Design and Characterization of a New pVII Combinatorial Phage Display Peptide Library for Protease Substrate Mining Using Factor VII Activating Protease (FSAP) as Model

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.     

Validation of a Method for Quantitation of Soybean Lectin in Commercial Varieties

Soybean agglutinin (SBA) protein, also known as soybean lectin, is regarded as an anti‐nutrient due to its negative effect on the ability of monogastric animals to gain weight following consumption of raw soybean seed. Historically, SBA has been measured using a time‐consuming and cumbersome hemagglutination procedure. The objective of our research was to obtain a validated methodology that is precise and accurate in the measurement of SBA while allowing minimally equipped laboratories to effectively conduct the analysis, thus our focus was on evaluating an existing commercially available ELISA, an enzyme‐linked‐lectin‐assay (ELLA), and a hybrid ELISA/ELLA. A new ELLA technique that can detect and quantify lectins was chosen and modified specifically for the quantitation of SBA in soybean seed. The proposed ELLA methodology is similar to a standard sandwich ELISA, and uses polyacrylamide‐linked N‐acetylgalactosamine (Gal–NAc–PAA) for a capture phase and the biotinylated version (Gal–NAc–PAA–Biotin) for detection. Based upon the validation data, the ELLA method can precisely and accurately determine soybean lectin levels in soybean seed. The validated ELLA method was used to quantify SBA in nine commercial soybean varieties introduced between 1972 and 2008 and demonstrated that the natural variability of SBA is subject to the effects of genotype and environment.     

Energy oriented simulation of manufacturing systems – Concept and application

Energy efficiency has become a key concern in industry due to the increased energy costs and the associated environmental impacts. Besides considering single processes/machines, the perspective on process chains and factories as a whole bears further potentials for improvement. In this context, dynamic interactions of different processes as well as auxiliary equipments (e.g. compressed air generation) need to be taken into account when planning and controlling manufacturing systems. This paper presents an innovative energy oriented simulation model for planning of manufacturing systems. A number of industry cases are used to demonstrate the potentials and applicability of the proposed methodology.     

Motility in the Hunt-Lawrence pouch after total gastrectomy

PURPOSE: The aim of this study was to evaluate motility patterns of the Hunt-Lawrence pouch and the jejunal limb of patients reconstructed with a pouch after total gastrectomy, and to compare the findings in symptomatic patients to those without symptoms after the operation. PATIENTS AND METHODS: Thirty-three patients who had undergone post-gastrectomy pouch reconstruction were studied using a water-perfused motility system. In 21, the pouch was connected by a Roux-en-Y, and, in 12, by a jejunal interposition. Twenty-eight patients were asymptomatic, including 17 connected by a Roux-en-Y and 11 by a jejunal interposition. Five patients were by a jejunal interposition. Five patients were symptomatic, including 4 connected by Roux-en-Y Y and 1 by jejunal interposition. A control group consisted of 5 healthy volunteers who had not undergone operation. RESULTS: The motility phases in the pouch and jejunal limb of asymptomatic patients were of shorter duration than those of controls, and they followed a random sequence instead of a normal progression from phase I to II to III. Motility features were similar in the pouch and the jejunal limb. Orthograde propagation of phase III-like activity was reduced and may contribute to the pouch storage function. Four of the 5 symptomatic patients showed highly abnormal motility with hypomotile or obstructive patterns. The technique of connecting the pouch--jejunal interposition of Roux-en-Y--did not affect the motility findings. CONCLUSIONS: The altered motility occurs after a Hunt-Lawrence pouch reconstruction in asymptomatic patients. Symptoms after gastrectomy are associated with further disturbed motility that can be differentiated from the motility changes in asymptomatic patients.     

35-GHz static and 48-GHz dynamic frequency divider ICs using0.2-μm AlGaAs/GaAs-HEMTs

Two static and two dynamic frequency dividers based on enhancement and depletion 0.2-μm gate length AlGaAs/GaAs-high electron mobility transistor (HEMT) (f_T=60 and 55 GHz) technology were designed and fabricated. High-speed operations up to 35 GHz for the static frequency dividers and 48 GHz for the dynamic dividers, respectively, have been achieved. The single-ended input and differential outputs to ground simplify many applications. The power consumption is 250 mW for the divide-by-two dividers and 350 mW for the divide-by-four dividers using two supply voltages of 4 and -2.5 V     

0.5-25 GHz inductorless single-ended resistive mixer in 0.13 μm CMOS

A wideband inductorless resistive down-conversion mixer in 0.13 μm CMOS technology is presented. The mixer provides a conversion loss of 9?11.7 dB over a frequency range of 0.5?25 GHz at LO power of 6 dBm. The circuit exhibits an input-referred 1 dB compression point and IIP3 of 4.7 and 11.5 dBm, respectively. The mixer consumes only 0.2 mA from 1.5 V for biasing. The isolation between the ports is higher than 10 dB for the whole frequency range. The circuit is realised without inductors, thus offering very wide bandwidth. The chip size including the pads is 0.23 mm², and the circuit active area is only 0.014 mm².     

FSAP Protects against Histone-Mediated Increase in Endothelial Permeability In Vitro

Factor-VII-activating protease (FSAP) is involved in the regulation of hemostasis and inflammation. Extracellular histones play a role in inflammation and the conversion of latent pro-FSAP into active FSAP. FSAP has been shown to regulate endothelial permeability, but the mechanisms are not clear. Here, we have investigated the effects of FSAP on endothelial permeability in vitro. A mixture of histones from calf thymus stimulated permeability, and the wild-type (WT) serine protease domain (SPD) of FSAP blocked this effect. WT–SPD–FSAP did not influence permeability on its own, nor that stimulated by thrombin or vascular endothelial growth factor (VEGF)-A₁₆₅. Histones induced a large-scale rearrangement of the junction proteins VE-cadherin and zona occludens-1 from a clear junctional distribution to a diffuse pattern. The presence of WT–SPD–FSAP inhibited these changes. Permeability changes by histones were blocked by both TLR-2 and TLR4 blocking antibodies. Histones upregulated the expression of TLR-2, but not TLR-4, in HUVEC cells, and WT–SPD–FSAP abolished the upregulation of TLR-2 expression. An inactive variant, Marburg I (MI)–SPD–FSAP, did not have any of these effects. The inhibition of histone-mediated permeability may be an important function of FSAP with relevance to sepsis, trauma, and stroke and the need to be investigated further in in vivo experiments.