High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88–100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp.     

A Regional Initiative to Reduce Skin Infections amongst Aboriginal Children Living in Remote Communities of the Northern Territory,Australia

Background

Linked to extreme rates of chronic heart and kidney disease, pyoderma is endemic amongst Aboriginal children in Australia''s Northern Territory (NT). Many of those with pyoderma will also have scabies. We report the results of a community-based collaboration within the East Arnhem Region, which aimed to reduce the prevalence of both skin infections in Aboriginal children.

Methodology/Principal Findings

Commencing September 2004, we conducted an ecological study that included active surveillance for skin infections amongst children aged <15 years in five remote East Arnhem communities over a three year period. Screening was undertaken by trained local community workers, usually accompanied by another project team member, using a standard data collection form. Skin infections were diagnosed clinically with the aid of a pictorial flip chart developed for the purpose. Topical 5% permethrin was provided for age-eligible children and all household contacts whenever scabies was diagnosed, whilst those with pyoderma were referred to the clinic for treatment in accordance with current guidelines. In addition, annual mass scabies treatment (5% permethrin cream) was offered to all community residents in accordance with current guidelines but was not directly observed. Pyoderma and scabies prevalence per month was determined from 6038 skin assessments conducted on 2329 children. Pyoderma prevalence dropped from 46.7% at baseline to a median of 32.4% (IQR 28.9%–41.0%) during the follow-up period – an absolute reduction of 14.7% (IQR 4.7%–16.8%). Compared to the first 18 months of observation, there was an absolute reduction in pyoderma prevalence of 18 cases per 100 children (95%CI −21.0, −16.1, p≤0.001) over the last 18 months. Treatment uptake increased over the same period (absolute difference 13.4%, 95%CI 3.3, 23.6). While scabies prevalence was unchanged, the prevalence of infected scabies (that is with superimposed pyoderma) decreased from 3.7% (95%CI 2.4, 4.9) to 1.5% (95%CI 0.7, 2.2), a relative reduction of 59%.

Conclusion

Although pyoderma prevalence remained unacceptably high, there was a substantial reduction overall with improvements in treatment uptake a critical factor. More acceptable alternatives, such as cotrimoxazole for pyoderma and ivermectin as a community-wide scabicide, warrant further investigation in these settings. We are encouraged by progress made through this work, where local action was led by local community members and primary health care providers with external training and support.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00884728","term_id":"NCT00884728"}}NCT00884728     

Responses of eight boreal flat bug (Heteroptera: Aradidae) species to clear-cutting and forest fire

Boreal flat bugs include a high proportion of species that are considered negatively affected by forestry. Knowledge on the biology and habitat demands of individual species is generally limited. We examined the influence on flat bugs of stand-age and clear-cutting, comparing five classes of spruce stands. The five classes were: clear-cut, unthinned, and thinned (all three products of current clear-cutting forestry), mature managed and old-growth stands (these two had never been clear-cut). We also compared unburned and recently burned mature pine forest. Fire, but not stand age, had a pronounced effect on species richness and total abundance. Aradus depressus showed a significant association with older forest stands. Aradus betulae occurred only in clear-cuts and burned forest indicating that this species is favored by disturbance in general. Aradus lugubris, Aradus crenaticollis and Aradus brevicollis were found only in the burned forest. Aradus brevicollis has not previously been shown to be associated with fire.     

Characterisation of the role of Vrp1 in cell fusion during the development of visceral muscle of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

In Drosophila muscle cell fusion takes place both during the formation of the somatic mesoderm and the visceral mesoderm, giving rise to the skeletal muscles and the gut musculature respectively. The core process of myoblast fusion is believed to be similar for both organs. The actin cytoskeleton regulator Verprolin acts by binding to WASP, which in turn binds to the Arp2/3 complex and thus activates actin polymerization. While Verprolin has been shown to be important for somatic muscle cell fusion, the function of this protein in visceral muscle fusion has not been determined.     

Functional genomics of cactus host shifts in Drosophila mojavensis

Understanding the genetic basis of adaptation to novel environments remains one of the major challenges confronting evolutionary biologists. While newly developed genomic approaches hold considerable promise for addressing this overall question, the relevant tools have not often been available in the most ecologically interesting organisms. Our study organism, Drosophila mojavensis, is a cactophilic Sonoran Desert endemic utilizing four different cactus hosts across its geographical range. Its well-known ecology makes it an attractive system in which to study the evolution of gene expression during adaptation. As a cactophile, D. mojavensis oviposits in the necrotic tissues of cacti, therefore exposing larvae and even adults to the varied and toxic compounds of rotting cacti. We have developed a cDNA microarray of D. mojavensis to examine gene expression associated with cactus host use. Using a population from the Baja California population we examined gene expression differences of third instar larvae when reared in two chemically distinct cactus hosts, agria (Stenocereus gummosus, native host) vs. organpipe (Stenocereus thurberi, alternative host). We have observed differential gene expression associated with cactus host use in genes involved in metabolism and detoxification.     

Microplastics alter composition of fungal communities in aquatic ecosystems

Despite increasing concerns about microplastic (MP) pollution in aquatic ecosystems, there is insufficient knowledge on how MP affect fungal communities. In this study, we explored the diversity and community composition of fungi attached to polyethylene (PE) and polystyrene (PS) particles incubated in different aquatic systems in north‐east Germany: the Baltic Sea, the River Warnow and a wastewater treatment plant. Based on next generation 18S rRNA gene sequencing, 347 different operational taxonomic units assigned to 81 fungal taxa were identified on PE and PS. The MP‐associated communities were distinct from fungal communities in the surrounding water and on the natural substrate wood. They also differed significantly among sampling locations, pointing towards a substrate and location specific fungal colonization. Members of Chytridiomycota, Cryptomycota and Ascomycota dominated the fungal assemblages, suggesting that both parasitic and saprophytic fungi thrive in MP biofilms. Thus, considering the worldwide increasing accumulation of plastic particles as well as the substantial vector potential of MP, especially these fungal taxa might benefit from MP pollution in the aquatic environment with yet unknown impacts on their worldwide distribution, as well as biodiversity and food web dynamics at large.     

Diversity-enhancing selection acts on a female reproductive protease family in four subspecies of Drosophila mojavensis

Protein components of the Drosophila male ejaculate are critical modulators of reproductive success, several of which are known to evolve rapidly. Recent evidence of adaptive evolution in female reproductive tract proteins suggests this pattern may reflect sexual selection at the molecular level. Here we explore the evolutionary dynamics of a five-paralog gene family of female reproductive proteases within geographically isolated subspecies of Drosophila mojavensis. Remarkably, four of five paralogs show exceptionally low differentiation between subspecies and unusually structured haplotypes that suggest the retention of old polymorphisms. These gene genealogies are accompanied by deviations from neutrality consistent with diversifying selection. While diversifying selection has been observed among the reproductive molecules of mammals and marine invertebrates, our study provides the first evidence of this selective regime in any Drosophila reproductive protein, male or female.     

Identification of an interaction between the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor with protein kinase C-related kinase (PRK) 1: implications for prostate cancer

In humans, thromboxane (TX) A(2) signals through the TPα and TPβ isoforms of the TXA(2) receptor or TP. Here, the RhoA effector protein kinase C-related kinase (PRK) 1 was identified as an interactant of both TPα and ΤPβ involving common and unique sequences within their respective C-terminal (C)-tail domains and the kinase domain of PRK1 (PRK1(640-942)). Although the interaction with PRK1 is constitutive, agonist activation of TPα/TPβ did not regulate the complex per se but enhanced PRK1 activation leading to phosphorylation of its general substrate histone H1 in vitro. Altered PRK1 and TP expression and signaling are increasingly implicated in certain neoplasms, particularly in androgen-associated prostate carcinomas. Agonist activation of TPα/TPβ led to phosphorylation of histone H3 at Thr(11) (H3 Thr(11)), a previously recognized specific marker of androgen-induced chromatin remodeling, in the prostate LNCaP and PC-3 cell lines but not in primary vascular smooth muscle or endothelial cells. Moreover, this effect was augmented by dihydrotestosterone in androgen-responsive LNCaP but not in nonresponsive PC-3 cells. Furthermore, PRK1 was confirmed to constitutively interact with TPα/TPβ in both LNCaP and PC-3 cells, and targeted disruption of PRK1 impaired TPα/TPβ-mediated H3 Thr(11) phosphorylation in, and cell migration of, both prostate cell types. Collectively, considering the role of TXA(2) as a potent mediator of RhoA signaling, the identification of PRK1 as a bona fide interactant of TPα/TPβ, and leading to H3 Thr(11) phosphorylation to regulate cell migration, has broad functional significance such as within the vasculature and in neoplasms in which both PRK1 and the TPs are increasingly implicated.     

Development of autoimmune diabetes in the absence of detectable IL-17A in a CD8-driven virally induced model

Recent studies have shown that IL-17 can contribute beneficially to pathogen defense but also that excessive IL-17 levels are associated with chronic inflammation and autoimmune disorders. To date, the role of IL-17 in viral infections and type 1 diabetes is ambiguous. In this study, we used IL-17A enhanced green fluorescent protein bicistronic reporter mouse strains to analyze in situ production of IL-17A. Upon Klebsiella pneumoniae bacterial infection, CD4(+) and γδ T cells produce IL-17A. In contrast, CD4(+) or CD8(+) T cells do not produce IL-17A in response to acute or protracted viral infection with lymphocytic choriomeningitis virus or during autoimmune diabetes development in the CD8-driven lymphocytic choriomeningitis virus-induced model of type 1 diabetes. We conclude that viral elimination and type 1 diabetes can occur in the absence of detectable IL-17A production, suggesting IL-17A is not essential in these settings.     

Evaluation of Agrilus planipennis (Coleoptera: Buprestidae) control provided by emamectin benzoate and two neonicotinoid insecticides, one and two seasons after treatment

Effective methods are needed to protect ash trees (Fraxinus spp.) from emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), an invasive buprestid that has killed millions of North American ash (Fraxinus spp.) trees. We randomly assigned 175 ash trees (11.5-48.1 cm in diameter) in 25 blocks located in three study sites in Michigan to one of seven insecticide treatments in May 2007. Treatments included 1) trunk-injected emamectin benzoate; 2) trunk-injected imidacloprid; 3) basal trunk spray of dinotefuran with or 4) without Pentra-Bark, an agricultural surfactant; 5) basal trunk spray of imidacloprid with or 6) without Pentra-Bark; or (7) control. Foliar insecticide residues (enzyme-linked immunosorbent assay) and toxicity of leaves to adult A. planipennis (4-d bioassays) were quantified at 3-4-wk intervals posttreatment. Seven blocks of trees were felled and sampled in fall 2007 to quantify A. planipennis larval density. Half of the remaining blocks were retreated in spring 2008. Bioassays and residue analyses were repeated in summer 2008, and then all trees were sampled to assess larval density in winter. Foliage from emamectin benzoate-treated trees was highly toxic to adult A. planipennis, and larval density was < 1% of that in comparable control trees, even two seasons posttreatment. Larval densities in trees treated with trunk-injected imidacloprid in 2007 + 2008 were similar to control trees. Dinotefuran and imidacloprid were effectively translocated within trees treated with the noninvasive basal trunk sprays; the surfactant did not appreciably enhance A. planipennis control. In 2008, larval densities were 57-68% lower in trees treated with dinotefuran or imidacloprid trunk sprays in 2007 + 2008 than on controls, but densities in trees treated only in 2007 were similar to controls. Highly effective control provided by emamectin benzoate for > or = 2 yr may reduce costs or logistical issues associated with treatment.