Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin

<p>Abstract</p> <p>Background</p> <p>Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD₅₀of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein.</p> <p>Results</p> <p>Secretion level of the fusion protein produced <it>in vitro </it>in BHK cells was ~30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04–1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of ~150 kDa by Western blot. The purified fusion protein produced <it>in vitro </it>was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (~32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (~3 h). <it>In vitro </it>nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested.</p> <p>Conclusion</p> <p>Both the pharmacokinetic study and the <it>in vitro </it>nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced <it>in vitro </it>and <it>in vivo</it>. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.</p

Screening of novel fungal Carbohydrate Esterase family 1 enzymes identifies three novel dual feruloyl/acetyl xylan esterases

Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important enzymes for plant biomass degradation and are both present in Carbohydrate Esterase family 1 (CE1) of the Carbohydrate-Active enZymes database. In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced and screened for their activity towards model and complex plant biomass substrates. CE1_1 enzymes possess AXE activity, while CE1_5 enzymes showed FAE activity. Two enzymes from CE1_2 and one from CE1_5 possess dual feruloyl/acetyl xylan esterase (FXE) activity, showing expansion of substrate specificity. The new FXEs from CE1 can efficiently release both feruloyl and acetyl residues from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation

Carbohydrate esterase family 16 contains fungal hemicellulose acetyl esterases (HAEs) with varying specificity

Acetyl esterases are an important component of the enzymatic machinery fungi use to degrade plant biomass and are classified in several Carbohydrate Esterase families of the CAZy classification system. Carbohydrate Esterase family 16 (CE16) is one of the more recently discovered CAZy families, but only a small number of its enzyme members have been characterized so far, revealing activity on xylan-derived oligosaccharides, as well as activity related to galactoglucomannan. The number of CE16 genes differs significantly in the genomes of filamentous fungi. In this study, four CE16 members were identified in the genome of Aspergillus niger NRRL3 and it was shown that they belong to three of the four phylogenetic Clades of CE16. Significant differences in expression profiles of the genes and substrate specificity of the enzymes were revealed, demonstrating the diversity within this family of enzymes. Detailed characterization of one of these four A. niger enzymes (HaeA) demonstrated activity on oligosaccharides obtained from acetylated glucuronoxylan, galactoglucomannan and xyloglucan, thus establishing this enzyme as a general hemicellulose acetyl esterase. Their broad substrate specificity makes these enzymes highly interesting for biotechnological applications in which deacetylation of polysaccharides is required.Peer reviewe

Carbohydrate esterase family 16 contains fungal hemicellulose acetyl esterases (HAEs) with varying specificity

Acetyl esterases are an important component of the enzymatic machinery fungi use to degrade plant biomass and are classified in several Carbohydrate Esterase families of the CAZy classification system. Carbohydrate Esterase family 16 (CE16) is one of the more recently discovered CAZy families, but only a small number of its enzyme members have been characterized so far, revealing activity on xylan-derived oligosaccharides, as well as activity related to galactoglucomannan. The number of CE16 genes differs significantly in the genomes of filamentous fungi. In this study, four CE16 members were identified in the genome of Aspergillus niger NRRL3 and it was shown that they belong to three of the four phylogenetic Clades of CE16. Significant differences in expression profiles of the genes and substrate specificity of the enzymes were revealed, demonstrating the diversity within this family of enzymes. Detailed characterization of one of these four A. niger enzymes (HaeA) demonstrated activity on oligosaccharides obtained from acetylated glucuronoxylan, galactoglucomannan and xyloglucan, thus establishing this enzyme as a general hemicellulose acetyl esterase. Their broad substrate specificity makes these enzymes highly interesting for biotechnological applications in which deacetylation of polysaccharides is required

Le rapport à l'identité chez les jeunes des écoles francophones de l'Ontario

En Ontario, l'école de langue française joue un rôle significatif dans le développement du rapport à l'identité francophone chez les jeunes, en raison du contexte minoritaire. À leur sortie de l'école, ces jeunes sont exposés à une langue et une culture différentes; leur réseau social, et même familial, évolue dans un environnement autre que leur contexte éducationnel. Ce texte vise à poser un regard critique sur la notion d'identité telle que définie dans les documents officiels afin d'examiner si cette forme correspond réellement au contexte franco-ontarien actuel, compte tenu que la clientèle de l'école de langue française est beaucoup plus diversifiée qu'auparavant. Les résultats de mon analyse suggèrent qu'au lieu de promouvoir le développement personnel et social propre à une seule culture et une langue d'enseignement, le système scolaire devrait plutôt permettre aux élèves de s'associer à la culture et la langue d'enseignement sans toutefois nier leurs origines ou leur sentiment d'appartenance à la culture dominante.M.A

Functional characterization of fungal lytic polysaccharide monooxygenases for cellulose surface oxidation

Abstract Background Microbial lytic polysaccharide monooxygenases (LPMOs) cleave diverse biomass polysaccharides, including cellulose and hemicelluloses, by initial oxidation at C1 or C4 of glycan chains. Within the Carbohydrate-Active Enzymes (CAZy) classification, Auxiliary Activity Family 9 (AA9) comprises the first and largest group of fungal LPMOs, which are often also found in tandem with non-catalytic carbohydrate-binding modules (CBMs). LPMOs originally attracted attention for their ability to potentiate complete biomass deconstruction to monosaccharides. More recently, LPMOs have been applied for selective surface modification of insoluble cellulose and chitin. Results To further explore the catalytic diversity of AA9 LPMOs, over 17,000 sequences were extracted from public databases, filtered, and used to construct a sequence similarity network (SSN) comprising 33 phylogenetically supported clusters. From these, 32 targets were produced successfully in the industrial filamentous fungus Aspergillus niger, 25 of which produced detectable LPMO activity. Detailed biochemical characterization of the eight most highly produced targets revealed individual C1, C4, and mixed C1/C4 regiospecificities of cellulose surface oxidation, different redox co-substrate preferences, and CBM targeting effects. Specifically, the presence of a CBM correlated with increased formation of soluble oxidized products and a more localized pattern of surface oxidation, as indicated by carbonyl-specific fluorescent labeling. On the other hand, LPMOs without native CBMs were associated with minimal release of soluble products and comparatively dispersed oxidation pattern. Conclusions This work provides insight into the structural and functional diversity of LPMOs, and highlights the need for further detailed characterization of individual enzymes to identify those best suited for cellulose saccharification versus surface functionalization toward biomaterials applications

Opioid-associated iatrogenic withdrawal in critically ill adult patients: a multicenter prospective observational study

Abstract Background Opioids and benzodiazepines are frequently used in the intensive care unit (ICU). Regular use and prolonged exposure to opioids in ICU patients followed by abrupt tapering or cessation may lead to iatrogenic withdrawal syndrome (IWS). IWS is well described in pediatrics, but no prospective study has evaluated this syndrome in adult ICU patients. The objective of this study was to determine the incidence of IWS caused by opioids in a critically ill adult population. This multicenter prospective cohort study was conducted at two level-1 trauma ICUs between February 2015 and September 2015 and included 54 critically ill patients. Participants were eligible if they were 18 years and older, mechanically ventilated and had received more than 72 h of regular intermittent or continuous intravenous infusion of opioids. For each enrolled patient and per each opioid weaning episode, presence of IWS was assessed by a qualified ICU physician or senior resident according to the 5th edition of Diagnostic and Statistical Manual of Mental Disorders criteria for opioid withdrawal. Results The population consisted mostly of males (74.1%) with a median age of 50 years (25th–75th percentile 38.2–64.5). The median ICU admission APACHE II score was 22 (25th–75th percentile 12.0–28.2). The overall incidence of IWS was 16.7% (95% CI 6–27). The median cumulative opioid dose prior to weaning was higher in patients with IWS (245.7 vs. 169.4 mcg/kg, fentanyl equivalent). Patients with IWS were also exposed to opioids for a longer period of time as compared to patients without IWS (median 151 vs. 125 h). However, these results were not statistically significant. Conclusions IWS was occasionally observed in this very specific population of mechanically ventilated, critically ill ICU patients. Further studies are needed to confirm these preliminary results and identify risk factors

Additional file 1 of Functional characterization of fungal lytic polysaccharide monooxygenases for cellulose surface oxidation

Additional file 1: Figure S1: Product released by 5 μM C1 LPMOs after 16 h on PASC (0.1%), Avicel (1%), and SA-Avicel (1%)with 1 mM ascorbic acid as the electron donor. For each substrate, T. reesei cellulase cocktail was used to convert all C1-oxidized products into cellobionic acid, which was then quantified by HPAEC-PAD and reported as the total C1-oxidized ends generated (nanomoles per mg of starting fiber). Each bar is the average of three independent assays measured singly by HPAEC-PAD, with error bars indicating the standard error of the mean. Figure S2: Brightfield (A) and confocal (B) images of untreated SA-Avicel labelled using rhodamine chloride. 1% SA-Avicel with 1 mM of gallic acid was incubated at 50 °C for 24 h. Insoluble products were separated, labelled with fluorescent dye, and visualized using confocal microscopy. Figure S3: Process schematic for LPMO treatment of cellulosic substrates and subsequent soluble and insoluble product analysis. PASC (0.1%), Avicel (1%), and SA-Avicel (1%) were treated with LPMOs using either ascorbic acid, gallic acid, or cysteine as the electron donor. Insoluble products were separated from soluble products using centrifugation. For the soluble products analysis, HPAEC-PAD was used to annotate native and oxidized cello-oligosaccharide peaks. To quantify the C1-oxidized products, T. reesei cellulase cocktail was used to convert all C1-oxidized products into cellobionic acid, which was then quantified by HPAEC-PAD and reported as the total C1-oxidized ends generated (nanomoles per mg of starting fiber). For insoluble product analysis, separated insoluble products were labelled with either C1-specific or C4-specific fluorescent dye and subsequently visualized using confocal microscopy