A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.     

Biosynthesis of silver nanoparticles in collagen gel improves their medical use in periodontitis treatment

Silver nanoparticles (AgNP) suspensions were biosynthesized by silver ions reduction in the presence of collagen, a nontoxic, organic polymer, intending to improve their medical use in periodontitis treatment. Spectrophotometric measurements showed a time- and concentration-dependent increase of AgNP formation in each suspension variant. Transmission electron microscopy revealed spherical morphology of AgNP in collagen and their mean diameter size was around 30?nm. The particle size distribution and zeta potential values of AgNP in collagen were determined by dynamic light scattering measurements. The surface charge of AgNP in collagen was positive, while commercial AgNP stabilized in citrate had negative surface charge. In vitro cytotoxicity testing of AgNP in collagen showed that they were biocompatible with human gingival fibroblasts in a wider range of concentrations than commercial nanoparticles. The antibacterial activity of AgNP in collagen against two pathogenic strains present in the periodontal pocket was dose-dependent and higher than that of AgNP in citrate. All these results demonstrated that AgNP prepared in collagen gel had improved properties, like small diameter, positive surface charge, high biocompatibility in human gingival fibroblasts, efficiency against bacterial growth and, thus, better therapeutic potential in periodontal disease treatment.     

Combination of freeze drying and molecular inclusion techniques improves the bioaccessibility of microencapsulated anthocyanins from black rice (Oryza sativa L.) and lavender (Lavandula angustifolia L.) essential oils in a model food system

Black rice and lavender are promising sources of bioactives, in terms of anthocyanins and essential oils. Their bioaccessibility were improved by microencapsulation, followed by mixing in order to benefit both from colour and flavour, along with radical scavenging and biological properties. The mixed powder showed a satisfactory anthocyanins of 2.55 mg g⁻¹ DW, leading to a radical scavenging activity of 65.14 mmol g⁻¹ DW. The powder displayed an inhibitory effect against α-glucosidase (~49%) and α-amylase (39%), respectively, with a controlled release in intestinal environment. To further examine the functional properties, the powder was added to a food model system. During storage, a release in anthocyanins and flavonoids were found, leading to an increase in radical scavenging activity. The sensorial analysis showed that supplemented biscuits were appreciated for colour and lavender aroma. The obtained results were promising in tailoring the health benefits of secondary metabolites, underutilised in human’s nutrition due to their low stability and bioavailability.     

Fluoroscopy-based navigation system in spine surgery

The variability in width, height, and spatial orientation of a spinal pedicle makes pedicle screw insertion a delicate operation. The aim of the current paper is to describe a computer-assisted surgical navigation system based on fluoroscopic X-ray image calibration and three-dimensional optical localizers in order to reduce radiation exposure while increasing accuracy and reliability of the surgical procedure for pedicle screw insertion. Instrumentation using transpedicular screw fixation was performed: in a first group, a conventional surgical procedure was carried out with 26 patients (138 screws); in a second group, a navigated surgical procedure (virtual fluoroscopy) was performed with 26 patients (140 screws). Evaluation of screw placement in every case was done by using plain X-rays and post-operative computer tomography scan. A 5 per cent cortex penetration (7 of 140 pedicle screws) occurred for the computer-assisted group. A 13 per cent penetration (18 of 138 pedicle screws) occurred for the non computer-assisted group. The radiation running time for each vertebra level (two screws) reached 3.5 s on average in the computer-assisted group and 11.5 s on average in the non computer-assisted group. The operative time for two screws on the same vertebra level reaches 10 min on average in the non computer-assisted group and 11.9 min on average in the computer-assisted group. The fluoroscopy-based (two-dimensional) navigation system for pedicle screw insertion is a safe and reliable procedure for surgery in the lower thoracic and lumbar spine.     

On analyzing user preference dynamics with temporal social networks

The preferences adopted by individuals are constantly modified as these are driven by new experiences, natural life evolution and, mainly, influence from friends. Studying these temporal dynamics of user preferences has become increasingly important for personalization tasks in information retrieval and recommendation systems domains. However, existing models are too constrained for capturing the complexity of the underlying phenomenon. Online social networks contain rich information about social interactions and relations. Thus, these become an essential source of knowledge for the understanding of user preferences evolution. In this work, we investigate the interplay between user preferences and social networks over time. First, we propose a temporal preference model able to detect preference change events of a given user. Following this, we use temporal networks concepts to analyze the evolution of social relationships and propose strategies to detect changes in the network structure based on node centrality. Finally, we look for a correlation between preference change events and node centrality change events over Twitter and Jam social music datasets. Our findings show that there is a strong correlation between both change events, specially when modeling social interactions by means of a temporal network.     

SULFUR CAPTURE DURING PARTIAL COAL COMBUSTION

A coal-water slurry is an econonical substitute for premium liquid fuels, but the amount of sulfur in coal is usually higher than in petroleum liquids. Lime has been used to capture sulfur in fluidized coal combustion as CaSO₄, but the latter decomposes above 1273 K. We report here on the possible capture of sulfur in the ash as CaS, which has higher thermal stability than CaSO₄. This can be accomplished using a fuel-rich first stage in a coal-water slurry combustor, or under the conditions normally encountered in some coal gasifiers. Thermodynamic calculations and some preliminary experiments suggest that the concept is promising for effective sulfur removal and, by extension, for NO_x, control.     

Complex Interactions among Sheep,Insects, Grass,and Fungi in a Simple New Zealand Grazing System

Epichloë fungi (Ascomycota) live within aboveground tissues of grasses and can have important implications for natural and managed ecosystems through production of alkaloids. Nonetheless, vertebrate herbivores may possess traits, like oral secretions, that mitigate effects of alkaloids. We tested if sheep saliva mitigates effects of Epichloë alkaloids on a beetle pest of perennial ryegrass (Lolium perenne L.) in a New Zealand pasture setting. Plants with one of several fungal isolates were clipped with scissors, grazed by sheep, or clipped with sheep saliva applied to cut ends of stems. We then assessed feeding damage by Argentine stem weevils on blade segments collected from experimental plants. We found that clipping plants induced synthesis of an alkaloid that reduces feeding by beetles and that sheep saliva mitigates this effect. Unexpectedly, the alkaloid (perloline) that explains variation in beetle feeding is one produced not by the endophyte, but rather by the plant. Yet, these effects depended upon fungal isolate. Such indirect, complex interactions may be much more common in both managed and natural grassland systems than typically thought and could have implications for managing grazing systems.     

Structural,morphological and magnetic characterization of metal-chitosan/poly (vinyl amine) complexes

Cu²⁺ and Ni²⁺ complexes of chitosan/poly(vinyl amine) (CS/PVAm) composites were prepared. The metal-CS/PVAm complexes were characterized by FT-IR spectroscopy, SEM-EDX, X-ray diffraction (XRD), and magnetic moment determination. FT-IR spectra of the metal-CS/PVAm complexes showed the characteristic bands of anhidroglucose unit were affected by the metal complexation, and new bands assigned to Me–N and Me-O bonds were observed. SEM images of the surface of metal-composite complexes show the influence of metal ion on the morphology of the complexes, the strong binding of Cu²⁺ and Ni²⁺, involving most of the amino groups, leading to a dense surface structure. The chemical composition on the surface of metal-CS/PVAm complexes was determined from EDX measurements. XRD provided information about the amorphous or crystalline nature of the composite and metal-composite complexes. Using magnetic susceptibility method, the oxidation degree of metal ions from the polymer phase, the homogeneous distribution of the ligand groups from the volume of the CS/PVAm beads, and the existence of the antiferromagnetic interactions between the metal ions were determined.     

Hybrid networks based on epoxidized camelina oil

Lately, renewable resources received great attention in the macromolecular compounds area, regarding the design of the monomers and polymers with different applications. In this study the capacity of several modified vegetable oil-based monomers to build competitive hybrid networks was investigate, taking into account thermal and mechanical behavior of the designed materials. In order to synthesize such competitive nanocomposites, the selected renewable raw material, camelina oil, was employed due to the non-toxicity and biodegradability behavior. General properties of epoxidized camelina oil-based materials were improved by loading of different types of organic-inorganic hybrid compounds – polyhedral oligomeric silsesquioxane (POSS) bearing one (POSS1Ep) or eight (POSS8Ep) epoxy rings on the cages. In order to identify the chemical changes occurring after the thermal curing reactions, FT-IR spectrometry was employed. The new synthesized nanocomposites based on epoxidized camelina oil (ECO) were characterized by dynamic mechanical analyze and thermogravimetric analyze. The morphology of the ECO-based materials was investigate by scanning electron microscopy and supplementary information regarding the presence of the POSS compounds were establish by energy dispersive X-ray analysis and X-ray photoelectron spectroscopy. The smooth materials without any separation phase indicates a well dispersion of the Si–O–Si cages within the organic matrix and the incorporation of this hybrid compounds into the ECO network demonstrates to be a well strategy to improve the thermal and mechanical properties, simultaneously.     

Controlled fabrication of nanopores using a direct focused ion beam approach with back face particle detection

We report a direct, ion drilling technique that enables the reproducible fabrication and placement of nanopores in membranes of different thickness. Using a 30?keV focused Ga ion beam column combined with an in situ, back face, multi-channelplate particle detector, nanopores are sputtered in Si(3)N(4) and W/Si(3)N(4) to have diameters as small as 12?nm. Transmission electron microscopy shows that focused ion beam-drilled holes are near-conical with the diameter decreasing from entry to exit side. By monitoring the detector signal during ion exposure, the drilled hole width can be minimized such that the exit-side diameter is smaller than the full width at half-maximum of the nominally Gaussian-shaped incident beam. Judicious choice of the beam defining aperture combined with back face particle detection allows for reproducible exit-side hole diameters between 18 and 100?nm. The nanopore direct drilling technique does not require potentially damaging broad area exposure to tailor hole sizes. Moreover, this technique successfully achieves breakthrough despite the effects of varying membrane thickness, redeposition, polycrystalline grain structure, and slight ion beam current fluctuations.