Polarimetric assay for the medium-throughput determination of alpha-amino acid racemase activity

A polarimetric assay has been developed for the identification of alpha-amino acid racemase activity. The setup consists of a microcuvette polarimeter (40 microL volume) connected to a pipetting robot for microtiter plates, a pump, and data processing. It could be demonstrated for a glutamate racemase from Lactobacillus fermentii, expressed in Escherichia coli, serving as model enzyme, that its activity can be determined from the time-dependent change of the optical rotation using l-glutamate as substrate. Thus, the specific activity was determined to 111.4 mdeg/min which corresponds to 45.7 micromol/min per mg purified enzyme. Moreover, a protocol was developed that allows the measurement of racemase activity from 96-well microtiter plates using purified enzymes. Thus, the method described can be used to determine racemase activity in an automatic manner. It should be also applicable for the screening of enzyme libraries created by directed evolution.     

Size distribution and waiting times for the avalanches of the Cell Network Model of Fracture

The Cell Network Model is a fracture model recently introduced that resembles the microscopical structure and drying process of the parenchymatous tissue of the Bamboo Guadua angustifolia. The model exhibits a power-law distribution of avalanche sizes, with exponent −3.0 when the breaking thresholds are randomly distributed with uniform probability density. Hereby we show that the same exponent also holds when the breaking thresholds obey a broad set of Weibull distributions, and that the humidity decrements between successive avalanches (the equivalent to waiting times for this model) follow in all cases an exponential distribution. Moreover, the fraction of remaining junctures shows an exponential decay in time. In addition, introducing partial breakings and cumulative damages induces a crossover behavior between two power-laws in the histogram of avalanche sizes. This results support the idea that the Cell Network Model may be in the same universality class as the Random Fuse Model.     

Characterization of the Gene Cluster CYP264B1‐geoA from Sorangium cellulosum So ce56: Biosynthesis of (+)‐Eremophilene and Its Hydroxylation

Terpenoids can be found in almost all forms of life; however, the biosynthesis of bacterial terpenoids has not been intensively studied. This study reports the identification and functional characterization of the gene cluster CYP264B1–geoA from Sorangium cellulosum So ce56. Expression of the enzymes and synthesis of their products for NMR analysis and X‐ray diffraction were carried out by employing an Escherichia coli whole‐cell conversion system that provides the geoA substrate farnesyl pyrophosphate through simultaneous overexpression of the mevalonate pathway genes. The geoA product was identified as a novel sesquiterpene, and assigned NMR signals unambiguously proved that geoA is an (+)‐eremophilene synthase. The very tight binding of (+)‐eremophilene (～0.40 μM ), which is also available in S. cellulosum So ce56, and its oxidation by CYP264B1 suggest that the CYP264B1–geoA gene cluster is required for the biosynthesis of (+)‐eremophilene derivatives.     

Phytins?ure in Getreide und Getreideerzeugnissen

Zusammenfassung Phytinsäure in Lebensmitteln wird für eine verringerte Bioverfügbarkeit von essentiellen Mineralstoffen verantwortlich gemacht; sie kann während der Verarbeitung durch Phytase teilweise abgebaut werden. Der durchschnittliche Phytinsäuregehalt betrug in Roggen 8,18 mg/g und im Mehl der Type 997 3,44 mg/g; im Ganzkornmaterial wurden durchschnittlich Phytaseaktivitäten von 3,7 U/g und im Mehl 2,6. U/g gefunden. In den beiden Kornhälften (quergeteilt) waren Phytat und Phytase etwa gleichmäßig verteilt. Während der dreitägigen Keimung blieb die Phytaseaktivität konstant, der Phytinsäuregehalt nahm um ein Drittel ab. Bei der Vermahlung und dem Schälen von Roggen wurden die größten Mengen an Phytinsäure und Phytase mit Schrot- und Grießkleien bzw. Schälfraktionen entfernt. Es wird daraus geschlossen, daß Substrat und Enzym in den gleichen morphologischen Strukturen, wenn auch intrazellulär getrennt, vorkommen. Küchentechnische Zubereitungsformen von Vollkornprodukten waren für eine Phytatreduzierung um so wirkungsvoller, 1.) je feiner das Getreide vermahlen war, 2.) je mehr Wasser zugegeben wurde und 3.) je länger Phytase im optimalen Temperaturbereich einwirken konnte. Extrusion von Vollkornschrot bewirkte erst bei einer hohen Temperatur ( 170 °C) eine 23%ige Reduktion, während Phytase schon bei 80 °C um etwa 80% geschädigt wurde.

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Phytic acid in cerealsI. Phytic acid and Phytase in rye and rye products

Summary Phytic acid in food is considered to be responsible for a reduced bioavailability of essential dietary minerals; its detrimental effects can be diminished by hydrolysis with phytase during processing. The average phytic acid content was 8.18 mg/g and 3.44 mg/g and average phytase activity was 3.7 U/g and 2.6 U/g in rye kernels and in flour (Type 997, 1.09 ash content), respectively. Phytate and Phytase were about equally distributed between the two kernel halves (cross sections). During the early stages of germination (3 days) phytase activity did not change, and phytic acid content was reduced to 67%. After milling most of the phytic acid and phytase activity were found in the bran fractions. It is concluded that substrate and enzyme are present in the same kernel structures but separate within the cells. Cooking of ground rye caused a phytate hydrolysis which was the more effective 1.) the smaller the particle sizes were, 2.) the more water was added, and 3.) the longer phytase worked at optimum temperature. Extrusion cooking of the rye whole flour at up to 100 °C did not influence the phytic acid level but caused a 23% reduction at 170 °C. Phytase activity was reduced by 80% by extrusion cooking at 80 °C.

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Veröffentl.-Nr. 5342 der Bundesforschungsanstalt für Getreide-und Kartoffelverarbeitung, Detmold     

Copper-zirconium tungstate composites exhibiting low and negative thermal expansion influenced by reinforcement phase transformations

A fully-dense Cu-75 vol pct ZrW₂O₈ metal matrix composite was fabricated by hot isostatic pressing of Cu-coated ZrW₂O₈ particles. A small amount of the high-pressure γ-ZrW₂O₈ phase was created during the cooldown and depressurization following densification; near complete transformation to γ-ZrW₂O₈ was achieved by subsequent cold isostatic pressing. The thermal expansion behavior of the composite between 25 °C and 325 °C was altered by the cold isostatic pressing treatment, and also depended on the length of time that had passed between thermal cycles. The measured thermal expansion coefficients within specific temperature ranges varied from −6 · 10⁻⁶ K⁻¹ to far above the thermal expansion coefficient of the copper matrix. The complex temperature-dependent expansion/contraction behavior could be justified by considering the evolution of phase transformations taking place in the ZrW₂O₈ phase, which were observed by in-situ synchrotron X-ray diffraction measurements.     

Middleware for the use of storage in communication

The Logistical Computing and Internetworking (LoCI) project is a reflection of the way that the next generation internetworking fundamentally changes our definition of high performance wide area computing. A key to achieving this aim is the development of middleware that can provide reliable, flexible, scalable, and cost-effective delivery of data with quality of service guarantees to support high performance applications of all types. The LoCI effort attacks this problem with a simple but innovative strategy. At the base of the LoCI project is a richer view of the use of storage in communication and information sharing.     

Solar photoassisted advanced oxidation of synthetic phenolic wastewaters using ferrioxalate complexes

In this work, the performance of advanced oxidation solar assisted photochemical treatments of synthetic phenolic type wastewaters was studied at natural pH conditions, and with phenol concentrations in the range of 180-733 mg/L. The photochemical treatment was mediated with ferrioxalate and peroxide in two CPC (compound parabolic collector) photoreactors of different volumes and operation conditions (batch and with closed flow). Phenol transformation efficiencies of 100% and total COD reduction percentages of 85% were reached within the first hour of phototreatment, having as final product an aromatics free effluent, in both types of reactors. The ferrioxalate type complexes using mass ratios of oxalate/phenol = 1.5, oxalate/Fe³⁺ = 15 and H₂O₂/phenol > 5.0 showed to be very effective in the treatment of these effluents, even at pH conditions close to neutrality, pH region in which the Fenton type processes begin to lose efficiency due to the precipitation of iron as a hydroxide.     

Genotype imputation in a tropical crossbred dairy cattle population

The objective of this study was to investigate different strategies for genotype imputation in a population of crossbred Girolando (Gyr × Holstein) dairy cattle. The data set consisted of 478 Girolando, 583 Gyr, and 1,198 Holstein sires genotyped at high density with the Illumina BovineHD (Illumina, San Diego, CA) panel, which includes ～777K markers. The accuracy of imputation from low (20K) and medium densities (50K and 70K) to the HD panel density and from low to 50K density were investigated. Seven scenarios using different reference populations (RPop) considering Girolando, Gyr, and Holstein breeds separately or combinations of animals of these breeds were tested for imputing genotypes of 166 randomly chosen Girolando animals. The population genotype imputation were performed using FImpute. Imputation accuracy was measured as the correlation between observed and imputed genotypes (CORR) and also as the proportion of genotypes that were imputed correctly (CR). This is the first paper on imputation accuracy in a Girolando population. The sample-specific imputation accuracies ranged from 0.38 to 0.97 (CORR) and from 0.49 to 0.96 (CR) imputing from low and medium densities to HD, and 0.41 to 0.95 (CORR) and from 0.50 to 0.94 (CR) for imputation from 20K to 50K. The CORR_anim exceeded 0.96 (for 50K and 70K panels) when only Girolando animals were included in RPop (S1). We found smaller CORR_anim when Gyr (S2) was used instead of Holstein (S3) as RPop. The same behavior was observed between S4 (Gyr + Girolando) and S5 (Holstein + Girolando) because the target animals were more related to the Holstein population than to the Gyr population. The highest imputation accuracies were observed for scenarios including Girolando animals in the reference population, whereas using only Gyr animals resulted in low imputation accuracies, suggesting that the haplotypes segregating in the Girolando population had a greater effect on accuracy than the purebred haplotypes. All chromosomes had similar imputation accuracies (CORR_snp) within each scenario. Crossbred animals (Girolando) must be included in the reference population to provide the best imputation accuracies.     

Functional Screen for microRNAs Suppressing Anchorage-Independent Growth in Human Cervical Cancer Cells

The progression of anchorage-dependent epithelial cells to anchorage-independent growth represents a critical hallmark of malignant transformation. Using an in vitro model of human papillomavirus (HPV)-induced transformation, we previously showed that acquisition of anchorage-independent growth is associated with marked (epi)genetic changes, including altered expression of microRNAs. However, the laborious nature of the conventional growth method in soft agar to measure this phenotype hampers a high-throughput analysis. We developed alternative functional screening methods using 96- and 384-well ultra-low attachment plates to systematically investigate microRNAs regulating anchorage-independent growth. SiHa cervical cancer cells were transfected with a microRNA mimic library (n = 2019) and evaluated for cell viability. We identified 84 microRNAs that consistently suppressed growth in three independent experiments. Further validation in three cell lines and comparison of growth in adherent and ultra-low attachment plates yielded 40 microRNAs that specifically reduced anchorage-independent growth. In conclusion, ultra-low attachment plates are a promising alternative for soft-agar assays to study anchorage-independent growth and are suitable for high-throughput functional screening. Anchorage independence suppressing microRNAs identified through our screen were successfully validated in three cell lines. These microRNAs may provide specific biomarkers for detecting and treating HPV-induced precancerous lesions progressing to invasive cancer, the most critical stage during cervical cancer development.     

A traverse inspection system for high precision visual on-loom fabric defect detection

A self-contained inspection system for vision-based on-loom fabric defect detection is presented in this paper. Design and loom integration of a traversing camera sled, a camera vibration damper and a complementary back-light illumination are presented and discussed. Image acquisition strategies and traverse control are described to complete the discussion on hardware and mechanics. The main part of the paper focuses on a novel algorithmic framework for woven fabric defect detection in highly resolved (1,000+ ppi) image data. Within this scope, single yarns are tracked and measured in terms of position, size, and appearance in real time. An inspection prototype has been mounted onto an industrial loom. Extensive on-line and off-line evaluations for various fabric materials gave precise and stable detection results with few false alarms. A brief cost analysis for the prototype system is provided and completes the presentation of the system.