Novel nevirapine-like inhibitors with improved activity against NNRTI-resistant HIV: 8-heteroarylthiomethyldipyridodiazepinone derivatives

A series of 8-heteroarylthiomethyldipyridodiazepinone derivatives were prepared and evaluated for their antiviral profile against wild type virus and the important K103N/Y181C mutant as an indicator for broad activity. 2,6-Dimethylpyridine derivative 16 was found to have a good pharmacokinetic profile in spite of poor metabolic stability in rat liver microsomes.     

Molecular and genealogical characterization of the R1443X BRCA1 mutation in high-risk French-Canadian breast/ovarian cancer families

The Quebec population contains about six-million French Canadians, descended from the French settlers who colonized Nouvelle-France between 1608 and 1765. Although the relative genetic contribution of each of these founders is highly variable, altogether they account for the major part of the contemporary French-Canadian gene pool. This study was designed to analyze the role of this founder effect in the introduction and diffusion of the BRCA1 recurrent R1443X mutant allele. A highly conserved haplotype, observed in 18 French-Canadian families and generated using 17 microsatellite markers surrounding the BRCA1 locus, supports the fact that the R1443X mutation is a founder mutation in the Quebec population. We also performed haplotyping analysis of R1443X carriers on 19 other families from seven different nationalities; although the same alleles are shared for three markers surrounding the BRCA1 gene, distinct haplotypes were obtained in four families, suggesting multiple origins for the R1443X mutation. Ascending genealogies of the 18 French Canadian families and of controls were reconstructed on an average depth of 10 generations. We identified the founder couple with the highest probability of having introduced the mutation in the population. Based on the descending genealogy of this couple, we detected the presence of geographical concentration in the diffusion pattern of the mutation. This study demonstrates how molecular genetics and demogenetic analyses can complement each other to provide findings that could have an impact on public health. Moreover, this approach is certainly not unique to breast cancer genetics and could be used to understand other complex traits.Other members of the BCLC Haplotype Group involved in this study are listed in Appendix 1Other members of INHERIT BRCAs involved in this study are listed in Appendix 2H. Vézina and F. Durocher contributed equally to this work and should be regarded as joint first author     

Evaluation for Hsp70 as a biomarker of effect of pollutants on the earthworm Lumbricus terrestris

Induction of heat shock proteins (Hsps) is often associated with a cellular response to a harmful stress or to adverse life conditions. The main aims of the present study were (1) to assess if stress-induced Hsp70 could be used to monitor exposure of the earthworm species Lumbricus terrestris to various soil pollutants, (2) to assess the specificity of pollutants in their tissue targeting and in Hsp70 induction, and (3) to evaluate if dose-response relationships could be established and if the stress-response observed was specific. The midgut/intestinal tissues of L. terrestris are shown to express an inducible member of the Hsp70 family after heat shock treatment in vitro and exposures to different soil toxicants in vivo (re: artificial soil). Short-term (24-72 hours) and long-term (14-16 days) exposures to the chemical standards chloroacetamide and pentachlorophenol and to heavy metals (Pb++, Cd++, Cu++, and Hg++) also affected the earthworms, and Hsp70 was induced in their midgut/intestinal tissues. After a 3-day exposure to heavy metals, the level of Hsp70 induction in the midgut/intestinal tissues appears to correlate well with the reported in vivo and in vitro toxicity data. Comparatively, in proximal and midbody wall muscle tissues of animals exposed to the heavy metals, a decrease in expression of Hsp70 was sometimes detected. Thus Hsp analysis by Western blot in L. terrestris tissues and particularly in the midgut/intestine proved to be a suitable and sensitive assay for adverse effects in earthworms and showed a good level of reproducibility despite some individual variations. The use of pristine/nonexposed animals transposed into contaminated environments as in the present study should therefore be of high ecological relevance. Induction of Hsp70 in earthworms should represent not only a good wide-spectrum biomarker of exposure but also a biomarker of effect since known toxicants altered gene expression in tissues of these animals, as contrasted with a simple accumulation of Hsp. Hence, the detection of Hsp70 in earthworms can constitute an early-warning marker for the presence of potentially deleterious agents in soils, with L. terrestris in particular and earthworms in general acting as potential sentinel animal species.     

Development of the Canadian beef reference herd for gene mapping studies

A project to map quantitative trait loci (QTL), in beef cattle using a full-sib design was initiated using six Bos taurus breeds. Embryo transfer was used in a large scale, short timeframe experiment to develop this herd for gene mapping. Full-sib families allowed for genetic information to be followed through both the sire and the dam and for both parents to be slaughtered so that carcass quality data could also be obtained from both of them at close to typical slaughter ages. Repeatability of response to superovulation was significant among the 3 flushes per female. Response to superovulation was negatively correlated with backfat of the donor. Crossbred embryos were found to have higher survival than purebred embryos.     

How mechanisms of habitat preference evolve and promote divergence with gene flow

Habitat preference may promote adaptive divergence and speciation, yet the conditions under which this is likely are insufficiently explored. We use individual‐based simulations to study the evolution and consequence of habitat preference during divergence with gene flow, considering four different underlying genetically based behavioural mechanisms: natal habitat imprinting, phenotype‐dependent, competition‐dependent and direct genetic habitat preference. We find that the evolution of habitat preference generally requires initially high dispersal, is facilitated by asymmetry in population sizes between habitats, and is hindered by an increasing number of underlying genetic loci. Moreover, the probability of habitat preference to emerge and promote divergence differs greatly among the underlying mechanisms. Natal habitat imprinting evolves most easily and can allow full divergence in parameter ranges where no divergence is possible in the absence of habitat preference. The reason is that imprinting represents a one‐allele mechanism of assortative mating linking dispersal behaviour very effectively to local selection. At the other extreme, direct genetic habitat preference, a two‐allele mechanism, evolves under restricted conditions only, and even then facilitates divergence weakly. Overall, our results indicate that habitat preference can be a strong reproductive barrier promoting divergence with gene flow, but that this is highly contingent on the underlying preference mechanism.     

Nα-Ipteroyltetra (γ-Glutamyl)]-Lysine as a Ligand for the Purification of Thymidylate Synthetase by Affinity Chromatography

N^α[Pteroyltetra (γ-glutamyl)]-lysine Sepharose was synthesized and shown to be a stable, high capacity affinity matrix capable of bringing about the purification of Lactobacillus casei thymidylate synthetase to maximum specific activity from crude extracts in high yield. Under conditions optimal for binding of thymidylate synthetase, dihydrofolate reductase was not bound.     

Evolution of mate choice and the so‐called magic traits in ecological speciation

Non‐random mating provides multiple evolutionary benefits and can result in speciation. Biological organisms are characterised by a myriad of different traits, many of which can serve as mating cues. We consider multiple mechanisms of non‐random mating simultaneously within a unified modelling framework in an attempt to understand better which are more likely to evolve in natural populations going through the process of local adaptation and ecological speciation. We show that certain traits that are under direct natural selection are more likely to be co‐opted as mating cues, leading to the appearance of magic traits (i.e. phenotypic traits involved in both local adaptation and mating decisions). Multiple mechanisms of non‐random mating can interact so that trait co‐evolution enables the evolution of non‐random mating mechanisms that would not evolve alone. The presence of magic traits may suggest that ecological selection was acting during the origin of new species.     

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts¹. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane². At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones^3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy². At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli^6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups ⁸. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium^9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation^12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice. Download video file.^{(54M, mov)}     

Improving estimates of maximal organic carbon stabilization by fine soil particles

Organic carbon (C) associated with fine soil particles (<20 μm) is relatively stable and accounts for a large proportion of total soil organic C (SOC). The soil C saturation concept proposes a maximal amount of SOC that can be stabilized in the fine soil fraction, and the soil C saturation deficit (i.e., the difference between current SOC and the maximal amount) is presumed to affect the capacity, magnitude, and rate of SOC storage. In this study, we argue that predictions using current models underestimate maximal organic C stabilization of fine soil particles due to fundamental limitations of using least-squares linear regression. The objective was to improve predictions of maximal organic C stabilization by using two alternative approaches; one mechanistic, based on organic C loadings, and one statistical, based on boundary line analysis. We collected 342 data points on the organic C content of fine soil particles, fine particle mass proportions in bulk soil, dominant soil mineral types, and land use types from 32 studies. Predictions of maximal organic C stabilization using linear regression models are questionable because of the use of data from soils that may not be saturated in SOC and because of the nature of regression itself, resulting in a high proportion of presumed over-saturated samples. Predictions of maximal organic C stabilization using the organic C loading approach fit the data for soils dominated by 2:1 minerals well, but not soils dominated by 1:1 minerals; suggesting that the use of a single value for specific surface area, and therefore a single organic C loading, to represent a large dataset is problematic. In boundary line analysis, only data representing soils having reached the maximal amount (upper tenth percentile) were used. The boundary line analysis estimate of maximal organic C stabilization (78 ± 4 g C kg^?1 fraction) was more than double the estimate by the linear regression approach (33 ± 1 g C kg^?1 fraction). These results show that linear regression models do not adequately predict maximal organic C stabilization. Soil properties associated with soil mineralogy, such as specific surface area and organic C loading, should be incorporated to generate more mechanistic models for predicting soil C saturation, but in their absence, statistical models should represent the upper envelope rather than the average value.     

Co-Culture of Human Bronchial Fibroblasts and CD4+ T Cells Increases Th17 Cytokine Signature

Background

Airway inflammation is an important characteristic of asthma and has been associated with airway remodelling and bronchial hyperreactivity. The mucosal microenvironment composed of structural cells and highly specialised extracellular matrix is able to amplify and promote inflammation. This microenvironment leads to the development and maintenance of a specific adaptive response characterized by Th2 and Th17. Bronchial fibroblasts produce multiple mediators that may play a role in maintaining and amplifying this response in asthma.

Objective

To investigate the role of bronchial fibroblasts obtained from asthmatic subjects and healthy controls in regulating Th17 response by creating a local micro-environment that promotes this response in the airways.

Methods

Human bronchial fibroblasts and CD4⁺T cells were isolated from atopic asthmatics and non-atopic healthy controls. CD4⁺T were co-cultured with bronchial fibroblasts of asthmatic subjects and healthy controls. RORc gene expression was detected by qPCR. Phosphorylated STAT-3 and RORγt were evaluated by western blots. Th17 phenotype was measured by flow cytometry. IL-22, IL17, IL-6 TGF-β and IL1-β were assessed by qPCR and ELISA.

Results

Co-culture of CD4⁺T cells with bronchial fibroblasts significantly stimulated RORc expression and induced a significant increase in Th17 cells as characterized by the percentage of IL-17⁺/CCR6⁺ staining in asthmatic conditions. IL-17 and IL-22 were increased in both normal and asthmatic conditions with a significantly higher amount in asthmatics compared to controls. IL-6, IL-1β, TGF-β and IL-23 were significantly elevated in fibroblasts from asthmatic subjects upon co-culture with CD4⁺T cells. IL-23 stimulates IL-6 and IL-1β expression by bronchial fibroblasts.

Conclusion

Interaction between bronchial fibroblasts and T cells seems to promote specifically Th17 cells profile in asthma. These results suggest that cellular interaction particularly between T cells and fibroblasts may play a pivotal role in the regulation of the inflammatory response in asthma.