Monocyte NOTCH2 expression predicts interferon-beta immunogenicity in multiple sclerosis patients

Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-β is an established treatment for MS; however, up to 30% of IFN-β–treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14+ monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-β. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-β administration. Reduced monocyte NOTCH2 expression in nADA+ MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA+ patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-β administration

Supplementary Material for: A Bagged, Partially Linear, Tree-Based Regression Procedure for Prediction and Variable Selection

<b><i>Objectives:</i></b> In genomics, variable selection and prediction accounting for the complex interrelationships between explanatory variables represent major challenges. Tree-based methods are powerful alternatives to classical regression models. We have recently proposed the generalized, partially linear, tree-based regression (GPLTR) procedure that integrates the advantages of generalized linear regression (allowing the incorporation of confounding variables) and of tree-based models. In this work, we use bagging to address a classical concern of tree-based methods: their instability. <b><i>Methods:</i></b> We present a bagged GPLTR procedure and three scores for variable importance. The prediction accuracy and the performance of the scores are assessed by simulation. The use of this procedure is exemplified by the analysis of a lung cancer data set. The aim is to predict the epidermal growth factor receptor (EGFR) mutation based on gene expression measurements, taking into account the ethnicity (confounder variable) and perform variable selection. <b><i>Results:</i></b> The procedure performs well in terms of prediction accuracy. The scores differentiate predictive variables from noise variables. Based on a lung adenocarcinoma data set, the procedure achieves good predictive performance for EGFR mutation and selects relevant genes. <b><i>Conclusion:</i></b> The proposed bagged GPLTR procedure performs well for prediction and variable selection

The PAF Complex and Prf1/Rtf1 Delineate Distinct Cdk9-Dependent Pathways Regulating Transcription Elongation in Fission Yeast

<div><p>Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast <i>Schizosaccharomyces pombe</i> that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.</p></div

Prf1 and PAF pathways have opposing biological effects.

<p>(<b>A</b>) Quantification of the abnormal septation patterns in the indicated strains. Strains carrying the <i>cdk9^as</i> allele were cultured in DMSO (−) or 2 µM 3-MB-PP1 (+) for 15 hours prior to fixation for microscopy. Error bars denote standard deviations from 3 independent experiments. (<b>B</b>) ChIP of RNAPII was carried out in the indicated strains and quantified by qPCR using primers specific to the <i>nup189</i>⁺ gene. Values were normalized to that for primer pair 1. Error bars denote standard deviations from three independent experiments. Significant differences from wild-type values (unpaired t-test) are indicated.</p

Prf1 does not stably associate with the PAF complex.

<p>(<b>A</b>) TAP purification was carried out using whole cell extracts from the indicated strains and purified material was analyzed by SDS-PAGE and silver staining. The proteins contained in the labeled bands were identified by mass spectrometry (as indicated on the right). “CBP” denotes the residual fusion to the calmodulin binding peptide resulting from the TAP procedure. Leo1 was detected in two bands that also contained either Paf1-CBP (in the <i>paf1-TAP</i> lane) or Paf1 (in the <i>tpr1-TAP</i> lane). Molecular weight standards (in kD) are denoted on the left. (<b>B</b>) Single-step TAP purifications were performed using extracts from the indicated strains. 5% of input fractions (“input”) and 50% of bead-bound fractions (“beads”) were analyzed by SDS-PAGE and western blotting.</p

Noncontiguous finished genome sequences and descriptions of Actinomyces ihuae, Actinomyces bouchesdurhonensis, Actinomyces urinae, Actinomyces marseillensis, Actinomyces mediterranea and Actinomyces oralis sp. nov. identified by culturomics

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