RESUMEN
The rise in the antibiotic resistance of bacteria has increased scientific interest in the study of materials with unique mechanisms of antimicrobial action. This paper presents the results of studies on the antimicrobial activity of carbon materials and textiles decorated with them. A comparative analysis of the bactericidal and fungicidal activities of graphene oxide, electrochemically exfoliated multigraphene, carbon dots, and their combinations was performed. Microbiological studies on reference strains of E. coli, S. aureus, and C. albicans showed that graphene oxide inhibited growth with up to 98% efficiency. Electrochemically exfoliated multigraphene was less effective (up to 40%). This study found no significant antimicrobial activity of carbon dots and the combination of carbon dots with graphene oxide significantly weakened their effectiveness. However, the combination of electrochemically exfoliated multigraphene and carbon dots exhibits a synergistic effect (up to 76%). A study on the antimicrobial activity of decorated cotton textiles demonstrated the effectiveness of antimicrobial textiles with graphene oxide, electrochemically exfoliated multigraphene, and a combination of carbon dots with electrochemically exfoliated multigraphene.
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Antiinfecciosos , Fibra de Algodón , Grafito , Grafito/química , Grafito/farmacología , Antiinfecciosos/farmacología , Antiinfecciosos/química , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Carbono/química , Staphylococcus aureus/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Textiles , Puntos Cuánticos/químicaRESUMEN
Facioscapulohumeral dystrophy (FSHD) is a neuromuscular disease with a prevalence that could reach 1 in 8,000 characterized by progressive asymmetric muscle weakness. Myoblasts isolated from FSHD muscles exhibit morphological differentiation defects and show a distinct transcription profile. These abnormalities may be linked to the muscle weakness in FSHD patients. We have tested whether fusion of FSHD myoblasts with primary myoblasts isolated from healthy individuals could correct the differentiation defects. Our results show that the number of hybrid myotubes with normal phenotype increased with the percentage of normal myoblasts initially cultured. We demonstrated that a minimum of 50% of normal nuclei is required for a phenotypic correction of the FSHD phenotype. Moreover, transcriptomic profiles of phenotypically corrected hybrid myotubes showed that the expression of deregulated genes in FSHD myotubes became almost normal. The number of deregulated pathways also decreased from 39 in FSHD myotubes to one in hybrid myotubes formed with 40% FSHD and 60% normal myoblasts. We thus propose that while phenotypical and functional correction of FSHD is feasible, it requires more than 50% of normal myoblasts, it creates limitations for cell therapy in the FSHD context.
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Diferenciación Celular/fisiología , Distrofia Muscular Facioescapulohumeral/metabolismo , Mioblastos/citología , Adulto , Diferenciación Celular/genética , Células Cultivadas , Femenino , Humanos , Masculino , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Fenotipo , Adulto JovenRESUMEN
Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy linked to a deletion of a subset of D4Z4 macrosatellite repeats accompanied by a chromatin relaxation of the D4Z4 array on chromosome 4q. In vitro, FSHD primary myoblasts show altered expression of oxidative-related genes and are more susceptible to oxidative stress. Double homeobox 4 (DUX4) gene, encoded within each D4Z4 unit, is normally transcriptionally silenced but is found aberrantly expressed in skeletal muscles of FSHD patients. Its expression leads to a deregulation of DUX4 target genes including those implicated in redox balance. Here, we assessed DNA repair efficiency of oxidative DNA damage in FSHD myoblasts and DUX4-transfected myoblasts. We have shown that the DNA repair activity is altered neither in FSHD myoblasts nor in immortalized human myoblasts transiently expressing DUX4. DNA damage caused by moderate doses of an oxidant is efficiently repaired while FSHD myoblasts exposed for 24 h to high levels of oxidative stress accumulated more DNA damage than normal myoblasts, suggesting that FSHD myoblasts remain more vulnerable to oxidative stress at high doses of oxidants.
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Daño del ADN , Reparación del ADN , Distrofia Muscular Facioescapulohumeral/metabolismo , Mioblastos Esqueléticos/metabolismo , Estrés Oxidativo , Células Cultivadas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Distrofia Muscular Facioescapulohumeral/patología , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/patología , Estrés Oxidativo/efectos de los fármacosRESUMEN
Muscular dystrophy is a condition potentially predisposing for cancer; however, currently, only Myotonic dystrophy patients are known to have a higher risk of cancer. Here, we have searched for a link between facioscapulohumeral dystrophy (FSHD) and cancer by comparing published transcriptome signatures of FSHD and various malignant tumours and have found a significant enrichment of cancer-related genes among the genes differentially expressed in FSHD. The analysis has shown that gene expression profiles of FSHD myoblasts and myotubes resemble that of Ewing's sarcoma more than that of other cancer types tested. This is the first study demonstrating a similarity between FSHD and cancer cell expression profiles, a finding that might indicate the existence of a common step in the pathogenesis of these two diseases.
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Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Mioblastos/metabolismo , Proteínas de Neoplasias/genética , Sarcoma de Ewing/genética , Transcriptoma , Aberraciones Cromosómicas , Cromosomas Humanos Par 4 , Epigénesis Genética , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Distrofia Muscular Facioescapulohumeral/metabolismo , Distrofia Muscular Facioescapulohumeral/patología , Mioblastos/patología , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Cultivo Primario de Células , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologíaRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant hereditary neuromuscular disorder linked to the deletion of an integral number of 3.3-kb-long macrosatellite repeats (D4Z4) within the subtelomeric region of chromosome 4q. Most genes identified in this region are overexpressed in FSHD myoblasts, including the double homeobox genes DUX4 and DUX4c. We have carried out a simultaneous miRNome/transcriptome analysis of FSHD and control primary myoblasts. Of 365 microRNAs (miRNAs) analyzed in this study, 29 were found to be differentially expressed between FSHD and normal myoblasts. Twenty-one microRNAs (miR-1, miR-7, miR-15a, miR-22, miR-30e, miR-32, miR-107, miR-133a, miR-133b, miR-139, miR-152, miR-206, miR-223, miR-302b, miR-331, miR-362, miR-365, miR-382, miR-496, miR-532, miR-654, and miR-660) were up-regulated, and eight were down-regulated (miR-15b, miR-20b, miR-21, miR-25, miR-100, miR-155, miR-345, and miR-594). Twelve of the miRNAs up-regulated in FHSD were also up-regulated in the cells ectopically expressing DUX4c, suggesting that this gene could regulate miRNA gene transcription. The myogenic miRNAs miR-1, miR-133a, miR-133b, and miR-206 were highly expressed in FSHD myoblasts, which nonetheless did not prematurely enter myogenic differentiation. This could be accounted for by the fact that in FSHD myoblasts, functionally important target genes, including cell cycle, DNA damage, and ubiquitination-related genes, escape myogenic microRNA-induced repression.
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MicroARNs/genética , MicroARNs/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/metabolismo , Mioblastos Esqueléticos/metabolismo , Adulto , Diferenciación Celular/genética , Células Cultivadas , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Masculino , Persona de Mediana Edad , Desarrollo de Músculos/genética , Distrofia Muscular Facioescapulohumeral/patología , Mioblastos Esqueléticos/patología , Regulación hacia Arriba , Adulto JovenRESUMEN
The rapid development of electric vehicles, unmanned aerial vehicles, and wearable electronic devices has led to great interest in research related to the synthesis of graphene with a high specific surface area for energy applications. However, the problem of graphene synthesis scalability, as well as the lengthy duration and high energy intensity of the activation processes of carbon materials, are significant disadvantages. In this study, a novel reactor was developed for the green, simple, and scalable electrochemical synthesis of graphene oxide with a low oxygen content of 14.1%. The resulting material was activated using the fast joule heating method. The processing of mildly oxidized graphene with a high-energy short electrical pulse (32 ms) made it possible to obtain a graphene-based porous carbon material with a specific surface area of up to 1984.5 m2/g. The increase in the specific surface area was attributed to the rupture of the original graphene flakes into smaller particles due to the explosive release of gaseous products. In addition, joule heating was able to instantly reduce the oxidized graphene and decrease its electrical resistance from >10 MΩ/sq to 20 Ω/sq due to sp2 carbon structure regeneration, as confirmed by Raman spectroscopy. The low energy intensity, simplicity, and use of environment-friendly chemicals rendered the proposed method scalable. The resulting graphene material with a high surface area and conductivity can be used in various energy applications, such as Li-ion batteries and supercapacitors.
RESUMEN
BACKGROUND: miRNA profiling performed in myogenic cells and biopsies from skeletal muscles has previously identified miRNAs involved in myogenesis. RESULTS: Here, we have performed miRNA transcriptome profiling in human affinity-purified CD56+ myoblasts induced to differentiate in vitro. In total, we have identified 60 miRNAs differentially expressed during myogenic differentiation. Many were not known for being differentially expressed during myogenic differentiation. Of these, 14 (miR-23b, miR-28, miR-98, miR-103, miR-107, miR-193a, miR-210, miR-324-5p, miR-324-3p, miR-331, miR-374, miR-432, miR-502, and miR-660) were upregulated and 6 (miR-31, miR-451, miR-452, miR-565, miR-594 and miR-659) were downregulated. mRNA transcriptome profiling performed in parallel resulted in identification of 6,616 genes differentially expressed during myogenic differentiation. CONCLUSIONS: This simultaneous miRNA/mRNA transcriptome profiling allowed us to predict with high accuracy target genes of myogenesis-related microRNAs and to deduce their functions.
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Diferenciación Celular/genética , MicroARNs/genética , Desarrollo de Músculos/genética , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , ARN Mensajero/metabolismo , Antígeno CD56/genética , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , MicroARNs/fisiología , ARN Mensajero/genética , Regulación hacia ArribaRESUMEN
In this study, cotton e-textiles were obtained using two types of graphene oxide. The first type of graphene oxide was synthesized using the Hummers' method. The second type was obtained by the electrochemical exfoliation of graphite in an ammonium salt solution. It was shown that e-textiles based on electrochemically exfoliated graphene have a higher electrical conductivity (2 kΩ/sq) than e-textiles based on graphene oxide obtained by the Hummers' method (585 kΩ/sq). In addition, textiles based on electrochemically exfoliated graphene exhibit better washing and mechanical stress stability. The electrical resistance of the e-textiles increased only 1.86 times after 10 cycles of washing, compared with 48 times for the Hummers' method graphene oxide textiles. The X-ray photoelectron spectra of the two types of graphene oxides showed similarity in their functional compositions after reduction. Studies of individual graphene flakes by atomic force microscopy showed that graphene oxide of the second type had a smaller lateral size. Raman spectroscopy showed a higher degree of sp2 structure regeneration after reduction for the second type of graphene. These properties and the tendency to form agglomerated particles determine the mechanochemical stability and high electrical conductivity of e-textiles based on electrochemically exfoliated graphene.
RESUMEN
Facioscapulohumeral muscular dystrophy (FSHD), a dominant hereditary disease with a prevalence of 7 per 100,000 individuals, is associated with a partial deletion in the subtelomeric D4Z4 repeat array on chromosome 4q. The D4Z4 repeat contains a strong transcriptional enhancer that activates promoters of several FSHD-related genes. We report here that the enhancer within the D4Z4 repeat binds the Krüppel-like factor KLF15. KLF15 was found to be up-regulated during myogenic differentiation induced by serum starvation or by overexpression of the myogenic differentiation factor MYOD. When overexpressed, KLF15 activated the D4Z4 enhancer and led to overexpression of DUX4c (Double homeobox 4, centromeric) and FRG2 (FSHD region gene 2) genes, whereas its silencing caused inactivation of the D4Z4 enhancer. In immortalized human myoblasts, the D4Z4 enhancer was activated by the myogenic factor MYOD, an effect that was abolished upon KLF15 silencing or when the KLF15-binding sites within the D4Z4 enhancer were mutated, indicating that the myogenesis-related activation of the D4Z4 enhancer was mediated by KLF15. KLF15 and several myogenesis-related factors were found to be expressed at higher levels in myoblasts, myotubes, and muscle biopsies from FSHD patients than in healthy controls. We propose that KLF15 serves as a molecular link between myogenic factors and the activity of the D4Z4 enhancer, and it thus contributes to the overexpression of the DUX4c and FRG2 genes during normal myogenic differentiation and in FSHD.
Asunto(s)
Cromosomas Humanos Par 4/metabolismo , Elementos de Facilitación Genéticos , Factores de Transcripción de Tipo Kruppel/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/metabolismo , Proteínas Nucleares/metabolismo , Animales , Cromosomas Humanos Par 4/genética , Cricetinae , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/genética , Células HeLa , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Desarrollo de Músculos/genética , Músculo Esquelético/patología , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/patología , Proteína MioD/genética , Proteína MioD/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The human thyroid gland acquires a differentiation program as early as weeks 3-4 of embryonic development. The onset of functional differentiation, which manifests by the appearance of colloid in thyroid follicles, takes place during gestation weeks 10-11. By 12-13 weeks functional differentiation is accomplished and the thyroid is capable of producing thyroid hormones although at a low level. During maturation, thyroid hormones yield increases and physiological mechanisms of thyroid hormone synthesis regulation are established. In the present work we traced the process of thyroid functional differentiation and maturation in the course of human development by performing transcriptomic analysis of human thyroids covering the period of gestation weeks 7-11 and comparing it to adult human thyroid. We obtained specific transcriptomic signatures of embryonic and adult human thyroids by comparing them to non-thyroid tissues from human embryos and adults. We defined a non-TSH (thyroid stimulating hormone) dependent transition from differentiation to maturation of thyroid. The study also sought to shed light on possible factors that could replace TSH, which is absent in this window of gestational age, to trigger transition to the emergence of thyroid function. We propose a list of possible genes that may also be involved in abnormalities in thyroid differentiation and/or maturation, hence leading to congenital hypothyroidism. To our knowledge, this study represent the first transcriptomic analysis of human embryonic thyroid and its comparison to adult thyroid.
RESUMEN
Systematic studies of the circulation of hepatitis C virus (HCV) recombinants in different parts of the world have been initiated only recently, and no detailed information on this subject is available. The aim of the current investigation was to determine the frequency of HCV recombinants in intravenous drug users (IVDU) from two European countries. HCV RNA from serum samples was tested by RT-PCR with primers derived from the core and NS5B regions with subsequent sequencing and genotype assignment. The 118 samples from Germany (100%) and 45 out of 47 (96%) sera from Russia demonstrated concordant genotyping results. In the two genotype discrepant sera from Russia 2k/1b recombinants were identified. In order to test the hypothesis that the individuals from the IVDU group might be multiply exposed to various genotypes, 145 out of 165 genotyped serum samples, which were found to be positive for anti-NS4 antibodies, were serotyped with the Murex HCV serotyping kit that is based on detection of antibodies to type-specific peptides derived from the NS4 proteins of different HCV genotypes. Discrepancy in genotype and serotype attributions was observed in 11% cases. Retesting of 99 type 1a or 3a samples with a set of type- and subtype-specific primers revealed the presence of a mixed infection only in one case (1a/3a). Thus, the cases of the mixed infection with different HCV genotypes as well as the recombinant forms of HCV are very rare even in such a highly exposed group as IVDU.
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Hepacivirus/genética , Hepatitis C/virología , Recombinación Genética , Adolescente , Adulto , Animales , Secuencia de Bases , Consumidores de Drogas , Femenino , Genotipo , Alemania , Hepacivirus/clasificación , Hepacivirus/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Fenotipo , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Federación de Rusia , Análisis de Secuencia de ADN , Homología de Secuencia , Serotipificación , Suero/virología , Abuso de Sustancias por Vía Intravenosa , Adulto JovenRESUMEN
Despite the discovery of the deletion on the long arm of the chromosome 4 specific for facioscapulohumeral muscular dystrophy (FSHD), the identity of the gene responsible for the disease still remains a mystery. In this review we focus on two genes, DUX4 and DUX4c, encoded by the D4Z4 repeats present in the 4q35 locus, which is affected in the disease.
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Cromosomas Humanos Par 4/genética , ADN Intergénico/genética , Predisposición Genética a la Enfermedad/genética , Distrofia Muscular Facioescapulohumeral/genética , Mutación/genética , Seudogenes/genética , Animales , Eliminación de Gen , Proteínas de Homeodominio/genética , Humanos , Distrofia Muscular Facioescapulohumeral/metabolismo , Distrofia Muscular Facioescapulohumeral/fisiopatología , Isoformas de Proteínas/genéticaRESUMEN
Facioscapulohumeral dystrophy (FSHD) is one of the three most common muscular dystrophies in the Western world, however, its etiology remains only partially understood. Here, we provide evidence of constitutive DNA damage in in vitro cultured myoblasts isolated from FSHD patients and demonstrate oxidative DNA damage implication in the differentiation of these cells into phenotypically-aberrant myotubes. Double homeobox 4 (DUX4), the major actor in FSHD pathology induced DNA damage accumulation when overexpressed in normal human myoblasts, and RNAi-mediated DUX4 inhibition reduced the level of DNA damage in FSHD myoblasts. Addition of tempol, a powerful antioxidant, to the culture medium of proliferating DUX4-transfected myoblasts and FSHD myoblasts reduced the level of DNA damage, suggesting that DNA alterations are mainly due to oxidative stress. Antioxidant treatment during the myogenic differentiation of FSHD myoblasts significantly reduced morphological defects in myotube formation. We propose that the induction of DNA damage is a novel function of the DUX4 protein affecting myogenic differentiation of FSHD myoblasts.
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Proteínas de Homeodominio/genética , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Mioblastos/metabolismo , Estrés Oxidativo , Antioxidantes/farmacología , Estudios de Casos y Controles , Diferenciación Celular , Óxidos N-Cíclicos/farmacología , Daño del ADN , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ontología de Genes , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/metabolismo , Humanos , Anotación de Secuencia Molecular , Familia de Multigenes , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Facioescapulohumeral/metabolismo , Distrofia Muscular Facioescapulohumeral/patología , Mioblastos/patología , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Marcadores de Spin , TransfecciónRESUMEN
We performed transcriptome profiling of human immortalized myoblasts (MB) transiently expressing double homeobox transcription factor 4 (DUX4) and double homeobox transcription factor 4 centromeric (DUX4c) and identified 114 and 70 genes differentially expressed in DUX4- and DUX4c-transfected myoblasts, respectively. A significant number of differentially expressed genes were involved in inflammation, cellular migration and chemotaxis suggesting a role for DUX4 and DUX4c in these processes. DUX4 but not DUX4c overexpression resulted in upregulation of the CXCR4 (C-X-C motif Receptor 4) and CXCL12 (C-X-C motif ligand 12 also known as SDF1) expression in human immortalized myoblasts. In a Transwell cell migration assay, human bone marrow-derived mesenchymal stem cells (BMSCs) were migrating more efficiently towards human immortalized myoblasts overexpressing DUX4 as compared to controls; the migration efficiency of DUX4-transfected BMSCs was also increased. DUX4c overexpression in myoblasts or in BMSCs had no impact on the rate of BMSC migration. Antibodies against SDF1 and CXCR4 blocked the positive effect of DUX4 overexpression on BMSC migration. We propose that DUX4 controls the cellular migration of mesenchymal stem cells through the CXCR4 receptor.
Asunto(s)
Movimiento Celular/fisiología , Quimiocina CXCL12/metabolismo , Proteínas de Homeodominio/metabolismo , Células Madre Mesenquimatosas/metabolismo , Receptores CXCR4/metabolismo , Células Cultivadas , Humanos , Mioblastos/metabolismo , TranscriptomaRESUMEN
Mechanisms that regulate attachment of the scaffold/matrix attachment regions (S/MARs) to the nuclear matrix remain largely unknown. We have studied the effect of simple sequence length polymorphism (SSLP), DNA methylation and chromatin organization in an S/MAR implicated in facioscapulohumeral dystrophy (FSHD), a hereditary disease linked to a partial deletion of the D4Z4 repeat array on chromosome 4q. This FSHD-related nuclear matrix attachment region (FR-MAR) loses its efficiency in myoblasts from FSHD patients. Three criteria were found to be important for high-affinity interaction between the FR-MAR and the nuclear matrix: the presence of a specific SSLP haplotype in chromosomal DNA, the methylation of one specific CpG within the FR-MAR and the absence of histone H3 acetylated on lysine 9 in the relevant chromatin fragment.
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Epigénesis Genética , Regiones de Fijación a la Matriz/genética , Repeticiones de Microsatélite/genética , Distrofia Muscular Facioescapulohumeral/genética , Matriz Nuclear/metabolismo , Polimorfismo Genético , Acetilación , Adulto , Secuencia de Bases , Línea Celular Tumoral , Células Cultivadas , Cromatina/metabolismo , Islas de CpG , Metilación de ADN , Femenino , Histonas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mioblastos/metabolismo , Unión ProteicaRESUMEN
Little is known about differences between induced pluripotent stem cells produced from tissues originating from the same germ layer. We have generated human myoblast-derived iPS cells by retroviral transduction of human primary myoblasts with the OCT3/4, SOX2, KLF4 and MYC coding sequences and compared them to iPS produced from human primary fibroblasts. When cultivated in vitro, these iPS cells proved similar to human embryonic stem cells in terms of morphology, expression of embryonic stemness markers and gene promoter methylation patterns. Embryonic bodies were derived that expressed endodermal, mesodermal as well as ectodermal markers. A comparative analysis of transcription patterns revealed significant differences in the gene expression pattern between myoblast- and fibroblast-derived iPS cells. However, these differences were reduced in the mesenchymal stem cells derived from the two iPS cell types were compared.
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Diferenciación Celular/genética , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Mioblastos/metabolismo , Animales , Células Cultivadas , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Desarrollo de Músculos/genética , Mioblastos/citología , Factor 3 de Transcripción de Unión a Octámeros/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-myc/genética , Retroviridae/genética , Factores de Transcripción SOXB1/genética , Transducción GenéticaRESUMEN
BACKGROUND: Introduction of new antibiotic resistance genes in the plasmids of interest is a frequent task in molecular cloning practice. Classical approaches involving digestion with restriction endonucleases and ligation are time-consuming. FINDINGS: We have created a set of insertion vectors (pINS) carrying genes that provide resistance to various antibiotics (puromycin, blasticidin and G418) and containing a loxP site. Each vector (pINS-Puro, pINS-Blast or pINS-Neo) contains either a chloramphenicol or a kanamycin resistance gene and is unable to replicate in most E. coli strains as it contains a conditional R6Kgamma replication origin. Introduction of the antibiotic resistance genes into the vector of interest is achieved by Cre-mediated recombination between the replication-incompetent pINS and a replication-competent target vector. The recombination mix is then transformed into E. coli and selected by the resistance marker (kanamycin or chloramphenicol) present in pINS, which allows to recover the recombinant plasmids with 100% efficiency. CONCLUSION: Here we propose a simple strategy that allows to introduce various antibiotic-resistance genes into any plasmid containing a replication origin, an ampicillin resistance gene and a loxP site.
RESUMEN
The number of D4Z4 repeats in the subtelomeric region of chromosome 4q is strongly reduced in patients with Facio-Scapulo-Humeral Dystrophy (FSHD). We performed chromosome conformation capture (3C) analysis to document the interactions taking place among different 4q35 markers. We found that the reduced number of D4Z4 repeats in FSHD myoblasts was associated with a global alteration of the three-dimensional structure of the 4q35 region. Indeed, differently from normal myoblasts, the 4qA/B marker interacted directly with the promoters of the FRG1 and ANT1 genes in FSHD cells. Along with the presence of a newly identified transcriptional enhancer within the 4qA allele, our demonstration of an interaction occurring between chromosomal segments located megabases away on the same chromosome 4q allows to revisit the possible mechanisms leading to FSHD.
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Translocador 1 del Nucleótido Adenina/genética , Cromosomas Humanos Par 4/genética , Regulación de la Expresión Génica , Reordenamiento Génico , Distrofia Muscular Facioescapulohumeral/genética , Proteínas Nucleares/genética , Células Cultivadas , Fibroblastos , Humanos , Proteínas de Microfilamentos , Regiones Promotoras Genéticas , Proteínas de Unión al ARN , Secuencias Repetitivas de Ácidos Nucleicos , Factores de TranscripciónRESUMEN
The regulation of telomerase action, and its coordination with conventional DNA replication and chromosome end "capping," are still poorly understood. Here we describe a genetic screen in yeast for mutants with relaxed telomere length regulation, and the identification of Pol12, the B subunit of the DNA polymerase alpha (Pol1)-primase complex, as a new factor involved in this process. Unlike many POL1 and POL12 mutations, which also cause telomere elongation, the pol12-216 mutation described here does not lead to either reduced Pol1 function, increased telomeric single-stranded DNA, or a reduction in telomeric gene silencing. Instead, and again unlike mutations affecting POL1, pol12-216 is lethal in combination with a mutation in the telomere end-binding and capping protein Stn1. Significantly, Pol12 and Stn1 interact in both two-hybrid and biochemical assays, and their synthetic-lethal interaction appears to be caused, at least in part, by a loss of telomere capping. These data reveal a novel function for Pol12 and a new connection between DNA polymerase alpha and Stn1. We propose that Pol12, together with Stn1, plays a key role in linking telomerase action with the completion of lagging strand synthesis, and in a regulatory step required for telomere capping.