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So far, the mechanisms that impede AAV transduction, especially in the human heart, are poorly understood, hampering the introduction of new, effective gene therapy strategies. Therefore, the aim of this study was to identify and overcome the main cellular barriers to successful transduction in the heart, using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs), iPSC-derived cardiac fibroblasts (iPSC-CFs), and primary endothelial cells to model vector-host interactions. Through phosphoproteome analysis we established that casein kinase 2 (CK2) signaling is one of the most significantly affected pathways upon AAV exposure. Transient inhibition of CK2 activity substantially enhanced the transduction rate of AAV2, AAV6, and AAV9 in all tested cell types. In particular, CK2 inhibition improved the trafficking of AAVs through the cytoplasm, impaired DNA damage response through destabilization of MRE11, and altered the RNA processing pathways, which were also highly responsive to AAV transduction. Also, it augmented transgene expression in already transduced iPSC-CFs, which retain AAV genomes in a functional, but probably silent form. In summary, the present study provides new insights into the current understanding of the host-AAV vector interaction, identifying CK2 activity as a key barrier to efficient transduction and transgene expression, which may translate to improving the outcome of AAV-based therapies in the future.
Assuntos
Caseína Quinase II , Células Endoteliais , Humanos , Transdução Genética , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Terapia Genética , Transgenes , Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos/genéticaRESUMO
Fusion proteins involving Nucleoporin 98 (NUP98) are recurrently found in acute myeloid leukemia (AML) and are associated with poor prognosis. Lack of mechanistic insight into NUP98-fusion-dependent oncogenic transformation has so far precluded the development of rational targeted therapies. We reasoned that different NUP98-fusion proteins deregulate a common set of transcriptional targets that might be exploitable for therapy. To decipher transcriptional programs controlled by diverse NUP98-fusion proteins, we developed mouse models for regulatable expression of NUP98/NSD1, NUP98/JARID1A, and NUP98/DDX10. By integrating chromatin occupancy profiles of NUP98-fusion proteins with transcriptome profiling upon acute fusion protein inactivation in vivo, we defined the core set of direct transcriptional targets of NUP98-fusion proteins. Among those, CDK6 was highly expressed in murine and human AML samples. Loss of CDK6 severely attenuated NUP98-fusion-driven leukemogenesis, and NUP98-fusion AML was sensitive to pharmacologic CDK6 inhibition in vitro and in vivo. These findings identify CDK6 as a conserved, critical direct target of NUP98-fusion proteins, proposing CDK4/CDK6 inhibitors as a new rational treatment option for AML patients with NUP98-fusions.
Assuntos
Quinase 6 Dependente de Ciclina/metabolismo , Sistemas de Liberação de Medicamentos , Leucemia Mieloide Aguda/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Animais , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/genética , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genéticaRESUMO
Mutations in the CEBPA gene are present in 10-15% of acute myeloid leukemia (AML) patients. The most frequent type of mutations leads to the expression of an N-terminally truncated variant of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), termed p30. While initial reports proposed that p30 represents a dominant-negative version of the wild-type C/EBPα protein, other studies show that p30 retains the capacity to actively regulate gene expression. Recent global transcriptomic and epigenomic analyses have advanced the understanding of the distinct roles of the p30 isoform in leukemogenesis. This review outlines direct and indirect effects of the C/EBPα p30 variant on oncogenic transformation of hematopoietic progenitor cells and discusses how studies of N-terminal CEBPA mutations in AML can be extrapolated to identify novel gain-of-function features in oncoproteins that arise from recurrent truncating mutations in transcription factors.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Mutação com Ganho de Função/genética , Leucemia Mieloide Aguda/genética , Animais , Regulação Neoplásica da Expressão Gênica/genética , Células-Tronco Hematopoéticas/fisiologia , HumanosRESUMO
Rivers are powerful systems supporting human civilization, but despite the enormous dependence on rivers by humans, this does not stop them to assault rivers in the most varied ways. Such dependency determines the establishment of strong river flow-human relationships, and river degradation the prompting of health and non-tangible complications for humans. This work assesses how river regulation, interacting with sociodemographic characteristics, influences the affinity for nature and the perception of humans regarding its effects on river systems. Increased affinity for nature and clearer perceptions about the effects of river regulation improve emotive connection with nature and promote pro-environmental concerns towards a more sustainable water management. Two case studies were selected with different river regulation types (run-of-river and storage reservoir). In each one, the affinity for nature and social perceptions were assessed via telephone-assisted questionnaire surveys carried out in 2020 using 402 randomly selected numbers of local human communities living in its influence areas. Results showed that despite river regulation, communities remain connected to the river system with well-established flow-human relationships. Nonetheless, these relationships have changed due to socioeconomic and cultural changes over time. Significant differences were found in educational attainment and age regarding the affinity for nature. On the other hand, gender differs significantly regarding both the affinity for nature and how the river regulation affect perception, highlighting a gender gap motivated by social and cultural customs passed throughout generations. The lower education level of women and less frequent use of the river acts as a barrier to their perception of river ecosystems and the regulation effects. The affinity for nature and the perception of ecosystems changes by local populations were also significantly different according to the river regulation type, where residents near the run-of-river dam present less affinity for nature. Notwithstanding, the perceptions of local communities were in general in accordance with the scientific knowledge on rivers' condition. Finally, this work highlights the necessity for education through schools, local communities, municipalities and families, providing conditions for dedication and time to nature and promoting environmental knowledge through direct experience.
Assuntos
Ecossistema , Rios , Feminino , Humanos , ConhecimentoRESUMO
Down's syndrome is the most frequent genetic cause of intellectual disability. As the risk for developing Alzheimer's disease is increased in Down's syndrome, comprehensive cognitive examination is essential, both in young adults (for baseline evaluation), as well as later for diagnosing dementia. So far, there are only a few recommendations for neuropsychological assessment in Down's syndrome. Here, we review (1) the development of cognition across the life span, (2) various causes of cognitive change in adults with Down's syndrome, and (3) procedures available for their evaluation. Furthermore, (4) we provide recommendations for the assessment and interpretation of diagnostic findings in adults with intellectual disabilities. We conclude with recommendations for cognitive assessment in intellectual disability in general.
Assuntos
Doença de Alzheimer , Síndrome de Down , Cognição , Síndrome de Down/complicações , Síndrome de Down/diagnóstico , Humanos , Longevidade , Testes Neuropsicológicos , Adulto JovemRESUMO
There have been increasing calls for triggering and sustaining a large-scale transition toward healthier and more sustainable food systems. To help materialize this transition, the present work aims to inform efforts for developing, marketing and promoting plant-based meals and plant-forward lifestyles, following a consumption-focused approach. The findings (Nparticipantsâ¯=â¯1600, Portugal; 52.6% female, Mageâ¯=â¯48.30) allowed to identify trends and differences on three sets of variables - (a) current eating habits (i.e., meat, fish, and plant-based meals), (b) consumer willingness to change (i.e., reduce meat consumption, follow a plant-based diet, maintain the status quo), and (c) enablers for eating plant-based meals more often (i.e., capability, opportunity, motivation) -, considering consumer orientations toward consumption in general, and food consumption in particular. Taken together, the results suggested that some consumption orientations were aligned with the transition to more plant-based diets (e.g., food orientation toward naturalness), others were open to - but not yet materialized in - the transition (e.g., general orientation toward consumption as exploration), and still others were in tension with the transition (e.g., food orientation toward pleasure). The discussion calls for developing and testing pathways to reduce meat consumption and increase plant-based eating which capture and build upon a range of consumption orientations, rather than against them.
Assuntos
Dieta Vegetariana/psicologia , Dieta/psicologia , Comportamento Alimentar/psicologia , Abastecimento de Alimentos , Motivação , Adulto , Comportamento de Escolha , Conservação dos Recursos Naturais , Dieta/métodos , Feminino , Humanos , Masculino , Marketing , Pessoa de Meia-Idade , PortugalRESUMO
The treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) induces granulocytic differentiation. This process renders APL cells resistant to cytotoxic chemotherapies. Epigenetic regulators of the histone deacetylases (HDACs) family, which comprise four classes (I-IV), critically control the development and progression of APL. We set out to clarify the parameters that determine the interaction between ATRA and histone deacetylase inhibitors (HDACi). Our assays included drugs against class I HDACs (MS-275, VPA, and FK228), pan-HDACi (LBH589, SAHA), and the novel HDAC6-selective compound Marbostat-100. We demonstrate that ATRA protects APL cells from cytotoxic effects of SAHA, MS-275, and Marbostat-100. However, LBH589 and FK228, which have a superior substrate-inhibitor dissociation constant (Ki) for the class I deacetylases HDAC1, 2, 3, are resistant against ATRA-dependent cytoprotective effects. We further show that HDACi evoke DNA damage, measured as induction of phosphorylated histone H2AX and by the comet assay. The ability of ATRA to protect APL cells from the induction of p-H2AX by HDACi is a readout for the cytoprotective effects of ATRA. Moreover, ATRA increases the fraction of cells in the G1 phase, together with an accumulation of the cyclin-dependent kinase inhibitor p21 and a reduced expression of thymidylate synthase (TdS). In contrast, the ATRA-dependent activation of the transcription factors STAT1, NF-κB, and C/EBP hardly influences the responses of APL cells to HDACi. We conclude that the affinity of HDACi for class I HDACs determines whether such drugs can kill naïve and maturated APL cells.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Leucemia/tratamento farmacológico , Leucemia/patologia , Tretinoína/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Leucemia/metabolismo , NF-kappa B/metabolismo , Piridinas/farmacologia , Fator de Transcrição STAT1/metabolismo , Tretinoína/administração & dosagemRESUMO
Morphological studies of skeletal muscle tissue provide insights into the architecture of muscle fibers, the surrounding cells, and the extracellular matrix (ECM). However, a spatial proteomics analysis of the skeletal muscle including the muscle-tendon transition zone is lacking. Here, we prepare cryotome muscle sections of the mouse soleus muscle and measure each slice using short liquid chromatography-mass spectrometry (LC-MS) gradients. We generate 3,000 high-resolution protein profiles that serve as the basis for a network analysis to reveal the complex architecture of the muscle-tendon junction. Among the protein profiles that increase from muscle to tendon, we find proteins related to neuronal activity, fatty acid biosynthesis, and the renin-angiotensin system (RAS). Blocking the RAS in cultured mouse tenocytes using losartan reduces the ECM synthesis. Overall, our analysis of thin cryotome sections provides a spatial proteome of skeletal muscle and reveals that the RAS acts as an additional regulator of the matrix within muscle-tendon junctions.
Assuntos
Músculo Esquelético , Proteômica , Tendões , Animais , Proteômica/métodos , Músculo Esquelético/metabolismo , Tendões/metabolismo , Camundongos , Matriz Extracelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Sistema Renina-Angiotensina/fisiologia , Adaptação Fisiológica , Proteoma/metabolismo , Losartan/farmacologiaRESUMO
In recent years, a novel treatment method for cancer has emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the l-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata. Previously isolated from the ink, the l-amino acid oxidase was described to oxidate the essential amino acids l-lysine and l-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of the amino acid oxidase Aplysia punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose-dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG, which led to deprivation of arginine, was well tolerated.
Assuntos
Aplysia , Arginina , Lisina , Polietilenoglicóis , Animais , Arginina/farmacologia , Arginina/química , Lisina/farmacologia , Lisina/química , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Toxinas Marinhas/farmacologia , Toxinas Marinhas/uso terapêutico , Toxinas Marinhas/química , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , L-Aminoácido Oxidase/farmacologia , L-Aminoácido Oxidase/metabolismo , L-Aminoácido Oxidase/química , Feminino , Linhagem Celular TumoralRESUMO
Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease. Although it leads to muscle weakness, affected individuals predominantly die from cardiomyopathy, which remains uncurable. Accumulating evidence suggests that an overexpression of utrophin may counteract some of the pathophysiological outcomes of DMD. The aim of this study was to investigate the role of utrophin in dystrophin-deficient human cardiomyocytes (CMs) and to test whether an overexpression of utrophin, implemented via the CRISPR-deadCas9-VP64 system, can improve their phenotype. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) lacking either dystrophin (DMD) or both dystrophin and utrophin (DMD KO/UTRN(+/-)). We carried out proteome analysis, which revealed considerable differences in the proteins related to muscle contraction, cell-cell adhesion, and extracellular matrix organization. Furthermore, we evaluated the role of utrophin in maintaining the physiological properties of DMD hiPSC-CMs using atomic force microscopy, patch-clamp, and Ca2+ oscillation analysis. Our results showed higher values of afterhyperpolarization and altered patterns of cytosolic Ca2+ oscillations in DMD; the latter was further disturbed in DMD KO/UTRN(+/-) hiPSC-CMs. Utrophin upregulation improved both parameters. Our findings demonstrate for the first time that utrophin maintains the physiological functions of DMD hiPSC-CMs, and that its upregulation can compensate for the loss of dystrophin.
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Mutations in the gene encoding the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) occur in 10-15% of acute myeloid leukemia (AML). Frameshifts in the CEBPA N-terminus resulting in exclusive expression of a truncated p30 isoform represent the most prevalent type of CEBPA mutations in AML. C/EBPα p30 interacts with the epigenetic machinery, but it is incompletely understood how p30-induced changes cause leukemogenesis. We hypothesized that critical effector genes in CEBPA-mutated AML are dependent on p30-mediated dysregulation of the epigenome. We mapped p30-associated regulatory elements (REs) by ATAC-seq and ChIP-seq in a myeloid progenitor cell model for p30-driven AML that enables inducible RNAi-mediated knockdown of p30. Concomitant p30-dependent changes in gene expression were measured by RNA-seq. Integrative analysis identified 117 p30-dependent REs associated with 33 strongly down-regulated genes upon p30-knockdown. CRISPR/Cas9-mediated mutational disruption of these genes revealed the RNA-binding protein MSI2 as a critical p30-target. MSI2 knockout in p30-driven murine AML cells and in the CEBPA-mutated human AML cell line KO-52 caused proliferation arrest and terminal myeloid differentiation, and delayed leukemia onset in vivo. In summary, this work presents a comprehensive dataset of p30-dependent effects on epigenetic regulation and gene expression and identifies MSI2 as an effector of the C/EBPα p30 oncoprotein.
Assuntos
Biomarcadores Tumorais/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/patologia , Mutação , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular , Hematopoese , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Prognóstico , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genéticaRESUMO
We conducted multiple laboratory trials in a robust and repeatable experimental layout to study dense non-aqueous phase liquid (DNAPL) source zone formation. We extended an image processing and analysis framework to derive DNAPL saturation distributions from reflective optical imaging data, with volume balance deviations <â¯5.07%. We used a multiphase flow model to simulate source zone formation in a Monte Carlo approach, where the parameter space was defined by the variation of retention curve parameters. Integral and geometric measures were used to characterize the source zones and implemented into a multi-criteria objective function. The latter showed good agreement between observation data and simulation results for effective DNAPL saturation values >â¯0.04, especially for early stages of DNAPL migration. The common hypothesis that parameters defining the DNAPL-water retention curves are constant over time was not confirmed. Once DNAPL pooling started, the optimal fit in the parameter space was significantly different compared to the earlier DNAPL migration stages. We suspect more complex processes (e.g., capillary hysteresis, adsorption) to become relevant during pool formation. Our results reveal deficits in the grayscale-DNAPL saturation relationship definition and laboratory estimation of DNAPL-water retention curve parameters to overcome current limitations to describe DNAPL source zone formation.
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The gene encoding the transcription factor C/EBPα is mutated in 10-15% of acute myeloid leukemia (AML) patients. N-terminal CEBPA mutations cause ablation of full-length C/EBPα without affecting the expression of a shorter oncogenic isoform, termed p30. The mechanistic basis of p30-induced leukemogenesis is incompletely understood. Here, we demonstrate that the MLL1 histone-methyltransferase complex represents a critical actionable vulnerability in CEBPA-mutated AML. Oncogenic C/EBPα p30 and MLL1 show global co-localization on chromatin and p30 exhibits robust physical interaction with the MLL1 complex. CRISPR/Cas9-mediated mutagenesis of MLL1 results in proliferation arrest and myeloid differentiation in C/EBPα p30-expressing cells. In line, CEBPA-mutated hematopoietic progenitor cells are hypersensitive to pharmacological targeting of the MLL1 complex. Inhibitor treatment impairs proliferation and restores myeloid differentiation potential in mouse and human AML cells with CEBPA mutations. Finally, we identify the transcription factor GATA2 as a direct critical target of the p30-MLL1 interaction. Altogether, we show that C/EBPα p30 requires the MLL1 complex to regulate oncogenic gene expression and that CEBPA-mutated AML is hypersensitive to perturbation of the MLL1 complex. These findings identify the MLL1 complex as a potential therapeutic target in AML with CEBPA mutations.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Sistemas CRISPR-Cas , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia Mieloide Aguda/patologia , Mutação , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Diferenciação Celular , Proliferação de Células , Fator de Transcrição GATA2 , Hematopoese , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células Tumorais CultivadasRESUMO
Mutations at the N- or C-terminus of C/EBPα are frequent in acute myeloid leukaemia (AML) with normal karyotype. Here, we investigate the role of the transcription factor Myb in AMLs driven by different combinations of CEBPA mutations. Using knockdown of Myb in murine cell lines modelling the spectrum of CEBPA mutations, we show that the effect of reduced Myb depends on the mutational status of the two Cebpa alleles. Importantly, Myb knockdown fails to override the block in myeloid differentiation in cells with biallelic N-terminal C/EBPα mutations, demonstrating for the first time that the dependency on Myb is much lower in AML with this mutational profile. By comparing gene expression following Myb knockdown and chromatin immunoprecipitation sequencing data for the binding of C/EBPα isoforms, we provide evidence for a functional cooperation between C/EBPα and Myb in the maintenance of AML. This co-dependency breaks down when both alleles of CEBPA harbour N-terminal mutations, as a subset of C/EBPα-regulated genes only bind the short p30 C/EBPα isoform and, unlike other C/EBPα-regulated genes, do so without a requirement for Myb.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação/genética , Proteínas Proto-Oncogênicas c-myb/genética , Alelos , Animais , Apoptose/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Fenótipo , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
Unresolved replication intermediates can block the progression of replication forks and become converted into DNA lesions, hence exacerbating genomic instability. The p53-binding protein 1 (53BP1) forms nuclear bodies at sites of unrepaired DNA lesions to shield these regions against erosion, in a manner dependent on the DNA damage kinase ATM. The molecular mechanism by which ATM is activated upon replicative stress to localize the 53BP1 protection complex is unknown. Here we show that the ATM-INteracting protein ATMIN (also known as ASCIZ) is partially required for 53BP1 localization upon replicative stress. Additionally, we demonstrate that ATM activation is impaired in cells lacking ATMIN and we define that ATMIN is required for initiating ATM signaling following replicative stress. Furthermore, loss of ATMIN leads to chromosomal segregation defects. Together these data reveal that chromatin integrity depends on ATMIN upon exposure to replication-induced stress.