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Methods Mol Biol ; 2047: 219-232, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31552657


The red flour beetle, Tribolium castaneum, is an emerging model system well suited to the study of embryonic brain development and evolution (see Chapters 11 and 13 ). Brain genesis is driven by specific gene products whose expression underlies a tight spatiotemporal control. Therefore, the analysis of gene expression in time and space provides valuable insights into the molecular mechanisms that govern brain development. Since Tribolium-specific antibodies are scarce, fluorescent RNA in situ hybridization is the method of choice to determine the dynamics of individual gene expression. We have modified common RNA in situ protocols to facilitate the concomitant detection of two gene-specific expression patterns (double fluorescent RNA in situ). In addition, we describe a procedure which combines fluorescent single RNA in situ and immunostaining with gene-specific antibodies. Conventional in situ using RNA probes that are complementary to mature mRNAs often produce diffuse signals. We demonstrate that RNA in situ probes complementary to intronic gene sequences facilitate single cell resolution because the fluorescent signal is restricted to the nucleus. We believe our protocols can be adapted easily to suit the analysis of brain development in other insect species.

Nat Genet ; 52(1): 29-34, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31844324


Extrachromosomal circularization of DNA is an important genomic feature in cancer. However, the structure, composition and genome-wide frequency of extrachromosomal circular DNA have not yet been profiled extensively. Here, we combine genomic and transcriptomic approaches to describe the landscape of extrachromosomal circular DNA in neuroblastoma, a tumor arising in childhood from primitive cells of the sympathetic nervous system. Our analysis identifies and characterizes a wide catalog of somatically acquired and undescribed extrachromosomal circular DNAs. Moreover, we find that extrachromosomal circular DNAs are an unanticipated major source of somatic rearrangements, contributing to oncogenic remodeling through chimeric circularization and reintegration of circular DNA into the linear genome. Cancer-causing lesions can emerge out of circle-derived rearrangements and are associated with adverse clinical outcome. It is highly probable that circle-derived rearrangements represent an ongoing mutagenic process. Thus, extrachromosomal circular DNAs represent a multihit mutagenic process, with important functional and clinical implications for the origins of genomic remodeling in cancer.

Rev. méd. Urug ; 35(4): 267-280, dic. 2019. tab, fig
Artigo em Espanhol | LILACS | ID: biblio-1026117


Introduccción: en Uruguay, el cáncer de cuello uterino ocupó el tercer lugar con una incidencia promedio de 312 nuevos casos por año; fallecieron 134 mujeres promedio por año en el período 2010 - 2014. Objetivo: el objetivo de este trabajo fue analizar los primeros resultados de una experiencia piloto con la aplicación del test de HPV captura híbrida HC2 (QiagenR) como test de tamizaje primario del cáncer de cuello uterino en una zona del departamento de Canelones. Método: se estudiaron 1.010 mujeres asintomáticas entre 30 y 64 años que concurrieron a realizarse el test de PAP a dos unidades de toma de muestras del Programa de prevención del cáncer de cuello uterino. Se realizó la extracción conjunta de la muestra para PAP, que fue derivada a uno de los laboratorios de citología de la Red de Atención Primaria de Salud y la muestra de HPV que fue procesada con la técnica de captura híbrida en el laboratorio de biología molecular del Centro Hospitalario Pereira Rossell. Las usuarias con resultados HPV + y PAP anormales (ASC-US+) fueron derivadas a colposcopia, con biopsia y posterior tratamiento cuando correspondió. Resultados: el test de HPV fue positivo en 126/1.010 mujeres (12,5%) y el PAP anormal en 167/1.010 (16,5%). El test de HPV fue positivo en todos los casos CIN2+ 13/13 (100%) mientras que el PAP fue anormal (ASCUS+) en 7/13 (54%) para CIN2+ por biopsia. Conclusión: el test de HPV resultó más eficaz y eficiente que el PAP para la detección de lesiones precancerosas de cuello uterino.

Introduction: in Uruguay, cervical cancer occupied the third place with an average incidence of 312 new cases per year. 134 women died in the 2010 -2014 period. Objective: the study aims to analyse the first results in a pilot experience with the application of Hybrid Capture® 2 High-Risk HPV DNA Test™ (hc2) (QiagenR) as the primary screening test for cervical cancer, in the Department of Canelones. Method: 1.010 asymptomatic women whose ages ranged between 30 and 64 years old were studied when they requested a pap smear at two units of sample taking within the Cervical Cancer Prevention Program. Samples were taken along with the pap smear, and sent to one of the cytology labs in the primary health care network, the HPV sample being processed with the hybrid capture technique in the molecular biology laboratory of the Pereira Rossell Hospital Center. Users with abnormal HPV+ and abnormal pap smear results (ASCUS+) were referred to colposcopy, with subsequent biopsy and treatment if required. Results: HPV test was positive in 126/1010 women (12.5%) and PAP was abnormal in 167/1010 cases (16.5%). HPV test was positive in all cases CIN2+ 13/13 (100%) whereas PAP was abnormal (ASCUS+) in 7/13 54%) for CIN2+ in biopsy. Conclusion: HPV test was more effective and efficient than pap smear to detect pre-cancer lesions in the cervix.

Introdução: no Uruguai, no período 2010 - 2014, o câncer de colo do útero foi a terceira causa com uma incidência média de 312 novos casos por ano e uma média de 134 mortes por ano. Objetivo: o objetivo deste trabalho foi analisar os primeiros resultados de uma experiência piloto com a utilização do exame de HPV Captura Híbrida HC2 (QiagenR) na tamizagem primária do câncer de colo do útero em una zona do departamento de Canelones. Método: foram estudadas 1010 mulheres assintomáticas, com idades entre 30 e 64 anos que foram a duas unidades do Programa de Prevenção do Câncer do Colo do Útero para a coleta de amostra para exame de Papanicolau (PAP). Realizou-se uma única extração para duas amostras: uma para PAP, que foi encaminhada a um laboratório de citologia da rede de atenção primária de saúde e outra para o exame de HPV que foi processada com a técnica de captura híbrida no laboratório de biologia molecular do Centro Hospitalario Pereira Rossell. As usuárias com resultados HPV + e PAP anormais (ASCUS+) foram encaminhadas para realização de colposcopia, com biopsia e tratamento quando fosse necessário. Resultados: o exame de HPV foi positivo em 126/1010 mulheres (12.5%) e o PAP foi anormal em 167/1010 (16.5%). O exame de HPV foi positivo em todos los casos CIN2+ 13/13 (100%) e o PAP foi anormal (ASCUS+) em 7/13 (54%) para CIN2+ por biopsia. Conclusão: o exame de HPV foi mais eficaz e eficiente que o PAP para detecção de lesões pré-cancerosas de colo do útero.

Humanos , Feminino , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/prevenção & controle , Programas de Rastreamento/métodos , Infecções por Papillomavirus/diagnóstico , Teste de Papanicolaou
Transbound Emerg Dis ; 66(4): 1674-1692, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30980699


Brucella-exposure and infection is increasingly recognized in marine mammals worldwide. To better understand the epidemiology and health impacts of Brucella spp. in marine mammals of Brazil, molecular (conventional PCR and/or real-time PCR), serological (Rose Bengal Test [RBT], Competitive [c]ELISA, Serum Agglutination Test [SAT]), pathological, immunohistochemical (IHC) and/or microbiological investigations were conducted in samples of 129 stranded or by-caught marine mammals (orders Cetartiodactyla [n = 124], Carnivora [n = 4] and Sirenia [n = 1]). Previous serological tests performed on available sera of 27 of the 129 animals (26 cetaceans and one manatee), indicated 10 seropositive cetaceans. Conventional PCR and/or real-time PCR performed in cases with available organs (n = 119) and/or blood or swabs (n = 10) revealed 4/129 (3.1%) Brucella-infected cetaceans (one of them with positive serology; the remaining three with no available sera). Pathological, IHC and/or microbiological analyses conducted in PCR/real-time PCR and/or seropositive cases (n = 13) revealed Brucella-type lesions, including meningitis/meningoencephalitis, pneumonia, necrotizing hepatitis, pericarditis and osteoarthritis in some of those animals, and positive IHC was found in all of them (excepting two live-stranded animals without available organs). Brucella spp. culture attempts were unsuccessful. Our results demonstrated exposure, asymptomatic, acute and chronic Brucella sp. infection in several cetacean species in the Brazilian coast, highlighting the role of this pathogen in stranding and/or death, particularly in Clymene dolphin (Stenella clymene) and short-finned pilot whale (Globicephala macrorhynchus) off Ceará State. Novel hosts susceptible to Brucella included the franciscana (Pontoporia blainvillei), the Guiana dolphin (Sotalia guianensis) and the spinner dolphin (Stenella longirostris). Additionally, three coinfection cases involving Brucella spp. and cetacean morbillivirus, Edwarsiella tarda and Proteus mirabilis were detected. To the best of our knowledge, this is the first long-term and large-scale survey of Brucella spp. in marine mammals of South America, widening the spectrum of susceptible hosts and geographical distribution range of this agent with zoonotic potential.

Brucella/fisiologia , Brucelose/veterinária , Cetáceos , Otárias , Sirênios , Animais , Brasil/epidemiologia , Brucelose/epidemiologia , Brucelose/microbiologia , Brucelose/patologia , Ensaio de Imunoadsorção Enzimática/veterinária , Reação em Cadeia da Polimerase/veterinária , Testes Sorológicos/veterinária
Neurobiol Dis ; 124: 248-262, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30472299


We recently identified a truncated and phosphorylated form of α-synuclein, pα-syn*, as a key neurotoxic α-synuclein species found in cultured neurons, as well as in mouse and Parkinson's disease patients' brains. Small pα-syn* aggregates localize to mitochondria and induce mitochondrial damage and fragmentation. Herein, we investigated the molecular basis of pα-syn*-induced toxicity. By immunofluorescence, we found phosphorylated MKK4, JNK, ERK5 and p38 MAPKs in pα-syn* inclusions. pJNK colocalized with pα-syn* at mitochondria and mitochondria-associated ER membranes where it was associated with BiP and pACC1, markers for the ER and energy deprivation, respectively. We also found that pα-syn* aggregates are tightly associated with small ptau aggregates of similar size. Pα-syn*/ptau inclusions localized to areas of mitochondrial damage and to mitophagic vesicles, showing their role in mitochondrial toxicity, mitophagy induction and their removal along with damaged mitochondrial fragments. Several MAPKs may act cooperatively to phosphorylate tau, notably JNK, p38 and GSK3ß, a non-MAPK that was also found phosphorylated in the vicinity of pα-syn*/ptau aggregates. These results add insight into the mechanisms by which pα-syn* exerts its toxic effects that include the phosphorylation of several kinases of the MAPK pathway, as well as the formation of ptau at the mitochondrial membrane, likely contributing to mitotoxicity. Thus pα-syn* appears to be the trigger of a series of kinase mediated pathogenic events and a link between α-syn pathology and tau, another protein known to aggregate in Parkinson's disease and other synucleinopathies.

MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Ativação Enzimática/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Fosforilação
Artigo em Espanhol | LILACS-Express | ID: biblio-978199


RESUMEN: Objetivo: El objetivo de este estudio fue establecer la prevalencia de signos y síntomas de trastornos temporomandibulares (TTM) en pacientes con maloclusión. Materiales y métodos: Estudio transversal descriptivo. Se revisaron de forma manual 951 fichas clínicas de pacientes ingresados en el Postítulo de Ortodoncia de la Facultad de Odontología de Universidad de Chile (FOUCH) entre los años 2013 y 2015. Se utilizaron los programas Excel 2013, Stata v. 12 y para la comparación entre grupos etarios se utilizó la prueba estadística de Kruskall Wallis. Resultados: Del total de pacientes con maloclusión, el 43,9% presentaron al menos un signo o síntoma de TTM, siendo la desviación mandibular el signo más prevalente con un 43,2%, seguido por el ruido articular con un 12,6%. El dolor fue el síntoma menos prevalente con un 2%. Los adultos fueron los que presentaron mayor prevalencia de signos y síntomas de TTM. No hubo diferencia estadística entre géneros. Conclusión: Debido a que los signos y síntomas de TTM son una condición tan común en pacientes con maloclusión, es necesaria la evaluación de la ATM siempre que se piense en iniciar un tratamiento ortodóncico, para así establecer un adecuado plan de tratamiento.

ABSTRACT: Objective: The aim of this study was to establish the prevalence of signs and symptoms of temporomandibular disorders (TMD) in patients with malocclusion. Materials and methods: Cross-sectional descriptive study. A total of 951 clinical records were reviewed, of patients admitted in the Postgraduate Program of Orthodontia from the Faculty of Dentistry of the University of Chile, between the years 2013 and 2015. The programs Excel 2013 and Stata v. 12 were used, and for the comparison between the age groups the statistical test of Kruskall Wallis was used. Results: Of the total number of patients with malocclusion, 43.85% had at least one sign or symptom of TMD, being the mandibular deviation the most prevalent sign with 43.22%, followed by joint noise with 12.62%. Pain was the least prevalent symptom with 2%. The adults group had the most prevalence of signs and symptoms of TMD. There was no statistical difference between genders. Conclusion: Since TMD signs and symptoms are such a common condition in patients with malocclusion, the assessment of TMJ is necessary to consider when planning an orthodontic treatment, to establish an adequate treatment plan.

Oncogenesis ; 7(9): 76, 2018 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-30250018


Carcinomas, such as colon cancer, initiate their invasion by rescuing the innate plasticity of both epithelial cells and stromal cells. Although Snail is a transcriptional factor involved in the Epithelial-Mesenchymal Transition, in recent years, many studies have also identified the major role of Snail in the activation of Cancer-Associated Fibroblast (CAF) cells and the remodeling of the extracellular matrix. In CAFs, Platelet-derived growth factor (PDGF) receptor signaling is a major functional determinant. High expression of both SNAI1 and PDGF receptors is associated with poor prognosis in cancer patients, but the mechanism(s) that underlie these connections are not understood. In this study, we demonstrate that PDGF-activated fibroblasts stimulate extracellular matrix (ECM) fiber remodeling and deposition. Furthermore, we describe how SNAI1, through the FAK pathway, is a necessary factor for ECM fiber organization. The parallel-oriented fibers are used by endothelial cells as "tracks", facilitating their activation and the creation of tubular structures mimicking in vivo capillary formation. Accordingly, Snail1 expression in fibroblasts was required for the co-adjuvant effect of these cells on matrix remodeling and neoangiogenesis when co-xenografted in nude mice. Finally, in tumor samples from colorectal cancer patients a direct association between stromal SNAI1 expression and the endothelial marker CD34 was observed. In summary, our results advance the understanding of PDGF/SNAI1-activated CAFs in matrix remodeling and angiogenesis stimulation.

Proc Natl Acad Sci U S A ; 115(11): E2634-E2643, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29487216


Exposure of cultured primary neurons to preformed α-synuclein fibrils (PFFs) leads to the recruitment of endogenous α-synuclein and its templated conversion into fibrillar phosphorylated α-synuclein (pα-synF) aggregates resembling those involved in Parkinson's disease (PD) pathogenesis. Pα-synF was described previously as inclusions morphologically similar to Lewy bodies and Lewy neurites in PD patients. We discovered the existence of a conformationally distinct, nonfibrillar, phosphorylated α-syn species that we named "pα-syn*." We uniquely describe the existence of pα-syn* in PFF-seeded primary neurons, mice brains, and PD patients' brains. Through immunofluorescence and pharmacological manipulation we showed that pα-syn* results from incomplete autophagic degradation of pα-synF. Pα-synF was decorated with autophagic markers, but pα-syn* was not. Western blots revealed that pα-syn* was N- and C-terminally trimmed, resulting in a 12.5-kDa fragment and a SDS-resistant dimer. After lysosomal release, pα-syn* aggregates associated with mitochondria, inducing mitochondrial membrane depolarization, cytochrome C release, and mitochondrial fragmentation visualized by confocal and stimulated emission depletion nanoscopy. Pα-syn* recruited phosphorylated acetyl-CoA carboxylase 1 (ACC1) with which it remarkably colocalized. ACC1 phosphorylation indicates low ATP levels, AMPK activation, and oxidative stress and induces mitochondrial fragmentation via reduced lipoylation. Pα-syn* also colocalized with BiP, a master regulator of the unfolded protein response and a resident protein of mitochondria-associated endoplasmic reticulum membranes that are sites of mitochondrial fission and mitophagy. Pα-syn* aggregates were found in Parkin-positive mitophagic vacuoles and imaged by electron microscopy. Collectively, we showed that pα-syn* induces mitochondrial toxicity and fission, energetic stress, and mitophagy, implicating pα-syn* as a key neurotoxic α-syn species and a therapeutic target.

Autofagia/efeitos dos fármacos , Neurotoxinas , Doença de Parkinson/metabolismo , alfa-Sinucleína , Acetil-CoA Carboxilase/química , Acetil-CoA Carboxilase/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Química Encefálica , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Lisossomos/metabolismo , Camundongos , Mitocôndrias , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade
An. pediatr. (2003. Ed. impr.) ; 87(4): 201-205, oct. 2017. tab
Artigo em Espanhol | IBECS | ID: ibc-167296


Introducción: Las técnicas de biología molecular han demostrado ser útiles en la detección del enterovirus en niños con meningitis aséptica. El objetivo de nuestro estudio fue analizar cambios en la práctica clínica tras la introducción de una técnica de RT-PCR a tiempo real, ensayo Xpert EV (Cepheid(R)), para la detección de enterovirus en muestras de líquido cefalorraquídeo de niños con sospecha de meningitis vírica. Métodos: Estudio retrospectivo de los niños mayores de 1año diagnosticados de meningitis por enterovirus en un hospital de tercer nivel desde noviembre de 2006 a febrero de 2013. Se comparó el periodo previo a la introducción del ensayo Xpert EV (Cepheid(R)) (grupo1: noviembre 2006-agosto de 2010) con el periodo posterior (grupo2: septiembre 2010-febrero 2013). Se compararon las características clínicas, los tiempos de estancia media y los costes por hospitalización. Resultados: Se incluyeron 41 pacientes con una mediana de edad de 64 meses (rango intercuartílico, 28-96). En el grupo 2 se incluyeron 26 pacientes (63,4%). No hubo diferencias epidemiológicas, de gravedad, ni de laboratorio estadísticamente significativas entre los pacientes valorados en ambos grupos. Se observó una disminución significativa en la duración de estancia media hospitalaria en el grupo 2 (48 h vs 40,5 h, p = 0,039) y una disminución significativa en el gasto por paciente hospitalizado (779,77 Euros vs. 656,05 Euros, p<0,05). Conclusiones: La incorporación de la técnica Xpert EV (Cepheid(R)) permitió disminuir la estancia y el gasto asociado a hospitalización en niños con meningitis por enterovirus (AU)

Introduction: Polymerase chain reaction (PCR) assays have shown to be useful and quick for the diagnosis of enterovirus in aseptic meningitis. The aim of our study was to analyse the changes in clinical practice after the introduction of a real-time polymerase chain reaction (RT-PCR) technique using the Xpert EV (Cepheid(R)) assay for the qualitative detection of enterovirus RNA in cerebrospinal fluid specimens from children with suspected viral meningitis. Methods: A retrospective study was performed in children older than 1 year, diagnosed with enterovirus meningitis in a third level hospital from November 2006 to February 2013. The first period, before the availability of Xpert EV (Cepheid(R)) (Group 1, November 2006-August 2010) was compared with the later period (Group 2, September 2010-February 2013). Clinical characteristics, the mean length of stay, and the cost per inpatient cases, were compared between the 2 periods. Results: Forty-one patients (60.9% male) were included, with a median age of 64 months (interquartile range 28-96). Twenty-six patients (63.4%) were included in Group 2. There were non-statistically significant differences in the epidemiological, disease severity, and laboratory characteristics between both periods of study. A significant difference was observed in the mean length of stay, with it being shorter in Group2 (48 hours vs 40.5 hours, P = .039), and a significant lower inpatient cost per case (Euros 779.77 vs Euros 656.05, P < .05). Conclusion: Xpert EV (Cepheid(R)) assay was useful for decreasing the length of hospital stay and the costs associated with hospitalisation in children with enterovirus meningitis (AU)

Humanos , Masculino , Feminino , Pré-Escolar , Criança , Técnicas de Diagnóstico Molecular/métodos , Meningite Asséptica/diagnóstico , Infecções por Enterovirus/líquido cefalorraquidiano , Enterovirus/patogenicidade , Estudos Retrospectivos , Reação em Cadeia da Polimerase/métodos , Punção Espinal
An Pediatr (Barc) ; 87(4): 201-205, 2017 Oct.
Artigo em Espanhol | MEDLINE | ID: mdl-27919639


INTRODUCTION: Polymerase chain reaction (PCR) assays have shown to be useful and quick for the diagnosis of enterovirus in aseptic meningitis. The aim of our study was to analyse the changes in clinical practice after the introduction of a real-time polymerase chain reaction (RT-PCR) technique using the Xpert EV (Cepheid®) assay for the qualitative detection of enterovirus RNA in cerebrospinal fluid specimens from children with suspected viral meningitis. METHODS: A retrospective study was performed in children older than 1year, diagnosed with enterovirus meningitis in a third level hospital from November 2006 to February 2013. The first period, before the availability of Xpert EV (Cepheid®) (Group1, November 2006-August 2010) was compared with the later period (Group2, September 2010-February 2013). Clinical characteristics, the mean length of stay, and the cost per inpatient cases, were compared between the 2periods. RESULTS: Forty-one patients (60.9% male) were included, with a median age of 64 months (interquartile range 28-96). Twenty-six patients (63.4%) were included in Group2. There were non-statistically significant differences in the epidemiological, disease severity, and laboratory characteristics between both periods of study. A significant difference was observed in the mean length of stay, with it being shorter in Group2 (48hours vs 40.5hours, P=.039), and a significant lower inpatient cost per case (€779.77 vs €656.05, P<.05). CONCLUSION: Xpert EV (Cepheid®) assay was useful for decreasing the length of hospital stay and the costs associated with hospitalisation in children with enterovirus meningitis.

Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/virologia , Enterovirus/isolamento & purificação , Meningite Viral/diagnóstico , Meningite Viral/virologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos
Nature ; 534(7605): 99-101, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27251283


The conduction of heat through minerals and melts at extreme pressures and temperatures is of central importance to the evolution and dynamics of planets. In the cooling Earth's core, the thermal conductivity of iron alloys defines the adiabatic heat flux and therefore the thermal and compositional energy available to support the production of Earth's magnetic field via dynamo action. Attempts to describe thermal transport in Earth's core have been problematic, with predictions of high thermal conductivity at odds with traditional geophysical models and direct evidence for a primordial magnetic field in the rock record. Measurements of core heat transport are needed to resolve this difference. Here we present direct measurements of the thermal conductivity of solid iron at pressure and temperature conditions relevant to the cores of Mercury-sized to Earth-sized planets, using a dynamically laser-heated diamond-anvil cell. Our measurements place the thermal conductivity of Earth's core near the low end of previous estimates, at 18-44 watts per metre per kelvin. The result is in agreement with palaeomagnetic measurements indicating that Earth's geodynamo has persisted since the beginning of Earth's history, and allows for a solid inner core as old as the dynamo.

PLoS One ; 11(2): e0149491, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26886938


Prevalent HSV-2 infection increases the risk of HIV acquisition both in men and women even in asymptomatic subjects. Understanding the impact of HSV-2 on the mucosal microenvironment may help to identify determinants of susceptibility to HIV. Vaginal HSV-2 infection increases the frequency of cells highly susceptible to HIV in the vaginal tissue of women and macaques and this correlates with increased susceptibility to vaginal SHIV infection in macaques. However, the effect of rectal HSV-2 infection on HIV acquisition remains understudied. We developed a model of rectal HSV-2 infection in macaques in combination with rectal SIVmac239Δnef (SIVΔnef) vaccination and our results suggest that rectal HSV-2 infection may increase the susceptibility of macaques to rectal SIVmac239 wild-type (wt) infection even in SIVΔnef-infected animals. Rectal SIVΔnef infection/vaccination protected 7 out of 7 SIVΔnef-infected macaques from SIVmac239wt rectal infection (vs 12 out of 16 SIVΔnef-negative macaques), while 1 out of 3 animals co-infected with SIVΔnef and HSV-2 acquired SIVmac239wt infection. HSV-2/SIVmac239wt co-infected animals had increased concentrations of inflammatory factors in their plasma and rectal fluids and a tendency toward higher acute SIVmac239wt plasma viral load. However, they had higher blood CD4 counts and reduced depletion of CCR5+ CD4+ T cells compared to SIVmac239wt-only infected animals. Thus, rectal HSV-2 infection generates a pro-inflammatory environment that may increase susceptibility to rectal SIV infection and may impact immunological and virological parameters during acute SIV infection. Studies with larger number of animals are needed to confirm these findings.

Produtos do Gene nef/metabolismo , Herpesvirus Humano 2/fisiologia , Reto/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Vacinação , Animais , Contagem de Linfócito CD4 , Coinfecção/sangue , Coinfecção/imunologia , Coinfecção/virologia , Citocinas/metabolismo , Humanos , Inflamação/patologia , Linfonodos/patologia , Macaca mulatta , Masculino , Fenótipo , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Carga Viral/imunologia
PLoS One ; 10(10): e0140048, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26457419


SUMMARY: A poly (A)-binding protein from Leishmania infantum (LiPABP) has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study. RESULTS: We have selected a ssDNA aptamer population against a recombinant 6xHIS-LiPABP protein (rLiPABP) that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP) protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A) in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes. CONCLUSION: Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be analyzed.

Aptâmeros de Nucleotídeos , DNA de Cadeia Simples , Proteínas de Ligação a DNA/metabolismo , Leishmania infantum/metabolismo , Proteínas de Protozoários/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Conformação de Ácido Nucleico , Poli A/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão , Técnica de Seleção de Aptâmeros
J Immunol ; 194(5): 2415-23, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25624458


The tissue microenvironment shapes the characteristics and functions of dendritic cells (DCs), which are important players in HIV infection and dissemination. Notably, DCs in the gut have the daunting task of orchestrating the balance between immune response and tolerance. They produce retinoic acid (RA), which imprints a gut-homing phenotype and influences surrounding DCs. To investigate how the gut microenvironment impacts the ability of DCs to drive HIV infection, we conditioned human immature monocyte-derived DCs (moDCs) with RA (RA-DCs), before pulsing them with HIV and mixing them with autologous T cells. RA-DCs showed a semimature, mucosal-like phenotype and released higher amounts of TGF-ß1 and CCL2. Using flow cytometry, Western blot, and microscopy, we determined that moDCs express the cell adhesion molecule mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and that RA increases its expression. MAdCAM-1 was also detected on a small population of DCs in rhesus macaque (Macaca mulata) mesenteric lymph node. RA-DCs formed more DC-T cell conjugates and promoted significantly higher HIV replication in DC-T cell mixtures compared with moDCs. This correlated with the increase in MAdCAM-1 expression. Blocking MAdCAM-1 partially inhibited the enhanced HIV replication. In summary, RA influences DC phenotype, increasing their ability to exacerbate HIV infection. We describe a previously unknown mechanism that may contribute to rapid HIV spread in the gut, a major site of HIV replication after mucosal exposure.

Células Dendríticas/efeitos dos fármacos , Infecções por HIV/imunologia , HIV-1/imunologia , Mucosa Intestinal/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Quimiocina CCL2/biossíntese , Quimiocina CCL2/metabolismo , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Feminino , Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Linfonodos/imunologia , Linfonodos/patologia , Linfonodos/virologia , Macaca mulatta , Mucoproteínas/genética , Mucoproteínas/imunologia , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Linfócitos T/virologia , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/metabolismo , Tretinoína/imunologia , Replicação Viral
PLoS Pathog ; 10(12): e1004567, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25521298


The availability of highly susceptible HIV target cells that can rapidly reach the mucosal lymphoid tissues may increase the chances of an otherwise rare transmission event to occur. Expression of α4ß7 is required for trafficking of immune cells to gut inductive sites where HIV can expand and it is expressed at high level on cells particularly susceptible to HIV infection. We hypothesized that HSV-2 modulates the expression of α4ß7 and other homing receptors in the vaginal tissue and that this correlates with the increased risk of HIV acquisition in HSV-2 positive individuals. To test this hypothesis we used an in vivo rhesus macaque (RM) model of HSV-2 vaginal infection and a new ex vivo model of macaque vaginal explants. In vivo we found that HSV-2 latently infected RMs appeared to be more susceptible to vaginal SHIVSF162P3 infection, had higher frequency of α4ß7high CD4+ T cells in the vaginal tissue and higher expression of α4ß7 and CD11c on vaginal DCs. Similarly, ex vivo HSV-2 infection increased the susceptibility of the vaginal tissue to SHIVSF162P3. HSV-2 infection increased the frequencies of α4ß7high CD4+ T cells and this directly correlated with HSV-2 replication. A higher amount of inflammatory cytokines in vaginal fluids of the HSV-2 infected animals was similar to those found in the supernatants of the infected explants. Remarkably, the HSV-2-driven increase in the frequency of α4ß7high CD4+ T cells directly correlated with SHIV replication in the HSV-2 infected tissues. Our results suggest that the HSV-2-driven increase in availability of CD4+ T cells and DCs that express high levels of α4ß7 is associated with the increase in susceptibility to SHIV due to HSV-2. This may persists in absence of HSV-2 shedding. Hence, higher availability of α4ß7 positive HIV target cells in the vaginal tissue may constitute a risk factor for HIV transmission.

Coinfecção/virologia , Suscetibilidade a Doenças/fisiopatologia , Infecções por HIV/fisiopatologia , Herpes Genital/complicações , Herpesvirus Humano 2/fisiologia , Integrinas/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Animais , Antígeno CD11c/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Coinfecção/patologia , Coinfecção/fisiopatologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças/metabolismo , Feminino , HIV/isolamento & purificação , HIV/fisiologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Herpes Genital/metabolismo , Herpes Genital/fisiopatologia , Herpesvirus Humano 2/isolamento & purificação , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia/isolamento & purificação , Vírus da Imunodeficiência Símia/fisiologia , Regulação para Cima , Vagina/metabolismo , Vagina/patologia , Vagina/virologia
Artigo em Inglês | MEDLINE | ID: mdl-25344107


More than 40 mutagenic and carcinogenic agents present in cigarette smoke have been identified as causative factors of human cancer, but no relation has been clearly documented in companion animals. In dogs, in addition to smoke inhalation and transdermic absorption, exposure to smoke includes oral ingestion of particles adhered to the animal's fur. This study evaluates the presence and type of histological alterations and DNA integrity in oropharyngeal tissue in dogs exposed and non-exposed to household cigarette smoke by means of histopathology and comet assay studies on biopsy and swab samples. A non-probabilistic convenience sample of 12 dogs were selected and classified in two groups: exposed and non-exposed to cigarette smoke. Non-parametric Kruskal-Wallis test was carried out on biopsy and swab data and a Chi(2) test was performed on the information obtained by histopathology. A significance level was set at P<0.05. Statistically significant differences were found between groups in comet assays carried out on biopsy samples. No differences (P>0.05) were found between groups based on comet assays swab samples and histopathology assessment. In conclusion, exposure to cigarette smoke causes DNA damage in dog oropharyngeal tissue. The use of dogs as sentinels for early DNA damage caused by exposure to environmental genotoxic agents like cigarette smoke is reported for the first time.

Dano ao DNA/efeitos dos fármacos , Doenças do Cão/etiologia , Exposição por Inalação/efeitos adversos , Melanose/etiologia , Orofaringe/efeitos dos fármacos , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Ensaio Cometa , Doenças do Cão/patologia , Cães , Humanos , Melanose/patologia , Fatores de Risco