Cereal β-glucan quantification with calcofluor-application to cell culture supernatants

The specific binding of the fluorescent dye calcofluor to cereal β-glucan results in increased fluorescence intensity of the formed complex and is in use for the quantification of β-glucan above a critical molecular weight (MW) by flow injection analysis. In this study, this method was applied in a fast and easy batch mode. In order to emphasize the spectral information of the emission spectra of the calcofluor/β-glucan complexes, derivative signals were calculated. A linear relationship was found between the amplitude of the second derivative signals and the β-glucan concentration between 0.1 and 0.4μg/mL. The low detection limit of this new method (0.045μg/mL) enabled its use to study the transport of cereal β-glucans over differentiated Caco-2 cell monolayers. Additionally, the method was applied to quantify β-glucan in arabinoxylan samples, which correlated well with data by an enzyme based method.     

An acidic phospholipase A₂ with antibacterial activity from Porthidium nasutum snake venom

Snake venoms are complex mixtures of proteins among which both basic and acidic phospholipases A₂ (PLA₂s) can be found. Basic PLA₂s are usually responsible for major toxic effects induced by snake venoms, while acidic PLA₂s tend to have a lower toxicity. A novel PLA₂, here named PnPLA₂, was purified from the venom of Porthidium nasutum by means of RP-HPLC on a C18 column. PnPLA₂ is an acidic protein with a pI of 4.6, which migrates as a single band under both non-reducing and reducing conditions in SDS-PAGE. PnPLA₂ had a molecular mass of 15,802.6 Da, determined by ESI-MS. Three tryptic peptides of this protein were characterized by HPLC-nESI-MS/MS, and N-terminal sequencing by direct Edman degradation showing homology to other acidic PLA₂s from viperid venoms. PnPLA₂ displayed indirect hemolytic activity in agarose erythrocyte-egg yolk gels and bactericidal activity against Staphylococcus aureus in a dose-dependent manner, with a MIC and MBC of 32 μg/mL. In addition, PnPLA₂ showed a potent inhibitory effect on platelet aggregation with doses up to 40 μg/mL. This acidic PLA₂, in contrast to basic enzymes isolated from other viperid snake venoms, was not cytotoxic to murine skeletal muscle myoblasts C₂C₁₂. This is the first report on a bactericidal protein of Porthidium nasutum venom.     

Snake venomics and antivenomics of Bothrops atrox venoms from Colombia and the Amazon regions of Brazil,Perú and Ecuador suggest the occurrence of geographic variation of venom phenotype by a trend towards paedomorphism

The venom proteomes of Bothrops atrox from Colombia, Brazil, Ecuador, and Perú were characterized using venomic and antivenomic strategies. Our results evidence the existence of two geographically differentiated venom phenotypes. The venom from Colombia comprises at least 26 different proteins belonging to 9 different groups of toxins. PI-metalloproteinases and K49-PLA₂ molecules represent the most abundant toxins. On the other hand, the venoms from Brazilian, Ecuadorian, and Peruvian B. atrox contain predominantly PIII-metalloproteinases. These toxin profiles correlate with the venom phenotypes of adult and juvenile B. asper from Costa Rica, respectively, suggesting that paedomorphism represented a selective trend during the trans-Amazonian southward expansion of B. atrox through the Andean Corridor. The high degree of crossreactivity of a Costa Rican polyvalent (Bothrops asper, Lachesis stenophrys, Crotalus simus) antivenom against B. atrox venoms further evidenced the close evolutionary kinship between B. asper and B. atrox. This antivenom was more efficient immunodepleting proteins from the venoms of B. atrox from Brazil, Ecuador, and Perú than from Colombia. Such behaviour may be rationalized taking into account the lower content of poorly immunogenic toxins, such as PLA₂ molecules and PI-SVMPs in the paedomorphic venoms. The immunological profile of the Costa Rican antivenom strongly suggests the possibility of using this antivenom for the management of snakebites by B. atrox in Colombia and the Amazon regions of Ecuador, Perú and Brazil.     

Health migration from Norway to Spain – ambiguous belonging

Abstract

In line with Gustafson (2008), most studies of transnationalism have investigated migration from south to north. This article examines health-, welfare- and lifestyle-related migration from Norway to Spain. The phenomenon is increasing, with consequences for national and municipal policies. The focus is on how Norwegian health migrants cope. Based on anthropological fieldwork and life story interviews we ask: Who are these migrants? How did the migration step come into being? How do they cope when the decision is effectuated? What kind of identity-related challenges do they face? The article also discusses implications for future comparative research on the phenomenon.     

Enhanced biodegradation of transformer oil in soils with cyclodextrin – from the laboratory to the field

The use cyclodextrins for the intensification of bioremediation by improving the mobility and bioavailability of contaminants has recently been studied. In this work, the role of randomly methylated -cyclodextrin in the bioremediation of soils contaminated with transformer oil was studied both in bench scale bioreactors and through field experiments. The aims of this research were to (a) establish the scientific background of a cyclodextrin-based soil bioremediation technology, (b) demonstrate its feasibility and effectiveness in the field, and (c) develop an integrated methodology, consisting of a combination of physical, chemical, biological and ecotoxicological analytical methods, for efficiently monitoring the technology performances. The stepwise increasing scale of the experiments and the application of the integrated analytical methodology supported the development of a scientifically established new technology and the identification of the advantages and the limitations of its application in the field. At each phase of the study, randomly methylated -cyclodextrin was found to significantly enhance the bioremediation and detoxification of the transformer oil-contaminated soils employed by increasing the bioavailability of the pollutants and the activity of indigenous microorganisms.     

The metabolic response to excitotoxicity – lessons from single-cell imaging

Journal of Bioenergetics and Biomembranes - Excitotoxicity is a pathological process implicated in neuronal death during ischaemia, traumatic brain injuries and neurodegenerative diseases....     

Spider silks from plants – a challenge to create native-sized spidroins

Silk threads from spiders exhibit extraordinary mechanical properties, such as superior toughness and elasticity. Spider silks consist of several different large repetitive proteins that act as the basic materials responsible for these outstanding features. The production of spider silk protein variants in plants opens up new horizons in the production and functional investigation that enable the use of spider silks in innovative material development, nanotechnology and biomedicine in the future. This review summarizes and discusses production of spider silk protein variants in plants, especially with regards to plant expression systems, purification strategies, and characteristics of spider silk variants. Furthermore, the challenge of producing native-sized recombinant spidroins in planta is outlined, presenting three different strategies for achieving these high repetitive proteins with the help of non-repetitive C-terminal domains, crosslinking transglutaminase, and self-linking inteins. The potential of these fascinating proteins in medicine is also highlighted.     

Toward Système International d'Unité-traceable protein quantification: from amino acids to proteins

Here we present a demonstration of the proof of principle that absolute concentration of a protein within a mixture of other proteins can be measured with SI traceability. The method used was based on tryptic digestion of a protein followed by quantification using double exact matching isotope dilution mass spectrometry (IDMS) of the peptides released. To provide full SI traceability to measurements of protein concentration we demonstrated a method of SI traceable peptide quantification in which the peptide standards used were quantified by an amino acid analysis method that incorporated double exact matching IDMS and amino acid standards of known purity. The concentration of the protein was therefore determined based upon the concentration of tryptic peptides, which in turn had been quantified based upon amino acid standards. This allowed fully SI-traceable measurements of protein concentration to be made. Important caveats in the implementation of this approach are also discussed and examples of how these can have detrimental effects on the measurements are shown.     

Neuronal Avalanches Differ from Wakefulness to Deep Sleep – Evidence from Intracranial Depth Recordings in Humans

Neuronal activity differs between wakefulness and sleep states. In contrast, an attractor state, called self-organized critical (SOC), was proposed to govern brain dynamics because it allows for optimal information coding. But is the human brain SOC for each vigilance state despite the variations in neuronal dynamics? We characterized neuronal avalanches – spatiotemporal waves of enhanced activity - from dense intracranial depth recordings in humans. We showed that avalanche distributions closely follow a power law – the hallmark feature of SOC - for each vigilance state. However, avalanches clearly differ with vigilance states: slow wave sleep (SWS) shows large avalanches, wakefulness intermediate, and rapid eye movement (REM) sleep small ones. Our SOC model, together with the data, suggested first that the differences are mediated by global but tiny changes in synaptic strength, and second, that the changes with vigilance states reflect small deviations from criticality to the subcritical regime, implying that the human brain does not operate at criticality proper but close to SOC. Independent of criticality, the analysis confirms that SWS shows increased correlations between cortical areas, and reveals that REM sleep shows more fragmented cortical dynamics.     

Druggable protein–protein interactions – from hot spots to hot segments

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A novel fibrinolytic metalloproteinase,barnettlysin-I from Bothrops barnetti (barnett´s pitviper) snake venom with anti-platelet properties

BackgroundViperid snake venoms contain active components that interfere with hemostasis. We report a new P-I class snake venom metalloproteinase (SVMP), barnettlysin-I (Bar-I), isolated from the venom of Bothrops barnetti and evaluated its fibrinolytic and antithrombotic potential.MethodsBar-I was purified using a combination of molecular exclusion and cation-exchange chromatographies. We describe some biochemical features of Bar-I associated with its effects on hemostasis and platelet function.ResultsBar-I is a 23.386 kDa single-chain polypeptide with pI of 6.7. Its sequence (202 residues) shows high homology to other members of the SVMPs. The enzymatic activity on dimethylcasein (DMC) is inhibited by metalloproteinase inhibitors e.g. EDTA, and by α2-macroglobulin. Bar-I degrades fibrin and fibrinogen dose- and time-dependently by cleaving their α-chains. Furthermore, it hydrolyses plasma fibronectin but not laminin nor collagen type I. In vitro Bar-I dissolves fibrin clots made either from purified fibrinogen or from whole blood. In contrast to many other P-I SVMPs, Bar-I is devoid of hemorrhagic activity. Also, Bar-I dose- and time-dependently inhibits aggregation of washed human platelets induced by vWF plus ristocetin and collagen (IC₅₀ = 1.3 and 3.2 μM, respectively), presumably Bar-I cleaves both vWF and GPIb. Thus, it effectively inhibits vWF-induced platelet aggregation. Moreover, this proteinase cleaves the collagen-binding α2-A domain (160 kDa) of α2β1-integrin. This explains why it additionally inhibits collagen-induced platelet activation.ConclusionA non-hemorrhagic but fibrinolytic metalloproteinase dissolves fibrin clots in vitro and impairs platelet function.General significanceThis study provides new opportunities for drug development of a fibrinolytic agent with antithrombotic effect.     

Bioremediation of PAH-contaminated soil with fungi – From laboratory to field scale

The purpose of this study was to develop a fungal bioremediation method that could be used for soils heavily contaminated with persistent organic compounds, such as polyaromatic hydrocarbons (PAHs). Sawmill soil, contaminated with PAHs, was mixed with composted green waste (1:1) and incubated with or without fungal inoculum. The treatments were performed at the laboratory and field scales. In the laboratory scale treatment (starting concentration 3500 mg kg⁻¹, sum of 16 PAH) the high molecular weight PAHs were degraded significantly more in the fungal-inoculated microcosms than in the uninoculated ones. In the microcosms inoculated with Phanerochaete velutina, 96% of 4-ring PAHs and 39% of 5- and 6-ring PAHs were removed in three months. In the uninoculated microcosms, 55% of 4-ring PAHs and only 7% of 5- and 6-ring PAHs were degraded. However, during the field scale (2 t) experiment at lower starting concentration (1400 mg kg⁻¹, sum of 16 PAH) the % degradation was similar in both the P. velutina-inoculated and the uninoculated treatments: 94% of the 16 PAHs were degraded in three months. In the field scale experiment the copy number of gram-positive bacteria PAH-ring hydroxylating dioxygenase genes was found to increase 1000 fold, indicating that bacterial PAH degradation also played an important role.