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This file contains Tables S1-S3. Table S1 shows number of injected mice and tumor incidence. Table S2 shows genes and fold changes of commonly up and down regulated genes in mouse Sox5/6/21fl/fl tumors and human GBM. Table S3 shows Sox21 ChIPseq peaks association to nearest gene.

This file contains Figures S1-S6. Figure S1 shows proliferation of neural stem cells after in utero electroporation of Sox5, Sox6 or Sox21, and the successful activation of viral vector expression by CRE enzyme. Figure S2 shows reduction of SOX5/6/21 levels after intracranial ARC injection. H

Molecular mechanisms preventing self-renewing brain stem cells from oncogenic transformation are poorly defined. We show that the expression levels of SOX5, SOX6, and SOX21 (SOX5/6/21) transcription factors increase in stem cells of the subventricular zone (SVZ) upon oncogenic stress, whereas their expression in human glioma decreases during malignant progression. Elevated levels of SOX5/6/21 promoted SVZ cells to exit the cell cycle, whereas genetic ablation of SOX5/6/21 dramatically increased the capacity of these cells to form glioma-like tumors in an oncogene-driven mouse brain tumor model. Loss-of-function experiments revealed that SOX5/6/21 prevent detrimental hyperproliferation of oncogene expressing SVZ cells by facilitating an antiproliferative expression profile. Consistently, restoring high levels of SOX5/6/21 in human primary glioblastoma cells enabled expression of CDK inhibitors and decreased p53 protein turnover, which blocked their tumorigenic capacity through cellular senescence and apoptosis. Altogether, these results provide evidence that SOX5/6/21 play a central role in driving a tumor suppressor response in brain stem cells upon oncogenic insult. Cancer Res; 77(18); 4985–97. ©2017 AACR.

This file contains additional description of western blot analysis, immunohistochemistry, antibodies, Sox21 targeting in mouse, bioinformatics, in utero electroporation, proliferation, senescence and cell death assays, NPC cultures and assays, and qRT-PCR.

Hydroconversion of 5-hydroxymethylfurfural (HMF) to 2,5-bis(hydoxymethyl)furan (BHMF) was studied over mono- and bimetallic supported catalysts. It was found that monometallic Ni/SiO2 catalysts exhibited superior performance with a total yield of BHMF of up to 99 wt%. This excellent performance may be attributed to higher Ni dispersion and low acidity of the support. Dimethyl sulfoxide (DMSO) is often present in HMF, due to the route used for its synthesis. DMSO adsorption caused a clear reduction of Ni/SiO2 performance for the HMF hydrodeoxygenation reaction. Characterization of the spent catalysts was performed using HAADF-STEM-EDX, Raman, ICP, and XPS spectroscopies, and showed the presence of sulfur and graphitic carbon, which could explain the deactivation.

How neurons in the CNS can maintain cellular identity over an entire lifespan remains largely unknown. Here we show that long-term maintenance of identity in differentiated dopaminergic and serotonergic neurons is critically reliant on the Polycomb repressive complex 2 (PRC2). Deletion of the obligate PRC2-component, Eed, in these neurons, resulted in global loss of H3K27me3, followed by a gradual activation of genes harbouring both H3K27me3 and H3K9me3 modifications. Notably, H3K9me3 was also lost at these PRC2-targets prior to gene activation. Neuronal survival was not compromised, instead there was a reduction in subtype specific gene expression as well as a progressive impairment of dopaminergic or serotonergic neuronal function leading to behavioural deficits characteristic of Parkinson’s disease (PD) or mood disorders, respectively. Single cell analysis revealed an unexpected subtype specific vulnerability to loss of PRC2-repression in dopamine neurons of the substantia nigra, the neurons primarily affected in PD. Taken together, our study reveals that a PRC2-dependent non-permissive chromatin state is essential to maintain subtype identity and function of dopaminergic and serotonergic neurons.

Background & Aims YAP (Yap1) and TAZ (Wwtr1) are transcriptional co-activators and downstream effectors of the Hippo pathway, which play crucial roles in organ size control and cancer pathogenesis. Genetic deletion of YAP/TAZ has shown their critical importance for embryonic development of the heart, vasculature, and gastrointestinal mesenchyme. The aim of this study was to determine the functional role of YAP/TAZ in adult smooth muscle cells in vivo. Methods Because YAP and TAZ are mutually redundant, we used YAP/TAZ double-floxed mice crossed with mice that express tamoxifen-inducible CreERT2 recombinase driven by the smooth muscle–specific myosin heavy chain promoter. Results Double-knockout of YAP/TAZ in adult smooth muscle causes lethality within 2 weeks, mainly owing to colonic pseudo-obstruction, characterized by severe distension and fecal impaction. RNA sequencing in colon and urinary bladder showed that smooth muscle markers and muscarinic receptors were down-regulated in the YAP/TAZ knockout. The same transcripts also correlated with YAP/TAZ in the human colon. Myograph experiments showed reduced contractility to depolarization by potassium chloride and a nearly abolished muscarinic contraction and spontaneous activity in colon rings of YAP/TAZ knockout. Conclusions YAP and TAZ in smooth muscle are guardians of colonic contractility and control expression of contractile proteins and muscarinic receptors. The knockout model has features of human chronic intestinal pseudo-obstruction and may be useful for studying this disease., Graphical abstract

Hypertension remains a major risk factor for cardiovascular diseases, but the underlying mechanisms are not well understood. We hypothesize that appropriate mechanotransduction and contractile function in vascular smooth muscle cells are crucial to maintain vascular wall integrity. The Hippo pathway effectors YAP (yes-associated protein 1) and TAZ (WW domain containing transcription regulator 1) have been identified as mechanosensitive transcriptional coactivators. However, their role in vascular smooth muscle cell mechanotransduction has not been investigated in vivo.We performed physiological and molecular analyses utilizing an inducible smooth muscle-specific YAP/TAZ knockout mouse model.Arteries lacking YAP/TAZ have reduced agonist-mediated contraction, decreased myogenic response, and attenuated stretch-induced transcriptional regulation of smooth muscle markers. Moreover, in established hypertension, YAP/TAZ knockout results in severe vascular lesions in small mesenteric arteries characterized by neointimal hyperplasia, elastin degradation, and adventitial thickening.This study demonstrates a protective role of YAP/TAZ against hypertensive vasculopathy.

Glioblastoma (GBM) is an aggressive nervous system tumor with a mean survival time of 12-14 months. Cells with neural stem cell-like properties can be derived from GBM tumors. These cells seem to escape conventional irradiation treatment, chemotherapy, and surgery, and may play a crucial role for relapse. It is therefore urgent to develop novel approaches for reliable detection of neural stem cell-like cells in GBM. Here we report a luminescent conjugated oligothiophene (LCO), named GlioStem (p-HTMI), for non-invasive and non-amplified real-time detection of live human patient-derived GBM cells and embryonic neural stem/progenitor cells (NSPCs). Within a maximum of 10 minutes after administration of the molecule in vitro, in the existing media, fluorescence emission was observed without any modulation of the cells or additional vehicle, resulting in efficient detection of cytoplasmic luminescent signal in NSPCs or GBM cells from rodents and humans, detectable at Alexa488/GFP wavelength. GlioStem is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. In vitro, GlioStem was shown to identify fetal cortical NSPCs from rat (FGF2-expanded), embryonic stem cell-derived NSPCs from mouse (FGF2/EGF-expanded), and FGF2-exposed C6 glioma cell cultures from rat, but not any other cell types investigated. Cell sorting experiments of patient-derived, FGF2/EGF-expanded GBM cells demonstrated that GlioStem in addition to NSPC-markers like Nestin and Sox2 labeled the same population (overlap > 90%) of cells as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO GlioStem is a versatile tool for immediate and selective detection of subpopulations of neural stem and glioma cells.

Childhood neuroblastoma has a remarkable variability in outcome. Age at diagnosis is one of the most important prognostic factors, with children less than 1 year old having favorable outcomes. Here we study single-cell and single-nuclei transcriptomes of neuroblastoma with different clinical risk groups and stages, including healthy adrenal gland. We compare tumor cell populations with embryonic mouse sympatho-adrenal derivatives, and post-natal human adrenal gland. We provide evidence that low and high-risk neuroblastoma have different cell identities, representing two disease entities. Low-risk neuroblastoma presents a transcriptome that resembles sympatho- and chromaffin cells, whereas malignant cells enriched in high-risk neuroblastoma resembles a subtype of TRKB+ cholinergic progenitor population identified in human post-natal gland. Analyses of these populations reveal different gene expression programs for worst and better survival in correlation with age at diagnosis. Our findings reveal two cellular identities and a composition of human neuroblastoma tumors reflecting clinical heterogeneity and outcome., Childhood neuroblastoma can be separated into high and low risk groups, with prognosis depending on age at diagnosis. Here, the authors show that low and high risk neuroblastoma tumours are composed of different cell types with different malignancy potential.

Background and aims Symptoms of dizziness and pain are both common complaints and the two symptoms often seem to coincide. When symptoms appear concomitant for sustained periods of time the symptoms might maintain and even exacerbate each other, sometimes leading to psychological distress. In order to evaluate such comorbidity we studied patients referred to a vestibular unit and to a psychiatric outpatient clinic with respectively balance disorders and psychological issues. Methods Consecutive patients referred to a vestibular unit (n = 49) and a psychiatric outpatient clinic (n = 62) answered the Dizziness Handicap Inventory (DHI) questionnaire and a questionnaire detailing occurrence of dizziness and pain. Results The experience of dizziness and pain often coincided within individuals across both clinical populations, especially if the pain was located to the neck/shoulder or the back (p = 0.006). Patients who reported dizziness had significantly more often pain (p = 0.024); in the head (p = 0.002), neck/shoulders (p = 0.003) and feet (p = 0.043). Moreover, patients who reported dizziness stated significantly higher scoring on emotional (p p p = 0.039) and pain (p p p p = 0.045) and they scored higher on the emotional (p = 0.004) and functional (p = 0.002) DHI sub-scales. Conclusions The findings suggest comorbidity to exist between dizziness and neck/shoulder or back pain in patients seeking health care for balance disorders or psychological issues. Patients suffering from dizziness and pain, or with both symptoms, also reported higher emotional and functional strain. Thus, healthcare professionals should consider comorbidity when determining diagnosis and consequent measures. Implications Clinicians need to have a broader “receptive scope” in both history and clinical examinations, and ask for all symptoms. Although the patients in this study visited a vestibular unit respectively a psychological clinic, they commonly reported pain conditions when explicitly asked for this symptom. A multimodal approach is thus to favor, especially when the symptoms persist, for the best clinical management.

The Work-related Acceptance and Action Questionnaire (WAAQ) has been developed to assess psychological flexibility in occupational settings. The aim of this study was to evaluate reliability and validity of the Swedish translation of WAAQ in a sample of 184 healthcare professionals. A principal component analysis supported a one-factor-solution, explaining 53.8% of the overall multivariate variability. WAAQ showed good internal consistency (Cronbach's alpha 0.85) and good test-retest reliability (ICC = 0.85). Validity was assessed by analysing the relationship between WAAQ and other work-related constructs. Spearman's rank correlations illustrated a significant negative relationship between WAAQ and perceived stress and a significant positive correlation with work engagement. Results from the study are consistent with previous studies on WAAQ, supporting the use of the Swedish translation of the instrument among healthcare professionals.

Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel–Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel–Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.

Endogenous retroviruses (ERVs) make up a large fraction of mammalian genomes and are thought to contribute to human disease, including brain disorders. In the brain, aberrant activation of ERVs is a potential trigger for an inflammatory response, but mechanistic insight into this phenomenon remains lacking. Using CRISPR/Cas9-based gene disruption of the epigenetic co-repressor protein Trim28, we found a dynamic H3K9me3-dependent regulation of ERVs in proliferating neural progenitor cells (NPCs), but not in adult neurons. In vivo deletion of Trim28 in cortical NPCs during mouse brain development resulted in viable offspring expressing high levels of ERVs in excitatory neurons in the adult brain. Neuronal ERV expression was linked to activated microglia and the presence of ERV-derived proteins in aggregate-like structures. This study demonstrates that brain development is a critical period for the silencing of ERVs and provides causal in vivo evidence demonstrating that transcriptional activation of ERV in neurons results in an inflammatory response.

Endogenous retroviruses (ERVs) make up a large fraction of mammalian genomes and are thought to contribute to human disease, including brain disorders. In the brain, aberrant activation of ERVs is a potential trigger for neuroinflammation, but mechanistic insight into this phenomenon remains lacking. Using CRISPR/Cas9-based gene disruption of the epigenetic co-repressor protein Trim28, we found a dynamic H3K9me3-dependent regulation of ERVs in proliferating neural progenitor cells (NPCs), but not in adult neurons.In vivodeletion ofTrim28in cortical NPCs during mouse brain development resulted in viable offspring expressing high levels of ERV expression in excitatory neurons in the adult brain. Neuronal ERV expression was linked to inflammation, including activated microglia, and aggregates of ERV-derived proteins. This study demonstrates that brain development is a critical period for the silencing of ERVs and provides causalin vivoevidence demonstrating that transcriptional activation of ERV in neurons results in neuroinflammation.

The hypoxia inducible transcription factor EPAS1/HIF2α has been described as an oncogene and a potential therapeutic target in neuroblastoma. Our analysis of several neuroblastoma tumour expression datasets does not support an oncogenic role, instead EPAS1 expression is associated with better patient outcome and characteristics of low-risk tumours. Treatment with HIF2α inhibitors did not block in vitro neuroblastoma cell proliferation nor xenograft growth. In addition, we analysed single cell sequencing data sets from the developing mouse sympathoadrenal lineage, wherein expression of Epas1 was a strong predictor of differentiated adrenal chromaffin cells and negatively correlated with progenitor characteristics. This was reflected in neuroblastoma tumours wherein genes co-expressed with Epas1 during sympathoadrenal development strongly predicts favourable patient outcome and features of low-risk tumours. Thus, our analysis suggest that with the current available data EPAS1/HIF2α should not be classified as a neuroblastoma oncogene and is less likely to represent a suitable drug target in this disease.

This JAMA Guide to Statistics and Methods describes collider bias, illustrates examples in directed acyclic graphs, and explains how it can threaten the internal validity of a study and the accurate estimation of causal relationships in randomized clinical trials and observational studies.

Nexilin, encoded by the NEXN gene, is expressed in striated muscle and localizes to Z-discs, influencing mechanical stability. We examined Nexilin/NEXN in smooth muscle cells (SMCs), and addressed if Nexilin localizes to dense bodies and dense bands and whether it is regulated by actin-controlled coactivators from the MRTF (MYOCD, MKL1, MKL2) and YAP/TAZ (YAP1 and WWTR1) families. NEXN expression in SMCs was comparable to that in striated muscles. Immunofluorescence and immunoelectron microscopy suggested that Nexilin localizes to dense bodies and dense bands. Correlations at the mRNA level suggested that NEXN expression might be controlled by actin polymerization. Depolymerization of actin using Latrunculin B repressed the NEXN mRNA and protein in bladder and coronary artery SMCs. Overexpression and knockdown supported involvement of both YAP/TAZ and MRTFs in the transcriptional control of NEXN. YAP/TAZ and MRTFs appeared equally important in bladder SMCs, whereas MRTFs dominated in vascular SMCs. Expression of NEXN was moreover reduced in situations of SMC phenotypic modulation in vivo. The proximal promoter of NEXN conferred control by MRTF-A/MKL1 and MYOCD. NEXN silencing reduced actin polymerization and cell migration, as well as SMC marker expression. NEXN targeting by actin-controlled coactivators thus amplifies SMC differentiation through the actin cytoskeleton, probably via dense bodies and dense bands.

Glioblastoma (GBM) is regarded as the most common malignant brain tumor but treatment options are limited. Thus, there is an unmet clinical need for compounds and corresponding targets that could inhibit GBM growth. We screened a library of 80 dopaminergic ligands with the aim of identifying compounds capable of inhibiting GBM cell line proliferation and survival. Out of 45 active compounds, 8 were further validated. We found that the dopamine receptor D2 antagonist trifluoperazine 2HCl inhibits growth and proliferation of GBM cells in a dose dependent manner. Trifluoperazine's inhibition of GBM cells is cell line dependent and correlates with variations in dopamine receptor expression profile. We conclude that components of the dopamine receptor signaling pathways are potential targets for pharmacological interventions of GBM growth.