Mutational Analysis of Hedgehog Signaling Pathway Genes in Human Malignant Mesothelioma

Background The Hedgehog (HH) signaling pathway is critical for embryonic development and adult homeostasis. Recent studies have identified regulatory roles for this pathway in certain cancers with mutations in the HH pathway genes. The extent to which mutations of the HH pathway genes are involved in the pathogenesis of malignant mesothelioma (MMe) is unknown. Methodology/Principal Findings Real-time PCR analysis of HH pathway genes PTCH1, GLI1 and GLI2 were performed on 7 human MMe cell lines. Exon sequencing of 13 HH pathway genes was also performed in cell lines and human MMe tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. GLI1, GLI2 and PTCH1 were highly expressed in MMe cells, indicative of active HH signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 MMe cell lines examined. A non-synonymous missense SUFU mutation (p.T411M) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.T411M mutation might alter protein function. However, we were unable to demonstrate any functional effect of this mutation on Gli activity. Deletion of exons of the PTCH1 gene was found in JU77 cells, resulting in loss of one of two extracellular loops implicated in HH ligand binding and the intracellular C-terminal domain. A 3-bp insertion (69_70insCTG) in SMO, predicting an additional leucine residue in the signal peptide segment of SMO protein was also identified in LO68 cells and a MMe tumour. Conclusions/Significance We identified the first novel mutations in PTCH1, SUFU and SMO associated with MMe. Although HH pathway mutations are relatively rare in MMe, these data suggest a possible role for dysfunctional HH pathway in the pathogenesis of a subgroup of MMe and help rationalize the exploration of HH pathway inhibitors for MMe therapy

Comparative Molecular Similarity Indices Analysis of 1-(Naphthylalky1)-1H-imidazole Analogs with Antiepileptic Activity

A three-dimensional quantitative structure-activity relationship (3D QSAR) of 44 structurally and functionally diverse series of 1- (Naphthylalkylimidazoles) as antiepileptic agents was studied using the Comparative molecular similarity indices analysis (CoMSIA) method. A training set containing 34 molecules served to establish the models. The optimum CoMSIA model obtained for the training set were all statistically significant, with cross-validated coefficients (q2) of 0.725 and conventional coefficients (r2ncv) of 0.998. The predictive capacities of the model were successfully validated by using a test set of 10 molecules that were not included in the training set. CoMSIA model (Model 1) obtained from the hydrophobic and Hbond acceptor field was found to have the best predictivity, with a predictive correlation coefficient (r2pred) of 0.67. The information obtained from this 3D-QSAR model can be used to guide the development of imidazoles as novel antiepileptic agents

Oral mucosal epithelial cells grown on porous silicon membrane for transfer to the rat eye

Dysfunction of limbal stem cells or their niche can result in painful, potentially sight-threatening ocular surface disease. We examined the utility of surface-modified porous-silicon (pSi) membranes as a scaffold for the transfer of oral mucosal cells to the eye. Male-origin rat oral mucosal epithelial cells were grown on pSi coated with collagen-IV and vitronectin, and characterised by immunocytochemistry. Scaffolds bearing cells were implanted into normal female rats, close to the limbus, for 8 weeks. Histology, immunohistochemistry and a multiplex nested PCR for sry were performed to detect transplanted cells. Oral mucosal epithelial cells expanded on pSi scaffolds expressed the corneal epithelial cell marker CK3/12. A large percentage of cells were p63⁺, indicative of proliferative potential, and a small proportion expressed ABCG2⁺, a putative stem cell marker. Cell-bearing scaffolds transferred to the eyes of live rats, were well tolerated, as assessed by endpoint histology. Immunohistochemistry for pan-cytokeratins demonstrated that transplanted epithelial cells were retained on the pSi membranes at 8 weeks post-implant, but were not detectable on the central cornea using PCR for sry. The pSi scaffolds supported and retained transplanted rat oral mucosal epithelial cells in vitro and in vivo and recapitulate some aspects of an artificial stem cell niche.Yazad D. Irani, Sonja Klebe, Steven J.P. McInnes, Marek Jasieniak, Nicolas H. Voelcker, Keryn A. William

Resource Utilization of Patients with Parkinson's Disease in the Late Stages of the Disease in Germany: Data from the CLaSP Study.

Objective: The Care of Late-Stage Parkinsonism (CLaSP) study aimed to collect qualitative and standardized patient data in six European countries (France, Germany, Netherlands, Portugal, UK, Sweden) to enable a detailed evaluation of the underexplored late stages of the disease (Hoehn and Yahr stage > 3) using clinical, neuropsychological, behavioral, and health economic data. The aim of this substudy was to provide a health economic evaluation for the German healthcare system. Methods: In Germany, 228 patients were included in the study. Costs were calculated from a societal perspective for a 3-month period. Univariate analyses were performed to identify cost-driving predictors. Total and direct costs were analyzed using a generalized linear model with a γ-distributed dependent variable and log link function. Indirect costs were analyzed using a binomial generalized linear model with probit link function. Results: The mean costs for the 3-month period were approximately €20,000. Informal care costs and hospitalization are approximately €11,000 and €5000. Direct costs amounted to 89% of the total costs, and the share of indirect costs was 11%. Independent predictors of total costs were the duration of the disease and age. The duration of the disease was the main independent predictor of direct costs, whereas age was an independent predictor of indirect costs. Discussion: Costs in the late stage of the disease are considerably higher than those found in earlier stages. Compared to the latter, the mean number of days in hospital and the need for care is increasing. Informal caregivers provide most of the care

Anti-prion drug mPPIg5 inhibits PrP(C) conversion to PrP(Sc).

Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The 'protein only hypothesis' advocates that PrP(Sc), an abnormal isoform of the cellular protein PrP(C), is the main and possibly sole component of prion infectious agents. Currently, no effective therapy exists for these diseases at the symptomatic phase for either humans or animals, though a number of compounds have demonstrated the ability to eliminate PrPSc in cell culture models. Of particular interest are synthetic polymers known as dendrimers which possess the unique ability to eliminate PrP(Sc) in both an intracellular and in vitro setting. The efficacy and mode of action of the novel anti-prion dendrimer mPPIg5 was investigated through the creation of a number of innovative bio-assays based upon the scrapie cell assay. These assays were used to demonstrate that mPPIg5 is a highly effective anti-prion drug which acts, at least in part, through the inhibition of PrP(C) to PrP(Sc) conversion. Understanding how a drug works is a vital component in maximising its performance. By establishing the efficacy and method of action of mPPIg5, this study will help determine which drugs are most likely to enhance this effect and also aid the design of dendrimers with anti-prion capabilities for the future

KCa1.1, a calcium-activated potassium channel subunit alpha 1, is targeted by miR-17-5p and modulates cell migration in malignant pleural mesothelioma

© 2016 Lin et al. Background: Malignant pleural mesothelioma (MPM) is an aggressive, locally invasive, cancer elicited by asbestos exposure and almost invariably a fatal diagnosis. To date, we are one of the leading laboratory that compared microRNA expression profiles in MPM and normal mesothelium samples in order to identify dysregulated microRNAs with functional roles in mesothelioma. We interrogated a significant collection of MPM tumors and normal pleural samples in our biobank in search for novel therapeutic targets. Methods: Utilizing mRNA-microRNA correlations based on differential gene expression using Gene Set Enrichment Analysis (GSEA), we systematically combined publicly available gene expression datasets with our own MPM data in order to identify candidate targets for MPM therapy. Results: We identified enrichment of target binding sites for the miR-17 and miR-30 families in both MPM tumors and cell lines. RT-qPCR revealed that members of both families were significantly downregulated in MPM tumors and cell lines. Interestingly, lower expression of miR-17-5p (P = 0.022) and miR-20a-5p (P = 0.026) was clearly associated with epithelioid histology. We interrogated the predicted targets of these differentially expressed microRNA families in MPM cell lines, and identified KCa1.1, a calcium-activated potassium channel subunit alpha 1 encoded by the KCNMA1 gene, as a target of miR-17-5p. KCa1.1 was overexpressed in MPM cells compared to the (normal) mesothelial line MeT-5A, and was also upregulated in patient tumor samples compared to normal mesothelium. Transfection of MPM cells with a miR-17-5p mimic or KCNMA1-specific siRNAs reduced mRNA expression of KCa1.1 and inhibited MPM cell migration. Similarly, treatment with paxilline, a small molecule inhibitor of KCa1.1, resulted in suppression of MPM cell migration. Conclusion: These functional data implicating KCa1.1 in MPM cell migration support our integrative approach using MPM gene expression datasets to identify novel and potentially druggable targets

Frag4Lead Growing crystallographic fragment hits by catalog using fragment guided template docking

In recent years, crystallographic fragment screening has matured into an almost routine experiment at several modern synchrotron sites. The hits of the screening experiment, i.e. small molecules or fragments binding to the target protein, are revealed along with their 3D structural information. Therefore, they can serve as useful starting points for further structure based hit to lead development. However, the progression of fragment hits to tool compounds or even leads is often hampered by a lack of chemical feasibility. As an attractive alternative, compound analogs that embed the fragment hit structurally may be obtained from commercial catalogs. Here, a workflow is reported based on filtering and assessing such potential follow up compounds by template docking. This means that the crystallographic binding pose was integrated into the docking calculations as a central starting parameter. Subsequently, the candidates are scored on their interactions within the binding pocket. In an initial proof of concept study using five starting fragments known to bind to the aspartic protease endothiapepsin, 28 follow up compounds were selected using the designed workflow and their binding was assessed by crystallography. Ten of these compounds bound to the active site and five of them showed significantly increased affinity in isothermal titration calorimetry of up to single digit micromolar affinity. Taken together, this strategy is capable of efficiently evolving the initial fragment hits without major synthesis efforts and with full control by X ray crystallograph