| 1. |
- Huang, Hua, 1986-, et al.
(author)
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ELTD1-deletion reduces vascular abnormality and improves T-cell recruitment after PD-1 blockade in glioma.
- 2021
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In: Neuro-Oncology. - : Oxford University Press. - 1522-8517 .- 1523-5866. ; 24:3, s. 398-411
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Journal article (peer-reviewed)abstract
- BACKGROUND: Tumor vessels in glioma are molecularly and functionally abnormal, contributing to treatment resistance. Proteins differentially expressed in glioma vessels can change vessel phenotype and be targeted for therapy. ELTD1 (Adgrl4) is an orphan member of the adhesion G-protein-coupled receptor family upregulated in glioma vessels, and has been suggested as a potential therapeutic target. However, the role of ELTD1 in regulating vessel function in glioblastoma is poorly understood.METHODS: ELTD1 expression in human gliomas and its association with patient survival was determined using tissue microarrays and public databases. The role of ELTD1 in regulating tumor vessel phenotype was analyzed using orthotopic glioma models and ELTD1 -/- mice. Endothelial cells isolated from murine gliomas were transcriptionally profiled to determine differentially expressed genes and pathways. The consequence of ELTD1-deletion on glioma immunity was determined by treating tumor bearing mice with PD-1-blocking antibodies.RESULTS: ELTD1 levels were upregulated in human glioma vessels, increased with tumor malignancy, and were associated with poor patient survival. Progression of orthotopic gliomas was not affected by ELTD1-deletion, however, tumor vascular function was improved in ELTD1 -/- mice. Bioinformatic analysis of differentially expressed genes indicated increased inflammatory response and decreased proliferation in tumor endothelium in ELTD1 -/- mice. Consistent with an enhanced inflammatory response, ELTD1-deletion improved T-cell infiltration in GL261-bearing mice after PD-1 checkpoint blockade.CONCLUSION: Our data demonstrate that ELTD1 participates in inducing vascular dysfunction in glioma, and suggests that targeting of ELTD1 may normalize the vessels and improve the response to immunotherapy.
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| 2. |
- Conze, Lei Liu, et al.
(author)
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Construction and deep sequencing of cDNA libraries representing medium-sized RNA
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Other publication (other academic/artistic)abstract
- Transcriptome analysis has recently become feasible with the application of RNA-seq, the high-throughput sequencing analysis of RNAs through cDNA synthesis. Current transcriptome analysis approaches are limited to analysis of long (>200 nts) or short (~20 nts) RNAs. Here we describe an approach that can be used for the analysis of the medium-sized (~200 nts) RNA transcriptome, which reveals the full-length sequence of RNAs at the single molecule level. Our approach can easily be adapted to generate libraries that are suitable for sequencing by several of the currently used next-generation platforms.
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| 3. |
- Conze, Lei Liu, et al.
(author)
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The primate spliceosomal snRNA transcriptome as revealed by deep sequencing
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Other publication (other academic/artistic)abstract
- Small nuclear RNAs (snRNAs) in combination with protein factors form the spliceosomes and are essential for pre-mRNA splicing. In this study we have characterized the spliceosomal snRNA (U snRNA) transcriptome in human, chimpanzee and rhesus using a recently developed high-throughput sequencing approach. Our study has revealed that the U snRNA transcriptomes of all three primates have a similar composition and that the vast majority of the U snRNAs originate from a few well-defined loci. We have detected some heterogeneity in the U snRNA transcriptomes that is primarily caused by the presence of SNPs in the few expressed loci that we have identified.
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| 4. |
- Conze, Lei Liu
(author)
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Understanding the Noise : Spliceosomal snRNA Profiling
- 2012
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Doctoral thesis (other academic/artistic)abstract
- The concept of the gene has been constantly challenged by new discoveries in the life sciences. Recent challenging observations include the high frequency of alternative splicing events and the common transcription of non-protein-coding-RNAs (ncRNAs) from the genome. The latter has long been considered noise in biological systems. Multiple lines of evidence from genomic studies indicate that alternative splicing and ncRNA play important roles in expanding proteome diversity in eukaryotes. Here, the aim is to find the link between alternative splicing and ncRNAs by studying the expression profile of the spliceosomal snRNAs (U snRNA).Spliceosomal snRNAs are essential for pre-mRNA splicing in eukaryotes. They participate in splice site selection, recruitment of protein factors and catalyzing the splicing reaction. Because of this, both the abundance and diversity of U snRNAs were expected to be large. In our study we deeply analyzed the U snRNA population in primates using a combination of bioinformatical, biochemical and high throughput sequencing approaches. This transcriptome profiling has revealed that human, chimpanzee and rhesus have similar U snRNA populations, i.e. the vast majority of U snRNAs originate from few well-defined gene loci and the heterogeneity observed in U snRNA populations was largely due to the presence of SNPs at these loci. It seems that the gene loci that could potentially encode a significantly heterogeneous population of U snRNAs are mostly silent. Only few minority transcripts were detected in our study, and among them three U1-like snRNAs might play a role in the regulation of alternative splicing by recognizing non-canonical splicing sites.Mutations of U snRNA have been shown to impact the splicing process. Therefore, our study provides a reference to study the biological significance of SNPs in U snRNA genes and their association with diseases.
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| 5. |
- Corada, Monica, et al.
(author)
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Fine-Tuning of Sox17 and Canonical Wnt Coordinates the Permeability Properties of the Blood-Brain Barrier
- 2019
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In: Circulation Research. - 0009-7330 .- 1524-4571. ; 124:4, s. 511-525
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Journal article (peer-reviewed)abstract
- Rationale: The microvasculature of the central nervous system includes the blood-brain barrier (BBB), which regulates the permeability to nutrients and restricts the passage of toxic agents and inflammatory cells. Canonical Wnt/β-catenin signaling is responsible for the early phases of brain vascularization and BBB differentiation. However, this signal declines after birth, and other signaling pathways able to maintain barrier integrity at postnatal stage are still unknown.Objective: Sox17 (SRY [sex-determining region Y]-box 17) constitutes a major downstream target of Wnt/β-catenin in endothelial cells and regulates arterial differentiation. In the present article, we asked whether Sox17 may act downstream of Wnt/β-catenin in inducing BBB differentiation and maintenance.Methods and Results: Using reporter mice and nuclear staining of Sox17 and β-catenin, we report that although β-catenin signaling declines after birth, Sox17 activation increases and remains high in the adult. Endothelial-specific inactivation of Sox17 leads to increase of permeability of the brain microcirculation. The severity of this effect depends on the degree of BBB maturation: it is strong in the embryo and progressively declines after birth. In search of Sox17 mechanism of action, RNA sequencing analysis of gene expression of brain endothelial cells has identified members of the Wnt/β-catenin signaling pathway as downstream targets of Sox17. Consistently, we found that Sox17 is a positive inducer of Wnt/β-catenin signaling, and it acts in concert with this pathway to induce and maintain BBB properties. In vivo, inhibition of the β-catenin destruction complex or expression of a degradation-resistant β-catenin mutant, prevent the increase in permeability and retina vascular malformations observed in the absence of Sox17.Conclusions: Our data highlight a novel role for Sox17 in the induction and maintenance of the BBB, and they underline the strict reciprocal tuning of this transcription factor and Wnt/β-catenin pathway. Modulation of Sox17 activity may be relevant to control BBB permeability in pathological conditions.
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| 6. |
- Jauhiainen, Suvi, et al.
(author)
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Proteomics on human cerebral cavernous malformations reveals novel biomarkers in neurovascular dysfunction for the disease pathology
- 2024
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In: Biochimica et Biophysica Acta - Molecular Basis of Disease. - : Elsevier. - 0925-4439 .- 1879-260X. ; 1870:5
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Journal article (peer-reviewed)abstract
- Background: Cerebral cavernous malformation (CCM) is a disease associated with an elevated risk of focal neurological deficits, seizures, and hemorrhagic stroke. The disease has an inflammatory profile and improved knowledge of CCM pathology mechanisms and exploration of candidate biomarkers will enable new non-invasive treatments. Methods: We analyzed protein signatures in human CCM tissue samples by using a highly specific and sensitive multiplexing technique, proximity extension assay. Findings: Data analysis revealed CCM specific proteins involved in endothelial dysfunction/inflammation/activation, leukocyte infiltration/chemotaxis, hemostasis, extracellular matrix dysfunction, astrocyte and microglial cell activation. Biomarker expression profiles matched bleeding status, especially with higher levels of inflammatory markers and activated astrocytes in ruptured than non-ruptured samples, some of these biomarkers are secreted into blood or urine. Furthermore, analysis was also done in a spatially resolving manner by separating the lesion area from the surrounding brain tissue. Our spatial studies revealed that although appearing histologically normal, the CCM border areas were pathological when compared to control brain tissues. Moreover, the functional relevance of CD93, ICAM-1 and MMP9, markers related to endothelial cell activation and extracellular matrix was validated by a murine pre-clinical CCM model. Interpretation: Here we present a novel strategy for proteomics analysis on human CCMs, offering a possibility for high-throughput protein screening acquiring data on the local environment in the brain. Our data presented here describe CCM relevant brain proteins and specifically those which are secreted can serve the need of circulating CCM biomarkers to predict cavernoma's risk of bleeding.
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| 7. |
- Keller, Maureen, et al.
(author)
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Altered cytoskeletal status in the transition from proneural to mesenchymal glioblastoma subtypes
- 2022
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In: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12:1, s. 9838-
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Journal article (peer-reviewed)abstract
- Glioblastoma is a highly aggressive brain tumor with poor patient prognosis. Treatment outcomes remain limited, partly due to intratumoral heterogeneity and the invasive nature of the tumors. Glioblastoma cells invade and spread into the surrounding brain tissue, and even between hemispheres, thus hampering complete surgical resection. This invasive motility can arise through altered properties of the cytoskeleton. We hypothesize that cytoskeletal organization and dynamics can provide important clues to the different malignant states of glioblastoma. In this study, we investigated cytoskeletal organization in glioblastoma cells with different subtype expression profiles, and cytoskeletal dynamics upon subtype transitions. Analysis of the morphological, migratory, and invasive properties of glioblastoma cells identified cytoskeletal components as phenotypic markers that can serve as diagnostic or prognostic tools. We also show that the cytoskeletal function and malignant properties of glioblastoma cells shift during subtype transitions induced by altered expression of the neurodevelopmental transcription factor SOX2. The potential of SOX2 re-expression to reverse the mesenchymal subtype into a more proneural subtype might open up strategies for novel glioblastoma treatments.
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| 8. |
- Liu, Lei Conze, et al.
(author)
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Transcriptome profiling of tobacco (Nicotiana tabacum) pollen and pollen tubes
- 2017
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In: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 18
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Journal article (peer-reviewed)abstract
- Background: Pollen tube growth is essential for plant reproduction and represents a widely employed model to investigate polarized cell expansion, a process important for plant morphogenesis and development. Cellular and regulatory mechanisms underlying pollen tube elongation are under intense investigation, which stands to greatly benefit from a comprehensive understanding of global gene expression profiles in pollen and pollen tubes. Here, RNA sequencing technology was applied to de novo assemble a Nicotiana tabacum male gametophytic transcriptome and to compare transcriptome profiles at two different stages of gametophyte development: mature pollen grains (MPG) and pollen tubes grown for six hours in vitro (PT6).Results: De novo assembly of data obtained by 454 sequencing of a normalized cDNA library representing tobacco pollen and pollen tube mRNA (pooled mRNA isolated from mature pollen grains [MPG] and from pollen tubes grown in vitro for 3 [PT3] or 6 [PT6] hours) resulted in the identification of 78,364 unigenes. Among these unigenes, which mapped to 24,933 entries in the Sol Genomics Network (SGN) N. tabacum unigene database, 24,672 were predicted to represent full length cDNAs. In addition, quantitative analyses of data obtained by Illumina sequencing of two separate non-normalized MPG and PT6 cDNA libraries showed that 8979 unigenes were differentially expressed (differentially expressed unigenes: DEGs) between these two developmental stages at a FDR q-value of <0.0001. Interestingly, whereas most of these DEGs were downregulated in PT6, the minor fraction of DEGs upregulated in PT6 was enriched for GO (gene ontology) functions in pollen tube growth or fertilization.Conclusions: A major output of our study is the development of two different high-quality databases representing the tobacco male gametophytic transcriptome and containing encompassing information about global changes in gene expression after pollen germination. Quantitative analyses of these databases 1) indicated that roughly 30% of all tobacco genes are expressed in the male gametophyte, and 2) support previous observations suggesting a global reduction of transcription after pollen germination. Interestingly, a small number of genes, many of which predicted to function in pollen tube growth or fertilization, were found to be upregulated in elongating pollen tubes despite globally reduced transcription.
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| 9. |
- Martinez-Arias, Germán, et al.
(author)
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Paternal easiRNAs regulate parental genome dosage in Arabidopsis
- 2018
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In: Nature Genetics. - : NATURE PUBLISHING GROUP. - 1061-4036 .- 1546-1718. ; 50:2, s. 193-198
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Journal article (peer-reviewed)abstract
- The regulation of parental genome dosage is of fundamental importance in animals and plants, as exemplified by X-chromosome inactivation and dosage compensation. The 'triploid block' is a classic example of dosage regulation in plants that establishes a reproductive barrier between species differing in chromosome number(1,2). This barrier acts in the embryo-nourishing endosperm tissue and induces the abortion of hybrid seeds through a yet unknown mechanism(3). Here we show that depletion of paternal epigenetically activated small interfering RNAs (easiRNAs) bypasses the triploid block in response to increased paternal ploidy in Arabidopsis thaliana. Paternal loss of the plant-specific RNA polymerase IV suppressed easiRNA formation and rescued triploid seeds by restoring small-RNA-directed DNA methylation at transposable elements (TEs), correlating with reduced expression of paternally expressed imprinted genes (PEGs). Our data suggest that easiRNAs form a quantitative signal for paternal chromosome number and that their balanced dosage is required for post-fertilization genome stability and seed viability.
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| 10. |
- Morini, Marco F., et al.
(author)
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VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression
- 2018
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In: Circulation Research. - : LIPPINCOTT WILLIAMS & WILKINS. - 0009-7330 .- 1524-4571. ; 122:2, s. 231-245
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Journal article (peer-reviewed)abstract
- Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability.Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization.Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and beta-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/beta-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP.Conclusions: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.
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