Qualitative assessment of an ultra-fast portable gas chromatograph (zNose™) for analyzing volatile organic chemicals and essential oils in laboratory and greenhouses

Qualitative performance of a portable gas chromatograph (zNose™) was assessed by comparing retention indices of major constituents of an essential oil-based insect repellent, and by comparing retention index of limonene, a major chemical in volatile blends of tomato plants, in the laboratory, a research greenhouse and a commercial greenhouse. Effects of temperature and relative humidity on the performance of the device were also assessed. In all experiments, the zNose™ produced consistent results comparable to that of a conventional GC–MS. Our results concur with previous studies confirming the zNose™ as a suitable device for analyzing plant volatiles in the field and for monitoring their rapid changes.     

Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering

The BioBrick™ paradigm for the assembly of enzymatic pathways is being adopted and becoming a standard practice in microbial engineering. We present a strategy to adapt the BioBrick™ paradigm to allow the quick assembly of multi-gene pathways into a number of vectors as well as for the quick mobilization of any cloned gene into vectors with different features for gene expression and protein purification. A primary BioBrick™ (BB-eGFP) was developed where the promoter/RBS, multiple cloning sites, optional protein purification affinity tags and reporter gene were all separated into discrete regions by additional restriction enzymes. This primary BB-eGFP then served as the template for additional BioBrick™ vectors with different origins of replication, antibiotic resistances, inducible promoters (arabinose, IPTG or anhydrotetracycline), N- or C-terminal Histidine tags with thrombin cleavage, a LacZα reporter gene and an additional origin of mobility (oriT). All developed BioBricks™ and BioBrick™ compatible vectors were shown to be functional by measuring reporter gene expression. Lastly, a C₃₀ carotenoid pathway was assembled as a model enzymatic pathway to demonstrate in vivo functionality and compatibility of this engineered vector system.     

High content selection of cells

Conventional methods for clonal analysis are mostly based on limiting dilution or FACS sorting. Purity of clones can only be verified retrospectively. This process is tedious and time-consuming. The Elektra™ improves and expedites the cell cloning process using IACS™ (Image Activated Cell Selection) for isolation of clonal cells providing high-content single cell information. A micro fluidic Sorter Chip within the Elektra™ uses CellProcessor technology, i. e. negative dielectrophoretic (nDEP) force to guide, cage and sort cells (about 500 to 1000 cells per run). The system is equipped with a highly sensitive CCD camera and a 40× lens. Cells are analyzed on-line and selected according to phase contrast, fluorescence and size while passing the micro device. Target cells are trapped and an image series can be acquired. Post process evaluation of the images enables quantitative analysis of e. g. subcellular fluorescence distribution and co-localization. Elektra™ produces micro titer plates containing single viable and 100% pure clones without the need for multiple iterations. This is demonstrated for different cell lines (e. g. CHO, U2-OS, hybridoma cells). The technology is shown to be compatible to label free detection techniques like impedance, electrorotation and dielectrophoretic cell deformation.     

A Tunable on-Chip Integrated Plasmonic Filter and Router Based on Metal/Dielectric Nanostructures

An on-chip integrated wavelength filter and router device is realized using two-dimensional metal/dielectric nanostructures. The device can filter wavelengths of light from an incident broadband beam, and further route the filtered signals to different ports on the same chip. The footprint of the entire device is only 3.4 μm × 7.3 μm. Both the number of wavelength channels and the central wavelength of each channel can be tuned by adjusting the structure parameters, or by using a pumped laser. This work demonstrates an ultracompact and robust integrated multifunctional device, and provides a novel and flexible method for the integration of nanophotonic devices.     

Comparison of the Rotorod to other air samplers for the determination of Ambrosia artemisiifolia pollen concentrations conducted in the Environmental Exposure Unit

The Environmental Exposure Unit (EEU) is a 924 m³ facility (Kingston General Hospital, Ontario) in which uniform concentrations of various pollens in HEPA-filtered air at known rates of laminar airflow can be maintained. This facility provided a unique opportunity to compare several air samplers without the environmental variation inherent in outdoor comparisons. The purpose of this study was to conduct a quantitative comparison of pollen measurements using the Rotorod, Burkard™ Personal Volumetric Air Sampler, Air-O-Cell™ and a 37 mm open-faced filter cassette with a microporous filter in the EEU. Pollen samples were taken during clinical trials being conducted in the Unit. Raw pollen counts/m³ obtained using the different methods were corrected using published particle collection efficiencies for the particle size (∼ ∼20 μm) and airflow. Data were analyzed by ANOVA/Tukey HSD. No statistically significant differences were found between pollen concentrations determined by Rotorod, Air-O-Cell and filter cassette. Pollen levels determined by the Burkard were up to 2 times higher than the other sampling methods. Relative standard deviations were similar for the Rotorod, Burkard, and filter cassette and higher for the Air-O-Cell. This study demonstrated that, under our conditions, the Rotorod sampler provides consistent and reliable measurements of ragweed pollen concentrations.     

Comparison of DNA Walking Methods for Isolation of Transgene-Flanking Regions in GM Potato

An important aspect in the safety assessment of transgenic plants is the exact location of transgene insertion sites within the host genome. However, robust standard operating procedures are not currently available. Using potato as a test species, different methodologies for the determination of insertion sites using a range of published protocols and commercially available kits were assessed in transgenic lines of varying degrees of complexity, from low copy number to complex re-transformed and co-transformed lines. Three commercial kits, APAgene™ GOLD Genome Walking Kit (BIO S&T), DNA Walking SpeedUp™ Kit II (Seegene), and Universal Vectorette™ System (Sigma) were compared with an adaptor-mediated PCR technique. Overall, the APAgene™ kit was used with a high success rate with low copy number potato lines, and also more complex co- and re-transformed lines, and adhering to key confirmation steps it was possible to obtain flanking sequence ranging in size from 300 to 2,500 bp and eliminate PCR artefacts from the analysis.     

A fluorometric assay for the measurement of endothelial cell density in vitro

Summary A fluorometric assay for determining endothelial cell numbers based on the endogenous enzyme acid phosphatase is described. In preliminary studies, three substrates—p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 2′-[2-benzthiazoyl]-6′-hydroxy-benthiazole phosphate (AttoPhos™)—were compared with respect to their kinetic, optimum assay conditions, sensitivity, and detection limits. Only AttoPhos™ was found to have a high degree of sensitivity, reliability, and reproducibility for measuring both high and low cell numbers in the same plate. In subsequent experiments, assay conditions were validated for measuring endothelial cell density in response to basic fibroblast growth factor and fumagillin. Furthermore, the AttoPhos™ assay revealed a linear correlation between acid phosphatase activity and cell number in many cell types, including BALB/3T3, CHO-K1, A431, MCF7, 2008, SK-OV-3, T47-D, and OVCAR-3. This assay is potentially valuable for use in many in vitro systems in which the quantitation of cell density and proliferation is necessary. The practical advantages of AttoPhos™ assay for measuring endothelial cell numbers include (1) nonradioactivity, (2) simplicity, (3) economy, (4) speed of assessment of proliferation of large number of samples, and (5) amenability to high-throughput drug screening.     

Synthesis of hydroxamic acids using SynPhase™ crowns: Development of the hydroxylamine trityl linker

Summary The synthesis of peptide and small molecule hydroxamic acids utilising SynPhase^™ crowns is demonstrated. A hydroxylamine trityl linker is generated from chlorotrityl derivatised Synphase^™ crowns by reaction with N-hydroxyphthalimide followed by subsequent deprotection. The loading of hydroxylamine crowns is quantified spectrophotometrically by measuring phthalhydrazide absorbance at 346 nm.     

A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites

A protocol is described for rapid RNA isolation from various plant species and tissues rich in polyphenolics and polysaccharides. The method is based on the Nucleon PhytoPure^™ system without the use of phenol. The procedure can be completed within 1 h and many samples can be processed at the same time. The yield ranged from 240 μg up to 3 mg per gram of tissue with an average purity measured as A_260/280 of 1.85. The RNA was of sufficient quality for use in RT-PCR reactions. Quantitation of single-stranded cDNA was carried out with the RiboGreen^™ reagent and of PCR products with the PicoGreen^™ reagent.     

The effect of a receptor layer on the measurement of rate constants

Many cellular reactions involve a reactant in solution binding to or dissociating from a reactant attached to a surface. Most studies assume that the reactions occur on this surface, when in actuality the receptors usually lie in a thin layer on top of it. The effect of this layer is considered, particularly as it relates to the BIAcore™ measurement device, though the results are applicable to biological systems. A dimensionless parameter measuring the strength of the effect of the receptor layer is found. Asymptotic and singular perturbation techniques are used to analyse association and dissociation kinetics, though the effect of the receptor layer need not be small. Linear and nonlinear integral equations result from the analysis; explicit and asymptotic solutions are constructed for physically realizable cases. In addition, effective rate constants are derived that illustrate the combined effects of transport and the receptor layer on the measured rate constants. All these expressions provide a direct way to estimate rate constants from BIAcore™ binding data.     

An improved assay to quantitate the invasiveness of cells in modified Boyden chambers

The most commonly used means of assessing the invasiveness of cultured cells is the Boyden chamber assay, which requires that cells lyse Matrigel™, followed by migration through pores in a filter in response to a chemotactic gradient. This report describes a simple method, which greatly increases the speed and accuracy by which Boyden chamber assays can be analyzed, and permits the concurrent analysis of distinct cell subpopulations within specimens containing multiple-cell types.     

Fast life cycle assessment of synthetic chemistry (FLASC™) tool

Background, Aim and Scope There is a clear need for simple methodology to deliver metrics that may be used to determine and benchmark the ‘greenness’ or relative sustainability of synthetic processes for Active Pharmaceutical Ingredients (APIs). Such methodology and metrics should facilitate more informed and sustainable business choices. This capability is particularly important at an early stage in R&D development activities when route and processes are being selected and detailed environmental data are not available. FLASC™ (Fast Life cycle Assessment of Synthetic Chemistry) is a web-based tool and methodology designed to meet these requirements. Materials and Methods FLASC™ was developed from a detailed assessment of the cradle-to-gate life cycle environmental impacts associated with the manufacture of materials used in a typical pharmaceutical process. Results This paper describes the methodology used to develop FLASC™ and provides examples of the type of information and guidance FLASC™ provides. Discussion Both Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA) were used for the statistical analysis during the development of FLASC™. Benchmarking within the pharmaceutical industry and use of normalization for molecular complexity were also integrated to the tool. Conclusions FLASC™ represents an important part of the overall efforts of GlaxoSmithKline (GSK) to incorporate and maintain sustainable business practices for manufacture of APIs used in its pharmaceutical products. Recommendations and Perspectives This tool is not intended to assess waste from GSK operations nor solvent recovery and currently does not incorporate specific chemical-related health and safety data. However, these are already routinely assessed within GSK R&D at appropriate milestones and the use of FLASC™ is complementary to these evaluations.     

LAMS™—A laboratory animal management system

The LAMS™ database stores data on a colony of breeding animals. Forms are hierarchial and show details of internal codes, matings, litters, and offspring. The identifier given to each animal can be subdivided as such. Each form shows an abbreviated list of the related data from the form one level down, and some special fields, when double-clicked, cause the related record to be displayed. The print button allows the user to print the current record and its related records. Other buttons on each form allow the user to amend, delete, find, and add records within certain rules. User-defined lists are created to allow the selection of various characteristics during data entry. The offspring form contains a section where the user can define the label of a comment and/or a text field. This name is then always subsequently available as an option in a list of user-defined labels. Reports are available for tailtipping dates (if applicable) and calculation of genetic ratios. A queryform allows the user to filter the records in the offspring form to the criteria specified, and a display of the actual query submitted is shown. An integrated HELP is available. The LAMS™ database is available at http://www.hgu.mrc.ac.uk/Softdata/Lams Received: 27 April 1998 / Accepted: 9 November 1998     

Efficient analysis and extraction of MS/MS result data from Mascot™ result files

Background  

Mascot™ is a commonly used protein identification program for MS as well as for tandem MS data. When analyzing huge shotgun proteomics datasets with Mascot™'s native tools, limits of computing resources are easily reached. Up to now no application has been available as open source that is capable of converting the full content of Mascot™ result files from the original MIME format into a database-compatible tabular format, allowing direct import into database management systems and efficient handling of huge datasets analyzed by Mascot™.     

"No-reflow" phenomenon during percutaneous carotid angioplasty in a scleroderma patient

Atherosclerosis remains the most common etiology of carotid artery stenosis, but other systemic diseases can also cause this disease. Percutaneous carotid angioplasty and stenting (CAS) with and without distal embolic protection devices is gaining acceptance as the treatment of choice. No-reflow phenomenon due to distal embolization has been previously described mainly during percutaneous intervention of degenerated saphenous vein graft. We describe a patient with systemic sclerosis who underwent CAS with distal embolic protection device with occurrence of no-reflow phenomenon during the procedure that resolved after retrieval of the filter device.     

Formation of embryoid bodies from mouse embryonic stem cells cultured on silicon-coated surfaces

Embryoid bodies (EBs) are primitive embryonic structures derived from differentiating embryonic stem cells (ESCs). Many techniques have been used to obtain EBs. Improving the technique of EB formation can help in achieving better results in ESCs differentiation into neurons, myocardiocytes, haemopoeitic cells, and others. We evaluated the use of Sigmacote™ as a hydrophobic substrate to improve EB formation. CCE and P19 cell lines were used to obtain EBs and retinoic acid was used to induce neural differentiation. The results revealed that Sigmacote™, as a hydrophobic substrate, can improve EB formation from ESCs. Our results demonstrate that the silicon-coating of glass petri dishes by Sigmacote™ is an easy and reproducible technique to enhance EB formation from murine ESCs and EC cells.     

Effectiveness of Metarhizium anisopliae formulations against dengue vectors under laboratory and field conditions

The oviposition behaviour of Aedes aegypti and the effectiveness of Metarhizium anisopliae conidia formulated in water or oil-in-water against A. aegypti adults and eggs were tested in multi-choice and no-choice tests in oviposition devices under laboratory conditions. Both females and males rested in the devices, regardless of the formulation, and were not repelled by the presence of conidia (up to 10⁶?conidia/cm²) without oil or formulated with oil on treated filter paper arranged in the device. However, at higher oil concentrations (≥0.1?μl/cm²), regardless of the presence of conidia, the number of eggs laid by gravid females on the filter paper dropped. The susceptibility of adults, especially of males, to fungal infection increased up to a 15-day incubation. An elevated number of larvae (≥41%) eclosed from eggs laid on the moistened filter paper in the device even without submersion of eggs in water, and these larvae subsequently died. In the laboratory, 1?μl/cm² oil combined with 10⁶?conidia/cm² clearly reduced eclosion to 1.8% after submersion of eggs in water compared to ≥13% eclosion in the control. In field tests in Goiânia, Brazil, eclosion of aedine larvae from eggs laid on filter paper previously treated with oil-in-water formulated conidia dropped to between 0% and 36% compared to 22–50% in the control. Promising results of laboratory and field tests with M. anisopliae formulated in water or oil-in-water and tested in a device emphasised the effectiveness of a fungus-based formulation for aedine mosquitoes in peridomestic areas.     

A nanoparticle-based immobilization assay for prion-kinetics study

A technique for permanently capturing a replica impression of biological cells has been developed to facilitate analysis using nanometer resolution imaging tools, namely the atomic force microscope (AFM). The method, termed Bioimprint™, creates a permanent cell 'footprint' in a non-biohazardous Poly (dimethylsiloxane) (PDMS) polymer composite. The transfer of nanometer scale biological information is presented as an alternative imaging technique at a resolution beyond that of optical microscopy. By transferring cell topology into a rigid medium more suited for AFM imaging, many of the limitations associated with scanning of biological specimens can be overcome. Potential for this technique is demonstrated by analyzing Bioimprint™ replicas created from human endometrial cancer cells. The high resolution transfer of this process is further detailed by imaging membrane morphological structures consistent with exocytosis. The integration of soft lithography to replicate biological materials presents an enhanced method for the study of biological systems at the nanoscale.