Immobilization and stabilization of a bimolecular aggregate of the lipase from Pseudomonas fluorescens by multipoint covalent attachment

The soluble lipase from Pseudomonas fluorescens (PFL) forms bimolecular aggregates in which the hydrophobic active centers of the enzyme monomers are in close contact. This bimolecular aggregate could be immobilized by multipoint covalent linkages on glyoxyl supports at pH 8.5. The monomer of PFL obtained by incubation of the soluble enzyme in the presence of detergent (0.5% TRITON X-100) could not be immobilized under these conditions. The bimolecular aggregate has two amino terminal residues in the same plane. A further incubation of the immobilized derivative under more alkaline conditions (e.g., pH 10.5) allows a further multipoint attachment of lysine (Lys) residues located in the same plane as the amino terminal residues. Monomeric PFL was immobilized at pH 10.5 in the presence of 0.5% TRITON X-100. The properties of both PFL derivatives were compared. In general, the bimolecular derivatives were more active, more selective and more stable both in water and in organic solvents than the monomolecular ones. The bimolecular derivative showed twice the activity and a much higher selectivity (100 versus 20) for the hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) in aqueous media at pH 5.0 compared to the monomeric derivative. In experiments measuring thermal inactivation at 75 °C, the bimolecular derivative was 5-fold more stable than the monomeric derivative (and 50-fold more stable than a one-point covalently immobilized PFL derivative), and it had a half-life greater than 4 h. In organic solvents (cyclohexane and tert-amyl alcohol), the bimolecular derivative was much more stable and more active than the monomeric derivative in catalyzing the transesterification of olive oil with benzyl alcohol.     

Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from Trametes versicolor

The hydrolysis of phenolic compounds using an immobilized and highly active and stable derivative of laccase from Trametes versicolor is presented. The enzyme was immobilized on aldehyde supports. For this, the enzyme was enriched in amino groups by chemical modification of its carboxyl groups. The aminated enzyme was immobilized with a high recovered activity (over 60%). Aldehyde derivatives were more stable than soluble or aminated-soluble enzyme and the reference derivatives after incubation in different inactivating conditions (high temperatures, different pH values or presence of organic cosolvents). The most stable derivative was obtained immobilizing the chemically aminated enzyme at pH 10 on aldehyde supports with a stabilization factor approximately 280 fold after incubation at pH 7 and 55 °C. In addition, it was possible to prepare immobilized derivatives with a maximal enzyme loading of 60 mg g^?1 of support. This derivative could be reused for 10 reaction cycles with negligible lost of activity.     

Beyond and within public engagement: a broadened approach to engagement in biobanking

Social studies on biobanking have traditionally focused on public engagement, that is, engagement with donors, patients and the general public as an important factor of sustainability. In this article, we claim that, in order to fully understand the way biobanks work, it is necessary to pay attention to a number of other actors, which have an equal, if not greater, impact on their practices and strategies. This means taking a broadened approach to biobank engagement. By using data collected from interviews with different biobank experts based in five different countries (UK, Canada, Finland, Spain and Iceland), we identify seven communities, including the public, that emerge as relevant. Such relationships condition the way biobanks develop, act and plan. The discussion illustrates how the relationships with those seven communities are articulated. We conclude that there is a need for a broadened approach to biobank engagement in order to understand biobank sustainability.     

Analyses of Extracellular Carbohydrates in Oomycetes Unveil the Existence of Three Different Cell Wall Types

Some of the most devastating plant and animal pathogens belong to the oomycete class. The cell walls of these microorganisms represent an excellent target for disease control, but their carbohydrate composition is elusive. We have undertaken a detailed cell wall analysis in 10 species from 2 major oomycete orders, the Peronosporales and the Saprolegniales, thereby unveiling the existence of 3 clearly different cell wall types: type I is devoid of N-acetylglucosamine (GlcNAc) but contains glucuronic acid and mannose; type II contains up to 5% GlcNAc and residues indicative of cross-links between cellulose and 1,3-β-glucans; type III is characterized by the highest GlcNAc content (>5%) and the occurrence of unusual carbohydrates that consist of 1,6-linked GlcNAc residues. These 3 cell wall types are also distinguishable by their cellulose content and the fine structure of their 1,3-β-glucans. We propose a cell wall paradigm for oomycetes that can serve as a basis for the establishment of cell wall architectural models and the further identification of cell wall subtypes. This paradigm is complementary to morphological and molecular criteria for taxonomic grouping and provides useful information for unraveling poorly understood cell wall carbohydrate biosynthetic pathways through the identification and characterization of the corresponding enzymes.     

Modulation of the Endocannabinoids N-Arachidonoylethanolamine (AEA) and 2-Arachidonoylglycerol (2-AG) on Executive Functions in Humans

Animal studies point to an implication of the endocannabinoid system on executive functions. In humans, several studies have suggested an association between acute or chronic use of exogenous cannabinoids (Δ9-tetrahydrocannabinol) and executive impairments. However, to date, no published reports establish the relationship between endocannabinoids, as biomarkers of the cannabinoid neurotransmission system, and executive functioning in humans. The aim of the present study was to explore the association between circulating levels of plasma endocannabinoids N-arachidonoylethanolamine (AEA) and 2-Arachidonoylglycerol (2-AG) and executive functions (decision making, response inhibition and cognitive flexibility) in healthy subjects. One hundred and fifty seven subjects were included and assessed with the Wisconsin Card Sorting Test; Stroop Color and Word Test; and Iowa Gambling Task. All participants were female, aged between 18 and 60 years and spoke Spanish as their first language. Results showed a negative correlation between 2-AG and cognitive flexibility performance (r = −.37; p<.05). A positive correlation was found between AEA concentrations and both cognitive flexibility (r = .59; p<.05) and decision making performance (r = .23; P<.05). There was no significant correlation between either 2-AG (r = −.17) or AEA (r = −.08) concentrations and inhibition response. These results show, in humans, a relevant modulation of the endocannabinoid system on prefrontal-dependent cognitive functioning. The present study might have significant implications for the underlying executive alterations described in some psychiatric disorders currently associated with endocannabinoids deregulation (namely drug abuse/dependence, depression, obesity and eating disorders). Understanding the neurobiology of their dysexecutive profile might certainly contribute to the development of new treatments and pharmacological approaches.     

Involvement of miRNAs in the Differentiation of Human Glioblastoma Multiforme Stem-Like Cells

Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.     

Profiles of Serum Cytokines in Acute Drug-Induced Liver Injury and Their Prognostic Significance

Drug-induced liver injury (DILI) is the most common cause of acute liver failure in the United-States. The aim of the study was to describe serum immune profiles associated with acute DILI, to investigate whether there are profiles associated with clinical features or types of DILI and/or with prognosis, and to assess temporal changes in levels. Twenty-seven immune analytes were measured in the sera of 78 DILI subjects in the Drug-Induced Liver Injury Network (DILIN) and compared with 40 healthy controls. Immune analytes (14 cytokines, 7 chemokines and 6 growth factors) were measured by BioPlex multiplex ELISA at DILI onset and after 6 months. A modeling process utilizing immune principles was used to select a final set of variables among 27 immune analytes and several additional clinical lab values for prediction of early death (within 6 months of DILI onset). Nineteen of the 27 immune analytes were differentially expressed among healthy control, DILI onset and 6-month cohorts. Disparate patterns of immune responses, especially innate and adaptive cellular (mostly TH17) immunity were evident. Low values of four immune analytes (IL-9, IL-17, PDGF-bb and RANTES) and serum albumin are predictive of early death [PPV = 88% (95% CI, 65%-100%), NPV = 97% (95% CI, 93%-100%), accuracy = 96% (95% CI, 92%-100%)].

Conclusions

Acute DILI is associated with robust and varying immune responses. High levels of expression of cytokines associated with innate immunity are associated with a poor prognosis, whereas high levels of expression of adaptive cytokines are associated with good long-term prognosis and eventual recovery. Serum immune analyte profiles at DILI onset appear to be of prognostic, and perhaps, diagnostic significance.