Cilia-driven rotational behavior in gastropod (Physa elliptica) embryos induced by serotonin and putative serotonin reuptake inhibitors (SSRIs)

We characterized the serotonin (5-hydroxytryptamine; 5-HT) receptor mediating cilia-driven rotational movement in embryos of the freshwater gastropod Physa elliptica. In addition, putative serotonin reuptake inhibitors (SSRIs), previously shown to induce other 5-HT-mediated processes in molluscs, were tested for their ability to induce rotation. As in previous studies with other freshwater gastropods, 5-HT induced a significant dose-dependent increase in rotation from 10(-6) to 10(-4) M. The 5-HT(1A) agonist 8-OH-DPAT produced a similar dose-dependent increase in rotation. However, the 5-HT(2) agonist alpha-CH3-serotonin evoked a significant rotational response only at the highest concentration of 10(-4) M. The 5-HT(2) receptor antagonist mianserin not only blocked 5-HT-induced rotation, it reduced rotation rate below that of baseline. However, two other antagonists, cyproheptadine (5-HT(2)) and propranolol (5-HT(1)), caused similar responses that consisted of an initial rotational surge followed by reduced rotation. Thus, these drugs appear to act as partial agonists. The putative SSRI fluvoxamine exhibited a significant dose-dependent increase in positive rotation as that seen with 5-HT. The SSRIs paroxetine and fluoxetine both caused an increase in rotation at 10(-6) and 10(-5) M but reduced rotation rate below that of baseline at 10(-4) M. These results agree with other studies on aquatic molluscs, suggest a 5-HT receptor with a mixed 5-HT(1)/5-HT(2) pharmacological profile and add to a now growing body of literature on the pharmacology of molluscan 5-HT receptors. In addition, all the tested putative SSRIs induced cilia-driven rotation in Physa embryos, indicating either the presence of 5-HT reuptake transporters or that these compounds act as 5-HT receptor ligands. J. Exp. Zool. 286:414-421, 2000.     

Where eagles soar: Fine‐resolution tracking reveals the spatiotemporal use of differential soaring modes in a large raptor

Unlike smaller raptors, which can readily use flapping flight, large raptors are mainly restricted to soaring flight due to energetic constraints. Soaring comprises of two main strategies: thermal and orographic soaring. These soaring strategies are driven by discrete uplift sources determined by the underlying topography and meteorological conditions in an area. High‐resolution GPS tracking of raptor flight allows the identification of these flight strategies and interpretation of the spatiotemporal occurrence of thermal and orographic soaring. In this study, we develop methods to identify soaring flight behaviors from high‐resolution GPS tracking data of Verreaux’s eagle Aquila verreauxii and analyze these data to understand the conditions that promote the use of thermal and orographic soaring. We use these findings to predict the use of soaring flight both spatially (across the landscape) and temporally (throughout the year) in two topographically contrasting regions in South Africa. We found that topography is important in determining the occurrence of soaring flight and that thermal soaring occurs in relatively flat areas which are likely to have good thermal uplift availability. The predicted use of orographic soaring was predominately determined by terrain slope. Contrary to our expectations, the topography and meteorology of eagle territories in the Sandveld promoted the use of soaring flight to a greater extent than in territories in the more mountainous Cederberg region. Spatiotemporal mapping of predicted flight behaviors can broaden our understanding of how large raptors like the Verreaux’s eagle use their habitat and how that links to energetics (as the preferential use of areas that maximize net energy gain is expected), reproductive success, and ultimately population dynamics. Understanding the fine‐scale landscape use and environmental drivers of raptor flight can also help to predict and mitigate potential detrimental effects of anthropogenic developments, such as mortality via collision with wind turbines.     

Assessment of an ultra-sensitive IFNγ immunoassay prototype for latent tuberculosis diagnosis

Worldwide there are about 1.7 billion individuals with latent tuberculosis infection (LTBI) and only 5% to 15% will develop active tuberculosis (TB). It is recommended to treat only those most at risk of developing active TB to avoid problems of drug resistance. LTBI diagnosis involves reviewing the individual’s medical history, physical examination, and biological tests. Interferon gamma release assays (IGRA) can yield “undeterminate” or “uncertain” results, which makes clinical management decisions difficult. We assessed an ultra-sensitive immunoassay prototype based on single molecule array (SiMoA) technology to evaluate its overall performance, and in particular, its performance for indeterminate and uncertain positive or negative samples, as classified by the results from the current ELISA technique used for IFNγ quantification. We analyzed samples from hospitalized or consulting patients and healthcare workers from three hospitals in Paris, previously classified as negative (n = 30), positive (n = 35), uncertain negative (n = 25), uncertain positive (n = 31), or indeterminate (n = 30). We observed that with the SiMoA assay 83.3% of the indeterminate samples became interpretable and could be classified as negative, whereas 74% of uncertain positive samples were classified as positive. Most uncertain negative samples (72%) were reclassified as uncertain positive (68%) or positive (4%). The results suggest that the ultra-sensitive SiMoA IFNγ assay could represent a useful tool for the identification of true positive and negative samples among those giving indeterminate or uncertain results with the TB IGRA assay currently used.     

Characterization of the Rust Fungus, Puccinia emaculata, and Evaluation of Genetic Variability for Rust Resistance in Switchgrass Populations

Several fungal pathogens have been identified on ornamental and native stands of switchgrass (Panicum virgatum L.). Diseases of switchgrass, particularly rust, have been largely neglected and are likely to become the major limiting factor to biomass yield and quality, especially when monocultured over a large acreage. Based on teliospore morphology and internal transcribed spacer-based diagnostic primers, the rust pathogen collected from switchgrass research fields in Oklahoma was identified as Puccinia emaculata. Furthermore, to identify genetically diverse source(s) of rust resistance, several switchgrass genotypes from both upland (cv. ‘Summer’ and ‘Cave-in-Rock’) and lowland (cv. ‘Alamo’ and ‘Kanlow’) ecotypes were evaluated in Ardmore, Oklahoma during 2008 and 2009 and in growth chamber assays. Field and growth chamber evaluations revealed a high degree of genetic variation within and among switchgrass cultivars. In general, Alamo and Kanlow showed moderate resistance to P. emaculata, while Summer was highly susceptible. Distinct ecotypic variations for reactions to rust were also prevalent with the lowlands maintaining a high level of resistance. These results suggest the potential for improvement of rust resistance via the selection of resistant individuals from currently available cultivars. Further, the selection pressure on the pathogen would also be reduced by employing several rust resistant cultivars in production-scale situations.     

Flexibility and constraints in the molt schedule of long‐distance migratory shorebirds: causes and consequences

Molt is a major component of the annual cycle of birds, the timing and extent of which can affect body condition, survival, and future reproductive success through carry‐over effects. The way in which molt is fitted into the annual cycle seems to be a somewhat neglected area which is both of interest and of importance. Study of the causes of annual variation in the timing of molt and its potential consequence in long‐distance migratory birds was examined using the Curlew Sandpiper, Calidris ferruginea, as a model species. Using the maximum likelihood molt models of Underhill and Zucchini (1988, Ibis 130:358–372), the relationship between annual variability in the start dates of molt at the population level with conditions on the breeding area was explored. Adult males typically started early in years when temperature in June on the Arctic breeding grounds were high compared to cold years while adult females molted later in years of high breeding success and/or warm July temperature and vice versa. When molt started later, the duration was often shorter, indicating that late completion of molt might have fitness consequences, probably jeopardizing survival. Evidence of this was seen in the low body condition of birds in years when molt was completed late. The results indicate that these migratory shorebirds follow a fine‐tuned annual life cycle, and disturbances at a certain stage can alter next biological events through carry‐over effects.     

Functional endemism: population connectivity,shifting baselines,and the scale of human experience

Quantifying population connectivity is important for visualizing the spatial and temporal scales that conservation measures act upon. Traditionally, migration based on genetic data has been reported in migrants per generation. However, the temporal scales over which this migration may occur do not necessarily accommodate the scales over which human perturbations occur, leaving the potential for a disconnect between population genetic data and conservation action based on those data. Here, we present a new metric called the “Rule of Memory”, which helps conservation practitioners to interpret “migrants per generation” in the context both of human modified ecosystems and the cultural memory of those doing the modification. Our rule states that clades should be considered functionally endemic regardless of their actual taxonomic designation if the migration between locations is insufficient to maintain a viable population over the timescales of one human generation (20 years). Since larger animals are more likely to be remembered, we quantify the relationship between migrants per human (N) and body mass of the organism in question (M) with the formula N = 10M⁻¹. We then use the coral reef fish Pomacentrus moluccensis to demonstrate the taxonomic and spatial scales over which this rule can be applied. Going beyond minimum viable population literature, this metric assesses the probability that a clade''s existence will be forgotten by people throughout its range during a period of extirpation. Because conservation plans are predicated on having well-established baselines, a loss of a species over the range of one human generation evokes the likelihood of that species no longer being recognized as a member of an ecosystem, and thus being excluded in restoration or conservation prioritization. [Correction added on 26 December 2012, after first online publication: this formula has been corrected to N=10M⁻¹].     

The Ketamine Analogue Methoxetamine and 3- and 4-Methoxy Analogues of Phencyclidine Are High Affinity and Selective Ligands for the Glutamate NMDA Receptor

In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as ‘designer drugs’ and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone) and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenyl)cyclohexanamine) and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine), were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.     

The effect of Glc6P uptake and its subsequent oxidation within pea root plastids on nitrite reduction and glutamate synthesis

In roots, nitrate assimilation is dependent upon a supply of reductant that is initially generated by oxidative metabolism including the pentose phosphate pathway (OPPP). The uptake of nitrite into the plastids and its subsequent reduction by nitrite reductase (NiR) and glutamate synthase (GOGAT) are potentially important control points that may affect nitrate assimilation. To support the operation of the OPPP there is a need for glucose 6-phosphate (Glc6P) to be imported into the plastids by the glucose phosphate translocator (GPT). Competitive inhibitors of Glc6P uptake had little impact on the rate of Glc6P-dependent nitrite reduction. Nitrite uptake into plastids, using (13)N labelled nitrite, was shown to be by passive diffusion. Flux through the OPPP during nitrite reduction and glutamate synthesis in purified plastids was followed by monitoring the release of (14)CO(2) from [1-(14)C]-Glc6P. The results suggest that the flux through the OPPP is maximal when NiR operates at maximal capacity and could not respond further to the increased demand for reductant caused by the concurrent operation of NiR and GOGAT. Simultaneous nitrite reduction and glutamate synthesis resulted in decreased rates of both enzymatic reactions. The enzyme activity of glucose 6-phosphate dehydrogenase (G6PDH), the enzyme supporting the first step of the OPPP, was induced by external nitrate supply. The maximum catalytic activity of G6PDH was determined to be more than sufficient to support the reductant requirements of both NiR and GOGAT. These data are discussed in terms of competition between NiR and GOGAT for the provision of reductant generated by the OPPP.     

Small-scale extracts for the study of nucleotide excision repair and non-homologous end joining

The repair of DNA by nucleotide excision repair (NER) and non-homologous end joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Examination of NHEJ and NER in vitro using cell-free extracts has led to a deeper understanding of the biochemical mechanisms that underlie these processes. Current methods for production of whole-cell extracts (WCEs) to investigate NER and NHEJ start with one or more liters of culture containing 1–5 × 10⁹ cells. Here, we describe a small-scale method for production of WCE that can be used to study NER. We also describe a rapid, small-scale method for the preparation of WCE that can be used in the study of NHEJ. These methods require less time, 20- to 1000-fold fewer cells than large-scale extracts, facilitate examination of numerous samples and are ideal for such applications as the study of host–virus interactions and analysis of mutant cell lines.     

Identification and expression of natriuretic peptide receptor type-A and -B mRNA in freshwater and seawater rainbow trout

Natriuretic peptide receptors mediate the physiological response of natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.