Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids,viral vectors and cell lines

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.     

Sand pile above the nest amplifies the sound emitted by the male sand goby

The male sand gobies calls and spawns inside cavities beneath stones, shells and other submerged objects (including artificial shelters) which he covers by piling sand on them. A previous study showed fish shelters likely act as impedance-matching devices enhancing sound frequencies below 500 Hz. This study examines the effect of the sand pile on sound amplification by shelters commonly used by Mediterranean sand gobies as nest sites in the field (bivalve shells, pebbles), or within aquarium tanks (tunnel-shaped plastic tiles, halves of clay flower-pots). Shelters were acoustically stimulated with white noise and artificial pulse-trains emitted by a small underwater buzzer placed inside the cavity. Results showed the sand pile increased the low-frequency gain by up to 12 dB. Conclusions were verified by examining the role of natural sand builds in sound amplification using data from a previous laboratory study on sound production in the male sand goby P. minutus. Implications for acoustic communication in sand gobies are discussed.     

Mapping of Alternaria and Pleospora concentrations in Central Italy using meteorological forecast and neural network estimator

Airborne particles (pollens and fungal spores) are recognized as important causes of allergies and many other pathologies whose main symptoms are usually associated with respiratory problems. In addition, these particles seem to be responsible for clinical symptoms of oculorhinitis and bronchial asthma. Many authors showed how pollen and spore concentrations are critically linked to meteorological conditions, while other studies investigated the possibility to estimate these concentrations through meteorological parameters. So, many different approaches have been proposed, and one of the most sophisticated is based on the use of a complex artificial neural network architecture. Once the neural device is calibrated using simultaneous time series of observed meteorological parameters and airborne biological particles, it is straightforward to use the Neural Network to predict spore concentrations using operational Limited Area Meteorological Model. In a previous work, it has been shown that the MM5 meteorological model developed by National Center for Atmospheric Research and Pennsylvania State University can be coupled with the above-cited neural predictor to provide a good prediction of Alternaria and Pleospora spore in the location of L’Aquila (Central Italy). Following the same approach, this work aims to provide the mapping of spore concentration over a wide area covered by high-resolution meteorological prediction in Central Italy. The complex patterns of fungal spore concentrations in selected areas will be described, and the high temporal variability of such fields will be discussed as well. The possibility to infer useful information from the predicted pattern of spore concentrations is discussed, as an example it appears that for people suffering from allergy to fungal spores is more comfortable to spend summertime close to the east coast of Italian Peninsula respect to the west coast. A further step of this work may easily lead to an operational use of the model for supporting the clinical management of allergies and for establishing a preventive strategy in agriculture to avoid unsafe and useless pollution of atmosphere, crops and fields.     

Temperature‐dependent conformational change affecting Tyr11 and sweetness loops of brazzein

The sweet protein brazzein, a member of the Csβα fold family, contains four disulfide bonds that lend a high degree of thermal and pH stability to its structure. Nevertheless, a variable temperature study has revealed that the protein undergoes a local, reversible conformational change between 37 and 3°C with a midpoint about 27°C that changes the orientations and side‐chain hydrogen bond partners of Tyr8 and Tyr11. To test the functional significance of this effect, we used NMR saturation transfer to investigate the interaction between brazzein and the amino terminal domain of the sweet receptor subunit T1R2; the results showed a stronger interaction at 7°C than at 37°C. Thus the low temperature conformation, which alters the orientations of two loops known to be critical for the sweetness of brazzein, may represent the bound state of brazzein in the complex with the human sweet receptor. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

A simple model of the hydrophobic effect for molecular simulation of interfacial phenomena

A coarse-grained model for simulation of interfacial phenomena in aqueous systems has been developed. The model captures the hydrophobic effect by only considering the structure and cohesiveness of water. Monte Carlo (MC) simulations of water-oil mixtures show that low concentrations of oil are solvated with little perturbation of the hydrogen bonding network structure of the water, while high concentrations of oil are excluded altogether. Analysis of the water structure in the simulations indicates that the water molecules maintain close to four coordination in the presence of solutes and the distribution of bond angles is not markedly affected by the presence of solutes. MC simulations of an alkane oligomer in water and a poly(ethylene oxide) (PEO) oligomer in water indicate that the chains are quite flexible and also do not perturb the network structure of the water phase.     

Hypersomnolence and Accidents in Truck Drivers: A Cross‐Sectional Study

Truck drivers are more likely to suffer severe injury and death due to certain truck driving characteristics. Identifying and preventing factors associated with accidents in this population is important to minimize damage and improve road safety. Excessive daytime sleepiness is a major public health problem, leading to impaired cognitive function, reduced alertness, and increased risk of motor vehicle crashes. The aim of this cross‐sectional study was to determine the prevalence and predictors of hypersomnolence (defined as an Epworth Sleepiness Scale score greater than 10) among truck drivers. Three hundred male truck drivers were studied. Quality of sleep was assessed by the Pittsburgh Sleep Quality Index, and the association between demographic, clinical, and occupational data with excessive sleepiness was analyzed. The mean daily sleep duration was 5.6±1.3 h, and poor quality of sleep was found in 46.3% of the individuals. Hypersomnolence was found in 46% of the drivers and was associated with younger age, snoring, and working >10 h without rest. A positive correlation between hypersomnolence and previous accidents was detected (p=0.005). These results show that sleep deprivation and hypersomnolence are frequent among truck drivers. The treatment of sleep‐disordered breathing and the implementation of educational programs, particularly targeting younger drivers and promoting increased awareness of the deleterious effects of sleep loss and work overload, may help to reduce hypersomnolence and accidents among truck drivers.     

Comparison of Two Different Actigraphs with Polysomnography in Healthy Young Subjects

The present study aimed to compare two commercially available actigraphs, with a concurrent polysomnographic (PSG) recording. Twelve healthy volunteers (six women; age range 19–28 yrs) simultaneously wore the Basic Mini‐Motionlogger® and Actiwatch® for seven overnight polysomnographic recordings. Comparisons of the following sleep measures were focused on: sleep onset latency (SOL), total sleep time, wake after sleep onset, and sleep efficiency. Both devices underestimated SOL in comparison to PSG, but they had similar performance compared to PSG for the other sleep measures. A limit of the study is that the results can be only generalized to healthy young subjects.