10,037 research outputs found

    Fanconi anaemia and LINE-1 retrotransposition in the mammalian genome

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    Transposable elements (TEs) are discrete, repetitive sequences of DNA that mobilise within genomes. For decades, TEs were dismissed as “junk DNA”, however, it is now clear that these elements have the potential to trigger genome instability, cause disease and shape the course of genome evolution. L1 elements constitute the only autonomous elements which remain active in the human genome and comprises approximately 17% of human DNA. As a retrotransposon, L1 canonically mobilises through a “cut and paste” mechanism called target primed reverse transcription (TPRT). Due to the deleterious impacts of L1 activity, mammalian cells have evolved a range of mechanisms to supress the mobilisation of these elements. The interactions between L1 elements and the host factors which regulate them are therefore an area of active research. Several DNA repair genes have shown potential as regulators of L1 activity. Moreover, in cell lines deficient in non-homologous end-joining, L1 has shown the potential to retrotranspose without its ORF2p endonuclease, which is usually a requirement for canonical TPRT. This retrotransposition has been termed endonuclease independent (ENi) retrotransposition, and takes place at unrepaired double stranded breaks in the DNA. Interestingly, several DNA repair factors have also been identified as potential regulators of L1 retrotransposition (both positive and negative), including a number of proteins from the Fanconi Anaemia pathway. The relationship between these factors and L1 has yet to be fully characterised, and it remains to be seen whether L1 can exploit other DNA lesions in the way that it utilises DSBs in ENi retrotransposition. This thesis aims to further investigate the relationship between L1 retrotransposition in the mammalian genome and DNA repair factors, particularly those comprising the Fanconi Anaemia pathway. Using cultured cell retrotransposition assays, I systematically tested a battery of mutant element in cells deficient in different proteins of the FANC pathway. In this way, I establish that ENi retrotransposition can be observed in a FANC background. I also demonstrate that FANC A deficient cells support retrotransposition of several L1 mutants which are immobile in parental cell lines. This includes elements with severe ORF1p mutations, mutations in the ORF2p endonuclease domain and mutations in the ORF2p PIP box. Despite testing a range of cell lines deficient in different DNA repair factors, including cells deficient in a range of FANC proteins, the retrotransposition of ORF1p, PIP and mutants appears to be unique to FANC A. My results are potentially indicative of a unique mechanism of retrotransposition in FANC A cells, a phenomena which has precedence in the ENi pathway of retrotransposition. Mass spectrometry of immunoprecipitated T7-tagged ORF1p, both in FANC A and parental cells, demonstrated that a different selection of host factors interact with ORF1p in the two cell lines. Several of these have not been previously identified as L1 interactors, including YTHDF2, a protein which binds and destabilises m6A-containing RNA. Previous reports suggest that YTHDF2 regulates the stability of RNA:DNA hybrids in vivo, and associates with R loop containing loci. Through co-immunoprecipitation of YTHDF2 with ORF1p, I confirm that the protein interacts with L1 elements in vitro

    A novel dilated contextual attention module for breast cancer mitosis cell detection

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    Background and object: Mitotic count (MC) is a critical histological parameter for accurately assessing the degree of invasiveness in breast cancer, holding significant clinical value for cancer treatment and prognosis. However, accurately identifying mitotic cells poses a challenge due to their morphological and size diversity.Objective: We propose a novel end-to-end deep-learning method for identifying mitotic cells in breast cancer pathological images, with the aim of enhancing the performance of recognizing mitotic cells.Methods: We introduced the Dilated Cascading Network (DilCasNet) composed of detection and classification stages. To enhance the model’s ability to capture distant feature dependencies in mitotic cells, we devised a novel Dilated Contextual Attention Module (DiCoA) that utilizes sparse global attention during the detection. For reclassifying mitotic cell areas localized in the detection stage, we integrate the EfficientNet-B7 and VGG16 pre-trained models (InPreMo) in the classification step.Results: Based on the canine mammary carcinoma (CMC) mitosis dataset, DilCasNet demonstrates superior overall performance compared to the benchmark model. The specific metrics of the model’s performance are as follows: F1 score of 82.9%, Precision of 82.6%, and Recall of 83.2%. With the incorporation of the DiCoA attention module, the model exhibited an improvement of over 3.5% in the F1 during the detection stage.Conclusion: The DilCasNet achieved a favorable detection performance of mitotic cells in breast cancer and provides a solution for detecting mitotic cells in pathological images of other cancers

    Advanced cellular models for rare disease study: exploring neural, muscle and skeletal organoids

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    Organoids are self-organized, three-dimensional structures derived from stem cells that can mimic the structure and physiology of human organs. Patient-specific induced pluripotent stem cells (iPSCs) and 3D organoid model systems allow cells to be analyzed in a controlled environment to simulate the characteristics of a given disease by modeling the underlying pathophysiology. The recent development of 3D cell models has offered the scientific community an exceptionally valuable tool in the study of rare diseases, overcoming the limited availability of biological samples and the limitations of animal models. This review provides an overview of iPSC models and genetic engineering techniques used to develop organoids. In particular, some of the models applied to the study of rare neuronal, muscular and skeletal diseases are described. Furthermore, the limitations and potential of developing new therapeutic approaches are discussed

    Single-cell time-series analysis of metabolic rhythms in yeast

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    The yeast metabolic cycle (YMC) is a biological rhythm in budding yeast (Saccharomyces cerevisiae). It entails oscillations in the concentrations and redox states of intracellular metabolites, oscillations in transcript levels, temporal partitioning of biosynthesis, and, in chemostats, oscillations in oxygen consumption. Most studies on the YMC have been based on chemostat experiments, and it is unclear whether YMCs arise from interactions between cells or are generated independently by each cell. This thesis aims at characterising the YMC in single cells and its response to nutrient and genetic perturbations. Specifically, I use microfluidics to trap and separate yeast cells, then record the time-dependent intensity of flavin autofluorescence, which is a component of the YMC. Single-cell microfluidics produces a large amount of time series data. Noisy and short time series produced from biological experiments restrict the computational tools that are useful for analysis. I developed a method to filter time series, a machine learning model to classify whether time series are oscillatory, and an autocorrelation method to examine the periodicity of time series data. My experimental results show that yeast cells show oscillations in the fluorescence of flavins. Specifically, I show that in high glucose conditions, cells generate flavin oscillations asynchronously within a population, and these flavin oscillations couple with the cell division cycle. I show that cells can individually reset the phase of their flavin oscillations in response to abrupt nutrient changes, independently of the cell division cycle. I also show that deletion strains generate flavin oscillations that exhibit different behaviour from dissolved oxygen oscillations from chemostat conditions. Finally, I use flux balance analysis to address whether proteomic constraints in cellular metabolism mean that temporal partitioning of biosynthesis is advantageous for the yeast cell, and whether such partitioning explains the timing of the metabolic cycle. My results show that under proteomic constraints, it is advantageous for the cell to sequentially synthesise biomass components because doing so shortens the timescale of biomass synthesis. However, the degree of advantage of sequential over parallel biosynthesis is lower when both carbon and nitrogen sources are limiting. This thesis thus confirms autonomous generation of flavin oscillations, and suggests a model in which the YMC responds to nutrient conditions and subsequently entrains the cell division cycle. It also emphasises the possibility that subpopulations in the culture explain chemostat-based observations of the YMC. Furthermore, this thesis paves the way for using computational methods to analyse large datasets of oscillatory time series, which is useful for various fields of study beyond the YMC

    Dissecting structural and biochemical features of DNA methyltransferase 1

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    DNA methylation is an epigenetic modification found in every branch of life. An essential enzyme for the maintenance of DNA methylation patterns in mammals is DNA methyltransferase 1 (DNMT1). Its recruitment is regulated through its large N-terminus, which contains six annotated domains. Although most of these have been assigned a function, we are still lacking a holistic understanding of the enzyme's spatio-temporal regulation. Interestingly, a large segment of the N-terminus is devoid of any known domain and appears to be disordered in its sequence. Over the past years, such disordered sequences have increasingly gained attention, due to their role in forming biomolecular condensates through liquid-liquid phase separation (LLPS). These liquid compartments offer specific environmental conditions distinct from the surrounding that can enhance protein recruitment and function. In this work, we explore a potential role for the intrinsically disordered domain (IDR) in the recruitment of DNMT1. Taking an evolutionary approach, we uncover that structural features of the region that are key for IDR function are highly conserved. Moreover, we find conserved biochemical signatures compatible with a role in LLPS. Using a reconstitution assay and an opto-genetic approach in cells, we for the first time show that the DNMT1 IDR is capable of undergoing LLPS in vitro and in vivo. In addition, we define a novel region of interest (ROI) of about 120 amino acids in the IDR that appears to have been inserted in the ancestor of eutherian mammals. Although the ROI has a distinct biochemical signature, we find no effect on the LLPS behavior of the IDR. Therefore, we discuss other potential roles of the ROI related to DNA methylation, for example, imprinting. Finally, we lay the foundation for investigating a biological function of the IDR and establish a system for screening DNMT1 mutant phenotypes in mouse embryonic stem cells. Swift depletion of the endogenous protein is enabled by degron-mediated degradation, while our optimized construct design and efficient derivation strategy ensure the robust expression of the large transgenes. In combination with different methods for DNA methylation read-out, this system can now be used to study the role of the IDR and ROI in maintaining the steady-state level of DNA methylation against mechanisms of passive and active demethylation, but also for studying phenotypes affecting the efficiency of DNMT1 recruitment in the future

    Effects of municipal smoke-free ordinances on secondhand smoke exposure in the Republic of Korea

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    ObjectiveTo reduce premature deaths due to secondhand smoke (SHS) exposure among non-smokers, the Republic of Korea (ROK) adopted changes to the National Health Promotion Act, which allowed local governments to enact municipal ordinances to strengthen their authority to designate smoke-free areas and levy penalty fines. In this study, we examined national trends in SHS exposure after the introduction of these municipal ordinances at the city level in 2010.MethodsWe used interrupted time series analysis to assess whether the trends of SHS exposure in the workplace and at home, and the primary cigarette smoking rate changed following the policy adjustment in the national legislation in ROK. Population-standardized data for selected variables were retrieved from a nationally representative survey dataset and used to study the policy action’s effectiveness.ResultsFollowing the change in the legislation, SHS exposure in the workplace reversed course from an increasing (18% per year) trend prior to the introduction of these smoke-free ordinances to a decreasing (−10% per year) trend after adoption and enforcement of these laws (β2 = 0.18, p-value = 0.07; β3 = −0.10, p-value = 0.02). SHS exposure at home (β2 = 0.10, p-value = 0.09; β3 = −0.03, p-value = 0.14) and the primary cigarette smoking rate (β2 = 0.03, p-value = 0.10; β3 = 0.008, p-value = 0.15) showed no significant changes in the sampled period. Although analyses stratified by sex showed that the allowance of municipal ordinances resulted in reduced SHS exposure in the workplace for both males and females, they did not affect the primary cigarette smoking rate as much, especially among females.ConclusionStrengthening the role of local governments by giving them the authority to enact and enforce penalties on SHS exposure violation helped ROK to reduce SHS exposure in the workplace. However, smoking behaviors and related activities seemed to shift to less restrictive areas such as on the streets and in apartment hallways, negating some of the effects due to these ordinances. Future studies should investigate how smoke-free policies beyond public places can further reduce the SHS exposure in ROK

    Molecular docking, ADMET and molecular dynamics simulation revealed metralindole as a multitargeted inhibitor for division kinases

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    Abstract Lung cancer is the most common type of cancer in the world, and alone, in 2020, almost 2.21 million new cases were diagnosed, with 1.80 million deaths, and are increasing daily. Non-small cell lung (NSCLC) is the primary type of lung cancer, predominantly forms around 80% of cases compared to small cell carcinoma, and about 75% of patients are already in an advanced state when diagnosed. Despite notable advances in early diagnosis and treatment, the five-year survival rate for NSCLC is not encouraging. Therefore, it is crucial to investigate the molecular causes of non-small cell lung cancer to create more efficient therapeutic approaches. Lung cancer showed a more significant and persistent binding affinity and energy landscape with the target CDK2 staurosporine and FGF receptor-1. In this study, we have picked two essential target proteins, human cyclin-dependent kinase-2 and Human Protein Kinase CK2 Holoenzyme and screened the entire prepared DrugBank prepared library of 1,55,888 compounds and identified 2-(2-methyl-5-nitroimidazole-1-yl) ethanol (Metralindole) as a major inhibitor. Metralindole has displayed high docking scores of -5.159 Kcal/mol and -5.99 Kcal/mol with good hydrogen bonding and other bonding topologies such as van der Waals force, and ADMET results shown excellent bioavailability, outstanding solubility, no side effects, and toxicity. The molecular dynamics simulation for 100ns in a water medium confirmed the compound's stability and interaction pattern with the lowest deviation and fluctuations. Our in-silico study suggests Metralindole, an experimental compound, can effectively cure lung cancer. Further, the experimental validation of the compound is a must before any prescription

    Exploring therapeutic vulnerabilities in tumours with GLI1 oncogene activation

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    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
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