The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid‐transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA‐induced silencing complexes), encounter of the target mRNA, and Ago2‐mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA‐ and siRNA‐loaded Ago2 populations co‐sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon‐induced protein kinase (PACT). Fractionation and membrane co‐immune precipitations further confirm that siRNA‐loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC‐associated double‐stranded siRNA, diagnostic of RISC loading, and RISC‐mediated mRNA cleavage products exclusively co‐sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA‐independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA‐mediated RNA silencing.     

Into the wild—WAMBAM goes to Canada

The sixth Wild Animal Models Bi‐Annual Meeting was held in July 2017 in Québec, with 42 participants. This report documents the evolution of questions asked and approaches used in evolutionary quantitative genetic studies of wild populations in recent decades, and how these questions and approaches were represented at the recent meeting. We explore how ideas from previous meetings in this series have developed to their present states, and consider how the format of the meetings may be particularly useful at fostering the rapid development and proliferation of ideas and approaches.     

Design, synthesis, and in vitro biological activity of benzimidazole based factor Xa inhibitors

Inhibitors based on the benzimidazole scaffold showed subnanomolar potency against Factor Xa with 500-1000-fold selectivity against thrombin and 50-100-fold selectivity against trypsin. The 2-substituent on the benzimidazole ring had a strong impact on the FXa inhibitory activity. Crystallography studies suggest that the 2-substituent may have a conformational effect favoring the extended binding conformation.     

New Loci in DICTYOSTELIUM DISCOIDEUM Determining Pigment Formation and Growth on BACILLUS SUBTILIS

Seventeen independently isolated pigmentless (white) mutations in Dictyostelium discoideum are all recessive and fall into three complementation groups identifying two new whi loci in addition to the previously characterized whiA locus. whiB and whiC map to linkage groups III and IV, respectively. In addition, it was discovered that our laboratory stock of NC4, the wild-type strain from which these mutants were derived, has spontaneously lost the ability to grow on Bacillus subtilis. This new mutation, bsgB500, maps to linkage group VII and is not allelic to bsgA. bsgB500 is the first spontaneously derived mutation in D. discoideum that can be used to select heterozygous diploids, and for the first time allows genetic analysis to be routinely performed on strains derived from an unmutagenized background.     

Interplay of robustness and plasticity of life‐history traits drives ecotypic differentiation in thermally distinct habitats

Phenotypic plasticity describes the ability of an individual to alter its phenotype in response to the environment and is potentially adaptive when dealing with environmental variation. However, robustness in the face of a changing environment may often be beneficial for traits that are tightly linked to fitness. We hypothesized that robustness of some traits may depend on specific patterns of plasticity within and among other traits. We used a reaction norm approach to study robustness and phenotypic plasticity of three life‐history traits of the collembolan Orchesella cincta in environments with different thermal regimes. We measured adult mass, age at maturity and growth rate of males and females from heath and forest habitats at two temperatures (12 and 22 °C). We found evidence for ecotype‐specific robustness of female adult mass to temperature, with a higher level of robustness in the heath ecotype. This robustness is facilitated by plastic adjustments of growth rate and age at maturity. Furthermore, female fecundity is strongly influenced by female adult mass, explaining the importance of realizing a high mass across temperatures for females. These findings indicate that different predicted outcomes of life‐history theory can be combined within one species' ontogeny and that models describing life‐history strategies should not assume that traits like growth rate are maximized under all conditions. On a methodological note, we report a systematic inflation of variation when standard deviations and correlation coefficients are calculated from family means as opposed to individual data within a family structure.     

Mutations upstream of fabI in triclosan resistant Staphylococcus aureus strains are associated with elevated fabI gene expression

Background

The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations.

Results

Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI.

Conclusions

In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1544-y) contains supplementary material, which is available to authorized users.     

The canine isolate Lactobacillus acidophilus LAB20 adheres to intestinal epithelium and attenuates LPS-induced IL-8 secretion of enterocytes in vitro

Background

For a good probiotic candidate, the abilities to adhere to intestinal epithelium and to fortify barrier function are considered to be crucial for colonization and functionality of the strain. The strain Lactobacillus acidophilus LAB20 was isolated from the jejunum of a healthy dog, where it was found to be the most pre-dominant lactobacilli. In this study, the adhesion ability of LAB20 to intestinal epithelial cell (IECs) lines, IECs isolated from canine intestinal biopsies, and to canine, porcine and human intestinal mucus was investigated. Further, we studied the ability of LAB20 to fortify the epithelial cell monolayer and to reduce LPS-induced interleukin (IL-8) release from enterocytes.

Results

We found that LAB20 presented higher adhesion to canine colonic mucus as compared to mucus isolated from porcine colon. LAB20 showed adhesion to HT-29 and Caco-2 cell lines, and importantly also to canine IECs isolated from canine intestinal biopsies. In addition, LAB20 increased the transepithelial electrical resistance (TER) of enterocyte monolayers and thus strengthened the intestinal barrier function. The strain showed also anti-inflammatory capacity in being able to attenuate the LPS-induced IL-8 production of HT-29 cells.

Conclusion

In conclusion, canine indigenous strain LAB20 is a potential probiotic candidate for dogs adhering to the host epithelium and showing intestinal barrier fortifying and anti-inflammatory effects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0337-9) contains supplementary material, which is available to authorized users.     

A note on simulating null distributions for G matrix comparisons

Genetic variances and covariances, summarized in G matrices, are key determinants of the course of adaptive evolution. Consequently, understanding how G matrices vary among populations is critical to answering a variety of questions in evolutionary biology. A method has recently been proposed for generating null distributions of statistics pertaining to differences in G matrices among populations. The general approach facilitated by this method is likely to prove to be very important in studies of the evolution of G . We have identified an issue in the method that will cause it to create null distributions of differences in G matrices that are likely to be far too narrow. The issue arises from the fact that the method as currently used generates null distributions of statistics pertaining to differences in G matrices across populations by simulating breeding value vectors based on G matrices estimated from data, randomizing these vectors across populations, and then calculating null values of statistics from G matrices that are calculated directly from the variances and covariances among randomized vectors. This calculation treats breeding values as quantities that are directly measurable, instead of predicted from G matrices that are themselves estimated from patterns of covariance among kin. The existing method thus neglects a major source of uncertainty in G matrices, which renders it anti‐conservative. We first suggest a correction to the method. We then apply the original and modified methods to a very simple instructive scenario. Finally, we demonstrate the use of both methods in the analysis of a real data set.     

Decreased growth of wild soil microbes after 15 years of transplant-induced warming in a montane meadow

The carbon stored in soil exceeds that of plant biomass and atmospheric carbon and its stability can impact global climate. Growth of decomposer microorganisms mediates both the accrual and loss of soil carbon. Growth is sensitive to temperature and given the vast biological diversity of soil microorganisms, the response of decomposer growth rates to warming may be strongly idiosyncratic, varying among taxa, making ecosystem predictions difficult. Here, we show that 15 years of warming by transplanting plant–soil mesocosms down in elevation, strongly reduced the growth rates of soil microorganisms, measured in the field using undisturbed soil. The magnitude of the response to warming varied among microbial taxa. However, the direction of the response—reduced growth—was universal and warming explained twofold more variation than did the sum of taxonomic identity and its interaction with warming. For this ecosystem, most of the growth responses to warming could be explained without taxon-specific information, suggesting that in some cases microbial responses measured in aggregate may be adequate for climate modeling. Long-term experimental warming also reduced soil carbon content, likely a consequence of a warming-induced increase in decomposition, as warming-induced changes in plant productivity were negligible. The loss of soil carbon and decreased microbial biomass with warming may explain the reduced growth of the microbial community, more than the direct effects of temperature on growth. These findings show that direct and indirect effects of long-term warming can reduce growth rates of soil microbes, which may have important feedbacks to global warming.