Importance of Post-Translational Modifications for Functionality of a Chloroplast-Localized Carbonic Anhydrase (CAH1) in Arabidopsis thaliana

Background: The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells. Methodology/Principal Findings: Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited. Conclusions/Significance: We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.This work was supported by the Swedish Research Council (VR), the Kempe Foundations and Carl Tryggers Foundation to GS, and grant numbers BIO2006-08946 and BIO2009-11340 from the Spanish Ministerio de Ciencia e Innovación (MICINN) to A

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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Non-standard errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence generating process (EGP). We claim that EGP variation across researchers adds uncertainty: Non-standard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for better reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

Phytoremediation of Heavy Metals and PAHs in Alkaline Slag Fill at a Wet Meadow Site

The feasibility of using phytoremediation to remediate an alkaline-slag-filled wet meadow site contaminated with polycyclic aromatic hydrocarbons (PAHs) and heavy metals has been studied. The objective of the present work is to investigate the ability of nine native grasses and trees to survive and remediate PAHs such as benzo(a) pyrene (BaP) and heavy metals such as arsenic (As), chromium (Cr), lead (Pb), and manganese (Mn) existing at the site during the three complete growing seasons. Replicate test plots were prepared by tilling and homogenizing the fill material to approximate depth of approximately 0.3 m. Soil samples from each plot were collected before and after preparing the test plots and analyzed for physicochemical properties and contaminant concentrations. The survival and growth of the plant species were monitored for two growing seasons. Only one plant species out of the total nine initially planted, specifically False Indigo Bush (Amorpha fruticosa) (FIB), survived the three growing seasons. Soil, plant root, and plant shoot samples were collected at the end of the second and third growing seasons at the plot where FIB was planted. PAHs and heavy metals were analyzed in the soil and plant samples. In addition, a sequential extraction procedure was followed to determine the fractionation of the heavy metals in soils before and after planting. The results showed no significant decrease in BaP, As, Cr, and Pb concentrations in the soil. In addition, there were no significant changes in heavy-metals fractions. However, Mn uptake in roots and shoots was observed, with a corresponding decrease in soil, at the end of the third growing season. The adaptability and survival of FIB and its high tolerance to harsh site conditions (high pH, fluctuating moisture, and contaminant toxicity) demonstrated the potential of this species for its use in the remediation of the study area. (C) 2017 American Society of Civil Engineers

Botulinum toxin type-A effect as a preemptive treatment in a model of acute trigeminal pain: a pre-clinical double-blind and placebo-controlled study

The purpose of this study was to investigate if botulinum neurotoxin type-A (BoNT/A) had a preemptive antinociceptive effect in a formalin-induced orofacial pain model (FT). To test this hypothesis, male Rattus norvegicus were injected with isotonic saline solution 0.9% or BoNT/A administered as a 40 μl bolus, lateral to their nose, at 24 hours, 8, 15, 22, 29 or 36 days pre-FT. The procedures were repeated 42 days later. Influence on motor activity was assessed through the open-field test. Pain scores corresponded to the time spent rubbing and flicking the injected area. Animals pre-treated with BoNT/A at the first protocol (8 days subgroup) showed reduced inflammatory scores (p=0.011). For the other groups no significant results were observed at any phase. Motor activity was similar in both groups. BoNT/A showed to be effective preventing inflammatory pain up to eight days after the first treatment, an effect not reproduced on the second dose administration