Subtype-specific neuronal differentiation of PC12 cells transfected with alpha2-adrenergic receptors

Cells of the PC12 rat pheochromocytoma cell line acquire characteristics of sympathetic neurons under appropriate treatment. Stably transfected PC12 cells expressing individual alpha2-adrenergic receptor (alpha2-AR) subtypes were used to assess the role of alpha2-ARs in neuronal differentiation and to characterise the signalling pathways activated by the alpha2-AR agonist epinephrine in these cells. The effects of alpha2-AR activation were compared with the differentiating action and the signalling mechanisms of nerve growth factor (NGF). Epinephrine induced neuronal differentiation of PC12alpha2 cells through alpha2-AR activation in a subtype-dependent manner, internalization of all human alpha2-AR subtypes, and activation of mitogen-activated protein kinase (MAPK) and the serine-threonine protein kinase Akt. Epinephrine and NGF showed synergism in their differentiating effects. The MAPK kinase (MEK-1) inhibitor PD 98059 abolished the differentiating effect of epinephrine indicating that the differentiation is dependent on MAPK activation. Activating protein-1 (AP-1) DNA-binding activity was increased after epinephrine treatment in all three PC12alpha2 subtype clones. Evaluation of the potential physiological consequences of these findings requires further studies on endogenously expressed alpha2-ARs in neuronal cells.     

Synthetic approaches for the synthesis of a cytostatic steroidal B-D bilactam

A new synthetic procedure and a modification of the original method described in the literature for the synthesis of the steroidal B-D bilactam, 3 beta-hydroxy-7 alpha,17 alpha-diaza-B,D-dihomo-5-androsten-7,17-dione are reported. The key step in the modified method involved protection of the D-lactamic nitrogen atom of 3 beta-acetoxy-17 alpha-aza-D-homo-5-androsten-17-one using a reagent of specific electrophilicity (due to the stereoelectronic properties of the cyclic amide), as Beckmann rearrangement of the B-steroidal ring was hindered, possibly via long range effects, by the presence of the unprotected D-lactamic moiety. Using the 3 beta-acetoxy-5-androsten-17-one as starting material, a new synthetic procedure was developed through ketalization of the 17-ketone and allylic oxidation to the 7-ketone, which was subsequently followed by Beckmann rearrangement of the B- and D-steroid rings. Both approaches resulted in 45 and 67% yields of the desired B,D-bilactam, respectively, in contrast to the 15% yield, which has been reported in the literature.     

Integration Analysis of Three Omics Data Using Penalized Regression Methods: An Application to Bladder Cancer

Omics data integration is becoming necessary to investigate the genomic mechanisms involved in complex diseases. During the integration process, many challenges arise such as data heterogeneity, the smaller number of individuals in comparison to the number of parameters, multicollinearity, and interpretation and validation of results due to their complexity and lack of knowledge about biological processes. To overcome some of these issues, innovative statistical approaches are being developed. In this work, we propose a permutation-based method to concomitantly assess significance and correct by multiple testing with the MaxT algorithm. This was applied with penalized regression methods (LASSO and ENET) when exploring relationships between common genetic variants, DNA methylation and gene expression measured in bladder tumor samples. The overall analysis flow consisted of three steps: (1) SNPs/CpGs were selected per each gene probe within 1Mb window upstream and downstream the gene; (2) LASSO and ENET were applied to assess the association between each expression probe and the selected SNPs/CpGs in three multivariable models (SNP, CPG, and Global models, the latter integrating SNPs and CPGs); and (3) the significance of each model was assessed using the permutation-based MaxT method. We identified 48 genes whose expression levels were significantly associated with both SNPs and CPGs. Importantly, 36 (75%) of them were replicated in an independent data set (TCGA) and the performance of the proposed method was checked with a simulation study. We further support our results with a biological interpretation based on an enrichment analysis. The approach we propose allows reducing computational time and is flexible and easy to implement when analyzing several types of omics data. Our results highlight the importance of integrating omics data by applying appropriate statistical strategies to discover new insights into the complex genetic mechanisms involved in disease conditions.     

Neuroendocrine immunity in patients with Alzheimer's disease: toward translational epigenetics

The emerging domain of epigenetics in molecular medicine finds application for a variety of patient populations. Here, we present fundamental neuroendocrine immune evidence obtained in patients with senile dementia of the Alzheimer's type (sDAT), and discuss the implications of these data from the viewpoint of translational epigenetics of Alzheimer's disease. We followed 18 subjects with mild sDAT treated with acetylcholinesterase inhibitors, and 10 control subjects matched for age in a repeated measure design every six months for 18 months. We monitored psychosocial profile (Mini-Mental State Examination, Functional Assessment Staging, Independence in Activities of Daily Living, Depression, Profile of Moods States) in parallel to immunophenotypic parameters of T cell subpopulations by flow cytometry. Based on change in the mini-mental state score at entry and at 18 months, patients with sDAT were assigned to a "fast progression" (delta greater than 2 points) or to a "slow progression" group (delta less than or equal to 2 points). The change in circulating activated T cells (CD3+Dr+) with time in patients with sDAT was significantly inversely correlated with the change in time in natural killer (NK) cytotoxic activity to cortisol modulation in these patients, which was greater in patients with fast progression, compared to slow progression sDAT. These data indicate underlying neuroendocrine immune processes during progression of sDAT. Our observations suggest that psychoimmune measures such as those we have monitored in this study provide relevant information about the evolving physiological modulation in patients with sDAT during progression of Alzheimer's disease, and point to new or improved translational epigenetic treatment interventions.