Integrated Computational Solution for Predicting Skin Sensitization Potential of Molecules.

INTRODUCTION: Skin sensitization forms a major toxicological endpoint for dermatology and cosmetic products. Recent ban on animal testing for cosmetics demands for alternative methods. We developed an integrated computational solution (SkinSense) that offers a robust solution and addresses the limitations of existing computational tools i.e. high false positive rate and/or limited coverage. RESULTS: The key components of our solution include: QSAR models selected from a combinatorial set, similarity information and literature-derived sub-structure patterns of known skin protein reactive groups. Its prediction performance on a challenge set of molecules showed accuracy = 75.32%, CCR = 74.36%, sensitivity = 70.00% and specificity = 78.72%, which is better than several existing tools including VEGA (accuracy = 45.00% and CCR = 54.17% with 'High' reliability scoring), DEREK (accuracy = 72.73% and CCR = 71.44%) and TOPKAT (accuracy = 60.00% and CCR = 61.67%). Although, TIMES-SS showed higher predictive power (accuracy = 90.00% and CCR = 92.86%), the coverage was very low (only 10 out of 77 molecules were predicted reliably). CONCLUSIONS: Owing to improved prediction performance and coverage, our solution can serve as a useful expert system towards Integrated Approaches to Testing and Assessment for skin sensitization. It would be invaluable to cosmetic/ dermatology industry for pre-screening their molecules, and reducing time, cost and animal testing.

Oncostatin-M differentially regulates mesenchymal and proneural signature genes in gliomas via STAT3 signaling.

Glioblastoma (GBM), the most malignant of the brain tumors is classified on the basis of molecular signature genes using TCGA data into four subtypes- classical, mesenchymal, proneural and neural. The mesenchymal phenotype is associated with greater aggressiveness and low survival in contrast to GBMs enriched with proneural genes. The proinflammatory cytokines secreted in the microenvironment of gliomas play a key role in tumor progression. The study focused on the role of Oncostatin-M (OSM), an IL-6 family cytokine in inducing mesenchymal properties in GBM. Analysis of TCGA and REMBRANDT data revealed that expression of OSMR but not IL-6R or LIFR is upregulated in GBM and has negative correlation with survival. Amongst the GBM subtypes, OSMR level was in the order of mesenchymal > classical > neural > proneural. TCGA data and RT-PCR analysis in primary cultures of low and high grade gliomas showed a positive correlation between OSMR and mesenchymal signature genes-YKL40/CHI3L1, fibronectin and vimentin and a negative correlation with proneural signature genes-DLL3, Olig2 and BCAN. OSM enhanced transcript and protein level of fibronectin and YKL-40 and reduced the expression of Olig2 and DLL3 in GBM cells. OSM-regulated mesenchymal phenotype was associated with enhanced MMP-9 activity, increased cell migration and invasion. Importantly, OSM induced mesenchymal markers and reduced proneural genes even in primary cultures of grade-III glioma cells. We conclude that OSM-mediated signaling contributes to aggressive nature associated with mesenchymal features via STAT3 signaling in glioma cells. The data suggest that OSMR can be explored as potential target for therapeutic intervention.

Secretory prostate apoptosis response (Par)-4 sensitizes multicellular spheroids (MCS) of glioblastoma multiforme cells to tamoxifen-induced cell death.

Glioblastoma multiforme (GBM) is the most malignant form of brain tumor and is associated with resistance to conventional therapy and poor patient survival. Prostate apoptosis response (Par)-4, a tumor suppressor, is expressed as both an intracellular and secretory/extracellular protein. Though secretory Par-4 induces apoptosis in cancer cells, its potential in drug-resistant tumors remains to be fully explored. Multicellular spheroids (MCS) of cancer cells often acquire multi-drug resistance and serve as ideal experimental models. We investigated the role of Par-4 in Tamoxifen (TAM)-induced cell death in MCS of human cell lines and primary cultures of GBM tumors. TCGA and REMBRANT data analysis revealed that low levels of Par-4 correlated with low survival period (21.85 ± 19.30 days) in GBM but not in astrocytomas (59.13 ± 47.26 days) and oligodendrogliomas (58.04 ± 59.80 days) suggesting low PAWR expression as a predictive risk factor in GBM. Consistently, MCS of human cell lines and primary cultures displayed low Par-4 expression, high level of chemo-resistance genes and were resistant to TAM-induced cytotoxicity. In monolayer cells, TAM-induced cytotoxicity was associated with enhanced expression of Par-4 and was alleviated by silencing of Par-4 using specific siRNA. TAM effectively induced secretory Par-4 in conditioned medium (CM) of cells cultured as monolayer but not in MCS. Moreover, MCS were rendered sensitive to TAM-induced cell death by exposure to conditioned medium (CM)-containing Par-4 (derived from TAM-treated monolayer cells). Also TAM reduced the expression of Akt and PKCζ in GBM cells cultured as monolayer but not in MCS. Importantly, combination of TAM with inhibitors to PI3K inhibitor (LY294002) or PKCζ resulted in secretion of Par-4 and cell death in MCS. Since membrane GRP78 is overexpressed in most cancer cells but not normal cells, and secretory Par-4 induces apoptosis by binding to membrane GRP78, secretory Par-4 is an attractive candidate for potentially overcoming therapy-resistance not only in malignant glioma but in broad spectrum of cancers.

Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data.

Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6-40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.

Involvement of G protein-coupled receptor kinase (GRK) 3 and GRK2 in down-regulation of the alpha2B-adrenoceptor.

Increasing the cellular levels of G protein-coupled receptor kinase (GRK) 2 or GRK3 renders the alpha2B-adrenoceptor (AR) more sensitive to agonist-induced down-regulation (J Pharmacol Exp Ther 312:767-773, 2005). However, an absolute requirement of GRK3 and GRK2 for alpha2B-AR down-regulation is controversial. In this study, using NG108 cells (endogenous alpha2B-AR), we provide strong evidence for a critical role of both GRK3 and GRK2 in down-regulation of the alpha2B-AR. Pretreatment of NG108 cells with 20 microM epinephrine (EPI) begins down-regulating the alpha2B-AR by 2 h. The translocation of GRK3 and GRK2 to the membrane peaks at 30 min, decreasing by 1 h. Although these results may implicate GRK3 and GRK2 in alpha2B-AR down-regulation, significant receptor down-regulation is not observed until 2 h, after GRK3 and GRK2 translocation has peaked and is declining. To more directly establish a role for GRK3 and GRK2 in alpha2B-AR down-regulation, NG108 cells were transfected to express GRK3ct, which binds to liberated Gbetagamma subunits, preventing GRK3 and GRK2 translocation to the membrane. Overexpression of GRK3ct prevented not only the translocation of GRK3 and GRK2 but also the down-regulation of the alpha2B-AR caused by 24-h pretreatment with 20 microM EPI. Taken together, these data provide direct evidence for a role of GRK3 and GRK2 in the down-regulation of the alpha2B-AR and contribute significantly to the increasing evidence in the literature for a pivotal role of GRKs in modulating the agonist-induced down-regulation of the alpha2-AR.

Two new behavioral QTLs, Emo4 and Reb1, map to mouse Chromosome 1: Congenic strains and candidate gene identification studies.

By use of newly developed subcongenic strains of mice from a parental B6.129-Il10-/- knockout/congenic strain, we have narrowed the critical region for a new behavioral QTL, called Emo4, for open-field activity to a segment of Chromosome 1 between Erbb4 (68.4Mb) and B3gnt7 (86.2 Mb). We have also uncovered an additional QTL governing repetitive beam breaks in the open field. This QTL, called Reb1, maps to the interval between Asb1 (91.4 Mb) and NM_172851 (100.0 Mb) and is one of the first QTLs mapped for this type of behavior. Genome-wide microarray expression analyses were then undertaken to help to identify candidate genes that may be the cause of these genetic differences in open-field performance. In this effort, we analyzed global gene expression differences in the amygdalae by use of Affymetrix GeneChips between B6, B6.129-Il10-/-, and B6.129R4. Several probe sets representing target Chr 1 genes were found that showed significantly differential expression in the subcongenic and congenic strains. Several candidate genes have been identified. One of these regions coincides with an homologous region in humans that has been associated with autism, a disease whose symptoms include repetitive actions. This study illustrates that the use of congenic strains combined with global gene expression analyses can produce a list of viable candidates. It further shows that caution should be observed when analyzing the effects of knockout/congenic strains because many of the gene expression differences in these comparisons could not be attributable to the ablated Il10 gene but rather to passenger gene effects.

Cellular G protein-coupled receptor kinase levels regulate sensitivity of the {alpha}2b-adrenergic receptor to undergo agonist-induced down-regulation.

Chronic coactivation of alpha(2B)- and beta(2)-adrenoceptors (AR) was recently reported to down-regulate the alpha(2B)-AR at a lower threshold epinephrine (EPI) concentration compared with the activation of alpha(2B)-AR alone. This is the result of a modest beta(2)-AR-dependent up-regulation of G protein-coupled receptor kinase 3 (GRK3). In the present study, we determined that increasing GRK2 or GRK3 levels, independent of beta(2)-AR activation, decreases the EC(50) concentration for agonist-induced down-regulation of the alpha(2B)-AR using NG108 cells with or without overexpression (2- to 10-fold) of GRK2 or GRK3. In parental NG108 cells, the EC(50) concentration of EPI required for down-regulation of the alpha(2B)-AR is 30 microM. A 2- to 3-fold overexpression of GRK3 in NG108 cells, however, reduces the EC(50) to 0.2 microM (a 150-fold decrease), whereas a comparable overexpression of GRK2 reduces it to 1 microM (a 30-fold decrease). However, when GRK3 or GRK2 in NG108 cells are overexpressed 8- to 10-fold, the EC(50) concentration (0.02 microM EPI) for alpha(2B)-AR down-regulation is reduced 1000-fold. These data clearly suggest that a modest (2- to 3-fold) up-regulation of GRK3 is more effective at enhancing the sensitivity of alpha(2B)-AR to down-regulation after exposure to EPI than a modest up-regulation of GRK2, but that both GRK2 and GRK3 are equally effective at inducing alpha(2B)-AR down-regulation when up-regulated 8- to 10-fold. To our knowledge, this is the first report to systematically demonstrate that GRKs, particularly GRK3, play a pivotal role in modulating the agonist EC(50) concentration that down-regulates the alpha(2B)-AR and thus adds a new dimension to an already intricate signaling network.

Simultaneous alpha2B- and beta2-adrenoceptor activation sensitizes the alpha2B-adrenoceptor for agonist-induced down-regulation.

We recently reported that alpha(2A)-adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 microM) in BE(2)-C cells that express both alpha(2)- and beta(2)-ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by beta(2)-AR blockade and is associated with an increase in G protein-coupled receptor kinase 3 (GRK3) expression. Because differences in agonist-induced down-regulation of the alpha(2A)-versus alpha(2B)-ARs have been reported, the present study examines the effects of simultaneous activation of alpha(2B)- and beta(2)-ARs on alpha(2B)-AR number and signaling. We studied NG108 cells that naturally express alpha(2B)-ARs, and BN17 cells, NG108 cells transfected to express the human beta(2)-AR. In NG108 cells, alpha(2B)-AR desensitization and down-regulation require treatment with 20 microM EPI or NE; GRK expression was not changed. In BN17 cells expressing beta(2)-ARs, the threshold EPI concentration for alpha(2B)-AR desensitization and down-regulation was reduced to 0.3 microM; 10 microM NE was required for the same effect. Furthermore, 24-h EPI or NE treatments that produced desensitization also resulted in a selective 2-fold up-regulation of GRK3; GRK2 was unchanged. The beta-AR antagonist alprenolol (1 microM) and GRK3 antisense (but not sense) DNA blocked 0.3 microM EPI- and 10 microM NE-induced desensitization and down-regulation of the alpha(2B)-AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of alpha(2B)- and beta(2)-ARs results in a 67-fold decrease in the threshold concentration of EPI required for alpha(2B)-AR down-regulation. This lower threshold for down-regulation is associated with alpha(2B)- and beta(2)-AR dependent up-regulation of GRK3 expression.