Gene expression silencing with 'specific' small interfering RNA goes beyond specificity - a study of key parameters to take into account in the onset of small interfering RNA off-target effects

RNA-mediated gene silencing (RNA interference) is a powerful way to knock down gene expression and has revolutionized the fields of cellular and molecular biology. Indeed, the transfection of cultured cells with small interfering RNAs (siRNAs) is currently considered to be the best and easiest approach to loss-of-function experiments. However, several recent studies underscore the off-target and potential cytotoxic effects of siRNAs, which can lead to the silencing of unintended mRNAs. In this study, we used a low-density microarray to assess gene expression modifications in response to five different siRNAs in various cell types and transfection conditions. We found major differences in off-target signature according to: (a) siRNA sequence; (b) cell type; (c) duration of transfection; and (d) post-transfection time before analysis. These results contribute to a better understanding of important parameters that could impact on siRNA side effects in knockdown experiments.     

Partial within-animal calibration: a new calibration approach in lead optimisation high-throughput bioanalysis

The lead optimization phase of drug discovery requires high-throughput analyses for quantification in biological matrices and in plasma in particular. Over the last decade, some technical innovations allowed the pharmaceutical industry to improve the quality of the results. However, there is room for improvement. In this context, a new calibration strategy is proposed in this paper. Experiments were performed on dog plasma samples and it was showed that a within-animal calibration strategy can reduce the bias up to 20% and improve the precision up to 20%. However, a partial within-animal calibration is preferred to the full approach in order to avoid to many sample preparations.     

MIP3 alpha stimulates the migration of Langerhans cells in models of human papillomavirus (HPV)-associated (pre)neoplastic epithelium

Although human papillomavirus (HPV) DNA is detected in the majority of cervical cancers and their precursors (squamous intraepithelial lesions; SIL), the persistence or progression of cervical lesions could be associated with quantitative and functional alterations of dendritic/Langerhans cells (DC/LC). As LC abnormalities have been associated with a decreased expression of macrophage inflammatory protein 3α (MIP3α) in cervical SIL, we tested the effect of exogenous MIP3α on the migration of LC in a (pre)neoplastic epithelium formed in vitro. By using a Boyden chamber assay, we first showed that the migratory capacity of LC generated in vitro is significantly increased in the presence of MIP3α compared to control medium. We next demonstrated that MIP3α is able to increase the 3D infiltration of LC in organotypic cultures of HPV-transformed keratinocytes. This property to stimulate LC migration was not altered after inclusion of MIP3α in a bioadhesive polycarbophil gel. Moreover, the function of DC to exert cytostatic effects and to present alloantigens was not altered in the presence of MIP3α. P. Hubert and L. Herman contributed equally to this work.     

A metabolic study of the regulation of proteolysis by sugars in maize root tips: effects of glycerol and dihydroxyacetone

Sugars, the main growth substrates of plants, act as physiological signals in the complex regulatory network of sugar metabolism. To investigate the function of different glycolytic steps in sugar sensing and signaling we compared the effects of carbon starvation with those of glucose, glycerol and dihydroxyacetone on carbon metabolism, proteolysis, and protease expression in excised maize (Zea mays L.) root tips. Respiration, soluble proteins, protein turnover and proteolytic activities were monitored as a function of time, along with in vitro and in vivo analysis of a variety of metabolites (sugars, amino and organic acids, phosphoesters, adenine nucleotides...) using ¹³C, ³¹P and ¹H NMR spectroscopy. Our results indicate that, in maize root tips, endopeptidase activities and protease expression are induced in response to a decrease in carbon supply to the upper part of the glycolytic pathway, i.e. at the hexokinase step. Proteolysis would be controlled downstream glycolysis, probably at the level of the respiratory substrate supply to mitochondria. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Two-dimensional fluorescence difference gel electrophoresis analysis of spermatogenesis in the rat

The molecular mechanisms underlying normal and pathological spermatogenesis remain poorly understood. We compared protein concentrations in different germ cell types to identify those proteins specifically or preferentially expressed at each stage of rat spermatogenesis. Crude cytosolic protein extracts and reversed-phase HPLC prefractionated cytosolic extracts from spermatogonia, pachytene spermatocytes, and early spermatids were subjected to two-dimensional difference gel electrophoresis (2-D DIGE). By comparing gels and carrying out statistical analyses, we were able to identify 1274 protein spots with relative abundances differing significantly between the three cell types. We found that 265 of these spots displaying highly differential expression (ratio > or = 2.5 between two cell types), identified by mass fingerprinting, corresponded to 123 nonredundant proteins. The proteins clustered into three clades, corresponding to mitotic, meiotic, and post-meiotic cell types. The differentially expressed proteins identified by 2-D DIGE were confirmed and validated by western blotting and immunohistochemistry, in the few cases in which antibodies were available. 2-D DIGE appears a relevant proteomics approach for studying rat germ cell differentiation, allowing the establishment of the precise expression profiles for a relatively large number of proteins during normal spermatogenesis.     

The kinetics of denitrification in permeable sediments

Permeable sediments comprise the majority of shelf sediments, yet the rates of denitrification remain highly uncertain in these environments. Computational models are increasingly being used to understand the dynamics of denitrification in permeable sediments, which are complex environments to study experimentally. The realistic implementation of such models requires reliable experimentally derived data on the kinetics of denitrification. Here we undertook measurements of denitrification kinetics as a function of nitrate concentration in carefully controlled flow through reactor experiments on sediments taken from six shallow coastal sites in Port Phillip Bay, Victoria, Australia. The results showed that denitrification commenced rapidly (within 30 min) after the onset of anoxia and the kinetics could be well described by Michaelis–Menten kinetics with half saturation constants (apparent K_m) ranging between 1.5 and 19.8 μM, and maximum denitrification rate (V_max) were in the range of 0.9–7.5 nmol mL^?1 h^?1. The production of N₂ through anaerobic ammonium oxidation (anammox) was generally found to be less than 10 % of denitrification. V_max were in the same range as previously reported in cohesive sediments despite organic carbon contents one order of magnitude lower for the sediments studied here. The ratio of sediment O₂ consumption to V_max was in the range of 0.02–0.09, and was on average much lower than the theoretical ratio of 0.8. As a consequence, models implemented with the theoretical ratio of 0.8 are likely to overestimate denitrification by a factor of ~3. The most likely explanation for this is that the microbial community is not able to instantaneously shift or optimally use a particular electron acceptor in the highly dynamic redox environment experienced in permeable sediments. In contrast to previous studies, we did not observe any significant rates of oxic denitrification.     

Histidine modification and mutagenesis point to the involvement of a large conformational change in the mechanism of action of phage lambda lysozyme

Phage lambda lysozyme (lambdaL) is structurally related to other known lysozymes but its mechanism of action is different from the classical lysozyme mechanism, acting as a transglycosidase rather than a hydrolase. As two conformations have been revealed by the crystal structure, we investigated the effect of mutating and modifying a histidine located near to or far from the active site in the respective closed and open conformations. Whereas its asparagine mutation has little or no effect on activity, its N-carbethoxylation inactivates the enzyme. This provide further evidence for the involvement of the closed conformation and for the need of conformational mobility in lambdaL function.     

Influence of macroinvertebrates and macrophytes on waterfowl utilization of wetlands in the Prairie Pothole Region of northwestern Wisconsin

Waterfowl and limnological data were monitored on Waterfowl Production Area (WPA) wetlands in northwestern Wisconsin over a 6-yr period (1983–88) to determine the impact of macroinvertebrates and macrophytes on waterfowl utilization. Interrelationships between limnological conditions and Waterfowl Breeding Pair Densities (BPDs reported as pairs/ha water surface) were analyzed using correlation and general linear model analysis techniques.Annual changes in waterfowl BPDs differed between wetlands according to differences in the structure of macrophyte communities and basin morphometry. The strength of associations differed between the two dominant waterfowl species. In a wetland dominated by dense stands of submersed vegetation, annual fluctuations in blue-winged teal (Anas discors) BPDs corresponded directly with changes in macrophyte biomass, but not with changes in macroinvertebrate density. In a nearby less densely vegetated wetland of similar water chemistry and trophic status, fluctuations in teal BPDs corresponded directly with changes in macroinvertebrate density, but not with changes in macrophyte biomass. These associations occurred despite a significant positive correlation between macroinvertebrates and macrophyte biomass in the latter habitat. Annual fluctuations in mallard (Anas platyrhynchos) BPDs were not correlated significantly with either macrophyte biomass or macroinvertebrate density in either wetland.