Sensitivity of Patterns of Molecular Evolution to Alterations in Methodology: A Critique of Hughes and Yeager

Employing a set of 43 othologous mouse and rat genes, Hughes and Yeager (J. Mol. Evol. 45:125–130, 1997) reported (1) no correlation between synonymous and nonsynonymous rates of nucleotide substitution, (2) a positive correlation between intronic GC contents (GC _i) and intronic substitution rates (K _i), (3) that the average K _i value was very similar to the average K _s value, and (4) that the compositional correlation between the rat and the mouse genes is stronger at the third codon position (GC₃) than at the first and second codon positions (GC₁₂). We have examined the robustness of these results to alterations in substitution rate estimation protocol, alignment protocol, and statistical procedure. We find that a significant correlation between K _a and K _s is observed either if a rank correlation statistic is used instead of regression analysis, if one outlier is excluded from the analysis, or if a regression weighted by gene size is employed. The correlation between K _i and GC _i we find to be sensitive to changes in alignment protocol and disappears on the use of weighted means. The finding that K _s and K _i are approximately the same is dependent on the method for estimating K _s values. Finally, the variance around the regression line of rat GC₃ versus mouse GC₃ we find to be significantly higher than that in GC₁₂. The source of the discrepancy between this and Hughes and Yeager's result is unclear. The variance around the line for GC₄ is higher still, as might be expected. Using a methodology that may be considered preferable to that of Hughes and Yeager, we find that all four of their results are contradicted. More importantly this analysis reinforces the need for caution in assembling and analyzing data sets, as the degree of sensitivity to what many might consider minor methodological alterations is unexpected. Received: 2 February 1998 / Accepted: 23 March 1998     

The competing countermarks hypothesis: reliable assessment of competitive ability by potential mates

Scent marking on top of (overmarking), or in the vicinity of, a scent mark already present is commonly termed countermarking. Scent marks and countermarks provide a continuous record of competitive challenges between conspecifics, thus providing a reliable advertisement of an individual's ability to dominate or defend an area to other competitors and potential mates. To test the hypothesis that females should prefer males that countermark competing scent marks in their territory over those whose own marks are partially countermarked by a competing male, we manipulated scent marks in the territories of neighbouring male house mice (captive-bred Mus domesticus). As predicted, oestrous females were more strongly attracted to approach territory owners that countermarked the scent mark challenges of competitors than those that had been countermarked, and females themselves deposited more scent marks near the scents of these males. To investigate whether female mice use scent age, overlap or intrinsic qualitative or quantitative differences between scent marks and countermarks to make this discrimination, we redeposited male scent marks artificially as marks and partially overlapping countermarks, with or without a 24-h age difference between them. While the intrinsic quality or quantity of countermarks did not affect discrimination, an age difference between the original mark and subsequent countermark was important for consistent discrimination. The ultimate function of such competitive scent signalling thus may be to provide potential mates with a reliable indicator of the competitive ability of individuals advertising their high status. Since scent marks remain in the environment and are continuously available to challenge and investigation, they may provide a particularly effective and reliable means of dominance advertisement. Copyright 1999 The Association for the Study of Animal Behaviour.     

Development of protegrins for the treatment and prevention of oral mucositis: structure-activity relationships of synthetic protegrin analogues

Protegrin antimicrobial peptides possess activity against gram-positive and gram-negative bacteria and yeasts. An extensive structure-activity relationship (SAR) study was conducted on several hundred protegrin analogues to gain understanding of the relationship between the primary and secondary structure of the protegrins and their antimicrobial activities, and to identify a protegrin analogue for clinical development. Native sequence protegrins are cationic, amphiphilic peptides that are characterized by the presence of a beta-sheet structure that is maintained by two disulfide bridges. The presence of the beta-sheet is key to the stability of the protegrin structure; linearized analogues or analogues that have amino acid substitutions that eliminate hydrogen bonding across the beta-sheet have reduced activity, especially in the presence of physiological concentrations of NaCl. Also, maintaining amphiphilicity of the beta-sheet is key; analogues with substitutions of polar amino acids in the hydrophobic face have reduced activity. Analogues with reduced positive charge tend to be less active, an observation that is more marked for gram-negative than gram-positive bacteria, and may implicate binding to lipopolysaccharide as a key mechanistic step in the killing of gram-negative bacteria. A very large number of amino acid substitutions are tolerated by the protegrin structure, implying that overall structural features such as amphiphilicity, charge, and shape are more important to activity than the presence of specific amino acids. This lack of importance of specific stereochemistry is supported by the fact that completely D-amino acid substituted protegrins are fully potent. Based on the SAR studies, and on the microbiological data from an animal model, one protegrin analogue, IB-367, was selected for clinical development as a topical agent to prevent the oral mucositis associated with cancer therapy.     

Systematic segregation to mutant mitochondrial DNA and accompanying loss of mitochondrial DNA in human NT2 teratocarcinoma Cybrids

In this study a well-characterized pathological mutation at nucleotide position 3243 of human mitochondrial DNA was introduced into human rho(0) teratocarcinoma (NT2) cells. In cloned and mixed populations of NT2 cells heteroplasmic for the mutation, mitotic segregation toward increasing levels of mutant mitochondrial DNA always occurred. Rapid segregation was frequently followed by complete loss of mitochondrial DNA. These findings support the idea that pathological mitochondrial DNA mutations are particularly deleterious in specific cell types, which can explain some of the tissue-specific aspects of mitochondrial DNA diseases. Moreover, these findings suggest that mitochondrial DNA depletion may be an important and widespread feature of mitochondrial DNA disease.     

Incidence of Male-Killing Rickettsia spp. (α-Proteobacteria) in the Ten-Spot Ladybird Beetle Adalia decempunctata L. (Coleoptera: Coccinellidae)

The diversity of endosymbiotic bacteria that kill male host offspring during embryogenesis and their frequencies in certain groups of host taxa suggest that the evolution of male killing and the subsequent spread of male-killing symbionts are primarily determined by host life history characteristics. We studied the 10-spot ladybird beetle, Adalia decempunctata L. (Coleoptera: Coccinellidae), in which male killing has not been recorded previously, to test this hypothesis, and we also assessed the evolution of the male killer identified by DNA sequence analysis. Our results show that A. decempunctata harbors male-killing Rickettsia (α-proteobacteria). Male-killing bacteria belonging to the genus Rickettsia have previously been reported only for the congeneric two-spot ladybird beetle, Adalia bipunctata L. Phylogenetic analysis of Rickettsia DNA sequences isolated from different populations of the two host species revealed a single origin of male killing in the genus Rickettsia. The data also indicated possible horizontal transfer of symbionts between host species. In addition, A. bipunctata is known to bear at least four different male-killing symbionts in its geographic range two of which coexist in the two locations from which A. decempunctata specimens were obtained for the present study. Since only a single male-killing taxon was found in A. decempunctata, we assume that the two closely related ladybird beetle species must differ in the number and/or geographic distribution of male killers. We discuss the importance of these findings to our understanding of the evolution and dynamics of symbiotic associations between male-killing bacteria and their insect hosts.     

Extensive public health initiatives drive the elimination of Aedes aegypti (Diptera,Culicidae) from a town in regional Queensland: A case study from Gin Gin,Australia

Aedes aegypti is the primary vector of exotic arboviruses (dengue, chikungunya and Zika) in Australia. Once established across much of Australia, this mosquito species remains prevalent in central and northern Queensland. In 2011, Ae. aegypti was re-discovered in the town of Gin Gin, Queensland, by health authorities during routine larval surveillance. This town is situated on a major highway that provides a distribution pathway into the highly vulnerable and populous region of the state where the species was once common. Following the detection, larval habitat and adult control activities were conducted as a public health intervention to eliminate the Ae. aegypti population and reduce the risk of exotic disease transmission. Importantly, genetic analysis revealed a homogenous cluster and small effective population vulnerable to an elimination strategy. By 2015, adult surveillance revealed the population had expanded throughout the centre of the town. In response, a collaboration between research agencies and local stakeholders activated a second control program in 2016 that included extensive community engagement, enhanced entomologic surveillance and vector control activities including the targeting of key containers, such as unsealed rainwater tanks. Here we describe a model of the public health intervention which successfully reduced the Ae. aegypti population below detection thresholds, using source reduction, insecticides and novel, intensive genetic surveillance methods. This outcome has important implications for future elimination work in small towns in regions sub-optimal for Ae. aegypti presence and reinforces the longstanding benefits of a partnership model for public health-based interventions for invasive urban mosquito species.     

Discovery of aryl ureas and aryl amides as potent and selective histamine H3 receptor antagonists for the treatment of obesity (Part I)

A series of structurally novel aryl ureas was derived from optimization of the HTS lead as selective histamine H₃ receptor (H₃R) antagonists. The SAR was explored and the data obtained set up the starting point and foundation for further optimization. The most potent tool compounds, as exemplified by compounds 2l, 5b, 5d, and 5e, displayed antagonism potencies in the subnanomolar range in in vitro human-H₃R FLIPR assays and rhesus monkey H₃R binding assays.     

Effects of environmental variables and soil characteristics on virus survival in soil.

Because of the increasing emphasis placed upon land application as a means of wastewater disposal, it is important to evaluate the influences of different factors upon virus survival in soil. The objective of this study was to measure the effects of various environmental variables on virus persistence. Test samples of soil were placed in vials, and the soil was wetted with suspensions of virus in either distilled water, unchlorinated secondary sewage effluent, or mixtures of effluent and water. The viruses used were coxsackieviruses A9 and B3, echovirus 1, poliovirus 2, rotavirus SA11, and bacteriophages T2 and MS2. The rate of virus inactivation was evaluated statistically with regard to conditions under which the vials were incubated and to the soil characteristics. The factors that were found to influence virus survival were temperature, soil moisture content, presence of aerobic microorganisms, degree of virus adsorption to the soil, soil levels of resin-extractable phosphorus, exchangeable aluminium, and soil pH. Overall, temperature and virus adsorption to soil appeared to be the most important factors affecting virus survival.     

Improving protein array performance: focus on washing and storage conditions

For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.