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.     

Getting off on the right foot: Subjective value versus economic value in predicting longitudinal job outcomes from job offer negotiations.

Although negotiation experiences can affect a negotiator's ensuing attitudes and behavior, little is known about their long-term consequences. Using a longitudinal survey design, the authors tested the degree to which economic and subjective value achieved in job offer negotiations predicts employees' subsequent job attitudes and intentions concerning turnover. Results indicate that subjective value predicts greater compensation satisfaction and job satisfaction and lower turnover intention measured 1 year later. Surprisingly, the economic outcomes that negotiators achieved had no apparent effects on these factors. Implications, limitations, and future directions are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

AHighResolvingPowerandMassAccuracyTOFMSforRapidDetectionandIdentificationofMicroorganisms

An exact mass measurement ESI TOF mass spectrometer has been developed for fast pathogen identification and strain typing. This unique technology relies on mass spectrometry-derived base composition signatures of amplicons from PCR (Polymerase Chain Reaction) products, and “intelligent PCR primers” that target broadly conserved regions of microbial genomes that flank variable regions. This rapid detection (30 seconds per sample) and identification of microorganism methodology also associates with a potential biological warfare attack or a natural outbreak of an emerging infectious disease. The strategy to build a high end and low cost ESI TOF machine will be discussed. The performance data particularly in accurate mass measurement, high resolving power and 24 hours stability test will be presented. The discovery of Swine flu virus H1N1 will be presented as an example to illustrate the procedure of detection and identification of microoganisms.     

Evolutionary cyber-psychology: Applying an evolutionary framework to Internet behavior

An evolutionary psychological perspective has much to offer the study of Internet behavior. However, cyber-psychologists have hitherto neglected this rich theoretical tradition and evolutionary psychologists have been slow to apply their perspective to computer-mediated behavior. This paper applies an evolutionary perspective to the study of Internet behavior in four relevant domains: (1) mating and sexual competition, (2) parenting and kinship, (3) trust and social exchange, and (4) personal information management. Both general and specific evolutionary theories are explored in relation to online behavior in each domain, with an emphasis on generating testable hypotheses for future research.     

Infrared multiphoton dissociation with a hollow fiber waveguide

A novel scheme for performing infrared multiphoton dissociation (IRMPD) is presented in which a hollow fiber waveguide (HFWG) is used to transmit IR radiation into the ion storage region of a mass spectrometer. Efficient dissociation of oligonucleotide and protein ions is demonstrated on an ESI-FTICR instrument in which IRMPD is performed in the external ion reservoir and on a quadrupole ion trap. Using a simple optical scheme consisting of a single focusing lens and an x, y translator, the 10.6-microm IR laser beam, initially 3.5 mm in diameter, is focused into the vacuum-sealed HFWG. The small internal diameter and the high transfer efficiency of the waveguide allow IR radiation of high power density to be employed for IRMPD. In studies performed on a quadrupole ion trap, a 500-microm-i.d. waveguide was used as a medium to transmit IR radiation directly through a 700-microm orifice in the ring electrode. Efficient IRMPD of both a 12-mer oligonucleotide and the protein melittin were performed at laser powers of 0.5 and 3.2 W, respectively.     

The formation of an apatite coating on carboxylated polyphosphazenes via a biomimetic process

The biomimetic deposition of an apatite coating on polyphosphazene was investigated. A polyphosphazene having carboxylatophenoxy side groups was synthesized as a candidate polymer with heterogeneous nucleation sites for apatite deposition. Poly[bis(carboxylatophenoxy)phosphazene] (PCPP) was synthesized by a two-step synthetic route involving the synthesis of poly(dichlorophosphazene) (PDP) followed by nucleophilic replacement of the chlorine atoms of PDP with carboxylatophenoxy groups. The surface of the carboxylated polyphosphazene induced the formation of a dense layer of apatite on the surface after incubation in simulated body fluid at 37 °C with an ionic concentration approximately the same as that of human blood plasma. The results suggested an early precipitation of octacalcium phosphate and calcium phosphate intermediates on the surface, both of which are known to promote osteoblast differentiation and proliferation.     

Optical Frequency Metrology of an Iodine-Stabilized He-Ne Laser Using the Frequency Comb of a Quantum-Interference-Stabilized Mode-Locked Laser

We perform optical frequency metrology of an iodine-stabilized He-Ne laser using a mode-locked Ti:sapphire laser frequency comb that is stabilized using quantum interference of photocurrents in a semiconductor. Using this technique, we demonstrate carrier-envelope offset frequency fluctuations of less than 5 mHz using a 1 s gate time. With the resulting stable frequency comb, we measure the optical frequency of the iodine transition [¹²⁷I₂ R(127) 11-5 i component] to be 473 612 214 712.96 ± 0.66 kHz, well within the uncertainty of the CIPM recommended value. The stability of the quantum interference technique is high enough such that it does not limit the measurements.     

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.     

Discovery of Affinity-Based Probes for Btk Occupancy Assays

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.