Multi-laboratory evaluations of the performance of Catellicoccus marimammalium PCR assays developed to target gull fecal sources

Here we report results from a multi-laboratory (n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR^® Green qPCR method, and two TaqMan^® qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.     

Business Activities of Not-For-Profit Organizations: Surviving the New Federalism?

All over the country community organizations are starting new community based businesses designed to fight unemployment, generate needed income, or respond to their own growth needs. This article addresses the questions of whether these new approaches can be used to plan and implement economic development efforts, what their political and ideological content is likely to be, what impact they will have on their neighborhoods, and what roles planners can play. The analysis is based on the authors' experiences as planners and technical assistants to such efforts, and on a detailed analysis of case studies.     

Heads or Tails: Genotyping of Hepatitis C Virus Concerning the 2k/1b Circulating Recombinant Form

As different hepatitis C virus (HCV) genotypes respond differently to initiated therapy, correct HCV genotyping is essential. A potential risk for misclassification of the intergenotypic HCV circulating recombinant form (CRF) 2k/1b strains exists, depending on the genotyping method used. The aim was to investigate the differences in HCV genotyping methods with regard to CRF 2k/1b and to gain insight in the prevalence of the CRF 2k/1b. Genotyping results by Versant HCV Genotype Assay were compared with nonstructural protein 5B (NS5B) sequencing. In total, from November 2001 until March 2015, 3296 serum samples were analyzed by Versant HCV Genotype Assay. As misclassified CRF is harbored among HCV genotype 2, we further focused our search on 142 (4.3%) samples positive for HCV genotype 2. On 116 (81.7%) retrieved samples, the NS5B sequencing was performed. Twelve out of the 116 retrieved samples (10.3%) were classified as CRF 2k/1b by sequencing of the NS5B region. Ten of these 12 samples were originally misclassified as genotype 2a or 2c, while 2 of them were misclassified as genotype 2. Our results show that the current prevalence of CRF 2k/1b is underestimated. The importance of correct HCV genotyping is emphasized, considering the tailored choice of treatment regimen and overall prognosis.     

Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms

Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure–function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-β-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure–function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms.     

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.     

Nanoparticle-filled silane films as chromate replacements for aluminum alloys

Silane surface treatments have been developed as an alternative for toxic and carcinogenic chromate-based treatments for years. It is consistently observed that ultra-thin silane films offer excellent corrosion protection as well as paint adhesion to metals. The silane performance is comparable to, or in some cases better than, that of chromate layers. The most recent studies also showed that the silane films can be thickened and strengthened by loading of a small amount of nanoparticles such as silica and alumina into the films resulting in enhanced corrosion protection of aluminum alloys.     

Preferentially oriented BaTiO3 thin films deposited on silicon with thin intermediate buffer layers

Barium titanate (BaTiO₃) thin films are prepared by conventional 2-methoxy ethanol-based chemical solution deposition. We report highly c-axis-oriented BaTiO₃ thin films grown on silicon substrates, coated with a lanthanum oxynitrate buffer layer of 8.9 nm. The influence of the intermediate buffer layer on the crystallization of BaTiO₃ film is investigated. The annealing temperature and buffer layer sintering conditions are optimized to obtain good crystal growth. X-ray diffraction measurements show the growth of highly oriented BaTiO₃ thin films having a single perovskite phase with tetragonal geometry. The scanning electron microscopy and atomic force microscopy studies indicate the presence of smooth, crack-free, uniform layers, with densely packed crystal grains on the silicon surface. A BaTiO₃ film of 150-nm thickness, deposited on a buffer layer of 7.2 nm, shows a dielectric constant of 270, remnant polarization (2P_r) of 5 μC/cm², and coercive field (E_c) of 60 kV/cm.     

Insights into the formation of microporous materials by in situ X-ray scattering techniques

Here we demonstrate the power of in situ scattering techniques in the understanding of formation of nanoporous aluminosilicate and aluminophosphate materials. We utilised a number of X-ray techniques, in particular, in situ energy dispersive X-ray diffraction (EDXRD) and small angle and wide angle X-ray scattering (SAXS/WAXS) techniques for this purpose. The in situ SAXS measurements show the formation of homogeneous precursors in the size of ca. 10 nm, prior to the crystallization of LTA. The crystal size is estimated by fitting the SAXS patterns with an equation for a cubic particle, and it is revealed that the final crystal size of the LTA depends on the synthesis temperature. Whereas, the crystallisation of CoAlPO-5, occurs through the formation of poly-dispersed particles with an average size of the precursor particle of ca. 50 nm. Also shown the effect of temperature and structure directing organic cations on the production of CoAlPO-5 materials.