent modification, this interference effect was nearly eliminated for C- peptide but remained about 20% decreased for hGH. CONCLUSION Premixing streptavidin beads and biotinylated capturing molecules is an effective approach to mitigate biotin interference for certain immunoassays. INTRODUCTION The appropriate use of laboratory diagnostics is increasingly at stake. The aim of this study was to depict some paradigmatic examples of under- and overutilization, as well as possible solutions across Europe. METHODS We collected six examples from five European countries where a rise or decline of orders for specific laboratory parameters was observed after organizational changes but without evidence of changes in patient collective characteristics as source of this variation. RESULTS The collected examples were the following 1-Germany) Switch from a Brain-Natriuretic-Peptide assay to NT-pro Brain-Natriuretic-Peptide assay, resulting in a 374% increase in these analytics; 2-Spain) Implementation of a gatekeeping strategy in tumor marker diagnostics, resulting in a 15-61% reduction of these diagnostics; 3-Croatia) Stepwise elimination of creatine-kinase-MB assay from the laboratory portfolio; 4-UK) Removal of γ-glutamyl transferase from a "liver function" profile, resulting in 82% reduction of orders; 5-Austria) Implementation of a new device for rapid Influenza-RNA detection, resulting in a 450% increase of Influenza testing; 6-Spain) Insourcing of 1,25-(OH)2-Vitamin D measurements, leading to a 378% increase of these analyses. CONCLUSION The six paradigmatic examples described in this manuscript show that availability of laboratory resources may considerably catalyze the demand, thus underscoring that inappropriate use of laboratory resources may be commonplace in routine laboratories all across Europe and most probably beyond. They also demonstrate that the application of simple strategies may assist in overcoming this issue. We believe that laboratory specialists need to refocus on the extra-analytical parts of the testing process and engage more in interdisciplinary patient-care. BACKGROUND Vitamin A and E are routinely monitored to assess nutritional status. The most commonly used approach for their measurement involves laborious liquid-liquid extraction followed by high-performance liquid chromatography (HPLC) analysis on dedicated instrumentation. We describe a simple, rapid protocol for measurement of vitamin A and E and their integration into an existing online sample preparation liquid chromatography tandem mass spectrometry (SPLC-MS/MS) workflow. METHODS We performed a method comparison between the SPLC-MS/MS and HPLC methods for vitamin A and E by measuring patient specimens across the concentration range 11-81 µg/dL for vitamin A and 1-18 mg/L for vitamin E. The analysis times on each platform were also compared. RESULTS SPLC-MS/MS and HPLC methods were comparable with regards to analytical performance; mean bias across the measured range was 2.54% (95% CL -11.56-16.64%) for vitamin A and -2.04% (95% CL -18.20-14.12%) for vitamin E. Total analysis times were 7 min and 15 min for SPLC-MS/MS and HPLC respectively.