Survival by genotype: patterns at Mc1r are not black and white at the White Sands ecotone.

Measuring links among genotype, phenotype and survival in the wild has long been a focus of studies of adaptation. We conducted a 4-year capture-recapture study to measure survival by genotype and phenotype in the Southwestern Fence Lizard (Sceloporus cowlesi) at the White Sands ecotone (transition area between white sands and dark soil habitats). We report several unanticipated findings. First, in contrast with previous work showing that cryptic blanched coloration in S. cowlesi from the heart of the dunes is associated with mutations in the melanocortin-1 receptor gene (Mc1r), ecotonal S. cowlesi showed minimal association between colour phenotype and Mc1r genotype. Second, the frequency of the derived Mc1r allele in ecotonal S. cowlesi appeared to decrease over time. Third, our capture-recapture data revealed a lower survival rate for S. cowlesi individuals with the derived Mc1r allele. Thus, our results suggest that selection at the ecotone may have favoured the wild-type allele in recent years. Even in a system where a genotype-phenotype association appeared to be black and white, our study suggests that additional factors - including phenotypic plasticity, epistasis, pleiotropy and gene flow - may play important roles at the White Sands ecotone. Our study highlights the importance of linking molecular, genomic and organismal approaches for understanding adaptation in the wild. Furthermore, our findings indicate that dynamics of natural selection can be particularly complex in transitional habitats like ecotones and emphasize the need for future research that examines the patterns of ongoing selection in other ecological 'grey' zones.

Comparative study of host response to chytridiomycosis in a susceptible and a resistant toad species.

In the past century, recently emerged infectious diseases have become major drivers of species decline and extinction. The fungal disease chytridiomycosis has devastated many amphibian populations and exacerbated the amphibian conservation crisis. Biologists are beginning to understand what host traits contribute to disease susceptibility, but more work is needed to determine why some species succumb to chytridiomycosis while others do not. We conducted an integrative laboratory experiment to examine how two toad species respond to infection with the pathogen Batrachochytrium dendrobatidis in a controlled environment. We selected two toad species thought to differ in susceptibility - Bufo marinus (an invasive and putatively resistant species) and Bufo boreas (an endangered and putatively susceptible species). We measured infection intensity, body weight, histological changes and genomewide gene expression using a custom assay developed from transcriptome sequencing. Our results confirmed that the two species differ in susceptibility with the more susceptible species, B. boreas, showing higher infection intensities, loss in body weight, more dramatic histological changes and larger perturbations in gene expression. We found key differences in skin expression responses in multiple pathways including upregulation of skin integrity-related genes in the resistant B. marinus. Together, our results show intrinsic differences in host response between related species, which are likely to be important in explaining variation in response to a deadly emerging pathogen in wild populations. Our study also underscores the importance of understanding differences among host species to better predict disease outcomes and reveal generalities about host response to emerging infectious diseases of wildlife.

Insights From Genomics Into Spatial and Temporal Variation in Batrachochytrium dendrobatidis.

Advances in genetics and genomics have provided new tools for the study of emerging infectious diseases. Researchers can now move quickly from simple hypotheses to complex explanations for pathogen origin, spread, and mechanisms of virulence. Here we focus on the application of genomics to understanding the biology of the fungal pathogen Batrachochytrium dendrobatidis (Bd), a novel and deadly pathogen of amphibians. We provide a brief history of the system, then focus on key insights into Bd variation garnered from genomics approaches, and finally, highlight new frontiers for future discoveries. Genomic tools have revealed unexpected complexity and variation in the Bd system suggesting that the history and biology of emerging pathogens may not be as simple as they initially seem.

Crossing the threshold: gene flow, dominance and the critical level of standing genetic variation required for adaptation to novel environments.

Genetic architecture plays an important role in the process of adaptation to novel environments. One example is the role of allelic dominance, where advantageous recessive mutations have a lower probability of fixation than advantageous dominant mutations. This classic observation, termed 'Haldane's sieve', has been well explored theoretically for single isolated populations adapting to new selective regimes. However, the role of dominance is less well understood for peripheral populations adapting to novel environments in the face of recurrent and maladaptive gene flow. Here, we use a combination of analytical approximations and individual-based simulations to explore how dominance influences the likelihood of adaptation to novel peripheral environments. We demonstrate that in the face of recurrent maladaptive gene flow, recessive alleles can fuel adaptation only when their frequency exceeds a critical threshold within the ancestral range.

Ecological release in White Sands lizards.

Ecological opportunity is any change that allows populations to escape selection from competition and predation. After encountering ecological opportunity, populations may experience ecological release: enlarged population size, broadened resource use, and/or increased morphological variation. We identified ecological opportunity and tested for ecological release in three lizard colonists of White Sands, New Mexico (Sceloporus undulatus, Holbrookia maculata, and Aspidoscelis inornata). First, we provide evidence for ecological opportunity by demonstrating reduced species richness and abundance of potential competitors and predators at White Sands relative to nearby dark soils habitats. Second, we characterize ecological release at White Sands by demonstrating density compensation in the three White Sands lizard species and expanded resource use in White Sands S. undulatus. Contrary to predictions from ecological release models, we observed directional trait change but not increased trait variation in S. undulatus. Our results suggest that ecological opportunity and ecological release can be identified in natural populations, especially those that have recently colonized isolated ecosystems.

Male territoriality and 'sex confusion' in recently adapted lizards at White Sands.

The evolution of intersexual interactions, like mate choice, during ecological speciation has received widespread attention. However, changes in intrasexual interactions, like male territoriality, during ecological divergence are largely unexamined. We conducted field experiments with adaptively diverged populations of the eastern fence lizard (Sceloporus undulatus) to determine whether territorial males behaved differently towards ecologically similar vs. dissimilar intruders. We performed trials with light-coloured males from White Sands, New Mexico and dark-coloured males from the surrounding desert. We found that intruders from White Sands elicited more aggression than intruders from dark-soil habitat. We also documented a case of 'sex confusion' where white-sand males courted dark-soil intruders. We found population differences in signalling patch size that can explain both aggression bias and sex misidentification. We argue that direct selection (for population recognition or optimal signal transmission) and indirect selection (by-products of ecological adaptation) should influence both intersexual and intrasexual interactions during ecological speciation.

A pediatric acute wound service: a novel approach in wound management.

INTRODUCTION: In 2001, in response to an overwhelming increase in patient visits for various pediatric abscesses, burns, and other wounds, an ambulatory burn and procedural sedation program (Pediatric Acute Wound Service, or PAWS) was developed to minimize operating room utilization. The purpose of this study is to report our initial 7-year experience with the PAWS program. METHODS: The hospital records of all children managed through PAWS from 2001 to 2007 were reviewed. Outcomes measured include patient demographics, number and location of visits per patient, procedure information, cause of wounds, and reimbursement. chi(2) test and linear regression were performed using GraphPad Prism (GraphPad Software Inc, San Diego, CA). RESULTS: Overall, 7620 children (age 0-18 years) received wound care through PAWS from 2001 to 2007. There were no differences in patient age, race, and sex during this time period. Between 2001 and 2007, the percentage of patients seen as outpatients increased from 51% to 68% (P < .05), and the average number of visits per patient decreased from 3.9 to 2.4 (P = .05). In, 2007, 46% of the children required only 1 visit. In 2007, 74% of the visits were for management of wound and soft tissue infections, compared with only 9% in 2001 (P < .05). The contribution margin of a PAWS visit and total contribution margin in 2007 were $1052 and $4.0 million, respectively. CONCLUSION: The creation of PAWS has allowed for the transition in management of most pediatric skin and soft tissue wounds and infections to an independent ambulatory setting, alleviating the need for operating room resources, while functioning at a profitable cost margin for the hospital.

Oligomeric structures of poliovirus polymerase are important for function.

Central to the replication of poliovirus and other positive-strand RNA viruses is the virally encoded RNA-dependent RNA polymerase. Previous biochemical studies have suggested that direct polymerase- polymerase interactions might be important for polymerase function, and the structure of poliovirus polymerase has revealed two regions of extensive polymerase-polymerase interaction. To explore potential functional roles for the structurally observed polymerase-polymerase interactions, we have performed RNA binding and extension studies of mutant polymerase proteins in solution, disulfide cross-linking studies, mutational analyses in cells, in vitro activity analyses and RNA substrate modeling studies. The results of these studies indicate that both regions of polymerase-polymerase interaction observed in the crystals are indeed functionally important and, furthermore, reveal specific functional roles for each. One of the two regions of interaction provides for efficient substrate RNA binding and the second is crucial for forming catalytic sites. These studies strongly support the hypothesis that the polymerase- polymerase interactions discovered in the crystal structure provide an exquisitely detailed structural context for poliovirus polymerase function and for poliovirus RNA replication in cells.

Optimized method for the determination of phosphoarginine in abalone tissue by high-performance liquid chromatography.

A rapid high-performance liquid chromatography method for the determination of phosphoarginine (PArg) in invertebrate tissue has been redeveloped and validated. The method employs a reversed-phase amino column and a KH2PO4-acetonitrile mobile phase. PArg peak identity was confirmed by comparison with a known standard and via enzymatic conversion. Additionally linear calibration data, low intra-assay variability (<4%), and a detection limit of 5 pmol were determined. The method was demonstrated using PArg extracted from red abalone (Haliotis rufescens) adductor muscle. Validation of the extraction procedure was also completed, including the measurement of a 100.2+/-0.9% extraction efficiency.

Aging, mating, and the evolution of mtDNA heteroplasmy in Drosophila melanogaster.

Heteroplasmy, the presence of more than one type of mtDNA within cells, is common in animals and has been associated with aging and disease in humans. Changes in the frequencies of mtDNA variants between cell and animal generations thus bears on the evolution of mtDNA and the progression of diverse pathologies. We have used densitometry of Southern blots of individual heteroplasmic Drosophila melanogaster to study the effects of age, increased egg production after mating, and temperature on evolution of heteroplasmy within and between generations. The frequency of the longer mtDNA variant consistently increased between early and late cohorts of F1 offspring derived from 18 independent heteroplasmic mothers as they aged. Neither temperature (flies maintained at 25 degrees C and 18 degrees C) nor the holding of flies as virgins for 10 days before mating had significant effects on transmission patterns. However, at the ends of their lives, flies that had laid eggs at 25 degrees C had a greater frequency of the long mtDNA than did their siblings who had laid eggs at 18 degrees C. The evolution of heteroplasmy within a generation was studied in samples of siblings that either were mated or held as virgins, and then scored for mtDNA haplotype frequencies at two different ages (day 2 and day 14). Mated flies showed a significantly greater increase in the frequency of the long mtDNA variant with age than did the unmated flies. This system provides a model for the joint analysis of generational and chronological age in the transmission dynamics of a molecular polymorphism.

The phytoestrogen congeners of alcoholic beverages: current status.

The idea that alcoholic beverages might contain biologically active phytoestrogenic congeners stemmed from findings of overt feminization observed in alcoholic men with alcohol-induced cirrhosis. Specifically, in addition to being hypogonadal, these chronically alcohol-abusing men with cirrhosis frequently manifest gynecomastia, palmar erythema, spider angiomata, and a female escutcheon. These physical signs of exposure to active estrogen occur in the presence of normal or only minimally elevated levels of endogenous steroid estrogens. Because levels of circulating steroid hormones failed to provide a satisfactory explanation for the feminization observed, alternate explanations were considered. If the estrogenization observed was not entirely a function of tissue expose to steroid estrogens produced endogenously, then perhaps tissues were being exposed to exogenous estrogenic substances from dietary sources. Given the degree of alcohol abuse in the population in which hypotheses for feminization were being formed, alcoholic beverages became a prime candidate as a dietary source of exogenous estrogenic substances.

Reoxygenation injury in isolated rat hepatocytes: relation to oxygen free radicals and lipid peroxidation.

Oxygen free radical (OFR) formation and lipid peroxidation (LP) were measured in freshly isolated perfused rat hepatocytes during 2-h reoxygenation after 2.5 h of anoxia. Superoxide anions and hydrogen peroxide (H2O2) were detected by enhanced chemiluminescence. LP and cell damage were assessed by measuring malondialdehyde (MDA) and lactic dehydrogenase (LDH) release, respectively. During anoxia, the chemiluminescence decreased to background levels and MDA remained constant, whereas LDH release increased progressively to 168 +/- 22 mU/min in 2.5 h. During reoxygenation after a 2.5-h period of anoxia, superoxide formation increased rapidly to 125 +/- 16 nA and then it declined progressively toward the control level. At the same time, H2O2 production exhibited a biphasic pattern with an initial peak reaching 78 +/- 16 nA at 15.5 +/- 1 min, followed by a slower increase to 92 +/- 14 nA during the 2nd h. LDH release increased from 168 +/- 22 to 286 +/- 32 mU/min in the first 30 min of reoxygenation and then declined toward the control rate during the 2nd h. MDA release increased continuously from 1.16 +/- 0.18 to 7.75 +/- 0.74 pmol/min. OFR generation occurred 15-30 min before the peak rise in LDH. Moreover, after shorter periods of anoxia (1-2 h), hepatocytes produced measurable amount of OFR but without a significant increase in LDH release. These results demonstrate that 1) isolated liver parenchymal cells generate measurable amounts of superoxide anions and of H2O2 during reoxygenation after 1-2.5 h of anoxia, 2) lipid peroxidation follows the formation of OFR, and 3) reoxygenation injury is correlated to OFR generation but not to lipid peroxidation.

Assessment of the estrogenic activity of phytoestrogens isolated from bourbon and beer.

Phytoestrogenic substances have previously been isolated and identified in two alcoholic beverages: bourbon and beer. To delineate the relative potencies of the estrogenic substances of plant origin thus far identified in these commonly consumed alcoholic beverages, we evaluated the ability of biochanin A, beta-sitosterol, genistein, and daidzein to bind to cytosolic estrogen receptor binding sites. The in vitro studies demonstrated that each of the contained substances was capable of effectively competing for cytosolic estrogen receptor binding sites of rat liver and uterus. Further, the two phytoestrogenic constituents of bourbon, beta-sitosterol and biochanin A, were less potent than those present in beer. Given the high concentration of beta-sitosterol in bourbon, we chose to evaluate the estrogenicity of beta-sitosterol in vivo using ovariectomized rats. beta-sitosterol was administered either daily or intermittently at 3 doses, based on amounts previously determined to be present in bourbon. The in vivo studies demonstrated that beta-sitosterol is capable of producing a weak estrogenic effect only at the lowest dose (6.2 micrograms/dl) administered intermittently. These responses suggest that beta-sitosterol may be weakly estrogenic at low doses, but is unable to maintain such an effect at higher doses.

Effect of ethanol on Kupffer cell function.

Kupffer cells are resident macrophages in the liver and are important in both local and systemic immune responses. We evaluated the ability of Kupffer cells in vitro to respond to immune stimulation after both acute exposure to ethanol and after long-term ethanol consumption of ethanol. Triplets of female Wistar rats were fed a liquid diet containing 0, 12, or 36% ethanol isocalorically for 112 days. When killed, the Kupffer cells were isolated by collagenase perfusion and adhered to plastic 24-well plates. They were then stimulated with 10 micrograms/ml lipopolysaccharide for 4.5 hr. Synthesis of procoagulant activity (PCA) and tumor necrosis factor (TNF), expressions of macrophage response to immune stimuli, were measured by a one-step clotting assay and L929 cytotoxicity assay, respectively. Within each of the 10 triplets, PCA and TNF levels were normalized and expressed as a percentage of the zero ethanol isocaloric control rat. The high ethanol group had significantly lower baseline and stimulated PCA and TNF levels than the low ethanol group. For evaluation of the effect of acute exposure to ethanol, Kupffer cells were stimulated with lipopolysaccharide and varying concentrations (0-400 mg/dl) of ethanol. Cells were incubated for 4.5 hr and assayed for PCA and TNF activity. There was dose-dependent inhibition of PCA and TNF, with increasing concentrations of ethanol. These results indicate that whereas exposure to high levels of ethanol depresses Kupffer cell function, lower levels may be immunostimulatory.

"Binge" versus steady drinking: effects on the liver in the ovariectomized rat.

Substantial interest exists as to whether or not differential effects in liver injury based on the pattern of alcohol intake exist; and further, if they do, are they simply a function of the total dose over time. A rat model in which ethanol (ETOH) at doses of 12%, 24%, or 36% of total calories was isocalorically administered for 4 months either daily or intermittently (4 days of ETOH, 3 days of control diet, repeatedly) was used to assess this question. There were significant differences in the two feeding pattern groups between 36% ETOH rats for the liver weight corrected for body weight, the fat infiltration score, the total amount of ETOH consumed/mg body weight, the proportion of animals with a fat infiltration score > 2, and albumin levels. There was a significant difference between 12% ETOH rats for the liver weight corrected for body weight. Of particular relevance is the comparison to be made between Daily 12% ETOH and Binge 24% ETOH animals, because these two groups consumed an identical total amount of ETOH/mg body weight (Daily: 445 +/- 5 vs. Binge: 468 +/- 15) and thus these animals are comparable in terms of ETOH dose over time but different in terms of the pattern of ETOH exposure. There were no differences in the liver/body ratio (Daily: 235 +/- 6 vs. Binge: 232 +/- 4), fat infiltration score (Daily: 2.5 +/- 4 vs. Binge: 2.4 +/- 0.3), the proportion of animals with a fat infiltration score > 2 (Daily: 5/10 vs. Binge: 4/8), or albumin levels (Daily: 3.0 +/- 0.1 vs. Binge: 3.1 +/- 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and identification of phytoestrogens from beer.

Two estrogenic substances of plant origin have been identified in beer using gas chromatography/mass spectrometry. These phytoestrogens, daidzein and genistein, have previously been shown to be biologically active in animals. Confirming the presence of biologically active phytoestrogens in beer and their possible presence in other beverages, suggests that there may be clinically significant effects related to sustained exposure to phytoestrogens contained in alcoholic beverages.

The phytoestrogens present in de-ethanolized bourbon are biologically active: a preliminary study in a postmenopausal woman.

Bourbon and some other alcoholic beverages have been shown to contain phytoestrogens which, as a result of an excessive intake and alcohol associated liver disease, have been suggested as contributing to the feminization of chronic alcoholic men. To evaluate this hypothesis directly, de-ethanolized bourbon was prepared and orally administered to a single postmenopausal woman. Serum or plasma levels of luteinizing hormone, follicle stimulating hormone, sex steroid binding globulin, high density lipoprotein cholesterol, and estradiol were monitored before, during, and after administration of the de-ethanolized bourbon at a dose equivalent to 4 ounces of bourbon for 20 days. The changes observed for each parameter during the injection of the bourbon extract are consistent with the presence of a biologically active estrogenic substance being present in the bourbon extract. These observations provide the first direct evidence in man for the presence of a biologically active estrogenic substance(s) in bourbon.

Paroxysmal tremor and orofacial dyskinesia secondary to a biopterin synthesis defect.

We report a child with a systemic biopterin synthesis defect due to an absence of 6-pyruvoyl-tetrahydropterin synthase who had an unusual presentation, with three episodes of coarse "rubral-like" tremor in arms and legs orofacial dyskinesia between the ages of 3 and 6 months. Response to levodopa therapy and CSF neurotransmitter metabolite concentrations before and after therapy suggests that his clinical syndrome resulted from a secondary dopamine deficiency.

Quantitation of beta-sitosterol in bourbon.

Four phytoestrogens have previously been isolated and identified in alcoholic beverages. Before studies to evaluate the degree of estrogenicity of these substances can be performed, it is necessary to determine appropriate doses to be administered to experimental animals. Because beta-sitosterol is the most plentiful, being present in bourbon at microgram/dl quantities, we have chosen to begin our quantitation work with this compound. The variability of the amount of beta-sitosterol contained in various brands of bourbon and in different lots of the same brand of bourbon have been assessed using combined gas chromatography/mass spectrometry (GC/MS) for quantitation following a simplified procedure to extract compounds of interest from bourbon.

The effect of portal-systemic shunting on hepatic sex hormone receptors in male rats.

Signs of feminization are seen in men with cirrhosis of alcoholic but also of nonalcoholic origin even in the absence of markedly increased plasma estrogen levels. Recently identified alterations of hepatic sex hormone receptor levels have provided a hypothetical mechanism for the pathogenesis of the feminization seen in cirrhotic men. The aim of the present study was to determine the effect of experimental portal-systemic shunting in adult male rats on hepatic sex hormone receptor levels, plasma sex hormones, and two markers for sex hormone action in the liver. The following alterations were found in male rats with surgically created portacaval shunts compared with sham-operated controls: the hepatic content of cytosolic estrogen receptors was reduced by 35% and the cytosolic androgen receptors content by 59%; plasma levels of estradiol increased 6.7-fold while those of testosterone were reduced by 71%; the estrogen-responsive ceruloplasmin levels were decreased by 31% and the androgen-responsive male-specific estrogen binder by 72%. Based on these data, it can be concluded that portal-systemic shunting reduces the hepatic cytoplasmic content of several sex hormone related proteins. These changes are paralleled by a decreased estrogen responsiveness of the liver, as evidenced by the plasma ceruloplasmin level.