Directin vivomapping of functional suppressors in glioblastoma genome

AbstractGlioblastoma (GBM) is one of the deadliest cancers, with limited effective treatments and single-digit five-year survival1-7. A causative understanding of genetic factors that regulate GBM formation is of central importance8-19. However, a global, quantitative and functional understanding of gliomagenesis in the native brain environment has been lacking due to multiple challenges. Here, we developed an adeno-associated virus (AAV) mediated autochthonous CRISPR screen and directly mapped functional suppressors in the GBM genome. Stereotaxic delivery of an AAV library targeting significantly mutated genes into fully immunocompetent conditional Cas9 mice robustly led to gliomagenesis, resulting in tumors that recapitulate features of human GBM. Targeted capture sequencing revealed deep mutational profiles with diverse patterns across mice, uncoveringin vivoroles of previously uncharacterized factors in GBM such as immune regulatorB2m,zinc finger proteinZc3h13,transcription repressorCic,epigenetic regulatorsMll2/3andArid1b,alongside canonical tumor suppressorsNf1andPten. Comparative cancer genomics showed that the mutation frequencies across all genes tested in mice significantly correlate with those in human from two independent patient cohorts. Co-mutation analysis identified frequently co-occurring driver combinations, which were validated using AAV minipools, such asMll2, B2m-Nf1,Mll3-Nf1andZc3h13-Rb1. Distinct fromNf1-oncotype tumors,Rb1-oncotype tumors exhibit undifferentiated histopathology phenotype and aberrant activation of developmental reprogramming signatures such asHomeoboxgene clusters. The secondary addition ofZc3h13orPtenmutations drastically altered the gene expression profiles ofRb1mutants and rendered them more resistant to the GBM chemotherapeutic temozolomide. Our study provides a systematic functional landscape of GBM suppressors directlyin vivo, opening new paths for high-throughput molecular mapping and cancer phenotyping.

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Transcriptome analysis of gravitational effects on mouse skeletal muscles under microgravity and artificial 1 g onboard environment

Scientific Reports ◽

10.1038/s41598-021-88392-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Risa Okada ◽

Shin-ichiro Fujita ◽

Riku Suzuki ◽

Takuto Hayashi ◽

Hirona Tsubouchi ◽

...

Keyword(s):

Gene Expression ◽

Muscle Mass ◽

Muscle Atrophy ◽

Transcriptome Analysis ◽

Fiber Type ◽

Expression Profiles ◽

Space Station ◽

Gene Expression Profiles

AbstractSpaceflight causes a decrease in skeletal muscle mass and strength. We set two murine experimental groups in orbit for 35 days aboard the International Space Station, under artificial earth-gravity (artificial 1 g; AG) and microgravity (μg; MG), to investigate whether artificial 1 g exposure prevents muscle atrophy at the molecular level. Our main findings indicated that AG onboard environment prevented changes under microgravity in soleus muscle not only in muscle mass and fiber type composition but also in the alteration of gene expression profiles. In particular, transcriptome analysis suggested that AG condition could prevent the alterations of some atrophy-related genes. We further screened novel candidate genes to reveal the muscle atrophy mechanism from these gene expression profiles. We suggest the potential role of Cacng1 in the atrophy of myotubes using in vitro and in vivo gene transductions. This critical project may accelerate the elucidation of muscle atrophy mechanisms.

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Integrated Genomic Analysis of Sézary Syndrome

Genetics Research International ◽

10.4061/2011/980150 ◽

2011 ◽

Vol 2011 ◽

pp. 1-13 ◽

Cited By ~ 1

Author(s):

Xin Mao ◽

Tracy Chaplin ◽

Bryan D. Young

Keyword(s):

Copy Number ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Genomic Analysis ◽

Gene Clusters ◽

Sézary Syndrome ◽

Sezary Syndrome ◽

Snp Microarray ◽

The Impact ◽

Chromosome Regions

Sézary syndrome (SS) is a rare variant of primary cutaneous T-cell lymphoma. Little is known about the underlying pathogenesis of S. To address this issue, we used Affymetrix 10K SNP microarray to analyse 13 DNA samples isolated from 8 SS patients and qPCR with ABI TaqMan SNP genotyping assays for the validation of the SNP microarray results. In addition, we tested the impact of SNP loss of heterozygosity (LOH) identified in SS cases on the gene expression profiles of SS cases detected with Affymetrix GeneChip U133A. The results showed: (1) frequent SNP copy number change and LOH involving 1, 2p, 3, 4q, 5q, 6, 7p, 8, 9, 10, 11, 12q, 13, 14, 16q, 17, and 20, (2) reduced SNP copy number at FAT gene (4q35) in 75% of SS cases, and (3) the separation of all SS cases from normal control samples by SNP LOH gene clusters at chromosome regions of 9q31q34, 10p11q26, and 13q11q12. These findings provide some intriguing information for our current understanding of the molecular pathogenesis of this tumour and suggest the possibility of presence of functional SNP LOH in SS tumour cells.

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Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow

Blood ◽

10.1182/blood-2010-04-278192 ◽

2010 ◽

Vol 116 (15) ◽

pp. e66-e73 ◽

Cited By ~ 104

Author(s):

Chih-Wen Ni ◽

Haiwei Qiu ◽

Amir Rezvan ◽

Kihwan Kwon ◽

Douglas Nam ◽

...

Keyword(s):

Carotid Artery ◽

Ex Vivo ◽

Expression Profiles ◽

Quantitative Polymerase Chain Reaction ◽

Gene Expression Profiles ◽

Disturbed Flow ◽

A Genome ◽

Pattern Comparison

Abstract Recently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.

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Recapitulating tumour microenvironment in chitosan–gelatin three-dimensional scaffolds: an improved in vitro tumour model

Journal of The Royal Society Interface ◽

10.1098/rsif.2012.0564 ◽

2012 ◽

Vol 9 (77) ◽

pp. 3288-3302 ◽

Cited By ~ 29

Author(s):

Neha Arya ◽

Viren Sardana ◽

Meera Saxena ◽

Annapoorni Rangarajan ◽

Dhirendra S. Katti

Keyword(s):

Cancer Progression ◽

Expression Profiles ◽

Three Dimensional ◽

Gene Expression Profiles ◽

Petri Dish ◽

Tumour Microenvironment ◽

Two Dimensional ◽

Tumour Model

Owing to the reduced co-relationship between conventional flat Petri dish culture (two-dimensional) and the tumour microenvironment, there has been a shift towards three-dimensional culture systems that show an improved analogy to the same. In this work, an extracellular matrix (ECM)-mimicking three-dimensional scaffold based on chitosan and gelatin was fabricated and explored for its potential as a tumour model for lung cancer. It was demonstrated that the chitosan–gelatin (CG) scaffolds supported the formation of tumoroids that were similar to tumours grown in vivo for factors involved in tumour-cell–ECM interaction, invasion and metastasis, and response to anti-cancer drugs. On the other hand, the two-dimensional Petri dish surfaces did not demonstrate gene-expression profiles similar to tumours grown in vivo . Further, the three-dimensional CG scaffolds supported the formation of tumoroids, using other types of cancer cells such as breast, cervix and bone, indicating a possible wider potential for in vitro tumoroid generation. Overall, the results demonstrated that CG scaffolds can be an improved in vitro tool to study cancer progression and drug screening for solid tumours.

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Abstract 268: Strain-specific Cardiac Fibroblast Response to Isoproterenol-induced Cardiac Fibrosis

Circulation Research ◽

10.1161/res.121.suppl_1.268 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Shuin Park ◽

Sara Ranjbarvaziri ◽

Fides Lay ◽

Peng Zhao ◽

Aldons J Lusis ◽

...

Keyword(s):

Gene Expression ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Expression Profiles ◽

Inbred Mouse ◽

Gene Expression Profiles ◽

Mouse Strains ◽

Sequencing Analysis ◽

Different Strains

Fibroblasts are a heterogeneous population of cells that function within the injury response mechanisms across various tissues. Despite their importance in pathophysiology, the effects of different genetic backgrounds on fibroblast contribution to the development of disease has yet to be addressed. It has previously been shown that mice in the Hybrid Mouse Diversity Panel, which consists of 110 inbred mouse strains, display a spectrum in severity of cardiac fibrosis in response to chronic treatment of isoproterenol (ISO). Here, we characterized cardiac fibroblasts (CFbs) from three different mouse strains (C57BL/6J, C3H/HeJ, and KK/HIJ) which exhibited varying degrees of fibrosis after ISO treatment. The select strains of mice underwent sham or ISO treatment via intraperitoneally-implanted osmotic pumps for 21 days. Masson’s Trichrome staining showed significant differences in fibrosis in response to ISO, with KK/HIJ mice demonstrating the highest levels, C3H/HeJ exhibiting milder levels, and C57BL/6J demonstrating little to no fibrosis. When CFbs were isolated and cultured from each strain, the cells demonstrated similar traits at the basal level but responded to ISO stimuli in a strain-specific manner. Likewise, CFbs demonstrated differential behavior and gene expression in vivo in response to ISO. ISO treatment caused CFbs to proliferate similarly across all strains, however, immunofluorescence staining showed differential levels of CFb activation. Additionally, RNA-sequencing analysis revealed unique gene expression profiles of all three strains upon ISO treatment. Our study depicts the phenotypic heterogeneity of CFbs across different strains of mice and our results suggest that ISO-induced cardiac fibrosis is a complex process that is independent of fibroblast proliferation and is mainly driven by the activation/inhibition of genes involved in pro-fibrotic pathways.

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Genomic Fabric Remodeling in Prostate Cancer

10.20944/preprints202103.0242.v1 ◽

2021 ◽

Author(s):

Sanda Iacobas ◽

Dumitru A. Iacobas

Keyword(s):

Prostate Cancer ◽

Normal Tissue ◽

Cancer Genomics ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Mathematical Object ◽

P53 Signaling ◽

Large Populations ◽

Genomic Characterization ◽

Meta Analyses

Prostate cancer is a leading cause of death among men but its genomic characterization and best therapeutic strategy are still under debate. The Genomic Fabric Paradigm (GFP) considers the transcriptome as a multi-dimensional mathematical object subjected to a dynamic set of expression correlations among the genes. Here, GFP is applied to gene expression profiles of three (one primary, and two secondary) cancer nodules and the surrounding normal tissue from a surgically removed prostate tumor. GFP was used to determine the regulation and rewiring of the P53 signaling, apoptosis, prostate cancer and several other pathways involved in survival and proliferation of the cancer cells. Genes responsible for the block of differentiation, evading apoptosis, immortality, insensitivity to anti-growth signals, proliferation, resistance to chemotherapy and sustained angiogenesis were found as differently regulated in the three cancer nodules with respect to the normal tissue. The analysis indicates that even histo-pathologically equally graded cancer nodules from the same tumor have substantially different transcriptomic organizations, raising legitimate questions about the validity of meta-analyses comparing large populations of healthy and cancer humans. The study suggests that GFP may provide a personalized alternative to the biomarkers’ approach of cancer genomics.

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TMOD-21. TARGETING ANAPLASTIC MENINGIOMA WITH PANOBINOSTAT IN PATIENT-DERIVED ORTHOTOPIC XENOGRAFT (PDOX) MODELS DERIVED FROM A MATCHING PAIR OF PRIMARY AND RECURRENT TUMORS

Neuro-Oncology ◽

10.1093/neuonc/noz175.1120 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi267-vi267

Author(s):

Huiyuan Zhang ◽

Lin Qi ◽

Yuchen Du ◽

Frank Braun ◽

Mari Kogiso ◽

...

Keyword(s):

Skull Base ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Small Animal ◽

Tumor Formation ◽

Anaplastic Meningioma ◽

Culture Cells ◽

Pathologic Features ◽

Two Samples

Abstract BACKGROUND Meningioma is the most common brain tumor in adults. Despite the overall benign nature of meningioma, skull base tumors can be difficult to completely resect while others exhibit progression and aggressive profiles. The lack of clinically relevant animal models is blocking the development of novel therapies. MATERIAL AND METHODS Twelve surgical specimens (1 × 105) from 11 adult meningioma patients were implanted into the frontal cranial-base of the brain of SCID mice. Mice were then followed and assessed for tumor formation. Tumor growth was confirmed by small animal MRI. Pathologic features of the PDOX models and the matched patient tumors were compared with standard H&E and immunohistochemical staining. RNAseq was performed to examine the molecular fidelity of PDOX tumors and to identify new therapeutic targets. A panel of 60 clinically-relevant drugs was developed for screening drug sensitivity. In vivo examination of therapeutic efficacy of Panobinostat was performed in two models by treating preformed PDOX tumors with i.p. injection (10 mg/kg), 5 days on, 5 days off for 2 cycles. RESULTS Intracranial xenograft formation was confirmed in two samples derived from the same patient, the first an atypical meningioma (K029MEN-P) and the second, which progressed to anaplastic meningioma at recurrence (K029MEN-R). MRI scanning revealed that the PDOX tumors grew from the skull base. These patient tumor cells can be cryopreserved for long-term maintenance of tumorigenicity. The xenograft tumors replicated histopathological features of parental tumors. Overall gene expression profiles of PDOX were similar to the original patient tumors. Using MEN primary culture cells, we screened 60 drugs and identified 12 (20%) active compounds. Panobinostat also significantly prolonged survival of mice bearing orthotopic meningiomas. CONCLUSION A set of meningioma PDOX models derived from primary and recurrent tumor was established. Our data further demonstrate that panobinostat exerts potent antitumor activity against high-grade meningioma.

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Major Latex Protein MdMLP423 Negatively Regulates Defense against Fungal Infections in Apple

International Journal of Molecular Sciences ◽

10.3390/ijms21051879 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1879 ◽

Cited By ~ 1

Author(s):

Shanshan He ◽

Gaopeng Yuan ◽

Shuxun Bian ◽

Xiaolei Han ◽

Kai Liu ◽

...

Keyword(s):

Transcription Factors ◽

Stress Responses ◽

Zinc Finger Protein ◽

Fungal Infections ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Genes Encoding ◽

Pathogenesis Related Gene ◽

Stress Related Genes ◽

Related Proteins

Major latex proteins (MLPs) play critical roles in plants defense and stress responses. However, the roles of MLPs from apple (Malus × domestica) have not been clearly identified. In this study, we focused on the biological role of MdMLP423, which had been previously characterized as a potential pathogenesis-related gene. Phylogenetic analysis and conserved domain analysis indicated that MdMLP423 is a protein with a ‘Gly-rich loop’ (GXGGXG) domain belonging to the Bet v_1 subfamily. Gene expression profiles showed that MdMLP423 is mainly expressed in flowers. In addition, the expression of MdMLP423 was significantly inhibited by Botryosphaeria berengeriana f. sp. piricola (BB) and Alternaria alternata apple pathotype (AAAP) infections. Apple calli overexpressing MdMLP423 had lower expression of resistance-related genes, and were more sensitive to infection with BB and AAAP compared with non-transgenic calli. RNA-seq analysis of MdMLP423-overexpressing calli and non-transgenic calli indicated that MdMLP423 regulated the expression of a number of differentially expressed genes (DEGs) and transcription factors, including genes involved in phytohormone signaling pathways, cell wall reinforcement, and genes encoding the defense-related proteins, AP2-EREBP, WRKY, MYB, NAC, Zinc finger protein, and ABI3. Taken together, our results demonstrate that MdMLP423 negatively regulates apple resistance to BB and AAAP infections by inhibiting the expression of defense- and stress-related genes and transcription factors.

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