Coliform dynamics and the implications for source tracking

In many parts of the world, coliform counts in recreational waters are unacceptably high. In an attempt to rectify this problem, programmes are under way to develop methods that will allow the sources of the faecal contamination thought to be responsible for these elevated counts to be identified. The success of these efforts depends on the validity of several assumptions that underlie many of the proposed methods. One of the critical assumptions is that the clonal composition of the coliform species being monitored in a water body reflects the clonal composition of the species in the host populations responsible for the faecal inputs into that water body. To determine the extent to which among-strain variation in a coliform species might invalidate this assumption, a series of simple mathematical models was proposed and analysed. The first series of models assumed that all cells of species were identical. The question posed was - is the density of a coliform species in a body of water linearly related to the rate at which cells of the species enter the water body via faecal production? The results of these models suggest that, over a wide range of conditions, cell densities in the water body are linearly related to the rate at which cells enter the water body as a result of faecal contamination. This outcome occurs whether or not cells are capable of division in the external environment. When the rate of cell division depends on the concentration of available nutrients then, when nutrient input rates are 'high' and rates of faecal contamination are 'low', this linear relationship does not hold. The second series of models assumed that the coliform species consists of different strains and that these strains differ in their performance in the external environment. The results of these multistrain models show that the relative abundance of strains in the external environment is unlikely to reflect their relative abundance in the faecal inputs to the environment. Consequently, statements such as - domestic animals are responsible for 30% and wildlife for 70% of the faecal inputs to a water body - may well be meaningless.     

Auto-validation of fluorescent primer extension genotyping assay using signal clustering and neural networks

Background

SNP genotyping typically incorporates a review step to ensure that the genotype calls for a particular SNP are correct. For high-throughput genotyping, such as that provided by the GenomeLab SNPstream^® instrument from Beckman Coulter, Inc., the manual review used for low-volume genotyping becomes a major bottleneck. The work reported here describes the application of a neural network to automate the review of results.

Results

We describe an approach to reviewing the quality of primer extension 2-color fluorescent reactions by clustering optical signals obtained from multiple samples and a single reaction set-up. The method evaluates the quality of the signal clusters from the genotyping results. We developed 64 scores to measure the geometry and position of the signal clusters. The expected signal distribution was represented by a distribution of a 64-component parametric vector obtained by training the two-layer neural network onto a set of 10,968 manually reviewed 2D plots containing the signal clusters.

Conclusion

The neural network approach described in this paper may be used with results from the GenomeLab SNPstream instrument for high-throughput SNP genotyping. The overall correlation with manual revision was 0.844. The approach can be applied to a quality review of results from other high-throughput fluorescent-based biochemical assays in a high-throughput mode.     

The "Goldilocks Effect" in Cystic Fibrosis: identification of a lung phenotype in the <Emphasis Type="Italic">cftr</Emphasis> knockout and heterozygous mouse

Background  

Cystic Fibrosis is a pleiotropic disease in humans with primary morbidity and mortality associated with a lung disease phenotype. However, knockout in the mouse of cftr, the gene whose mutant alleles are responsible for cystic fibrosis, has previously failed to produce a readily, quantifiable lung phenotype.     

Kin or self‐recognition? Colonial fusibility of the bryozoan Celleporella hyalina

We estimated fusion frequency with respect to coancestry in the bryozoan Celleporella hyalina, whose briefly planktonic sexually produced larvae settle on algal substrata and proceed to form encrusting colonies by iterative budding. Frequency of fusion between paired colonies growing on an artificial substratum was positively correlated with coefficient of relatedness, with allorecognition ability increasing during the early stages of colonial growth after larval settlement. Parents repressed the growth of F1 progeny with which they had fused. The results are concordant with the Feldgarden-Yund model of selection for self-recognition, which regards fusion with kin as an inevitable source of error whose cost diminishes with increasing relatedness. Contrary to fusion compatibility, gametic compatibility is negatively correlated with coancestry, indicating a need for further research on the possibility of common or linked genetic control that has opposite effect at somatic and gametic levels.     

Blood oxygen transport in common map turtles during simulated hibernation

We assessed the effects of cold and submergence on blood oxygen transport in common map turtles (Graptemys geographica). Winter animals were acclimated for 6-7 wk to one of three conditions at 3 degrees C: air breathing (AB-3 degrees C), normoxic submergence (NS-3 degrees C), and hypoxic (PO2=49 Torr) submergence (HS-3 degrees C). NS-3 degrees C turtles exhibited a respiratory alkalosis (pH 8.07; PCO2=7.9 Torr; [lactate]=2.2 mM) relative to AB-3 degrees C animals (pH 7.89; PCO2=13.4 Torr; [lactate]=1.1 mM). HS-3 degrees C animals experienced a profound metabolic acidosis (pH 7.30; PCO2=7.9 Torr; [lactate]=81 mM). NS-3 degrees C turtles exhibited an increased blood O2 capacity; however, isoelectric focusing revealed no seasonal changes in the isohemoglobin (isoHb) profile. Blood O2 affinity was significantly increased by cold acclimation; half-saturation pressures (P50's) for air-breathing turtles at 3 degrees and 22 degrees C were 6.5 and 18.8 Torr, respectively. P50's for winter animals submerged in normoxic and hypoxic water were 5.2 and 6.5 Torr, respectively. CO2 Bohr slopes (Delta logP50/Delta pH) were -0.15, -0.16, and -0.07 for AB-3 degrees C, NS-3 degrees C, and HS-3 degrees C turtles, respectively; the corresponding value for AB-22 degrees C was -0.37. The O2 equilibrium curve (O2EC) shape was similar for AB-3 degrees C and NS-3 degrees C turtles; Hill plot n coefficients ranged from 1.8 to 2.0. The O2EC shape for HS-3 degrees C turtles was anomalous, exhibiting high O2 affinity below P50 and a right-shifted segment above half-saturation. We suggest that increases in Hb-O2 affinity and O2 capacity enhance extrapulmonary O2 uptake by turtles overwintering in normoxic water. The anomalous O2EC shape and reduced CO2 Bohr effect of HS-3 degrees C turtles may also promote some aerobic metabolism in hypoxic water.     

The evolution of endothermy and its diversity in mammals and birds

Many elements of mammalian and avian thermoregulatory mechanisms are present in reptiles, and the changes involved in the transition to endothermy are more quantitative than qualitative. Drawing on our experience with reptiles and echidnas, we comment on that transition and on current theories about how it occurred. The theories divide into two categories, depending on whether selection pressures operated directly or indirectly on mechanisms producing heat. Both categories of theories focus on explaining the evolution of homeothermic endothermy but ignore heterothermy. However, noting that hibernation and torpor are almost certainly plesiomorphic (=ancestral, primitive), and that heterothermy is very common among endotherms, we propose that homeothermic endothermy evolved via heterothermy, with the earliest protoendotherms being facultatively endothermic and retaining their ectothermic capacity for "constitutional eurythermy." Thus, unlike current models for the evolution of endothermy that assume that hibernation and torpor are specialisations arising from homeothermic ancestry, and therefore irrelevant, we consider that they are central. We note the sophistication of thermoregulatory behavior and control in reptiles, including precise control over conductance, and argue that brooding endothermy seen in some otherwise ectothermic Boidae suggests an incipient capacity for facultative endothermy in reptiles. We suggest that the earliest insulation in protoendotherms may have been internal, arising from redistribution of the fat bodies that are typical of reptiles. We note that short-beaked echidnas provide a useful living model of what an (advanced) protoendotherm may have been like. Echidnas have the advantages of endothermy, including the capacity for homeothermic endothermy during incubation, but are very relaxed in their thermoregulatory precision and minimise energetic costs by using ectothermy facultatively when entering short- or long-term torpor. They also have a substantial layer of internal dorsal insulation. We favor theories about the evolution of endothermy that invoke direct selection for the benefits conferred by warmth, such as expanding daily activity into the night, higher capacities for sustained activity, higher digestion rates, climatic range expansion, and, not unrelated, control over incubation temperature and the benefits for parental care. We present an indicative, stepwise schema in which observed patterns of body temperature are a consequence of selection pressures, the underlying mechanisms, and energy optimization, and in which homeothermy results when it is energetically desirable rather than as the logical endpoint.     

The greatest step in vertebrate history: a paleobiological review of the fish-tetrapod transition

Recent discoveries of previously unknown fossil forms have dramatically transformed understanding of many aspects of the fish-tetrapod transition. Newer paleobiological approaches have also contributed to changed views of which animals were involved and when, where, and how the transition occurred. This review summarizes major advances made and reevaluates alternative interpretations of important parts of the evidence. We begin with general issues and concepts, including limitations of the Paleozoic fossil record. We summarize important features of paleoclimates, paleoenvironments, paleobiogeography, and taphonomy. We then review the history of Devonian tetrapods and their closest stem group ancestors within the sarcopterygian fishes. It is now widely accepted that the first tetrapods arose from advanced tetrapodomorph stock (the elpistostegalids) in the Late Devonian, probably in Euramerica. However, truly terrestrial forms did not emerge until much later, in geographically far-flung regions, in the Lower Carboniferous. The complete transition occurred over about 25 million years; definitive emergences onto land took place during the most recent 5 million years. The sequence of character acquisition during the transition can be seen as a five-step process involving: (1) higher osteichthyan (tetrapodomorph) diversification in the Middle Devonian (beginning about 380 million years ago [mya]), (2) the emergence of "prototetrapods" (e.g., Elginerpeton) in the Frasnian stage (about 372 mya), (3) the appearance of aquatic tetrapods (e.g., Acanthostega) sometime in the early to mid-Famennian (about 360 mya), (4) the appearance of "eutetrapods" (e.g., Tulerpeton) at the very end of the Devonian period (about 358 mya), and (5) the first truly terrestrial tetrapods (e.g., Pederpes) in the Lower Carboniferous (about 340 mya). We discuss each of these steps with respect to inferred functional utility of acquired character sets. Dissociated heterochrony is seen as the most likely process for the evolutionarily rapid morphological transformations required. Developmental biological processes, including paedomorphosis, played important roles. We conclude with a discussion of phylogenetic interpretations of the evidence.     

Bluegill (Lepomis macrochirus) vitellogenin: purification and enzyme-linked immunosorbent assay for detection of endocrine disruption by papermill effluent

Vitellogenin (VTG) is a highly specific marker of exposure to environmental estrogens and has been used extensively in field and laboratory studies of estrogenic endocrine disruption in fishes. The purpose of this study was to develop and validate a sensitive, competitive, enzyme-linked immunosorbent assay (ELISA) specific for bluegill (Lepomis macrochirus) vitellogenin. Bluegill VTG was purified by anion exchange chromatography on DEAE-agarose. The polypeptide had an apparent mass of 170 kDa and was specifically recognized by the rabbit antiserum raised against bluegill female-specific plasma protein. Plasma samples from vitellogenic females diluted in parallel with the purified VTG standard curve in the ELISA. The detection limit of the assay was 29 ng/ml and the working range extended to 2700 ng/ml. Recovery of purified VTG was 85.8+/-9.5%, intra-assay variation was 6.4% and interassay variation was 12.3%. We used this ELISA to analyze the seasonal cycle of vitellogenesis in female bluegill and to evaluate potential disruption of this process by exposure to bleached kraft mill effluent (BKME). Captive female bluegill stocked in outdoor experimental streams in New Bern, NC had the lowest levels of VTG, estradiol-17beta (E2), and testosterone (T) and the smallest oocyte diameters in January, but these variables increased in March and remained elevated through August, suggesting an extended spawning season. Plasma VTG, E2, T and oocyte diameter were unaffected by exposure to BKME concentrations as high as 30%. Development of the VTG ELISA allowed rapid and convenient analysis of plasma samples to evaluate exposure to potential endocrine disrupting compounds.     

The consequences of exhaustive antiestrogen therapy in breast cancer: estrogen-induced tumor cell death

Forty years ago, the endocrine treatment for breast cancer was a last resort at palliation before the disease overwhelmed the patient (1). Ovarian ablation was the treatment of choice for the premenopausal patient, whereas either adrenalectomy or, paradoxically, high-dose synthetic estrogen therapy were used for treatment in postmenopausal patients. A reduction or an excess of estrogen provoked objective responses in one out of three women. Unfortunately, there was no way of predicting who would respond to endocrine ablation, and because so few patients responded there was no enthusiasm for developing new endocrine agents. All hopes for a cure for breast cancer turned to appropriate combinations of cytotoxic chemotherapy. Today tamoxifen, a nonsteroidal antiestrogen (2), has proven to be effective in all stages of premenopausal and postmenopausal breast cancer, and several new endocrine strategies, including aromatase inhibitors, luteinizing-hormone releasing hormone (LHRH) superagonists, and a pure antiestrogen (fulvestrant), are now available for breast cancer treatment. Additionally, tamoxifen and raloxifene, a related compound, are used to reduce the risk of breast cancer and osteoporosis, respectively, in high-risk groups (3). Hormonal modulation and strategies to prevent the actions of estrogen in the breast are ubiquitous. However, with successful changes in treatment strategies comes the consequence of change. This minireview will describe the current strategies for the treatment and prevention of breast cancer and present emerging new concepts about the consequences of exhaustive antiestrogen treatment on therapeutic resistance.