Reed canary grass yield and fuel quality in Estonian farmers’ fields

Reed canary grass (Phalaris arundinacea) is one of the possible raw materials for bioenergy production in northern Europe. Its cultivation is favoured because its high productivity and local origin. However, problems with the biomass quality for combustion have been reported. Usually delayed harvest in spring is suggested to improve the quality and decrease the moisture content of biomass. On the other hand, the feasibility of spring harvest depends on local climatic conditions and may cause yield losses. In current paper we studied reed canary grass fields in Estonia locating on different soil types and cultivated with various varieties. The influence of several fertilisation schemes on biomass yield was analysed. Our results indicated that production was higher on mineral soils than on the organic soil of abandoned peat extraction sites. Even different types of fertilisation did not increase the production on organic soils to the level comparable to those on mineral soils. Among studied varieties ‘Venture’ had the highest production. The highest yield per area was obtained late in the autumn (12.7 t d.w. per ha and 7.2 t d.w. per ha on mineral and organic soils, respectively). By spring the amount of biomass had decreased in all studied sites. Due to wet soil some of the fields remained unharvested, the others had high yield losses during practical harvesting. The chemical analyses did not reveal significant differences in the composition of biomass between late autumn and spring. Therefore we conclude that late autumn harvest should be preferred in local climatic conditions.     

Highly adaptable and sensitive protease assay based on fluorescence resonance energy transfer

Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.     

Wasserbindungskapazit?t und Makrostruktur von Apfelgewebepartikeln

Zusammenfassung Haufwerke aus trockenen Apfelgewebepartikeln mit mittleren Partikelvolumina zwischen 0,3 und 5 mm³ können bis zu 80 g/g Wasser aufnehmen. Der überwiegende Teil dieses Wassers ist in makrocapillaren Haufwerks- und Partikelhohlräumen eingelagert und nur durch sehr geringe Kräfte gebunden. Bereits bei Einwirkung eines Gasdrucks von 2 kPa werden die Partikelhaufwerke stark entwässert. Die Wasserbeladung sinkt auf 20-15 g/g. Die Rehydratation einzelner getrockneter Apfelgewebepartikel ist vom Ausmaß der bei der Zerkleinerung eingetretenen Gewebezerstörung abhängig und kann durch eine der Trocknung vorgelagerte Entwässerung mit Ethanol verbessert werden. Vorentwässerte Partikel mit einem hohen Ethanolgehalt schrumpfen bei der Trocknung im geringeren Ausmaß und besitzen eine hohe Porosität. Solche Partikel binden im rehydratisierten Zustand bis zu 30 g/g Wasser. Nicht mit Ethanol behandelte Partikel binden unter 11 g/g Wasser.

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Water binding capacity and macrostructure of apple tissue particles

Samples (bulk materials) of dry apple tissue particles in a range of particle volume from 0.3 to 5 mm³ can absorb up to 80 g/g water. Most of this water is included in macrocapillary bulk and particle spaces and is bound by very small forces. An external gas pressure of 2 kPa decreases the water content to 20-15 g/g. The rehydration properties of single apple tissue particles depend on the degree of tissue disintegration and can be improved by a dehydration treatment with ethanol before drying. Such pre-dehydrated particles with a high ethanol concentration in the liquid phase show little shrinkage during the drying process and have a high porosity. They retain up to 30 g/g water after rehydration. The water uptake of non-ethanol-treated particles is less than 11 g/g.

     

Three generations of cyclosporine a formulations: an in vitro comparison

When the microemulsion formulation of the critical dose drug cyclosporine A (CsA) (Sandimmun Optoral) was introduced in the mid-1990s, it became clear that this new formulation improves the oral bioavailability of CsA and has a positive influence on its pharmacokinetic variability. Previous studies with the original CsA formulation (Sandimmun) showed that the size of the emulsion droplets and concomitant food intake has an effect on the absorption of CsA from the small intestine when orally administered. It was suggested that these effects might have an influence on the drugs' pharmacokinetic parameters.In this study, we focused on the two above-mentioned aspects and compared the first and second generations of CsA products (Sandimmun, Sandimmun Optoral) to generic CsA formulations by analyzing the contents of cyclosporine A gel capsules with respect to their emulsion droplet and micelle sizes using photon correlation spectroscopy (PCS). We tried to discern any differences in droplet size between different generations of CsA formulations, primarily the second and third generation, through simple physical tests. Because a high fat content food may influence the absorption of CsA, we also determined the distribution of CsA between hydrophilic and lipophilic phases using high-performance liquid chromatography analysis.It became clear that when compared under simple physical conditions, established cyclosporine formulations and new generic products show significant differences in droplet size and distribution between an aqueous phase and a high fat content food. Whether these differences are of clinical relevance remains to be investigated.     

Heat and mass transfer during the coffee drying process

For a better comprehension of heat and mass transfer during the coffee drying process and optimization of the industrial application transport coefficients and coffee properties were determined. Heat transfer coefficients were measured for different air velocities and were found to follow the known dimensionless equations for the flow surrounding a sphere. Thermal conductivities and effective diffusion coefficients were measured as a function of moisture content as well as volume and densities of the coffee beans. The mentioned properties depend directly on the humidity of the coffee beans rather than on the drying conditions. Sorption behaviour was investigated and temperature dependent parameters for the Guggenheimer–Anderson–deBoer-isotherm (GAB) were determined according to the Arrhenius relationship.     

An Engineered Mannoside Presenting Platform: Escherichia coli Adhesion under Static and Dynamic Conditions

The study of the adhesion mechanisms of pathogens to host tissues has gained increased interest as bacterial adhesion is involved in the early stages of surface colonization and infection. Here we describe a platform to study the specific binding of the bacterium Escherichia coli (E. coli) K‐12 strain to molecularly well‐defined surfaces mimicking cellular interfaces. This approach uses a poly(ethylene glycol) brush interface, which displays synthetic determinants of the high mannose N‐linked glycans in a range of densities (3.8 × 10⁴–1.6 × 10⁵ mannosides µm^?2) for the investigation of multivalent interactions with bacteria. The bacterial attachment is mediated by specific interactions between the adhesive protein FimH located on the tip of the bacterial type 1 pili and the mannosylated surfaces. With synthetically engineered mannoses, it is found that the number of strongly adhering bacteria is co‐regulated by many structural physical parameters. Beyond the dependency on carbohydrate density, higher numbers of E. coli attach to the branched trimannose Man(α1–3)(Man(α1–6))Man compared to the monomannose, while larger oligomannoses exposing Man(α1–2) Man at their non reducing end show low binding capacity. The linker used between the mannose moiety and PEG is also affecting the binding efficacy of E. coli. The (hydrophobic) propyl linker results in higher bacteria numbers in comparison to the (hydrophilic) tri(EG), likely a consequence of additional stabilization of the binding complex by hydrophobic interactions. Furthermore, differences are observed in bacteria attachment between stagnant and flow conditions that depend on the type of mannose ligand. Finally, a photolithographic resist lift‐off combined with site‐selective assembly of the glycopolymers is used to produce micropatterns with bacteria colonies confined to defined areas and at controlled colony numbers.     

Validation of the performance of a GMO multiplex screening assay based on microarray detection

A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct hybridisation of the amplicons on a predefined microarray (DualChip^® GMO, Eppendorf, Germany). The validation was performed within the framework of a European project (Co-Extra, contract no 007158) and in collaboration with 12 laboratories specialised in GMO detection. The present study reports the strategy and the results of an ISO complying validation of the method carried out through an inter-laboratory study. Sets of blind samples were provided consisting of DNA reference materials covering all the elements detectable by specific probes present on the array. The GMO concentrations varied from 1% down to 0.045%. In addition, a mixture of two GMO events (0.1% RRS diluted in 100% TOPAS19/2) was incorporated in the study to test the robustness of the assay in extreme conditions. Data were processed according to ISO 5725 standard. The method was evaluated with predefined performance criteria with respect to the EC CRL method acceptance criteria. The overall method performance met the acceptance criteria; in particular, the results showed that the method is suitable for the detection of the different target elements at 0.1% concentration of GMO with a 95% accuracy rate. This collaborative trial showed that the method can be considered as fit for the purpose of screening with respect to its intra- and inter-laboratory accuracy. The results demonstrated the validity of combining multiplex PCR with array detection as provided by the DualChip^® GMO (Eppendorf, Germany) for the screening of GMO. The results showed that the technology is robust, practical and suitable as a screening tool.     

Plant-derived polyphenols attenuate lipopolysaccharide-induced nitric oxide and tumour necrosis factor production in murine microglia and macrophages

Lipopolysaccharides released during bacterial infections induce the expression of pro-inflammatory cytokines and lead to complications such as neuronal damage in the CNS and septic shock in the periphery. While the initial infection is treated by antibiotics, anti-inflammatory agents would be advantageous add-on medications. In order to identify such compounds, we have compared 29 commercially available polyphenol-containing plant extracts and pure compounds for their ability to prevent LPS-induced up-regulation of NO production. Among the botanical extracts, bearberry and grape seed were the most active preparations, exhibiting IC(50) values of around 20 mug/mL. Among the pure compounds, IC(50) values for apigenin, diosmetin and silybin were 15, 19 and 12 muM, in N-11 murine microglia, and 7, 16 and 25 muM, in RAW 264.7 murine macrophages, respectively. In addition, these flavonoids were also able to down-regulate LPS-induced tumour necrosis factor production. Structure-activity relationships of the flavonoids demonstrated three distinct principles: (i) flavonoid-aglycons are more potent than the corresponding glycosides, (ii) flavonoids with a 4'-OH substitution in the B-ring are more potent than those with a 3'-OH-4'-methoxy substitution, (iii) flavonoids of the flavone type (with a C2=C3 double bond) are more potent than those of the flavanone type (with a at C2-C3 single bond).     

Comparative studies of a real-time PCR method and three enzyme-linked immunosorbent assays for the detection of central nervous system tissues in meat products

The removal of certain central nervous system (CNS) tissues (part of the bovine spongiform encephalopathy risk material) from the food chain is one of the highest priority tasks associated with avoiding contamination of the human food chain with the agent of bovine spongiform encephalopathy. A recently developed real-time PCR assay and three commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of CNS tissues in minced meat and three types of heat-treated sausages were evaluated. Bovine brain was used for spiking of internal reference material, and its detectability was examined during storage times of 12 months (for frozen minced meat and liver sausage) and 24 months (for sausages treated with medium and high heat). The real-time PCR method and both ELISA kits detected 0.1% CNS tissue in frozen minced meat and 0.1 or 1% CNS tissue in heat-treated meat products. The detectability of the amplified mRNA target region with the PCR assay was similar to the detectability of antigen by the ELISAs. Because the real-time PCR method also can be used to distinguish cattle, ovine, and caprine CNS tissues from porcine CNS tissues, it seems to be suitable as a routine diagnostic test for the sensitive and specific detection of CNS tissues in meat and meat products.     

Deepening our understanding of bioactive glass crystallization using TEM and 3D nano-CT

One key issue influencing a broader application of Bioglass 45S5 in tissue engineering is its inherent crystallization tendency, severely limiting the mechanical strength of 3D porous scaffolds. Despite numerous studies, Bioglass 45S5 crystallization is not yet fully understood with regard to the mechanisms involved or morphology of the crystal phases forming. Here we show how two cutting-edge imaging techniques, state-of-the-art transmission electron microscopy (TEM) with image correction including energy dispersive X-ray spectroscopy and X-ray nano-computed tomography (nano-CT), allowed us to visualize changes in microstructure from near-nucleation to almost full crystallization in bulk Bioglass 45S5. At early times of heat treatment at 660 °C the formation of phase-separated nano-droplets within the glassy matrix was observed. Later, besides surface crystallization, bulk crystallization of combeite spheres was predominant. The formation of the first combeite spheres, their coarsening with time and finally their merging at near full crystallization were recorded by in situ high-temperature optical microscopy videos. The 3D nature of these spheres was confirmed by nano-CT, while TEM showed that their internal structure was composed of sub-micron grains. X-ray diffraction analysis at early time points showed a much higher crystalline fraction in bulk samples compared to powder samples, highlighting the influence of processing and sample morphology. These results show the importance of using complementary techniques for gaining insight into the crystallization process in the volume. In addition, we show that TEM and nano-CT are suitable characterization techniques to visualize the crystallization even in fast crystallizing systems, such as bioactive glasses.