1,118 research outputs found

    The interplay between Natural Killer cells and Pancreatic Stellate cells in Pancreatic Ductal Adenocarcinoma

    Get PDF
    Pancreatic ductal adenocarcinoma (PDAC) is a disease with dismal prognosis. With five-year survival rates of less than 11%, PDAC is set to become the second leading cause of cancer related deaths by 2040. The role of pancreatic stellate cells in pancreatic ductal adenocarcinoma has been well established. However, to date, little remains know about the interaction between these crucial stromal cells and the innate lymphocytes, natural killer (NK) cells, in PDAC. Herein we demonstrate that naĂŻve NK cells possess the functional efficacy to target and kill both quiescent (qPSC) and activated (aPSC) pancreatic stellate cells. Furthermore, qPSC, but not aPSC education of NK cells resulted in decreased NK cell-mediated cancer cell cytotoxicity. NK-PSC direct co-culture was found to modulate both PSC and NK phenotype, as well as functional changes within NK cells, an effect not observed with TranswellTM separation. Multiplex Luminex ELISA further revealed upregulation of IFN-Îł and related chemokines in NK cells co-cultured with PSC (activated/quiescent), suggesting that this pathway may be involved in phenotypic modulation. Through global proteomic analysis we demonstrate NK cell-induced differential protein changes in aPSC versus qPSC. Furthermore, we demonstrate changes in intracellular NK pathways as a result of direct contact with PSCs, indicating a dynamic, bidirectional interaction between these two key players. Using multiplex immunohistochemical analysis, we demonstrate that NK cell proximity to CAFs, and not total NK cell infiltrate is correlated with overall survival in PDAC. Consequently, we suggest that the spatial biology of NK/CAFs may play a prognostic role in PDAC and may potentially be used as a tool for patient stratification Taken together, our results demonstrate a significant bidirectional relationship between NK cells and PSC/CAFs in the context of PDAC, providing novel insight into this crucial cell-cell interaction

    Advanced sequencing technologies applied to human cytomegalovirus

    Get PDF
    The betaherpesvirus human cytomegalovirus (HCMV) is a ubiquitous viral pathogen. It is the most common cause of congenital infection in infants and of opportunistic infections in immunocompromised patients worldwide. The large double-stranded DNA genome of HCMV (236 kb) contains several genes that exhibit a high degree of variation among strains within an otherwise highly conserved sequence. These hypervariable genes encode immune escape, tropism or regulatory factors that may affect virulence. Variation arising from these genes and from an evolutionary history of recombination between strains has been hypothesised to be linked to disease severity. To investigate this, the HCMV genome has been scrutinised in detail over the years using a variety of molecular techniques, most looking only at one or a few of these genes at a time. The advent of high-throughput sequencing (HTS) technology 20 years ago then started to enable more in-depth whole-genome analyses. My study extends this field by using both HTS and the more recently developed long-read nanopore technology to determine HCMV genome sequences directly from clinical samples. Firstly, I used an Illumina HTS pipeline to sequence HCMV strains directly from formalin-fixed, paraffin-embedded (FFPE) tissues. FFPE samples are a valuable repository for the study of relatively rare diseases, such as congenital HCMV (cCMV). However, formalin fixation induces DNA fragmentation and cross-linking, making this a challenging sample type for DNA sequencing. I successfully sequenced five whole HCMV genomes from FFPE tissues. Next, I developed a pipeline utilising the single-molecule, long-read sequencer from Oxford Nanopore Technologies (ONT) to sequence HCMV initially from high-titre cellcultured laboratory strains and then from clinical samples with high HCMV loads. Finally, I utilised a direct RNA sequencing protocol with the ONT sequencer to characterise novel HCMV transcripts produced during infection in cell culture, demonstrating the existence of transcript isoforms with multiple splice sites. Overall, my findings demonstrate how advanced sequencing technologies can be used to characterise the genome and transcriptome of a large DNA virus, and will facilitate future studies on HCMV prognostic factors, novel antiviral targets and vaccine development

    The Role of the HUSH complex and LINE-1 elements in the Regulation of Type 1 Interferon

    Get PDF
    Although Transposable Elements (TEs) have been a reliable source of genetic variation throughout evolution (Rishishwar et al., 2018), host genomes have simultaneously coevolved with TEs to employ a variety of strategies to regulate their aberrant activity. In fact, mounting evidence indicates the deleterious consequences of dysregulated TE expression in a number of human diseases including autoimmune and inflammatory disorders (M. K. Crow, 2010), monogenic diseases (Cordaux & Batzer, 2009; Nakamura et al., 2015) and various cancers (Helman et al., 2014; Lee et al., 2012). The goal of this project is to explore how the human genome continues to adapt to the ongoing evolutionary arms race between TEs and the innate immune defence system and to uncover novel epigenetic pathways that help suppress their aberrant activity and characterise how these pathways regulate innate immune genes. The Human Silencing Hub (HUSH) is an epigenetic silencing complex that is necessary for the repression of LINE-1 elements (Fukuda et al., 2018; N. Liu et al., 2018; Robbez-Masson et al., 2018). Here, we reveal the depletion of the HUSH complex component, MPP8, in human cell lines and primary fibroblasts leads to the induction of interferon-stimulated genes (ISGs) through JAK/STAT signalling and demonstrate that this effect is mainly attributable to MDA5 and RIG-I-mediated sensing of double-stranded RNAs (dsRNAs). This response coincides with the upregulation of primate-conserved LINE-1 elements, as well as increased expression of a subset of full-length hominid-specific LINE-1s that produce sense and antisense transcribed RNA products, which may form dsRNAs. Furthermore, we show that LINE-1 shRNAs could abrogate the HUSH-dependent response, while overexpression of an engineered full-length LINE-1 construct activates interferon signalling in somatic cells. Finally, we provide some insights into the physiological regulation of HUSH and HUSH-regulated LINE-1s during the normal immune response in primary human fibroblasts. Taken together, our results suggest that endogenous LINE-1s drive physiological and autoinflammatory responses through dsRNA sensing and gene-regulatory roles that are ultimately under HUSH control. Our work thus serves to highlight HUSH/MPP8 as a potential drug target for future cancer immunotherapies, where MPP8 inactivation may be harnessed to drive type 1 IFNs and anti-tumour immunity

    Exploring therapeutic vulnerabilities in tumours with GLI1 oncogene activation

    Get PDF
    Deregulation of oncogene expression is one of the main drivers in tumorigenesis. Genetic alterations, such as gene amplification and structural variation, or epigenetic mechanisms based on the chemical modification of DNA or histones, facilitate the activation of proto-oncogenes that convey growth and survival advantages to the cells. Previously, our group identified focal amplification of the chromosome arm 12q in 14 of 60 glioblastoma patients (23.3 %) of which 4 patients harboured fusion genes with the oncogene GLI Family Zinc Finger 1 (GLI1). In this study, I investigated the frequency and structure of GLI1 fusion genes, mechanisms of GLI1 transcriptional activation, GLI1-dependent tumour cell phenotype, and the potential value of GLI1 as a therapeutic target in precision-oncology in glioblastoma and liposarcoma. Initially, I identified GLI1 fusion genes linked with focal amplification on chromosome arm 12q in three independent glioblastoma cohorts (HIPO016, HIPO043, and TCGA-GB). GLI1 fusion genes were associated with high expression of GLI1 and its target genes, such as HHIP, PTCH1, and FOXS1. The boundary of the 12q amplification region often coincided with the GLI1 locus, presumably causing the breakage within the gene and the formation of fusion transcripts. The analysis of sarcoma tumours of the NCT MASTER study revealed high GLI1 expression in subtypes of osteosarcoma and soft tissue sarcoma. In addition, GLI1 fusion genes were found in liposarcoma and leiomyosarcoma. Furthermore, the disruption of a CTCF binding site upstream of the GLI1 locus upregulated the RNA expression of GLI1 and its target genes and increased cell proliferation. These data suggest that fusion-related genetic and epigenetic mechanisms regulate GLI1 expression. To explore its oncogenic function, I conducted phenotypic assays with and without GLI1 suppression and observed a reduction in tumour cell proliferation, anchorage-independent growth and increased apoptosis upon shRNA depletion or inhibition with the GLI1 inhibitor GlaB. The downregulation of several DNA repair pathways upon GLI1 depletion suggested that patients with aberrant GLI1 expression might benefit from combined GLI1 and DNA repair inhibitor therapy. To address this question, I performed a pre-clinical drug combination screen of GLI1 and DNA repair/cell cycle checkpoint inhibitors in glioblastoma and liposarcoma cell lines. In the primary screen, I tested inhibitors individually to identify effective and selective drugs of which the most promising candidates were tested in combination in the subsequent secondary screen. Both glioblastoma and liposarcoma showed high sensitivities to the SHH inhibitor JK184 and the GLI1 inhibitor GlaB. Synergistic effects were observed when GLI1 inhibitors were combined with inhibitors of the ATR/CHK1 axis, i.e., the CHK1 inhibitor LY2606368 or the ATR inhibitor Berzosertib. The independent validation of the screening results in cellular assays showed an increased effect of the combination treatment compared to the single agents on short- and long-term tumour cell proliferation. I furthermore confirmed the reduction in tumour growth upon treatment with GlaB and LY2606368 in a glioblastoma cerebral organoid model. In conclusion, these data suggest that concurrent targeting of the SHH/GLI1 and ATR/CHK1 axes provides a possible precision-therapy approach for tumours with high GLI1 expression

    Design of new algorithms for gene network reconstruction applied to in silico modeling of biomedical data

    Get PDF
    Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Ingeniería, Ciencia de Datos y BioinformáticaClave Programa: DBICódigo Línea: 111The root causes of disease are still poorly understood. The success of current therapies is limited because persistent diseases are frequently treated based on their symptoms rather than the underlying cause of the disease. Therefore, biomedical research is experiencing a technology-driven shift to data-driven holistic approaches to better characterize the molecular mechanisms causing disease. Using omics data as an input, emerging disciplines like network biology attempt to model the relationships between biomolecules. To this effect, gene co- expression networks arise as a promising tool for deciphering the relationships between genes in large transcriptomic datasets. However, because of their low specificity and high false positive rate, they demonstrate a limited capacity to retrieve the disrupted mechanisms that lead to disease onset, progression, and maintenance. Within the context of statistical modeling, we dove deeper into the reconstruction of gene co-expression networks with the specific goal of discovering disease-specific features directly from expression data. Using ensemble techniques, which combine the results of various metrics, we were able to more precisely capture biologically significant relationships between genes. We were able to find de novo potential disease-specific features with the help of prior biological knowledge and the development of new network inference techniques. Through our different approaches, we analyzed large gene sets across multiple samples and used gene expression as a surrogate marker for the inherent biological processes, reconstructing robust gene co-expression networks that are simple to explore. By mining disease-specific gene co-expression networks we come up with a useful framework for identifying new omics-phenotype associations from conditional expression datasets.In this sense, understanding diseases from the perspective of biological network perturbations will improve personalized medicine, impacting rational biomarker discovery, patient stratification and drug design, and ultimately leading to more targeted therapies.Universidad Pablo de Olavide de Sevilla. Departamento de Deporte e Informátic

    Targeting organogenesis and beta cell survival: role of the LRH1/NR5A2-PTGS2/COX2 signaling axis in pancreatic islet physiology and pathophysiology

    Get PDF
    Programa de Doctorado en BiotecnologĂ­a, IngenierĂ­a y TecnologĂ­a QuĂ­micaLĂ­nea de InvestigaciĂłn: BiotecnologĂ­a, Biomedicina y Ciencias de la SaludClave Programa: DBICĂłdigo LĂ­nea: 110Type 1 Diabetes Mellitus (T1DM) is a disease caused by the selective destruction of pancreatic islet beta cells by aberrant activation of the immune system, characterized by a subsequent chronic unresolved proinflammatory status within the pancreas. Up to date, no effective therapies have been developed to cure this autoimmune disorder, which indeed, apart from the beta cell death and subsequent lack of insulin, leads to long-term complications that substantially impact on life quality and shorten life expectancy. However, our laboratory recently reported promising outcomes from the in vivo activation of a nuclear receptor, denoted as Liver Receptor Homolog 1 (also known as (a.k.a.) Nuclear Receptor Subfamily 5 Group A Member 2, LRH1/NR5A2), using different preclinical mouse models of autoimmune diabetes, and also in vitro, by mimicking the stress/proinflammatory conditions that characterize T1DM in both, mouse and human primary islet-cell cultures. These beneficial effects derived from the treatment with a chemical agonist of LRH1/NR5A2, codename BL001, which potentially favoured a crosstalk between the immune system and islet cells, aimed at protecting the beta cell mass via increasing its survival. Understanding the molecular signaling and consequences derived from LRH1/NR5A2 expression and activation in beta cells was the following step to exploit its therapeutic value within T1DM conditions. In this Thesis, we first uncovered the essential role of LRH1/NR5A2 expression in beta cells during neonatal development. We found that the LRH1/NR5A2 constitutive ablation in the beta cell mass caused a significant reduction of this cell type, mainly characterized by blunted proliferation, along with detrimental consequences in the metabolic and physical health of mouse pups that culminated in early death. We next demonstrated that the LRH1/NR5A2 specific activation in beta cells was the responsible of the beneficial effects observed in vivo, after BL001 treatment. Using an inducible approach, LRH1/NR5A2 ablation in adult beta cells abolished the protective effect of BL001 in streptozotocin (STZ)-treated mice, correlating with an almost complete beta cell mass destruction. In order to get insight into the mode of action of this potential anti-diabetic drug in beta cells, we next explored the molecular branches of the BL001-LRH1/NR5A2 axis, focusing on the inducible Prostaglandin Endoperoxidase Synthase-2 gene (a.k.a. Cyclooxygenase-2, Ptgs2/Cox2), previously shown to be upregulated by BL001, and which plays a role in immunomodulation. Ptgs2/Cox2 downstream signaling involves the secretion of Prostaglandin E2 (PGE2) and activation of one or several Prostaglandin G-protein coupled receptors (a.k.a. E-Prostanoid receptors, PTGERs/EPs). We found that mouse islets treated in vitro with BL001 upon a proinflammatory cytokine (CTK) challenge produced PGE2 massively. Importantly, both silencing of Ptgs2/Cox2 gene or downstream blockade of the anti-apoptotic PTGER1/EP1 receptor negated BL001-mediated increased islet-cell survival upon the CTK treatment, establishing the molecular survival signaling axis in mouse beta cells as follows: BL001-LRH1/NR5A2-Ptgs2/Cox2-PGE2-PTGER1/EP1. In parallel, we uncovered the deleterious role of the pro-apoptotic PTGER3/EP3 in an in vivo context, using the RIP-B7.1 mouse model of autoimmune diabetes. We found that PTGER3/EP3 antagonism reduced insulitis and protected the beta cell mass in these animals. Finally, as a future therapy for T1DM, it was mandatory to translate our survival cascade to a human setting. As such, we successfully recapitulated part of this pathway in human induced-Pluripotent Stem Cells (hiPSCs) derived islet-like organoids. This research work provides a complete molecular characterization of LRH1/NR5A2 activation specifically in the beta cell mass, which could be further fine-tuned to finally develop a successful therapy for T1DM.Universidad Pablo de Olavide de Sevilla. Departamento de BiologĂ­a Molecular e IngenierĂ­a BioquĂ­mic

    Human VDAC pseudogenes: an emerging role for VDAC1P8 pseudogene in acute myeloid leukemia

    Get PDF
    Background Voltage-dependent anion selective channels (VDACs) are the most abundant mitochondrial outer membrane proteins, encoded in mammals by three genes, VDAC1, 2 and 3, mostly ubiquitously expressed. As ’mitochondrial gatekeepers’, VDACs control organelle and cell metabolism and are involved in many diseases. Despite the presence of numerous VDAC pseudogenes in the human genome, their significance and possible role in VDAC protein expression has not yet been considered. Results We investigated the relevance of processed pseudogenes of human VDAC genes, both in physiological and in pathological contexts. Using high-throughput tools and querying many genomic and transcriptomic databases, we show that some VDAC pseudogenes are transcribed in specific tissues and pathological contexts. The obtained experimental data confirm an association of the VDAC1P8 pseudogene with acute myeloid leukemia (AML). Conclusions Our in-silico comparative analysis between the VDAC1 gene and its VDAC1P8 pseudogene, together with experimental data produced in AML cellular models, indicate a specific over-expression of the VDAC1P8 pseudogene in AML, correlated with a downregulation of the parental VDAC1 gene. Keywords Pseudogene, Voltage-dependent anion selective channels (VDAC

    STUDIES OF EPIGENETICS AND PROPERTIES OF B CELL RECEPTOR IN WALDENSTRĂ–M MACROGLOBULINEMIA (WM)

    Get PDF
    Waldenström macroglobulinemia (WM) is an indolent lymphoproliferative disorder with aberrant monoclonal immunoglobulin M (IgM) production that is associated with disease symptoms. Despite significant advances in our understanding of disease biology, the cell of origin remains largely unknown. Although WM therapy options have improved over the last decade, WM is still an incurable disease. Many studies have identified epigenetic dysregulation as a regulatory factor in WM malignancy. Here, we investigated the role of Mixed-lineage leukemia 1 (MLL1) histone methyltransferase in WM. We showed that MLL1 and its partner Menin are upregulated in WM patients. We also found that MLL1 knockdown and pharmacological inhibition of MLL1 complex using the menin-MLL1 inhibitor (MI-2) significantly reduced IgM levels in-vitro and in-vivo. Further we showed that MLL1 binds to multiple sites in the 5\u27 Eμ enhancer of the IgM heavy chain (IGMH). We also found that MLL1 binding to IGH region was associated with increased histone 3 lysine 4 trimethylation (H3K4me3) enrichment at multiple MLL1 binding sites. Moreover, we described B cell receptor (BCR) repertoires of the currently available cell lines for WM (BCWM.1, MWCL-1 and RPCI-WM1). We also found abnormal transcription of immunoglobulin heavy chain (H-chain) and light chain (L-chain) transcripts with increased L:H-chain ratio in WM cell lines compared to normal B cells and this correlated with the amount of secreted IgM by each WM cell line. Finally, we identified that MI-2 inhibitor significantly reduced light chain expression in WM cells suggesting a role for MLL1 in regulation of light chain. Taken together, these results identify MLL1 as a regulator of IgM light and heavy chains that suggest MLL1 as a new therapeutic target for WM

    Live-cell imaging of non-coding RNAs dynamics in ALS condensates

    Get PDF
    In the last few years, interest around non-coding RNAs (ncRNAs) has been growing as they have been found to be involved in several physiological and pathological processes. In fact, their expression is highly enriched in neuronal tissues and, thanks to their complex and modular secondary structure, they can work as scaffold for other RNAs and proteins for the assembly of ribonucleoparticles (RNPs). These supramolecular structures are known to participate in axonal trafficking, a process usually impaired in neurodegenerative diseases such as Amyotrophic Lateral Sclerosis and in particular in the presence of mutations of several RNA binding proteins, among which FUS. In this context, it is crucial to investigate ncRNA dynamics and kinetics in live cells, in order to unveil novel mechanisms for the understanding of neurodegeneration. For this purpose, we managed to engineer the motor neuron enriched circRNA circ-Hdgfrp3 and the lncRNA HOTAIRM1 with an array of Pepper, a novel fluorescent aptamer that shows enhanced stability and brightness if compared with previously described fluorescent RNAs, allowing robust RNA imaging with minimal target perturbation. Combining widefield and structured illumination microscopy, we were able to confirm in live mammalian cells that circ-Hdgfrp3 is loaded in G3BP1 and FUSmut RNPs, possibly determining the mechanism through which it is recruited in pathological aggregates in motor neurons. Moreover, we also observed its interaction with DCP1A-tagged processing bodies, raising promising insight about its function and metabolism. Notably, we also determined HOTAIRM1 constitutive participation in stress granules, while we observed its involvement in the dynamics of FUSmut aggregation. Moreover, we were able to follow its behavior throughout the induction of oxidative stress, an event that leads to the production of aggregates containing several RNA binding proteins, including FUSmut. Via live imaging assays, we determined that its recruitment in stress granules is not mediated neither by G3BP1 nor by FUS, consistent with the observation that it does not interfere with stress granules assembly and that it preferentially joins the outer layers of such structures. Overall, as fluorescent RNA technologies are rapidly spreading and are improved for live-imaging applications, our work provides a novel approach for the investigation of ncRNAs’ implication in neurodegenerative diseases with a super-resolution potential in live neuronal cells
    • …
    corecore