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The tangerine pathotype of Alternaria alternata produces the Alternaria citri toxin (ACT), which elicits a host immune response characterized by the increase in harmful reactive oxygen species (ROS) production. ROS detoxification in A. alternata relies on the degradation of peroxisomes through autophagy and iron acquisition using siderophores. In this study, we investigated the role of autophagy in regulating siderophore and iron homeostasis in A. alternata. Our results showed that autophagy positively influences siderophore production and iron uptake. The A. alternata strains deficient in autophagy-related genes 1 and 8 (ΔAaatg1 and ΔAaatg8) could not thrive without iron, and their adaptability to high-iron environments was also reduced. Furthermore, the ability of autophagy-deficient strains to withstand ROS was compromised. Notably, autophagy deficiency significantly reduced the production of dimerumic acid (DMA), a siderophore in A. alternata, which may contribute to ROS detoxification. Compared to the wild-type strain, ΔAaatg8 was defective in cellular iron balances. We also observed iron-induced autophagy and lipid peroxidation in A. alternata. To summarize, our study indicates that autophagy and maintaining iron homeostasis are interconnected and contribute to the stress resistance and the virulence of A. alternata. These results provide new insights into the complex interplay connecting autophagy, iron metabolism, and fungal pathogenesis in A. alternata.
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Alternaria , Autofagia , Ferro , Espécies Reativas de Oxigênio , Autofagia/genética , Sideróforos , HomeostaseRESUMO
Bone regeneration involves multiple factors such as tissue interactions, an inflammatory response, and vessel formation. In the event of diseases, old age, lifestyle, or trauma, bone regeneration can be impaired which could result in a prolonged healing duration or requiring an external intervention for repair. Currently, bone grafts hold the golden standard for bone regeneration. However, several limitations hinder its clinical applications, e.g., donor site morbidity, an insufficient tissue volume, and uncertain post-operative outcomes. Bone tissue engineering, involving stem cells seeded onto scaffolds, has thus been a promising treatment alternative for bone regeneration. Adipose-derived mesenchymal stem cells (AD-MSCs) are known to hold therapeutic value for the treatment of various clinical conditions and have displayed feasibility and significant effectiveness due to their ease of isolation, non-invasive, abundance in quantity, and osteogenic capacity. Notably, in vitro studies showed AD-MSCs holding a high proliferation capacity, multi-differentiation potential through the release of a variety of factors, and extracellular vesicles, allowing them to repair damaged tissues. In vivo and clinical studies showed AD-MSCs favoring better vascularization and the integration of the scaffolds, while the presence of scaffolds has enhanced the osteogenesis potential of AD-MSCs, thus yielding optimal bone formation outcomes. Effective bone regeneration requires the interplay of both AD-MSCs and scaffolds (material, pore size) to improve the osteogenic and vasculogenic capacity. This review presents the advances and applications of AD-MSCs for bone regeneration and bone tissue engineering, focusing on the in vitro, in vivo, and clinical studies involving AD-MSCs for bone tissue engineering.
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Tecido Adiposo , Regeneração Óssea , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Osteogênese , Engenharia Tecidual , Alicerces Teciduais , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Tecido Adiposo/citologia , Animais , Transplante de Células-Tronco Mesenquimais/métodos , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Diferenciação CelularRESUMO
BACKGROUND: Umbilical cord blood (UCB) is a source of hematopoietic stem cells for transplantation, offering an alternative for patients unable to find a matched adult donor. UCB is also a versatile source of hematopoietic stem and progenitor cells (hCD34 + HSPCs) for research into hematologic diseases, in vitro expansion, ex vivo gene therapy, and adoptive immunotherapy. For these studies, there is a need to isolate hCD34 + HSPCs from cryopreserved units, and protocols developed for isolation from fresh cord blood are unsuitable. STUDY DESIGN: This study describes a modified method for isolating hCD34 + HSPCs from cryopreserved UCB. It uses the Plasmatherm system for thawing, followed by CD34 microbead magnetic-activated cell sorting isolation with a cell separation kit (Whole Blood Columns, Miltenyi Biotec). hCD34 + HSPC phenotypes and functionality were assessed in vitro and hematologic reconstitution determined in vivo in immunodeficient mice. RESULTS: Total nucleated cell recovery after thawing and washing was 44.7 ± 11.7%. Recovery of hCD34 + HSPCs after application of thawed cells to Whole Blood Columns was 77.5 ± 22.6%. When assessed in two independent laboratories, the hCD34+ cell purities were 71.7 ± 10.7% and 87.8 ± 2.4%. Transplantation of the enriched hCD34 + HSPCs into NSG mice revealed the presence of repopulating hematopoietic stem cells (estimated frequency of 0.07%) and multilineage engraftment. CONCLUSION: This provides a simplified protocol for isolating high-purity human CD34 + HSPCs from banked UCB adaptable to current Good Manufacturing Practice. This protocol reduces the number of steps and associated risks and thus total production costs. Importantly, the isolated CD34 + HSPCs possess in vivo repopulating activity in immunodeficient mice, making them a suitable starting population for ex vivo culture and gene editing.
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Antígenos CD34/metabolismo , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco/citologia , Animais , Criopreservação , Edição de Genes , Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoterapia , Camundongos , Células-Tronco/metabolismoRESUMO
The management and reconstruction of critical-sized segmental bone defects remain a major clinical challenge for orthopaedic clinicians and surgeons. In particular, regenerative medicine approaches that involve incorporating stem cells within tissue engineering scaffolds have great promise for fracture management. This narrative review focuses on the primary components of bone tissue engineering-stem cells, scaffolds, the microenvironment, and vascularisation-addressing current advances and translational and regulatory challenges in the current landscape of stem cell therapy for critical-sized bone defects. To comprehensively explore this research area and offer insights for future treatment options in orthopaedic surgery, we have examined the latest developments and advancements in bone tissue engineering, focusing on those of clinical relevance in recent years. Finally, we present a forward-looking perspective on using stem cells in bone tissue engineering for critical-sized segmental bone defects.
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Background: Pulmonary arterial hypertension (PAH) is a condition that limits the quality of life and life expectancy. The predicted mortality at 1 year is estimated at 30-40% without treatment. Of the types of PAH, chronic thromboembolic pulmonary hypertension (CTEPH) is most amenable to treatment and guidelines recommend pulmonary endarterectomy (PEA) surgery for 'operable' patients (where disease is found in the proximal pulmonary vessels). Traditionally these patients were referred to a European centre with the complexities of international travel, pre- and post-operative care, and funding. We sought to establish a national PEA programme to serve the Bulgarian population and avoid some of the problems of international healthcare. Case Description: A total of 11 patients underwent PEA in 2 cardiac centres in Bulgaria (Acibadem Hospital and Government Hospital Lozenetz Sofia). The age of patients ranged from 22 to 80. The preoperative pulmonary vascular resistance (PVR) ranged from 309 to 1,906 dynes/sec/cm-5. For the surviving patients the average PVR reduction was 615 dynes/sec/cm-5 at 6 months, the average intensive care unit (ICU) stay 6.7 days, and hospitalisation 15.2 days. Nine out of 11 patients survived to hospital discharge and 6 months follow, all with normalised PVR and exercise tolerance. Conclusions: We present our results of initial experience with PEA in Bulgaria with encouraging results. Our work shows that inter-European relationship for healthcare can be productive and offer safe treatment on local level.
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The main challenge in the treatment of acute myeloid leukemia (AML) is relapse, as it has no good treatment options and 90% of relapsed patients die as a result. It is now well accepted that relapse is due to a persisting subset of AML cells known as leukemia-initiating cells or leukemic stem cells (LSCs). Hematopoietic stem cells (HSCs) reside in the bone marrow microenvironment (BMM), a specialized niche that coordinates HSC self-renewal, proliferation, and differentiation. HSCs are divided into two types: long-term HSCs (LT-HSCs) and short-term HSCs, where LT-HSCs are typically quiescent and act as a reserve of HSCs. Like LT-HSCs, a quiescent population of LSCs also exist. Like LT-HSCs, quiescent LSCs have low metabolic activity and receive pro-survival signals from the BMM, making them resistant to drugs, and upon discontinuation of therapy, they can become activated and re-establish the disease. Several studies have shown that the activation of quiescent LSCs may sensitize them to cytotoxic drugs. However, it is very difficult to experimentally model the quiescence-inducing BMM. Here we report that culturing AML cells with bone marrow stromal cells, transforming growth factor beta-1 and hypoxia in a three-dimensional system can replicate the quiescence-driving BMM. A quiescent-like state of the AML cells was confirmed by reduced cell proliferation, increased percentage of cells in the G0 cell cycle phase and a decrease in absolute cell numbers, expression of markers of quiescence, and reduced metabolic activity. Furthermore, the culture could be established as co-axial microbeads, enabling high-throughput screening, which has been used to identify combination drug treatments that could break BMM-mediated LSC quiescence, enabling the eradication of quiescent LSCs.
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OBJECTIVE: High-dose tranexamic acid (TXA) can cause seizures in patients who have undergone pulmonary endarterectomy (PTE). Seizures secondary to TXA will resolve once the drug is excreted from the body, and the patients do not have to be on long-term anticonvulsants. The aim of the study is to find out if medication review in the hospital has led to deprescribing of anticonvulsants for TXA-associated seizures on discharge from the critical care unit (CCU) and hospital. METHODS: This is a single-centre retrospective study conducted at a tertiary cardiothoracic hospital between 2012 and 2017. The inclusion criteria consisted of all adult patients who have undergone PTE surgery. Patients who were started on anticonvulsants preoperatively or postoperatively for seizures secondary to organic causes were excluded. RESULTS: A total of 933 patients underwent PTE from January 2012 to August 2017. 25 patients had TXA-related seizures postoperatively and were started on anticonvulsant therapy, giving an incidence of 2.7%. 15 patients were discharged from the CCU without anticonvulsants. A further three patients had their anticonvulsants deprescribed in the ward before being discharged from the hospital. CONCLUSION: Deprescribing of anticonvulsants after benign seizures secondary to high-dose TXA is facilitated by verbal and written handover, which can be improved in our hospital. A detailed handover summary, as well as a discharge letter with clearly defined instructions for drug review, is needed to make deprescribing a more robust process.
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Anticonvulsivantes/administração & dosagem , Antifibrinolíticos/efeitos adversos , Desprescrições , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Centros de Atenção Terciária , Antifibrinolíticos/administração & dosagem , Relação Dose-Resposta a Droga , Endarterectomia/tendências , Humanos , Artéria Pulmonar/cirurgia , Estudos Retrospectivos , Convulsões/epidemiologia , Centros de Atenção Terciária/tendências , Ácido Tranexâmico/administração & dosagem , Ácido Tranexâmico/efeitos adversos , Reino Unido/epidemiologiaRESUMO
Hybrid organic-inorganic perovskites with appealing optoelectronic properties have attracted significant interest for photovoltaic application. The use of chloride (Cl-)-containing species to induce improved perovskite thin-film microstructures and improved optoelectronic properties is well-established. However, the mechanism for the formation of perovskite films with highly textured, micron-sized grains in the presence of Cl- is not well established. Using synchrotron-based in situ two-dimensional grazing incidence wide-angle X-ray scattering complemented by scanning electron microscopy imaging, we present an oriented attachment mechanism via mineral bridge formation for the microstructural evolution of perovskite films post-treated with methylammonium chloride. We have identified the crucial role of the chlorine-containing intermediate phase as the mineral bridge, which enables the reorientation of primary, nanoscale perovskite grains followed by fusion into uniaxial oriented quasi-single crystal grains. The resulting perovskite films exhibit micron-sized grains with preferential orientation of the tetragonal (110) direction perpendicular to the substrate, resulting in improved solar cell efficiency attributed to improved charge collection. Our findings help to understand the fundamental mechanisms of microstructure evolution via soft processing in hybrid perovskite films.
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Priming haematopoietic stem/progenitor cells (HSPCs) in vitro with specific chromatin modifying agents and cytokines under serum-free-conditions significantly enhances engraftable HSC numbers. We extend these studies by culturing human CD133+ HSPCs on nanofibre scaffolds to mimic the niche for 5-days with the HDAC inhibitor Scriptaid and cytokines. Scriptaid increases absolute Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSPC numbers, while concomitantly decreasing the Lin-CD38-CD34+CD45RA-CD90- subset. Hypothesising that Scriptaid plus cytokines expands the CD90+ subset without differentiation and upregulates CD90 on CD90- cells, we sorted, then cultured Lin-CD34+CD38-CD45RA-CD90- cells with Scriptaid and cytokines. Within 2-days and for at least 5-days, most CD90- cells became CD90+. There was no significant difference in the transcriptomic profile, by RNAsequencing, between cytokine-expanded and purified Lin-CD34+CD38-CD45RA-CD49f+CD90+ cells in the presence or absence of Scriptaid, suggesting that Scriptaid maintains stem cell gene expression programs despite expansion in HSC numbers. Supporting this, 50 genes were significantly differentially expressed between CD90+ and CD90- Lin-CD34+CD38-CD45RA-CD49f+ subsets in Scriptaid-cytokine- and cytokine only-expansion conditions. Thus, Scriptaid treatment of CD133+ cells may be a useful approach to expanding the absolute number of CD90+ HSC, without losing their stem cell characteristics, both through direct effects on HSC and potentially also conversion of their immediate CD90- progeny into CD90+ HSC.
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Doenças Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hidroxilaminas/farmacologia , Quinolinas/farmacologia , Transcriptoma/efeitos dos fármacos , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Meios de Cultura Livres de Soro , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , RNA-Seq , Análise de Célula Única , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismoRESUMO
Embryonic Stem (ES) cells-derived cardiomyocytes can possibly be applied for cell therapy of diseases such as heart failure. Biodegradable scaffolds will significantly improve the expansion of sufficient functional ES cell-derived cardiomyocytes and may also increase the survival rate of cardiomyocytes after their transplantation. In the present study, we cultivated cardiomyocytes isolated from a transgenic a-myosin heavy chain (alpha-MHC) ES cell lineage expressing both puromycin resistance and enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter in macroporous gelatine microspheres using small-scale bioreactors and proved that cardiomyocytes function after their cultivation in micropsperes. The average number of cultivated cells per microsphere was optimised once the most suitable agitation conditions and the optimal timeframe of cultivation were identified. Our study shows that 72% of CultiSpher-S beads were colonised by cardiomyocytes under optimal conditions. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) showed that colonization of the beads was not limited to the surface, but that cells also invaded the inner surfaces of the microspheres. Electrophysiological experiments demonstrated that the action potentials (APs) of alpha-MHC(+) cardiomyocytes entrapped in microspheres were identical to action potentials of control cells. This attractive approach for cultivation and expansion of functional cardiomyocytes in biodegradable macroporous may offer a perspective for higher transplantation efficiencies of ES cell-derived cardiomyocytes.
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Materiais Biocompatíveis/metabolismo , Células-Tronco Embrionárias/citologia , Microesferas , Miócitos Cardíacos/citologia , Potenciais de Ação , Animais , Linhagem Celular , Células-Tronco Embrionárias/ultraestrutura , Camundongos , Microscopia Confocal , Miócitos Cardíacos/ultraestrutura , PorosidadeRESUMO
We have exploited emulsion-based in vitro compartmentalization (IVC) to devise a method for the selection of zinc finger proteins (ZFPs) on the basis of their DNA-binding specificity. A library of ZFPs fused to a C-terminal peptide tag is encoded by a set of DNA cassettes that are prepared wholly in vitro. In addition to the ZFP gene, each DNA cassette also carries a given DNA target binding site sequence for which one wishes to isolate ZFP binders. An aliquot of the library is added to bacterial S30 extract and emulsified in mineral oil so that most of the aqueous droplets contain, on average, no more than one gene. If an intra-compartmentally expressed ZFP binds specifically to its encoding DNA via the target binding site, the complex can be purified by affinity capture via the peptide tag after breaking the emulsion, thus rescuing the gene. We present proof-of-principle for this IVC selection method by selecting a specific high-affinity ZFP gene from a high background of a related gene. We also propose that high-affinity ZFPs can be used as genotype-phenotype linkages to enable selection of other proteins using IVC.
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Sistema Livre de Células/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dedos de Zinco , Sítios de Ligação , Compartimento Celular , Primers do DNA , Técnicas In Vitro , Biblioteca de Peptídeos , Transcrição GênicaRESUMO
The use of artificial zinc finger chimeras to manipulate the expression of a gene of interest is a promising approach because zinc finger proteins can be engineered to bind any given DNA sequence in the genome. We have previously shown that a zinc finger chimera with a VP16 activation domain can activate a reporter gene in transgenic Arabidopsis thaliana (Sánchez, J.P., Ullman, C., Moore, M., Choo, Y. and Chua, N.H. (2002) Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol. 43, 1465-1472). Here, we report the use of artificial zinc finger chimeras to specifically regulate the 4-coumarate:coenzyme-A ligase-1 (At4CL1) gene in A. thaliana. At4CL1 is a key enzyme in lignin biosynthesis and the down-regulation of At4CL1 can lead to a decrease in lignin content, which has a significant commercial value for the paper industry. To this end, we designed zinc finger chimeras containing either an activation or a repression domain, which bind specifically to the At4CL1 promoter region. Transgenic lines expressing a zinc finger chimera with the VP16 activation domain showed an increase in At4CL1 expression and enzyme activity. In contrast, transgenic lines expressing a chimera with the KOX (KRAB) repression domain displayed repression of At4CL1 expression and enzyme activity. The activation of At4CL1 expression produced an increase in lignin content, and transgenic plant stems showed ectopic lignin distribution. Repression of the At4CL1 gene resulted in reduced lignin content, and lignin distribution in transgenic stems was severely diminished. Our results confirm and extend previous studies of gene regulation using various artificial zinc finger chimeras in animal and plant systems, and show that this system can be used to up- and down-regulate the expression of an endogenous plant gene such as At4CL1.
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Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Regulação da Expressão Gênica de Plantas , Dedos de Zinco/genética , Arabidopsis/química , Arabidopsis/crescimento & desenvolvimento , Celulose/análise , Regulação para Baixo , Lignina/análise , Lignina/biossíntese , Fenótipo , Caules de Planta/química , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão , Fatores de Transcrição/genética , Transformação Genética , Regulação para CimaRESUMO
Photocatalytic CO2 reduction over the UV-Vis-NIR broad spectrum was realized for the first time. The presence of surface oxygen vacancy defects on Bi2WO6 resulted in significant photocatalytic enhancement over the pristine counterpart under UV and visible light irradiation. Meanwhile, the photocatalytic responsiveness of Bi2WO6-OV was successfully extended to the NIR region.
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The main limitations of hematopoietic cord blood (CB) transplantation, viz, low cell dosage and delayed reconstitution, can be overcome by ex vivo expansion. CB expansion under conventional culture causes rapid cell differentiation and depletion of hematopoietic stem and progenitor cells (HSPCs) responsible for engraftment. In this study, we use combinatorial cell culture technology (CombiCult®) to identify medium formulations that promote CD133+ CB HSPC proliferation while maintaining their phenotypic characteristics. We employed second-generation CombiCult screens that use electrospraying technology to encapsulate CB cells in alginate beads. Our results suggest that not only the combination but also the order of addition of individual components has a profound influence on expansion of specific HSPC populations. Top protocols identified by the CombiCult screen were used to culture human CD133+ CB HSPCs on nanofiber scaffolds and validate the expansion of the phenotypically defined CD34+CD38lo/-CD45RA-CD90+CD49f+ population of hematopoietic stem cells and their differentiation into defined progeny.
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Citocinas/farmacologia , Células-Tronco Hematopoéticas/citologia , Ensaios de Triagem em Larga Escala/métodos , Antígeno AC133/metabolismo , Alginatos/farmacologia , Algoritmos , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Ácido Glucurônico/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Ácidos Hexurônicos/farmacologia , Humanos , Microesferas , Nanofibras/química , Reprodutibilidade dos Testes , Alicerces Teciduais/químicaRESUMO
We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.
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Diferenciação Celular , Linhagem da Célula , Meios de Cultura Livres de Soro/química , Células-Tronco Embrionárias/citologia , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Técnicas de Química Combinatória/métodos , Meios de Cultura Livres de Soro/farmacologia , Neurônios Dopaminérgicos/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Imunofenotipagem , Células Progenitoras Linfoides/metabolismo , Camundongos , Microesferas , Neurônios/citologia , Neurônios/metabolismo , Fagócitos/citologia , Fagócitos/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5' UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity.
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Regulação Fúngica da Expressão Gênica , Glicoproteínas de Membrana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Transcrição , Dedos de Zinco , Sequência de Bases , Adesão Celular , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Biblioteca de Peptídeos , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The G-quadruplex nucleic acid structural motif is a target for designing molecules that could potentially modulate telomere length or have anticancer properties. We have recently described an engineered zinc finger protein (Gq1) that binds with specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5'-(GGTTAG)(5)-3' (Isalan et al. (2001) Biochemistry 40, 830-836). Here, we report that Gq1 is able to arrest the action of a DNA polymerase on a template-containing telomeric sequence. Inhibition occurs in a concentration-dependent manner, probably by forming a stabilized G-quadruplex.protein complex. Furthermore, Gq1 inhibits the apparent activity of the enzyme telomerase in vitro, with an IC(50) value of 74.3 +/- 11.1 nM. Possible molecular mechanisms of inhibition are discussed, together with the potential for using engineered zinc fingers to interfere with the cellular processes associated with telomere function.
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Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Conformação de Ácido Nucleico , Engenharia de Proteínas , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Dedos de Zinco , DNA Polimerase I/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Quadruplex G , Humanos , Ligação Proteica , Engenharia de Proteínas/métodos , Inibidores da Síntese de Proteínas/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Telomerase/biossíntese , Telômero/enzimologia , Moldes Genéticos , Dedos de Zinco/genéticaRESUMO
The artificial regulation of endogenous gene expression in plants is limited to only a few approaches. Here, we describe the use of artificial zinc finger chimeras to regulate the expression of a known reporter construct. The artificial zinc finger chimera TFIIIAZif is a fusion protein consisting of the four zinc fingers of TFIIIA linked through a spacer region to the three zinc fingers of Zif268. This artificial zinc finger chimera is able to bind specifically to a target DNA sequence (ZBS, zinc finger binding site) of 27 base pairs (bp). TFIIIAZif was fused to a transactivation domain from the herpes simplex virus VP16 or its tetramer VP64 to give ZF-VP16 or ZF-VP64, respectively. In transient expression assays, these two transcription activators were able to activate a target reporter gene (luc and GFP) expressed from a minimal -46 35S promoter linked to four copies of ZBS. The activation was confirmed in transgenic plants using an inducible XVE system [Zuo et al. (2000) Plant J. 24: 265] to express ZF-VP16 or ZF-VP64. Furthermore, to test the specificity of ZF-VP64 we have compared reporter gene expression from a wild type (1xZBS) and a mutant (1xZBSmu) binding site in transgenic plants. The 1xZBS was used to express green fluorescent protein (GFP) whereas the 1xZBSmu was used to express red fluorescent protein (RFP). Upon induction of ZF-VP64 we found a much higher expression of GFP (about 33-fold) as compared to RFP expression. These results suggest that artificial zinc finger chimeras can be used to target specific DNA sequences and to regulate gene expression in plants.
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Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Dedos de Zinco/genética , Arabidopsis/metabolismo , Genes Reporter/genética , Proteínas de Fluorescência Verde , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismoRESUMO
The herpes simplex virus 1 (HSV-1) replicative cycle begins by binding of the viral activator, VP16, to a set of sequences in the immediate-early (IE) gene promoters. With the aim of inhibiting this cycle, we have constructed a number of synthetic zinc-finger DNA-binding peptides by using recently reported methods. Peptides containing either three or six fingers, targeted to a viral promoter, were engineered as fusions with a KOX-1 transcription repression domain. These proteins bound to the HSV-1 IE175k (ICP4) promoter, in vitro, with nanomolar or subnanomolar binding affinity. However, in a chloramphenicol acetyltransferase reporter system, only the six-finger protein was found to repress VP16-activated transcription significantly. Thus the longer array of zinc fingers is required to compete successfully against VP16, one of the most powerful natural activators known. We found that the HSV-1 replication cycle can be partially repressed by the six-finger peptide with the viral titer reduced by 90%.
Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Simplexvirus/genética , Fatores de Transcrição/fisiologia , Dedos de Zinco , Animais , Sequência de Bases , Células COS , Cloranfenicol O-Acetiltransferase/genética , Primers do DNA , Imunofluorescência , Genes Virais , Células HeLa , Humanos , Fatores de Transcrição/químicaRESUMO
Zinc finger domains are small DNA-binding modules that can be engineered to bind desired target sequences. Functional transcription factors can be made from these DNA-binding modules, by fusion with an appropriate effector domain. In this study, eight three-zinc-finger proteins (ZFPs) that bound HIV-1 sequences in vitro were engineered into transcription repressors by linking them to the Krüppel-associated box (KRAB) repressor domain (KOX1). One protein, ZFP HIVB-KOX, which bound to a 9-bp region overlapping two Sp1 sites, was found to repress a Tat-activated 5' LTR cellular HIV-reporter assay to almost basal levels. A related six-finger protein, HIVBA'-KOX, was made to target all three Sp1 sites in the 5' LTR promoter and efficiently inhibited both basal and Tat-activated transcription in unstimulated and mitogen-stimulated T cells. In contrast, a combination of two unlinked three-finger ZFPs, HIVA'-KOX and HIVB-KOX, which bind over the same region of DNA, resulted in less effective repression. Finally, HIVBA'-KOX was tested for its capacity to block viral replication in a cellular infection assay using the HIV-1 HXB2 strain. This ZFP was found to inhibit HIV-1 replication by 75% compared with control constructs, thus demonstrating the potential of this approach for antiviral therapy.