Engineering highly thermostable xylanase variants using an enhanced combinatorial library method

A new directed evolution method was used to enhance the thermostability of the wild-type GH11 xylanase 2 (known as BD-11) from Hypocrea jecorina (Trichoderma reesei). Both Look-Through Mutagenesis (LTM?), which is a method for rapidly screening selected positions in the protein sequence for amino acids that introduce favorable properties, and Combinatorial Beneficial Mutagenesis (CBM?), which is a method for identifying the best ensemble of individual mutations, were employed to enhance the stability of an enzyme that has been thoroughly engineered by various means during the past 20 years. A diverse set of novel mutations was discovered, including N71D, Y73G, T95G and Y96Q. When these mutations were combined into a single construct (Hjx-81), the purified protein was active even after heating at 100°C for 20 min. This time-effective method should be generally applicable for quickly improving the physico-chemical properties of other industrial and therapeutic enzymes in only several months time.     

Classification of Intact Cereal Flours by Front-Face Synchronous Fluorescence Spectroscopy

Synchronous fluorescence spectroscopy, a technique that measures both the absorption and the emission properties of a sample in a single measurement, was used for the analysis and classification of intact cereal flours (wheat, corn, rye, buckwheat, rice, and barley). Total synchronous fluorescence spectra recorded in constant wavelength mode show clear differences in the emission spectra of different flours due to variances in intrinsic fluorophore concentrations and their microenvironments. Principal component analysis, cluster analysis, and partial least squares discriminant analysis are used to assess the ability of synchronous fluorescence measurements to differentiate and classify intact samples of different flour types. The flour specimens were obtained directly from a market in Belgrade and had different expiration dates to provide a more representative set of samples. The results of the current analysis suggest that chemometric methods applied on synchronous fluorescence data can discriminate and classify flour types and that the best results are achieved using a combination of synchronous fluorescence measurements at synchronous intervals of 7 and 20 nm. The quality of results, the high speed of measurements, and the avoidance of extensive sample preparation make synchronous fluorescence spectroscopy a promising technique for cereal research.     

Antioxidant, antimicrobial and anticancer activities of three Parmelia species

BACKGROUND: Lichens are symbiotic organisms consisting of algae and fungi. They are used for human and animal nutrition and in the production of colours, perfumes and alcohol. Lichens have also been used in traditional medicine to treat diseases such as jaundice, pulmonary, stomach and cranial diseases. In this study the acetone extracts of three lichens, Parmelia caperata, Parmelia sulcata and Parmelia saxatilis, were tested for their antioxidant, antimicrobial and anticancer potential. RESULTS: Of the lichens tested, P. saxatilis had the highest free radical‐scavenging activity (55.3% inhibition). Moreover, all tested extracts showed effective reducing power and superoxide anion radical scavenging. Strong relationships between total phenolic and flavonoid contents and antioxidant effects of the tested extracts were observed. The extract of P. sulcata was most active in terms of antimicrobial ability, with minimum inhibitory concentration values ranging from 0.78 to 12.5 mg L^?1. All extracts were found to have strong anticancer activity, with IC₅₀ values ranging from 9.55 to 22.95 µg mL^?1. CONCLUSION: The present study showed that the tested lichen extracts exhibited strong antioxidant, antimicrobial and anticancer effects. This suggests that lichens may be used as possible natural antioxidant, antimicrobial and anticancer agents. Copyright © 2012 Society of Chemical Industry     

Spark plasma sintering of ZrC–SiC ceramics with LiYO2 additive

Spark plasma sintering (SPS) of ZrC–SiC composite powders in the presence of LiYO₂ sintering additive was studied. The starting powders were obtained by a carbothermal reduction (CTR) of natural mineral zircon (ZrSiO₄), which provided an intimate mixing of in-situ created ZrC and SiC powders. This composite powder and LiYO₂ as additive were densified by spark plasma sintering. Microstructural features of the composite were investigated by XRD, SEM/EDS and AFM analysis. The sintered composite material possesses promising mechanical properties and excellent cavitation resistance which was observed with a cavitation erosion test. The values of Vickers microhardness and fracture toughness of the composite material are 20.7 GPa and 5.07 MPam^1/2, respectively.     

Labeling TiO2 Nanoparticles with Dyes for Optical Fluorescence Microscopy and Determination of TiO2–DNA Nanoconjugate Stability

Visualization of nanoparticles without intrinsic optical fluorescence properties is a significant problem when performing intracellular studies. Such is the case with titanium dioxide (TiO₂) nanoparticles. These nanoparticles, when electronically linked to single‐stranded DNA oligonucleotides, have been proposed to be used both as gene knockout devices and as possible tumor imaging agents. By interacting with complementary target sequences in living cells, these photoinducible TiO₂–DNA nanoconjugates have the potential to cleave intracellular genomic DNA in a sequence specific and inducible manner. The nanoconjugates also become detectable by magnetic resonance imaging with the addition of gadolinium Gd(III) contrast agents. Herein two approaches for labeling TiO₂ nanoparticles and TiO₂–DNA nanoconjugates with optically fluorescent agents are described. This permits direct quantification of fluorescently labeled TiO₂ nanoparticle uptake in a large population of living cells (>10⁴ cells). X‐ray fluorescence microscopy (XFM) is combined with fluorescent microscopy to determine the relative intracellular stability of the nanoconjugates and used to quantify intracellular nanoparticles. Imaging the DNA component of the TiO₂–DNA nanoconjugate by fluorescent confocal microscopy within the same cell shows an overlap with the titanium signal as mapped by XFM. This strongly implies the intracellular integrity of the TiO₂–DNA nanoconjugates in malignant cells.     

Effect of thermal oxidative ageing on the morphology of natural rubber networks as viewed by ESR

The change of network morphology of unfilled sulfur crosslinked natural rubber exposed to thermal oxidative ageing has been studied. Three model networks with different sulfur contents (efficient network, semi‐efficient and conventional vulcanizate) have been analysed by applying spin probe ESR, equilibrium swelling measurements and mechanical measurements. By combining the density distribution deduced from the complex ESR spectra and the equilibrium swelling density, it is shown that the major network changes occur in the networks with predominantly polysulfidic bonds (conventional network) after 96 h of ageing at 373 K. The effect of ageing on the network with predominantly monosulfidic bonds is much less pronounced. Both effects (main chain scission and additional local crosslinking) have been analysed in the course of prolonged ageing up to 240 h. A correlation between network structure and its mechanical performance is established. © 2000 Society of Chemical Industry     

欧洲单一天空空中交通管理研究——欧洲空中交通管理的过去、现在与未来

欧洲单一天空空中交通管理研究(SESAR)项目是欧盟委员会欧洲单一天空倡议的技术支柱,旨在实现空中交通管理(ATM)的现代化.在本文中,我们介绍了SESAR的建立过程和该项目的主要部分:研发部分(由SESAR联合执行)、部署部分(由SESAR部署管理方管理),以及欧洲空中交通管理总体规划(汇集并说明了各种研发和部署需求...     

DNA Demethylation Induces Tree Peony Flowering with a Low Deformity Rate Compared to Gibberellin by Inducing PsFT Expression under Forcing Culture Conditions

Gibberellin (GA) is frequently used in tree peony forcing culture, but inappropriate application often causes flower deformity. Here, 5-azacytidine (5-azaC), an efficient DNA demethylating reagent, induced tree peony flowering with a low deformity rate by rapidly inducing PsFT expression, whereas GA treatment affected various flowering pathway genes with strong pleiotropy. The 5-azaC treatment, but not GA, significantly reduced the methylation level in the PsFT promoter with the demethylation of five CG contexts in a 369 bp CG-rich region, and eight light-responsive related cis-elements were also predicted in this region, accompanied by enhanced leaf photosynthetic efficiency. Through GO analysis, all methylation-closer differentially expressed genes (DEGs) were located in the thylakoid, the main site for photosynthesis, and were mainly involved in response to stimulus and single-organism process, whereas GA-closer DEGs had a wider distribution inside and outside of cells, associated with 12 categories of processes and regulations. We further mapped five candidate DEGs with potential flowering regulation, including three kinases (SnRK1, WAK2, and 5PTase7) and two bioactive enzymes (cytochrome P450 and SBH1). In summary, 5-azaC and GA may have individual roles in inducing tree peony flowering, and 5-azaC could be a preferable regulation approach; DNA demethylation is suggested to be more focused on flowering regulation with PsFT playing a core role through promoter demethylation. In addition, 5-azaC may partially undertake or replace the light-signal function, combined with other factors, such as SnRK1, in regulating flowering. This work provides new ideas for improving tree peony forcing culture technology.     

Norepinephrine Inhibits Lipopolysaccharide-Stimulated TNF-α but Not Oxylipin Induction in n-3/n-6 PUFA-Enriched Cultures of Circumventricular Organs

Sensory circumventricular organs (sCVOs) are pivotal brain structures involved in immune-to-brain communication with a leaky blood–brain barrier that detect circulating mediators such as lipopolysaccharide (LPS). Here, we aimed to investigate the potential of sCVOs to produce n-3 and n-6 oxylipins after LPS-stimulation. Moreover, we investigated if norepinephrine (NE) co-treatment can alter cytokine- and oxylipin-release. Thus, we stimulated rat primary neuroglial sCVO cultures under n-3- or n-6-enriched conditions with LPS or saline combined with NE or vehicle. Supernatants were assessed for cytokines by bioassays and oxylipins by HPLC-MS/MS. Expression of signaling pathways and enzymes were analyzed by RT-PCR. Tumor necrosis factor (TNF)α bioactivity and signaling, IL-10 expression, and cyclooxygenase (COX)2 were increased, epoxide hydroxylase (Ephx)2 was reduced, and lipoxygenase 15-(LOX) was not changed by LPS stimulation. Moreover, LPS induced increased levels of several n-6-derived oxylipins, including the COX-2 metabolite 15d-prostaglandin-J2 or the Ephx2 metabolite 14,15-DHET. For n-3-derived oxylipins, some were down- and some were upregulated, including 15-LOX-derived neuroprotectin D1 and 18-HEPE, known for their anti-inflammatory potential. While the LPS-induced increase in TNFα levels was significantly reduced by NE, oxylipins were not significantly altered by NE or changes in TNFα levels. In conclusion, LPS-induced oxylipins may play an important functional role in sCVOs for immune-to-brain communication.