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In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.
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Cromatina , Epigénesis Genética , Genotipo , Mutación , Análisis de la Célula Individual , Animales , Femenino , Humanos , Masculino , Ratones , Antígenos CD34/metabolismo , Diferenciación Celular/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Epigénesis Genética/genética , Epigenoma/genética , Genoma Mitocondrial/genética , Técnicas de Genotipaje , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Inflamación/genética , Inflamación/patología , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Megacariocitos/metabolismo , Megacariocitos/patología , Proteínas de la Membrana/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , ARN/genética , Células Clonales/metabolismoRESUMEN
Transmissible cancers are malignant cell lineages that spread clonally between individuals. Several such cancers, termed bivalve transmissible neoplasia (BTN), induce leukemia-like disease in marine bivalves. This is the case of BTN lineages affecting the common cockle, Cerastoderma edule, which inhabits the Atlantic coasts of Europe and northwest Africa. To investigate the evolution of cockle BTN, we collected 6,854 cockles, diagnosed 390 BTN tumors, generated a reference genome and assessed genomic variation across 61 tumors. Our analyses confirmed the existence of two BTN lineages with hemocytic origins. Mitochondrial variation revealed mitochondrial capture and host co-infection events. Mutational analyses identified lineage-specific signatures, one of which likely reflects DNA alkylation. Cytogenetic and copy number analyses uncovered pervasive genomic instability, with whole-genome duplication, oncogene amplification and alkylation-repair suppression as likely drivers. Satellite DNA distributions suggested ancient clonal origins. Our study illuminates long-term cancer evolution under the sea and reveals tolerance of extreme instability in neoplastic genomes.
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Bivalvos , Cardiidae , Leucemia , Neoplasias , Animales , Humanos , Cardiidae/genética , Evolución ClonalRESUMEN
Current therapies against prostate cancer (PCa) disease, such as surgery, radiotherapy, or in last term chemical castration by androgen deprivation, have led to significant reduction of the incidence of PCa throughout the world. Worse prognosis is found in those patients which exhibit castration resistance, relapsing into the disease with even greater aggressiveness. Hypoxia cancer cell adaption has been observed to be closely connected to fatal prognostic tumor features. Therefore, hypoxia adaptive mechanisms of cancer cells have attracted large interest as a relevant biological target for treatment-resistant patients. Dendrimers have been established as a promising nanotechnological tool owing to their beneficial physicochemical features such as multivalency and monodispersity. Herein, we have completed a thorough study to better understand the effect within the cell of the already published ruthenium(II)-N-heterocyclic carbene metallodendrimer (G2Ru) that was able to drastically reduce HIF-1α stabilization and exhibited antiproliferative capability against androgen-sensitive (LNCaP) and androgen-resistant prostate cancer cells (LNFLU) in vitro. G2Ru, as well as its cationic imidazolium precursor (G2P), displayed scavenging properties against intracellular and externally stimulated ROS levels, which would presumably hinder the stabilization of HIF-1α by prolyl hydroxylase (PHD) inhibition. Furthermore, these dendrimers have shown considerably beneficial properties against tumor progression capability in terms of apoptosis, cell cycle, CSCs expression, and epithelial phenotype promotion. Taken all together, in this study we could demonstrate the extraordinary anticancer properties of NHC-based carbosilane dendrimers against androgen-resistant prostate cancer cells in vitro.
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Andrógenos , Neoplasias de la Próstata , Humanos , Masculino , Andrógenos/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Factor A de Crecimiento Endotelial Vascular , Antagonistas de Andrógenos , Línea Celular Tumoral , Transducción de Señal , Ciclo Celular , HipoxiaRESUMEN
With the purpose of obtaining a new dendritic system against cancer, this paper is focused on the synthesis of spherical carbosilane metallodendrimers of different generations holding Ru(II) N-heterocyclic carbene (NHC) on the periphery from the imidazolium precursors. Both imidazolium salt dendrimers and their metallodendrimers counterparts showed promising anticancer activity, similar to cisplatin, mainly at high generations. In addition, both families of second and third generations were able to form dendriplexes with anticancer small interfering RNA (siRNA), protecting the cargo against RNAse and being able to internalize it in HEPG2 (human liver cancer) tumour cells. The characterization and effectiveness of the dendriplexes were evaluated by various analytical techniques such as zeta potential, electrophoresis and circular dichroism, the stability of the system and the protective nature of the dendrimer estimated using RNAse and the internalization of dendriplexes by confocal microscopy. The major advantage observed with the ruthenium metallodendrimers with respect to the imidazolium salts precursors was in cellular uptake, where the internalization of Mcl-1-FITC siRNA (myeloid cell leukaemia-1 fluorescein labelled siRNA) proceeded more efficiently. Therefore, we propose here that both imidazolium and Ru metallodendrimers are interesting candidates in cancer due to their double action, as anticancer per se and as carrier for anticancer siRNA, providing in this way a combined action.
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Antineoplásicos/farmacología , Complejos de Coordinación/farmacología , Dendrímeros/farmacología , Portadores de Fármacos/farmacología , Compuestos Organometálicos/farmacología , ARN Interferente Pequeño/farmacología , Antineoplásicos/síntesis química , Complejos de Coordinación/síntesis química , Dendrímeros/síntesis química , Portadores de Fármacos/síntesis química , Ensayos de Selección de Medicamentos Antitumorales , Células Hep G2 , Humanos , Imidazoles/síntesis química , Imidazoles/farmacología , Compuestos Organometálicos/síntesis química , Rutenio/químicaRESUMEN
This work is focused on the electroanalytical study of a family of five imidazolium-terminated carbosilane dendrimers (from generation G1 to G3) at the polarized liquid-liquid interface formed between water and 1,2-dichloroethane solutions. All dendrimers with permanently and positively charged imidazolium groups located at the periphery within the branched carbosilane core were found to be electrochemically active. Based on the concentration and scan rate dependencies we have concluded that these molecules undergo interfacial ion transfer processes accompanied by interfacial adsorption/desorption rather than the electrochemically induced interfacial formation of the macromolecule-anion (tetrakis(4-chlorophenyl)borate) from the organic phase complex. Also, we report several physicochemical and electroanalytical parameters (e.g. diffusion coefficients, LODs, and detection sensitivities) for the studied family of dendrimers. Our work aims to contribute to the understating of the interaction between branched macromolecules and biomimetic interfaces.
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Dendrímeros , Adsorción , Dicloruros de Etileno , SilanosRESUMEN
Antibodies are macromolecules that specifically recognize their target, making them good candidates to be employed in various therapies. The possibility of attaching a drug to an immunoglobulin makes it possible to release it specifically into the affected tissue as long as it overexpresses the target. However, chemical coupling could affect the functionality (specificity and affinity) of the antibody. It has been observed that the use of intermediaries, such as dendrimers, could resolve this issue. Because carbosilane dendrimers have aroused great interest in the field of biomedicine, this report describes the synthesis of an anionic carbosilane dendrimer with a fluorochrome on its surface that then forms a conjugate with an antibody. It has been used as immunoglobulin and infliximab, whose target is TNF-α, which is a cytokine that is overexpressed in the inflamed area or even in the blood of patients with autoimmune diseases, such as rheumatoid arthritis. In addition, the integrity and functionality of the antibody has been studied to see if they have been affected after the chemical coupling process.
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Artritis Reumatoide/tratamiento farmacológico , Dendrímeros/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Infliximab/uso terapéutico , Nanomedicina Teranóstica , Dendrímeros/síntesis química , Infliximab/farmacología , Espectroscopía de Protones por Resonancia Magnética , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Spherical dendrimers and dendrons containing silver(I) N-heterocyclic carbenes (Ag(I)-NHC) and additionally bow-tie metal-free dendritic systems were synthesized in a simple and straightforward synthetic procedure and subsequently characterized. The antibacterial activity was evaluated, and in parallel, a comparative study with the cationic analogue precursors was performed to explore the effect of silver ions in the dendritic structure. Other parameters, such as topology, generation, and hydrophobicity, of the imidazole substituents were also studied. All these dendritic systems presented antibacterial activity against three different bacterial strains, two Gram-positive (Staphylococcus aureus and Bacillus subtilis) and one Gram-negative (Escherichia coli). Several assays were conducted to elucidate their mechanism of action against Bacillus subtilis, by using bacterial biosensors or specific probes and fluorescent proteins sensitive to changes in the cell membrane potential. These studies are specially focused on the role of the polyvalence of our systems containing silver atoms, which may provoke interesting effects in the mode of action.
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The theory of evolution is one of the greatest scientific achievements in the intellectual history of humankind, yet it is still contentious within certain social groups. Despite being as robust and evidence-based as any other notable scientific theory, some people show a strong reluctance to accept it. In this study, we used the Measure of Acceptance of the Theory of Evolution (MATE) and Knowledge of Evolution Exam (KEE) questionnaires with university students from four academic degree programs (Chemistry, English, History, and Biology) of ten universities from Spain to measure, respectively, acceptance and knowledge of evolutionary theory among third-year undergraduate students (nMATE = 978; nKEE = 981). Results show that acceptance of evolution is relatively high (87.2%), whereas knowledge of the theory is moderate (5.4 out of 10) although there are differences across degrees (Biology>Chemistry>History>English), and even among various universities (ranging from 4.71 to 5.81). Statistical analysis reveals that knowledge of evolutionary theory among Biology students is partially explained by the relative weight of evolutionary themes within the curriculum, suggesting that an increase in the number of hours dedicated to this topic could have a direct influence on students' knowledge of it. We also found that religion may have a significant-although relatively small-negative influence on evolutionary theory acceptance. The moderate knowledge of evolution in our undergraduate students, together with the potential problem of acceptance in certain groups, suggests the need for a revision of the evolutionary concepts in the teaching curricula of our students since primary school.
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Evolución Biológica , Conocimiento , Estudiantes/psicología , Curriculum , Humanos , España , Encuestas y Cuestionarios , Universidades , Adulto JovenRESUMEN
Tumor samples most often comprise a mixture of different cell lineages. Multiregional trees built from bulk mutational profiles do not consider this heterogeneity and can potentially lead to erroneous evolutionary inferences, including biased timing of somatic mutations, spurious parallel mutation events, and/or incorrect chronological ordering of metastatic events.