A microtubule interactome: complexes with roles in cell cycle and mitosis.

addresses: Department of Zoology, University of Oxford, Oxford, United Kingdom.notes: PMCID: PMC2323305types: Journal Article; Research Support, Non-U.S. Gov'tCopyright: © 2008 Hughes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division

Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which "amplifies" spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle

Small molecules, big targets: drug discovery faces the protein-protein interaction challenge.

Protein-protein interactions (PPIs) are of pivotal importance in the regulation of biological systems and are consequently implicated in the development of disease states. Recent work has begun to show that, with the right tools, certain classes of PPI can yield to the efforts of medicinal chemists to develop inhibitors, and the first PPI inhibitors have reached clinical development. In this Review, we describe the research leading to these breakthroughs and highlight the existence of groups of structurally related PPIs within the PPI target class. For each of these groups, we use examples of successful discovery efforts to illustrate the research strategies that have proved most useful.JS, DES and ARB thank the Wellcome Trust for funding.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nrd.2016.2

Enabling large-scale design, synthesis and validation of small molecule protein-protein antagonists

Although there is no shortage of potential drug targets, there are only a handful known low-molecular-weight inhibitors of protein-protein interactions (PPIs). One problem is that current efforts are dominated by low-yield high-throughput screening, whose rigid framework is not suitable for the diverse chemotypes present in PPIs. Here, we developed a novel pharmacophore-based interactive screening technology that builds on the role anchor residues, or deeply buried hot spots, have in PPIs, and redesigns these entry points with anchor-biased virtual multicomponent reactions, delivering tens of millions of readily synthesizable novel compounds. Application of this approach to the MDM2/p53 cancer target led to high hit rates, resulting in a large and diverse set of confirmed inhibitors, and co-crystal structures validate the designed compounds. Our unique open-access technology promises to expand chemical space and the exploration of the human interactome by leveraging in-house small-scale assays and user-friendly chemistry to rationally design ligands for PPIs with known structure. © 2012 Koes et al

Conventional and molecular cytogenetics of human non-medullary thyroid carcinoma: characterization of eight cell line models and review of the literature on clinical samples

<p>Abstract</p> <p>Background</p> <p>Cell lines are often poorly characterized from a genetic point of view, reducing their usefulness as tumor models. Our purpose was to assess the genetic background of eight commonly used human thyroid carcinoma models and to compare the findings with those reported for primary tumors of the gland.</p> <p>Methods</p> <p>We used chromosome banding analysis and comparative genomic hybridization to profile eight non-medullary thyroid carcinoma cell lines of papillary (TPC-1, FB2, K1 and B-CPAP), follicular (XTC-1) or anaplastic origin (8505C, C643 and HTH74). To assess the representativeness of the findings, we additionally performed a thorough review of cytogenetic (n = 125) and DNA copy number information (n = 270) available in the literature on clinical samples of thyroid carcinoma.</p> <p>Results</p> <p>The detailed characterization of chromosomal markers specific for each cell line revealed two cases of mistaken identities: FB2 was shown to derive from TPC-1 cells, whereas K1 cells have their origin in cell line GLAG-66. All cellular models displayed genomic aberrations of varying complexity, and recurrent gains at 5p, 5q, 8q, and 20q (6/7 cell lines) and losses at 8p, 13q, 18q, and Xp (4/7 cell lines) were seen. Importantly, the genomic profiles were compatible with those of the respective primary tumors, as seen in the meta-analysis of the existing literature data.</p> <p>Conclusion</p> <p>We provide the genomic background of seven independent thyroid carcinoma models representative of the clinical tumors of the corresponding histotypes, and highlight regions of recurrent aberrations that may guide future studies aimed at identifying target genes. Our findings further support the importance of routinely performing cytogenetic studies on cell lines, to detect cross-contamination mishaps such as those identified here.</p

Kebab: Kinetochore and EB1 Associated Basic Protein That Dynamically Changes Its Localisation during Drosophila Mitosis

Microtubule plus ends are dynamic ends that interact with other cellular structures. Microtubule plus end tracking proteins are considered to play important roles in the regulation of microtubule plus ends. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. Here we report the identification of a novel Drosophila protein, which we call Kebab (kinetochore and EB1 associated basic protein), through in vitro expression screening for EB1-interacting proteins. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. In telophase, it associates with microtubules in central-spindle and centrosomal regions. The localisation to kinetochores depends on microtubules. The protein has a domain most similar to the atypical CH domain of Ndc80, and a coiled-coil domain. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localisation. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility

Measuring Adherence to Inhaled Control Medication in Patients with Asthma: Comparison Among an Asthma App, Patient Self‐Report and Physician Assessment

Background: Previous studies have demonstrated the feasibility of using an asthma app to support medication management and adherence but failed to compare with other measures currently used in clinical practice. However, in a clinical setting, any additional adherence measurement must be evaluated in the context of both the patient and physician perspectives so that it can also help improve the process of shared decision making. Thus, we aimed to compare different measures of adherence to asthma control inhalers in clinical practice, namely through an app, patient self-report and physician assessment. Methods: This study is a secondary analysis of three prospective multicentre observational studies with patients (≥13 years old) with persistent asthma recruited from 61 primary and secondary care centres in Portugal. Patients were invited to use the InspirerMundi app and register their inhaled medication. Adherence was measured by the app as the number of doses taken divided by the number of doses scheduled each day and two time points were considered for analysis: 1-week and 1-month. At baseline, patients and physicians independently assessed adherence to asthma control inhalers during the previous week using a Visual Analogue Scale (VAS 0-100). Results: A total of 193 patients (72% female; median [P25-P75] age 28 [19-41] years old) were included in the analysis. Adherence measured by the app was lower (1 week: 31 [0-71]%; 1 month: 18 [0-48]%) than patient self-report (80 [60-95]) and physician assessment (82 [51-94]) (p 0.05). There was a moderate correlation between patient self-report and physician assessment (ρ = 0.596, p < 0.001). Conclusions: Adherence measured by the app was lower than that reported by the patient or the physician. This was expected as objective measurements are commonly lower than subjective evaluations, which tend to overestimate adherence. Nevertheless, the low adherence measured by the app may also be influenced by the use of the app itself and this needs to be considered in future studies.info:eu-repo/semantics/publishedVersio

Proliferation and survival molecules implicated in the inhibition of BRAF pathway in thyroid cancer cells harbouring different genetic mutations

<p>Abstract</p> <p>Background</p> <p>Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds.</p> <p>Methods</p> <p>Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot.</p> <p>Results</p> <p>Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAF^V600Emutation. In BRAF^V600Emutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27^Kip1. Specific inhibition of BRAF by RNAi in cells with BRAF^V600Emutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAF^V600Emutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2.</p> <p>Conclusion</p> <p>Our results in thyroid cancer cells, namely those harbouring BRAF^V600Emutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAF^V600Emutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and harbouring BRAF mutations.</p

Mutation Analysis of NR5A1 Encoding Steroidogenic Factor 1 in 77 Patients with 46, XY Disorders of Sex Development (DSD) Including Hypospadias

BACKGROUND: Mutations of the NR5A1 gene encoding steroidogenic factor-1 have been reported in association with a wide spectrum of 46,XY DSD (Disorder of Sex Development) phenotypes including severe forms of hypospadias. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the frequency of NR5A1 gene mutations in a large series of patients presenting with 46,XY DSD and hypospadias. Based on their clinical presentation 77 patients were classified either as complete or partial gonadal dysgenesis (uterus seen at genitography and/or surgery, n = 11), ambiguous external genitalia without uterus (n = 33) or hypospadias (n = 33). We identified heterozygous NR5A1 mutations in 4 cases of ambiguous external genitalia without uterus (12.1%; p.Trp279Arg, pArg39Pro, c.390delG, c140_141insCACG) and a de novo missense mutation in one case with distal hypospadias (3%; p.Arg313Cys). Mutant proteins showed reduced transactivation activity and mutants p.Arg39Pro and p.Arg313Cys did not synergize with the GATA4 cofactor to stimulate reporter gene activity, although they retained their ability to physically interact with the GATA4 protein. CONCLUSIONS/SIGNIFICANCE: Mutations in NR5A1 were observed in 5/77 (6.5%) cases of 46,XY DSD including hypospadias. Excluding the cases of 46,XY gonadal dysgenesis the incidence of NR5A1 mutations was 5/66 (7.6%). An individual with isolated distal hypopadias carried a de novo heterozygous missense mutation, thus extending the range of phenotypes associated with NR5A1 mutations and suggesting that this group of patients should be screened for NR5A1 mutations

First Report of 13 Species of Culicoides (Diptera: Ceratopogonidae) in Mainland Portugal and Azores by Morphological and Molecular Characterization

The genus Culicoides (Diptera: Ceratopogonidae) contains important vectors of animal and human diseases, including bluetongue, African horse sickness and filariosis. A major outbreak of bluetongue occurred in mainland Portugal in 2004, forty eight years after the last recorded case. A national Entomological Surveillance Plan was initiated in mainland Portugal, Azores and the Madeira archipelagos in 2005 in order to better understand the disease and facilitate policy decisions. During the survey, the most prevalent Culicoides species in mainland Portugal was C. imicola (75.3%) and species belonging to the Obsoletus group (6.5%). The latter were the most prevalent in Azores archipelago, accounting for 96.7% of the total species identified. The Obsoletus group was further characterized by multiplex Polymerase Chain Reaction to species level showing that only two species of this group were present: C. obsoletus sensu strictu (69.6%) and C. scoticus (30.4%). Nine species of Culicoides were detected for the first time in mainland Portugal: C. alazanicus, C. bahrainensis, C. deltus, C. lupicaris, C. picturatus, C. santonicus, C. semimaculatus, C. simulator and C. subfagineus. In the Azores, C. newsteadi and C. circumscriptus were identified for the first time from some islands, and bluetongue vectors belonging to the Obsoletus group (C. obsoletus and C. scoticus) were found to be widespread