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1.
Int J Equity Health ; 18(1): 18, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31155006

RESUMO

BACKGROUND: Despite representing 70 million people in Latin America, access to comprehensive rehabilitation and participation in the community remains a challenge for persons with disability (PWDs) in the region. Through enactment of the Disability Law, Colombia has made improvements to recognize and address some of the barriers for PWDs, including access to comprehensive rehabilitation. However access remains limited with significant disconnect between perspectives of various stakeholders and the needs of the population. We examined the unique perceptions on access to comprehensive rehabilitation services and participation of PWDs. We also explored the perspective of caregivers of PWDs, rehabilitation professionals, and other stakeholders on the experiences of PWDs. Our goal was to identify gaps in the implementation of comprehensive rehabilitation programs, and barriers to access resources for comprehensive rehabilitation or services that would impact participation of PWDs. METHODS: Qualitative study conducted in 2017. Data was collected from a purposive sample of adults with physical disability, aged 18-44 years, who had received rehabilitation services at a local partner organization and with different backgrounds and experiences with disability. Purposive sampling was also conducted with caregivers, rehabilitation professionals, and other stakeholders. Socio-demographic information was collected and semi-structured interviews were conducted by a research team member, recorded, transcribed and analyzed using a thematic analysis method to identify main themes related to our aim. CES University ethical review board approved this study. RESULTS: Thirty-two participants were interviewed: eight were male, 42.1 ± 11.1 years old, and 44% (n = 14) were PWDs. Three main themes were identified among all the participants: the meaning of rehabilitation, challenges to access services, and participation. Challenges to access services had three sub-themes: barriers to personal mobility, perceptions and knowledge on disability, and navigating the system. CONCLUSION: The main focus of rehabilitation as perceived by stakeholders is still on functional rehabilitation. If healthcare personnel is better trained on disability and if those with disabilities are actively involved in the developing these programs, the focus may evolve to a comprehensive and equitable rehabilitation process that fosters full participation.

2.
Disabil Rehabil Assist Technol ; : 1-6, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31094586

RESUMO

Purpose: Access to an appropriate wheelchair is a human right. Only between 5-15% of people who need a wheelchair have access to one. One of the key barriers to access is the lack of appropriately trained rehabilitation professionals. The objective of this study was to evaluate basic manual wheelchair provision knowledge in final-year physiotherapy undergraduate students in two programs in Colombia. Materials and methods: Students took the International Society of Wheelchair Professionals Wheelchair Service Provision - Basic Test which was administered online and in Spanish. The minimum score to pass the test is 70%; it assesses seven domains: Assessment; Prescription; Products; Fitting; User training; Follow-up, maintenance, and repairs; and Process. Results and conclusions: One-hundred sixteen students took the test and no one passed the test. The highest median domain scores were in Assessment and Process while the lowest were in Fitting and Products. The limitations of this study include that this sample does not represent all physiotherapy programmes or students in Colombia, there may be potential errors in the Spanish translation of the outcome measure, and students encountered Internet connectivity issues during the test that may have impacted their scores. Immediate interventions are required to improve teaching and students' learning outcomes related to basic manual wheelchair provision in these two programs. This study may serve as a foundation for future regional or national studies that assess the situation of wheelchair provision training in rehabilitation programs that will inform improvement actions. This manuscript is also available in Spanish as Supplemental Material . Implications for rehabilitation This study indicates that students' current knowledge on basic appropriate manual wheelchair provision from two physiotherapy programs in Colombia is insufficient. Students' knowledge does not align with the minimum guidelines recommended for wheelchair service provision by the World Health Organization. Objectively identifying the gap in knowledge in rehabilitation trainees (i.e., physiotherapy students) is a strategy to promote the inclusion of assistive technology related content in formal academic training. The need to include formal training of appropriate wheelchair provision persists and without this training, people with disabilities who require a wheelchair for mobility will continue to face barriers to full participation in society.

3.
Antioxidants (Basel) ; 7(12)2018 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-30486406

RESUMO

Oxidative stress is associated with obstetric complications during ovarian hyperstimulation in women undergoing in vitro fertilization. The follicular fluid contains high levels of proteins, which are the main targets of free radicals. The aim of this work was to determine specific biomarkers of non-enzymatic oxidative modifications of proteins from follicular fluid in vivo, and the effect of ovarian stimulation with gonadotropins on these biomarkers. For this purpose, 27 fertile women underwent both a natural and a stimulated cycle. The biomarkers, glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), Nε-(carboxymethyl)lysine (CML), and Nε-(carboxyethyl)lysine (CEL), were measured by gas-liquid chromatography coupled to mass spectrometry. Results showed that follicular fluid contained products of protein modifications by direct metal-catalyzed oxidation (GSA and AASA), glycoxidation (CML and CEL), and lipoxidation (CML). GSA was the most abundant biomarker (91.5%). The levels of CML amounted to 6% of the total lesions and were higher than AASA (1.3%) and CEL (1.2%). In the natural cycle, CEL was significantly lower (p < 0.05) than in the stimulated cycle, suggesting that natural cycles are more protected against protein glycoxidation. These findings are the basis for further research to elucidate the possible relevance of this follicular biomarker of advanced glycation end product in fertility programs.

4.
PLoS One ; 13(10): e0204769, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30286127

RESUMO

INTRODUCTION: People with mobility impairments often rely on wheelchairs as their primary means of mobility. Untrained wheelchair service providers may provide inappropriate wheelchairs and services which result in negative consequences in wheelchair users' health, quality of life, safety, and social participation. This study aimed to evaluate the influence of the Spanish Hybrid Course on Basic Wheelchair Service Provision, a training based on the World Health Organization's Wheelchair Service Training Program-Basic Level, to increase knowledge in basic level wheelchair service provision among a group of wheelchair service providers from Colombia. In addition, we developed a satisfaction survey which participants completed after the training to understand levels of satisfaction with the training intervention. METHODS: A quasi-experimental study was conducted to evaluate changes in basic level wheelchair knowledge using the Wheelchair Service Provision-Basic Test. Paired sample t tests were used to assess pre-and post-training changes in basic level wheelchair knowledge. The Hybrid Satisfaction Survey was developed in collaboration with a multidisciplinary, international stakeholders' group. The survey's construct of interest was level of satisfaction determined by interaction, instructor, instruction methodology, content, and technology, using a five-point Likert scale (0 = strongly disagree to 4 = strongly agree). The survey was completed anonymously after the training intervention and analyzed using frequencies and percentages. RESULTS: Fifteen wheelchair service providers in Colombia completed the Spanish Hybrid Course. Mean post-scores were significantly higher (Mean (M) = 56.13, Standard Deviation (SD) = 7.8), than pre-assessment scores (M = 50.07, SD = 8.38, t(14) = 4.923, p = <0.0001). Participants who completed the surveys (N = 15) reported that the Spanish Hybrid Course was well received, with 98.66% of responses distributed in favorable levels (>3). CONCLUSIONS: The Spanish Hybrid Course proved to be effective in increasing basic level wheelchair knowledge with a high satisfaction level among participants. Further testing is needed to evaluate the effectiveness of this course across different individuals and countries as a potential tool to build professional capacity in basic level wheelchair provision.

5.
BMC Bioinformatics ; 19(1): 239, 2018 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-29940840

RESUMO

BACKGROUND: The adaptive immune response intrinsically depends on hypervariable human leukocyte antigen (HLA) genes. Concomitantly, correct HLA phenotyping is crucial for successful donor-patient matching in organ transplantation. The cost and technical limitations of current laboratory techniques, together with advances in next-generation sequencing (NGS) methodologies, have increased the need for precise computational typing methods. RESULTS: We tested two widespread HLA typing methods using high quality full genome sequencing data from 150 individuals in 50 family trios from the Genome Denmark project. First, we computed descendant accuracies assessing the agreement in the inheritance of alleles from parents to offspring. Second, we compared the locus-specific homozygosity rates as well as the allele frequencies; and we compared those to the observed values in related populations. We provide guidelines for testing the accuracy of HLA typing methods by comparing family information, which is independent of the availability of curated alleles. CONCLUSIONS: Although current computational methods for HLA typing generally provide satisfactory results, our benchmark - using data with ultra-high sequencing depth - demonstrates the incompleteness of current reference databases, and highlights the importance of providing genomic databases addressing current sequencing standards, a problem yet to be resolved before benefiting fully from personalised medicine approaches HLA phenotyping is essential.

6.
Eur Heart J ; 39(25): 2390-2397, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29750272

RESUMO

Aims: The gut microbiome influences metabolic syndrome (MetS) and inflammation and is therapeutically modifiable. Arterial stiffness is poorly correlated with most traditional risk factors. Our aim was to examine whether gut microbial composition is associated with arterial stiffness. Methods and results: We assessed the correlation between carotid-femoral pulse wave velocity (PWV), a measure of arterial stiffness, and gut microbiome composition in 617 middle-aged women from the TwinsUK cohort with concurrent serum metabolomics data. Pulse wave velocity was negatively correlated with gut microbiome alpha diversity (Shannon index, Beta(SE)= -0.25(0.07), P = 1 × 10-4) after adjustment for covariates. We identified seven operational taxonomic units associated with PWV after adjusting for covariates and multiple testing-two belonging to the Ruminococcaceae family. Associations between microbe abundances, microbe diversity, and PWV remained significant after adjustment for levels of gut-derived metabolites (indolepropionate, trimethylamine oxide, and phenylacetylglutamine). We linearly combined the PWV-associated gut microbiome-derived variables and found that microbiome factors explained 8.3% (95% confidence interval 4.3-12.4%) of the variance in PWV. A formal mediation analysis revealed that only a small proportion (5.51%) of the total effect of the gut microbiome on PWV was mediated by insulin resistance and visceral fat, c-reactive protein, and cardiovascular risk factors after adjusting for age, body mass index, and mean arterial pressure. Conclusions: Gut microbiome diversity is inversely associated with arterial stiffness in women. The effect of gut microbiome composition on PWV is only minimally mediated by MetS. This first human observation linking the gut microbiome to arterial stiffness suggests that targeting the microbiome may be a way to treat arterial ageing.

7.
Physiol Genomics ; 50(2): 117-126, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29341867

RESUMO

Disruption in the metabolism of lipids is broadly classified under dyslipidemia and relates to the concentration of lipids in the blood. Dyslipidemia is a predictor of cardio-metabolic disease including obesity. Traditionally, the large interindividual variation has been related to genetic factors and diet. Genome-wide association studies have identified over 150 loci related to abnormal lipid levels, explaining ~40% of the total variation. Part of the unexplained variance has been attributed to environmental factors including diet, but the extent of the dietary contribution remains unquantified. Furthermore, other factors are likely to influence lipid metabolism including the gut microbiome, which plays an important role in the digestion of different dietary components including fats and polysaccharides. Here we describe the contributing role of host genetics and the gut microbiome to dyslipidemia and discuss the potential therapeutic implications of advances in understanding the gut microbiome to the treatment of dyslipidemia.

8.
Rev. biol. trop ; 65(4): 1609-1624, Oct.-Dec. 2017. tab, graf
Artigo em Espanhol | LILACS-Express | ID: biblio-897646

RESUMO

Resumen Dioon holmgrenii esta catalogada en peligro de extinción y es endémica del sur de Oaxaca, México. A pesar de la situación de esta especie, no existe información específica sobre aspectos ecológicos para cada población. Esto dificulta la autorización de planes de manejo para su conservación. El objetivo de este trabajo fue determinar los atributos dendrométricos, la estructura poblacional y la diversidad de estadios en diez poblaciones de D. holmgrenii localizadas en la Sierra Madre del Sur de Oaxaca. Cuatro unidades de muestreo (UM) de 25 x 50 m cada una (1 250 m2) se establecieron por población, 40 UM en total (50 000 m2). La densidad de plantas, el diámetro basal (DB), área basal (AB), altura de tallo (AT), diámetro de copa (DC), área de copa (AC) y número de hojas (NH) de los individuos se evaluó de enero a mayo 2013. Con estos datos se determinó la estructura poblacional, índice de dominancia estructural (IDE), heterogeneidad (H´), uniformidad (E) y semejanza (ISct) de estadios. La densidad en las poblaciones varió de 148 a 954 plantas ha-1. El área basal total fluctuó de 1.3 a 17.6 m2 ha-1 y área de copa total osciló de 78.3 a 1 136.3 m2 ha-1. Los atributos dendrométricos de los individuos (DB = 7.7 a 13.4 cm, AB = 0.010 a 0.023 m2, AT = 0.20 a 0.47 m, DC = 0.49 a 1.09 m, AC = 0.55 a 1.55 m2 y NH = 7.06 a 14.90) fueron diferentes entre las poblaciones (p ˂ 0.05). Adulto 1 y adulto 2 tuvieron mayor IDE (0.01 a 5.63) en todas las poblaciones. La estructura poblacional más común fue aquella donde juvenil 1 y juvenil 2 tuvieron las proporciones más bajas de individuos, mientras que adulto 1 tuvo la más alta. La heterogeneidad (H´= 1.05 a 1.71) y uniformidad (E = 0.57 a 0.88) de estadios fueron significativamente diferentes entre las poblaciones (p˂0.05), con al menos cuatro estadios (3.91 = 48.9x8/100) diferentes entre ellas para la mitad de las comparaciones (22 de 45) de semejanza (ISct < 0.68). La diversidad alfa de estadios fue alta en poblaciones donde no se dañaron las plantas, en contraste a la diversidad beta que fue baja entre poblaciones con presencia de actividad humana. Estadios con alto vigor reproductivo (adulto 1 y adulto 2) presentaron una mayor dominancia estructural en todas las poblaciones. Estos resultados pueden ayudar a la conservación in-situ de D. holmgrenii y se recomienda sean considerados en la elaboración y aplicación de planes de manejo de sus poblaciones.


Abstract Dioon holmgrenii is an endangered species and found in Southern Oaxaca, Mexico. Notwithstanding the extintion condition of this species, there is no relevant information on the ecological aspects of its populations, and this situation complicates the approval of management plans for its conservation. Our aim was to study D. holmgrenii forest characteristics, population structure and life-stages diversity in ten populations located in Sierra Madre del Sur, Oaxaca. For this, four sampling units (SU) measuring 25 x 50 m each (1 250 m2) were established per population, 40 SU in total (50 000 m2). Basal diameter (BD), basal area (BA), stem height (AT), crown diameter (DC), crown area (AC), and number of leaves (NH), were measured for each individual, from January to May 2013. Additionally, plant density, population structure, structural dominance index (IDE), heterogeneity (H'), evenness (E) and similarity (ISct) of life-stages (seedling, juvenile 1, juvenile 2, pre-reproductive, adult 1, adult 2, adult 3, and adult 4) were assessed. Plant density ranged from 148 to 954 individuals ha-1. Total basal area ranged from 1.3 to 17.6 m2.ha-1, and total crown area varied from 78.3 to 1 136.3 m2.ha-1. Forest traits of each individual (DB= 7.7 to 13.4 cm, AB= 0.010 to 0.023 m2, AT= 0.20 to 0.47 m, DC= 0.49 to 1.09 m, AC= 0.55 to 1.55 m2 and NH= 7.06 to 14.90) were different (p˂0.05) among populations. The highest values of IDE (0.01 to 5.63) were found in adult 1 and adult 2 in all populations. The most common population structure was found in those populations with the lowest proportions in juvenile 1 and juvenile 2, and the highest proportion in adult 1. Heterogeneity (H'= 1.05 to 1.71) and uniformity (E= 0.57 to 0.88) of life-stages were significantly different among populations (p˂0.05), and at least four life-stages (3.91= 48.9x8/100) were different among populations for half of the comparisons (22 of 45) of similarity (ISct < 0.68). Alfa diversity of life-stages was high in populations without plant damage, while beta diversity was low in those populations with human presence. Life-stages with the highest reproductive vigour (adult 1, adult 2) showed the highest structural dominance in all populations. These results may support the design and operation of management plans of D. holmgrenii populations, to enhance its protection in the area.

9.
Nature ; 548(7665): 87-91, 2017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28746312

RESUMO

Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits. Genetic variation is identified mainly by mapping short reads to the reference genome or by performing local assembly. However, these approaches are biased against discovery of structural variants and variation in the more complex parts of the genome. Hence, large-scale de novo assembly is needed. Here we show that it is possible to construct excellent de novo assemblies from high-coverage sequencing with mate-pair libraries extending up to 20 kilobases. We report de novo assemblies of 150 individuals (50 trios) from the GenomeDenmark project. The quality of these assemblies is similar to those obtained using the more expensive long-read technology. We use the assemblies to identify a rich set of structural variants including many novel insertions and demonstrate how this variant catalogue enables further deciphering of known association mapping signals. We leverage the assemblies to provide 100 completely resolved major histocompatibility complex haplotypes and to resolve major parts of the Y chromosome. Our study provides a regional reference genome that we expect will improve the power of future association mapping studies and hence pave the way for precision medicine initiatives, which now are being launched in many countries including Denmark.


Assuntos
Variação Genética/genética , Genética Populacional/normas , Genoma Humano/genética , Genômica/normas , Análise de Sequência de DNA/normas , Adulto , Alelos , Criança , Cromossomos Humanos Y/genética , Dinamarca , Feminino , Haplótipos/genética , Humanos , Complexo Principal de Histocompatibilidade/genética , Masculino , Idade Materna , Taxa de Mutação , Idade Paterna , Mutação Puntual/genética , Padrões de Referência
10.
BMC Genomics ; 17 Suppl 2: 396, 2016 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-27357839

RESUMO

BACKGROUND: The association between aberrant signal processing by protein kinases and human diseases such as cancer was established long time ago. However, understanding the link between sequence variants in the protein kinase superfamily and the mechanistic complex traits at the molecular level remains challenging: cells tolerate most genomic alterations and only a minor fraction disrupt molecular function sufficiently and drive disease. RESULTS: KinMutRF is a novel random-forest method to automatically identify pathogenic variants in human kinases. Twenty six decision trees implemented as a random forest ponder a battery of features that characterize the variants: a) at the gene level, including membership to a Kinbase group and Gene Ontology terms; b) at the PFAM domain level; and c) at the residue level, the types of amino acids involved, changes in biochemical properties, functional annotations from UniProt, Phospho.ELM and FireDB. KinMutRF identifies disease-associated variants satisfactorily (Acc: 0.88, Prec:0.82, Rec:0.75, F-score:0.78, MCC:0.68) when trained and cross-validated with the 3689 human kinase variants from UniProt that have been annotated as neutral or pathogenic. All unclassified variants were excluded from the training set. Furthermore, KinMutRF is discussed with respect to two independent kinase-specific sets of mutations no included in the training and testing, Kin-Driver (643 variants) and Pon-BTK (1495 variants). Moreover, we provide predictions for the 848 protein kinase variants in UniProt that remained unclassified. A public implementation of KinMutRF, including documentation and examples, is available online ( http://kinmut2.bioinfo.cnio.es ). The source code for local installation is released under a GPL version 3 license, and can be downloaded from https://github.com/Rbbt-Workflows/KinMut2 . CONCLUSIONS: KinMutRF is capable of classifying kinase variation with good performance. Predictions by KinMutRF compare favorably in a benchmark with other state-of-the-art methods (i.e. SIFT, Polyphen-2, MutationAssesor, MutationTaster, LRT, CADD, FATHMM, and VEST). Kinase-specific features rank as the most elucidatory in terms of information gain and are likely the improvement in prediction performance. This advocates for the development of family-specific classifiers able to exploit the discriminatory power of features unique to individual protein families.


Assuntos
Biologia Computacional/métodos , Mutação , Proteínas Quinases/genética , Bases de Dados de Proteínas , Árvores de Decisões , Variação Genética , Humanos , Software
11.
Rev. obstet. ginecol. Venezuela ; 76(1): 53-59, mar. 2016. ilus, tab
Artigo em Espanhol | LILACS-Express | ID: lil-788163

RESUMO

Objetivo: Identificar la mutación ΔF508 en pacientes con íleo meconial. Ambiente: En el Instituto de Investigaciones Genéticas de la Facultad de Medicina de la Universidad del Zulia. Maracaibo. Métodos: Se estudiaron diez pacientes con ileo meconial. La detección de la mutación ΔF508 se realizó a partir de amplificación por reacción en cadena de la polimerasa de un segmento del gen de fibrosis quística de 98 pares de bases que contiene el codón que codifica a la fenilalanina en la posición 508 y el cual está ausente en los que tienen la mutación. Resultados: Se detectó la mutación ΔF508 en ambos alelos del gen de la fibrosis quística en tres pacientes, en un solo alelo en cinco y en dos no se identificó el alelo ΔF508 en su patrón molecular. Conclusión: El íleo meconial fue el marcador que sugirió el diagnóstico de fibrosis quística y permitió el asesoramiento genético de las familias al confirmar la presencia de la mutación ΔF508.


Objective: To perform ΔF508 mutation in patients with meconium ileus. Setting: In the Genetic Research Institute of the Faculty of Medicine. University of Zulia. Maracaibo. Methods: We studied 10 patients with meconium ileus. Detection of the mutation was performed from the amplification of a 98 pair of bases cystic fibrosis gene segment which contains the codon that encodes fenilalanine in the 508 position by polymerase chain reaction. This amplified product is absent in those who have the mutation. Results: The ΔF508 mutation was detected in both alleles of the cystic fibrosis gene in 3 patients, 5 were heterozygous for this mutation and in two patients were undetectable. Conclusion: Meconium ileus was the marker that suggested the diagnosis of cystic fibrosis and allowed the genetic counseling in this family to confirm the presence of the ΔF508 mutation.

12.
Biochem Soc Trans ; 44(1): 197-201, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26862206

RESUMO

Phospholipase C (PLC)-mediated hydrolysis of the limited pool of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] requires replenishment from a larger pool of phosphatidylinositol (PtdIns) via sequential phosphorylation by PtdIns 4-kinases and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinases. Since PtdIns is synthesized in the endoplasmic reticulum (ER) and PtdIns(4,5)P2 is generated in the PM, it has been postulated that PtdIns transfer proteins (PITPs) provide the means for this lipid transfer function. Recent studies identified the large PITP protein, Nir2 as important for PtdIns transfer from the ER to the PM. It was also found that Nir2 was required for the transfer of phosphatidic acid (PtdOH) from the PM to the ER. In Nir2-depleted cells, activation of PLC leads to PtdOH accumulation in the PM and PtdIns synthesis becomes severely impaired. In quiescent cells, Nir2 is localized to the ER via interaction of its FFAT domain with ER-bound VAMP-associated proteins VAP-A and-B. After PLC activation, Nir2 also binds to the PM via interaction of its C-terminal domains with diacylglycerol (DAG) and PtdOH. Through these interactions, Nir2 functions in ER-PM contact zones. Mutations in VAP-B that have been identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrig's disease) cause aggregation of the VAP-B protein, which then impairs its binding to several proteins, including Nir2. These findings have shed new lights on the importance of non-vesicular lipid transfer of PtdIns and PtdOH in ER-PM contact zones with a possible link to a devastating human disease.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfatidilinositóis/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Transporte Biológico , Humanos
13.
Biochim Biophys Acta ; 1861(3): 213-26, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26724696

RESUMO

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its derivatives diphosphoinositol phosphates (DPIPs) play key signaling and regulatory roles. However, a direct function of these molecules in lipid and membrane homeostasis remains obscure. Here, we have studied the cold tolerance phenotype of yeast cells lacking the Inp51-mediated phosphoinositide-5-phosphatase. Genetic and biochemical approaches showed that increased metabolism of PI(4,5)P2 reduces the activity of the Pho85 kinase by increasing the levels of the DPIP isomer 1-IP7. This effect was key in the cold tolerance phenotype. Indeed, pho85 mutant cells grew better than the wild-type at 15 °C, and lack of this kinase abolished the inp51-mediated cold phenotype. Remarkably, reduced Pho85 function by loss of Inp51 affected the activity of the Pho85-regulated target Pah1, the yeast phosphatidate phosphatase. Cells lacking Inp51 showed reduced Pah1 abundance, derepression of an INO1-lacZ reporter, decreased content of triacylglycerides and elevated levels of phosphatidate, hallmarks of the pah1 mutant. However, the inp51 phenotype was not associated to low Pah1 activity since deletion of PAH1 caused cold sensitivity. In addition, the inp51 mutant exhibited features not shared by pah1, including a 40%-reduction in total lipid content and decreased membrane fluidity. These changes may influence the activity of membrane-anchored and/or associated proteins since deletion of INP51 slows down the transit to the vacuole of the fluorescent dye FM4-64. In conclusion, our work supports a model in which changes in the PI(4,5)P2 pool affect the 1-IP7 levels modulating the activity of Pho85, Pah1 and likely additional Pho85-controlled targets, and regulate lipid composition and membrane properties.


Assuntos
Membrana Celular/enzimologia , Temperatura Baixa , Fluidez de Membrana , Lipídeos de Membrana/metabolismo , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Adaptação Fisiológica , Transporte Biológico , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Corantes Fluorescentes/metabolismo , Regulação Fúngica da Expressão Gênica , Genótipo , Fenótipo , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Monoéster Fosfórico Hidrolases/genética , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Sistemas do Segundo Mensageiro , Fatores de Tempo , Triglicerídeos/metabolismo
14.
Glia ; 64(3): 440-56, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26539695

RESUMO

The spatial organization of vascular endothelial growth factor (VEGF) signaling is a key determinant of vascular patterning during development and tissue repair. How VEGF signaling becomes spatially restricted and the role of VEGF secreting astrocytes in this process remains poorly understood. Using a VEGF-GFP fusion protein and confocal time-lapse microscopy, we observed the intracellular routing, secretion and immobilization of VEGF in scratch-activated living astrocytes. We found VEGF to be directly transported to cell-extracellular matrix attachments where it is incorporated into fibronectin fibrils. VEGF accumulated at ß1 integrin containing fibrillar adhesions and was translocated along the cell surface prior to internalization and degradation. We also found that only the astrocyte-derived, matrix-bound, and not soluble VEGF decreases ß1 integrin turnover in fibrillar adhesions. We suggest that polarized VEGF release and ECM remodeling by VEGF secreting cells is key to control the local concentration and signaling of VEGF. Our findings highlight the importance of astrocytes in directing VEGF functions and identify these mechanisms as promising target for angiogenic approaches.


Assuntos
Astrócitos/metabolismo , Polaridade Celular/fisiologia , Matriz Extracelular/metabolismo , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Astrócitos/ultraestrutura , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hidrazonas/metabolismo , Antígeno Ki-67/metabolismo , Microscopia Confocal , Neurônios/metabolismo , Fotodegradação , Puromicina/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/genética , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Fatores de Tempo , Transfecção
15.
Invest Clin ; 56(3): 284-95, 2015 Sep.
Artigo em Espanhol | MEDLINE | ID: mdl-26710543

RESUMO

Neural tube defects (NTD) are the most common congenital anomalies of the central nervous system, with a multifactorial pattern of inheritance, presumably involving the interaction of several genetic and environmental factors. The methylenetetrahydrofolate reductase (MTHFR) gene 677C>T polymorphism has been implicated as a risk factor for NTD. The main objective of this research was to investigate the association of the 677C>T polymorphism of the MTHFR gene as a genetic risk factor for NTD. Molecular analysis was performed in DNA samples from 52 mothers with antecedent of NTD offspring and from 119 healthy control mothers. Using the Polymerase Chain Reaction, a 198 bases pairs fragment was digested with the restriction enzyme Hinfi. 677T MTHFR allele frequencies for the problem and the control groups were 51.92% and 34.45%, respectively, and 677C MTHFR allele frequencies were 48.08% and 65.55%, respectively. There were significant differences in allele (p: 0.002) and genotype (p: 0.007) frequencies between these two groups. The odds ratio (OR) to the TT genotype vs. the CC genotype was estimated as OR: 4.9 [95% CI: 1,347-6.416] p: 0.002; CT+TT vs. CC: OR: 2.9 [95% CI: 1.347-6.416] p: 0.005; TT vs. CT+CC: OR: 2.675 [95% CI: 1,111-6.441] p: 0.024. The data presented in this study support the relationship between MTHFR 677C>T polymorphism and risk in mothers with antecedent of NTD offspring.


Assuntos
Predisposição Genética para Doença , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Defeitos do Tubo Neural/genética , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Frequência do Gene , Genótipo , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Defeitos do Tubo Neural/epidemiologia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Gravidez , Fatores de Risco , Adulto Jovem
16.
Invest. clín ; 56(3): 284-295, sep. 2015. ilus, tab
Artigo em Espanhol | LILACS-Express | ID: biblio-841086

RESUMO

Los defectos del tubo neural (DTN) son las alteraciones congénitas más frecuentes del sistema nervioso central. El mecanismo de transmisión hereditario de los DTN aislados es multifactorial, se debe a la interacción de factores ambientales y genéticos. El polimorfismo 677C>T del gen de la metilentetrahidrofolato reductasa (MTHFR) ha sido implicado como factor de riesgo para DTN. El objetivo de este trabajo fue investigar la asociación del polimorfismo 677C>T del gen de la MTHFR como factor de riesgo en los DTN. Se analizaron muestras de ADN de 52 madres con antecedente de al menos un hijo con DTN y de 119 madres controles. A través de la reacción en cadena de la polimerasa se amplificó un fragmento de 198 pb, el cual se sometió a digestión con la enzima HinfI. La frecuencia alélica de la MTHFR en los grupos problema y control fue de 51,92% y 34,45%; para el alelo T y 48,08% y 65,55%; para el C respectivamente. Se encontró diferencia significativa entre las frecuencias del alelo T y del alelo C (p: 0,002), así como entre las frecuencias genotípicas (p: 0,007) al ser comparadas en ambos grupos. El odds ratio (OR) para el genotipo TT vs CC se estimó como OR: 4,9 [IC 95%: 1,347-6,416] p: 0,002; CT+TT vs CC: OR: 2,9 [IC 95%: 1,347-6,416] p: 0,005; TT vs CT+CC: OR: 2,675 [IC 95%: 1,111-6,441] p: 0,024. Los presentes datos aportan una asociación significativa entre el polimorfismo 677C>T de la MTHFR y riesgo aumentado en las madres con antecedente de hijos con DTN.


Neural tube defects (NTD) are the most common congenital anomalies of the central nervous system, with a multifactorial pattern of inheritance, presumably involving the interaction of several genetic and environmental factors. The methylenetetrahydrofolate reductase (MTHFR) gene 677C>T polymorphism has been implicated as a risk factor for NTD. The main objective of this research was to investigate the association of the 677C>T polymorphism of the MTHFR gene as a genetic risk factor for NTD. Molecular analysis was performed in DNA samples from 52 mothers with antecedent of NTD offspring and from 119 healthy control mothers. Using the Polymerase Chain Reaction, a 198 bases pairs fragment was digested with the restriction enzyme HinfI. 677T MTHFR allele frequencies for the problem and the control groups were 51.92% and 34.45%, respectively, and 677C MTHFR allele frequencies were 48.08% and 65.55%, respectively. There were significant differences in allele (p: 0.002) and genotype (p: 0.007) frequencies between these two groups. The odds ratio (OR) to the TT genotype vs the CC genotype was estimated as OR: 4.9 [95% CI: 1,347-6.416] p: 0.002; CT+TT vs CC: OR: 2.9 [95% CI: 1.347-6.416] p: 0.005; TT vs CT+CC: OR: 2.675 [95% CI: 1,111-6.441] p: 0.024. The data presented in this study support the relationship between MTHFR 677C>T polymorphism and risk in mothers with antecedent of NTD offspring.

17.
Dev Cell ; 33(5): 549-61, 2015 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-26028218

RESUMO

Sustained agonist-induced production of the second messengers InsP3 and diacylglycerol requires steady delivery of phosphatidylinositol (PtdIns) from its site of synthesis in the ER to the plasma membrane (PM) to maintain PtdIns(4,5)P2 levels. Similarly, phosphatidic acid (PtdOH), generated from diacylglycerol in the PM, has to reach the ER for PtdIns resynthesis. Here, we show that the Drosophila RdgB homolog, Nir2, a presumed PtdIns transfer protein, not only transfers PtdIns from the ER to the PM but also transfers PtdOH to the opposite direction at ER-PM contact sites. PtdOH delivery to the ER is impaired in Nir2-depleted cells, leading to limited PtdIns synthesis and ultimately to loss of signaling from phospholipase C-coupled receptors. These studies reveal a unique feature of Nir2, namely its ability to serve as a highly localized lipid exchanger that ensures that PtdIns synthesis is matched with PtdIns(4,5)P2 utilization so that cells maintain their signaling competence.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Membrana/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfatidilinositóis/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Imunofluorescência , Células HEK293 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Fosfatos de Fosfatidilinositol/metabolismo , RNA Interferente Pequeno/genética
18.
J Cell Sci ; 128(1): 118-28, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25380825

RESUMO

The yeast Efr3p protein is a main regulator of the Stt4p phosphatidylinositol 4-kinase at contact sites between the endoplasmic reticulum and the plasma membrane. A mutation in its fly homologue Rbo, leads to diminished light responses in the eye attributed to progressively impaired PLC signaling. Here, we find that Efr3s plays a role in maintaining responsiveness to the type-I angiotensin II (AngII) receptors. siRNA-mediated depletion of EFR3A and EFR3B impaired the sustained phase of cytosolic Ca(2+) response to high concentration of AngII in HEK293 cells that express wild type but not truncated AGTR1 (AT1a receptor), missing the phosphorylation sites. Efr3 depletion had minimal effect on the recovery of plasma membrane phosphoinositides during stimulation, and AT1 receptors still underwent ligand-induced internalization. A higher level of basal receptor phosphorylation and a larger response was observed after stimulation. Moreover, Gq activation more rapidly desensitized after AngII stimulation in Efr3 downregulated cells. A similar but less pronounced effect of EFR3 depletion was observed on the desensitization of the cAMP response after stimulation with isoproterenol. These data suggest that mammalian Efr3s contribute to the control of the phosphorylation state and, hence, desensitization of AT1a receptors, and could affect responsiveness of G-protein-coupled receptors in higher eukaryotes.


Assuntos
AMP Cíclico/metabolismo , Lipoilação/fisiologia , Receptor Tipo 1 de Angiotensina/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Agonistas Adrenérgicos beta/farmacologia , AMP Cíclico/genética , Células HEK293 , Humanos , Isoproterenol/farmacologia , Lipoilação/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/genética , Sistemas do Segundo Mensageiro/efeitos dos fármacos
19.
Mol Biol Cell ; 25(7): 1061-72, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24501421

RESUMO

Vascular endothelial growth factor (VEGF) is a critical regulator of endothelial cell differentiation and vasculogenesis during both development and tumor vascularization. VEGF-165 is a major form that is secreted from the cells via a poorly characterized pathway. Here we use green fluorescent protein- and epitope-tagged VEGF-165 and find that its early trafficking between the endoplasmic reticulum and the Golgi requires the small GTP-binding proteins Sar1 and Arf1 and that its glycosylation in the Golgi compartment is necessary for efficient post-Golgi transport and secretion from the cells. The relative temperature insensitivity of VEGF secretion and its Sar1 and Arf1 inhibitory profiles distinguish it from other cargoes using the "constitutive" secretory pathway. Prominent features of VEGF secretion are the retention of the protein on the outer surface of the plasma membrane and the stimulation of its secretion by Ca(2+) and protein kinase C. Of importance, shedding of VEGF-165 from the cell surface together with other membrane components appears to be a unique feature by which some VEGF is delivered to the surroundings to exert its known biological actions. Understanding VEGF trafficking can reveal additional means by which tumor vascularization can be inhibited by pharmacological interventions.


Assuntos
Membrana Celular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Células COS , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Cercopithecus aethiops , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Glicosilação/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Sirolimo/farmacologia , Fator A de Crescimento do Endotélio Vascular/ultraestrutura
20.
Plant Physiol ; 164(3): 1237-49, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24406791

RESUMO

A transcriptomic approach has been used to identify genes predominantly expressed in maize (Zea mays) scutellum during maturation. One of the identified genes is oil body associated protein1 (obap1), which is transcribed during seed maturation predominantly in the scutellum, and its expression decreases rapidly after germination. Proteins similar to OBAP1 are present in all plants, including primitive plants and mosses, and in some fungi and bacteria. In plants, obap genes are divided in two subfamilies. Arabidopsis (Arabidopsis thaliana) genome contains five genes coding for OBAP proteins. Arabidopsis OBAP1a protein is accumulated during seed maturation and disappears after germination. Agroinfiltration of tobacco (Nicotiana benthamiana) epidermal leaf cells with fusions of OBAP1 to yellow fluorescent protein and immunogold labeling of embryo transmission electron microscopy sections showed that OBAP1 protein is mainly localized in the surface of the oil bodies. OBAP1 protein was detected in the oil body cellular fraction of Arabidopsis embryos. Deletion analyses demonstrate that the most hydrophilic part of the protein is responsible for the oil body localization, which suggests an indirect interaction of OBAP1 with other proteins in the oil body surface. An Arabidopsis mutant with a transfer DNA inserted in the second exon of the obap1a gene and an RNA interference line against the same gene showed a decrease in the germination rate, a decrease in seed oil content, and changes in fatty acid composition, and their embryos have few, big, and irregular oil bodies compared with the wild type. Taken together, our findings suggest that OBAP1 protein is involved in the stability of oil bodies.


Assuntos
Arabidopsis/metabolismo , Estruturas Citoplasmáticas/metabolismo , Evolução Molecular , Proteínas de Plantas/metabolismo , Zea mays/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/metabolismo , Western Blotting , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Mutagênese Insercional/genética , Tamanho do Órgão , Proteínas de Plantas/genética , Transporte Proteico , Interferência de RNA , Sementes/metabolismo , Sementes/ultraestrutura , Frações Subcelulares/metabolismo , Zea mays/genética
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