Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode <it>Caenorhabditis elegans</it>, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from <it>C. elegans</it>.</p> <p>Results</p> <p>We report improved and effective <it>in vitro </it>RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, <it>Brugia malayi</it>. The cellular disorganization observed in <it>B. malayi </it>embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their <it>C. elegans </it>orthologs. Targeting the <it>B. malayi </it>cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in <it>C. elegans</it>. Cellular phenotypes induced by our <it>in vitro </it>RNAi procedure can be observed by immunofluorescence in as little as one week.</p> <p>Conclusions</p> <p>We observed cytological defects following RNAi targeting all seven <it>B. malayi </it>transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism <it>C. elegans</it>. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.</p

IGF2 stimulates fetal growth in a sex- and organ-dependent manner

BackgroundInsulin-like growth factor 2 (IGF2) is a key determinant of fetal growth, and the altered expression of IGF2 is implicated in fetal growth disorders and maternal metabolic derangements including gestational diabetes. Here we studied how increased levels of IGF2 in late pregnancy affect fetal growth.MethodsWe employed a rat model of repeated intrafetal IGF2 administration in late pregnancy, i.e., during GD19-GD21, and measured the consequences on fetal organ weight and expression of insulin/IGF-axis components.ResultsIGF2 treatment tended to increase fetal weight, but only weight increase of the fetal stomach reached significance (+33±9%; P<0.01). Sex-dependent data analysis revealed a sexual dimorphism of IGF2 action. In male fetuses, IGF2 administration significantly increased fetal weight (+13±3%; P<0.05) and weight of fetal stomach (+42±10%; P<0.01), intestine (+26±5%; P<0.05), liver (+13±4%; P<0.05), and pancreas (+25±8%; P<0.05). Weights of heart, lungs, and kidneys were unchanged. In female fetuses, IGF2 increased only stomach weight (+26±9%; P<0.05). Furthermore, gene expression of insulin/IGF axis in the heart, lungs, liver, and stomach was more sensitive toward IGF2 treatment in male than in female fetuses.ConclusionData suggest that elevated circulating IGF2 in late pregnancy predominantly stimulates organ growth of the digestive system, and male fetuses are more susceptible toward the IGF2 effects than female fetuses.Fil: White, Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Jawerbaum, Alicia Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Mazzucco, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Gauster, Martin. Medizinische Universität Graz; AustriaFil: Desoye, Gernot. Medizinische Universität Graz; AustriaFil: Hiden, Ursula. Medizinische Universität Graz; Austri

Individual differences in reproductive strategy are related to views about recreational drug use in Belgium, the Netherlands and Japan

Individual differences in moral views are often explained as the downstream effect of ideological commitments, such as political orientation and religiosity. Recent studies in the U.S. suggest that moral views about recreational drug use are also influenced by attitudes towards sex and that this relationship cannot be explained by ideological commitments. In this study, we investigate student samples from Belgium, the Netherlands, and Japan. We find that, in all samples, sexual attitudes are strongly related to views about recreational drug use, even after controlling for various ideological variables. We discuss our results in the light of reproductive strategies as determinants of moral views

Selection of modalities, prescription, and technical issues in children on peritoneal dialysis

Peritoneal dialysis (PD) is widely employed as a dialytic therapy for uraemic children, especially in its automated form (APD), that is associated with less burden of care on patient and family than continuous ambulatory PD. Since APD offers a wide range of treatment options, based on intermittent and continuous regimens, prescription can be individualized according to patient’s age, body size, residual renal function, nutritional intake, and growth-related metabolic needs. Transport capacity of the peritoneal membrane of each individual patient should be assessed, and regularly monitored, by means of standardized peritoneal function tests validated in pediatric patients. To ensure maximum recruitment of peritoneal exchange area, fill volume should be scaled to body surface area and adapted to each patient, according to clinical tolerance and intraperitoneal pressure. PD solutions should be employed according to their biocompatibility and potential ultrafiltration capacity; new pH-neutral, glucose-free solutions can be used in an integrated way in separate dwells, or by appropriately mixing during the same dialytic session. Kinetic modelling software programs may help in the tailoring of PD prescription to individual patients’ characteristics and needs. Owing to advances in the technology of new APD machines, greater programming flexibility, memorized delivery control, and tele-dialysis are currently possible

Environmental Influences on Mate Preferences as Assessed by a Scenario Manipulation Experiment

Many evolutionary psychology studies have addressed the topic of mate preferences, focusing particularly on gender and cultural differences. However, the extent to which situational and environmental variables might affect mate preferences has been comparatively neglected. We tested 288 participants in order to investigate the perceived relative importance of six traits of an ideal partner (wealth, dominance, intelligence, height, kindness, attractiveness) under four different hypothetical scenarios (status quo/nowadays, violence/post-nuclear, poverty/resource exhaustion, prosperity/global well-being). An equal number of participants (36 women, 36 men) was allotted to each scenario; each was asked to allocate 120 points across the six traits according to their perceived value. Overall, intelligence was the trait to which participants assigned most importance, followed by kindness and attractiveness, and then by wealth, dominance and height. Men appraised attractiveness as more valuable than women. Scenario strongly influenced the relative importance attributed to traits, the main finding being that wealth and dominance were more valued in the poverty and post-nuclear scenarios, respectively, compared to the other scenarios. Scenario manipulation generally had similar effects in both sexes, but women appeared particularly prone to trade off other traits for dominance in the violence scenario, and men particularly prone to trade off other traits for wealth in the poverty scenario. Our results are in line with other correlational studies of situational variables and mate preferences, and represent strong evidence of a causal relationship of environmental factors on specific mate preferences, corroborating the notion of an evolved plasticity to current ecological conditions. A control experiment seems to suggest that our scenarios can be considered as realistic descriptions of the intended ecological conditions

Environmental Influences on Mate Preferences as Assessed by a Scenario Manipulation Experiment

Many evolutionary psychology studies have addressed the topic of mate preferences, focusing particularly on gender and cultural differences. However, the extent to which situational and environmental variables might affect mate preferences has been comparatively neglected. We tested 288 participants in order to investigate the perceived relative importance of six traits of an ideal partner (wealth, dominance, intelligence, height, kindness, attractiveness) under four different hypothetical scenarios (status quo/nowadays, violence/post-nuclear, poverty/resource exhaustion, prosperity/global well-being). An equal number of participants (36 women, 36 men) was allotted to each scenario; each was asked to allocate 120 points across the six traits according to their perceived value. Overall, intelligence was the trait to which participants assigned most importance, followed by kindness and attractiveness, and then by wealth, dominance and height. Men appraised attractiveness as more valuable than women. Scenario strongly influenced the relative importance attributed to traits, the main finding being that wealth and dominance were more valued in the poverty and post-nuclear scenarios, respectively, compared to the other scenarios. Scenario manipulation generally had similar effects in both sexes, but women appeared particularly prone to trade off other traits for dominance in the violence scenario, and men particularly prone to trade off other traits for wealth in the poverty scenario. Our results are in line with other correlational studies of situational variables and mate preferences, and represent strong evidence of a causal relationship of environmental factors on specific mate preferences, corroborating the notion of an evolved plasticity to current ecological conditions. A control experiment seems to suggest that our scenarios can be considered as realistic descriptions of the intended ecological conditions

Instability of aquaglyceroporin (Aqp) 2 contributes to drug resistance in trypanosoma brucei

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic-oriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance

The potential of antisense oligonucleotide therapies for inherited childhood lung diseases.

Antisense oligonucleotides are an emerging therapeutic option to treat diseases with known genetic origin. In the age of personalised medicines, antisense oligonucleotides can sometimes be designed to target and bypass or overcome a patient's genetic mutation, in particular those lesions that compromise normal pre-mRNA processing. Antisense oligonucleotides can alter gene expression through a variety of mechanisms as determined by the chemistry and antisense oligomer design. Through targeting the pre-mRNA, antisense oligonucleotides can alter splicing and induce a specific spliceoform or disrupt the reading frame, target an RNA transcript for degradation through RNaseH activation, block ribosome initiation of protein translation or disrupt miRNA function. The recent accelerated approval of eteplirsen (renamed Exondys 51™) by the Food and Drug Administration, for the treatment of Duchenne muscular dystrophy, and nusinersen, for the treatment of spinal muscular atrophy, herald a new and exciting era in splice-switching antisense oligonucleotide applications to treat inherited diseases. This review considers the potential of antisense oligonucleotides to treat inherited lung diseases of childhood with a focus on cystic fibrosis and disorders of surfactant protein metabolism