Differential genetic regulation of motor activity and anxiety-related behaviors in mice using an automated home cage task.

Traditional behavioral tests, such as the open field test, measure an animal's responsiveness to a novel environment. However, it is generally difficult to assess whether the behavioral response obtained from these tests relates to the expression level of motor activity and/or to avoidance of anxiogenic areas. Here, an automated home cage environment for mice was designed to obtain independent measures of motor activity levels and of sheltered feeding preference during three consecutive days. Chronic treatment with the anxiolytic drug chlordiazepoxide (5 and 10 mg/kg/day) in C57BL/6J mice reduced sheltered feeding preference without altering motor activity levels. Furthermore, two distinct chromosome substitution strains, derived from C57BL/6J (host strain) and A/J (donor strain) inbred strains, expressed either increased sheltering preference in females (chromosome 15) or reduced motor activity levels in females and males (chromosome 1) when compared to C57BL/6J. Longitudinal behavioral monitoring revealed that these phenotypic differences maintained after adaptation to the home cage. Thus, by using new automated behavioral phenotyping approaches, behavior can be dissociated into distinct behavioral domains (e.g., anxiety-related and motor activity domains) with different underlying genetic origin and pharmacological responsiveness. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Probing Nanoscale Coassembly with Dual Mechanochromic Sensors

Attractive electrostatic forces between polymers can be exploited to create well‐defined and responsive nanoscale structures. In the process of charge‐driven coassembly, the polymers involved undergo subtle conformational changes. However, ascertaining these conformational transitions, and relating this to the nanostructures that are formed, has remained elusive to date. Here it is shown how the force‐optical response of tailored mechanochromic polymers can be used to detect structural transitions that occur at the nanoscale during assembly. It is shown that at low‐charge stoichiometry, electrostatic binding causes individual macromolecules to stretch and stiffen. Remarkably, at stoichiometries close to full charge compensation a gradual transition from single molecular complexes to multimolecular micelles is observed. Moreover, the same macromolecular sensors reveal how the assembly pathways are fully reversible as the binding strength is weakened. These results highlight how mechanochromic polymer sensors can be used to detect the molecular transitions occur during supramolecular structure formation with high precision.     

Thiolated chitosan nanoparticles: transfection study in the Caco-2 differentiated cell culture

The aim of this study was to monitor the expression of secreted protein in differentiated Caco-2 cells after transfection with nanoparticles, in order to improve gene delivery. Based on unmodified chitosan and thiolated chitosan conjugates, nanoparticles with the gene reporter pSEAP (recombinant Secreted Alkaline Phosphatase) were generated at pH 4.0. Transfection studies of thiolated chitosan in Caco-2 cells during the exponential growth phase and differentiation growth phase of the cells led to a 5.0-fold and 2.0-fold increase in protein expression when compared to unmodified chitosan nanoparticles. The mean particle size for both unmodified chitosan and cross-linked thiolated chitosan nanoparticles is 212.2 ± 86 and 113.6 ± 40?nm, respectively. The zeta potential of nanoparticles was determined to be 7.9 ± 0.38?mV for unmodified chitosan nanoparticles and 4.3 ± 0.74?mV for cross-linked thiolated chitosan nanoparticles. Red blood cell lysis evaluation was used to evaluate the membrane damaging properties of unmodified and thiolated chitosan nanoparticles and led to 4.61 ± 0.36% and 2.29 ± 0.25% lysis, respectively. Additionally, cross-linked thiolated chitosan nanoparticles were found to exhibit higher stability toward degradation in gastric juices. Furthermore the reversible effect of thiolated chitosan on barrier properties was monitored by measuring the transepithelial electrical resistance (TEER) and is supported by immunohistochemical staining for the tight junction protein claudin. According to these results cross-linked thiolated chitosan nanoparticles have the potential to be used as a non-viral vector system for gene therapy.     

Diachronic Analysis of Fuzzy Objects

This paper presents a method to monitor the behavior of fuzzy spatial objects through time. The method consists of two steps. Firstly the spatial extents of objects are determined at several sequential epochs. The method explains the case where objects are not crisply defined, so that the identified spatial extents will be fuzzy. Secondly, to detect dynamic changes in fuzzy objects, a method was proposed to identify objects and their state transitions from fuzzy spatial extents (regions) at different epochs. Similarity indicators of fuzzy regions were calculated based upon overlap between regions at consecutive epochs. Different combinations of indicator values imply different relationships between regions. Regions that are very similar represent the consecutive states of one object. By linking the regions, the historic lifelines of objects are built automatically. Then the relationship between regions became the relationship or interactions between objects, which were expressed in terms of processes, such as shift, merge or split. This approach will be illustrated by means of a coastal monitoring study of a barrier island in The Netherlands.     

Overproduction of bovine {beta}-casein in Escherichia coli and engineering of its main chymosin cleavage site

A cDNA clone containing the entire coding region for bovineß-casein A³ flanked by 53 base pairs of 5' non-codingand 358 base pairs of 3' non-coding sequences was isolated froma bovine mammary cDNA phagemid library. The coding segment formature ß-casein was subcloned into the T7 expressionsystem, in which the expression of recombinant ß-caseinwas controlled by the T7 gene 10 promoter and ribosome bindingsite. High level expression of Met-ß-casein to 20%of the total soluble proteins was obtained in Escherichia coliwithin 2 h after induction of T7 RNA-polymerase synthesis. Inan attempt to induce secretion the coding segment for matureß-casein was coupled to the ompA translations initiationsignal and signal peptide coding sequence but no secretion ofthe fusion protein and no processing of the signal peptide fromthe fusion protein was observed. Instead, the Met-ß-caseincould be isolated in asoluble form from E.coli cells after anosmotic shock, indicative of a periplasmic location. This proceduredid not lyse the cells. The protein was purified to homogeneityafter a pH 4.8 isoelectric precipitation followed by reversed-phasehigh-performance liquid chromatography. The ß-caseincDNA was altered to change the main chymosin cleavage siteinß-casein at position 192–193 in two ways, namelyfrom Leu–Tyr to Pro–Pro and to Leu–stop. Thesemutations were designed to prevent generation of the bitterpeptide ßcasein(193–209) by chymosin cleavage.The mutant Met-ß-caseins were expressed in E.colito the same level as wild-type Met-ß-casein. Purifiedmutant Met-ß-casein(Prol92– Prol93) was no longerhydrolysed by chymosin at the 192–193 bond.     

A Fast Quantitative Multi-analyte Method for Growth Promoters in Bovine Meat Using Bead-Disruption, 96-well SPE Clean-up and Narrow-Bore UHPLC-MS/MS Analysis

A new method for detecting low levels of growth promoters in bovine meat was developed with the following goal: easy, fast and sensitive analysis of a wide range of compounds, with reduced consumption of chemicals and disposables. Several classes of growth promoters were included, i.e. resorcylic acid lactones (RALs) and steroids, the latter including corticosteroids and gestagens. For sample treatment, 0.5 g of homogenised bovine meat was simultaneously disrupted and extracted in a bead-ruptor machine. The organic extraction solvent was further processed by solid-phase extraction (SPE) clean-up using 96-Well Oasis® HLB Plates. Six SPE washing steps were applied to remove matrix compounds after which the growth promoters were eluted and analysed using UHPLC-MS/MS. To achieve lower detection levels and to reduce LC-solvent consumption, a narrow-bore column with an internal diameter of 1 mm was used, instead of the conventional 2.1 mm. During analysis, the mass spectrometer was operated in negative and positive ionisation mode (ion switching). The newly developed method was validated according to the Commission Decision 2002/657. The results demonstrate that the method meets the criteria as established in this Commission Decision. The precision of the method for exogenous steroids varies between 85 and 115%, the CCα for the compounds ranges from 0.1–0.9 μg kg^?1 and the expanded measurement uncertainty was lower than 36%. Compared to our current in-house methods with analysis times of 2 days for a maximum of 24 samples, the new method offers improved sample throughput (96 samples in less than 24 h) and lower detection limits.     

LC-MS/MS multi-method for mycotoxins after single extraction, with validation data for peanut, pistachio, wheat, maize, cornflakes, raisins and figs

Mycotoxin analysis is usually carried out by high performance liquid chromatography after immunoaffinity column cleanup or in enzyme-linked immunosorbent assay tests. These methods normally involve determination of single compounds only. EU legislation already exists for the aflatoxins, ochratoxin A and patulin in food, and legislation will come into force for deoxynivalenol, zearalenone and the fumonisins in 2007. To enforce the various legal limits, it would be preferable to determine all mycotoxins by routine analysis in different types of matrices in one single extract. This would also be advantageous for HACCP control purposes. For this reason, a multi-method was developed with which 33 mycotoxins in various products could be analysed simultaneously. The mycotoxins were extracted with an acetonitrile/water mixture, diluted with water and then directly injected into a LC-MS/MS system. The mycotoxins were separated by reversed-phase HPLC and detected using an electrospray ionisation interface (ESI) and tandem MS, using MRM in the positive ion mode, to increase specificity for quality control. The following mycotoxins could be analysed in a single 30-min run: Aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin, alpha-zearalenol, alpha-zearalanol, beta-zearalanol, sterigmatocystin, cyclopiazonic acid, penicillic acid, fumonisins B1, B2 and B3, diacetoxyscirpenol, 3- and 15-acetyl-deoxynivalenol, zearalanone, ergotamin, ergocornin, ergocristin, alpha-ergocryptin, citrinin, roquefortin C, fusarenone X, nivalenol, mycophenolic acid, alternariol and alternariol monomethyl ether. The limit of quantification for the aflatoxins and ochratoxin A was 1.0 microg kg(-1) and for deoxynivalenol 50 microg kg(-1). The quantification limits for the other mycotoxins were in the range 10-200 microg kg(-1). The matrix effect and validation data are presented for between 13 and 24 mycotoxins in peanuts, pistachios, wheat, maize, cornflakes, raisins and figs. The method has been compared with the official EU method for the determination of aflatoxins in food and relevant FAPAS rounds. The multi-mycotoxin method has been proven by the detection of more than one mycotoxin in maize, buckwheat, figs and nuts. The LC-MS/MS technique has also been applied to baby food, which is subject to lower limits for aflatoxin B1 and ochratoxin A, ergot alkaloids in naturally contaminated rye and freeze-dried silage samples.     

Building pattern recognition in topographic data: examples on collinear and curvilinear alignments

Building patterns are important features that should be preserved in the map generalization process. However, the patterns are not explicitly accessible to automated systems. This paper proposes a framework and several algorithms that automatically recognize building patterns from topographic data, with a focus on collinear and curvilinear alignments. For both patterns two algorithms are developed, which are able to recognize alignment-of-center and alignment-of-side patterns. The presented approach integrates aspects of computational geometry, graph-theoretic concepts and theories of visual perception. Although the individual algorithms for collinear and curvilinear patterns show great potential for each type of the patterns, the recognized patterns are neither complete nor of enough good quality. We therefore advocate the use of a multi-algorithm paradigm, where a mechanism is proposed to combine results from different algorithms to improve the recognition quality. The potential of our method is demonstrated by an application of the framework to several real topographic datasets. The quality of the recognition results are validated in an expert survey.     

Antimicrobial lysozyme-containing starch microgel to target and inhibit amylase-producing microorganisms

The aim of this study is to determine the release of lysozyme from oxidized starch microgels and subsequently test its antimicrobial activity. The gels are made of oxidized potato starch polymers, which are chemically cross-linked by sodium trimetaphosphate (STMP). The microgel is negatively charged and interacts with positively charged lysozyme by electrostatic attraction. Application of the lysozyme-containing starch particles to environments contaminated with microbes, may lead to hydrolysis of the starch by microbial enzymes. As a result, lysozyme is released in the environment where it inhibits microbial growth. In this study, first bacteria were screened for amylase production and lysozyme sensitivity. Then, the bacteria were mixed with empty gel particles (i.e., without lysozyme) in a Nutrient Broth liquid medium to test whether the bacteria that can produce amylase are also able to degrade oxidized starch gel. Subsequently the amylase-producing lysozyme sensitive bacteria, Bacillus licheniformis 7558 and Bacillus subtilis 168, were selected for further quantification of the antimicrobial activity of the gel-lysozyme particles after incubation with these bacteria in Nutrient Broth liquid suspensions. The results prove that the starch microgel has a potential as antimicrobial carrier targeting amylase-producing and lysozyme-sensitive bacteria. The controlled antimicrobial delivery for inactivating undesired microorganisms may find applications in food related systems, where amylase-producing bacteria may be abundantly present.