A patient specific point mutation (c.1288G>T) of Men1 gene was introduced into wide type iPSC line with CRISPR/Cas9 and single-stranded donor oligonucleotides carrying the mutation. The mutated iPSC line has a heterozygous c.1288G>T mutation on exon-9 of Men1 that was confirmed by sequencing analysis. The karyotype of this line was normal and the pluripotency was demonstrated by its ability to differentiate into three germ layers. These artificially created Men1 mutation in wild type iPSC line will help to dissect out the molecular basis of two patients carried the same mutation from one family who were differentially represented hypoglycemia.
Urine resource cells were collected from a 59-year-old female patient with multiple endocrine neoplasia type 1 syndrome (MEN1) for generating iPS cells with episomal plasmids carrying Oct4, Sox2, Klf4 and miR-302-367. The patient sustained a heterozygous G>T transition mutation on the exon 9 of Men1 gene that was confirmed by sequencing analysis on the obtained iPSC lines. Karyotyping indicated the chromosomes with normal appearances and numbers. Their pluripotency was demonstrated by gene expression, as well as their abilities for differentiating into three germ layers. This cell line provides an ideal model for studying MEN1.
The gene of ATP-binding cassette subfamily C member 8 (Abcc8) is cytogenetically located at 11p15.1 and encodes the sulfonylurea receptor (SUR1). SUR1 is a subunit of ATP-sensitive potassium channel (KAPT) in the β-cell regulating insulin secretion. Mutations of ABCC8 are responsible for congenital hyperinsulinism (CHI). Here we reported that an Abcc8 heterozygous mutant cell line was generated by CRISPR/Cas9 technique with 1 bp insertion resulting in abnormal splicing on human embryonic stem cell line H1. The phenotypic characteristics of this cell line reveal defective KATP channel and diazoxide-responsive that provides ideal model for molecular pathology research and drug screening for CHI.
EPN-30. YAP1-MAMLD1 FUSIONS ALONE ARE SUFFICIENT TO FORM SUPRATENTORIAL EPENDYMOMA-LIKE TUMORS IN MICE Kristian W. Pajtler1,2, Sebastian Brabetz1, Monika Mauermann1, Norman Mack1, Laura Sieber1, David T. W. Jones1, Hendrik Witt1,2, Huiqin Korkel-Qu3, Marc Zuckermann4, Jan Gronych4, Andrey Korshunov5,6, David Capper5,6, Hai-Kun Liu3, Stefan M. Pfister1,2, Marcel Kool1, and Daisuke Kawauchi1; German Cancer Research Center, Division of Pediatric Neurooncology, Heidelberg, Germany; University Hospital Heidelberg, Department of Pediatric Oncology, Hematology and Immunology, Heidelberg, Germany; German Cancer Research Center, Molecular Neurogenetics, Heidelberg, Germany; German Cancer Research Center, Molecular Genetics, Heidelberg, Germany; GermanCancer Research Center, Clinical Cooperation Unit Neuropathology, Heidelberg, Germany; University Hospital Heidelberg, Department of Neuropathology, Heidelberg, Germany Oncogenic fusions containing RELA orYAP1have recentlybeen identified as genetic hallmarks of distinct molecular subgroups of supratentorial ependymomas (ST-EPN), designated ST-EPN-RELA and ST-EPN-YAP1, respectively. ST-EPN-YAP1 tumors, exclusively found in pediatric patients, are molecularly andclinicallydifferentfromST-EPN-RELAtumors, suggesting that theyhave to be treated differently. YAP1 acts as a transcriptional regulator in the HIPPO tumor suppressor pathway. The lack of adequate models for ST-EPN-YAP1 has so far hindered efforts to develop effective targeted therapies for these tumors. In an attempt to model this subgroup, the most frequent fusion type, YAP1-MAMLD1, was constructed and cloned upstream of IRES-Luciferase into the pT2K transposable vector. The resulting vector was injected together with the Tol2 transposase into the lateral ventricle of E13.5 wildtype mouse embryos followed by transfection using an electroporation-based in vivo gene transferapproach.Afterbirth,YAP1-MAMLD1-expressing tumors,monitored using luciferase-based in vivo bioluminescence imaging, developed rapidly with 100% penetrance. The animals had to be sacrificed due to severe neurological symptoms on average at P20. Resulting tumors resembled molecular characteristics of their human counterparts. The YAP1-MAMLD1 protein was predominantly compartmentalized to the nuclei of tumor cells even when phosphorylated at serine residue 127, which normally retains YAP1 in the cytoplasm, implying constitutive activation of the YAP1 fusion protein. Conserved tumorigenic potential of transformed cells was confirmed by subsequent orthotopic transplantation of these tumor cells into immunocompromised recipient mice. We thus provide a novel model for ST-EPN-YAP1, which is currently entering first preclinical drug testings. Neuro-Oncology 18:iii30–iii39, 2016. doi:10.1093/neuonc/now070.29 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Micro Deformation Monitoring Radar has been widely used in the field of surface deformation and displacement monitoring.However, limited by radar imaging geometry, the deformation measurement by existing radar technology can only extract the deformation and displacement of the target line of sight (LoS), which cannot directly reflect the actual deformation and displacement of the landslide direction and easily results in misjudgment or omission of the surface deformation monitoring information.In this paper, the relationship model and mapping between radar data and three-dimensional coordinate system were analyzed to perform three-dimensional analysis of the LoS displacement.Combined with the landslide displacement direction, the mapping angle between the LoS direction at any point in the observation area and the landslide direction was solved, and then the deformation displacement in the landslide direction was obtained by solving the LoS direction displacement.Finally, taking the measured data of one slope as the research object, the feasibility and accuracy of the method were analyzed and verified.The conclusion shows that the method proposed in this paper can be effectively applied to calculate the true value of landslide deformation. INTRODUCTIONChina is a country with frequent landslide disasters, which poses a serious threat to human life and may cause serious losses in property, infrastructure, and environmental degradation.For example, on March 15, 2019, a landslide occurred in Xiangning County, Linfen City, Shanxi Province, resulting in 20 deaths and 13 injuries, and a direct economic loss of 7.135 million yuan.According to the statistical data released by the Geological Disaster Technical Guidance Center of the Ministry of Natural Resources, there were 6181 geological disasters in 2019, including 4220 landslides, accounting for 68.27% of the total number of geological disasters [1,2].The early prevention, monitoring, and prediction of landslides have always been a common concern.At present, the calculation of deformation displacement in the direction of landslide mainly depends on the single-point high-precision measurement method.However, this method must be deployed to the site, and the network must be selected.It is difficult to meet the monitoring requirements for the personnel who cannot set foot in the wide area and large area.Especially when the calculation of deformation displacement needs to estimate the landslide direction, it is unable to effectively grasp the macroscopic trend of surface deformation [3][4][5].The micro-variable monitoring radar benefits from its all-weather imaging ability and has the advantages of wide observation area, high accuracy, high resolution, and areas that cannot be accessed by non-contact monitoring personnel.It effectively makes up for the shortcomings of traditional monitoring
Mutations in chromatin modifier genes are frequently associated with neurodevelopmental diseases. We herein demonstrate that the chromodomain helicase DNA-binding protein 7 (Chd7), frequently associated with CHARGE syndrome, is indispensable for normal cerebellar development. Genetic inactivation of Chd7 in cerebellar granule neuron progenitors leads to cerebellar hypoplasia in mice, due to the impairment of granule neuron differentiation, induction of apoptosis and abnormal localization of Purkinje cells, which closely recapitulates known clinical features in the cerebella of CHARGE patients. Combinatory molecular analyses reveal that Chd7 is required for the maintenance of open chromatin and thus activation of genes essential for granule neuron differentiation. We further demonstrate that both Chd7 and Top2b are necessary for the transcription of a set of long neuronal genes in cerebellar granule neurons. Altogether, our comprehensive analyses reveal a mechanism with chromatin remodellers governing brain development via controlling a core transcriptional programme for cell-specific differentiation.
Abstract A network of communicating tumour cells established by tumour microtubes (TMs) is supposed to mediate relevant aspects of progression and resistance of incurable gliomas. Moreover, neuronal activity has been shown to foster malignant behavior of glioma cells by non-synaptic paracrine and autocrine mechanisms. Here, we report an unexpected direct communication channel between neurons and glioma cells in multiple disease models as well as in astrocytomas and glioblastomas (GBs) of adult patients: functional bona fide chemical synapses formed between presynaptic neurons and postsynaptic glioma cells. These neurogliomal synapses (NGS) show a typical synaptic ultrastructure, are located on TM networks, and produce depolarizing postsynaptic currents mediated by glutamate receptors of the AMPA subtype. AMPA-type glutamate receptors (AMPAR) are expressed by a molecularly and morphologically distinct subpopulation of network-integrated glioma cells. Increased neuronal activity under epileptic conditions ex vivo or neuronal optogenetic stimulation in vivo enhanced, while general anesthesia diminished synchronized calcium transients in TM-connected glioma networks. Accordingly, anesthesia reduced invasiveness of TM-positive tumour cells in mice. Genetic perturbation of AMPAR or chronic AMPAR inhibition by perampanel decreased glioma invasion and proliferation in mice and deletion of GluRII in Drosophila glioma increased survival. These findings reveal a hitherto unappreciated direct synaptic communication between neurons and glioma cells that appears relevant for brain tumour biology, implying new avenues for glioma treatment.
Abstract Recent advances in the genomics of glioblastoma (GBM) led to the introduction of molecular neuropathology but failed to translate into treatment improvement. This is largely attributed to the genetic and phenotypic heterogeneity of GBM, which are considered the major obstacle to GBM therapy. Here, we use advanced human GBM-like organoid (LEGO: L aboratory E ngineered G lioblastoma-like O rganoid) models and provide an unprecedented comprehensive characterization of LEGO models using single-cell transcriptome, DNA methylome, metabolome, lipidome, proteome, and phospho-proteome analysis. We discovered that genetic heterogeneity dictates functional heterogeneity across molecular layers and demonstrates that NF1 mutation drives mesenchymal signature. Most importantly, we found that glycerol lipid reprogramming is a hallmark of GBM, and several targets and drugs were discovered along this line. We also provide a genotype-based drug reference map using LEGO-based drug screen. This study provides new human GBM models and a research path toward effective GBM therapy.
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