The discovery of furo[2,3-c]pyridine-based indanone oximes as potent and selective B-Raf inhibitors

Virtual and high-throughput screening identified imidazo[1,2-a]pyrazines as inhibitors of B-Raf. We describe the rationale, SAR, and evolution of the initial hits to a series of furo[2,3-c]pyridine indanone oximes as highly potent and selective inhibitors of B-Raf.     

Differential expression, function and response to inflammatory stimuli of 11β-hydroxysteroid dehydrogenase type 1 in human fibroblasts: a mechanism for tissue-specific regulation of inflammation

Stromal cells such as fibroblasts play an important role in defining tissue-specific responses during the resolution of inflammation. We hypothesized that this involves tissue-specific regulation of glucocorticoids, mediated via differential regulation of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Expression, activity and function of 11β-HSD1 was assessed in matched fibroblasts derived from various tissues (synovium, bone marrow and skin) obtained from patients with rheumatoid arthritis or osteoarthritis. 11β-HSD1 was expressed in fibroblasts from all tissues but mRNA levels and enzyme activity were higher in synovial fibroblasts (2-fold and 13-fold higher mRNA levels in dermal and synovial fibroblasts, respectively, relative to bone marrow). Expression and activity of the enzyme increased in all fibroblasts following treatment with tumour necrosis factor-α or IL-1β (bone marrow: 8-fold and 37-fold, respectively, compared to vehicle; dermal fibroblasts: 4-fold and 14-fold; synovial fibroblasts: 7-fold and 31-fold; all P < 0.01 compared with vehicle). Treatment with IL-4 or interferon-γ was without effect, and there was no difference in 11β-HSD1 expression between fibroblasts (from any site) obtained from patients with rheumatoid arthritis or osteoarthritis. In the presence of 100 nmol/l cortisone, IL-6 production – a characteristic feature of synovial derived fibroblasts – was significantly reduced in synovial but not dermal or bone marrow fibroblasts. This was prevented by co-treatment with an 11β-HSD inhibitor, emphasizing the potential for autocrine activation of glucocorticoids in synovial fibroblasts. These data indicate that differences in fibroblast-derived glucocorticoid production (via the enzyme 11β-HSD1) between cells from distinct anatomical locations may play a key role in the predeliction of certain tissues to develop persistent inflammation.     

Testosterone, and winning and losing in human competition

Testosterone and cortisol were measured in six university tennis players across six matches during their varsity season. Testosterone rose just before most matches, and players with the highest prematch testosterone had the most positive improvement in mood before their matches. After matches, mean testosterone rose for winners relative to losers, especially for winners with very positive moods after their victories and who evaluated their own performance highly. Winners with rising testosterone had higher testosterone before their next match, in contrast to losers with falling testosterone, who had lower testosterone before their next match. Cortisol was not related to winning or losing, but it was related to seed (top players having low cortisol), and cortisol generally declined as the season progressed. These results are consistent with a biosocial theory of status.     

Expression of a Leishmaniadonovani nucleotide sugar transporter in Leishmaniamajor enhances survival in visceral organs

Leishmania donovani and Leishmaniainfantum infections cause fatal visceral leishmaniasis, and Leishmaniamajor causes self healing cutaneous lesions. It is poorly understood what genetic differences between these Leishmania species are responsible for the different pathologies of infection. To investigate whether L.donovani species-specific genes are involved in visceral Leishmania infection, we have examined a L.donovani species-specific gene Ld1590 (ortholog of LinJ15_V3.0900) that is a pseudogene in L.major. We have previously shown that transgenic expression of L.donovani Ld1590 in L.major significantly increased the liver and spleen parasite burdens in infected BALB/c mice. In this study we report that Ld1590 potentially encodes a nucleotide sugar transporter (NST) which localizes in the L.donovani Golgi apparatus. Surprisingly, although transgenic expression of the Ld1590 NST increased L.major survival in visceral organs, deletion of Ld1590 NST in L.donovani had no significant effect on L.donovani survival in mice. These observations suggest that loss of the functional Ld1590 gene in L.major may have been associated with reduced virulence in visceral organs in its animal reservoir and could have contributed to L.major’s tropism for cutaneous infections.     

Association between history and physical examination factors and change in lumbar multifidus muscle thickness after spinal manipulation in patients with low back pain

Understanding the clinical characteristics of patients with low back pain (LBP) who display improved lumbar multifidus (LM) muscle function after spinal manipulative therapy (SMT) may provide insight into a potentially synergistic interaction between SMT and exercise. Therefore, the purpose of this study was to identify the baseline historical and physical examination factors associated with increased contracted LM muscle thickness one week after SMT. Eighty-one participants with LBP underwent a baseline physical examination and ultrasound imaging assessment of the LM muscle during submaximal contraction before and one week after SMT. The relationship between baseline examination variables and 1-week change in contracted LM thickness was assessed using correlation analysis and hierarchical multiple linear regression. Four variables best predicted the magnitude of increases in contracted LM muscle thickness after SMT. When combined, these variables suggest that patients with LBP, (1) that are fairly acute, (2) have at least a moderately good prognosis without focal and irritable symptoms, and (3) exhibit signs of spinal instability, may be the best candidates for a combined SMT and lumbar stabilization exercise (LSE) treatment approach.     

Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a <Emphasis Type="Italic">Quercus</Emphasis> woodland

The Internal Transcribed Spacer (ITS) regions of ribosomal DNA are widely used as markers for phylogenetic analyses and environmental sampling from a variety of organisms including fungi, plants, and animals. In theory, concerted evolution homogenizes multicopy genes so that little or no variation exists within populations or individuals. However, contrary to theory, ITS variation has been confirmed in populations and individuals from a diverse range of eukaryotes. The presence of intraspecific and intra-individual variation in multicopy genes has important implications for ecological and phylogenetic studies, yet relatively little is known about natural variation of these genes, particularly at the community level. In this study, we examined intraspecific and intra-sporocarp ITS variation by DNA sequencing from sporocarps and pooled roots from 68 species of ectomycorrhizal fungi collected at a single site in a Quercus woodland. We detected ITS variation in 27 species, roughly 40% of the taxa examined. Although intraspecific ITS variation was generally low (0.16–2.85%, mean = 0.74%), it was widespread within this fungal community. We detected ITS variation in both sporocarps and ectomycorrhizal roots, and variation was present within species of Ascomycota and Basidiomycota, two distantly related lineages within the Fungi. We discuss the implications of such widespread ITS variability with special reference to DNA-based environmental sampling from diverse fungal communities. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies

Doa4 is a ubiquitin-specific protease in Saccharomyces cerevisiae that deubiquitinates integral membrane proteins sorted into the lumenal vesicles of late-endosomal multivesicular bodies (MVBs). We show that the non-catalytic N terminus of Doa4 mediates its recruitment to endosomes through its association with Bro1, which is one of several highly conserved class E Vps proteins that comprise the core MVB sorting machinery. In turn, Bro1 directly stimulates deubiquitination by interacting with a YPxL motif in the catalytic domain of Doa4. Mutations in either Doa4 or Bro1 that disrupt catalytic activation of Doa4 impair deubiquitination and sorting of MVB cargo proteins and lead to the formation of lumenal MVB vesicles that are predominantly small compared with the vesicles seen in wild-type cells. Thus, by recruiting Doa4 to late endosomes and stimulating its catalytic activity, Bro1 fulfills a novel dual role in coordinating deubiquitination in the MVB pathway.