Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity

HBZ Stimulates Brain-Derived Neurotrophic Factor/TrkB Autocrine/Paracrine Signaling To Promote Survival of Human T-Cell Leukemia Virus Type 1-Infected T Cells

International audienc

Proteogenomic characterization of 5-azacytidine effects on acute myeloid leukemia immunopeptidome

peer reviewedBackground: Hypomethylating agents like 5-azacytidine (AZA) are licensed for the treatment of acute myeloid leukemia (AML) patients ineligible for allogeneic hematopoietic cell transplantation. While previous reports suggested that AZA promotes the recognition of AML blasts by cytotoxic T cells, the mechanism behind this improved recognition is not fully understood. Specifically, AZA is assumed to promote the expression of transcripts repressed by genomic methylation, namely cancer-testis antigens (CTA) and endogenous retroelements (ERE), resulting in the presentation of immunogenic MHC-I-associated peptides (MAPs, collectively referred to as the immunopeptidome) derived from the translation of these transcripts. However, the presentation of such MAPs after AZA treatment has not been firmly demonstrated thus far. Aims: Our study aims to characterize how AZA treatment shapes the identity of MAPs presented by AML cells. Methods: Four different AML cell lines, THP-1, OCI-AML3, SKM-1, and MOLM-13 were treated with a non-cytotoxic dose of AZA for 3 days. Their transcriptome has been characterized by high-coverage RNA sequencing (RNA-seq) and their immunopeptidome by shotgun mass spectrometry (MS). To identify MAPs deriving from unannotated genomic regions, we have designed a cutting-edge proteogenomic pipeline using the RNA-seq data to build personalized MS databases that enabled the identification of non-canonical MAPs such as ERE-derived MAPs. Results: Paired transcriptomic comparisons between treated and untreated cells showed that AZA induces a large-scale gene upregulation (87% differentially-expressed transcripts were upregulated). Among them, 38% were EREs and 6% were CTAs, suggesting that AZA-induced MAPs have greater chances of deriving from EREs than from CTAs. However, we could not identify a single ERE-derived upregulated MAP among the immunopeptidome of AZA-treated AML cells while multiple CTA-derived MAPs (0.4 - 0.7% of the upregulated MAPs) were presented at greater levels by AZA. Because a GO-term analysis of the upregulated protein-coding genes evidenced a robust innate immune response in AZA-treated cells, we conclude that AZA-induced enhanced CTL recognition is more dependent on CTA- than on ERE-derived MAP presentation and that ERE enhanced expression could rather trigger the observed innate immune response. An in-depth analysis of the immunopeptidome showed that MAPs having an altered presentation following AZA treatment derived only partially from transcripts whose expression was affected by AZA. The analysis of the other AZA-induced MAPs showed that they resulted preferentially from the activity of the constitutive proteasome (vs the immunoproteasome) and derived preferentially from proteins having less aromatic residues (and therefore more prone to misfolding). Because the degradation of misfolded proteins by the constitutive proteasome can indicate that AZA induces an unfolded protein response, we have examined whether AZA stimulates autophagy, a process frequently triggered in association with such stress. Accordingly, flow cytometry assays on AML cells treated with AZA for 24h evidenced a robust autophagy induction. Summary/Conclusion: Altogether our results show that AZA promotes the presentation of CTA-derived rather than ERE-derived MAPs and that autophagy induction could enable the survival of AML cells to AZA-induced proteotoxic stress. Our findings suggest that autophagy inhibitors could synergize with AZA in AML therapy

NLRC4 GOF Mutations, a Challenging Diagnosis from Neonatal Age to Adulthood

International audienceThe NLRC4 inflammasome is part of the human immune innate system. Its activation leads to the cleavage of pro-inflammatory cytokines IL-1β and IL-18, promoting inflammation. NLRC4 gain-of-function (GOF) mutations have been associated with early-onset recurrent fever, recurrent macrophagic activation syndrome and enterocolitis. Herein, we describe two new patients with NLRC4 mutations. The first case presented with recurrent fever and vasoplegic syndrome, gut symptoms and urticarial rashes initially misdiagnosed as a severe protein-induced enterocolitis syndrome. The second case had recurrent macrophage activation syndrome (MAS) and shock, suggesting severe infection. We identified two NLRC4 mutations, on exon 4, within the nucleotide-binding protein domain (NBD). After a systematic review of NLRC4 GOF mutations, we highlight the wide spectrum of this disease with a limited genotype-phenotype correlation. Vasoplegic shock was only reported in patients with mutation in the NBD. Diagnosing this new entity combined with gastrointestinal symptoms and vasoplegic shocks is challenging. It mimics severe allergic reaction or sepsis. The plasma IL-18 level and genetic screening are instrumental to make a final diagnosis

Parietal Damage Dissociates Saccade Planning from Presaccadic Perceptual Facilitation

Khan AZ, Blangero A, Rossetti Y, et al. Parietal Damage Dissociates Saccade Planning from Presaccadic Perceptual Facilitation. CEREBRAL CORTEX. 2009;19(2):383-387.A well-known theory in the field of attention today is the premotor theory of attention which suggests that the mechanisms involved in eye movements are the same as those for spatial attention shifts. We tested a parietal damaged patient with unilateral optic ataxia and 4 controls on a dual saccade/attentional task and show a dissociation between saccadic eye movements and presaccadic perceptual enhancement at the saccade goal. Remarkably, though the patient was able to make the appropriate saccades to the left, impaired visual field (undistinguishable from saccades to his right, intact visual field), he was unable to discriminate the letter at the saccade goal (whereas his performance was like controls for letter discrimination in his right visual field). This suggests that saccade planning and presaccadic perceptual facilitation are separable-025EFplanning a saccade to a location does not necessitate that the processing of this location is enhanced. Based on these results, we suggest that the parietal cortex is necessary for the coupling between saccade planning and presaccadic perceptual facilitation

Asymmetric segregation and self-renewal of hematopoietic stem and progenitor cells with endocytic Ap2a2

The stem cell-intrinsic model of self-renewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. Interestingly, live-cell videomicroscopy of the endocytic protein AP2A2 shows instances of asymmetric segregation during HSC/progenitor cell cytokinesis. These results contribute further evidence that ACD is functional in HSC self-renewal, suggest a role for Ap2a2 in HSC activity, and provide a unique opportunity to prospectively analyze progeny from HSC asymmetric divisions

Supplementary Material for: Target Door-to-Needle Time for Tissue Plasminogen Activator Treatment with Magnetic Resonance Imaging Screening Can Be Reduced to 45 min

<b><i>Objective:</i></b> The purpose of this study was to demonstrate that the median door-to-needle (DTN) time for intravenous tissue plasminogen activator (tPA) treatment can be reduced to 45 min in a primary stroke centre with MRI-based screening for acute ischaemic stroke (AIS). <b><i>Methods:</i></b> From February 2015 to February 2017, the stroke unit of Perpignan general hospital, France, implemented a quality-improvement (QI) process. During this period, patients who received tPA within 4.5 h after AIS onset were included in the QI cohort. Their clinical characteristics and timing metrics were compared each semester and also with those of 135 consecutive patients with AIS treated by tPA during the 1-year pre-QI period (pre-QI cohort). <b><i>Results:</i></b> In the QI cohort, 274 patients (92.5%) underwent MRI screening. While the demographic and baseline characteristics were not significantly different between cohorts, the median DTN time was significantly lower in the QI than in the pre-QI cohort (52 vs. 84 min; <i>p</i> < 0.00001). Within the QI cohort, the median DTN time for each semester decreased from 65 to 44 min (<i>p</i> < 0.00001) and the proportion of treated patients with a DTN time ≤45 min increased from 25 to 58.9% (<i>p</i> < 0.0001). Overall, DTN time improvement was associated with a better outcome at 3 months (patients with a modified Rankin Scale score between 0 and 2: 61.8% in the QI vs. 39.3% in the pre-QI cohort; <i>p</i> < 0.0001). <b><i>Conclusions:</i></b> A QI process can reduce the DTN within 45 min with MRI as a screening tool