miR-212/132 expression and functions: within and beyond the neuronal compartment

During the last two decades, microRNAs (miRNAs) emerged as critical regulators of gene expression. By modulating the expression of numerous target mRNAs mainly at the post-transcriptional level, these small non-coding RNAs have been involved in most, if not all, biological processes as well as in the pathogenesis of a number of diseases. miR-132 and miR-212 are tandem miRNAs whose expression is necessary for the proper development, maturation and function of neurons and whose deregulation is associated with several neurological disorders, such as Alzheimer's disease and tauopathies (neurodegenerative diseases resulting from the pathological aggregation of tau protein in the human brain). Although their involvement in neuronal functions is the most described, evidences point towards a role of these miRNAs in many other biological processes, including inflammation and immune functions. Incidentally, miR-132 was recently classified as a ‘neurimmiR’, a class of miRNAs operating within and between the neural and immune compartments. In this review, we propose an outline of the current knowledge about miR-132 and miR-212 functions in neurons and immune cells, by describing the signalling pathways and transcription factors regulating their expression as well as their putative or demonstrated roles and validated mRNA targets

Unbiased proteomic analysis of proteins interacting with the HIV-1 5′LTR sequence:Role of the transcription factor Meis

To depict the largest picture of a core promoter interactome, we developed a one-step DNA-affinity capture method coupled with an improved mass spectrometry analysis process focused on the identification of low abundance proteins. As a proof of concept, this method was developed through the analysis of 230 bp contained in the 5′long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1). Beside many expected interactions, many new transcriptional regulators were identified, either transcription factors (TFs) or co-regulators, which interact directly or indirectly with the HIV-1 5′LTR. Among them, the homeodomain-containing TF myeloid ectopic viral integration site was confirmed to functionally interact with a specific binding site in the HIV-1 5′LTR and to act as a transcriptional repressor, probably through recruitment of the repressive Sin3A complex. This powerful and validated DNA-affinity approach could also be used as an efficient screening tool to identify a large set of proteins that physically interact, directly or indirectly, with a DNA sequence of interest. Combined with an in silico analysis of the DNA sequence of interest, this approach provides a powerful approach to select the interacting candidates to validate functionally by classical approaches

Development and validation of a Luminex assay for detection of a predictive biomarker for PROSTVAC-VF therapy

<div><p>Cancer therapies can provide substantially improved survival in some patients while other seemingly similar patients receive little or no benefit. Strategies to identify patients likely to respond well to a given therapy could significantly improve health care outcomes by maximizing clinical benefits while reducing toxicities and adverse effects. Using a glycan microarray assay, we recently reported that pretreatment serum levels of IgM specific to blood group A trisaccharide (BG-A_tri) correlate positively with overall survival of cancer patients on PROSTVAC-VF therapy. The results suggested anti-BG-A_tri IgM measured prior to treatment could serve as a biomarker for identifying patients likely to benefit from PROSTVAC-VF. For continued development and clinical application of serum IgM specific to BG-A_tri as a predictive biomarker, a clinical assay was needed. In this study, we developed and validated a Luminex-based clinical assay for measuring serum IgM specific to BG-A_tri. IgM levels were measured with the Luminex assay and compared to levels measured using the microarray for 126 healthy individuals and 77 prostate cancer patients. This assay provided reproducible and consistent results with low %CVs, and tolerance ranges were established for the assay. IgM levels measured using the Luminex assay were found to be highly correlated to the microarray results with R values of 0.93–0.95. This assay is a Laboratory Developed Test (LDT) and is suitable for evaluating thousands of serum samples in CLIA certified laboratories that have validated the assay. In addition, the study demonstrates that discoveries made using neoglycoprotein-based microarrays can be readily migrated to a clinical assay.</p></div

Assay specificity^a.

<p>Assay specificity<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182739#t002fn001" target="_blank">^a</a>.</p

Overview of neoglycoproteins and assay formats.

<p>Glycans or glycopeptides are covalently coupled to albumin to produce neoglycoproteins, which are then printed onto a microarray surface and used to detect IgM to BG-A_tri. Neoglycoproteins immobilized on Luminex microspheres mimic glycan presentation on the microarray surface.</p

Comparison of BG-A_tri signals measured using the Luminex assay and the microarray assay.

<p>Comparison of BG-A_tri signals measured using the Luminex assay and the microarray assay.</p

Analytical validation summary.

<p>For the analytical validation, for each sample dilution, we ran eight plates with twelve replicates on each plate (n = 96).</p

Selection of sample dilution.^a

<p>Selection of sample dilution.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182739#t001fn001" target="_blank">^a</a></p