48 results on '"Coelho PS"'
Search Results
2. Preimplantation development of in vitro-produced bovine embryos treated with hydroxychloroquine.
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Coelho PS, Sousa AJO, Rodrigues ISR, Nascimento HS, Pantoja LC, and Miranda MS
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- Animals, Cattle, Female, Antimalarials toxicity, Fertilization in Vitro, Embryo Culture Techniques, Hydroxychloroquine toxicity, Embryonic Development drug effects, Autophagy drug effects, Apoptosis drug effects, Blastocyst drug effects
- Abstract
Hydroxychloroquine (HCQ) is a safe antimalarial drug but its overdosage or inappropriate use, such as during the pandemic, may cause adverse effects once this drug is considered a potent inhibitor of autophagy. Information about HCQ's effects on the reproductive field, including gametes and initial embryos, is limited. In this study, we evaluated the effect of HCQ (1, 6, 12, and 24 μM) on pre-implantation embryo development, autophagy, and apoptosis of bovine embryos produced in vitro. A dose-response experiment showed a reduction (p < 0.05) in cleavage only at the highest concentration. Blastocyst rate was gradually reduced (p < 0.05) with the increase of HCQ dosage starting at 6 μM, with no embryo formation occurring at 24 μM. Further analysis showed that embryos treated with 12 μM of HCQ had a higher (p < 0.05) accumulation of acidic autophagic vesicles on Days 5 and 7 of development and a higher (p < 0.01) apoptotic index on Day 7. To our knowledge, this is the first study to evaluate the effects of HCQ on embryo pre-implantation development in mammals. The results contribute with more information related to the study of autophagy in embryology as well as add some discussion on HCQ toxicology and its effects on reproductive cells., Competing Interests: Declaration of competing interest Authors have no competing interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)
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- 2024
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3. Role of serum-specific immunoglobulin E in egg allergy: a comprehensive study of Portuguese pediatric patients.
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Coelho PS, Santos GMD, Sangalho I, Rosa S, and Pinto PL
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- Humans, Male, Female, Retrospective Studies, Child, Preschool, Child, Infant, Portugal epidemiology, Adolescent, Prevalence, Eggs adverse effects, Egg Hypersensitivity diagnosis, Egg Hypersensitivity immunology, Egg Hypersensitivity epidemiology, Egg Hypersensitivity blood, Immunoglobulin E blood, Immunoglobulin E immunology, Allergens immunology, Biomarkers blood
- Abstract
Introduction: Food allergies represent a growing public health concern, particularly among children. This study aims to examine egg allergy in pediatric patients and analyze the value of serum-specific immunoglobulin E (sIgE) levels as predictive biomarkers for oral food challenge (OFC) outcomes., Methods: Retrospective study, involving pediatric patients with suspected IgE-mediated egg allergy, conducted at a tertiary hospital., Results: Data from 176 pediatric patients were analyzed, revealing a higher male prevalence (59.1%). Most cases (40.3%) presented symptoms in the first year of life, predominantly mucocutaneous symptoms (46%). OFC results varied across various forms of egg presentation, with cooked egg being the most frequently tested food. Positive OFCs were observed in 14.6% (n = 36) of cases. The study identified specific egg protein biomarkers for positive OFC, with ovalbumin for raw egg (sIgE > 1.28 KUA/L; area under the curve [AUC] = 0.917; sensitivity [S] 100%; and specificity [Sp] 92%), ovomucoid for cooked egg (sIgE > 0.99 KUA/L; AUC = 0.788, 95%; S: 79%; and Sp: 74%), and ovomucoid for baked egg (sIgE> 4.63 KUA/L; AUC = 0.870; S: 80%; and Sp: 85%) showing predictive capacities., Conclusions: The findings underscore the importance of considering various forms of egg presentation in the diagnosis and management of egg allergy. The findings highlight the valuable discriminatory capacity and provided reliable biomarkers, such as ovalbumin for raw egg and ovomucoid for cooked and baked egg in risk assessment, aiding in predicting OFC outcomes and helping clinicians to make informed decisions in diagnosing and managing egg allergies, thus improving patient care and quality of life., Competing Interests: The authors declared that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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- 2024
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4. The burden of atopic dermatitis in Portuguese patients: an observational study.
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Coelho PS, Apalhão M, Victorino G, Cardoso C, Camilo J, and Silva JM
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- Humans, Young Adult, Adult, Quality of Life, Portugal epidemiology, Comorbidity, Skin, Severity of Illness Index, Dermatitis, Atopic diagnosis
- Abstract
Atopic dermatitis (AD) is a common inflammatory skin condition that significantly affects patients' lives and imposes both economic and non-economic burdens. The precise societal and individual consequences of AD remain incompletely understood. This study aimed to characterize AD in Portuguese patients and assess its personal, familial, and societal implications, including health status and quality of life. The research, conducted from June 2019 to January 2020, involved 204 confirmed AD patients in Portugal, who completed a 70-question questionnaire. Results show that, on average, patients experienced a two-year delay in diagnosis, with two-thirds having allergic comorbidities. Late-onset AD (after age 20) was found to be correlated with worsening symptoms post-diagnosis. Globally, patients reported substantial effects on health, quality of life, and mental well-being. Effects include significant levels of anxiety, frustration and sleep disorders. Severe AD correlated with more suffering and reduced perceived health, indicating a link between disease severity and quality of life. Remarkably, despite questionable effectiveness, 92% of severe AD patients were prescribed antihistamines, while only 19% received biological treatments. In Portugal, delayed AD diagnosis hinders timely treatment, and despite its profound impact and high comorbidity rates, AD patients tend to remain undertreated. Recognizing the personal and societal repercussions is crucial for enhancing care, contributing to improving QoL, social functioning and global well-being., (© 2024. The Author(s).)
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- 2024
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5. Non-pharmacological treatment-related changes of molecular biomarkers in major depressive disorder: A systematic review and meta-analysis.
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Irwin CL, Coelho PS, Kluwe-Schiavon B, Silva-Fernandes A, Gonçalves ÓF, Leite J, and Carvalho S
- Abstract
Background: Major depressive disorder (MDD) is a serious mood disorder and leading cause of disability. Despite treatment advances, approximately 30% of individuals with MDD do not achieve adequate clinical response. Better understanding the biological mechanism(s) underlying clinical response to specific psychopharmacological interventions may help fine tune treatments in order to further modulate their underlying mechanisms of action. However, little is known regarding the effect of non-pharmacological treatments (NPTs) on candidate molecular biomarker levels in MDD. This review aims to identify molecular biomarkers that may elucidate NPT response for MDD., Methods: We performed a systematic review and a multilevel linear mixed-effects meta-analyses, and a meta-regression. Searches were performed in PubMed, Scopus, and PsycINFO in October 2020 and July 2021., Results: From 1387 retrieved articles, 17 and six studies were included in the systematic review and meta-analyses, respectively. Although there was little consensus associating molecular biomarker levels with symptomology and/or treatment response, brain metabolites accessed via molecular biomarker-focused neuroimaging techniques may provide promising information on whether an individual with MDD would respond positively to NPTs. Furthermore, non-invasive brain stimulation interventions significantly increased the expression of neurotrophic factors (NTFs) compared to sham/placebo, regardless of add-on pharmacological treatment., Conclusions: NTFs are candidate biomarkers to fine-tune NIBS for MDD treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 The Authors.)
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- 2023
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6. The effect of non-pharmaceutical interventions on COVID-19 outcomes: A heterogeneous age-related generalisation of the SEIR model.
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Mendes JM and Coelho PS
- Abstract
Successive generalisations of the basic SEIR model have been proposed to accommodate the different needs of the organisations handling the SARS-CoV-2 epidemic and the assessment of the public health measures adopted and named under the common umbrella of Non-Pharmaceutical Interventions (NPIs). So far, these generalisations have not been able to assess the ability of these measures to avoid infection by the SARS-CoV-2 and thus their contribution to contain the spread of the disease. This work proposes a new generalisation of SEIR model and includes a heterogeneous and age-related generation of infections that depends both on a probability that a contact generates the transmission of the disease and a contact rate. The results show (1) thanks to the universal wearing of facial coverings, the probability that a contact provokes the transmission of the disease was reduced by at least 50% and (2) the impact of the other NPI is so significant that otherwise Portugal would have gone into a non-sustainable situation of having 80% of its population infected in the first 300 days of the pandemic. This situation would have led to a number of deaths almost twenty times higher than the number that was actually recorded by December 26th, 2020. Moreover, the results suggest that even if the requirement of universal wearing of facial coverings was adopted sooner jointly with closing workplaces and resorting to teleworking would have postponed the peak of the incidence, altought the epidemic path would have result in a number of infections hardly managed by the National Health System. Complementary, results confirm that (3) the health authorities adopted a conservative approach on the criteria to consider an infected individual not infective any longer; and (4) the most effective NPIs and stringency levels either impacting on self-protection against infection or reducing the contacts that would eventually result in infection are, in decreasing order of importance, the use of Facial coverings , Workplace closing and Stay at home requirements ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)
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- 2023
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7. Dystonic posturing with athetoid movements in stroke without thalamic lesion.
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Ferreira A, Carvalho V, and Coelho PS
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- Humans, Athetosis etiology, Thalamus diagnostic imaging, Basal Ganglia, Movement Disorders diagnosis, Stroke complications
- Abstract
Complex hyperkinetic movement disorders are a rare complication of stroke, frequently involving posterolateral contralateral thalamic lesions. One of the proposed mechanisms for these presentations is proprioceptive impairment, hence not involving deregulation of the basal ganglia-thalamocortical circuits. We report a patient who presented with dystonic posturing and athetoid movements with onset 2 years after right frontoparietotemporal stroke. Brain MRI showed no thalamic lesion. Based on the phenomenology, a diagnosis of pseudochoreoathetosis was proposed. To our knowledge, this is the first case report of poststroke pseudochoreoathetosis without thalamic involvement., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2023. No commercial re-use. See rights and permissions. Published by BMJ.)
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- 2023
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8. Assessment of Wild Rocket ( Diplotaxis tenuifolia (L.) DC.) Germplasm Accessions by NGS Identified SSR and SNP Markers.
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Reis JM, Pereira RJ, Coelho PS, and Leitão JM
- Abstract
Rocket is the common designation for two baby-leaf salad crops of the Brassicaceae family: Eruca sativa (L.) Cav., usually referred to as annual garden rocket, and Diplotaxis tenuifolia (L.) DC. commonly named to as perennial wild rocket. E. sativa is used for human consumption since antiquity. However, the growing consumer preference for D. tenuifolia is being accompanied by the fast increase in its production area and commercialization of new cultivars. Nevertheless, the worldwide number of wild rocket accessions maintained in germplasm collections is very reduced, the solution for which situation the project “REMIRucula” intends to contribute, establishing a germplasm collection at the INIAV, Oeiras, Portugal. Herein, we report on the establishment via next generation sequencing (NGS) of the first genome assembly of D. tenuifolia and the identification of specific single sequence repeat (SSR) and single nucleotide polymorphisms (SNP) loci for the establishment of specific DNA-markers for this species. A representative set of 87 D. tenuifolia and 3 E. sativa accessions were assessed by 5 SSR and 9 SNP-CAPS markers, allowing a drastic discrimination between both species and the establishment of unequivocal molecular fingerprints for the analyzed accessions. The non-discrimination within six pairs and one trio of D. tenuifolia accessions is discussed.
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- 2022
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9. Recurrent pyogenic infections caused by a novel Gln1420* mutation in the C3 gene.
- Author
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Coelho PS, Gouveia C, Pinto MV, Neves C, Cordeiro AI, and Neves JF
- Abstract
C3 is a crucial protein of the complement system. Congenital C3 deficiency is extremely rare and manifests through recurrent, severe infections and should always be considered as a differential diagnosis of recurrent pyogenic infections. We report a case of a patient with a novel C3 gene mutation, responsible for complete C3 deficiency with impaired complement system activation and recurrent infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Coelho, Gouveia, Pinto, Neves, Cordeiro and Neves.)
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- 2022
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10. Structural disconnectome analysis of acute freezing of gait due to parietal stroke: implications for FOG pathogenesis.
- Author
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Ferreira A, Coelho PS, Alves PN, and Carvalho V
- Subjects
- Aged, 80 and over, Brain, Female, Gait, Humans, Gait Disorders, Neurologic etiology, Parkinson Disease, Stroke complications, Stroke diagnostic imaging
- Abstract
Acute onset isolated freezing of gait (FOG) is a rare and defying diagnosis. Its pathophysiology is not yet totally understood and several brain regions seem to be involved. Postlesional FOG can help to shed light on the networks involved.We report a case of an 80-year-old woman who presented to the emergency department with acute onset impairment of gait. Her medical history was unremarkable. On neurological examination she was presented with difficulties in gait initiation, turning and progressing through narrow spaces. Her remaining neurological examination was unremarkable. Brain CT showed a subacute cortico-subcortical right parieto-occipital infarction. Acute isolated FOG due to parieto-occipital stroke was diagnosed. A structural disconnectome analysis showed that the lesion disrupted structural connections with the ipsilateral ventral striatum. This case highlights that acute onset FOG might be a consequence of a parietal cortical lesion., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2022. No commercial re-use. See rights and permissions. Published by BMJ.)
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- 2022
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11. Addressing hospitalisations with non-error-free data by generalised SEIR modelling of COVID-19 pandemic.
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Mendes JM and Coelho PS
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- Bayes Theorem, COVID-19 pathology, COVID-19 virology, Humans, Pandemics, Portugal epidemiology, Quarantine, SARS-CoV-2 isolation & purification, COVID-19 epidemiology, Hospitalization statistics & numerical data, Models, Statistical
- Abstract
Successive generalisations of the basic SEIR model have been proposed to accommodate the different needs of the organisations handling the SARS-CoV-2 epidemic. These generalisations have not been able until today to represent the potential of the epidemic to overwhelm hospital capacity until today. This work builds on previous generalisations, including a new compartment for hospital occupancy that allows accounting for the infected patients that need specialised medical attention. Consequently, a deeper understanding of the hospitalisations rate and probability as well as of the recovery rates for hospitalised and non-hospitalised individuals is achieved, offering new information and predictions of crucial importance for the planning of the health systems and global epidemic response. Additionally, a new methodology to calibrate epidemic flows between compartments is proposed. We conclude that the two-step calibration procedure is able to recalibrate non-error-free data and showed crucial to reconstruct the series in a specific situation characterised by significant errors over the official recovery cases. The performed modelling also allowed us to understand how effective the several interventions (lockdown or other mobility restriction measures) were, offering insight for helping public authorities to set the timing and intensity of the measures in order to avoid the implosion of the health systems., (© 2021. The Author(s).)
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- 2021
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12. Music streaming services: understanding the drivers of customer purchase and intention to recommend.
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Barata ML and Coelho PS
- Abstract
The music industry has undergone tremendous changes in relation to its production, distribution, and consumption habits due to the exponential development of new technologies, namely streaming platforms. The fact that sales of physical copies continue to decline significantly made it mandatory for this industry to reinvent itself by introducing music streaming services as a key part of its business development. This study aims to understand the factors that influence music consumption through streaming platforms, particularly studying the intention to adopt premium (paid) versions of a music streaming service and recommend them. An extension of the UTAUT2 model (version of the Unified Theory of Acceptance and Use of Technology, applied to the consumer side) was created. Based on data collected from 324 music streaming services users, the framework of this study was tested using structural equation modelling (SEM). Research also included in-depth semi-structured interviews in order to generate a more profound knowledge about the profile, behaviours and motivations of the new music consumer. Our findings confirm that habit, performance expectancy and price value play the most important role in influencing the intention to use a paid music streaming service. Simultaneously, new dimensions such as personalisation, attitude towards piracy and perceived freemium-premium fit arise as having an additional relevant role in adopting this type of service. The research contributes insights into music streaming services consumer behaviour, providing several theoretical and practical implications to music streaming services providers., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)
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- 2021
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13. In silico identification of glycosylphosphatidylinositol-anchored proteins in Paracoccidioides spp.
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Gonçales RA, Salamanca AL, Júnior LR, E Silva KS, de Vasconcelos EJ, Dos Reis TF, Castro RC, C Ruy P, Romagnoli B, Ruiz J, Pereira M, de A Soares CM, and Coelho PS
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- Amino Acid Sequence, Cell Wall genetics, Cell Wall metabolism, Computational Biology, Conserved Sequence, Fungal Proteins chemistry, Fungal Proteins metabolism, Genome, Fungal genetics, Membrane Proteins chemistry, Membrane Proteins metabolism, Open Reading Frames, Paracoccidioides genetics, Paracoccidioides pathogenicity, Paracoccidioidomycosis microbiology, Proteomics, Virulence, Fungal Proteins genetics, Glycosylphosphatidylinositols metabolism, Membrane Proteins genetics, Paracoccidioides metabolism
- Abstract
Aim: To predict glycosylphosphatidylinositol (GPI)-anchored proteins in the genome of Paracoccidioides brasiliensis and Paracoccidioides lutzii . Materials & methods: Five different bioinformatics tools were used for predicting GPI-anchored proteins; we considered as GPI-anchored proteins those detected by at least two in silico analysis methods. We also performed the proteomic analysis of P. brasiliensis cell wall by mass spectrometry. Results: Hundred GPI-anchored proteins were predicted in P. brasiliensis and P. lutzii genomes. A series of 57 proteins were classified in functional categories and 43 conserved proteins were reported with unknown functions. Four proteins identified by in silico analyses were also identified in the cell wall proteome. Conclusion: The data obtained in this study are important resources for future research of GPI-anchored proteins in Paracoccidioides spp. to identify targets for new diagnostic tools, drugs and immunological tests.
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- 2021
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14. CORRELATION STUDY BETWEEN DRUSEN MORPHOLOGY AND FUNDUS AUTOFLUORESCENCE.
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Flores R, Carneiro A, Serra J, Gouveia N, Pereira T, Mendes JM, Coelho PS, Tenreiro S, and Seabra MC
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- Aged, Aged, 80 and over, Female, Follow-Up Studies, Fundus Oculi, Humans, Male, Middle Aged, Prospective Studies, Fluorescein Angiography methods, Ophthalmoscopy methods, Optical Imaging, Retinal Drusen diagnosis, Retinal Pigment Epithelium diagnostic imaging, Tomography, Optical Coherence methods
- Abstract
Purpose: To correlate drusen morphology and outer retinal status with autofluorescence (AF) imaging in patients with intermediate age-related macular degeneration., Methods: Drusen type and morphology were analyzed using color fundus photography and spectral-domain optic coherence tomography, whereas fundus AF was used for drusen AF evaluation. Additional structural changes on spectral-domain optic coherence tomography, such as disruption of external limiting membrane, ellipsoid zone, and retinal pigment epithelium/Bruch membrane complex, as well as the presence of choroidal hypertransmission at correspondent locations were also evaluated and correlated with fundus AF findings. Spearman's correlation coefficient was used to analyze the correlation between spectral-domain optic coherence tomography morphological characteristics of drusen and AF appearance of the corresponding drusen. Strength of correlation was calculated (r), and a P value < 0.05 was considered statistically significant., Results: Two hundred and twenty-eight drusen from 53 eyes of 53 patients were analyzed, 130 soft drusen (57.02%) and 98 cuticular drusen (42.98%). Sixty percent of the drusen were isoautofluorescent (n = 136), 35% hyperautofluorescent (n = 80), and 5% hypoautofluorescent (n = 12). We found positive correlation between drusen AF and hyperreflective foci (r = 0.4). Outer retinal layers morphology (external limiting membrane and ellipsoid zone status and hypertransmission) also correlates with autofluorescent findings (r = 0.3)., Conclusion: Multimodal imaging reveals a broad spectrum of ultrastructural changes, which may reflect different stages in the evolution of drusen. Our results suggest that drusen morphological characteristics and autofluorescent findings are correlated but other factors or cofactors may be involved. The described correlations will help us understand new progression biomarkers of nonexudative age-related macular degeneration.
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- 2021
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15. Validation of the Telephone-Administered Version of the Mediterranean Diet Adherence Screener (MEDAS) Questionnaire.
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Gregório MJ, Rodrigues AM, Salvador C, Dias SS, de Sousa RD, Mendes JM, Coelho PS, Branco JC, Lopes C, Martínez-González MA, Graça P, and Canhão H
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- Adolescent, Adult, Aged, Female, Humans, Male, Middle Aged, Nutrition Assessment, Portugal, Reproducibility of Results, Surveys and Questionnaires, Young Adult, Diet, Mediterranean psychology, Patient Compliance psychology, Telephone
- Abstract
A 14-Item Mediterranean Diet Adherence Screener (MEDAS) questionnaire was developed and validated in face-to-face interviews, but not via telephone. The aims of this study were to evaluate the validity and reliability of a telephone-administered version of the MEDAS as well as to validate the Portuguese version of the MEDAS questionnaire. A convenience community-based sample of adults ( n = 224) participated in a three-stage survey. First, trained researchers administered MEDAS via a telephone. Second, the Portuguese version of Food Frequency Questionnaire (FFQ), and MEDAS were administered in a semi-structured face-to-face interview. Finally, MEDAS was again administered via telephone. The telephone-administered MEDAS questionnaire was compared with the face-to-face-version using several metrics. The telephone-administered MEDAS was significantly correlated with the face-to-face-administered MEDAS [r = 0.805, p < 0.001; interclass correlation coefficient (ICC) = 0.803, p < 0.001] and showed strong agreement (k = 0.60). The MEDAS scores that were obtained in the first and second telephone interviews were significantly correlated (r = 0.661, p < 0.001; ICC = 0.639, p < 0.001). The overall agreement between the Portuguese version of MEDAS and the FFQ-derived Mediterranean diet adherence score had a Cohen's k = 0.39. The telephone-administered version of MEDAS is a valid tool for assessing the adherence to the Mediterranean diet and acquiring data for large population-based studies.
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- 2020
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16. Persistent trigeminal artery in a patient with posterior circulation stroke treated with rt-PA: case report.
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Ferreira A, Coelho PS, and Cruz VT
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- Aged, 80 and over, Computed Tomography Angiography, Fibrinolytic Agents therapeutic use, Humans, Male, Plasminogen Activators therapeutic use, Stroke drug therapy, Stroke pathology, Vertebrobasilar Insufficiency complications, Vertebrobasilar Insufficiency pathology, Vertigo etiology, Stroke etiology, Vertebrobasilar Insufficiency congenital
- Abstract
Background: A persistent trigeminal artery (PTA) is a non-involuted embryonic vessel that connects the cavernous part of the internal carotid artery with the posterior circulation. In the adult it is associated with multiple pathological conditions including trigeminal neuralgia, ophthalmoplegia, hypopituitarism, intracavernous fistula, brain aneurysms and posterior circulation strokes. The latter may occur through steal phenomena or thrombosis in the anterior circulation. PTA associated vertebrobasilar hypoplasia has yet to be associated to TIA like events, however, in the reported case, that seems to be the case with reported vertigo being probably linked to vertebrobasilar insufficiency., Case Report: We present a case of an 82-year-old man with sudden onset neurological deficits, including left hemiparesis with crural predominance, vertical nystagmus, right internuclear ophthalmoplegia, dysarthria and dysmetria on the left arm. CT angiography disclosed basilar artery hypoplasia in the proximal two thirds and a persistent trigeminal artery. He was diagnosed with acute ischemic stroke. He was submitted to rt-PA with partial reversion of deficits., Conclusion: The ischemic events related to PTA remain a rare cause of stroke with specific pathophysiological mechanisms and implications. They may occur through steal phenomena or thrombosis in the anterior circulation. Upon literature review, in the described case both mechanisms seem possible, however the transient episodes of vertigo could have been the first sign of vertebrobasilar insufficiency.
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- 2019
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17. Low Serum Levels of DKK2 Predict Incident Low-Impact Fracture in Older Women.
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Rodrigues AM, Eusébio M, Rodrigues AB, Caetano-Lopes J, Lopes IP, Lopes A, Mendes JM, Coelho PS, Fonseca JE, Branco JC, and Canhão H
- Abstract
There are currently no robust noninvasive markers of fragility fractures. Secreted frizzled related protein-1 (sFRP-1), dickkopf-related protein 1 (DKK1) and DKK2, and sclerostin (SOST) inhibit Wnt signaling and interfere with osteoblast-mediated bone formation. We evaluated associations of serum levels of sFRP-1, DKK1, DKK2, and SOST with incident low-impact fracture and BMD in 828 women aged ≥65 years from EpiDoC, a longitudinal population-based cohort. A structured questionnaire during a baseline clinical appointment assessed prevalent fragility fractures and clinical risk factors (CRFs) for fracture. Blood was collected to measure serum levels of bone turnover markers and Wnt regulators. Lumbar spine and hip BMD were determined by DXA scanning. Follow-up assessment was performed through a phone interview; incident fragility fracture was defined by any new self-reported low-impact fracture. Multivariate Cox proportional hazard models were used to analyze fracture risk adjusted for CRFs and BMD. During a mean follow-up of 2.3 ± 1.0 years, 62 low-impact fractures were sustained in 58 women. A low serum DKK2 level (per 1 SD decrease) was associated with a 1.5-fold increase in fracture risk independently of BMD and CRFs. Women in the two lowest DKK2 quartiles had a fracture incidence rate of 32 per 1000 person-years, whereas women in the two highest quartiles had 14 fragility fractures per 1000 person-years. A high serum sFRP1 level was associated with a 1.6-fold increase in fracture risk adjusted for CRFs, but not independently of BMD. Serum levels of SOST ( r = 0.191; p = 0.0025) and DKK1( r = -0.1725; p = 0.011) were correlated with hip BMD, but not with incident fragility fracture. These results indicate that serum DKK2 and sFRP1 may predict low-impact fracture. The low number of incident fractures recorded is a limitation and serum levels of Wnt regulators should be further studied in other populations as potential noninvasive markers of fragility fractures. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
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- 2019
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18. The burden and undertreatment of fragility fractures among senior women.
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Rodrigues AM, Eusébio M, Santos MJ, Gouveia N, Tavares V, Coelho PS, Mendes JM, Branco JC, and Canhão H
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- Absorptiometry, Photon methods, Aged, Bone Density, Female, Humans, Needs Assessment, Portugal epidemiology, Prevalence, Risk Factors, Women's Health, Bone Density Conservation Agents therapeutic use, Cost of Illness, Health Services Misuse statistics & numerical data, Osteoporosis, Postmenopausal complications, Osteoporosis, Postmenopausal diagnosis, Osteoporosis, Postmenopausal drug therapy, Osteoporosis, Postmenopausal epidemiology, Osteoporotic Fractures epidemiology, Osteoporotic Fractures physiopathology, Osteoporotic Fractures prevention & control, Osteoporotic Fractures psychology, Quality of Life
- Abstract
Using a large population database, we showed that fragility fractures were highly prevalent in senior women and were associated with significant physical disability. However, treatment rates were low because osteoporosis treatment was not prescribed or not agreed to by the majority of women with prevalent fragility fractures., Purpose: The purpose of the study is to estimate prevalence of fragility fractures (FF), risk factors, and treatment rates in senior women and to assess impact of FF on physical function and quality of life., Methods: Women aged 65 years and older from the EpiReumaPt study (2011-2013) were evaluated. Rheumatologists collected data regarding FF, clinical risk factors for fractures, and osteoporosis (OP) treatment. Health-related quality of life (EQ5D) and physical function (HAQ) were analyzed. Peripheral dual-energy X-ray absorptiometry was performed. FF was defined as any self-reported low-impact fracture that occurred after 40 years of age. Prevalence estimates of FF were calculated., Results: Among 3877 subjects evaluated in EpiReumaPt, 884 were senior women. The estimated prevalence of FF was 20.7%. Lower leg was the most frequent fracture site reported (37.8%) followed by wrist (18.6%). Only 7.1% of the senior women reporting a prevalent FF were under treatment for OP, and 13.9% never had treatment. OP treatment was not prescribed in 47.7% of FF women, and 23.4% refused treatment. Age (OR = 2.46, 95% CI 1.11-5.47), obesity (OR = 2.05, 95% CI 1.14-3.70), and low wrist BMD (OR = 2.29; 95% CI 1.20, 4.35; p = 0.012) were positively associated with prevalent FF. A significantly higher proportion of women in the lowest quintile of wrist bone mineral density reported FF (OR = 2.29, 95% CI 1.20-4.35). FF were associated with greater physical disability (β = 0.33, 95% CI 0.13-0.51) independent of other comorbidities., Conclusion: FF was frequently reported among senior women as an important cause of physical disability. However, the prevalence of OP treatment was low, which constitutes a public health problem in this vulnerable group.
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- 2018
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19. Challenges of Ageing in Portugal: Data from the EpiDoC Cohort.
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Rodrigues AM, Gregório MJ, Sousa RD, Dias SS, Santos MJ, Mendes JM, Coelho PS, Branco JC, and Canhão H
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- Aged, Aged, 80 and over, Aging, Cohort Studies, Cross-Sectional Studies, Female, Humans, Male, Portugal epidemiology, Quality of Life, Multimorbidity
- Abstract
Introduction: Portuguese adults have a long lifespan, but it is unclear whether they live a healthy life in their final years. We aimed to determine the prevalence of multimorbidity and characterize lifestyle and other health outcomes among older Portuguese adults., Material and Methods: We performed a cross-sectional evaluation of 2393 adults, aged 65 and older, during the second wave of follow-up of the EpiDoC cohort, a population-based study involving long-term follow-up of a representative sample of the Portuguese population. Subjects completed a structured questionnaire during a telephone interview. Socioeconomic, demographic, lifestyle behaviours, chronic diseases, and health resources consumption were assessed. Cluster analysis was done to identify dietary patterns. Descriptive and analytic analysis was performed to estimate multimorbidity prevalence and its associated factors., Results: Multimorbidity prevalence among older adults was 78.3%, increased with age strata (72.8% for 65 - 69 years to 83.4% for ≥ 80 years), and was highest in Azores (84.9%) and Alentejo (83.6%). The most common chronic diseases were hypertension (57.3%), rheumatic disease (51.9%), hypercholesterolemia (49.4%), and diabetes (22.7%). Depression symptoms were frequent (11.8%) and highest in the oldest strata. The mean health-related quality of life (EQ-5D-3L) score was 0.59 ± 0.38. Hospitalization in the previous 12 months was reported by 25.8% of individuals. Overall, 66.6% of older adults were physically inactive. 'Fruit and vegetables dietary pattern' was followed by 85.4% of individuals; however, regional inequalities were found (69% in Azores). Obesity prevalence was 22.3% overall and was highest among Azoreans (33%)., Conclusion: The high prevalence of multimorbidity, combined with unhealthy lifestyle behaviours, suggests that the elderly populationconstitutes a vulnerable group warranting dedicated intervention.
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- 2018
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20. Dietary Patterns Characterized by High Meat Consumption Are Associated with Other Unhealthy Life Styles and Depression Symptoms.
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Gregório MJ, Rodrigues AM, Eusébio M, Sousa RD, Dias S, André B, Grønning K, Coelho PS, Mendes JM, Graça P, Espnes GA, Branco JC, and Canhão H
- Abstract
Objective: We aimed to identify dietary patterns (DPs) of Portuguese adults, to assess their socioeconomic, demographic, lifestyle determinants, and to identify their impact on health., Design: EpiDoC 2 study included 10,153 Portuguese adults from the EpiDoC Cohort, a population-based study. In this study, trained research assistants using computer-assisted telephone interview collected socioeconomic, demographic, dietary, lifestyles, and health information from March 2013 to July 2015. Cluster analysis was performed, based on questions regarding the number of meals, weekly frequency of soup consumption, vegetables, fruit, meat, fish, dairy products, and daily water intake. Factors associated with DP were identified through logistic regression models., Results: Two DPs were identified: the "meat dietary pattern" and the "fruit & vegetables dietary pattern." After multivariable adjustment, women (OR = 0.52; p < 0.001), older adults (OR = 0.97; p < 0.001), and individuals with more years of education (OR = 0.96; p = 0.025) were less likely to adopt the "meat dietary pattern," while individuals in a situation of job insecurity/unemployment (OR = 1.49; p = 0.013), Azores island residents (OR = 1.40; p = 0.026), current smoking (OR = 1.58; p = 0.001), daily alcohol intake (OR = 1.46; p = 0.023), and physically inactive (OR = 1.86; p < 0.001) were positively and significantly associated with "meat dietary pattern." Moreover, individuals with depression symptoms (OR = 1.50; p = 0.018) and the ones who did lower number of medical appointments in the previous year (OR = 0.98; p = 0.025) were less likely to report this DP., Conclusion: Our results suggest that unhealthy DPs (meat DP) are part of a lifestyle behavior that includes physical inactivity, smoking habits, and alcohol consumption. Moreover, depression symptoms are also associated with unhealthy DPs.
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- 2017
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21. Home-Based Intervention Program to Reduce Food Insecurity in Elderly Populations Using a TV App: Study Protocol of the Randomized Controlled Trial Saúde.Come Senior.
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Rodrigues AM, Gregório MJ, Gein P, Eusébio M, Santos MJ, de Sousa RD, Coelho PS, Mendes JM, Graça P, Oliveira P, Branco JC, and Canhão H
- Abstract
Background: The limited or uncertain access to adequate food in elderly people includes not only economic restrictions but also inability of food utilization due to functional or cognitive impairment, health problems, and illiteracy., Objective: The aim of this work is to present the protocol of the randomized controlled trial Saúde.Come Senior, an educational and motivational television (TV)-based intervention to promote healthy lifestyles and decrease food insecurity in elderly people., Methods: A randomized controlled study will be conducted in subjects aged 60 years and older with food insecurity, identified at 17 primary care centers in the Lisboa e Vale do Tejo health region in Lisbon, Portugal. The primary outcome will be the changes in participants' food insecurity score (evaluated by the Household Food Insecurity Scale) at 3 months. Change in other outcomes will be assessed (dietary habits, nutritional status, physical activity, health status, and clinical outcomes). Subjects will be followed over 6 months; the intervention will last 3 months. Data collection will be performed at 3 different time points (baseline, end of intervention at 3 months, and follow-up at 6 months). The intervention is based on an interactive TV app with an educational and motivational program specifically developed for the elderly that has weekly themes and includes daily content in video format: (1) nutrition and diet tips for healthy eating, (2) healthy, easy to cook and low-cost recipes, and (3) physical exercise programs. Furthermore, brief reminders on health behaviors will also be broadcasted through the TV app. The total duration of the study will be 6 months. The intervention is considered to be effective and meaningful if 50% of the individuals in the experimental group have a decrease of 1 point in the food insecurity score, all the remaining being unchanged. We expect to include and randomize 282 (141 experimental and 141 control) elderly with food insecurity. We will recruit a total of 1,128 subjects considering that 50% of the target individuals are food insecure (based on INFOFAMÍLIA Survey) (567) and about 50% of those will adhere to the study (282)., Results: The randomized controlled trial with the 12-week home-based intervention with a comprehensive program on healthy eating and physical activity delivery is planned to start recruiting participants at the end of 2017., Conclusions: This study will assess the efficacy of this innovative tool (Saúde.Come Senior) for disseminating relevant health information, modifying behaviors, and decreasing food insecurity in an easy, low-cost, and massive way., (©Ana Maria Rodrigues, Maria João Gregório, Pierre Gein, Mónica Eusébio, Maria José Santos, Rute Dinis de Sousa, Pedro S Coelho, Jorge M Mendes, Pedro Graça, Pedro Oliveira, Jaime C Branco, Helena Canhão. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 13.03.2017.)
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- 2017
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22. Prevalence of rheumatic and musculoskeletal diseases and their impact on health-related quality of life, physical function and mental health in Portugal: results from EpiReumaPt- a national health survey.
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Branco JC, Rodrigues AM, Gouveia N, Eusébio M, Ramiro S, Machado PM, da Costa LP, Mourão AF, Silva I, Laires P, Sepriano A, Araújo F, Gonçalves S, Coelho PS, Tavares V, Cerol J, Mendes JM, Carmona L, and Canhão H
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Objectives: To estimate the national prevalence of rheumatic and musculoskeletal diseases (RMDs) in the adult Portuguese population and to determine their impact on health-related quality of life (HRQoL), physical function, anxiety and depression., Methods: EpiReumaPt is a national health survey with a three-stage approach. First, 10 661 adult participants were randomly selected. Trained interviewers undertook structured face-to-face questionnaires that included screening for RMDs and assessments of health-related quality of life, physical function, anxiety and depression. Second, positive screenings for ≥1 RMD plus 20% negative screenings were invited to be evaluated by a rheumatologist. Finally, three rheumatologists revised all the information and confirmed the diagnoses according to validated criteria. Estimates were computed as weighted proportions, taking the sampling design into account., Results: The disease-specific prevalence rates (and 95% CIs) of RMDs in the adult Portuguese population were: low back pain, 26.4% (23.3% to 29.5%); periarticular disease, 15.8% (13.5% to 18.0%); knee osteoarthritis (OA), 12.4% (11.0% to 13.8%); osteoporosis, 10.2% (9.0% to 11.3%); hand OA, 8.7% (7.5% to 9.9%); hip OA, 2.9% (2.3% to 3.6%); fibromyalgia, 1.7% (1.1% to 2.1%); spondyloarthritis, 1.6% (1.2% to 2.1%); gout, 1.3% (1.0% to 1.6%); rheumatoid arthritis, 0.7% (0.5% to 0.9%); systemic lupus erythaematosus, 0.1% (0.1% to 0.2%) and polymyalgia rheumatica, 0.1% (0.0% to 0.2%). After multivariable adjustment, participants with RMDs had significantly lower EQ5D scores (β=-0.09; p<0.001) and higher HAQ scores (β=0.13; p<0.001) than participants without RMDs. RMDs were also significantly associated with the presence of anxiety symptoms (OR=3.5; p=0.006)., Conclusions: RMDs are highly prevalent in Portugal and are associated not only with significant physical function and mental health impairment but also with poor HRQoL, leading to more health resource consumption. The EpiReumaPt study emphasises the burden of RMDs in Portugal and the need to increase RMD awareness, being a strong argument to encourage policymakers to increase the amount of resources allocated to the treatment of rheumatic patients.
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- 2016
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23. Evaluating Common Humoral Responses against Fungal Infections with Yeast Protein Microarrays.
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Coelho PS, Im H, Clemons KV, Snyder MP, and Stevens DA
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- Animals, Female, Male, Mice, Mycoses microbiology, Antibodies, Fungal blood, Antigens, Fungal immunology, Mycoses immunology, Protein Array Analysis methods, Saccharomyces cerevisiae Proteins immunology
- Abstract
We profiled the global immunoglobulin response against fungal infection by using yeast protein microarrays. Groups of CD-1 mice were infected systemically with human fungal pathogens (Coccidioides posadasii, Candida albicans, or Paracoccidioides brasiliensis) or inoculated with PBS as a control. Another group was inoculated with heat-killed yeast (HKY) of Saccharomyces cerevisiae. After 30 days, serum from mice in the groups were collected and used to probe S. cerevisiae protein microarrays containing 4800 full-length glutathione S-transferase (GST)-fusion proteins. Antimouse IgG conjugated with Alexafluor 555 and anti-GST antibody conjugated with Alexafluor 647 were used to detect antibody-antigen interactions and the presence of GST-fusion proteins, respectively. Serum after infection with C. albicans reacted with 121 proteins: C. posadasii, 81; P. brasiliensis, 67; and after HKY, 63 proteins on the yeast protein microarray, respectively. We identified a set of 16 antigenic proteins that were shared across the three fungal pathogens. These include retrotransposon capsid proteins, heat shock proteins, and mitochondrial proteins. Five of these proteins were identified in our previous study of fungal cell wall by mass spectrometry (Ann. N. Y. Acad. Sci. 2012, 1273, 44-51). The results obtained give a comprehensive view of the immunological responses to fungal infections at the proteomic level. They also offer insight into immunoreactive protein commonality among several fungal pathogens and provide a basis for a panfungal vaccine.
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- 2015
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24. rPbPga1 from Paracoccidioides brasiliensis Activates Mast Cells and Macrophages via NFkB.
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Valim CX, da Silva EZ, Assis MA, Fernandes FF, Coelho PS, Oliver C, and Jamur MC
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- Animals, Fungal Proteins genetics, Humans, Interleukin-6 genetics, Interleukin-6 immunology, Male, Mice, Mice, Inbred BALB C, NF-kappa B genetics, Paracoccidioides genetics, Paracoccidioidomycosis genetics, Paracoccidioidomycosis microbiology, Fungal Proteins immunology, Macrophages immunology, Mast Cells immunology, NF-kappa B immunology, Paracoccidioides immunology, Paracoccidioidomycosis immunology
- Abstract
Background: The fungus Paracoccidioides brasiliensis is the leading etiological agent of paracoccidioidomycosis (PCM), a systemic granulomatous disease that typically affects the lungs. Cell wall components of P. brasiliensis interact with host cells and influence the pathogenesis of PCM. In yeast, many glycosylphosphatidylinositol (GPI)-anchored proteins are important in the initial contact with the host, mediating host-yeast interactions that culminate with the disease. PbPga1 is a GPI anchored protein located on the surface of the yeast P. brasiliensis that is recognized by sera from PCM patients., Methodology/principal Findings: Endogenous PbPga1 was localized to the surface of P. brasiliensis yeast cells in the lungs of infected mice using a polyclonal anti-rPbPga1 antibody. Furthermore, macrophages stained with anti-CD38 were associated with P. brasiliensis containing granulomas. Additionally, rPbPga1 activated the transcription factor NFkB in the macrophage cell line Raw 264.7 Luc cells, containing the luciferase gene downstream of the NFkB promoter. After 24 h of incubation with rPbPga1, alveolar macrophages from BALB/c mice were stimulated to release TNF-α, IL-4 and NO. Mast cells, identified by toluidine blue staining, were also associated with P. brasiliensis containing granulomas. Co-culture of P. Brasiliensis yeast cells with RBL-2H3 mast cells induced morphological changes on the surface of the mast cells. Furthermore, RBL-2H3 mast cells were degranulated by P. brasiliensis yeast cells, but not by rPbPga1, as determined by the release of beta-hexosaminidase. However, RBL-2H3 cells activated by rPbPga1 released the inflammatory interleukin IL-6 and also activated the transcription factor NFkB in GFP-reporter mast cells. The transcription factor NFAT was not activated when the mast cells were incubated with rPbPga1., Conclusions/significance: The results indicate that PbPga1 may act as a modulator protein in PCM pathogenesis and serve as a useful target for additional studies on the pathogenesis of P. brasiliensis.
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- 2015
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25. EpiReumaPt- the study of rheumatic and musculoskeletal diseases in Portugal: a detailed view of the methodology.
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Rodrigues AM, Gouveia N, da Costa LP, Eusébio M, Ramiro S, Machado P, Mourão AF, Silva I, Laires P, Sepriano A, Araújo F, Coelho PS, Gonçalves S, Zhao A, Fonseca JE, de Almeida JM, Tavares V, da Silva JA, Barros H, Cerol J, Mendes J, Carmona L, Canhão H, and Branco JC
- Subjects
- Humans, Portugal epidemiology, Prevalence, Musculoskeletal Diseases epidemiology, Rheumatic Diseases epidemiology
- Abstract
Rheumatic and musculoskeletal diseases (RMD) are prevalent and leading causes of disability and consumption of healthcare and social resources. EpiReumaPt is a national population-based survey developed by the Portuguese Society of Rheumatology that aimed to estimate the prevalence of RMDs and determine their impact on function, quality of life, mental health and use of healthcare resources. This article describes in detail the design, methodology and planned analyses of EpiReumaPt. Recruitment started in September 2011 and finished in December 2013. This study involved a three-stage approach. The first step was a face-to-face survey performed by trained interviewers at the household of 10,661 subjects who where randomly selected by a stratified multistage sampling. A highly sensitive screening questionnaire for RMDs was used. Secondly, participants who screened positive (64%) for at least one RMD as well as 20% of individuals with a negative screening were invited for assessment by a rheumatologist. In total, 3,877 subjects participated in this second phase, where they were also invited to donate a blood sample to be stored at the Biobanco-IMM. History and physical examination, followed by appropriate laboratory and imaging tests were performed. At the end of the visit, the rheumatologist established a diagnosis. Finally, a team of three experienced rheumatologists reviewed all the clinical data and defined the diagnoses according to previously validated criteria. The EpiReumaPt dataset, containing data from several questionnaires, various clinical measurements and information from laboratory and imaging tests, comprises an invaluable asset for research. The large amount of information collected from each participant and the large number of participants, with a wide age range covering and being representative of the adults from the entire country, makes EpiReumaPt the largest study of RMDs performed in Portugal.
- Published
- 2015
26. Saccharomyces cerevisiae expressing Gp43 protects mice against Paracoccidioides brasiliensis infection.
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Assis-Marques MA, Oliveira AF, Ruas LP, dos Reis TF, Roque-Barreira MC, and Coelho PS
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- Animals, Antigens, Fungal genetics, Antigens, Fungal immunology, Fungal Proteins genetics, Fungal Proteins immunology, Fungal Vaccines immunology, Glycoproteins genetics, Glycoproteins immunology, Interferon-gamma metabolism, Interleukin-2 metabolism, Lung drug effects, Lung microbiology, Lung pathology, Male, Mice, Paracoccidioides genetics, Paracoccidioidomycosis immunology, Paracoccidioidomycosis pathology, Saccharomyces cerevisiae genetics, Spleen drug effects, Spleen microbiology, Spleen pathology, Vaccination, Antigens, Fungal metabolism, Fungal Proteins metabolism, Glycoproteins metabolism, Paracoccidioides immunology, Paracoccidioidomycosis prevention & control, Paracoccidioidomycosis veterinary, Saccharomyces cerevisiae physiology
- Abstract
The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM). It is believed that approximately 10 million people are infected with the fungus and approximately 2% will eventually develop the disease. Unlike viral and bacterial diseases, fungal diseases are the ones against which there is no commercially available vaccine. Saccharomyces cerevisiae may be a suitable vehicle for immunization against fungal infections, as they require the stimulation of different arms of the immune response. Here we evaluated the efficacy of immunizing mice against PCM by using S. cerevisiae yeast expressing gp43. When challenged by inoculation of P. brasiliensis yeasts, immunized animals showed a protective profile in three different assays. Their lung parenchyma was significantly preserved, exhibiting fewer granulomas with fewer fungal cells than found in non-immunized mice. Fungal burden was reduced in the lung and spleen of immunized mice, and both organs contained higher levels of IL-12 and IFN-γ compared to those of non-vaccinated mice, a finding that suggests the occurrence of Th1 immunity. Taken together, our results indicate that the recombinant yeast vaccine represents a new strategy to confer protection against PCM.
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- 2015
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27. Improved cyclopropanation activity of histidine-ligated cytochrome P450 enables the enantioselective formal synthesis of levomilnacipran.
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Wang ZJ, Renata H, Peck NE, Farwell CC, Coelho PS, and Arnold FH
- Subjects
- Bacillus megaterium enzymology, Catalytic Domain, Cyclopropanes chemistry, Cytochrome P-450 Enzyme System genetics, Enzymes, Histidine chemistry, Milnacipran, Mutagenesis, Site-Directed, Protein Engineering, Stereoisomerism, Cyclopropanes chemical synthesis, Cytochrome P-450 Enzyme System metabolism, Histidine metabolism
- Abstract
Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative-scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active-site residues of the cytochrome P450 enzyme from Bacillus megaterium (P450-BM3), a highly active His-ligated variant of P450-BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This enzyme, BM3-Hstar, catalyzes the cyclopropanation of N,N-diethyl-2-phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non-natural enzyme activity., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
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- 2014
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28. Enantioselective intramolecular C-H amination catalyzed by engineered cytochrome P450 enzymes in vitro and in vivo.
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McIntosh JA, Coelho PS, Farwell CC, Wang ZJ, Lewis JC, Brown TR, and Arnold FH
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- Amination, Biocatalysis, Cytochrome P-450 Enzyme System chemistry, Molecular Structure, Protein Engineering, Stereoisomerism, Sulfonamides chemistry, Cytochrome P-450 Enzyme System metabolism, Sulfonamides metabolism
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- 2013
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29. A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo.
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Coelho PS, Wang ZJ, Ener ME, Baril SA, Kannan A, Arnold FH, and Brustad EM
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- Alkenes chemistry, Biocatalysis, Cytochrome P-450 Enzyme System chemistry, Cytochrome P-450 Enzyme System genetics, Escherichia coli chemistry, Escherichia coli cytology, Escherichia coli metabolism, Methane chemistry, Methane metabolism, Mutation, Serine chemistry, Alkenes metabolism, Cytochrome P-450 Enzyme System metabolism, Methane analogs & derivatives, Serine metabolism
- Abstract
Whole-cell catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a cytochrome 'P411' with unique serine-heme ligation that catalyzes efficient and selective olefin cyclopropanation in intact Escherichia coli cells. The mutation C400S in cytochrome P450(BM3) gives a signature ferrous CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting-state Fe(III)-to-Fe(II) reduction potential and substantially improves NAD(P)H-driven activity.
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- 2013
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30. Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes.
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Coelho PS, Brustad EM, Kannan A, and Arnold FH
- Subjects
- Bacillus megaterium enzymology, Catalysis, Cytochrome P-450 Enzyme System genetics, Methane chemistry, Models, Chemical, Protein Engineering, Stereoisomerism, Alkenes chemistry, Cyclopropanes chemistry, Cytochrome P-450 Enzyme System chemistry, Methane analogs & derivatives
- Abstract
Transition metal-catalyzed transfers of carbenes, nitrenes, and oxenes are powerful methods for functionalizing C=C and C-H bonds. Nature has evolved a diverse toolbox for oxene transfers, as exemplified by the myriad monooxygenation reactions catalyzed by cytochrome P450 enzymes. The isoelectronic carbene transfer to olefins, a widely used C-C bond-forming reaction in organic synthesis, has no biological counterpart. Here we report engineered variants of cytochrome P450(BM3) that catalyze highly diastereo- and enantioselective cyclopropanation of styrenes from diazoester reagents via putative carbene transfer. This work highlights the capacity to adapt existing enzymes for the catalysis of synthetically important reactions not previously observed in nature.
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- 2013
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31. Cyclic parenteral nutrition does not change the intestinal microbiota in patients with short bowel syndrome.
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Furtado Ede C, Marchini JS, Fonseca CK, Coelho PS, Menegueti MG, Auxiliadora-Martins M, Basile-Filho A, and Suen VM
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- Case-Control Studies, Cross-Sectional Studies, DNA, Bacterial, Energy Intake, Humans, Intestinal Absorption, Intestine, Small surgery, Nutritional Status, Enterobacteriaceae isolation & purification, Feces microbiology, Intestine, Small microbiology, Microbiota, Parenteral Nutrition methods, Short Bowel Syndrome microbiology
- Abstract
Purpose: To characterize of the intestinal microbiota of patients with short bowel syndrome (SBS) admitted to the Metabolic Unit of a University Hospital., Methods: Fecal samples were evaluated, and biochemical tests were conducted only in the case of SBS patients. The nutritional status was assessed via anthropometric measurements and evaluation of food intake by means of a food questionnaire. The pathogenic strains were detected with the aid of cultures and specific biochemical tests in aerobic medium, for determination of species belonging to the Family enterobacteriaceae. Anti-sera were applied to each isolated E. coli strain, for determination of their possible pathogenicity. Molecular methodology was employed for establishment of the intestinal bacterial microbiota profile, Results: A lower amount of microorganisms of the family enterobacteriaceae per gram of stool was observed in the case of patients with SBS. However, molecular analysis showed maintenance of the bacterial species ratio, which is equivalent to a healthy intestinal microbiota., Conclusion: Despite the massive removal of the small bowel, frequent use of antibiotics, immune system depression, presence of non-digested food in the gastrointestinal tract, and accelerated intestinal transit, the ratio between intestinal bacterial species remain similar to normality.
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- 2013
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32. Characterization of PbPga1, an antigenic GPI-protein in the pathogenic fungus Paracoccidioides brasiliensis.
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Valim CX, Basso LR Jr, dos Reis Almeida FB, Reis TF, Damásio AR, Arruda LK, Martinez R, Roque-Barreira MC, Oliver C, Jamur MC, and Coelho PS
- Subjects
- Animals, Antibodies immunology, Chickens, Fungal Proteins genetics, GPI-Linked Proteins genetics, Gene Expression Regulation, Fungal, Gene Order, Glycosylation, Humans, Macrophages immunology, Macrophages metabolism, Nitric Oxide biosynthesis, Paracoccidioides genetics, Paracoccidioides metabolism, Pichia genetics, Pichia metabolism, Protein Transport, Tumor Necrosis Factor-alpha biosynthesis, Fungal Proteins immunology, GPI-Linked Proteins immunology, Paracoccidioides immunology, Paracoccidioidomycosis immunology
- Abstract
Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.
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- 2012
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33. Physical Mapping in a Triplicated Genome: Mapping the Downy Mildew Resistance Locus Pp523 in Brassica oleracea L.
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Carlier JD, Alabaça CS, Sousa NH, Coelho PS, Monteiro AA, Paterson AH, and Leitão JM
- Abstract
We describe the construction of a BAC contig and identification of a minimal tiling path that encompass the dominant and monogenically inherited downy mildew resistance locus Pp523 of Brassica oleracea L. The selection of BAC clones for construction of the physical map was carried out by screening gridded BAC libraries with DNA overgo probes derived from both genetically mapped DNA markers flanking the locus of interest and BAC-end sequences that align to Arabidopsis thaliana sequences within the previously identified syntenic region. The selected BAC clones consistently mapped to three different genomic regions of B. oleracea. Although 83 BAC clones were accurately mapped within a ∼4.6 cM region surrounding the downy mildew resistance locus Pp523, a subset of 33 BAC clones mapped to another region on chromosome C8 that was ∼60 cM away from the resistance gene, and a subset of 63 BAC clones mapped to chromosome C5. These results reflect the triplication of the Brassica genomes since their divergence from a common ancestor shared with A. thaliana, and they are consonant with recent analyses of the C genome of Brassica napus. The assembly of a minimal tiling path constituted by 13 (BoT01) BAC clones that span the Pp523 locus sets the stage for map-based cloning of this resistance gene.
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- 2011
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34. Septin localization in the dimorphic fungus Paracoccidioides brasiliensis.
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Reis TF, Basso LR Jr, Oliveira RR, and Coelho PS
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- Cell Division, Escherichia coli genetics, Fluorescent Antibody Technique, Fungal Proteins chemistry, Fungal Proteins genetics, Hyphae metabolism, Paracoccidioides ultrastructure, Phylogeny, Septins chemistry, Septins genetics, Fungal Proteins analysis, Paracoccidioides metabolism, Septins analysis
- Abstract
Septins are evolutionarily conserved proteins that contain a GTPase domain and are capable of forming filaments at the cell periphery. Septins are involved in many essential cellular processes, such as cytokinesis and cell polarization, and are used as markers of morphogenesis in several fungi. Dimorphism in fungi enables cells to switch between morphologies (yeast or filament forms), due to changes in the temperature of the environment. We analysed the localization of septin proteins in yeast and filamentous cells of the dimorphic fungus Paracoccidioides brasiliensis, a common cause of granulomatous mycosis. In order to determine septin localization, we first cloned Cdc12p, a septin homolog from P. brasiliensis, and expressed it in Escherichia coli. Following PbCdc12p purification, specific serum against PbCdc12p were raised for use in immunofluorescence assays. We observed the hourglass and ring forms of septin filaments during cell division in yeast. Septin filaments were also simultaneously localized in the necks of multiple budding cells. A distinctive pattern of punctuate and/or diffuse localization was also seen in the periphery of multinucleate yeast cells and at the tips and septa of filamentous cells. A more diffuse and punctuate pattern of localization observed in P. brasiliensis cells seems to be unique to filamentous and dimorphic fungi and may be related to their specialization in cell wall deposition, morphogenesis and cell cycle control., (Copyright © 2011 John Wiley & Sons, Ltd.)
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- 2011
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35. Enzymatic functionalization of carbon-hydrogen bonds.
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Lewis JC, Coelho PS, and Arnold FH
- Subjects
- Biocatalysis, Hydro-Lyases metabolism, Hydroxylation, Stereoisomerism, Carbon chemistry, Hydrogen chemistry, Mixed Function Oxygenases metabolism
- Abstract
The development of new catalytic methods to functionalize carbon-hydrogen (C-H) bonds continues to progress at a rapid pace due to the significant economic and environmental benefits of these transformations over traditional synthetic methods. In nature, enzymes catalyze regio- and stereoselective C-H bond functionalization using transformations ranging from hydroxylation to hydroalkylation under ambient reaction conditions. The efficiency of these enzymes relative to analogous chemical processes has led to their increased use as biocatalysts in preparative and industrial applications. Furthermore, unlike small molecule catalysts, enzymes can be systematically optimized via directed evolution for a particular application and can be expressed in vivo to augment the biosynthetic capability of living organisms. While a variety of technical challenges must still be overcome for practical application of many enzymes for C-H bond functionalization, continued research on natural enzymes and on novel artificial metalloenzymes will lead to improved synthetic processes for efficient synthesis of complex molecules. In this critical review, we discuss the most prevalent mechanistic strategies used by enzymes to functionalize non-acidic C-H bonds, the application and evolution of these enzymes for chemical synthesis, and a number of potential biosynthetic capabilities uniquely enabled by these powerful catalysts (110 references).
- Published
- 2011
- Full Text
- View/download PDF
36. Transformation of Candida albicans with a synthetic hygromycin B resistance gene.
- Author
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Basso LR Jr, Bartiss A, Mao Y, Gast CE, Coelho PS, Snyder M, and Wong B
- Subjects
- Candida albicans metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Molecular Sequence Data, Phosphotransferases (Alcohol Group Acceptor) genetics, Phosphotransferases (Alcohol Group Acceptor) metabolism, Protein Engineering, Candida albicans drug effects, Candida albicans genetics, Drug Resistance, Fungal, Genes, Synthetic, Hygromycin B pharmacology, Transformation, Genetic
- Abstract
Synthetic genes that confer resistance to the antibiotic nourseothricin in the pathogenic fungus Candida albicans are available, but genes conferring resistance to other antibiotics are not. We found that multiple C. albicans strains were inhibited by hygromycin B, so we designed a 1026 bp gene (CaHygB) that encodes Escherichia coli hygromycin B phosphotransferase with C. albicans codons. CaHygB conferred hygromycin B resistance in C. albicans transformed with ars2-containing plasmids or single-copy integrating vectors. Since CaHygB did not confer nourseothricin resistance and since the nourseothricin resistance marker SAT-1 did not confer hygromycin B resistance, we reasoned that these two markers could be used for homologous gene disruptions in wild-type C. albicans. We used PCR to fuse CaHygB or SAT-1 to approximately 1 kb of 5' and 3' noncoding DNA from C. albicans ARG4, HIS1 and LEU2, and introduced the resulting amplicons into six wild-type C. albicans strains. Homologous targeting frequencies were approximately 50-70%, and disruption of ARG4, HIS1 and LEU2 alleles was verified by the respective transformants' inabilities to grow without arginine, histidine and leucine. CaHygB should be a useful tool for genetic manipulation of different C. albicans strains, including clinical isolates., (Copyright © 2010 John Wiley & Sons, Ltd.)
- Published
- 2010
- Full Text
- View/download PDF
37. New transposons to generate GFP protein fusions in Candida albicans.
- Author
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Dias MV, Basso LR Jr, and Coelho PS
- Subjects
- Cell Cycle Proteins biosynthesis, Fungal Proteins biosynthesis, Recombinant Fusion Proteins biosynthesis, Transformation, Genetic, Artificial Gene Fusion methods, Candida albicans genetics, DNA Transposable Elements, Green Fluorescent Proteins genetics, Mutagenesis, Insertional
- Abstract
Transposon elements are important tools for gene function analysis, for example they can be used to easily create genome-wide collections of insertion mutants. Transposons may also carry sequences coding for an epitope or fluorescent marker useful for protein expression and localization analysis. We have developed three new Tn5-based transposons that incorporate a GFP (green fluorescent protein) coding sequence to generate fusion proteins in the important fungal pathogen Candida albicans. Each transposon also contains the URA3 and Kan(R) genes for yeast and bacterial selection, respectively. After in vitro transposition, the insertional allele is transferred to the chromosomal locus by homologous recombination. Transposons Tn5-CaGFP and Tn5-CaGFP-URA3::FLIP can generate C-terminal truncated GFP fusions. A URA3 flipper recycling cassette was incorporated into the transposon Tn5-CaGFP-URA3::FLIP. After the induction of Flip recombinase to excise the marker, the heterozygous strain is transformed again in order to obtain a GFP-tagged homozygous strains. In the Tn5-CaGFP-FL transposon the markers are flanked by a rare-cutting enzyme. After in vitro transposition into a plasmid-borne target gene, the markers are eliminated by restriction digestion and religation, resulting in a construct coding for full-length GFP-fusion proteins. This transposon can generate plasmid libraries of GFP insertions in proteins where N- or C-terminal tagging may alter localization. We tested our transposon system by mutagenizing the essential septin CDC3 gene. The results indicate that the Cdc3 C-terminal extension is important for correct septin filament assembly. The transposons described here provide a new system to obtain global gene expression and protein localization data in C. albicans.
- Published
- 2008
- Full Text
- View/download PDF
38. A novel mitochondrial protein, Tar1p, is encoded on the antisense strand of the nuclear 25S rDNA.
- Author
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Coelho PS, Bryan AC, Kumar A, Shadel GS, and Snyder M
- Subjects
- Amino Acid Sequence, Molecular Sequence Data, RNA, Ribosomal genetics, Sequence Alignment, DNA, Ribosomal genetics, Genome, Fungal, Mitochondrial Proteins genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics
- Abstract
In eukaryotes, it is widely assumed that genes coding for proteins and structural RNAs do not overlap. Using a transposon-tagging strategy to globally analyze the Saccharomyces cerevisiae genome for expressed genes, we identified multiple insertions in an open reading frame that is contained fully within and transcribed antisense to the 25S rRNA gene in the nuclear rDNA repeat region on Chromosome XII. Expression of this gene, TAR1 (Transcript Antisense to Ribosomal RNA), can be detected at the RNA and protein levels, and the primary sequence of the corresponding 124-amino-acid protein is conserved in several yeast species. Tar1p was found to localize to mitochondria, and overexpression of the protein suppresses the respiration-deficient petite phenotype of a point mutation in mitochondrial RNA polymerase that affects mitochondrial gene expression and mtDNA stability. These findings indicate that coding information for protein and structural RNAs can overlap, raising issues regarding the coevolution of such complex genes, and also suggest that rDNA transcription and mitochondrial function are coordinately regulated in eukaryotic cells.
- Published
- 2002
- Full Text
- View/download PDF
39. The control of BhB10-1 gene expression in the salivary gland of Bradysia hygida (Diptera, Sciaridae) is disrupted in vivo by a delayed effect of cycloheximide.
- Author
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Conacci ME, Coelho PS, Valente V, de F Sousa J, Paçó-Larson ML, and de Almeida JC
- Subjects
- Animals, Polyadenylation, RNA, Salivary Glands metabolism, Cycloheximide pharmacology, Diptera genetics, Gene Expression Regulation drug effects, Genes, Insect, Protein Synthesis Inhibitors pharmacology
- Abstract
BhB10-1 is an amplified gene present in DNA puff B10. This gene is very active in the salivary gland regions S1 and S3 at the end of the larval development. Two transcripts of this gene, 1.3 and 1.1 kb in size, were detected. A secretory protein, SP23, is the product of BhB10-1. In this work, we present evidence supporting the hypothesis that a biphasic process of mRNA degradation is an important component in the control of BhB10-1 gene expression. The 1.3 kb transcript, by a process of poly(A) tail shortening, is converted to the inactive transcript of 1.1 kb which is detected during and after the period of SP23 expression. Cycloheximide in very low concentration, if applied at a proper time, can disrupt this process leading to extended periods of 1.3 kb RNA detection and SP23 synthesis. A tentative model is proposed to explain this phenomenon.
- Published
- 2002
- Full Text
- View/download PDF
40. Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes induce in vivo leukocyte recruitment dependent on MCP-1 production by IFN-gamma-primed-macrophages.
- Author
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Coelho PS, Klein A, Talvani A, Coutinho SF, Takeuchi O, Akira S, Silva JS, Canizzaro H, Gazzinelli RT, and Teixeira MM
- Subjects
- Animals, Cells, Cultured, Chemokines genetics, Chemokines physiology, Glycosylphosphatidylinositols analysis, Interferon-gamma genetics, Interferon-gamma pharmacology, Interferon-gamma physiology, Kinetics, Macrophages drug effects, Macrophages immunology, Male, Membrane Glycoproteins physiology, Mice, Mice, Inbred BALB C, Mice, Inbred C3H, Mice, Knockout, Mucins chemistry, Mucins isolation & purification, Protozoan Proteins chemistry, Protozoan Proteins isolation & purification, Receptors, Cell Surface physiology, Toll-Like Receptor 2, Toll-Like Receptors, Trypanosoma cruzi growth & development, Chemokine CCL2 biosynthesis, Chemotaxis, Leukocyte, Drosophila Proteins, Mucins pharmacology, Protozoan Proteins pharmacology, Trypanosoma cruzi physiology
- Abstract
Glycosylphosphatidylinositol-anchored mucin-like glycoproteins from Trypanosoma cruzi trypomastigotes (tGPI-mucins) activate macrophages in vitro to produce proinflammatory cytokines, chemokines, and nitric oxide. These effects of tGPI-mucins may be important in the ensuing immune response to T. cruzi. Here, we have sought evidence for a role of tGPI-mucins in mediating leukocyte recruitment in vivo. tGPI-mucins are highly effective in promoting cell recruitment in the pleural cavity of mice primed with IFN-gamma-inducing agents but not in naïve mice. Maximal recruitment was observed at a dose between 250 and 1250 ng tGPI-mucins. There was a significant elevation in the levels of MCP-1 in the pleural cavity of primed animals injected with tGPI-mucins, and in vivo neutralization of MCP-1 abolished leukocyte recruitment. Pretreatment with anti-MIP-1alpha or anti-RANTES had no effect on the recruitment induced by tGPI-mucins. MCP-1 immunoreactivity was detected in pleural macrophages, and macrophages produced MCP-1 in vitro, especially after priming with IFN-gamma. Finally, tGPI-mucins induced significant leukocyte recruitment in primed C3H/HeJ but not in TLR2-deficient mice. Together, our results suggest that T. cruzi-derived GPI-mucins in conjunction with IFN-gamma may drive tissue chemokine production and inflammation and bear a significant role in the pathogenesis of Chagas disease.
- Published
- 2002
41. The induction of DNA puff BhC4-1 gene is a late response to the increase in 20-hydroxyecdysone titers in last instar dipteran larvae.
- Author
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Basso LR Jr, Vasconcelos C, Fontes AM, Hartfelder K, Silva JA Jr, Coelho PS, Monesi N, and Paçó-Larson ML
- Subjects
- Animals, Animals, Genetically Modified, Chromosomes drug effects, Chromosomes ultrastructure, Diptera drug effects, Diptera metabolism, Drosophila genetics, Drosophila Proteins genetics, Ecdysterone metabolism, Ecdysterone pharmacology, Gene Expression Regulation, Developmental drug effects, Genes, Insect drug effects, Hemolymph metabolism, Larva drug effects, Larva growth & development, Salivary Glands drug effects, Salivary Glands ultrastructure, Diptera genetics, Diptera growth & development, Insect Proteins genetics, Salivary Proteins and Peptides genetics
- Abstract
The characterization of DNA puff BhC4-1 expression was extended and its response to 20-hydroxyecdysone investigated in Bradysia hygida and in transgenic Drosophila carrying the BhC4-1 gene. In both organisms the activation of BhC4-1 in salivary glands occurs at the end of the larval stage coinciding with the peak in ecdysone titers which induces metamorphosis. Injections of 20-hydroxyecdysone into mid-fourth instar larvae of B. hygida show that the induction of BhC4-1 expression, as well as amplification and puff C4 expansion, are late events induced by the hormone. This late response of BhC4-1 expression was also observed in transgenic salivary glands cultivated in the presence of 20-hydroxyecdysone. In vitro studies using transgenic Drosophila indicate that both repressor and activator factors regulate the timing of BhC4-1 expression in salivary glands.
- Published
- 2002
- Full Text
- View/download PDF
42. A dual role of 20-hydroxyecdysone in the control of protein synthesis related to DNA puff activity in the anterior region of Bradysia hygida (Diptera, Sciaridae) salivary gland.
- Author
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de Carvalho DP, Coelho PS, and de Almeida JC
- Subjects
- Animals, DNA genetics, Gene Expression Regulation, Developmental, Larva growth & development, Protein Biosynthesis, Transcription, Genetic, Diptera genetics, Ecdysterone pharmacology, Gene Amplification, Salivary Glands physiology
- Abstract
During the last 30 h of the larval stage, the salivary glands of Bradysia hygida show the amplification of some genes, resulting in the formation of two successive groups of DNA puffs, which direct the synthesis of two different sets of polypeptides. Incubation of anterior (S1) salivary gland regions, at age E7, beginning of first group of DNA puffs activity, in culture medium for 2 to 10 h results in a decrease in the synthesis of the polypeptides characteristic of this period. However, during subsequent incubation (from E7 to E7+12 h-24 h), when the second group of DNA puffs is active, S1 regions were able to synthesize some polypeptides characteristic of this period. The role of 20-OH ecdysone was studied, in vitro and in vivo, during these two periods of protein synthesis in S1 regions. The presence of the hormone was shown to be necessary to maintain, in vitro, the synthesis of the first set of polypeptides and was strongly inhibitory, in vitro and in vivo, to the synthesis of the second set of polypeptides. Thus, it is likely that the activity of the two distinct groups of DNA puffs is under opposite 20-OH-ecdysone control mechanisms.
- Published
- 2000
- Full Text
- View/download PDF
43. Genome-wide mutant collections: toolboxes for functional genomics.
- Author
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Coelho PS, Kumar A, and Snyder M
- Subjects
- Animals, Genome, Mutagenesis, Insertional methods, Polymerase Chain Reaction methods
- Abstract
The sequencing of entire genomes has led to the identification of many genes. A future challenge will be to determine the function of all of the genes of an organism. One of the best ways to ascertain function is to disrupt genes and determine the phenotype of the resulting organism. Novel large-scale approaches for generating gene disruptions and analyzing the resulting phenotype are underway in the budding yeast Saccharomyces cerevisiae and other organisms including flies, Mycoplasma, worms, plants and mice. These approaches and mutant collections will be extremely valuable to the scientific community and will dramatically alter the manner in which science is performed in the future.
- Published
- 2000
- Full Text
- View/download PDF
44. TRIPLES: a database of gene function in Saccharomyces cerevisiae.
- Author
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Kumar A, Cheung KH, Ross-Macdonald P, Coelho PS, Miller P, and Snyder M
- Subjects
- DNA Transposable Elements, Gene Expression, Open Reading Frames, Phenotype, Databases, Factual, Genes, Fungal, Saccharomyces cerevisiae genetics
- Abstract
Using a novel multipurpose mini-transposon, we have generated a collection of defined mutant alleles for the analysis of disruption phenotypes, protein localization, and gene expression in Saccharomyces cerevisiae. To catalog this unique data set, we have developed TRIPLES, a Web-accessible database of TRansposon-Insertion Phenotypes, Localization and Expression in Saccharomyces. Encompassing over 250 000 data points, TRIPLES provides convenient access to information from nearly 7800 transposon-mutagenized yeast strains; within TRIPLES, complete data reports of each strain may be viewed in table format, or if desired, downloaded as tab-delimited text files. Each report contains external links to corresponding entries within the Saccharomyces Genome Database and International Nucleic Acid Sequence Data Library (GenBank). Unlike other yeast databases, TRIPLES also provides on-line order forms linked to each clone report; users may immediately request any desired strain free-of-charge by submitting a completed form. In addition to presenting a wealth of information for over 2300 open reading frames, TRIPLES constitutes an important medium for the distribution of useful reagents throughout the yeast scientific community. Maintained by the Yale Genome Analysis Center, TRIPLES may be accessed at http://ycmi.med.yale.edu/ygac/triples.htm
- Published
- 2000
- Full Text
- View/download PDF
45. High-throughput methods for the large-scale analysis of gene function by transposon tagging.
- Author
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Kumar A, des Etages SA, Coelho PS, Roeder GS, and Snyder M
- Subjects
- Base Sequence, Escherichia coli enzymology, Gene Library, Integrases metabolism, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Plasmids, Recombinant Fusion Proteins analysis, Recombinant Fusion Proteins biosynthesis, Saccharomyces cerevisiae genetics, beta-Galactosidase analysis, DNA Transposable Elements, Escherichia coli genetics, Mutagenesis, Insertional methods, Sequence Tagged Sites, Viral Proteins, beta-Galactosidase genetics
- Published
- 2000
- Full Text
- View/download PDF
46. Large-scale analysis of the yeast genome by transposon tagging and gene disruption.
- Author
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Ross-Macdonald P, Coelho PS, Roemer T, Agarwal S, Kumar A, Jansen R, Cheung KH, Sheehan A, Symoniatis D, Umansky L, Heidtman M, Nelson FK, Iwasaki H, Hager K, Gerstein M, Miller P, Roeder GS, and Snyder M
- Subjects
- Algorithms, Escherichia coli genetics, Fungal Proteins genetics, Fungal Proteins physiology, Gene Expression Profiling, Molecular Sequence Data, Mutagenesis, Oligonucleotide Array Sequence Analysis, Open Reading Frames, Phenotype, Polymerase Chain Reaction, Transformation, Genetic, DNA Transposable Elements, Genetic Techniques, Genome, Fungal, Saccharomyces cerevisiae genetics
- Abstract
Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background--a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.
- Published
- 1999
- Full Text
- View/download PDF
47. DNA puff C4 of Bradysia hygida (Diptera: Sciaridae) contains genes unequally amplified and differentially expressed during development.
- Author
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Coelho PS, De Almeida JC, Toledo F, Buttin G, and Paçó-Larson ML
- Subjects
- Animals, Blotting, Southern, Chromosomes ultrastructure, DNA genetics, DNA isolation & purification, Deoxyribonuclease EcoRI, Female, In Situ Hybridization, Fluorescence, Larva, Peptide Mapping, RNA isolation & purification, Salivary Glands cytology, Diptera genetics, Gene Amplification
- Abstract
We report here the isolation and characterization of a 2.3 kb genomic EcoRI fragment that co-localizes in the DNA puff C4 of Bradysia hygida with a 4 kb EcoRI fragment previously characterized as containing part of a gene amplified and expressed in the salivary gland at the time when puff C4 expands. Verification of the relative amount of DNA complementary to these two genomic fragments shows that they are unequally amplified in the salivary gland. The fragment containing part of the gene expressed when puff C4 expands amplifies about eight times more than the 2.3 kb fragment. This 2.3 kb fragment also carries sequences complementary to RNA species present in the gland in a period when puff C4 has already receded. Based on these data we discuss the nature of the DNA puff and the possible way in which amplification is occurring at these sites.
- Published
- 1993
- Full Text
- View/download PDF
48. Agenesis of the right lobe of the liver with supra-hepatic gallbladder.
- Author
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d'Araújo JM and Coelho PS
- Subjects
- Aged, Humans, Male, Radiography, Abnormalities, Multiple diagnostic imaging, Gallbladder abnormalities, Liver abnormalities
- Published
- 1992
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