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1.
Int J Mol Sci ; 25(10)2024 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-38791553

RESUMO

Long non-coding RNAs (lncRNAs) have been shown to modulate gene expression and are involved in the initiation and progression of various cancer types. Despite the wealth of studies describing transcriptome changes upon lncRNA knockdown, there is limited information describing lncRNA-mediated effects on regulatory elements (REs) modulating gene expression. In this study, we investigated how the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA regulates primary target genes using time-resolved MALAT1 knockdown followed by parallel RNA-seq and ATAC-seq assays. The results revealed that MALAT1 primarily regulates specific protein-coding genes and a substantial decrease in the accessibility downstream of the NR4A1 gene that was associated with a decreased NR4A1 expression. Moreover, the presence of an NR4A1-downstream RE was demonstrated by CRISPR-i assays to define a functional MALAT1/NR4A1 axis. By analyzing TCGA data, we identified a positive correlation between NR4A1 expression and NR4A1-downstream RE accessibility in breast cancer but not in pancreatic cancer. Accordingly, this regulatory mechanism was experimentally validated in breast cancer cells (MCF7) but not in pancreatic duct epithelial carcinoma (PANC1) cells. Therefore, our results demonstrated that MALAT1 is involved in a molecular mechanism that fine-tunes NR4A1 expression by modulating the accessibility of a downstream RE in a cell type-specific manner.


Assuntos
Regulação Neoplásica da Expressão Gênica , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , RNA Longo não Codificante , RNA Longo não Codificante/genética , Humanos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Linhagem Celular Tumoral , Células MCF-7 , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Feminino , Sequências Reguladoras de Ácido Nucleico
2.
Biol. Res ; 56: 6-6, 2023. ilus, tab, graf
Artigo em Inglês | LILACS | ID: biblio-1429907

RESUMO

BACKGROUND: Despite representing the largest fraction of animal life, the number of insect species whose genome has been sequenced is barely in the hundreds. The order Dermaptera (the earwigs) suffers from a lack of genomic information despite its unique position as one of the basally derived insect groups and its importance in agroecosystems. As part of a national educational and outreach program in genomics, a plan was formulated to engage the participation of high school students in a genome sequencing project. Students from twelve schools across Chile were instructed to capture earwig specimens in their geographical area, to identify them and to provide material for genome sequencing to be carried out by themselves in their schools. RESULTS: The school students collected specimens from two cosmopolitan earwig species: Euborellia annulipes (Fam. Anisolabididae) and Forficula auricularia (Fam. Forficulidae). Genomic DNA was extracted and, with the help of scientific teams that traveled to the schools, was sequenced using nanopore sequencers. The sequence data obtained for both species was assembled and annotated. We obtained genome sizes of 1.18 Gb (F. auricularia) and 0.94 Gb (E. annulipes) with the number of predicted protein coding genes being 31,800 and 40,000, respectively. Our analysis showed that we were able to capture a high percentage (≥ 93%) of conserved proteins indicating genomes that are useful for comparative and functional analysis. We were also able to characterize structural elements such as repetitive sequences and non-coding RNA genes. Finally, functional categories of genes that are overrepresented in each species suggest important differences in the process underlying the formation of germ cells, and modes of reproduction between them, features that are one of the distinguishing biological properties that characterize these two distant families of Dermaptera. CONCLUSIONS: This work represents an unprecedented instance where the scientific and lay community have come together to collaborate in a genome sequencing project. The versatility and accessibility of nanopore sequencers was key to the success of the initiative. We were able to obtain full genome sequences of two important and widely distributed species of insects which had not been analyzed at this level previously. The data made available by the project should illuminate future studies on the Dermaptera.


Assuntos
Animais , Insetos/genética , Chile , Análise de Sequência de DNA
3.
Lupus ; 31(5): 637-645, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35382633

RESUMO

OBJECTIVE: The objective is to describe the main characteristics of patients with systemic lupus erythematosus (SLE) in Argentina and to examine the influence of ethnicity on the expression of the disease. PATIENTS AND METHODS: RELESSAR is a multicentre register carried out by 106 researchers from 67 rheumatologic Argentine centres. It is a cross-sectional study of SLE (1982/1997 ACR) patients. RELESSAR electronic database includes demographic, cumulative SLE manifestations, SELENA-SLEDAI, SLICC-SDI, Katz's severity and Charlson's comorbidity indexes and treatment patterns. RESULTS: We included 1,610 patients, 91.7% were female with a median age at diagnosis of 28.1 ± 12.8; 96.2% met ≥4 ACR 1982/97 criteria. Frequent manifestations were arthritis (83.5%), malar rash (79.5%), photosensitivity (75.3%), haematological (63.8%) and renal disease (47.4%), antinuclear antibodies (96%), anti-dsDNA (66.5%) and anti-Smith antibodies (29%). The mean Selena-SLEDAI score at last visit was 3.18 (SD 4.3) and mean SDI was 1 (SD 1.3). The accumulated treatments most frequently used were antimalarials (90.4%), corticosteroids (90%), azathioprine (31.8%), intravenous cyclophosphamide (30.2%), mycophenolate mofetil or mycophenolic acid (24.5%), methotrexate (19.3%), belimumab 5.3% and rituximab 5.1%. Refractory lupus was diagnosed in 9.3% of the cases. The main causes of death were lupus activity (25.0%), activity and concomitant infections (25.0%), infections (18.2%), vascular disease (13.6%) and cancer (4.5%). Mortality was associated with higher SLEDAI, Katz, damage indexes and comorbidities. Of the 1610 patients included, 44.6% were Caucasian, 44.5% Mestizo, 8.1% Amerindian and 1.2% Afro-Latin American. Mestizo patients had higher male representation, low socioeconomic status, more inadequate medical coverage, fewer formal years of education and shorter disease duration. Polyadenopathies and Raynaud's phenomenon were more frequent in Caucasians. In the logistic regression analysis higher damage index (OR 1.28, CI 95% 1.02-1.61, p = 0.03) remained associated to mestizo ethnicity. CONCLUSIONS: This study represents the largest number of adult patients with SLE studied in Argentina. Caucasian patients were differentiated by having Raynaud's phenomenon and polyadenopathy more frequently, while patients of Mestizo origin had higher damage indexes.


Assuntos
Etnicidade , Lúpus Eritematoso Sistêmico , Argentina/epidemiologia , Estudos Transversais , Feminino , Humanos , Lúpus Eritematoso Sistêmico/complicações , Masculino , Fenótipo , Índice de Gravidade de Doença
4.
Rev. argent. reumatolg. (En línea) ; 33(1): 14-25, ene. - mar. 2022. tab
Artigo em Espanhol | LILACS, BINACIS | ID: biblio-1394706

RESUMO

Introducción: el lupus es una enfermedad compleja y varias veces de difícil abordaje. Alcanzar la remisión es uno de los objetivos, incorporando opciones terapéuticas. Objetivos: describir las características generales de los pacientes según el estado de la enfermedad y el uso de belimumab. Materiales y métodos: estudio de corte transversal, registro RELESSAR. Se definió el estado de la enfermedad como: remisión: SLEDAI=0 y sin corticoides; baja actividad de la enfermedad: SLEDAI >0 y ≤4 y sin corticoides; control no óptimo: SLEDAI >4 y cualquier dosis de corticoides. Resultados: se incluyeron 1.277 pacientes, 23,4% en remisión, 12,6% en baja actividad y 63,8% con control no óptimo. En este último grupo eran más jóvenes y con menor duración de la enfermedad; presentaban mayores índices de actividad y cronicidad, y mayor empleo de inmunosupresores. Solo el 22,3% de los pacientes con criterio potencial de uso de belimumab (lupus eritematoso sistémico activo a pesar del tratamiento estándar) lo recibía en ese momento. Las variables asociadas a hospitalizaciones fueron: terapia con corticoides, ciclofosfamida y mayor SLICC. Conclusiones: se refleja la complejidad del manejo de estos pacientes y se visualizan aspectos estructurales como la desigualdad. El uso del belimumab resultaría beneficioso en los pacientes seleccionados.


Introduction: lupus is a complex disease and often difficult to approach. Achieving remission is one of the objectives, incorporating therapeutic options. Objectives: to describe the characteristics of the patients and the use of belimumab, according to the status of the disease. Materials and methods: cross-sectional study. Patients of the RELESSAR registry. Stratification: Remission: SLEDAI=0 and without corticosteroids. Low disease activity SLEDAI> 0 and ≤4 and without corticosteroids and non-optimal control: SLEDAI> 4 and any dose of corticosteroids. Results: a total of 1,277 patients were included, 23.4% in remission, 12.6% in low disease activity and 63.8% in non-optimal control. The last group was younger and had a shorter duration of the disease. They had higher activity and chronicity indices and greater use of immunosuppressants. Only 22.3% of the patients with potential criteria for the use of belimumab (activity disease despite standard treatment) were receiving it. The variables associated with hospitalizations were: corticosteroids, cyclophosphamide and higher SLICC. Those associated with severe infection: mycophenolate mofetil, azathioprine, corticosteroids, and higher SLICC. Conclusions: the complexity of the management of these patients is reflected, visualizing structural aspects such as inequality. The use of belimumab could be beneficial in selected patients.

5.
Rev. argent. reumatolg. (En línea) ; 33(1): 14-25, ene. - mar. 2022. tab
Artigo em Espanhol | LILACS, BINACIS | ID: biblio-1392898

RESUMO

Introducción: el lupus es una enfermedad compleja y varias veces de difícil abordaje. Alcanzar la remisión es uno de los objetivos, incorporando opciones terapéuticas. Objetivos: describir las características generales de los pacientes según el estado de la enfermedad y el uso de belimumab. Materiales y métodos: estudio de corte transversal, registro RELESSAR. Se definió el estado de la enfermedad como: remisión: SLEDAI=0 y sin corticoides; baja actividad de la enfermedad: SLEDAI >0 y ≤4 y sin corticoides; control no óptimo: SLEDAI >4 y cualquier dosis de corticoides. Resultados: se incluyeron 1.277 pacientes, 23,4% en remisión, 12,6% en baja actividad y 63,8% con control no óptimo. En este último grupo eran más jóvenes y con menor duración de la enfermedad; presentaban mayores índices de actividad y cronicidad, y mayor empleo de inmunosupresores. Solo el 22,3% de los pacientes con criterio potencial de uso de belimumab (lupus eritematoso sistémico activo a pesar del tratamiento estándar) lo recibía en ese momento. Las variables asociadas a hospitalizaciones fueron: terapia con corticoides, ciclofosfamida y mayor SLICC. Conclusiones: se refleja la complejidad del manejo de estos pacientes y se visualizan aspectos estructurales como la desigualdad. El uso del belimumab resultaría beneficioso en los pacientes seleccionados.


Introduction: lupus is a complex disease and often difficult to approach. Achieving remission is one of the objectives, incorporating therapeutic options. Objectives: to describe the characteristics of the patients and the use of belimumab, according to the status of the disease. Materials and methods: cross-sectional study. Patients of the RELESSAR registry. Stratification: Remission: SLEDAI=0 and without corticosteroids. Low disease activity SLEDAI> 0 and ≤4 and without corticosteroids and non-optimal control: SLEDAI> 4 and any dose of corticosteroids. Results: a total of 1,277 patients were included, 23.4% in remission, 12.6% in low disease activity and 63.8% in non-optimal control. The last group was younger and had a shorter duration of the disease. They had higher activity and chronicity indices and greater use of immunosuppressants. Only 22.3% of the patients with potential criteria for the use of belimumab (activity disease despite standard treatment) were receiving it. The variables associated with hospitalizations were: corticosteroids, cyclophosphamide and higher SLICC. Those associated with severe infection: mycophenolate mofetil, azathioprine, corticosteroids, and higher SLICC. Conclusions: the complexity of the management of these patients is reflected, visualizing structural aspects such as inequality. The use of belimumab could be beneficial in selected patients.


Assuntos
Humanos , Lúpus Eritematoso Sistêmico , Encaminhamento e Consulta , Terapêutica
6.
Rev. argent. reumatolg. (En línea) ; 32(4): 12-20, dic. 2021. tab
Artigo em Espanhol | LILACS, BINACIS | ID: biblio-1376439

RESUMO

Introducción: las limitaciones laborales son un punto importante a considerar en el tratamiento de la espondiloartritis axial (EspAax) dado que esta enfermedad afecta a las personas en la etapa más productiva de la vida. Objetivos: describir la situación laboral en pacientes con EspAax de Argentina, incluyendo la espondilitis anquilosante (EA) y la espondiloartritis axial no radiográfica (EspAax-nr), y evaluar los factores asociados a la pérdida de productividad laboral (PPL) en esta cohorte nacional y los factores asociados a estar empleado. Materiales y métodos: en este estudio transversal y multicéntrico se incluyeron pacientes con diagnóstico de EA y EspAax-nr según los criterios de clasificación de la Assessment of SpondyloArthritis international Society (ASAS 2009) y en edad laboral (≤65 años). Los objetivos principales fueron evaluar la situación laboral, el ausentismo y el presentismo, valorados por el cuestionario Work Productivity and Activity Impairment Spondyloarthritis (WPAI-SpA). Se utilizó el coeficiente de Spearman para evaluar la correlación entre las medidas de la enfermedad y la PPL. Se realizó un análisis bivariado y multivariado para evaluar los factores asociados a estar empleado. Resultados: se incluyeron 129 pacientes con EspAax, 95 (73,6 %) con EA y 34 (26,4%) con EspAax-nr. La mediana (p25-75) de edad fue de 45 (35-55) años. La duración mediana de la enfermedad fue de 62 (24-123) meses y el retraso en el diagnóstico fue de 24 (6-72) meses. Sesenta (46,5%) pacientes estaban empleados. La mediana (p25-75) de presentismo de los pacientes con EA fue del 29,6% (0-57) y del 30% (20-40) para los pacientes con EspAax-nr (p=0,02). Asimismo, la mediana (p25-75) de PPL fue del 30% en ambos grupos de pacientes. Se encontró una correlación positiva entre la PPL y las siguientes variables: ASDAS (Rho:0.60), BASDAI (Rho:0.50), BASFI (Rho:0.60), ASQoL (Rho:0.60) y ASAS health index (Rho:0.54). En el análisis bivariado, los factores asociados al desempleo fueron el diagnóstico de EA, la edad avanzada, la mayor duración de la enfermedad, las comorbilidades (hipertensión y diabetes), el menor número de años de educación, la peor calidad de vida y la menor capacidad funcional. En el análisis multivariado, una mejor función física (evaluada por BASFI) se asoció de forma independiente a estar empleado. Conclusiones: este estudio demostró que la PPL en esta cohorte nacional fue del 30% en la EspAax. Se asoció con la actividad de la enfermedad, el estado de salud, la calidad de vida y la capacidad funcional. Una mejor función física se relacionó en forma independiente con una mayor probabilidad de mantener a los pacientes con EspAax empleados.


Introduction: work disability is an important outcome in the treatment of spondyloarthritis (SpA) since this disease affects people in the most productive stage of life. Objectives: to investigate working status in patients with axial spondyloarthritis (axSpA) from Argentina, including ankylosing spondylitis (AS) and nonradiographic axial SpA (nr-axSpA), and to evaluate factors associated with work productivity loss (WPL) in this national cohort and factors associated with being employed. Materials and methods: patients with a diagnosis of AS and nr-axSpA according to Assessment of SpondyloArthritis international Society (ASAS 2009) classification criteria and in working age (≤65 years) were included in this multicentric cross-sectional study. Outcomes of interest were employment status, absenteeism and presenteeism, assessed by the Work Productivity and Activity Impairment Spondyloarthritis (WPAI-SpA) questionnaire. Spearman's coefficient was used to assess the correlation between disease measures and WPL. Bivariate and multivariate analysis were performed in order to evaluate factors associated with being employed. Results: 129 patients with axSpA were included, 95 (73.6%) with AS and 34 (26.4%) with nr-axSpA. Median (p25-75) age of 45 (35-55) years. Median (p25-75) disease duration was 62 (24-123) months and diagnosis delay was 24 (6-72) months. 60 (46.5%) of the patients were employed. Median (p25-75) presenteeism of AS patients was 29.6% (0-57) and 30% (20-40) for patients with EspAax-nr (p=0.02). Median (p25-75) WPL was 30% in both groups of patients. A positive correlation was found between WPL and the following variables: ASDAS (Rho:0.60), BASDAI (Rho:0.50), BASFI (Rho:0.60), ASQoL (Rho:0.60) and ASAS health index (Rho:0.54). In the bivariate analysis, the factors associated with unemployment were AS diagnosis, older age, longer disease duration, comorbidities (hypertension and diabetes), fewer years of education, worse quality of life and lower functional capacity. In the multivariate analysis, better physical function (assessed by BASFI) was independently associated with being employed. Conclusions: this study showed that WPL in this national cohort was 30% in axSpA. It was associated with disease activity, health status, quality of life and functional capacity. Better physical function was independently associated with a higher likelihood of keeping patients with axSpA employed.


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Espondiloartrite Axial/epidemiologia , Doenças Profissionais/epidemiologia , Qualidade de Vida , Fatores Socioeconômicos , Modelos Logísticos , Nível de Saúde , Estudos Transversais , Estudos de Coortes , Absenteísmo , Eficiência , Presenteísmo , Espondiloartrite Axial/etiologia , Espondiloartrite Axial não Radiográfica/etiologia , Espondiloartrite Axial não Radiográfica/epidemiologia
7.
Rev. argent. reumatolg. (En línea) ; 32(1): 21-25, mar. 2021. ilus, tab
Artigo em Espanhol | LILACS, BINACIS | ID: biblio-1279755

RESUMO

Introducción: Diversas entidades clínicas, como enfermedades autoinmunes, infecciones, neoplasias y fármacos pueden manifestarse con lesiones vasculíticas en la piel. Debido a la heterogeneidad de las causas, suelen representar un desafío diagnóstico. El objetivo de este estudio es describir la etiología de las vasculitis cutáneas (VC) y evaluar las características clínicas, histológicas y de laboratorio halladas en estos pacientes. Material y métodos: Se realizó un estudio retrospectivo con revisión de historias clínicas de pacientes mayores de 16 años con VC por diagnóstico clínico y/o histológico evaluados en el período 2010-2018. Resultados: Se incluyeron 74 pacientes. El 69% son mujeres con una edad media al diagnóstico de 41 años (DE 16.5, rango 16-75). Las causas más frecuentes asociadas a las VC fueron las enfermedades autoinmunes (EAI) en un 50% de los pacientes evaluados. En el 29.7% de los casos no pudo identificarse una causa subyacente. En el 2.7% de los casos se asoció a neoplasias, otro 2.7% a fármacos y un 12% a otras etiologías. El 76% de los pacientes presentaron formas clínicas no severas, predominando la púrpura palpable en el 65% de los casos. Entre los pacientes biopsiados, el 76% fueron vasculitis leucocitoclásticas (VLC). Como manifestaciones extracutáneas asociadas, predominó el compromiso articular (43,2%). En las vasculitis asociadas a EAI, el 33 % presentó compromiso renal, en tanto que éste no se observó en ninguno de los pacientes con vasculitis idiopáticas. El 78% de los pacientes recibieron glucocorticoides sistémicos. Conclusión: En nuestro centro, la etiología más común de VC fue la asociada a EAI. La mayoría de los pacientes eran mujeres. Clínicamente predominaron las manifestaciones cutáneas no severas y la VLC fue el hallazgo más frecuente en las biopsias.


Background: Various clinical entities, such as autoimmune diseases, infections, neoplasms and drugs can manifest with vasculitic lesions on the skin. Due to the heterogeneity of causes, they often represent a diagnostic challenge. The aim of this study is to describe the etiology of cutaneous vasculitis (CV) and to assess the clinical, histological and laboratory characteristics found in these patients. Material and methods: A retrospective study was carried out with a review of the medical records of patients over 16 years old with CV by clinical and/or histological diagnosis evaluated in the period 2010-2018. Results: 74 patients were included. 69% are women with a mean age at diagnosis of 41 years (SD 16.5, range 16-75). The most frequent causes associated with CVs were autoimmune diseases (AID) in 50% of the patients evaluated. In 29.7% of the cases, an underlying cause could not be identified. In 2.7% of the cases it was associated with neoplasms, another 2.7% with drugs, and 12% with other etiologies. 76% of the patients presented non-severe clinical forms, with palpable purpura predominant in 65% of the cases. Among the biopsied patients, 76% were leukocytoclastic vasculitis (LCV). As associated extracutaneous manifestations, joint involvement predominated (43.2%). In vasculitis associated with AID, 33% presented renal involvement, while this was not observed in any of the patients with idiopathic vasculitis. 78% of the patients received systemic glucocorticoids. Conclusion: In our center, the most common etiology of CV was associated with AID. Most of the patients were women. Clinically, non-severe skin manifestations predominated and VLC was the most frequent finding in biopsies.


Assuntos
Vasculite , Manifestações Cutâneas , Diagnóstico Clínico
8.
Biol. Res ; 54: 41-41, 2021. ilus
Artigo em Inglês | LILACS | ID: biblio-1505797

RESUMO

Cellular fate and gene expression patterns are modulated by different epigenetic factors including non-coding RNAs (ncRNAs) and chromatin organization. Both factors are dynamic throughout male germ cell differentiation on the seminiferous tubule, despite the transcriptional inactivation in the last stages of spermatogenesis. Sperm maturation during the caput-to-cauda transit on the epididymis involves changes in chromatin organization and the soma-to-germ line transference of ncRNAs that are essential to obtain a functional sperm for fertilization and embryo development. Here, the male environment (diseases, drugs, mental stress) is crucial to modulate these epigenetic factors throughout sperm maturation, affecting the corresponding offspring. Paternal transgenerational inheritance has been directly related to sperm epigenetic changes, most of them associated with variations in the ncRNA content and chromatin marks. Our aim is to give an overview about how epigenetics, focused on ncRNAs and chromatin, is pivotal to understand spermatogenesis and sperm maturation, and how the male environment impacts the sperm epigenome modulating the offspring gene expression pattern.


Assuntos
Humanos , Masculino , Cromatina/genética , Epigênese Genética/genética , Espermatogênese/genética , Expressão Gênica , Diferenciação Celular
9.
J Am Chem Soc ; 141(39): 15605-15610, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31536338

RESUMO

The formation of oligomeric soluble aggregates is related to the toxicity of amyloid peptides and proteins. In this manuscript, we report the use of a ruthenium polypyridyl complex ([Ru(bpy)2(dpqp)]2+) to track the formation of amyloid oligomers at different times using photoluminescence anisotropy. This technique is sensitive to the rotational correlation time of the molecule under study, which is consequently related to the size of the molecule. [Ru(bpy)2(dpqp)]2+ presents anisotropy values of zero when free in solution (due to its rapid rotation and long lifetime) but larger values as the size and concentration of amyloid-ß (Aß) oligomers increase. Our assays show that Aß forms oligomers immediately after the assay is started, reaching a steady state at ∼48 h. SDS-PAGE, DLS, and TEM were used to confirm and characterize the formation of oligomers. Our experiments show that the rate of formation for Aß oligomers is temperature dependent, with faster rates as the temperature of the assay is increased. The probe was also effective in monitoring the formation of α-synuclein oligomers at different times.


Assuntos
Amiloide/química , Medições Luminescentes/métodos , Polímeros/química , Anisotropia , Processos Fotoquímicos , Compostos de Rutênio/química
10.
RNA ; 24(3): 371-380, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29222118

RESUMO

Triplexes are noncanonical DNA structures, which are functionally associated with regulation of gene expression through ncRNA targeting to chromatin. Based on the rules of Hoogsteen base-pairing, polypurine sequences of a duplex can potentially form triplex structures with single-stranded oligonucleotides. Prediction of triplex-forming sequences by bioinformatics analyses have revealed enrichment of potential triplex targeting sites (TTS) at regulatory elements, mainly in promoters and enhancers, suggesting a potential function of RNA-DNA triplexes in transcriptional regulation. Here, we have quantitatively evaluated the potential of different sequences of human and mouse ribosomal RNA genes (rDNA) to form triplexes at different salt and pH conditions. We show by biochemical and biophysical approaches that some of these predicted sequences form triplexes with high affinity, following the canonical rules for triplex formation. We further show that RNA triplex-forming oligos (TFOs) are more stable than their DNA counterpart, and point mutations strongly affect triplex formation. We further show differential sequence requirements of pyrimidine and purine TFO sequences for efficient binding, depending on the G-C content of the TTS. The unexpected sequence specificity, revealing distinct sequence requirements for purine and pyrimidine TFOs, shows that in addition to the Hoogsteen pairing rules, a sequence code and mutations have to be taken into account to predict genomic TTS.


Assuntos
DNA Ribossômico/genética , DNA/genética , Oligonucleotídeos/genética , Animais , Pareamento de Bases , Sítios de Ligação , DNA/química , DNA Ribossômico/química , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Camundongos , Oligonucleotídeos/química , Mutação Puntual , Regiões Promotoras Genéticas/genética , Purinas/química , Pirimidinas/química , Sequências Reguladoras de Ácido Nucleico/genética
11.
Nucleic Acids Res ; 45(18): 10534-10554, 2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-28977666

RESUMO

CHD3 and CHD4 (Chromodomain Helicase DNA binding protein), two highly similar representatives of the Mi-2 subfamily of SF2 helicases, are coexpressed in many cell lines and tissues and have been reported to act as the motor subunit of the NuRD complex (nucleosome remodeling and deacetylase activities). Besides CHD proteins, NuRD contains several repressors like HDAC1/2, MTA2/3 and MBD2/3, arguing for a role as a transcriptional repressor. However, the subunit composition varies among cell- and tissue types and physiological conditions. In particular, it is unclear if CHD3 and CHD4 coexist in the same NuRD complex or whether they form distinct NuRD complexes with specific functions. We mapped the CHD composition of NuRD complexes in mammalian cells and discovered that they are isoform-specific, containing either the monomeric CHD3 or CHD4 ATPase. Both types of complexes exhibit similar intranuclear mobility, interact with HP1 and rapidly accumulate at UV-induced DNA repair sites. But, CHD3 and CHD4 exhibit distinct nuclear localization patterns in unperturbed cells, revealing a subset of specific target genes. Furthermore, CHD3 and CHD4 differ in their nucleosome remodeling and positioning behaviour in vitro. The proteins form distinct CHD3- and CHD4-NuRD complexes that do not only repress, but can just as well activate gene transcription of overlapping and specific target genes.


Assuntos
Autoantígenos/metabolismo , DNA Helicases/metabolismo , Regulação da Expressão Gênica , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Animais , Linhagem Celular Tumoral , Galinhas , Reparo do DNA , Humanos , Nucleossomos/metabolismo , Transcrição Gênica
12.
J Cell Biochem ; 116(10): 2283-92, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25833220

RESUMO

The development and survival of male germ cells depend on the antioxidant capacity of the seminiferous tubule. Glutathione (GSH) plays an important role in the antioxidant defenses of the spermatogenic epithelium. Autophagy can act as a pro-survival response during oxidative stress or nutrient deficiency. In this work, we evaluated whether autophagy is involved in spermatogonia-type germ cell survival during severe GSH deficiency. We showed that the disruption of GSH metabolism with l-buthionine-(S,R)-sulfoximine (BSO) decreased reduced (GSH), oxidized (GSSG) glutathione content, and GSH/GSSG ratio in germ cells, without altering reactive oxygen species production and cell viability, evaluated by 2',7'-dichlorodihydrofluorescein (DCF) fluorescence and exclusion of propidium iodide assays, respectively. Autophagy was assessed by processing the endogenous protein LC3I and observing its sub-cellular distribution. Immunoblot and immunofluorescence analysis showed a consistent increase in LC3II and accumulation of autophagic vesicles under GSH-depletion conditions. This condition did not show changes in the level of phosphorylation of AMP-activated protein kinase (AMPK) or the ATP content. A loss in S-glutathionylated protein pattern was also observed. However, inhibition of autophagy resulted in decreased ATP content and increased caspase-3/7 activity in GSH-depleted germ cells. These findings suggest that GSH deficiency triggers an AMPK-independent induction of autophagy in germ cells as an adaptive stress response.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glutationa/metabolismo , Estresse Oxidativo/genética , Espermatogônias/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Trifosfato de Adenosina/biossíntese , Animais , Antioxidantes/metabolismo , Autofagia/genética , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Glutationa/deficiência , Dissulfeto de Glutationa/metabolismo , Masculino , Camundongos , Propídio/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Túbulos Seminíferos/crescimento & desenvolvimento , Túbulos Seminíferos/metabolismo , Espermatogônias/crescimento & desenvolvimento
13.
Rev. chil. cardiol ; 33(3): 165-172, dic. 2014. tab
Artigo em Espanhol | LILACS | ID: lil-743818

RESUMO

Antecedentes: El infarto agudo del miocardio (IAM) es consecuencia de la disrupción del endotelio vascular que lleva a oclusión del vaso coronario por un trombo. En la coronariografía de algunos de estos pacientes es posible encontrar arterias coronarias sin lesiones significativas. Objetivo: Comparar características basales, presentación clínica, tratamiento y mortalidad intrahospitalaria de pacientes con IAM, con y sin lesiones significativas. Métodos: Se incluyeron 3.194 pacientes registrados en la ficha electrónica GEMI que además del diagnóstico de IAM, hubiesen sido sometidos a estudio coronariográfico. Se consideró "sin lesiones significativas" si ninguna estenosis era > al 50 por ciento. Resultados: El grupo sin lesiones significativas correspondió a 116 pacientes (3,6 por ciento). La edad promedio en este grupo fue menor (60+/-14 años vs 62+/-12 años, p<0,024) y con una mayor proporción de mujeres. En este grupo se observó menor prevalencia de dislipidemia (30,2 por ciento vs 42,8 por ciento, p<0.007), diabetes mellitus (14,7 por ciento vs 28,9 por ciento, p<0.001) y tabaquismo (40,5 por ciento vs 61,3 por ciento, p<0.0001). En el ECG de ingreso predominó el IAM sin SDST y menor elevación de CK y CK-MB, junto a una mejor fracción de eyección. Durante la hospitalización se utilizó menos aspirina, clopidogrel, betabloqueadores, heparina ev y HBPM. Existió un mayor uso de antagonistas de Calcio. Conclusiones: Los pacientes con IAM sin lesiones significativas tienen una menor carga de factores de riesgo coronario, es más frecuente encontrar al ingreso ECG SSDST, menor grado de daño miocárdico y mejor fracción de eyección. Además, se observan diferencias en el tratamiento farmacológico en este grupo de pacientes.


Background: Acute myocardial infarction (AMI) is a consequence of disruption of coronary artery lesions leading to intracoronary thrombosis. Some patients with AMI show no significant lesions in a coronary angiography. Aim: To compare clinical characteristics, treatment and intrahospital mortality in patients with AMI with or without coronary artery lesions. Methods: 3194 patients from a clinical registry of AMI (GEMI) were included. AMI with no significant coronary artery lesions was defined as the abscense of any lesion >50 percent. Results: 116 patients (3.6 percent) had no significant coronary lesions (NSL). Compared to the group with significant lesions (SL), they were younger (60+/-14 years vs 62+/-12 years, p<0,024) and the proportion of women was higher. NSL patients lower prevalence of hyperlipidemia (30,2 percent vs 42,8 percent, p<0.007), diabetes mellitus (14,7 percent vs 28,9 percent, p<0.001) and smoking habit (40,5 percent vs 61,3 percent, p<0.0001). NSL patients had a higher prevalence of non ST elevation AMI and lower levels of CK and CK-MB, as well as a lower ejection fraction. During hospitalization, NSL patients received aspirin, clopidogrel, betablockers, iv heparin and LMW heparin, less frequently tan SL patients. In contrast, they received calcium antagonists more frequently. Conclusion: Patients with AMI and no significant coronary lesions have a lower prevalence of coronary artery disease risk factors. A greater frequency of non ST elevation AMI, lower indices of myocardial necrosis and better EF was also observed. The intensity of pharmacologic therapy was lower.


Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Infarto do Miocárdio/mortalidade , Infarto do Miocárdio/tratamento farmacológico , Biomarcadores , Estenose Coronária , Estudos Transversais , Mortalidade Hospitalar , Análise Multivariada , Fatores de Risco , Volume Sistólico
14.
Biol. Res ; 44(2): 169-180, 2011. ilus, tab
Artigo em Inglês | LILACS | ID: lil-602973

RESUMO

In this article, we focus on the fundamental role of vitamin C transporters for the normal delivery of vitamin C to germ cells in the adluminal compartment of seminiferous tubules. We argue that the redox status within spermatozoa or in semen is partly responsible for the etiology of infertility. In this context, antioxidant defence plays a critical role in male fertility. Vitamin C, a micronutrient required for a wide variety of metabolic functions, has long been associated with male reproduction. Two systems for vitamin C transport have been described in mammals. Facilitative hexose transporters (GLUTs), with 14 known isoforms to date, GLUT1-GLUT14, transport the oxidized form of vitamin C (dehydroascorbic acid) into the cells. Sodium ascorbic acid co-transporters (SVCTs), SVCT1 and SVCT2 transport the reduced form of vitamin C (ascorbic acid). Sertoli cells control germ cell proliferation and differentiation through cell-cell communication and form the blood-testis barrier. Because the blood-testis barrier limits direct access of molecules from the plasma into the adluminal compartment of the seminiferous tubule, one important question is the method by which germ cells obtain vitamin C. Some interesting results have thrown light on this matter. Expression of SVCT2 and some isoforms of GLUT transporters in the testis have previously been described. Our group has demonstrated that Sertoli cells express functionally active vitamin C transporters. Kinetic characteristics were described for both transport systems (SVCT and GLUT systems). Sertoli cells are able to transport both forms of vitamin C. These findings are extremely relevant, because Sertoli cells may control the amount of vitamin C in the adluminal compartment, as well as regulating the availability of this metabolite throughout spermatogenesis.


Assuntos
Animais , Humanos , Masculino , Camundongos , Ratos , Ácido Ascórbico/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Estresse Oxidativo/fisiologia , Epitélio Seminífero/citologia , Epitélio Seminífero/metabolismo , Células de Sertoli/metabolismo , Transportadores de Sódio Acoplados à Vitamina C/metabolismo , Transporte Biológico , Infertilidade Masculina/metabolismo , Mamíferos
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