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Abstract Background Microsatellite instability (MSI) due to mismatch repair deficiency (dMMR) is common in colorectal cancer (CRC). These cancers are associated with somatic coding events, but the noncoding pathophysiological impact of this genomic instability is yet poorly understood. Here, we perform an analysis of coding and noncoding MSI events at the different steps of colorectal tumorigenesis using whole exome sequencing and search for associated splicing events via RNA sequencing at the bulk-tumor and single-cell levels. Results Our results demonstrate that MSI leads to hundreds of noncoding DNA mutations, notably at polypyrimidine U2AF RNA-binding sites which are endowed with cis-activity in splicing, while higher frequency of exon skipping events are observed in the mRNAs of MSI compared to non-MSI CRC. At the DNA level, these noncoding MSI mutations occur very early prior to cell transformation in the dMMR colonic crypt, accounting for only a fraction of the exon skipping in MSI CRC. At the RNA level, the aberrant exon skipping signature is likely to impair colonic cell differentiation in MSI CRC affecting the expression of alternative exons encoding protein isoforms governing cell fate, while also targeting constitutive exons, making dMMR cells immunogenic in early stage before the onset of coding mutations. This signature is characterized by its similarity to the oncogenic U2AF1-S34F splicing mutation observed in several other non-MSI cancer. Conclusions Overall, these findings provide evidence that a very early RNA splicing signature partly driven by MSI impairs cell differentiation and promotes MSI CRC initiation, far before coding mutations which accumulate later during MSI tumorigenesis.

Recently, interest in transcriptomic assessment of kidney biopsies has been growing. This study investigates the use of NGS to identify gene expression changes and analyse the pathways involved in rejection. An Illumina bulk RNA sequencing on the polyadenylated RNA of 770 kidney biopsies was conducted. Differentially-expressed genes (DEGs) were determined for AMR and TCMR using DESeq2. Genes were segregated according to their previous descriptions in known panels (microarray or the Banff Human Organ Transplant (B-HOT) panel) to obtain NGS-specific genes. Pathway enrichment analysis was performed using the Reactome and Kyoto Encyclopaedia of Genes and Genomes (KEGG) public repositories. The differential gene expression using NGS analysis identified 6,141 and 8,478 transcripts associated with AMR and TCMR. While most of the genes identified were included in the microarray and the B-HOT panels, NGS analysis identified 603 (9.8%) and 1,186 (14%) new specific genes. Pathways analysis showed that the B-HOT panel was associated with the main immunological processes involved during AMR and TCMR. The microarrays specifically integrated metabolic functions and cell cycle progression processes. Novel NGS-specific based transcripts associated with AMR and TCMR were discovered, which might represent a novel source of targets for drug designing and repurposing.

Abstract Background Advanced prostate cancer (PC) is characterized by insensitivity to androgen deprivation therapy and chemotherapy, resulting in poor outcome for most patients. Thus, advanced PC urgently needs novel therapeutic strategies. Mounting evidence points to splicing dysregulation as a hallmark of advanced PC. Moreover, pharmacologic inhibition of the splicing process is emerging as a promising option for this disease. Method By using a representative androgen-insensitive PC cell line (22Rv1), we have investigated the genome-wide transcriptomic effects underlying the cytotoxic effects exerted by three splicing-targeting drugs: Pladienolide B, indisulam and THZ531. Bioinformatic analyses were performed to uncover the gene structural features underlying sensitivity to transcriptional and splicing regulation by these treatments. Biological pathways altered by these treatments were annotated by gene ontology analyses and validated by functional experiments in cell models. Results Although eliciting similar cytotoxic effects on advanced PC cells, Pladienolide B, indisulam and THZ531 modulate specific transcriptional and splicing signatures. Drug sensitivity is associated with distinct gene structural features, expression levels and cis-acting sequence elements in the regulated exons and introns. Importantly, we identified PC-relevant genes (i.e. EZH2, MDM4) whose drug-induced splicing alteration exerts an impact on cell survival. Moreover, computational analyses uncovered a widespread impact of splicing-targeting drugs on intron retention, with enrichment in genes implicated in pre-mRNA 3’-end processing (i.e. CSTF3, PCF11). Coherently, advanced PC cells displayed high sensitivity to a specific inhibitor of the cleavage and polyadenylation complex, which enhances the effects of chemotherapeutic drugs that are already in use for this cancer. Conclusions Our study uncovers intron retention as an actionable vulnerability for advanced PC, which may be exploited to improve therapeutic management of this currently incurable disease.

Cholangiocarcinoma (CCA) results from the malignant transformation of cholangiocytes. Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are chronic diseases in which cholangiocytes are primarily damaged. Although PSC is an inflammatory condition predisposing to CCA, CCA is almost never found in the autoimmune context of PBC. Here, we hypothesized that PBC might favor CCA immunosurveillance. In preclinical murine models of cholangitis challenged with syngeneic CCA, PBC (but not PSC) reduced the frequency of CCA development and delayed tumor growth kinetics. This PBC-related effect appeared specific to CCA as it was not observed against other cancers, including hepatocellular carcinoma. The protective effect of PBC was relying on type 1 and type 2 T cell responses and, to a lesser extent, on B cells. Single-cell TCR/RNA sequencing revealed the existence of TCR clonotypes shared between the liver and CCA tumor of a PBC host. Altogether, these results evidence a mechanistic overlapping between autoimmunity and cancer immunosurveillance in the biliary tract.

Liver cirrhosis is characterised by extensive fibrosis of the liver, and understanding the underpinning immunological processes is important in designing intervention. Here authors show that Mucosal-Associated Invariant T cells are instrumental to controlling the balance between profibrogenic and restorative macrophages and inhibiting their activation might reverse liver fibrosis.

Abstract Background Most human genes produce several transcripts with different exon contents by using alternative promoters, alternative polyadenylation sites and alternative splice sites. Much effort has been devoted to describing known gene transcripts through the development of numerous databases. Nevertheless, owing to the diversity of the transcriptome, there is a need for interactive databases that provide information about the potential function of each splicing variant, as well as its expression pattern. Description After setting up a database in which human and mouse splicing variants were compiled, we developed tools (1) to predict the production of protein isoforms from these transcripts, taking account of the presence of open reading frames and mechanisms that could potentially eliminate transcripts and/or inhibit their translation, i.e. nonsense-mediated mRNA decay and microRNAs; (2) to support studies of the regulation of transcript expression at multiple levels, including transcription and splicing, particularly in terms of tissue specificity; and (3) to assist in experimental analysis of the expression of splicing variants. Importantly, analyses of all features from transcript metabolism to functional protein domains were integrated in a highly interactive, user-friendly web interface that allows the functional and regulatory features of gene transcripts to be assessed rapidly and accurately. Conclusion In addition to identifying the transcripts produced by human and mouse genes, fast DB http://www.fast-db.com provides tools for analyzing the putative functions of these transcripts and the regulation of their expression. Therefore, fast DB has achieved an advance in alternative splicing databases by providing resources for the functional interpretation of splicing variants for the human and mouse genomes. Because gene expression studies are increasingly employed in clinical analyses, our web interface has been designed to be as user-friendly as possible and to be readily searchable and intelligible at a glance by the whole biomedical community.

Summary: Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of UM. To improve our molecular understanding of UM, we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis using Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.

Background & Aims: Liver regeneration is a repair process in which metabolic reprogramming of parenchymal and inflammatory cells plays a major role. Monoacylglycerol lipase (MAGL) is an ubiquitous enzyme at the crossroad between lipid metabolism and inflammation. It converts monoacylglycerols into free fatty acids and metabolises 2-arachidonoylglycerol into arachidonic acid, being thus the major source of pro-inflammatory prostaglandins in the liver. In this study, we investigated the role of MAGL in liver regeneration. Methods: Hepatocyte proliferation was studied in vitro in hepatoma cell lines and ex vivo in precision-cut human liver slices. Liver regeneration was investigated in mice treated with a pharmacological MAGL inhibitor, MJN110, as well as in animals globally invalidated for MAGL (MAGL-/-) and specifically invalidated in hepatocytes (MAGLHep-/-) or myeloid cells (MAGLMye-/-). Two models of liver regeneration were used: acute toxic carbon tetrachloride injection and two-thirds partial hepatectomy. MAGLMye-/- liver macrophages profiling was analysed by RNA sequencing. A rescue experiment was performed by in vivo administration of interferon receptor antibody in MAGLMye-/- mice. Results: Precision-cut human liver slices from patients with chronic liver disease and human hepatocyte cell lines exposed to MJN110 showed reduced hepatocyte proliferation. Mice with global invalidation or mice treated with MJN110 showed blunted liver regeneration. Moreover, mice with specific deletion of MAGL in either hepatocytes or myeloid cells displayed delayed liver regeneration. Mechanistically, MAGLHep-/- mice showed reduced liver eicosanoid production, in particular prostaglandin E2 that negatively impacts on hepatocyte proliferation. MAGL inhibition in macrophages resulted in the induction of the type I interferon pathway. Importantly, neutralising the type I interferon pathway restored liver regeneration of MAGLMye-/- mice. Conclusions: Our data demonstrate that MAGL promotes liver regeneration by hepatocyte and macrophage reprogramming. Impact and Implications: By using human liver samples and mouse models of global or specific cell type invalidation, we show that the monoacylglycerol pathway plays an essential role in liver regeneration. We unveil the mechanisms by which MAGL expressed in both hepatocytes and macrophages impacts the liver regeneration process, via eicosanoid production by hepatocytes and the modulation of the macrophage interferon pathway profile that restrains hepatocyte proliferation.

IntroductionThe molecular changes leading to Alzheimer’s disease (AD) progression are poorly understood. A decisive factor in the disease occurs when neurofibrillary tangles (NFT) composed of microtubule associated protein tau (MAPT) form in the entorhinal cortex and then spread throughout the brain.MethodsWe therefore determined mRNA and circular RNA changes during AD progression, comparing Braak NFT stages I-VI. Total RNA was isolated from human brain (entorhinal and frontotemporal cortex). Poly(A)+ RNA was subjected to Nanopore sequencing, and total RNA was analyzed by standard Illumina sequencing. Circular RNAs were sequenced from RNase R treated and rRNA depleted total RNA. The sequences were analyzed using different bioinformatic tools, and expression constructs for circRNAs were analyzed in transfection experiments.ResultsWe detected 11,873 circRNAs of which 276 correlated with Braak NFT stages. Adenosine to inosine RNA editing increased about threefold in circRNAs during AD progression. Importantly, this correlation cannot be detected with mRNAs. CircMAN2A1 expression correlated with AD progression and transfection experiments indicated that RNA editing promoted its translation using start codons out of frame with linear mRNAs, which generates novel proteins.DiscussionThus, we identified novel regulated retained introns that correlate with NFT Braak stages and provide evidence for a role of translated circRNAs in AD development.

To determine molecular changes that correlate with long-term physiological changes after spinal cord injury associated with spasticity, we used a complete transection model with an injury at sacral spinal level S2, wherein tail spasms develop in rats weeks to months post-injury. Using Illumina and nanopore sequencing, we found that from 12,266 expressed genes roughly 11% (1,342) change expression levels in the rats with spasticity. The transcription factor PU.1 (Spi-1 proto-oncogene) and several of its known regulated genes were upregulated during injury, possibly reflecting changes in cellular composition. In contrast to widespread changes in gene expression, only a few changes in alternative exon usage could be detected because of injury. There were more than 1,000 changes in retained intron usage, however. Unexpectedly, most of these retained introns have not been described yet but could be validated using direct RNA nanopore sequencing. In addition to changes from injury, our model allowed regional analysis of gene expression. Comparing the segments rostral and caudal to the injury site in na?ve animals showed 525 differentially regulated genes and differential regional use of retained introns. We did not detect changes in the serotonin receptor 2C editing that were implicated previously in this spinal cord injury model. Our data suggest that regulation of intron retention of polyadenylated pre-mRNA is an important regulatory mechanism in the spinal cord under both physiological and pathophysiological conditions.

Introduction: Inflammatory Breast Cancer (IBC) is the most aggressive form of breast carcinoma characterized by rapid onset of inflammatory signs and its molecular fingerprint has not yet been elucidated. Objectives: The objective of this study was to detect both gene expression levels and alternate RNA splice variants specific for IBC. Methods: W e performed splice-sensitive array profiling using Affymetrix Exon Array and quantitative RT-PCR analyses in 177 IBC compared to 183 non-IBC. We also assessed the prognostic value of the identified candidate genes and splice variants. Results: A 5-splice signature (HSPA8, RPL10, RPL4, DIDO1 and EVL) was able to distinguish IBC from non-IBC tumors (p

Abstract Background The adult mammary epithelium is composed of basal and luminal cells. The luminal lineage comprises two major cell populations, positive and negative for estrogen and progesterone receptors (ER and PR, respectively), both containing clonogenic progenitor cells. Deregulated ER/PR− luminal progenitor cells are suspected to be at the origin of basal-type triple-negative (TNBC) breast cancers, a subtype frequently associated with loss of P53 function and MET signaling hyperactivation. Using mouse models, we recently reported that p53 restricts luminal progenitor cell amplification whereas paracrine Met activation stimulates their growth and favors a luminal-to-basal switch. Here, we analyzed how these two critical pathways interact to control luminal progenitor function. Methods We have (i) established and analyzed the gene expression profile of luminal progenitors isolated by ICAM-1, a robust surface marker we previously identified; (ii) purified luminal progenitors from p53-deficient and p53-proficient mouse mammary epithelium to compare their functional and molecular characteristics; and (iii) analyzed their response to HGF, the major Met ligand, in three-dimensional cultures. Results We found that luminal progenitors, compared to non-clonogenic luminal cells, overexpress Trp53 and numerous p53 target genes. In vivo, loss of Trp53 induced the expansion of luminal progenitors, affecting expression of several important p53 target genes including those encoding negative regulators of cell cycle progression. Consistently, p53-deficient luminal progenitors displayed increased proliferative and self-renewal activities in culture. However, they did not exhibit perturbed expression of luminal-specific markers and major regulators, such as Hey1, Elf5, and Gata3. Moreover, although expressing Met at higher level than p53-proficient luminal progenitors, p53-deficient luminal progenitors failed to acquire basal-specific features when stimulated by HGF, showing that p53 promotes the plastic behavior of luminal progenitors downstream of Met activation. Conclusions Our study reveals a crosstalk between Met- and p53-mediated signaling pathways in the regulation of luminal progenitor function. In particular, it shows that neither p53 loss alone nor p53 loss combined with Met signaling activation caused an early detectable cell fate alteration in luminal progenitors. Conceivably, additional events are required to confer basal-specific characteristics to luminal-derived TNBCs.

Background and AimsPatients with cirrhosis and acute-on-chronic liver failure (ACLF) have immunosuppression, indicated by an increase in circulating immune-deficient monocytes. The aim of this study was to investigate simultaneously the major blood-immune cell subsets in these patients.Material and MethodsBlood taken from 67 patients with decompensated cirrhosis (including 35 critically ill with ACLF in the intensive care unit), and 12 healthy subjects, was assigned to either measurements of clinical blood counts and microarray (genomewide) analysis of RNA expression in whole-blood; microarray (genomewide) analysis of RNA expression in blood neutrophils; or assessment of neutrophil antimicrobial functions.ResultsSeveral features were found in patients with ACLF and not in those without ACLF. Indeed, clinical blood count measurements showed that patients with ACLF were characterized by leukocytosis, neutrophilia, and lymphopenia. Using the CIBERSORT method to deconvolute the whole-blood RNA-expression data, revealed that the hallmark of ACLF was the association of neutrophilia with increased proportions of macrophages M0-like monocytes and decreased proportions of memory lymphocytes (of B-cell, CD4 T-cell lineages), CD8 T cells and natural killer cells. Microarray analysis of neutrophil RNA expression revealed that neutrophils from patients with ACLF had a unique phenotype including induction of glycolysis and granule genes, and downregulation of cell-migration and cell-cycle genes. Moreover, neutrophils from these patients had defective production of the antimicrobial superoxide anion.ConclusionsGenomic analysis revealed that, among patients with decompensated cirrhosis, those with ACLF were characterized by dysregulation of blood immune cells, including increases in neutrophils (that had a unique phenotype) and macrophages M0-like monocytes, and depletion of several lymphocyte subsets (including memory lymphocytes). All these lymphocyte alterations, along with defective neutrophil superoxide anion production, may contribute to immunosuppression in ACLF, suggesting targets for future therapies.

Physiological low oxygen (O2) concentration (2 concentration, via the stabilization of hypoxia-induced transcription factors (HIFs), intervenes with Notch signaling in the control of cell fate. In addition, Notch activation is implicated in the regulation of erythroid differentiation. We test here if the favorable effects of a physiological O2 concentration (3%) on the amplification of erythroid progenitors implies a cooperation between HIFs and the Notch pathway. To this end, we utilized a model of early erythropoiesis ex vivo generated from cord blood CD34+ cells transduced with shHIF1α and shHIF2α at 3% O2 and 20% O2 in the presence or absence of the Notch pathway inhibitor. We observed that Notch signalization was activated by Notch2R–Jagged1 ligand interaction among progenitors. The inhibition of the Notch pathway provoked a modest reduction in erythroid cell expansion and promoted erythroid differentiation. ShHIF1α and particularly shHIF2α strongly impaired erythroid progenitors’ amplification and differentiation. Additionally, HIF/NOTCH signaling intersects at the level of multipotent progenitor erythroid commitment and amplification of BFU-E. In that, both HIFs contribute to the expression of Notch2R and Notch target gene HES1. Our study shows that HIF, particularly HIF2, has a determining role in the early erythroid development program, which includes Notch signaling.

Abstract Background Atypical Myeloproliferative Neoplasms (aMPN) share characteristics of MPN and Myelodysplastic Syndromes. Although abnormalities in cytokine signaling are common in MPN, the pathophysiology of atypical MPN still remains elusive. Since deregulation of microRNAs is involved in the biology of various cancers, we studied the miRNome of aMPN patients. Methods MiRNome and mutations in epigenetic regulator genes ASXL1, TET2, DNMT3A, EZH2 and IDH1/2 were explored in aMPN patients. Epigenetic regulation of miR-10a and HOXB4 expression was investigated by treating hematopoietic cell lines with 5-aza-2’deoxycytidine, valproic acid and retinoic acid. Functional effects of miR-10a overexpression on cell proliferation, differentiation and self-renewal were studied by transducing CD34+ cells with lentiviral vectors encoding the pri-miR-10a precursor. Results MiR-10a was identified as the most significantly up-regulated microRNA in aMPN. MiR-10a expression correlated with that of HOXB4, sitting in the same genomic locus. The transcription of these two genes was increased by DNA demethylation and histone acetylation, both necessary for optimal expression induction by retinoic acid. Moreover, miR-10a and HOXB4 overexpression seemed associated with DNMT3A mutation in hematological malignancies. However, overexpression of miR-10a had no effect on proliferation, differentiation or self-renewal of normal hematopoietic progenitors. Conclusions MiR-10a and HOXB4 are overexpressed in aMPN. This overexpression seems to be the result of abnormalities in epigenetic regulation mechanisms. Our data suggest that miR-10a could represent a simple marker of transcription at this genomic locus including HOXB4, widely recognized as involved in stem cell expansion.

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.

Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect—the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.

Abstract Background In spite of the recent discovery of genetic mutations in most myelodysplasic (MDS) patients, the pathophysiology of these disorders still remains poorly understood, and only few in vivo models are available to help unravel the disease. Methods We performed global specific gene expression profiling and functional pathway analysis in purified Sca1+ cells of two MDS transgenic mouse models that mimic human high-risk MDS (HR-MDS) and acute myeloid leukemia (AML) post MDS, with NRASD12 and BCL2 transgenes under the control of different promoters MRP8NRASD12/tethBCL-2 or MRP8[NRASD12/hBCL-2], respectively. Results Analysis of dysregulated genes that were unique to the diseased HR-MDS and AML post MDS mice and not their founder mice pointed first to pathways that had previously been reported in MDS patients, including DNA replication/damage/repair, cell cycle, apoptosis, immune responses, and canonical Wnt pathways, further validating these models at the gene expression level. Interestingly, pathways not previously reported in MDS were discovered. These included dysregulated genes of noncanonical Wnt pathways and energy and lipid metabolisms. These dysregulated genes were not only confirmed in a different independent set of BM and spleen Sca1+ cells from the MDS mice but also in MDS CD34+ BM patient samples. Conclusions These two MDS models may thus provide useful preclinical models to target pathways previously identified in MDS patients and to unravel novel pathways highlighted by this study.

The feasibility of ex vivo expansion allows us to consider the steady-state peripheral blood as an alternative source of hematopoietic stem progenitor cells for transplantation when growth factor-induced cell mobilization is contraindicated or inapplicable. Ex vivo expansion dramatically enhances the in vivo reconstituting cell population from steady-state blood. In order to investigate phenotype and the expression of homing molecules, the expression of CD34, CD133, CD90, CD45RA, CD26 and CD9 was determined on sorted CD34+ cells according to CXCR4 (“neg”, “low” “bright”) and CD133 expression before and after ex vivo expansion. Hematopoietic stem cell activity was determined in vivo on the basis of hematopoietic repopulation of primary and secondary recipients - NSG immuno-deficient mice. In vivo reconstituting cells in the steady-state blood CD34+ cell fraction before expansion belong to the CD133+ population and are CXCR4low or, to a lesser extent, CXCR4neg, while after ex vivo expansion they are contained only in the CD133+CXCR4low cells. The failure of the CXCR4bright population to engraft is probably due to the exclusive expression of CD26 by these cells. The limiting-dilution analysis showed that both repopulating cell number and individual proliferative capacity were enhanced by ex vivo expansion. Thus, steady-state peripheral blood cells exhibit a different phenotype compared to mobilized and cord blood cells, as well as to those issued from the bone marrow. These data represent the first phenotypic characterization of steady-state blood cells exhibiting short- and long-term hematopoietic reconstituting potential, which can be expanded ex vivo, a sine qua non for their subsequent use for transplantation.

Abstract In Huntington disease (HD), polyglutamine expansion in the huntingtin protein causes specific neuronal death. The consequences of the presence of mutant huntingtin in other tissues are less well understood. Here we propose that mutant huntingtin influences breast cancer progression. Indeed, we show that mammary tumours appear earlier in mouse breast cancer models expressing mutant huntingtin as compared to control mice expressing wild‐type huntingtin. Tumours bearing mutant huntingtin have a modified gene expression pattern that reflects enhanced aggressiveness with the overexpression of genes favouring invasion and metastasis. In agreement, mutant huntingtin accelerates epithelial to mesenchymal transition and enhances cell motility and invasion. Also, lung metastasis is higher in HD conditions than in control mice. Finally, we report that in HD, the dynamin dependent endocytosis of the ErbB2/HER2 receptor tyrosine kinase is reduced. This leads to its accumulation and to subsequent increases in cell motility and proliferation. Our study may thus have important implications for both cancer and HD.

Urticarial lesions are observed in both cutaneous and systemic disorders. Familial forms of urticarial syndromes are rare and can be encountered in systemic autoinflammatory diseases. We sought to investigate a large family with dominantly inherited chronic urticarial lesions associated with hypercytokinemia. We performed a genetic linkage analysis in 14 patients from a 5-generation family, as well as whole-exome sequencing, cytokine profiling, and transcriptomic analyses on samples from 2 patients. The identified candidate protein was studied after in vitro expression of the corresponding normal and mutated recombinant proteins. An unsupervised proteomic approach was used to unveil the associated protein network. The disease phenotype of the most affected family members is characterized by chronic urticarial flares associated with extremely high plasma levels of proinflammatory (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-10 and IL-1 receptor antagonist [IL-1RA]) cytokines, with no secondary organ dysfunction, no susceptibility to infections, no fever, and normal C-reactive protein levels. Monocyte transcriptomic analyses identified an immunotolerant profile in the most affected patient. The affected family members carried a loss-of-function mutation in RNF213 that encodes mysterin, a protein with a poorly known physiologic role. We identified the deubiquitinase CYLD, a major regulator of inflammation, as an RNF213 partner and showed that CYLD expression is inhibited by wild-type but not mutant RNF213. We identified a new entity characterized by chronic urticarial lesions associated with a clinically blunted hypercytokinemia. This disease, which is due to loss of function of RNF213, reveals mysterin's key role in the complex molecular network of innate immunity. [ABSTRACT FROM AUTHOR]

It is widely accepted that most colorectal cancers (CRCs) arise from colorectal adenomas (CRAs), but transcriptomic data characterizing the progression from colorectal normal mucosa to adenoma, and then to adenocarcinoma are scarce. These transition steps were investigated using microarrays, both at the level of gene expression and alternative pre-mRNA splicing. Many genes and exons were abnormally expressed in CRAs, even more than in CRCs, as compared to normal mucosae. Known biological pathways involved in CRC were altered in CRA, but several new enriched pathways were also recognized, such as the complement and coagulation cascades. We also identified four intersectional transcriptional signatures that could distinguish CRAs from normal mucosae or CRCs, including a signature of 40 genes differentially deregulated in both CRA and CRC samples. A majority of these genes had been described in different cancers, including FBLN1 or INHBA, but only a few in CRC. Several of these changes were also observed at the protein level. In addition, 20% of these genes (i.e. CFH, CRYAB, DPT, FBLN1, ITIH5, NR3C2, SLIT3 and TIMP1) showed altered pre-mRNA splicing in CRAs. As a global variation occurring since the CRA stage, and maintained in CRC, the expression and splicing changes of this 40-gene set may mark the risk of cancer occurrence from analysis of CRA biopsies.

BACKGROUND: Xenotransplantation models allowing the identification and quantification of human Hematopoietic stem cells (HSC) in immunodeficient mice remain the only way to appropriately address human HSC function despite the recent progress in phenotypic characterization. However, these in vivo experiments are technically demanding, time consuming and expensive. Indeed, HSCs engraftment in mouse requires pre-conditioning of animals either by irradiation or cytotoxic drugs to allow homing of injected cells in specific stem cell niches and their subsequent expansion and differentiation in bone marrow. Recently, the development of busulfan pre-conditioning of animals improved the flexibility of experimentation in comparison with irradiation. DESIGN AND METHODS: In order to further facilitate the organization of these complex experiments we investigated the effect of extending the period between mice pre-conditioning and cell injection on the engraftment efficiency. In the meantime, we also explored the role of busulfan doses, mouse gender and intravenous injection route (caudal or retro orbital) on engraftment efficiency. RESULTS AND CONCLUSION: We showed that a period of up to 7 days did not modify engraftment efficiency of human HSCs in NSG model. Moreover, retro orbital cell injection to female mice pre-conditioned with 2x25 mg/kg of busulfan seems to be the best adapted schema to detect the human HSC in xenotransplantation experiments.

Prader-Willi syndrome (PWS) is caused by the loss of RNA expression from an imprinted region on chromosome 15 that includes SNRPN, SNORD115, and SNORD116. Currently, there are no mouse models that faithfully reflect the human phenotype and investigations rely on human post-mortem material. During molecular characterization of tissue deposited in a public brain bank from a patient diagnosed with Prader-Willi syndrome, we found RNA expression from SNRPN, SNORD115, and SNORD116 which does not support a genetic diagnosis of Prader-Willi syndrome. The patient was a female, Caucasian nursing home resident with history of morbid obesity (BMI 56.3) and mental retardation. She died at age of 56 from pulmonary embolism. SNORD115 and SNORD116 are unexpectedly stable in post mortem tissue and can be used for post-mortem diagnosis. Molecular characterization of PWS tissue donors can confirm the diagnosis and identify those patients that have been misdiagnosed.