15N discrimination and the sensitivity of nitrogen fixation to changes in dietary nitrogen in Reticulitermes flavipes (Isoptera: Rhinotermitidae)

Xylophagous termites possess symbiotic bacteria that fix atmospheric nitrogen (N(2)). Although symbiotic N(2) fixation is central to termite nutrition and ecologically important, it is energetically costly. Using stable isotopes, we tested the hypothesis that symbiotic N(2) fixation would decrease in workers of the eastern subterranean termite, Reticulitermes flavipes Kollar, which were exposed to high nitrogen diets. To calculate the isotope discrimination factor occurring as a result of digestion, Δ(dig), and which occurs as the result of N(2) fixation, Δ(fix), symbiotic N(2) fixation was inhibited via force feeding termites the antibiotic kanamycin. Antibiotic-treated termites and control (N(2)-fixing) termites were exposed to different concentrations of dietary N (0, 0.21, and 0.94% N), their (15)N signatures were obtained, and the percent nitrogen derived from the atmosphere within termite samples was calculated. As we hypothesized, symbiotic N(2) fixation rates were negatively correlated with dietary N, suggesting that high concentrations of dietary N suppressed symbiotic N(2) fixation in R. flavipes. A comparison of the (15)N isotope signatures of antibiotic-treated termites with their food sources demonstrated that Δ(dig) = 2.284‰, and a comparison of the (15)N isotope signatures of antibiotic-treated termites with control termites indicated that Δ(fix) = -1.238‰. These are the first estimates of Δ(dig) for R. flavipes, and the first estimate of Δ(fix) for any N(2)-fixing termite species.     

Host utilization of field-caged native and introduced thistle species by Rhinocyllus conicus

Rhinocyllus conicus Fr?elich was introduced from Europe into North America as a biological control agent of the exotic weed Carduus nutans L. Concern exists over the feeding of this weevil on at least 25 species of native Cirsium thistles. Beginning in 2008, cage studies isolating adults of R. conicus on buds and flower heads of all eight thistle species (native and introduced) recorded from Tennessee were conducted to test if R. conicus could use these species for reproduction and what impacts larval feeding of R. conicus may have on seed production. Larvae of R. conicus completed development in heads of the native species C. carolinianum (Walter) Fernald and Schubert. and C. horridulum Michaux, and significant reductions in seed numbers of both species occurred during 2008. Rhinocyllus conicus oviposited on both C. carolinianum and C. horridulum at significantly greater levels than the introduced species C. arvense (L.) Scopoli and C. vulgare (Savi) Tenore. Infested heads of C. carolinianum contained numbers of R. conicus per centimeter of plant head width similar to Ca. nutans in 2008, and both native species contained numbers of R. conicus per centimeter of plant head width similar to C. arvense and C. vulgare in 2009. Body length was similar between R. conicus reared on native thistles and its target host Ca. nutans. This report is the first documentation of R. conicus feeding and reproducing on C. carolinianum and C. horridulum. Although R. conicus has been observed only on introduced thistles in naturally occurring populations in this region, the utilization of C. carolinianum and C. horridulum as host species in controlled conditions warrants continued monitoring of field populations and further investigation into factors that may influence nontarget feeding in the future.     

Pinnacle Point Cave 13B (Western Cape Province, South Africa) in context: The Cape Floral kingdom, shellfish, and modern human origins

Genetic and anatomical evidence suggests that Homo sapiens arose in Africa between 200 and 100ka, and recent evidence suggests that complex cognition may have appeared between ~164 and 75ka. This evidence directs our focus to Marine Isotope Stage (MIS) 6, when from 195-123ka the world was in a fluctuating but predominantly glacial stage, when much of Africa was cooler and drier, and when dated archaeological sites are rare. Previously we have shown that humans had expanded their diet to include marine resources by ~164ka (±12ka) at Pinnacle Point Cave 13B (PP13B) on the south coast of South Africa, perhaps as a response to these harsh environmental conditions. The associated material culture documents an early use and modification of pigment, likely for symbolic behavior, as well as the production of bladelet stone tool technology, and there is now intriguing evidence for heat treatment of lithics. PP13B also includes a later sequence of MIS 5 occupations that document an adaptation that increasingly focuses on coastal resources. A model is developed that suggests that the combined richness of the Cape Floral Region on the south coast of Africa, with its high diversity and density of geophyte plants and the rich coastal ecosystems of the associated Agulhas Current, combined to provide a stable set of carbohydrate and protein resources for early modern humans along the southern coast of South Africa during this crucial but environmentally harsh phase in the evolution of modern humans. Humans structured their mobility around the use of coastal resources and geophyte abundance and focused their occupation at the intersection of the geophyte rich Cape flora and coastline. The evidence for human occupation relative to the distance to the coastline over time at PP13B is consistent with this model.     

Development of the AGREE II,part 1: performance,usefulness and areas for improvement

Background

We undertook research to improve the AGREE instrument, a tool used to evaluate guidelines. We tested a new seven-point scale, evaluated the usefulness of the original items in the instrument, investigated evidence to support shorter, tailored versions of the tool, and identified areas for improvement.

Method

We report on one component of a larger study that used a mixed design with four factors (user type, clinical topic, guideline and condition). For the analysis reported in this article, we asked participants to read a guideline and use the AGREE items to evaluate it based on a seven-point scale, to complete three outcome measures related to adoption of the guideline, and to provide feedback on the instrument’s usefulness and how to improve it.

Results

Guideline developers gave lower-quality ratings than did clinicians or policy-makers. Five of six domains were significant predictors of participants’ outcome measures (p < 0.05). All domains and items were rated as useful by stakeholders (mean scores > 4.0) with no significant differences by user type (p > 0.05). Internal consistency ranged between 0.64 and 0.89. Inter-rater reliability was satisfactory. We received feedback on how to improve the instrument.

Interpretation

Quality ratings of the AGREE domains were significant predictors of outcome measures associated with guideline adoption: guideline endorsements, overall intentions to use guidelines, and overall quality of guidelines. All AGREE items were assessed as useful in determining whether a participant would use a guideline. No clusters of items were found more useful by some users than others. The measurement properties of the seven-point scale were promising. These data contributed to the refinements and release of the AGREE II.Evidence-based guidelines are systematically developed statements aimed at assisting clinicians and patients in decisions about appropriate health care for specific clinical circumstances. ¹ Guidelines assist decision-makers in solving system-level and population-level challenges. ^2,3 The potential benefits of guidelines, however, are only as good as the quality of the guidelines themselves. Rigorous development and strategies for reporting are important precursors to successful implementation of the resulting recommendations. ⁴The quality of guidelines is variable, often falling short of basic standards.^5–7 To address this variability, an international team of guideline developers and researchers, the AGREE Collaboration (Appraisal of Guidelines, Research and Evaluation), created a generic instrument to assess the process of guideline development and reporting. The result of this work was the AGREE instrument, a 23-item tool targeting six quality-related domains.^8,9 It became accepted by many as the standard for guideline evaluation.¹⁰As with any new assessment tool, ongoing development of the instrument is required. The AGREE Next Steps Consortium was established to conduct a program of research aimed at improving the AGREE and advancing the overall guideline enterprise. We report on the first of two studies designed to achieve these goals. This study focused on four key issues related to methodology and implementation (Figure 1).Open in a separate windowFigure 1Program of research.First, the original four-point response scale was not in keeping with standards for test construction that are intended to maximize the reliability and discriminability of an instrument and minimize the number of appraisers required to evaluate a guideline.¹¹ To address this issue, we introduced a seven-point response scale, tested its performance and conducted a preliminary analysis of some of its measurement properties.Second, to be of value, the AGREE instrument needs to be easy to apply and needs to generate information that is useful. To generate a reliable estimate of guideline quality, it is recommended that the 23 items of the AGREE instrument be applied by four independent reviewers.^8,9 This process can be cumbersome and resource-intensive. However, no systematic analysis has been undertaken previously to determine if all items of the AGREE instrument generate information that is equally useful across different groups. This fact opens the possibility that fewer than 23 items, and varying combinations of items unique to different users, may be sufficient for evaluation purposes. Therefore, we explored whether evidence exists to inform the development of abridged versions of the AGREE that could be tailored to the unique priorities of different user groups.Third, to be useful as well, the AGREE ratings should be associated with outcomes that are relevant to guideline usage. In keeping with previous findings,⁴ guidelines of higher quality should be more attractive, endorsed or used than those of lower quality. We therefore explored whether these relationships existed and whether they were consistent across different types of users.Finally, given that the AGREE had been in the field for some time, we systematically collected feedback from users on how the items and domains might be improved, updated and refined.In combination with the results of the second study,¹² the data were used by the consortium to craft the AGREE II,¹³ the next version of the AGREE instrument.     

ChIPing the cistrome of PXR in mouse liver

The pregnane X receptor (PXR) is a key regulator of xenobiotic metabolism and disposition in liver. However, little is known about the PXR DNA-binding signatures in vivo, or how PXR regulates novel direct targets on a genome-wide scale. Therefore, we generated a roadmap of hepatic PXR bindings in the entire mouse genome [chromatin immunoprecipitation (ChIP)-Seq]. The most frequent PXR DNA-binding motif is the AGTTCA-like direct repeat with a 4bp spacer [direct repeat (DR)-4)]. Surprisingly, there are also high motif occurrences with spacers of a periodicity of 5 bp, forming a novel DR-(5n + 4) pattern for PXR binding. PXR-binding overlaps with the epigenetic mark for gene activation (histone-H3K4-di-methylation), but not with epigenetic marks for gene suppression (DNA methylation or histone-H3K27-tri-methylation) (ChIP-on-chip). After administering a PXR agonist, changes in mRNA of most PXR-direct target genes correlate with increased PXR binding. Specifically, increased PXR binding triggers the trans-activation of critical drug-metabolizing enzymes and transporters. The mRNA induction of these genes is absent in PXR-null mice. The current work provides the first in vivo evidence of PXR DNA-binding signatures in the mouse genome, paving the path for predicting and further understanding the multifaceted roles of PXR in liver.     

Confirmation of Anopheles (Anopheles) calderoni Wilkerson, 1991 (Diptera: Culicidae) in Colombia and Ecuador through molecular and morphological correlation with topotypic material

The morphologically similar taxa Anopheles calderoni, Anopheles punctimacula, Anopheles malefactor and Anopheles guarao are commonly misidentified. Isofamilies collected in Valle de Cauca, Colombia, showed morphological characters most similar to An. calderoni, a species which has never previously been reported in Colombia. Although discontinuity of the postsubcostal pale spots on the costa (C) and first radial (R1) wing veins is purportedly diagnostic for An. calderoni, the degree of overlap of the distal postsubcostal spot on C and R1 were variable in Colombian specimens (0.003-0.024). In addition, in 98.2% of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7% of specimens examined. Correlation of DNA sequences of the second internal transcribed spacer and mtDNA cytochrome c oxidase subunit I gene (COI) barcodes (658 bp of the COI gene) generated from Colombian progeny material and wild-caught mosquitoes from Ecuador with those from the Peruvian type series of An. calderoni confirmed new country records. DNA barcodes generated for the closely related taxa, An. malefactor and An. punctimacula are also presented for the first time. Examination of museum specimens at the University of the Valle, Colombia, revealed the presence of An. calderoni in inland localities across Colombia and at elevations up to 1113 m.     

Evolution in fossil lineages: paleontology and The Origin of Species

Of all of the sources of evidence for evolution by natural selection, perhaps the most problematic for Darwin was the geological record of organic change. In response to the absence of species-level transformations in the fossil record, Darwin argued that the fossil record was too incomplete, too biased, and too poorly known to provide strong evidence against his theory. Here, this view of the fossil record is evaluated in light of 150 years of subsequent paleontological research. Although Darwin's assessment of the completeness and resolution of fossiliferous rocks was in several ways astute, today the fossil record is much better explored, documented, and understood than it was in 1859. In particular, a reasonably large set of studies tracing evolutionary trajectories within species can now be brought to bear on Darwin's expectation of gradual change driven by natural selection. An unusually high-resolution sequence of stickleback-bearing strata records the transformation of this lineage via natural selection. This adaptive trajectory is qualitatively consistent with Darwin's prediction, but it occurred much more rapidly than he would have guessed: almost all of the directional change was completed within 1,000 generations. In most geological sequences, this change would be too rapid to resolve. The accumulated fossil record at more typical paleontological scales (10(4)-10(6) years) reveals evolutionary changes that are rarely directional and net rates of change that are perhaps surprisingly slow, two findings that are in agreement with the punctuated-equilibrium model. Finally, Darwin's view of the broader history of life is reviewed briefly, with a focus on competition-mediated extinction and recent paleontological and phylogenetic attempts to assess diversity dependence in evolutionary dynamics.     

VEGF signaling has distinct spatiotemporal roles during heart valve development

Heart valve malformations are one of the most common types of birth defects, illustrating the complex nature of valve development. Vascular endothelial growth factor (VEGF) signaling is one pathway implicated in valve formation, however its specific spatial and temporal roles remain poorly defined. To decipher these contributions, we use two inducible dominant negative approaches in mice to disrupt VEGF signaling at different stages of embryogenesis. At an early step in valve development, VEGF signals are required for the full transformation of endocardial cells to mesenchymal cells (EMT) at the outflow tract (OFT) but not atrioventricular canal (AVC) endocardial cushions. This role likely involves signaling mediated by VEGF receptor 1 (VEGFR1), which is highly expressed in early cushion endocardium before becoming downregulated after EMT. In contrast, VEGFR2 does not exhibit robust cushion endocardium expression until after EMT is complete. At this point, VEGF signaling acts through VEGFR2 to direct the morphogenesis of the AVC cushions into mature, elongated valve leaflets. This latter role of VEGF requires the VEGF-modulating microRNA, miR-126. Thus, VEGF roles in the developing valves are dynamic, transitioning from a differentiation role directed by VEGFR1 in the OFT to a morphogenetic role through VEGFR2 primarily in the AVC-derived valves.