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We identify a population of Protogenin-positive (PRTG+ve) MYChigh NESTINlow stem cells in the four-week-old human embryonic hindbrain that subsequently localizes to the ventricular zone of the rhombic lip (RLVZ). Oncogenic transformation of early Prtg+ve rhombic lip stem cells initiates group 3 medulloblastoma (Gr3-MB)-like tumors. PRTG+ve stem cells grow adjacent to a human-specific interposed vascular plexus in the RLVZ, a phenotype that is recapitulated in Gr3-MB but not in other types of medulloblastoma. Co-culture of Gr3-MB with endothelial cells promotes tumor stem cell growth, with the endothelial cells adopting an immature phenotype. Targeting the PRTGhigh compartment of Gr3-MB in vivo using either the diphtheria toxin system or chimeric antigen receptor T cells constitutes effective therapy. Human Gr3-MBs likely arise from early embryonic RLVZ PRTG+ve stem cells inhabiting a specific perivascular niche. Targeting the PRTGhigh compartment and/or the perivascular niche represents an approach to treat children with Gr3-MB.
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Meningiomas are the most common primary intracranial tumour in adults1. Patients with symptoms are generally treated with surgery as there are no effective medical therapies. The World Health Organization histopathological grade of the tumour and the extent of resection at surgery (Simpson grade) are associated with the recurrence of disease; however, they do not accurately reflect the clinical behaviour of all meningiomas2. Molecular classifications of meningioma that reliably reflect tumour behaviour and inform on therapies are required. Here we introduce four consensus molecular groups of meningioma by combining DNA somatic copy-number aberrations, DNA somatic point mutations, DNA methylation and messenger RNA abundance in a unified analysis. These molecular groups more accurately predicted clinical outcomes compared with existing classification schemes. Each molecular group showed distinctive and prototypical biology (immunogenic, benign NF2 wild-type, hypermetabolic and proliferative) that informed therapeutic options. Proteogenomic characterization reinforced the robustness of the newly defined molecular groups and uncovered highly abundant and group-specific protein targets that we validated using immunohistochemistry. Single-cell RNA sequencing revealed inter-individual variations in meningioma as well as variations in intrinsic expression programs in neoplastic cells that mirrored the biology of the molecular groups identified.
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Biomarcadores de Tumor/metabolismo , Meningioma/clasificación , Meningioma/metabolismo , Proteogenómica , Metilación de ADN , Análisis de Datos , Descubrimiento de Drogas , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Meningioma/tratamiento farmacológico , Meningioma/genética , Mutación , RNA-Seq , Reproducibilidad de los Resultados , Análisis de la Célula IndividualRESUMEN
Mutations in the pivotal metabolic isocitrate dehydrogenase (IDH) enzymes are recognized to drive the molecular footprint of diffuse gliomas, and patients with IDH mutant gliomas have overall favorable outcomes compared to patients with IDH wild-type tumors. However, survival still varies widely among patients with IDH mutated tumors. Here, we aimed to characterize molecular signatures that explain the range of IDH mutant gliomas. By integrating matched epigenome-wide methylome, transcriptome, and global metabolome data in 154 patients with gliomas, we identified a group of IDH mutant gliomas with globally altered metabolism that resembled IDH wild-type tumors. IDH-mutant gliomas with altered metabolism have significantly shorter overall survival from their IDH mutant counterparts that is not fully accounted for by recognized molecular prognostic markers of CDKN2A/B loss and glioma CpG Island Methylator Phenotype (GCIMP) status. IDH-mutant tumors with dysregulated metabolism harbored distinct epigenetic alterations that converged to drive proliferative and stem-like transcriptional profiles, providing a window to target novel dependencies in gliomas.
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Glioma , Isocitrato Deshidrogenasa , Humanos , Isocitrato Deshidrogenasa/genética , Glioma/genética , Epigenómica , Mutación/genética , TranscriptomaRESUMEN
Homozygous deletion of CDKN2A/B was recently incorporated into the World Health Organization classification for grade 3 meningiomas. While this marker is overall rare in meningiomas, its relationship to other CDKN2A alterations on a transcriptomic, epigenomic, and copy number level has not yet been determined. We therefore utilized multidimensional molecular data of 1577 meningioma samples from 6 independent cohorts enriched for clinically aggressive meningiomas to comprehensively interrogate the spectrum of CDKN2A alterations through DNA methylation, copy number variation, transcriptomics, and proteomics using an integrated molecular approach. Homozygous CDKN2A/B deletions were identified in only 7.1% of cases but were associated with significantly poorer outcomes compared to tumors without these deletions. Heterozygous CDKN2A/B deletions were identified in 2.6% of cases and had similarly poor outcomes as those with homozygous deletions. Among tumors with intact CDKN2A/B (without a homozygous or heterozygous deletion), we found a distinct difference in outcome based on mRNA expression of CDKN2A, with meningiomas that had elevated mRNA expression (CDKN2Ahigh) having a significantly shorter time to recurrence. The expression of CDKN2A was independently prognostic after accounting for copy number loss and consistently increased with WHO grade and more aggressive molecular and methylation groups irrespective of cohort. Despite the discordant and mutually exclusive status of the CDKN2A gene in these groups, both CDKN2Ahigh meningiomas and meningiomas with CDKN2A deletions were enriched for similar cell cycle pathways but at different checkpoints. High mRNA expression of CDKN2A was also associated with gene hypermethylation, Rb-deficiency, and lack of response to CDK inhibition. p16 immunohistochemistry could not reliably differentiate between meningiomas with and without CDKN2A deletions but appeared to correlate better with mRNA expression. These findings support the role of CDKN2A mRNA expression as a biomarker of clinically aggressive meningiomas with potential therapeutic implications.
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Neoplasias Meníngeas , Meningioma , Humanos , Genes p16 , Meningioma/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Transcriptoma , Variaciones en el Número de Copia de ADN , Homocigoto , Eliminación de Secuencia , Neoplasias Meníngeas/genéticaRESUMEN
PURPOSE: Meningiomas are the most common primary brain tumor in adults. Traditionally they have been understudied compared to other central nervous system (CNS) tumors. However over the last decade, there has been renewed interest in uncovering the molecular topography of these tumors, with landmark studies identifying key driver alterations contributing to meningioma development and progression. Recent work from several independent research groups have integrated different genomic and epigenomic platforms to develop a molecular-based classification scheme for meningiomas that could supersede histopathological grading in terms of diagnostic accuracy, biological relevance, and outcome prediction, keeping pace with contemporary grading schemes for other CNS tumors including gliomas and medulloblastomas. METHODS: Here we summarize the studies that have uncovered key alterations in meningiomas which builds towards the discovery of consensus molecular groups in meningiomas by integrating these findings. These groups supersede WHO grade and other clinical factors in being able to accurately predict tumor biology and clinical outcomes following surgery. RESULTS: Despite differences in the nomenclature of recently uncovered molecular groups across different studies, the biological similarities between these groups enables us to likely reconciliate these groups into four consensus molecular groups: two benign groups largely dichotomized by NF2-status, and two clinically aggressive groups defined by their hypermetabolic transcriptome, and by their preponderance of proliferative, cell-cycling pathways respectively. CONCLUSION: Future work, including by our group and others are underway to validate these molecular groups and harmonize the nomenclature for routine clinical use.
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Neoplasias del Sistema Nervioso Central , Neoplasias Cerebelosas , Neoplasias Meníngeas , Meningioma , Adulto , Humanos , Meningioma/patología , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/terapia , MultiómicaRESUMEN
OBJECTIVE: Isocitrate dehydrogenase (IDH) mutation status is a key diagnostic and prognostic feature of gliomas. It is thought to occur early in glioma tumorigenesis and remain stable over time. However, there are reports documenting a loss of IDH mutation status in a subset of patients with glioma recurrence. Here, we identified patients with a documented loss of IDH mutation status longitudinally and performed multi-platform analysis in order to determine if IDH mutations are stable throughout glioma evolution. METHODS: We retrospectively identified patients from our institution from 2009 to 2018 with immunohistochemistry (IHC)-recorded IDH mutation status changes longitudinally. Archived formalin-fixed paraffin-embedded and frozen tissue samples from these patients were collected from our institution's tumour bank. Samples were analysed using methylation profiling, copy number variation, Sanger sequencing, droplet digital PCR (ddPCR) and IHC. RESULTS: We reviewed 1491 archived glioma samples including 78 patients with multiple IDH mutant tumour samples collected longitudinally. In all instances of documented loss of IDH mutation status, multi-platform profiling identified a mixture of low tumour cell content and non-neoplastic tissue including perilesional, reactive or inflammatory cells. CONCLUSIONS: All patients with a documented loss of IDH mutation status longitudinally were resolved through multi-platform analysis. These findings support the hypothesis that IDH mutations occur early in gliomagenesis and in the absence of copy number changes at the IDH loci and are stable throughout tumour treatment and evolution. Our study highlights the importance of accurate surgical sampling and the role of DNA methylome profiling in diagnostically uncertain cases for integrated pathological and molecular diagnosis.
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Schwannomatosis (SWNTS) is a genetic cancer predisposition syndrome that manifests as multiple and often painful neuronal tumors called schwannomas (SWNs). While germline mutations in SMARCB1 or LZTR1, plus somatic mutations in NF2 and loss of heterozygosity in chromosome 22q have been identified in a subset of patients, little is known about the epigenomic and genomic alterations that drive SWNTS-related SWNs (SWNTS-SWNs) in a majority of the cases. We performed multiplatform genomic analysis and established the molecular signature of SWNTS-SWNs. We show that SWNTS-SWNs harbor distinct genomic features relative to the histologically identical non-syndromic sporadic SWNs (NS-SWNS). We demonstrate the existence of four distinct DNA methylation subgroups of SWNTS-SWNs that are associated with specific transcriptional programs and tumor location. We show several novel recurrent non-22q deletions and structural rearrangements. We detected the SH3PXD2A-HTRA1 gene fusion in SWNTS-SWNs, with predominance in LZTR1-mutant tumors. In addition, we identified specific genetic, epigenetic, and actionable transcriptional programs associated with painful SWNTS-SWNs including PIGF, VEGF, MEK, and MTOR pathways, which may be harnessed for management of this syndrome.
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Epigénesis Genética , Genómica , Neoplasias de la Vaina del Nervio/genética , Neurilemoma/genética , Neurofibromatosis/genética , Neoplasias Cutáneas/genética , Transcriptoma , Proteínas Adaptadoras del Transporte Vesicular/genética , Estudios de Cohortes , Metilación de ADN , Fusión Génica , Predisposición Genética a la Enfermedad/genética , Mutación de Línea Germinal , Serina Peptidasa A1 que Requiere Temperaturas Altas/genética , Humanos , Proteínas Quinasas Activadas por Mitógenos/genética , Neurofibromina 2/genética , Factores de Transcripción/genéticaRESUMEN
Amorpha-4,11-diene (AMD4,11) is a precursor to artemisinin, a potent antimalarial drug that is traditionally extracted from the shrubs of Artemisia annua. Despite significant prior efforts to produce artemisinin and its precursors through biotechnology, there remains a dire need for more efficient biosynthetic routes for its production. Here, we describe the optimization of key process conditions for an Escherichia coli strain producing AMD4,11 via the native methylerythritol phosphate (MEP) pathway. By studying the interplay between glucose uptake rates and oxygen demand, we were able to identify optimal conditions for increasing carbon flux through the MEP pathway by manipulating the availability of NADPH required for terpenoid production. Installation of an optimal qO2 /qglucose led to a 6.7-fold increase in product titers and a 6.5-fold increase in carbon yield.
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Antimaláricos/metabolismo , Eritritol/análogos & derivados , Escherichia coli/metabolismo , Glucosa/metabolismo , Consumo de Oxígeno , Oxígeno/metabolismo , Sesquiterpenos Policíclicos/metabolismo , Fosfatos de Azúcar/metabolismo , Eritritol/metabolismo , Escherichia coli/genéticaRESUMEN
Aim: To describe practices and outcomes in veterans with relapsed/refractory diffuse large B-cell lymphoma. Patients & methods: Using Veteran Affairs Cancer Registry System and electronic health record data, we identified relapsed/refractory diffuse large B-cell lymphoma patients completing second-line treatment (2L) in 2000-2016. Treatments were classified as aggressive/nonaggressive. Analyses included descriptive statistics and the Kaplan-Meier estimation of progression-free survival and overall survival. Results: Two hundred and seventy patients received 2L. During median 9.7-month follow-up starting from 2L, 470 regimens were observed, averaging 2.7 regimens/patient: 219 aggressive, 251 nonaggressive. One hundred and twenty-one patients proceeded to third-line, 50 to fourth-line and 18 to fifth-line treatment. Median progression-free survival in 2L was 5.2 months. Median overall survival was 9.5 months. Forty-four patients (16.3%) proceeded to bone marrow transplant. Conclusion: More effective, less toxic treatments are needed and should be initiated earlier in treatment trajectory.
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Linfoma de Células B Grandes Difuso/terapia , Adulto , Anciano , Anciano de 80 o más Años , Trasplante de Médula Ósea , Femenino , Humanos , Linfoma de Células B Grandes Difuso/mortalidad , Masculino , Persona de Mediana Edad , Recurrencia , VeteranosRESUMEN
Two Gram-stain positive, endospore forming, non-motile, rod shaped bacterial strains SN6T and SN6b were isolated from scats of a mildly venomous vine snake (Ahaetulla nasuta). Strains were phenotypically resistant to multiple antibiotics of four different classes i.e. aminoglycosides, ß-lactams, fluoroquinolones and sulphonamides. Cells of both the strains were catalase positive and oxidase negative. Phylogenetic analysis based on 16S rRNA gene sequence analysis of these two strains showed closest similarity (99.2% and 99.3%) with Savagea faecisuis Con12T, the only species of the genus Savagea and ≤ 94.9% with the species of other closest genera of the family Planococcaceae. The 16S rRNA gene sequence similarity (99%), DNA-DNA relatedness (95%) and similar phenotypic characteristics between the strains SN6T and SN6b revealed their phylogenetic affiliation to the same species. Hence, strain SN6b is an additional strain of the type strain SN6T. DNA-DNA relatedness of strain SN6T with S. faecisuis Con12T was 32.8%. Predominant fatty acids were iso-C15:0 (32.0%), iso-C16:1 ω11c (19.2%) and iso-C17:1 ω10c (12.1%). MK-6 (100%) was the only respiratory quinone of strain SN6T. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were the major polar lipids. Cell wall peptidoglycan was A4α; L-Lys-Gly-D-Glu type. The DNA G + C content (mol%) of SN6T was 40.8. Whole genome sequence of SN6T consisted of 26,37,389 base pairs in length with 2667 annotated genes, out of which 1021 corresponds to hypothetical proteins and 1646 with functional assignments including antibiotic resistance, multidrug resistance efflux pumps, invasion and virulence factors. Comparative polyphasic study of the strains SN6T, SN6b and S. faecisuis Con12T elucidated the differentiating characteristics which led to describing strain SN6T and SN6b as a novel species of the genus Savagea for which the name Savagea serpentis sp. nov is proposed. The type strain of Savagea serpentis is SN6T (= KCTC 33546T = CCUG 6786T).
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Fosfolípidos , Planococcaceae , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/análisis , Genómica , Hibridación de Ácido Nucleico , Fosfolípidos/análisis , Filogenia , Planococcaceae/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , SerpientesRESUMEN
For decades, bacterial natural products have served as valuable resources for developing novel drugs to treat several human diseases. Recent advancements in the integrative approach of using genomic and functional tools have proved beneficial in obtaining a comprehensive understanding of these biomolecules. This study presents an in-depth characterization of the anti-diabetic activity exhibited by a bacterial isolate SW1, isolated from an effluent treatment plant. As a primary screening, we assessed the isolate for its potential to inhibit alpha-amylase and alpha-glucosidase enzymes. Upon confirmation, we further utilized LC-MS, ESI-MS/MS, and NMR spectroscopy to identify and characterize the biomolecule. These efforts were coupled with the genomic assessment of the biosynthetic gene cluster involved in the anti-diabetic compound production. Our investigation discovered that the isolate SW1 inhibited both α-amylase and α-glucosidase activity. The chemical analysis suggested the production of acarbose, an anti-diabetic biomolecule, which was further confirmed by the presence of biosynthetic gene cluster "acb" in the genome. Our in-depth chemical characterization and genome mining approach revealed the potential of bacteria from an unconventional niche, an effluent treatment plant. To the best of our knowledge, it is one of the first few reports of acarbose production from the genus Arthrobacter.
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Arthrobacter , Acarbosa , Arthrobacter/genética , Genómica , Inhibidores de Glicósido Hidrolasas , Humanos , Espectrometría de Masas en Tándem , alfa-Glucosidasas/genéticaRESUMEN
Liquid biopsy, as a non-invasive technique for cancer diagnosis, has emerged as a major step forward in conquering tumors. Current practice in diagnosis of central nervous system (CNS) tumors involves invasive acquisition of tumor biopsy upon detection of tumor on neuroimaging. Liquid biopsy enables non-invasive, rapid, precise and, in particular, real-time cancer detection, prognosis and treatment monitoring, especially for CNS tumors. This approach can also uncover the heterogeneity of these tumors and will likely replace tissue biopsy in the future. Key components of liquid biopsy mainly include circulating tumor cells (CTC), circulating tumor nucleic acids (ctDNA, miRNA) and exosomes and samples can be obtained from the cerebrospinal fluid, plasma and serum of patients with CNS malignancies. This review covers current progress in application of liquid biopsies for diagnosis and monitoring of CNS malignancies.
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Neoplasias del Sistema Nervioso Central/diagnóstico , Biopsia Líquida/métodos , Biopsia Líquida/tendencias , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/líquido cefalorraquídeo , Neoplasias del Sistema Nervioso Central/metabolismo , ADN Tumoral Circulante/sangre , Exosomas/patología , Humanos , MicroARNs , Células Neoplásicas Circulantes/patología , PronósticoRESUMEN
Glioblastoma (GBM) is the most aggressive type of glioma. Temozolomide (TMZ) is currently the drug of choice used for post-operative chemotherapy of GBM. However, the presence of intrinsic and acquired resistance hinders the success of chemotherapy. To understand the TMZ resistant mechanisms in glioma, we investigated the alterations in cellular signaling pathways by performing transcriptome analysis of TMZ treated glioma cells. Gene Set Enrichment Analysis (GSEA) indicated a significant enrichment of Wnt/ß-catenin signaling besides many other pathways in TMZ treated cells. Further, we demonstrate that TMZ treatment increased the activity from TOPflash reporter, (a Wnt responsive reporter), enhanced the levels of pGSK-3ß (S9) and reduced the levels of p-ß-catenin (S33/37/T41) with a concomitant increase in transcript and protein levels of Wnt targets in a concentration and time-dependent manner. While TMZ treated cells did not show alteration in any of the Wnt ligands, PI3K inhibitor (LY294002) treatment repressed Akt activation and abolished the TMZ-mediated induction of Wnt/ß-catenin pathway. In addition, we show that Wnt/ß-catenin signaling activation by TMZ is independent of ATM/Chk2 pathway. Further, we also demonstrate the activation of mTOR pathway after TMZ treatment. Thus, our results demonstrate that activation of Wnt/ß-catenin pathway involves an ATM/Chk2- independent PI3K/Akt/GSK-3 cascade in TMZ treated cells and further provides mechanistic basis for the chemoresistance of glioma to TMZ.
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Glioma/metabolismo , Temozolomida/farmacología , Vía de Señalización Wnt/fisiología , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/metabolismo , Glioma/tratamiento farmacológico , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Temozolomida/metabolismo , Proteínas Wnt/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/metabolismoRESUMEN
Naringinase has high industrial importance, and the progress in naringinase research is still quite slow. The unavailability of an effective, simple screening method, which will be applicable to different microorganisms such as bacteria, fungi, and actinomycetes, is one of the main reasons for this gap. Therefore, a simple plate assay was developed for effective screening of microorganisms for naringinase by exposing to iodine vapors. This plate assay will fill the technological void for simple screening method and will lead to screen more potent industrially important naringinase-producing microorganisms.
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Bacterias/enzimología , Hongos/enzimología , Complejos Multienzimáticos/biosíntesis , beta-Glucosidasa/biosíntesis , Bacterias/metabolismo , Cromatografía Líquida de Alta Presión , Hongos/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , beta-Glucosidasa/química , beta-Glucosidasa/metabolismoRESUMEN
This study aims to explore the fermentative production and physicochemical properties of an exopolysaccharide (EPS) produced from agricultural isolate, Bacillus subtilis S1 in submerged culture. The structural characterization (Ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, and 13 C Nuclear magnetic resonance spectrometry) revealed that the EPS is an acidic heteropolymer consisting of glucose, glucuronic acid, pyruvic acid, and succinic acid. The non-Newtonian shear thickening nature of EPS with a 1.55 × 107 Da molecular weight is confirmed by rheology analysis. The extracted EPS was 61.3% amorphous with partial crystallinity (38.7%) as confirmed by X-ray diffraction analysis. The EPS shows two-step decomposition and thermal stability up to 300 °C as confirmed by thermogravimetric analysis and differential scanning calorimetry analysis. The EPS has a small Z-average particle size (74.29 nm), high porosity (92.99%), high water holding (92.39%), and absorption capacity (1,198%). The biocompatible nature is confirmed by cytotoxic testing on the human keratinocytes cell line. The demonstrated unique characteristics of Bacillus EPS presents it as a choice of biomaterial for diverse applications.
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Bacillus subtilis/química , Materiales Biocompatibles/farmacología , Productos Biológicos/farmacología , Queratinocitos/efectos de los fármacos , Polisacáridos Bacterianos/farmacología , Bacillus subtilis/metabolismo , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fermentación , Humanos , Queratinocitos/metabolismo , Peso Molecular , Tamaño de la Partícula , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/química , ReologíaRESUMEN
A novel and efficient metal free, redox-neutral method for the synthesis of secondary thiocarbamates by cross-coupling of readily available thiophenol and isocyanides has been developed. The present methodology exhibits a broad substrate scope with good to excellent yields without an additive/extra oxidant under mild reaction conditions catalyzed by inexpensive iodine as the catalyst.
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Enhancer of zeste homolog-2(EZH2) is a key epigenetic regulator that functions as oncogene and also known for inducing altered trimethylation of histone at lysine-27 (H3K27me3) mark in various tumors. However, H3K27me3 targets and their precise relationship with gene expression are largely unknown in astrocytic tumors. In this study, we checked EZH2 messenger RNA and protein expression in 90 astrocytic tumors of different grades using quantitative PCR and immunohistochemistry, respectively. Further, genome-wide ChIP-seq analysis for H3K27me3 modification was also performed on 11 glioblastomas (GBMs) and 2 diffuse astrocytoma (DA) samples. Our results showed EZH2 to be highly overexpressed in astrocytic tumors with a significant positive correlation with grade. Interestingly, ChIP-seq mapping revealed distinct differences in genes and pathways targeted by these H3K27me3 modifications between GBM versus DA. Neuroactive ligand receptor pathway was found most enriched in GBM (P = 9.4 × 10-25), whereas DA were found to be enriched in metabolic pathways. Also, GBM showed a higher enrichment of H3K27me3 targets reported in embryonic stem cells and glioma stem cells as compared with DAs. Our results show majority of these H3K27me3 target genes were downregulated, not only due to H3K27me3 modification but also due to concomitant DNA methylation. Further, H3K27me3 modification-associated gene silencing was not restricted to promoter but also present in gene body and transcription start site regions. To the best of our knowledge, this is the first high-resolution genome-wide mapping of H3K27me3 modification in adult astrocytic primary tissue samples of human, highlighting the differences between grades. Interestingly, we identified SLC25A23 as important target of H3K27me3 modification, which was downregulated in GBM and its low expression was associated with poor prognosis in GBMs.
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Antiportadores/genética , Astrocitoma/genética , Metilación de ADN/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Glioblastoma/genética , N-Metiltransferasa de Histona-Lisina/genética , Proteínas Mitocondriales/genética , Astrocitoma/patología , Línea Celular Tumoral , Elementos de Facilitación Genéticos/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Glioblastoma/patología , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Masculino , Clasificación del Tumor , Regiones Promotoras GenéticasRESUMEN
Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NFκB pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NFκB-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Adulto , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Femenino , Glioblastoma/genética , Glioblastoma/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor Estimulante de Colonias de Macrófagos/genética , Masculino , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas de Neoplasias/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/genética , Quinasa SykRESUMEN
BACKGROUND: Glioblastomas (GBM) continue to remain one of the most dreaded tumours that are highly infiltrative in nature and easily preclude comprehensive surgical resection. GBMs pose an intricate etiology as they are being associated with a plethora of genetic and epigenetic lesions. Misregulation of the PI3 kinase pathway is one of the most familiar events in GBM. While the PI3 kinase signalling regulated pathways and genes have been comprehensively studied, its impact on the miRNome is yet to be explored. The objective of this study was to elucidate the PI3 kinase pathway regulated miRNAs in GBM. METHODS: miRNA expression profiling was conducted to monitor the differentially regulated miRNAs upon PI3 kinase pathway abrogation. qRT-PCR was used to measure the abundance of miR-326 and its host gene encoded transcript. Proliferation assay, colony suppression assay and wound healing assay were carried out in pre-miR transfected cells to investigate its role in malignant transformation. Potential targets of miR-326 were identified by transcriptome analysis of miR-326 overexpressing cells by whole RNA sequencing and selected targets were validated. Several publically available data sets were used for various investigations described above. RESULTS: We identified several miRNA that were regulated by PI3 kinase pathway. miR-326, a GBM downregulated miRNA, was validated as one of the miRNAs whose expression was alleviated upon abrogation of the PI3 kinase pathway. Overexpression of miR-326 resulted in reduced proliferation, colony suppression and hindered the migration capacity of glioma cells. Arrestin, Beta 1 (ARRB1), the host gene of miR-326, was also downregulated in GBM and interestingly, the expression of ARRB1 was also alleviated upon inhibition of the PI3 kinase pathway, indicating similar regulation pattern. More importantly, miR-326 exhibited a significant positive correlation with ARRB1 in terms of its expression. Transcriptome analysis upon miR-326 overexpression coupled with integrative bioinformatics approach identified several putative targets of miR-326. Selected targets were validated and interestingly found to be upregulated in GBM. CONCLUSIONS: Taken together, our study uncovered the PI3 kinase regulated miRNome in GBM. miR-326, a PI3 kinase pathway inhibited miRNA, was demonstrated as a tumour suppressor miRNA in GBM.