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A retrospective study was conducted to determine the patterns of strong opioid use in patients admitted to a hospice inpatient unit, with special attention to the use of the transdermal fentanyl patch. This study was conducted to validate or negate the subjective feeling that many patients, treated at admittance with the fentanyl patch, received inappropriately high doses compared to patients treated with oral or parenteral opioids. The case notes of 1154 patients were reviewed and data collected on age, sex, diagnosis, care settings, opioid form and dose on referral, maximal dose during admission and opioid use during the last 24 hours of their life. At admission opioids had been prescribed for 47% of patients. Thirty-two percent of these patients received oral morphine. The median dose at admission of those patients was 60 mg (oral morphine equivalent (OME)). Thirty-six percent of the patients on opioids were using the fentanyl patch. The median dose at admission was triple that of the orally treated patients (median 180 mg OME). In the 199 patients using transdermal fentanyl at admission, in most patients the dose of the patch was gradually diminished and finally stopped in 58% of patients. Only 83 kept it until the last 24 hours. We would like to draw attention to the fact that (sometimes inappropriately) high doses of fentanyl were used at admission, probably due to lack of knowledge of the relative strength of the opioid involved and to the failure to recognize the phenomenon of opioid-induced hyperalgesia. In addition, in our experience the long action of the patch can be a disadvantage during the last days and weeks of life, due to the difficulty of dose adjustment and the risk for toxicity.

Background Only a few studies have examined fatigue in Osteoarthritis, but all revealed fatigue as ubiquitously present and related to several physical and mental health aspects among OA patients. Objectives The goal of this study was to examine whether different groups of fatigue trajectories can be identified among middle-age patients with early symptomatic osteoarthritis (OA), to establish the level of fatigue severity within each of these fatigue groups, and to assess the role of age, gender and OA severity in relation to group classification. Methods Data on fatigue and patient characteristics were collected from the CHECK cohort. Growth mixture modeling (GMM) was applied in order to identify distinct fatigue groups as well as to take into account the influence of radiographic OA severity (Kellgren-Lawrence grading), age and gender on group classification. Results A stable, a U-shape, and an inverted U-shape trajectory were identified. The fatigue levels of the latter two groups (i.e. approximately two-thirds of the sample) were comparable to the Dutch cancer population. Females and younger OA patients experienced higher levels of fatigue within each group and females were more likely than males to exhibit the inverted U-shape fatigue pattern. The covariates were not related to the interindividual differences in fatigue change over time within each fatigue group. Conclusions Three different fatigue patterns were found among a sample of early OA patients. These patterns indicate that a large number of OA patients experience elevated levels of fatigue already at an early stage of OA. This finding asks for more attention regarding fatigue in OA research, and warrants tailored psychosocial interventions for patients with elevated levels of fatigue. Acknowledgements The CHECK study is financed by the Dutch Arthritis Foundation. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5062

We have produced human alpha1-antitrypsin (A1AT), a major therapeutic protein, in genetically engineered tobacco plastids. Four different expression vectors have been evaluated which encode A1AT under the control of various 5' and 3' plastid expression elements. The use of heterologous promoter and terminator sequences derived from the corn and soybean plastid genomes leads to simpler and predictable recombinant genome patterns, avoiding unwanted recombination products between introduced and resident tobacco sequences. High level expression of unglycosylated A1AT, representing up to 2% of total soluble proteins, has been measured in leaves of transgenic tobacco lines. Some heterogeneity in the recombinant A1AT is detected after 2D protein separation, but the chloroplast-made protease inhibitors are fully active and bind to porcine pancreatic elastase.

To improve the digestibility of the forage crop alfalfa (Medicago sativa L.), cinnamyl alcohol dehydrogenase (CAD), which catalyses the last step in the biosynthesis of the lignin monomers, was down-regulated by using an antisense approach. A subset of six transgenic lines with reduced CAD activity and control lines were analysed when grown in the greenhouse and in the field. The down-regulation of the CAD enzyme was associated with a red coloration of the stem. The lignin quantity remained unchanged, but the lignin composition, as determined by thioacidolysis, was altered. The highest reduction of CAD activity was associated with a lower syringyl/guaiacyl (S/G) ratio and a lower S+G yield, mainly because of a decreased amount of S units. An increase in in situ disappearance of dry matter and of cell wall residue was detected in one of the transgenic lines grown in the greenhouse, and for two of the lines grown in the field the rate of disappearance of dry matter slightly improved. Furthermore, these two lines had a higher solubility in alkali as shown by the lower yield of saponified residue. This study opens perspectives for improving forage crop digestibility by the modulation of enzymes involved in lignin biosynthesis.

Transgenic maize plants have been generated by particle gun bombardment that overproduce a Nicotiana plumbaginifolia L. manganese superoxide dismutase (MnSOD). To target this mitochondrial enzyme into chloroplasts, the mature MnSOD-coding sequence was fused to a chloroplast transit peptide from a pea ribulose-1,5-bisphosphate carboxylase gene, whereas expression of the chimeric gene was driven by the CaMV 35S promoter. Transgenic MnSOD activity contributed to 20% of the total SOD activity. The presence of transgenic MnSOD had clear effects on foliar tolerance to chilling and oxidative stress. The results suggest that overproduction of MnSOD in the chloroplasts increases the antioxidant capacity of the leaves.

Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses that were found to be noncompeting. Unlike vectors derived from the same virus, noncompeting vectors effectively coexpress the heavy and light chains in the same cell throughout the plant body, resulting in yields of up to 0.5 g of assembled mAbs per kg of fresh-leaf biomass. This technology allows production of gram quantities of mAbs for research purposes in just several days, and the same protocol can be used on an industrial scale in situations requiring rapid response, such as pandemic or terrorism events.

To study protein secretion in plant cells, we established and evaluated a model system based on transient synthesis of heterologous proteins in tobacco protoplasts. We show that the nonsecretory enzymes phosphinothricin acetyl transferase, neomycin phosphotransferase II, and beta-glucuronidase are secreted when targeted to the lumen of the endoplasmic reticulum by signal peptide-mediated translocation. These data are consistent with the view that secretion can occur independent of active sorting mechanisms by nonspecific migration through the exocytic pathway. However, the rate of secretion differs significantly among these enzymes. Furthermore, the presence of signal sequences was found to be correlated with a reduction of the levels of the encoded gene products. This is the result of post-transcriptional events that limit either synthesis or stability of the proteins in vivo

Enzymes that catalyze the condensation of acetyl coenzyme A and 2-oxo acids are likely to be important in two distinct metabolic pathways in Arabidopsis. These are the synthesis of isopropylmalate, an intermediate of Leu biosynthesis in primary metabolism, and the synthesis of methylthioalkylmalates, intermediates of Met elongation in the synthesis of aliphatic glucosinolates (GSLs), in secondary metabolism. Four Arabidopsis genes in the ecotype Columbia potentially encode proteins that could catalyze these reactions. MAM1 and MAML are adjacent genes on chromosome 5 at the Gsl-elong locus, while MAML-3 and MAML-4 are at opposite ends of chr 1. The isopropylmalate synthase activity of each member of the MAM-like gene family was investigated by heterologous expression in an isopropylmalate synthase-null Escherichia coli mutant. Only the expression of MAML-3 restored the ability of the mutant to grow in the absence of Leu. A MAML knockout line (KO) lacked long-chain aliphatic GSLs, which were restored when the KO was transformed with a functional MAML gene. Variation in expression of MAML did not alter the total levels of Met-derived GSLs, but just the ratio of chain lengths. MAML overexpression in Columbia led to an increase in long-chain GSLs, and an increase in 3C GSLs. Moreover, plants overexpressing MAML contained at least two novel amino acids. One of these was positively identified via MS/MS as homo-Leu, while the other, with identical mass and fragmentation patterns, was likely to be homo-Ile. A MAML-4 KO did not exhibit any changes in GSL profile, but had perturbed soluble amino acid content.

We have cloned cDNAs of the tobacco homolog of the luminal binding protein (BiP) that has been described in other higher eukaryotes. In contrast to the mammalian and yeast protein, tobacco BiP is encoded by a multigene family. The gene products of all the cloned members of this family contain a carboxy-terminal His-Asp-Glu-Leu peptide that may form the signal for retention in the endoplasmic reticulum. Analysis of expression patterns revealed that BiP transcripts are predominantly present in tissues with high rates of cell divisions, in secretory tissues, and in cells treated with tunicamycin. We also show that a chimeric gene containing the coding region of one of the tobacco BiP genes is able to complement a mutation in the Saccharomyces cerevisiae BiP gene.