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The epitranscriptome represents the more than 140 types of chemically varying and reversable RNA modifications affecting RNA fate. Among these, the most relevant for this review are the mRNA modifications N6-methyladenosine and N6,2'-O-dimethyladenosine. Epitranscriptomic mRNA biology involves RNA methyltransferases (so-called "writers"), RNA demethylases ("erasers"), and RNA-binding proteins ("readers") that interact with methylation sites to determine the functional outcome of the modification. In this review, we discuss the role of a specific RNA demethylase encoded by the fat mass and obesity-associated gene (FTO) in cancer. FTO initially became known as the strongest genetic link for human obesity. Only in 2010, 16 years after its discovery, was its enzymatic function as a demethylase clarified, and only recently has its role in the development of cancer been revealed. FTO functions are challenging to study and interpret because of its genome-wide effects on transcript turnover and translation. We review the discovery of FTO and its enzymatic function, the tumor-promoting and suppressive roles of FTO in selected cancer types, and its potential as a therapeutic target.

Universal influenza vaccines have the potential to protect against continuously evolving and newly emerging influenza viruses. T cells may be an essential target of such vaccines as they can clear infected cells through recognition of conserved influenza virus epitopes. We evaluated a novel T cell-inducing nucleoside-modified mRNA vaccine that encodes the conserved nucleoprotein, matrix protein 1 and polymerase basic protein 1 of an H1N1 influenza virus. To mimic the human situation, we applied the mRNA vaccine as a prime-boost regimen in naïve ferrets (mimicking young children) and as a booster in influenza-experienced ferrets (mimicking adults). The vaccine induced and boosted broadly-reactive T cells in the circulation, bone marrow and respiratory tract. Booster vaccination enhanced protection against heterosubtypic infection with potential pandemic H7N9 influenza virus in influenza-experienced ferrets. Our findings show that mRNA vaccines encoding internal influenza virus proteins are a promising strategy to induce broadly-protective T-cell immunity against influenza viruses.

Apart from controlling hematopoiesis, the bone marrow (BM) also serves as a secondary lymphoid organ, as it can induce naïve T cell priming by resident dendritic cells (DC). When analyzing DCs in murine BM, we uncovered that they are localized around sinusoids, can (cross)-present antigens, become activated upon intravenous LPS-injection, and for the most part belong to the cDC2 subtype which is associated with Th2/Th17 immunity. Gene-expression profiling revealed that BM-resident DCs are enriched for several c-type lectins, including Dectin-1, which can bind beta-glucans expressed on fungi and yeast. Indeed, DCs in BM were much more efficient in phagocytosis of both yeast-derived zymosan-particles and Aspergillus conidiae than their splenic counterparts, which was highly dependent on Dectin-1. DCs in human BM could also phagocytose zymosan, which was dependent on β1-integrins. Moreover, zymosan-stimulated BM-resident DCs enhanced the differentiation of hematopoietic stem and progenitor cells towards neutrophils, while also boosting the maintenance of these progenitors. Our findings signify an important role for BM DCs as translators between infection and hematopoiesis, particularly in anti-fungal immunity. The ability of BM-resident DCs to boost neutrophil formation is relevant from a clinical perspective and contributes to our understanding of the increased susceptibility for fungal infections following BM damage.

Genetic evidence indicates disrupted epigenetic regulation as a major risk factor for psychiatric disorders, but the molecular mechanisms that drive this association remain to be determined. EHMT1 is an epigenetic repressor that is causal for Kleefstra Syndrome (KS), a genetic disorder linked with neurodevelopmental disorders and associated with schizophrenia. Here, we show that reduced EHMT1 activity decreases NRSF/REST protein leading to abnormal neuronal gene expression and progression of neurodevelopment in human iPSC. We further show that EHMT1 regulates NRSF/REST indirectly via repression of miRNA and leads to aberrant neuronal gene regulation and neurodevelopment timing. Expression of a NRSF/REST mRNA that lacks the miRNA-binding sites restores neuronal gene regulation to EHMT1 deficient cells. Significantly, the EHMT1-regulated miRNA gene set not only controls NRSF/REST but is enriched for association for Intellectual Disability (ID) and schizophrenia. This reveals a broad molecular interaction between H3K9 demethylation, NSRF/REST regulation and risk for ID and Schizophrenia.

A major goal of methodologies related to large scale gene expression analyses is to initiate comprehensive information on transcript signatures in single cells within the tissue's anatomy. Until now, this could be achieved in a stepwise experimental approach: (1) identify the majority of transcripts in a single cell (single cell transcriptome); (2) provide information on transcripts on multiple cell subtypes in a complex sample (cell heterogeneity); and (3) give information on each cell's spatial location within the tissue (zonation transcriptomics). Such genetic information will allow construction of functionally relevant gene expression maps of single cells of a given anatomically defined tissue compartment and thus pave the way for subsequent analyses, including their epigenetic modifications. Until today these aims have not been achieved in the area of cardiovascular disease research though steps toward these goals become apparent: laser capture microdissection (LCM)-based mRNA expression microarrays of atherosclerotic plaques were applied to gain information on local gene expression changes during disease progression, providing limited spatial resolution. Moreover, while LCM-derived tissue RNA extracts have been shown to be highly sensitive and covers a range of 10-16,000 genes per array/small amount of RNA, its original promise to isolate single cells from a tissue section turned out not to be practicable because of the inherent contamination of the cell's RNA of interest with RNA from neighboring cells. Many shortcomings of LCM-based analyses have been overcome using single-cell RNA sequencing (scRNA-seq) technologies though scRNA-seq also has several limitations including low numbers of transcripts/cell and the complete loss of spatial information. Here, we describe a protocol toward combining advantages of both techniques while avoiding their flaws.

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin β4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies., ZEB2 transcription factor is increased in a subset of cardiomyocytes during stress to induce cardioprotective effects after injury. Here the authors show that therapeutic delivery of ZEB2 prevents cardiac dysfunction after ischemic damage and promotes the activation of pro-angiogenic signals.

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome in which carriers are at an increased risk of developing a variety of tumors in multiple organ systems. A clinical diagnosis of VHL is determined by the presence of specific clinical manifestations while a molecular genetic diagnosis results from a pathogenic variant in the VHL gene. The majority of mutations occur in VHL coding exons and DNA analysis of these regions has a reported sensitivity of nearly 100%. However, rare variants in the VHL gene promoter may be detected in some cases of suspected VHL disease. We report two cases where VHL promoter variants were detected and describe the role of multi-step mRNA and protein analysis in the diagnostic evaluation of these cases.

Inhibitors of DNA methylation and orexin type-1 receptor antagonists modulate the neurobiological effects driving drugs of abuse and natural reinforcers by activating common brain structures of the mesolimbic reward system. In this study, we applied a self-administration paradigm to assess the involvement of factors regulating DNA methylation processes and satiety or appetite signals. These factors include Dnmts and Tets, miR-212/132, orexins, and orx-R1 genes. The study focused on dopamine projection areas such as the prefrontal cortex (PFCx) and caudate putamen (CPu) and in the hypothalamus (HP) that is interconnected with the reward system. Striking changes were observed in response to both reinforcers, but differed depending on contingent and non-contingent delivery. Expression also differed in the PFCx and the CPu. Cocaine and food induced opposite effects on Dnmt3a expression in both brain structures, whereas they repressed both miRs to a different extent, without affecting their primary transcript in the CPu. Unexpectedly, orexin mRNAs were found in the CPu, suggesting a transport from their transcription site in the HP. The orexin receptor1 gene was found to be induced by cocaine in the PFCx, consistent with a regulation by DNA methylation. Global levels of 5-methylcytosines in the PFCx were not significantly altered by cocaine, suggesting that it is rather their distribution that contributes to long-lasting behaviors. Together, our data demonstrate that DNA methylation regulating factors are differentially altered by cocaine and food. At the molecular level, they support the idea that neural circuits activated by both reinforcers do not completely overlap.

The first therapeutic application of messenger RNA (mRNA) was suggested more than two decades ago. However, its application was constrained by the ability of mRNA to activate the innate immune response, cytotoxicity, and poor potency. We and others recently demonstrated that these undesirable properties of mRNA may be overcome by alterating its structure. In this study, we developed a new chemically modified mRNA coding for BMP-2 with improved osteogenic features. To develop this new construct, we removed from the mRNA sequence the following undesirable elements: an upstream open reading frame in the 5-untranslated region (UTR) and a polyadenylation element together with an AU-rich tract in the 3UTR. In addition, a translation initiator of short UTRs (TISU) was introduced together with 5-iodo modified pyrimidine nucleotides. The new TISU BMP-2 chemically modified RNA (cmRNA) showed robust BMP-2 production in vitro in cell lines (HEK293 and MC3T3) and primary cells (muscle-derived mesenchymal stem cells). Stem cells additionally showed upregulation of osteogenic and angiogenic genes as a result of the TISU BMP-2 cmRNA transfection. The in vivo osteogenic properties of TISU BMP-2 cmRNA were explored in a critical-sized femoral defect in the rat. For this, the TISU BMP-2 cmRNA was loaded into collagen sponges to form transcript-activated matrices. Animals treated with TISU BMP-2 cmRNA showed superior bone formation that seemed to recapitulate endochondral ossification. The higher of the two doses examined in this model showed more robust new tissue formation. Finally, improved vascularization was detected in the healing area for animals treated with TISU BMP-2 cmRNA.

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2.

The use of tagged RNA probes to directly hybridize frozen sections of chemically fixed tissues, followed by the tag detection with specific antibodies and gold conjugates form the core of the in situ hybridization (ISH)-immunoelectron microscopy (IEM) method that we have developed and successfully used to detect endogenous gurken and bicoid mRNAs in Drosophila oocytes.

mRNA translation is a key step in decoding the genetic information stored in DNA. Regulation of translation efficiency contributes to gene expression control and is therefore important for cell fate and function. Here, we describe a recently developed microscopy-based method that allows for visualization of translation of single mRNAs in live cells. The ability to measure translation dynamics of single mRNAs will enable a better understanding of spatiotemporal control of translation, and will provide unique insights into translational heterogeneity of different mRNA molecules in single cells.

Growing evidence indicates that protein synthesis is deregulated in cancer onset and progression and targeting this process might be a selective way to combat cancers. While harmine is known to inhibit DYRK1A and intercalate into the DNA, tri-substitution was shown previously to modify its activity profile in favor of protein synthesis inhibition. In this study, we thus evaluated the optimized derivative CM16 in vitro anti-cancer effects unfolding its protein synthesis inhibition activity. Indeed, the growth inhibitory profile of CM16 in the NCI 60-cancer-cell-line-panel correlated with those of other compounds described as protein synthesis inhibitors. Accordingly, CM16 decreased in a time- and concentration-dependent manner the translation of neosynthesized proteins in vitro while it did not affect mRNA transcription. CM16 rapidly penetrated into the cell in the perinuclear region of the endoplasmic reticulum where it appears to target translation initiation as highlighted by ribosomal disorganization. More precisely, we found that the mRNA expression levels of the initiation factors EIF1AX, EIF3E and EIF3H differ when comparing resistant or sensitive cell models to CM16. Additionally, CM16 induced eIF2α phosphorylation. Those effects could explain, at least partly, the CM16 cytostatic anti-cancer effects observed in vitro while neither cell cycle arrest nor DNA intercalation could be demonstrated. Therefore, targeting protein synthesis initiation with CM16 could represent a new promising alternative to current cancer therapies due to the specific alterations of the translation machinery in cancer cells as recently evidenced with respect to EIF1AX and eIF3 complex, the potential targets identified in this present study.

Bone regeneration using stem cells and growth factors has disadvantages while needing to use supraphysiological growth factors concentrations. Gene therapy has been proposed as alternative, but also has limitation. Messenger RNA (mRNA)-based transcript therapy is a novel approach that may solve plasmid DNA-based gene therapy limitations. Although much more efficient in delivering genes into the cell, mRNA is unfortunately unstable and immunogenic. However, recent reports indicated that chemical modifications of the mRNA molecule can improve stability and toxicity. In this study, we have combined biomaterials and chemically modified mRNA (cmRNA) encoding Metridia luciferase, eGFP, and bone morphogenetic protein (BMP)-2 to develop transcript-activated matrices (TAMs) for gene transfer to stem cells. BMP-2 cmRNA was produced to evaluate its feasibility in stimulating osteogenic differentiation. Fibrin gel and micro-macro biphasic calcium phosphate (MBCP) granules were used as biomaterials. A sustained release of hBMP-2 cmRNA from both biomaterials was observed during 7 days. This occurred significantly faster from the MBCP granules compared to fibrin gels (92% from MBCP and 43% from fibrin after 7 days). Stem cells cultured in hBMP-2 cmRNA/fibrin or on hBMP-2 cmRNA/MBCP were transfected and able to secrete significant amounts of hBMP-2. Furthermore, transfected cells expressed osteogenic markers in vitro. Interestingly, although both TAMs promoted gene expression at the same level, hBMP-2 cmRNA/MBCP granules induced significantly higher collagen I and osteocalcin gene expression. This matrix also induced more mineral deposition. Overall, our results demonstrated the feasibility of developing efficient TAMs for bone regeneration by combining biomaterials and cmRNAs. MBCP synergistically enhances the hBMP-2 cmRNA-induced osteogenic pathway.

Successful delivery of mRNA into the cytosol of professional antigen-presenting cells (APCs) poses one of the biggest challenges in developing effective mRNA vaccines to treat various cancers and viral infectious diseases. However, most polymeric mRNA delivery systems fail to transfect APCs. We have discovered that decoration of pH-sensitive endosome-disruptive GALA peptides on the surface of mRNA polyplexes leads to efficient targeting and transfection of APCs. GALA peptides not only enhance specific uptake in APCs through binding to sialic acid moieties, they also facilitate the endosomal escape of mRNA especially in dendritic cells (DCs). Here, we describe in detail the production of stabilized mRNA polyplexes post-conjugated with GALA peptides via copper-free click chemistry. Methods described here include the synthesis and purification of GALA peptides and its conjugation to mRNA polyplexes.

UNLABELLED: Gene expression analysis in the tumor microenvironment using archived clinical samples is challenging because of formalin fixation, RNA degradation, and limiting sample volume. NanoString gene expression profiling is a RNA-DNA hybrid capture technology that does not require PCR and can accurately quantify the expression of to 800 transcripts in a single reaction. The technology requires 50-100 ng of RNA, which can be degraded ( EDITOR: is this correct?) to a 200 bp fragment size. In contrast to amplification technologies, nanoString counts the actual numbers of transcripts that are captured with transcript-specific and fluorescently-barcoded probes. This chapter describes protocols for RNA extraction, quantification, mRNA and miRNA profiling and data analysis.

AIMS/HYPOTHESIS: The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment. METHODS: Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice. RESULTS: At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean ± SEM: 0.118 ± 0.01 [n = 3] in Vegf-A mRNA transfected group (VEGF) vs 0.056 ± 0.01 [n = 3] in no RNA [p

Erythropoiesis is regulated at many levels, including control of mRNA translation. Changing environmental conditions, such as hypoxia or the availability of nutrients and growth factors, require a rapid response enacted by the enhanced or repressed translation of existing transcripts. Cold shock domain protein e1 (Csde1/Unr) is an RNA-binding protein required for erythropoiesis and strongly upregulated in erythroblasts relative to other hematopoietic progenitors. The aim of this study is to identify the Csde1-containing protein complexes and investigate their role in post-transcriptional expression control of Csde1-bound transcripts. We show that Serine/Threonine kinase receptor-associated protein (Strap/Unrip), was the protein most strongly associated with Csde1 in erythroblasts. Strap is a WD40 protein involved in signaling and RNA splicing, but its role when associated with Csde1 is unknown. Reduced expression of Strap did not alter the pool of transcripts bound by Csde1. Instead, it altered the mRNA and/or protein expression of several Csde1-bound transcripts that encode for proteins essential for translational regulation during hypoxia, such as Hmbs, eIF4g3 and Pabpc4. Also affected by Strap knockdown were Vim, a Gata-1 target crucial for erythrocyte enucleation, and Elavl1, which stabilizes Gata-1 mRNA. The major cellular processes affected by both Csde1 and Strap were ribosome function and cell cycle control.

To better understand the tissue-specific regulation of chromatin state in cell-fate determination and animal development, we defined the tissue-specific expression of all 36 C. elegans presumptive lysine methyltransferase (KMT) genes using single-molecule fluorescence in situ hybridization (smFISH). Most KMTs were expressed in only one or two tissues. The germline was the tissue with the broadest KMT expression. We found that the germline-expressed C. elegans protein SET-17, which has a SET domain similar to that of the PRDM9 and PRDM7 SET-domain proteins, promotes fertility by regulating gene expression in primary spermatocytes. SET-17 drives the transcription of spermatocyte-specific genes from four genomic clusters to promote spermatid development. SET-17 is concentrated in stable chromatin-associated nuclear foci at actively transcribed msp (major sperm protein) gene clusters, which we term msp locus bodies. Our results reveal the function of a PRDM9/7-family SET-domain protein in spermatocyte transcription. We propose that the spatial intranuclear organization of chromatin factors might be a conserved mechanism in tissue-specific control of transcription., National Institutes of Health (U.S.) (grant GM24663), NIH/National Cancer Institute Physical Sciences Oncology Center (U54CA1438 74), National Institutes of Health (U.S.) Pioneer Award (8 DP1 CA174420- 05)

G-protein coupled receptor (GPCR) mediated activation of the MAPK signalling cascade is a key pathway in the induction of hypertrophic remodelling of the heart – a response to pathological cues including hypertension and myocardial infarction. While levels of pro-hypertrophic hormone agonists of GPCRs increase during periods of greater workload to enhance cardiac output, hypertrophy does not necessarily result. Here we investigated the relationship between the duration of exposure to the pro-hypertrophic GPCR agonist endothelin-1 (ET-1) and the induction of hypertrophic remodelling in neonatal rat ventricular myocytes (NRVM) and in the adult rat heart in vivo. Notably, a 15 min pulse of ET-1 was sufficient to induce markers of hypertrophy that were present when measured at 24 h in vivo and 48 h in vitro. The persistence of ET-1 action was insensitive to ET type A receptor (ETA receptor) antagonism with BQ123. The extended effects of ET-1 were dependent upon sustained MAPK signalling and involved persistent transcription. Inhibitors of endocytosis however conferred sensitivity upon the hypertrophic response to BQ123, suggesting that endocytosis of ETA receptors following ligand binding preserves their active state by protection against antagonist. Contrastingly, α1 adrenergic-induced hypertrophic responses required the continued presence of agonist and were sensitive to antagonist. These studies shed new light on strategies to pharmacologically intervene in the action of different pro-hypertrophic mediators., Highlights • Acute ET-1 exposure elicits a long-lasting cardiac myocyte hypertrophic response. • ET-1 effects depend on persistent MAPK signalling and active transcription. • ET-1 elicited hypertrophy is insensitive to subsequent ETA receptor antagonism. • Endocytosis inhibition potentiates ET-1-induction of hypertrophy markers. • Endocytosis inhibition sensitises effects of ET-1 to ETA receptor antagonist.

RBP4 is produced mainly by hepatocytes. In type 2 diabetes and obesity, circulating RBP4 is increased and may act systemically to cause insulin resistance and glucose intolerance. Observations that adipocyte RBP4 mRNA increases in parallel with circulating RBP4 in these conditions, whereas liver RBP4 mRNA does not, led to a widely held hypothesis that elevated circulating RBP4 is a direct result of increased production by adipocytes. To test this, we generated mice with hepatocyte-specific deletion of RBP4 (liver RBP4 knockout or LRKO mice). Adipose tissue RBP4 expression and secretion remained intact in LRKO mice and increased as expected in the setting of diet-induced insulin resistance. However, circulating RBP4 was undetectable in LRKO mice. We conclude that adipocyte RBP4 is not a significant source of circulating RBP4, even in the setting of insulin resistance. Adipocyte RBP4, therefore, may have a more important autocrine or paracrine function that is confined within the adipose tissue compartment.

Contains fulltext : 171962.PDF (Publisher’s version ) (Open Access) The Wnts can be considered as candidates for the Congenital Anomaly of Kidney and Urinary Tract, CAKUT diseases since they take part in the control of kidney organogenesis. Of them Wnt5a is expressed in ureteric bud (UB) and its deficiency leads to duplex collecting system (13/90) uni- or bilateral kidney agenesis (10/90), hypoplasia with altered pattern of ureteric tree organization (42/90) and lobularization defects with partly fused ureter trunks (25/90) unlike in controls. The UB had also notably less tips due to Wnt5a deficiency being at E15.5 306 and at E16.5 765 corresponding to 428 and 1022 in control (p

This study evaluated the suitability of adipose-derived stem cells (ASCs) combined with fibrin matrix of variable composition for adipose tissue-equivalent formation in vitro. Therefore, undifferentiated ASCs were embedded in fibrin clots composed of 2 IU/ml thrombin and fibrinogen of varying concentrations (6.25, 12.5 and 25 mg/ml) and kept under control or adipogenic conditions. Fibrin-cell composites were evaluated by scanning electron microscopy, live/dead staining, lactate-dehydrogenase (LDH) assay, quantitative PCR for the adipogenic markers fatty acid binding protein 4 (FABP4), peroxisome proliferative activated receptor-γ (PPARγ) and leptin, leptin ELISA and oil red O staining. Cells were found homogeneously distributed throughout the clot. Their number increased to day 7 (up to 3.62-fold median) and decreased thereafter until day 28. The proliferation was unaffected by fibrinogen concentration in the control. Adipogenic conditions generally yielded higher cell numbers, which were in addition increasing with increasing fibrinogen concentrations. FABP4, PPARγ and leptin mRNA expression was strongly upregulated by adipogenic medium, which was confirmed by the levels of leptin secretion and lipid vesicles formation demonstrated by oil red O staining. When embedded in 25 mg/ml fibrinogen clots, ASCs showed the highest expression levels of FABP4 (up to 629.0-fold), PPARγ (up to 1.6-fold) and leptin (up to 57.9-fold), corroborated by significantly elevated leptin secretion (median 33.29 ng/ml) on day 14. Constructs composed of fibrin matrix of low component concentrations-allowing homogeneous cell distribution-with ASCs should represent a suitable strategy for adipose tissue formation in vivo.

Light is among the most important exogenous factors that regulate plant development. To sense light quality, intensity, direction, and duration, plants have evolved multiple photoreceptors that enable the detection of photons from the ultraviolet B (UV-B) to the far-red spectrum. To study the effect of different light qualities on early gene expression, dark-grown Arabidopsis (Arabidopsis thaliana) seedlings were either irradiated with continuous far-red, red, or blue light or received pulses of red, UV-A, or UV-A/B light. The expression profiles of seedlings harvested at 45 min and 4 h were determined on a full genome level and compared with the profiles of dark controls. Data were used to identify light-regulated genes and to group these genes according to their light responses. While most of the genes were regulated by more than one light quality, a considerable number of UV-B-specific gene expression responses were obtained. An extraordinarily high similarity in gene expression patterns was obtained for samples that perceived continuous irradiation with either far-red or blue light for 4 h. Mutant analyses hint that this coincidence is caused by a convergence of the signaling cascades that regulate gene expression downstream of cryptochrome blue light photoreceptors and phytochrome A. Whereas many early light-regulated genes exhibited uniform responses to all applied light treatments, highly divergent expression patterns developed at 4 h. These data clearly indicate that light signaling during early deetiolation undergoes a switch from a rapid, but unspecific, response mode to regulatory systems that measure the spectral composition and duration of incident light.

International audience; Multiple endocrine neoplasia type 1 (MEN1) results from the mutation of the predisposing gene, MEN1. Heterozygous Men1 mutant mice previously generated by several laboratories, including ours, mimic largely MEN1 pathology. Interestingly, our heterozygous Men1 mutant mice exhibit not only the endocrine tumours commonly seen in MEN1 patients, but also Leydig cell tumours (LCT) with high frequency, accompanied systematically by loss of the wild-type Men1 allele. As there exists a similarity of tumour phenotype between these mice and those mutated for the components of anti-Mullerian hormone (AMH)/bone morphogenic protein (BMP) pathway belonging to transforming growth factor-beta (TGF-beta) family, we investigated the expression and the activity of this pathway, known to have an important biological role in Leydig cells. Here, we report that the expression of AMH receptor type 2 is reduced in Men1 LCTs. Both immunostaining and western blot analyses also demonstrate a markedly decreased nuclear expression of Smad1, 3, 4 and 5 in the tumours. More interestingly, we show that the reconstituted menin expression in Men1-deficient Leydig cells derived from LCTs can significantly increase the transcriptional activity of a BMP pathway target promoter, XVent2. Furthermore, we found that the expression of p18, p27 and cyclin dependant kinase 4 (Cdk4), targets of TGF-beta pathways, is altered in the Leydig cell lesions. Our data provide the evidence of the deregulation of AMH/BMP and TGF-beta pathways in mouse Men1 LCTs, highlighting their involvement in tumorigenesis of Leydig cells due to Men1 inactivation.

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is characterized by chromosomal rearrangements possibly enforcing arrest at specific development stages. We studied the relationship between molecular- cytogenetic abnormalities and T-cell development stage to investigate whether arrest at specific stages can explain the prognostic significance of specific abnormalities. We extensively studied 72 pediatric T-ALL cases for genetic abnormalities and expression of transcription factors, NOTCH1 mutations and expression of specific CD markers. HOX11 cases were CD1 positive consistent with a cortical stage, but as 4/5 cases lacked cytoplasmatic-beta expression, developmental arrest may precede beta-selection. HOX11L2 was especially confined to immature and pre- AB developmental stages, but 3/17 HOX11L2 mature cases were restricted to the gamma delta-lineage. TAL1 rearrangements were restricted to the alpha beta-lineage with most cases being TCR-alpha beta positive. NOTCH1 mutations were present in all molecular-cytogenetic subgroups without restriction to a specific developmental stage. CALM-AF10 was associated with early relapse. TAL1 or HOX11L2 rearrangements were associated with trends to good and poor outcomes, respectively. Also cases with high vs low TAL1 expression levels demonstrated a trend toward good outcome. Most cases with lower TAL1 levels were HOX11L2 or CALM-AF10 positive. NOTCH1 mutations did not predict for outcome. Classification into T-cell developmental subgroups was not predictive for outcome.

International audience; The necessary overall coordination of cardiac cellular functions is little known at the mRNA level. Focusing on energy production and cardiac contraction, we analyzed microarray data from heart tissue obtained in groups of mice and rats in normal conditions and with a left ventricular dysfunction. In each group and for each function, we identified genes positively or negatively correlated with numerous genes of the function, which were called coordinated or inversely coordinated with the function. The genes coordinated with energy production or cardiac contraction showed the coupling of these functions in all groups. Among coordinated or inversely coordinated genes common to the two functions, we proposed a fair number of transcriptional regulators as potential determinants of the energy production and cardiac contraction coupling. Although this coupling was constant across the groups and unveiled a stable gene core, the combinations of transcriptional regulators were very different between the groups, including one half that has never been linked to heart function. These results highlighted the stable coordination of energy production or cardiac contraction at the mRNA level, and the combinatorial and versatile nature of potential transcriptional regulation. In addition, this work unveiled new transcriptional regulators potentially involved in normal or altered cardiac functional coupling.

Oncogene-induced senescence (OIS) can occur in response to hyperactive oncogenic signals and is believed to be a fail-safe mechanism protecting against tumorigenesis. To identify new factors involved in OIS, we performed a screen for microRNAs that can overcome or inhibit OIS in human diploid fibroblasts. This screen led to the identification of miR-378a-5p and in addition several other miRNAs that have previously been shown to play a role in senescence. We show that ectopic expression of miR-378a-5p reduces the expression of several senescence markers, including p16(INK4A) and senescence-associated β-galactosidase. Moreover, cells with ectopic expression of miR-378a-5p retain proliferative capacity even in the presence of an activated Braf oncogene. Finally, we identified several miR-378a-5p targets in diploid fibroblasts that might explain the mechanism by which the microRNA can delay OIS. We speculate that miR-378a-5p might positively influence tumor formation by delaying OIS, which is consistent with a known pro-oncogenic function of this microRNA.

International audience; The identification of galectin-7 as a p53-induced gene and its ability to induce apoptosis in many cell types support the hypothesis that galectin-7 has strong antitumor activity. This has been well documented in colon cancer. However, in some cases, such as breast cancer and lymphoma, its high expression level correlates with aggressive subtypes of cancer, suggesting that galectin-7 may have a dual role in cancer progression. In fact, in breast cancer, overexpression of galectin-7 alone is sufficient to promote metastasis to the bone and lung. In the present work, we investigated the expression and function of galectin-7 in melanoma. An analysis of datasets obtained from whole-genome profiling of human melanoma tissues revealed that galectin-7 mRNA was detected in more than 90% of biopsies of patients with nevi while its expression was more rarely found in biopsies collected from patients with malignant melanoma. This frequency, however, was likely due to the presence of normal epidermis tissues in biopsies, as shown our studies at the protein level by immunohistochemical analysis. Using the experimental melanoma B16F1 cell line, we found that melanoma cells can express galectin-7 at the primary tumor site and in lung metastasis. Moreover, we found that overexpression of galectin-7 increased the resistance of melanoma cells to apoptosis while inducing de novo egr-1 expression. Overexpression of galectin-7, however, was insufficient to modulate the growth of tumors induced by the subcutaneous injection of B16F1 cells. It also failed to modulate the dissemination of B16F1 cells to the lung.

Contains fulltext : 107835.pdf (Author’s version preprint ) (Open Access) PURPOSE: Hypospadias is a common congenital malformation of the male external genitalia. Association studies for single nucleotide polymorphisms in genes encoding steroid 5alpha-reductase, estrogen receptors 1 and 2, and activating transcription factor 3 have been equivocal. We examined whether nonreplication of findings for 4 single nucleotide polymorphisms in these genes could be due to interaction with environmental exposures. MATERIALS AND METHODS: We genotyped 712 Dutch hypospadias case-parent triads for the 4 single nucleotide polymorphisms, used questionnaire information to determine exposures and performed association tests using the log-linear approach. We studied gene-environment interactions for the 4 single nucleotide polymorphisms with exposure to estrogens, cytokines or cigarette smoke, multiple birth, being born small for gestational age, maternal hypertension or preeclampsia, high body mass index or primiparity. In addition, the presence of maternal genetic and parent of origin effects was tested. RESULTS: Gene-environment interactions were identified for rs523349 in SRD5A2 with estrogen exposure and maternal hypertension or preeclampsia, as well as for rs11119982 in ATF3 with exposure to cytokines. Both single nucleotide polymorphisms seemed to influence hypospadias risk only in exposed cases. For rs6932902 in ESR1 only maternally derived alleles appeared to increase hypospadias risk in offspring. CONCLUSIONS: Interactions between genetic and environmental factors may help to explain nonreplication in genetic studies of hypospadias.

The differentiation of distinct cell types in the ventral neural tube depends on local inductive signals from the notochord. We have isolated a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh) from zebrafish and rat, termed vhh-1. vhh-1 is expressed in the node, notochord, floor plate, and posterior limb bud mesenchyme. Each of these cell groups has floor plate inducing activity, suggesting that the vhh-1 gene may encode a floor plate-inducing molecule. Widespread expression of rat vhh-1 in frog embryos leads to ectopic floor plate differentiation in the neural tube. In vitro tests for the signaling functions of vhh-1 demonstrate that COS cells expressing the rat vhh-1 gene induce floor plate and motor neuron differentiation in neural plate explants. vhh-1 may, therefore, contribute to the floor plate and motor neuron inducing activities of the notochord.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.

Metallothioneins (MTs) are small cysteine-rich proteins whose structure is conserved from fungi to man. MTs strongly bind heavy metals, notably zinc, copper and cadmium. Upon exposure of cells to heavy metal and other adverse treatments, MT gene transcription is strongly enhanced. Metal induction is mediated by several copies of a 15 bp consensus sequence (metal-responsive element, MRE) present in the promoter region of MT genes. We and others have demonstrated the presence of an MRE-binding factor in HeLa cell nuclear extracts. We found that this factor, termed MTF-1 (MRE-binding transcription factor) is inactivated/reactivated in vitro by zinc withdrawal/addition. Here we report that the amounts of MTF-1-DNA complexes are elevated several-fold in zinc-treated cells, as measured by bandshift assay. We have also cloned the cDNA of mouse MTF-1, a 72.5 kDa protein. MTF-1 contains six zinc fingers and separate transcriptional activation domains with high contents of acidic and proline residues. Ectopic expression of MTF-1 in primate or rodent cells strongly enhances transcription of a reporter gene that is driven by four consensus MREd sites, or by the complete mouse MT-I promoter, even at normal zinc levels.

Detection of osteogenic differentiation is crucial for bone tissue engineering. Despite established standard end point assays, there is increasing demand for methods allowing noninvasive kinetic differentiation monitoring. Reporter gene assays employing tissue-specific promoters and suitable reporter genes fulfill these requirements. Many promoters, however, exhibit only weak cis-activating potential, thus limiting their application to generate sensitive reporter gene assays. Therefore, the aim of this study was to design a reporter gene assay employing elements of the murine osteocalcin promoter coupled to a viral enhancer for signal amplification. Additionally, the system's practicability was enhanced by introducing a secreted luciferase as a quantifiable reporter gene. The constructs were tested in C2C12 cells stimulated with recombinant human bone morphogenetic protein 2 for osteogenic differentiation in two-dimensional and three-dimensional culture. Osteogenic differentiation was confirmed by standard assays for osteogenesis. The reporter gene signal was detected through a secreted luciferase or fluorescence microscopy for enhanced yellow fluorescent protein. The constructs exhibited strong activation upon treatment with recombinant human bone morphogenetic protein 2. Weak background expression was observable in negative controls, attributed to the pan-active viral enhancer. In conclusion, a novel enhancer/tissue-specific promoter combination allows specific signal-amplified, kinetic monitoring of osteogenic differentiation in a nonsample-destructive manner.

Aim: To investigate which phosphodiesterase (PDE) is involved in regulating cyclic 3′5′ guanosine monophosphate breakdown in retinal pigment epithelium (RPE) cells. Methods: cGMP content in the cultured RPE cells (D407 cell line) was evaluated by immunocytochemistry in the presence of non-selective or isoform-selective PDE inhibitors in combination with the particulate guanylyl cyclase stimulator atrial natriuretic peptide (ANP) or the soluble guanylyl cyclase stimulator sodium nitroprusside (SNP). mRNA expression of PDE2, PDE5 and PDE9 was studied in cultured human RPE cells and rat RPE cell layers using non-radioactive in situ hybridisation. Results: In the absence of PDE inhibitors, cGMP levels in cultured RPE cells are very low. cGMP accumulation was readily detected in cultured human RPE cells after incubation with Bay60–7550 as a selective PDE2 inhibitor, sildenafil as a selective PDE5 inhibitor or Sch51866 as a selective PDE9 inhibitor. In the presence of PDE inhibition, cGMP content increased markedly after stimulation of the particulate guanylyl cyclase. mRNA of PDE2,PDE5 and PDE9 was detected in all cultured human RPE cells and also in rat RPE cell layers. Conclusions: PDE2, PDE5 and PDE9 have a role in cGMP metabolism in RPE cells.

COMMD [copper metabolism gene MURR1 (mouse U2af1-rs1 region 1) domain] proteins constitute a recently identified family of NF-kappaB (nuclear factor kappaB)-inhibiting proteins, characterized by the presence of the COMM domain. In the present paper, we report detailed investigation of the role of this protein family, and specifically the role of the COMM domain, in NF-kappaB signalling through characterization of protein-protein interactions involving COMMD proteins. The small ubiquitously expressed COMMD6 consists primarily of the COMM domain. Therefore COMMD1 and COMMD6 were analysed further as prototype members of the COMMD protein family. Using specific antisera, interaction between endogenous COMMD1 and COMMD6 is described. This interaction was verified by independent techniques, appeared to be direct and could be detected throughout the whole cell, including the nucleus. Both proteins inhibit TNF (tumour necrosis factor)-induced NF-kappaB activation in a non-synergistic manner. Mutation of the amino acid residues Trp24 and Pro41 in the COMM domain of COMMD6 completely abolished the inhibitory effect of COMMD6 on TNF-induced NF-kappaB activation, but this was not accompanied by loss of interaction with COMMD1, COMMD6 or the NF-kappaB subunit RelA. In contrast with COMMD1, COMMD6 does not bind to IkappaBalpha (inhibitory kappaBalpha), indicating that both proteins inhibit NF-kappaB in an overlapping, but not completely similar, manner. Taken together, these data support the significance of COMMD protein-protein interactions and provide new mechanistic insight into the function of this protein family in NF-kappaB signalling.

To investigate whether high glucose (HG)-induced downregulation of connexin 43 (Cx43), a gap junction protein, alters ZO-1 and occludin expression and cell monolayer permeability.Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM) medium, high glucose (HG; 30 mM) medium, N medium transfected with Cx43 siRNA, or N medium transfected with scrambled siRNA. To determine Cx43, occludin, and ZO-1 protein expression, Western blot (WB) analysis and immunostaining were performed. Gap junction intercellular communication (GJIC) was determined using scrape load dye transfer (SLDT) assay. In parallel, cell monolayer permeability was assessed in the four groups of cells, and in cells transfected with Cx43 plasmid or dominant negative Cx43 plasmid.Connexin 43 protein expression was significantly reduced in cells grown in HG (67 ± 15% of control), and a significant reduction in Cx43 was achieved when cells grown in N medium were transfected with Cx43 siRNA (76 ± 12% of control), with concomitant decrease in GJIC activity. Cells grown in HG showed significant reduction in occludin (77 ± 9% of control) and ZO-1 (80 ± 11% of control) protein level compared with cells grown in N media. Importantly, cells transfected with Cx43 siRNA and grown in N medium showed significant downregulation in occludin (78 ± 8% of control) and ZO-1 (81 ± 6% of control) expression, and exhibited increased cell monolayer permeability. Furthermore, Cx43 upregulation protected cells against HG-induced excess cell monolayer permeability.Our findings indicate that HG-induced downregulation of Cx43 expression and GJIC may contribute to the breakdown of endothelial barrier tight junctions associated with diabetic retinopathy.

The dystrophin-glycoprotein complex, which comprises alpha- and beta-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against alpha- and beta-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane.

The mesencephalic and rhombencephalic levels of origin of the hypobranchial skeleton (lower jaw and hyoid bone) within the neural fold have been determined at the 5-somite stage with a resolution corresponding to each single rhombomere, by means of the quail-chick chimera technique. Expression of certain Hox genes (Hoxa-2, Hoxa-3 and Hoxb-4) was recorded in the branchial arches of chick and quail embryos at embryonic days 3 (E3) and E4. This was a pre-requisite for studying the regeneration capacities of the neural crest, after the dorsal neural tube was resected at the mesencephalic and rhombencephalic level. We found first that excisions at the 5-somite stage extending from the mid-mesencephalon down to r8 are followed by the regeneration of neural crest cells able to compensate for the deficiencies so produced. This confirmed the results of previous authors who made similar excisions at comparable (or older) developmental stages. When a bilateral excision was followed by the unilateral homotopic graft of the dorsal neural tube from a quail embryo, thus mimicking the situation created by a unilateral excision, we found that the migration of the grafted unilateral neural crest (quail-labelled) is bilateral and compensates massively for the missing crest derivatives. The capacity of the intermediate and ventral neural tube to yield neural crest cells was tested by removing the chick rhombencephalic neural tube and replacing it either uni- or bilaterally with a ventral tube coming from a stage-matched quail. No neural crest cells exited from the ventral neural tube but no deficiency in neural crest derivatives was recorded. Crest cells were found to regenerate from the ends of the operated region. This was demonstrated by grafting fragments of quail neural fold at the extremities of the excised territory. Quail neural crest cells were seen migrating longitudinally from both the rostral and caudal ends of the operated region and filling the branchial arches located inbetween. Comparison of the behaviour of neural crest cells in this experimental situation with that showed by their normal fate map revealed that crest cells increase their proliferation rate and change their migratory behaviour without modifying their Hox code.

The Xenopus laevis genome was screened for putative DNA-binding gene products by using the 'finger' region of the Drosophila gene Krüppel as a probe. The one gene detected, named Xfin, codes for a protein with 37 finger domains that comprise nearly 90% of the protein. In the light of studies by Rhodes and Klug (Cell, 46, 123-132, 1986), these data suggest that the Xfin protein has the capacity to bind an unusually large stretch (185 bases) of DNA. The Xfin gene is expressed as a maternal and zygotic mRNA that undergoes extensive polyadenylation changes during early development. The Xfin mRNA expression pattern and the potential DNA binding activity of the protein point to the possibility that the Xfin gene may have a role in controlling gene activity during early embryonic development.

The expression of the Xenopus homoeobox gene xhox3 is an early response to mesoderm induction by peptide growth factors and the level of xhox3 expression marks the antero-posterior character of the induced mesoderm. Different peptide growth factors specify different antero-posterior mesodermal cell fates as seen by the level of xhox3 expression and the capacity to induce specific secondary neural/epidermal structures. These factors and homoeobox genes thus form part of the mechanism necessary for establishing antero-posterior polarity in the frog embryo.

Intestinal organoids as an infection model Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes an influenza-like disease with a respiratory transmission route; however, patients often present with gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain. Moreover, the virus has been detected in anal swabs, and cells in the inner-gut lining express the receptor that SARS-CoV-2 uses to gain entry to cells. Lamers et al. used human intestinal organoids, a “mini-gut” cultured in a dish, to demonstrate that SARS-CoV-2 readily replicates in an abundant cell type in the gut lining—the enterocyte—resulting in the production of large amounts of infective virus particles in the intestine. This work demonstrates that intestinal organoids can serve as a model to understand SARS-CoV-2 biology and infectivity in the gut. Science , this issue p. 50

ObjectivesSystemic lupus erythematosus (SLE) diagnosis and treatment remain empirical and the molecular basis for its heterogeneity elusive. We explored the genomic basis for disease susceptibility and severity.MethodsmRNA sequencing and genotyping in blood from 142 patients with SLE and 58 healthy volunteers. Abundances of cell types were assessed by CIBERSORT and cell-specific effects by interaction terms in linear models. Differentially expressed genes (DEGs) were used to train classifiers (linear discriminant analysis) of SLE versus healthy individuals in 80% of the dataset and were validated in the remaining 20% running 1000 iterations. Transcriptome/genotypes were integrated by expression-quantitative trail loci (eQTL) analysis; tissue-specific genetic causality was assessed by regulatory trait concordance (RTC).ResultsSLE has a ‘susceptibility signature’ present in patients in clinical remission, an ‘activity signature’ linked to genes that regulate immune cell metabolism, protein synthesis and proliferation, and a ‘severity signature’ best illustrated in active nephritis, enriched in druggable granulocyte and plasmablast/plasma–cell pathways. Patients with SLE have also perturbed mRNA splicing enriched in immune system and interferon signalling genes. A novel transcriptome index distinguished active versus inactive disease—but not low disease activity—and correlated with disease severity. DEGs discriminate SLE versus healthy individuals with median sensitivity 86% and specificity 92% suggesting a potential use in diagnostics. Combined eQTL analysis from the Genotype Tissue Expression (GTEx) project and SLE-associated genetic polymorphisms demonstrates that susceptibility variants may regulate gene expression in the blood but also in other tissues.ConclusionSpecific gene networks confer susceptibility to SLE, activity and severity, and may facilitate personalised care.

A mouse embryonic cDNA containing two opa-like (CAX)n repeats was isolated on the basis of its cross-hybridization with a Drosophila K10 cDNA. Such repeated sequences were present in different murine mRNAs, some of which were specifically expressed during fetal life or in different adult tissues. This suggests that, as already described for Drosophila, opa-like sequences are parts of proteins involved in ontogenic or cell-type-specific functions in vertebrates. However, unlike Drosophila, such repeated sequences were not found within the murine homeo-boxes containing genes of the Hox-1 complex.