Defining the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 inhibition.

BubR1 is essential for the mitotic checkpoint that prevents aneuploidy in cellular progeny by triggering anaphase delay in response to kinetochores incorrectly/not attached to the mitotic spindle. Here, we define the molecular architecture of the functionally significant N-terminal region of human BubR1 and present the 1.8 A crystal structure of its tetratricopeptide repeat (TPR) domain. The structure reveals divergence from the classical TPR fold and is highly similar to the TPR domain of budding yeast Bub1. Shared distinctive features include a disordered loop insertion, a 3(10)-helix, a tight turn involving glycine positive Phi angles, and noncanonical packing of and between the TPR motifs. We also define the molecular determinants of the interaction between BubR1 and kinetochore protein Blinkin. We identify a shallow groove on the concave surface of the BubR1 TPR domain that forms multiple discrete and potentially cooperative interactions with Blinkin. Finally, we present evidence for a direct interaction between BubR1 and Bub1 mediated by regions C-terminal to their TPR domains. This interaction provides a mechanism for Bub1-dependent kinetochore recruitment of BubR1. We thus present novel molecular insights into the structure of BubR1 and its interactions at the kinetochore-microtubule interface. Our studies pave the way for future structure-directed engineering aimed at dissecting the roles of kinetochore-bound and other pools of BubR1 in vivo

Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases.

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region

Automated Analysis of Cryptococcal Macrophage Parasitism Using GFP-Tagged Cryptococci

The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic immune effector cells, a phenomenon that correlates strongly with virulence in rodent models of infection. Despite the importance of phagocyte/Cryptococcus interactions to disease progression, current methods for assaying virulence in the acrophage system are both time consuming and low throughput. Here, we introduce the first stable and fully characterised GFP–expressing derivatives of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Both strains show unaltered responses to environmental and host stress conditions and no deficiency in virulence in the macrophage model system. In addition, we report the development of a method to effectively and rapidly investigate macrophage parasitism by flow cytometry, a technique that preserves the accuracy of current approaches but offers a four-fold improvement in speed

Synthesis of some nucleosides derivatives from L- rhamnose with expected biological activity

Practical procedures for production of variously blocked compounds from L-rhamnose have been developed. These compounds are highly useful as indirect β-L-rhamnosyl donors. This approach represents a new method for the synthesis of aromatic nucleoside analogues and the synthesis of (3S, 4S, 5S, 6R) 3, 4, 5-triacetoxy-2-methyl-7,9-diaza-1-oxa-spiro [4,5]decane-10-one-8-thione (7)

Testing matter effects in propagation of atmospheric and long-baseline neutrinos

We quantify our current knowledge of the size and flavor structure of the matter effects in the evolution of atmospheric and long-baseline neutrinos based solely on the analysis of the corresponding neutrino data. To this aim we generalize the matter potential of the Standard Model by rescaling its strength, rotating it away from the e-e sector, and rephasing it with respect to the vacuum term. This phenomenological parametrization can be easily translated in terms of non-standard neutrino interactions in matter. We show that in the most general case, the strength of the potential cannot be determined solely by atmospheric and long-baseline data. However its flavor composition is very much constrained and the present determination of the neutrino masses and mixing is robust under its presence. We also present an update of the constraints arising from this analysis in the particular case in which no potential is present in the e-mu and e-tau sectors. Finally we quantify to what degree in this scenario it is possible to alleviate the tension between the oscillation results for neutrinos and antineutrinos in the MINOS experiment and show the relevance of the high energy part of the spectrum measured at MINOS.Comment: PDFLaTeX file using JHEP3 class, 25 pages, 7 figures included. Accepted for publication in JHE

Weekly platinum chemotherapy for recurrent ovarian cancer

British Journal of Cancer (2002) 86, 2–4. DOI: 10.1038/sj/bjc/6600062 www.bjcancer.co

Persistence on therapy and propensity matched outcome comparison of two subcutaneous interferon beta 1a dosages for multiple sclerosis

To compare treatment persistence between two dosages of interferon β-1a in a large observational multiple sclerosis registry and assess disease outcomes of first line MS treatment at these dosages using propensity scoring to adjust for baseline imbalance in disease characteristics. Treatment discontinuations were evaluated in all patients within the MSBase registry who commenced interferon β-1a SC thrice weekly (n = 4678). Furthermore, we assessed 2-year clinical outcomes in 1220 patients treated with interferon β-1a in either dosage (22 µg or 44 µg) as their first disease modifying agent, matched on propensity score calculated from pre-treatment demographic and clinical variables. A subgroup analysis was performed on 456 matched patients who also had baseline MRI variables recorded. Overall, 4054 treatment discontinuations were recorded in 3059 patients. The patients receiving the lower interferon dosage were more likely to discontinue treatment than those with the higher dosage (25% vs. 20% annual probability of discontinuation, respectively). This was seen in discontinuations with reasons recorded as “lack of efficacy” (3.3% vs. 1.7%), “scheduled stop” (2.2% vs. 1.3%) or without the reason recorded (16.7% vs. 13.3% annual discontinuation rate, 22 µg vs. 44 µg dosage, respectively). Propensity score was determined by treating centre and disability (score without MRI parameters) or centre, sex and number of contrast-enhancing lesions (score including MRI parameters). No differences in clinical outcomes at two years (relapse rate, time relapse-free and disability) were observed between the matched patients treated with either of the interferon dosages. Treatment discontinuations were more common in interferon β-1a 22 µg SC thrice weekly. However, 2-year clinical outcomes did not differ between patients receiving the different dosages, thus replicating in a registry dataset derived from “real-world” database the results of the pivotal randomised trial. Propensity score matching effectively minimised baseline covariate imbalance between two directly compared sub-populations from a large observational registry