Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

Is Demography Destiny? Application of Machine Learning Techniques to Accurately Predict Population Health Outcomes from a Minimal Demographic Dataset

For years, we have relied on population surveys to keep track of regional public health statistics, including the prevalence of non-communicable diseases. Because of the cost and limitations of such surveys, we often do not have the up-to-date data on health outcomes of a region. In this paper, we examined the feasibility of inferring regional health outcomes from socio-demographic data that are widely available and timely updated through national censuses and community surveys. Using data for 50 American states (excluding Washington DC) from 2007 to 2012, we constructed a machine-learning model to predict the prevalence of six non-communicable disease (NCD) outcomes (four NCDs and two major clinical risk factors), based on population socio-demographic characteristics from the American Community Survey. We found that regional prevalence estimates for non-communicable diseases can be reasonably predicted. The predictions were highly correlated with the observed data, in both the states included in the derivation model (median correlation 0.88) and those excluded from the development for use as a completely separated validation sample (median correlation 0.85), demonstrating that the model had sufficient external validity to make good predictions, based on demographics alone, for areas not included in the model development. This highlights both the utility of this sophisticated approach to model development, and the vital importance of simple socio-demographic characteristics as both indicators and determinants of chronic disease.     

Hematology, plasma biochemistry, and antibodies to select viruses in wild-caught Eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) from Illinois

During the 2004 field season, blood was collected from Eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) in the Carlyle Lake (Carlyle, Illinois, USA) and Allerton Park (Monticello, Illinois, USA) populations to derive baseline complete blood count and plasma biochemistry data and to assess the prevalence of antibodies to West Nile virus (WNV) and ophidian paramyxovirus (OPMV). Massasaugas were located for sampling through visual encounter surveys. Body weight, snout-vent length, total protein, globulins, sodium, and potassium were normally distributed among the survey population. Aspartate aminotransferase, creatine kinase, albumin, calcium, uric acid, white blood cell count, heterophils, lymphocytes, monocytes, eosinophils, and basophils were non-normally distributed within these animals. Female snakes had significantly shorter tail lengths; lower blood glucose, packed cell volumes, and absolute azurophil counts; and higher plasma calcium and phosphorus concentrations than did males. None of the snakes tested (n=21) were seropositive for WNV, whereas all (n=20) were seropositive for OPMV.     

Regulation to Create Environments Conducive to Physical Activity: Understanding the Barriers and Facilitators at the Australian State Government Level

Introduction

Policy and regulatory interventions aimed at creating environments more conducive to physical activity (PA) are an important component of strategies to improve population levels of PA. However, many potentially effective policies are not being broadly implemented. This study sought to identify potential policy/regulatory interventions targeting PA environments, and barriers/facilitators to their implementation at the Australian state/territory government level.

Methods

In-depth interviews were conducted with senior representatives from state/territory governments, statutory authorities and non-government organisations (n = 40) to examine participants'': 1) suggestions for regulatory interventions to create environments more conducive to PA; 2) support for preselected regulatory interventions derived from a literature review. Thematic and constant comparative analyses were conducted.

Results

Policy interventions most commonly suggested by participants fell into two areas: 1) urban planning and provision of infrastructure to promote active travel; 2) discouraging the use of private motorised vehicles. Of the eleven preselected interventions presented to participants, interventions relating to walkability/cycling and PA facilities received greatest support. Interventions involving subsidisation (of public transport, PA-equipment) and the provision of more public transport infrastructure received least support. These were perceived as not economically viable or unlikely to increase PA levels. Dominant barriers were: the powerful ‘road lobby’, weaknesses in the planning system and the cost of potential interventions. Facilitators were: the provision of evidence, collaboration across sectors, and synergies with climate change/environment agendas.

Conclusion

This study points to how difficult it will be to achieve policy change when there is a powerful ‘road lobby’ and government investment prioritises road infrastructure over PA-promoting infrastructure. It highlights the pivotal role of the planning and transport sectors in implementing PA-promoting policy, however suggests the need for clearer guidelines and responsibilities for state and local government levels in these areas. Health outcomes need to be given more direct consideration and greater priority within non-health sectors.     

The toxicity of dithiocarbamate fungicides to soil nematodes, assessed using a stress-inducible transgenic strain of Caenorhabditis elegans.

The dithiocarbamate fungicides maneb and mancozeb induce a short-term stress response in a transgenic Caenorhabditis elegans strain (PC72) carrying a reporter lacZ gene under the control of a homologous heat shock (hsp16) promoter. This response can be readily monitored as induced beta-galactosidase activity, either by in situ staining or by a quantitative fluorometric enzyme assay. Particularly strong responses are induced by mancozeb (three- to fivefold above controls at 500 microg mL(-1)), causing acute toxicity at concentrations comparable to those recommended for field application (2 mg mL(-1)). Although much of this fungicide is adsorbed by soil, sufficient (ca. 6%) enters the soil water compartment to cause mild stress in the transgenic worm assay. Among possible metabolites from mancozeb breakdown, neither Mn2+ nor ethylenethiourea (ETU) is particularly toxic even at 10% of the optimum mancozeb dosage. Stress responses to a range of other pesticides are also reported, and in several cases it is clear that a nontarget soil species (here, transgenic C. elegans) may be sensitive to low-level contamination.     

Development of Snake Fungal Disease after Experimental Challenge with Ophidiomyces ophiodiicola in Cottonmouths (Agkistrodon piscivorous)

Snake fungal disease (SFD) is a clinical syndrome associated with dermatitis, myositis, osteomyelitis, and pneumonia in several species of free-ranging snakes in the US. The causative agent has been suggested as Ophidiomyces ophiodiicola, but other agents may contribute to the syndrome and the pathogenesis is not understood. To understand the role of O. ophiodiicola in SFD, a cottonmouth snake model of SFD was designed. Five cottonmouths (Agkistrodon piscivorous) were experimentally challenged by nasolabial pit inoculation with a pure culture of O. ophiodiicola. Development of skin lesions or facial swelling at the site of inoculation was observed in all snakes. Twice weekly swabs of the inoculation site revealed variable presence of O. ophiodiicola DNA by qPCR in all five inoculated snakes for 3 to 58 days post-inoculation; nasolabial flushes were not a useful sampling method for detection. Inoculated snakes had a 40% mortality rate. All inoculated snakes had microscopic lesions unilaterally on the side of the swabbed nasolabial pit, including erosions to ulcerations and heterophilic dermatitis. All signs were consistent with SFD; however, the severity of lesions varied in individual snakes, and fungal hyphae were only observed in 3 of 5 inoculated snakes. These three snakes correlated with post-mortem tissue qPCR evidence of O. ophiodiicola. The findings of this study conclude that O. ophiodiicola inoculation in a cottonmouth snake model leads to disease similar to SFD, although lesion severity and the fungal load are quite variable within the model. Future studies may utilize this model to further understand the pathogenesis of this disease and develop management strategies that mitigate disease effects, but investigation of other models with less variability may be warranted.