Reagent decontamination to eliminate false-positives in Escherichia coli qPCR

The application of real-time quantitative PCR (qPCR) for the detection of low concentrations of Escherichia coli as well as universal 16S rDNA has been hindered by false-positives due to endogenous contamination of PCR reagents with E. coli and other bacterial DNA. We optimized a DNase I decontamination method to eliminate false-positives in a qPCR assay targeting the uidA gene in E. coli. In contrast to previous methods reported in the literature, our decontamination method did not cause PCR inhibition. We determined that residual DNase I activity was the cause of the inhibition in the previous methods, and eliminated it by ensuring complete inactivation prior to qPCR. DNase inactivation was accomplished by adding dithiothreitol (DTT) and then heating for 30 min at 80 degrees C. The optimized DNase method was compared to another decontamination method, ultrafiltration, and to untreated controls. We detected contamination in 85% of the untreated commercial PCR master mix samples at a level of about 10 copies per well (12.5 microL of master mix). Both decontamination methods could eliminate up to 100 copies of added contaminant DNA and did not cause PCR inhibition, resulting in a reduction of the detection limit to 10 copies per reaction well.     

Impact of Weight‐Cycling History on Bone Density in Obese Women

Objective: The purpose of this study was to examine the effect of weight cycling (as defined by the frequency and magnitude of intentional weight loss) on bone mineral density and bone mineral content in obese sedentary women. Research Methods and Procedures: Bone mineral content and density measured by DXA, submaximal physical fitness assessment, nutrient intake, oral contraceptive use, and weight‐cycling history were assessed in 195 healthy, overweight sedentary women (age, 21 to 45 years; body mass index, 27 to 40 kg/m²) before beginning a behavioral weight‐loss intervention. Results: After controlling for body weight, multivitamin use, oral contraceptive/estrogen use, and calcium and magnesium intake, women who had a history of weight cycling did not have significantly lower total‐body bone mineral content or density or total femur bone mineral density. In addition, 99% of subjects were above or within one SD of age and gender normative data for total femur bone mineral density. Discussion: It does not seem that a history of weight cycling has an adverse affect on total femur and total‐body bone mineral density in overweight sedentary premenopausal women.     

Characterization of Hydrogenobacter thermophilus cytochromes c 552 expressed in the cytoplasm and periplasm of Escherichia coli

Hydrogenobacter thermophilus cytochrome c(552) ( Ht cyt c(552)) is a small monoheme protein in the cytochrome c(551) family. Ht cyt c(552) is unique because it is hypothesized to undergo spontaneous cytoplasmic maturation (covalent heme attachment) when expressed in Escherichia coli. This is in contrast to the usual maturation route for bacterial cytochromes c that occurs in the cellular periplasm, where maturation factors direct heme attachment. Here, the expression of Ht cyts c(552) in the periplasm as well as the cytoplasm of E. coli is reported. The products are characterized by absorption, circular dichroism, and NMR spectroscopy as well as mass spectrometry, proteolysis, and denaturation studies. The periplasmic product's properties are found to be indistinguishable from those reported for protein isolated from Ht cells, while the major cytoplasmic product exhibits structural anomalies in the region of the N-terminal helix. These anomalies are shown to result from the retention of the N-terminal methionine in the cytoplasmic product, and not from heme attachment errors. The (1)H NMR chemical shifts of the heme methyls of the oxidized ( S=1/2) expression products display a unique pattern not previously reported for a cytochrome c with histidine-methionine axial ligation, although they are consistent with native-like heme ligation. These results support the hypothesis that proper heme attachment can occur spontaneously in the E. coli cytoplasm for Ht cyt c(552).     

Non-peptide alpha(v)beta(3) antagonists. Part 3: identification of potent RGD mimetics incorporating novel beta-amino acids as aspartic acid replacements.

Potent non-peptidic alpha(v)beta(3) antagonists have been prepared incorporating various beta-amino acids as aspartic acid mimetics. Modification of the beta-alanine 3-substituents alters the potency and physicochemical properties of these receptor antagonists and in some cases provides orally bioavailable alpha(v)beta(3) inhibitors.     

Extrapair paternity is influenced by breeding synchrony and density in the common yellowthroat

The effects of breeding synchrony and density on levels of extrapair paternity in birds are controversial. We used multilocus DNAfingerprinting and microsatellite analysis to examine the effectsof breeding synchrony and density on levels of extrapair paternityin the common yellowthroat (Geothlypis trichas). As in manyNeotropical migrants, breeding synchrony was greatest at thebeginning of the breeding season. Levels of extrapair paternitywere higher after the peak in synchrony, leading to an overallnegative relationship between extrapair paternity and breeding synchrony. However, there was a significant interaction betweenbreeding synchrony and density, as levels of extrapair paternitywere higher only for males breeding when both synchrony anddensity were low. We discuss several possible explanationsfor this interaction, including lower quality males or territoriesin low density areas and greater demands on mate guarding among males with larger territories. Most studies have not consideredsimultaneously the effects of breeding synchrony and densityon extrapair paternity. Our results suggest that ecologicalcorrelates of paternity may be revealed only after testingfor interactions in multivariate analyses.     

Assessing conservation attitudes and behaviors of Congolese children neighboring the world's first bonobo (Pan paniscus) release site

Poaching and habitat destruction in the Congo Basin threaten African great apes including the bonobo (Pan paniscus), chimpanzees (Pan troglodytes), and gorillas (Gorilla spp.) with extinction. One way to combat extinction is to reintroduce rescued and rehabilitated apes and repopulate native habitats. Reintroduction programs are only successful if they are supported by local populations. Ekolo ya Bonobo, located in Equateur province of the Democratic Republic of Congo (DRC), is the world's only reintroduction site for rehabilitated bonobos. Here we assess whether children, of the Ilonga‐Pôo, living adjacent to Ekolo ya Bonobo demonstrate more pro‐ape conservation attitudes than children living in, Kinshasa, the capital city. We examined children's attitudes toward great apes because children are typically the focus of conservation education programs. We used the Great Ape Attitude Questionnaire to test the Contact Hypothesis, which posits that proximity to great ape habitat influences pro‐conservation attitudes toward great apes. Ilonga‐Pôo children who live in closer contact with wild bonobos felt greater responsibility to protect great apes compared to those in Kinshasa who live outside the natural habitat of great apes. These results suggest that among participants in the DRC, spatial proximity to a species fosters a greater sense of responsibility to protect and conserve. These results have implications for the successful implementation of great ape reintroduction programs in the Congo Basin. The data analyzed in this study were collected in 2010 and therefore provide a baseline for longitudinal study of this reintroduction site.     

Breaking dormancy during flood and drought: sublethal growth and physiological responses of three emergent wetland herbs used in bioretention basins

Bioretention basins are man-made topographic depressions designed to collect and retain surface water runoff. In most cases these basins are used to prevent flooding and/or remove environmentally harmful pollutants and sediments from entering natural aquatic systems. To maximize environmental benefits, these systems are often planted with flood-tolerant wetland hydrophytes that are capable of withstanding extended periods of drought. Unfortunately, little is known about how these plants respond to extreme hydrologies while breaking seasonal dormancy. The purpose of this study was to better understand the morphological and physiological responses of three wetland species (Pontederia cordata L., Saururus cernuus L., and Schoenoplectus tabernaemontani C.C. Gmel. Palla) often used in bioretention basins while emerging from dormancy in either flooded or drought conditions. Results indicate that only S. tabernaemontani was affected morphologically by drought with lower leaf area and aboveground biomass. While significant reductions in stomatal surface indices were also observed in drought-treated S. tabernaemontani, all three species had reductions in stomatal conductance (g) when grown in drier soils. Moreover, drought conditions promoted decreases in leaf water potential (Ψ _leaf) for all three species, and reductions in tissue water content (θ) for P. cordata and S. tabernaemontani. Based on the overall morphological and physiological responses, S. cernuus maintained the lowest productivity, and appeared to be the best suited for tolerating sustained soil water deficits. If high plant productivity is desired, however, S. tabernaemontani was able to maintain high plant growth while making necessary modifications that facilitated greater drought-resistance.     

Distinct Contributions of the Dorsolateral Prefrontal and Orbitofrontal Cortex during Emotion Regulation

The lateral prefrontal and orbitofrontal cortices have both been implicated in emotion regulation, but their distinct roles in regulation of negative emotion remain poorly understood. To address this issue we enrolled 58 participants in an fMRI study in which participants were instructed to reappraise both negative and neutral stimuli. This design allowed us to separately study activations reflecting cognitive processes associated with reappraisal in general and activations specifically related to reappraisal of negative emotion. Our results confirmed that both the dorsolateral prefrontal cortex (DLPFC) and the lateral orbitofrontal cortex (OFC) contribute to emotion regulation through reappraisal. However, activity in the DLPFC was related to reappraisal independently of whether negative or neutral stimuli were reappraised, whereas the lateral OFC was uniquely related to reappraisal of negative stimuli. We suggest that relative to the lateral OFC, the DLPFC serves a more general role in emotion regulation, perhaps by reflecting the cognitive demand that is inherent to the regulation task.     

Combined Flow Cytometric Analysis of Surface and Intracellular Antigens Reveals Surface Molecule Markers of Human Neuropoiesis

Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, we present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. We have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f^-/CD200^high surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology.