Michael P. O'Connor

THERMAL ECOLOGY OF DESERT TORTOISES IN THE EASTERN MOJAVE DESERT: SEASONAL PATTERNS OF OPERATIVE AND BODY TEMPERATURES, AND MICROHABITAT UTILIZATION

We monitored meteorological variables, daily and seasonal patterns of body tem- perature, corresponding operative temperatures, and microhabitat utilization by desert tortoises (Gopherus agassizii) during the 1991 and 1992 activity seasons of tortoises in the eastern Mojave desert. We studied tortoises in enclosures of natural habitat at the Desert Tortoise Conservation Center (DTCC) near Las Vegas, Nevada and a population of free-ranging tortoises in a field site adjacent to the DTCC. Air, ground and operative temperatures coincided with daily and monthly patterns of incident solar radiation. Variation in body temperature was primarily a consequence of microhabitat selection, principally use of burrows. During July-October, in the morning, body temperatures of tortoises in burrows were cooler than those of individuals on the surface. During midday, tortoises remained in burrows where body temperatures were cooler than extreme surface operative temperatures. While tortoises remained in burrows during much of the day, tortoises typically did not sleep in burrows at night. Microhabitat utilization was dictated by avoidance of extreme temperatures during midday, and microhabitat selection corresponded qualitatively to maintenance of energy and water balances. Effective conservation efforts to preserve habitat of desert tortoises will focus upon managing variables associated with integrity of burrows. ulation

The leatherback turtle, Dermochelys coriacea exhibits both polyandry and polygyny

The leatherback turtle (Dermochelys coriacea) is an endangered species, and world‐wide populations are declining. To understand better the mating structure of this pelagic and fragile species, we investigated paternity in nearly 1000 hatchlings from Playa Grande in Parque Marino Nacional Las Baulas, Costa Rica. We collected DNA samples from 36 adult female leatherbacks and assessed allele frequency distributions for three microsatellite loci. For 20 of these 36 females, we examined DNA from hatchlings representing multiple clutches, and in some cases assessed up to four successive clutches from the same female. We inferred paternal alleles by comparing maternal and hatchling genotypes. We could not reject the null hypothesis of single paternity in 12 of 20 families (31 of 50 clutches), but we did reject the null hypothesis in two families (eight of 50 clutches). In the remaining six families, the null hypothesis could not be accepted or rejected with certainty because the number of hatchlings exhibiting extra nonmaternal alleles was small, and could thus be a result of mutation or sample error. Successive clutches laid by the same female had the same paternal allelic contribution, indicating sperm storage or possibly monogamy. None of 20 females shared the same three‐locus genotype whereas there were two instances of shared genotypes among 17 inferred paternal three‐locus genotypes. We conclude that both polyandry and polygyny are part of the mating structure of this leatherback sea turtle population.

Variability in P-Glycoprotein Inhibitory Potency (IC50) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug-Drug Interaction Risk Assessment Decision Criteria

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells—Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

Patterns, causes and consequences of defensive microbiome dynamics across multiple scales

The microbiome can significantly impact host phenotypes and serve as an additional source of heritable genetic variation. While patterns across eukaryotes are consistent with a role for symbiotic microbes in host macroevolution, few studies have examined symbiont‐driven host evolution or the ecological implications of a dynamic microbiome across temporal, spatial or ecological scales. The pea aphid, Acyrthosiphon pisum, and its eight heritable bacterial endosymbionts have served as a model for studies on symbiosis and its potential contributions to host ecology and evolution. But we know little about the natural dynamics or ecological impacts of the heritable microbiome of this cosmopolitan insect pest. Here we report seasonal shifts in the frequencies of heritable defensive bacteria from natural pea aphid populations across two host races and geographic regions. Microbiome dynamics were consistent with symbiont responses to host‐level selection and findings from one population suggested symbiont‐driven adaptation to seasonally changing parasitoid pressures. Conversely, symbiont levels were negatively correlated with enemy‐driven mortality when measured across host races, suggesting important ecological impacts of host race microbiome divergence. Rapid drops in symbiont frequencies following seasonal peaks suggest microbiome instability in several populations, with potentially large costs of ‘superinfection’ under certain environmental conditions. In summary, the realization of several laboratory‐derived, a priori expectations suggests important natural impacts of defensive symbionts in host‐enemy eco‐evolutionary feedbacks. Yet negative findings and unanticipated correlations suggest complexities within this system may limit or obscure symbiont‐driven contemporary evolution, a finding of broad significance given the widespread nature of defensive microbes across plants and animals.

Kinetic Identification of Membrane Transporters That Assist P-glycoprotein-Mediated Transport of Digoxin and Loperamide through a Confluent Monolayer of MDCKII-hMDR1 Cells

A robust screen for compound interaction with P-glycoprotein (P-gp) has some obvious requirements, such as a cell line expressing P-gp and a probe substrate that is transported solely by P-gp and passive permeability. It is actually difficult to prove that a particular probe substrate interacts only with P-gp in the chosen cell line. Using a confluent monolayer of MDCKII-hMDR1 cells, we have determined the elementary rate constants for the P-gp efflux of amprenavir, digoxin, loperamide, and quinidine. For amprenavir and quinidine, transport was fitted with just P-gp and passive permeability. For digoxin and loperamide, fitting required a basolateral transporter (p < 0.01), which was inhibited by the P-gp inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). This means that when digoxin is used as a probe substrate and a compound is shown to inhibit digoxin flux, it could be that the inhibition occurs at the basolateral transporter rather than at P-gp. Digoxin basolateral>apical efflux also required an apical importer (p < 0.05). We propose that amprenavir and quinidine are robust probe substrates for assessing P-gp interactions using the MDCKII-hMDR1 confluent cell monolayer. Usage of another cell line, e.g., LLC-hMDR1 or Caco-2, would require the same kinetic validation to ensure that the probe substrate interacts only with P-gp. Attempts to identify the additional digoxin and loperamide transporters using a wide range of substrates/inhibitors of known epithelial transporters (organic cation transporters, organic anion transporters, organic ion-transporting polypeptide, uric acid transporter, or multidrug resistance-associated protein) failed to inhibit the digoxin or loperamide transport through their basolateral transporter.

Biophysical constraints on the thermal ecology of dinosaurs

A physical, model-based approach to body temperatures in dinosaurs allows us to pre- dict what ranges of body temperatures and what thermoregulatory strategies were available to those dinosaurs. We argue that 1. The huge range of body sizes in the dinosaurs likely resulted in very different thermal problems and strategies for animals at either end of this size continuum. 2. Body temperatures of the smallest adult dinosaurs and of hatchlings and small juveniles would have been largely insensitive to metabolic rates in the absence of insulation. The smallest an- imals in which metabolic heating resulted in predicted body temperatures 2 2?C above oper- ative temperatures (TJ) weigh 10 kg. Body temperature would respond rapidly enough to

An AICD-based functional screen to identify APP metabolism regulators

BackgroundA central event in Alzheimers disease (AD) is the regulated intramembraneous proteolysis of the β-amyloid precursor protein (APP), to generate the β-amyloid (Aβ) peptide and the APP intracellular domain (AICD). Aβ is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered.ResultsTo validate this experimental approach we modulated α-, β-, and γ-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis.ConclusionTaken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y.

Phylogeny, Regression, and the Allometry of Physiological Traits

Physiological and ecological allometries often pose linear regression problems characterized by (1) noncausal, phylogenetically autocorrelated independent (x) and dependent (y) variables (characters); (2) random variation in both variables; and (3) a focus on regression slopes (allometric exponents). Remedies for the phylogenetic autocorrelation of species values (phylogenetically independent contrasts) and variance structure of the data (reduced major axis [RMA] regression) have been developed, but most functional allometries are reported as ordinary least squares (OLS) regression without use of phylogenetically independent contrasts. We simulated Brownian diffusive evolution of functionally related characters and examined the importance of regression methodologies and phylogenetic contrasts in estimating regression slopes for phylogenetically constrained data. Simulations showed that both OLS and RMA regressions exhibit serious bias in estimated regression slopes under different circumstances but that a modified orthogonal (least squares variance‐oriented residual [LSVOR]) regression was less biased than either OLS or RMA regressions. For strongly phylogenetically structured data, failure to use phylogenetic contrasts as regression data resulted in overestimation of the strength of the regression relationship and a significant increase in the variance of the slope estimate. Censoring of data sets by simulated extinction of taxa did not affect the importance of appropriate regression models or the use of phylogenetic contrasts.

Potential Hematological and Biochemical Indicators of Stress in Free-Ranging Desert Tortoises and Captive Tortoises Exposed to a Hydric Stress Gradient

Hematologic and plasma biochemical parameters were monitored in free-ranging desert tortoises, Gopherus agassizii, near Las Vegas, NV and on a population of captive tortoises maintained in 4 ha pens with natural physiography and vegetation and subjected to varying levels of water supplementation to mimic different levels of hydric stress. Significant increases in plasma electrolyte and urea nitrogen concentrations and white blood cell counts (compared to free-ranging and water-supplemented, captive animals) occurred in captive animals without water supplementation. Differences in electrolytes and BUN among treatments were consistent with altered water balance. Free-ranging animals received no supplemental water. Blood profiles from free-ranging animals, however, more closely resembled profiles from captive animals that received supplemental water than profiles from animals that did not receive supplemental water. Captivity, thus, altered blood profiles in a manner similar to that seen with decreased water availability. The electrolyte, urea nitrogen, and white blood cell differences among water supplementation treatments, while significant, were not sufficient to distinguish among animals from different treatments. No reliably predictive indicators of water stress-or other stresses-were found. Several biochemical assays suggested specific stresses (other than water stress) which some of the tortoises faced. We suggest that blood profiles might be more profitably used to search for specific hypothesized stressors in populations rather than as assays of unspecified generic stress on populations of tortoises.

Application of receiver operating characteristic analysis to refine the prediction of potential digoxin drug interactionss

In the 2012 Food and Drug Administration (FDA) draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits P-glycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when the maximum concentration of inhibitor at steady state divided by IC50 ([I1]/IC50) is ≥0.1 or concentration of inhibitor based on highest approved dose dissolved in 250 ml divide by IC50 ([I2]/IC50) is ≥10. In this article, refined criteria are presented, determined by receiver operating characteristic analysis, using IC50 values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for P-gp overexpressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this article take into account variability in IC50 values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of [I1]/IC50 ≥ 0.03 and [I2]/IC50 ≥ 45 and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, whereas the number of false positives (DDI predicted but not observed) substantially decreased from 51 to 40%, relative to the FDA criteria. On the basis of estimated overall variability in IC50 values, a theoretical 95% confidence interval calculation was developed for single laboratory IC50 values, translating into a range of [I1]/IC50 and [I2]/IC50 values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, attributable to variability in IC50 values.