Inhibition of feline leukemia virus replication in chronically infected cell line utilizing RNA interference

Feline Leukemia virus (FeLV) is a pathogenic retrovirus endemic among domestic cats, remaining a serious disease since its discovery in 1964. RNA interference (RNAi) is a process in which double-stranded RNA induces the post-transcriptional sequence-specific degradation of homologous messenger RNA. At present, RNAi technology is regarded as a potential strategy for the treatment of various diseases as it can be used to inhibit the expression of desired peptides/proteins. This study aimed to apply RNAi technology to inhibit the replication of FeLV. We examined the effect of vector-mediated transfer and expression of FeLV specific short hairpin RNA (shRNA) against p27 protein expression and replication of FeLV in a feline T-cell line chronically infected with FeLV (3201-EECC). Three shRNA homologous to the FeLV gag gene were synthesized, cloned and transfected into a feline fibroblastic cell line (CrFK) expressing FeLV which efficiently reduced FeLV p27 protein expression, consequently decreasing and inhibiting the viral replication in a chronically FeLV infected feline T-cell line (3201-EECC). The expression of shRNA against FeLV gag gene showed markedly lower p27 levels and viral replication in both cell lines, 3201-EECC and CrFK. These results provide useful information to pave the road for the development of a gene therapy strategy to control FeLV and related pathogenic retrovirus infections in the future

EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples

AbstractObjetivesThe extracellular DNA occurring in plasma-EDTA and serum is a biomarker of growing interest, especially in prenatal diagnosis and oncology. The objectives of the present study were to compare the DNase activity in these specimens and to investigate its ex-vivo impact over the circulating cell-free DNA yield (ccfDNA), using the circulating cell-free fetal DNA (ccffDNA) as a tool.Design and methodsEDTA-plasma and serum from women bearing male fetus were submitted to an endogenous DNase activity assay based on qPCR hydrolysis probe degradation, they were treated with DNAse I to investigate the action of an exogenous nuclease and also submitted to different temperature conditions to investigate the temperature-dependent degradation of the ccffDNA. In all instances, all male ccffDNA were quantified by qPCR targeting the Y chromosome-specific sequence DYS-14. Moreover, a serial dilution of EDTA was added to nonanticoagulated plasma and serum before the endogenous DNAse activity assay, to investigate the EDTA-mediated inhibition of the blood's DNase.ResultsThe endogenous nuclease activity was 14.9-fold higher in serum compared to EDTA-plasma. The DNAse I treatment did not alter the ccffDNA yields in EDTA-plasma, but completely degraded it in serum. The addition of increasing doses of EDTA to nonanticoagulated plasma and serum resulted in a stepwise inhibition of their nucleases activity. Finally, we observed a much more pronounced temperature-mediated decrease on the ccffDNA amount in serum compared to EDTA-plasma.ConclusionThe exogenous and endogenous DNases are more active in serum, the anticoagulant EDTA indirectly inhibits blood DNases, and consequently ccfDNA is protected from the blood's DNase preanalytical impact in EDTA-plasma

Associação do polimorfismo de nucleotídeo único rs7903146 no locus do TCF7L2 com diabetes tipo 2 em indivíduos brasileiros

OBJECTIVE:To investigate the association of the T allele of the single nucleotide polymorphism (SNP) rs7903146 of TCF7L2 with the occurrence of T2D in a sample of subjects followed up at the Brasilia University Hospital. SUBJECTS AND METHODS: The SNP rs7903146 of TCF7L2 was genotyped by allele-specific PCR in 113 patients with known T2D and in 139 non-diabetic controls in Brasilia, Brazil. RESULTS:We found that the T allele of the SNP rs7903146 of TCF7L2 was significantly associated with T2D risk (odds ratio of 3.92 for genotype TT in the recessive genetic model, p = 0.004 and 1.5 for T allele, p = 0.032). CONCLUSION:These results reinforce previous findings on the consistent association of this genetic factor and the risk of T2D in populations of diverse ethnic backgrounds. Arq Bras Endocrinol Metab. 2012;56(8):479-84OBJETIVO: Investigar a associação do alelo T do polimorfismo de nucleotídeo único (SNP) rs7903146 do TCF7L2 com a ocorrência de DM2 em uma amostra de indivíduos acompanhados no Hospital Universitário de Brasília. SUJEITOS E MÉTODOS: O SNP 7903146 do TCF7L2 foi genotipado por PCR alelo-específica em 113 pacientes portadores de DM2 e em 139 controles não diabéticos em Brasília, Brasil. RESULTADOS: Foi observada associação significativa do alelo T do SNP rs7903146 do TCF7L2 com a ocorrência de DM2 (razão de chances de 3,92 para o genótipo TT utilizando o modelo genético recessivo, p = 0,003; e de 1,5 para o alelo T, p = 0,032). CONCLUSÃO: Esse resultado reforça os achados prévios de associação consistente desse fator genético com o risco de diabetes em populações de origens étnicas diversas. Arq Bras Endocrinol Metab. 2012;56(8):479-8

Lack of association between the CC genotype of the rs7903146 polymorphism in the TCF7L2 gene and rheumatoid arthritis

INTRODUÇÃO: TCF7L2 é um fator de transcrição envolvido na sinalização Wnt/beta-catenina e tem uma variante conhecida por associar-se consistentemente com o risco de diabetes tipo 2. Alguns estudos também relataram sua associação com o risco de alguns tipos de câncer. OBJETIVO: Como essa via pode também estar envolvida na fisiopatologia de outras doenças inflamatórias crônicas, tais como artrite reumatoide, o objetivo deste estudo foi investigar o efeito do polimorfismo rs7903146 do gene TCF7L2 na gravidade da artrite reumatoide em uma população brasileira. PACIENTES E MÉTODOS: Esse polimorfismo foi genotipado em 208 pacientes com artrite reumatoide e em 104 controles saudáveis. Analisou-se também a associação desse polimorfismo com história de tabagismo, classe funcional e indicadores radiológicos de gravidade da doença. RESULTADOS: A distribuição dos genótipos CC, CT e TT do polimorfismo rs7903146 do gene TCF7L2 não diferiu entre pacientes e controles, nem se encontrou qualquer associação entre o genótipo e os indicadores de gravidade da doença ou história de tabagismo. Quando os dados foram avaliados usando-se o modelo dominante, no qual portadores dos genótipos CT e TT foram agrupados, observou-se um aumento do alelo T em pacientes com fator reumatoide positivo e erosões, embora não significativo. A frequência do alelo T também estava aumentada nos pacientes com classe funcional II quando comparados àqueles com classe I (P = 0,032). CONCLUSÃO: É possível que o pequeno número de pacientes incluído neste estudo tenha dificultado achados adicionais. Outros estudos são, portanto, necessários para que se investigue o papel das variantes do gene TCF7L2 no risco de artrite reumatoide e sua gravidade.INTRODUCTION: TCF7L2 is a transcription factor involved in Wnt/beta-catenin signaling and which has a variant known to be consistently associated with type 2 diabetes risk and some studies have also indicated its association with risk of certain types of cancer. OBJECTIVE: Since this pathway may be involved in the pathophysiology of other chronic inflammatory diseases such as rheumatoid arthritis, we aimed to investigate the effect of TCF7L2 polymorphism rs7903146 on rheumatoid arthritis severity in a Brazilian population. PATIENTS AND METHODS: This polymorphism was genotyped in 208 patients with rheumatoid arthritis and in 104 healthy controls. We also analyzed the association of this polymorphism to smoking history, functional status classification and radiological indicators of disease severity. RESULTS: The distribution of CC, CT and TT genotypes of SNP rs7903146 of the TCF7L2 gene was not different between patients and controls, and no association between the genotype and indicators of disease severity or smoking history was found. When data were evaluated using the dominant model, in which carriers of the CT and TT genotypes were grouped, an increase in the T allele was observed in patients positive for rheumatoid factor and erosions, although this was not significant. The frequency of T allele was also increased in patients with functional class II compared to class I (P = 0.032). CONCLUSION: It is possible that the small number of patients included in this study may have restrained additional findings. Further studies are therefore needed to investigate the role of TCF7L2 gene variants in the risk of rheumatoid arthritis and its severity

Thyroid hormone response element organization dictates the composition of active receptor

Thyroid hormone (triiodothyronine, T3) is known to activate transcription by binding heterodimers of thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). RXR-TRs bind to T3 response elements (TREs) composed of direct repeats of the sequence AGGTCA spaced by four nucleotides (DR-4). In other TREs, however, the half-sites can be arranged as inverted palindromes and palindromes (Pal). Here we show that TR homodimers and monomers activate transcription from representative TREs with alternate half-site placements. TRβ activates transcription more efficiently than TRα at an inverted palindrome (F2), and this correlates with preferential TRβ homodimer formation at F2 in vitro. Furthermore, reconstruction of TR transcription complexes in yeast indicates that TRβ homodimers are active at F2, whereas RXR-TRs are active at DR-4 and Pal. Finally, analysis of TRβ mutations that block homodimer and/or heterodimer formation reveal TRE-selective requirements for these surfaces in mammalian cells, which suggest that TRβ homodimers are active at F2, RXR-TRs at DR-4, and TR monomers at Pal. TRβ requires higher levels of hormone for activation at F2 than other TREs, and this differential effect is abolished by a dimer surface mutation suggesting that it is related to composition of the TR·TRE complex. We propose that interactions of particular TR oligomers with different elements play unappreciated roles in TRE-selective actions of liganded TRs in vivo

Síndrome de Pendred causada por mutação em homozigoze no gene SLC26A4 em uma família brasileira consangüínea

ABSTRACTPendred Syndrome (PS) is an autossomal recessive disorder characterized by sensorineural deafness, goiter and iodide organification defect. The hearing loss is associated with inner ear abnormalities, ranging from an isolated enlarged vestibular aqueduct (EVA) to a typical coclear dysplasia. Mutations in the gene that encodes pendrin (SLC26A4), a chloride/iodide transporter, have been shown to be associated with PS. We describe the clinical and molecular characteristics of a large consanguineous family harboring a mutation in the SLC26A4 gene. The proband was a 26-year-old deaf Brazilian woman who presented a bulky multinodular goiter and hypothyroidism since puberty. Five other siblings were deaf: one brother had a similar phenotype, three siblings also had goiters but normal thyroid function tests, and one brother had only a subtle thyroid enlargement. Other 4 siblings had no thyroid or hearing disorder. Parents were first degree cousins and had normal hearing. The mother was healthy, except for subclinical hypothyroidism; the father was deceased. A perchlorate test in the proband showed a discharge of 21% of the incorporated iodide 2h after the administration of 1g of KClO4. Audiological examinations showed profound hearing loss in all deaf subjects; CT and MRI of the temporal bones showed EVA in all of them. Genomic DNA was isolated from whole blood, from the 6 affected and 4 unaffected siblings, the mother and control. The coding region of the PDS gene (exons 2-21), including exon/intron boundaries, were amplified by PCR and sequenced. A single base-pair (T) deletion at position 1197 of exon 10 was detected in homozygous state in the 6 deaf siblings. The mother and 2 unaffected siblings were heterozygous for this mutation, which has been described by Everett et al. The 1197delT mutation is predicted to result in a frameshift and a truncated protein. The existence of PS phenocopies and intrafamilial phenotypic variability are well documented. The definite diagnosis requires molecular analysis. Our study illustrates the value and challenges of mutational analysis in selected patients with PS. __________________________________________________________________________________ RESUMOA syndrome de Pendred (SP) é uma doença autossômica recessiva caracterizada por surdez neurossensorial, bócio e defeito de organificação do iodo. A perda auditiva está associada a anormalidades do ouvido interno, desde a dilatação isolada do aqueduto vestibular (DAV) até uma típica displasia coclear. Mutações no gene que codifica a pendrina (SLC26A4), um transportador de cloreto/iodeto, têm sido associadas à SP. Descrevemos as características clínicas e moleculares de uma grande família consangüínea portadora de uma mutação no gene SLC26A4. O caso-índice era uma paciente do sexo feminino, brasileira, 26 anos, portadora de surdez congênita, que apresentava um volumoso bócio multinodular e hipotireoidismo desde a puberdade. Outros cinco irmãos eram surdos: um irmão tinha fenotipo semelhante, três também tinham bócio, porém com função tiroideana normal e um irmão tinha apenas um discreto aumento da tiróide. Outros quatro irmãos não apresentavam alteração tiroideana ou auditiva. Os pais eram primos de primeiro grau e tinham audição normal. A mãe era saudável, exceto por hipotireoidismo subclínico; o pai era falecido. O teste do perclorato no caso-índice revelou a liberação de 21% do iodo incorporado duas horas após a administração de 1 g de KClO4. Os exames audiológicos mostraram perda auditiva profunda em todos os indivíduos afetados; TC e RMN dos ossos temporais mostraram DAV em todos eles. O DNA genômico foi isolado do sangue total dos seis irmãos afetados e dos quatro não-afetados, da mãe e do controle. A região codificante do gene PDS (éxons 2-21), incluindo as junções éxon/íntron, foram amplificadas por PCR e seqüenciadas. Foi detectada a deleção de uma base (T) na posição 1197 do éxon 10, em homozigoze, nos seis irmãos afetados. A mãe e dois irmãos não-afetados eram heterozigotos para a mutação, que foi descrita inicialmente por Everett e cols. A mutação 1197delT provavelmente resulta em um erro de fase de leitura (frameshift) e em uma proteína truncada. A existência de fenocópias da SP e a variabilidade fenotípica intrafamiliar são bem conhecidas. O diagnóstico definitivo requer análise molecular. O presente estudo ilustra o valor e os desafios da análise mutacional em pacientes selecionados com SP

Farmacologia do Receptor do Hormônio Tiroidiano, Nova mutação no sítio de "splicing" do gene do hormônio luteinizante e Determinação da atividade da Luciferase : um novo método para medida in vitro de interação proteína-proteína

Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, 2008.Os hormônios tireoideanos (HTs) são necessários para a diferenciação, crescimento e metabolismo de diversos tecidos de mamíferos. Seus efeitos são mediados pelos receptores do hormônio tireoideano (TRs), que pertencem à superfamília dos receptores nucleares. Os TRs são fatores de transcrição que se ligam ao DNA nos promotores dos genes alvos, em regiões denominadas de elementos responsivos ao TR (TRE). Para modular a atividade transcricional (repressão ou ativação) TR se interage com correpressores e co-ativadores, na ausência e na presença de T3, respectivamente. Para melhor se entender a relação entre estrutura e função do TR, investigamos o papel da isoleucina 280 na interação deste com o correpressor SMRT e com os coativadores SRC1 e GRIP. Para isso utilizamos TRs mutantes onde a isoleucina 280 foi substituída por uma arginina (I280R), metionina (I280M), ou lisina (I280K). A atividade transcricional destes mutantes foi avaliada nos elementos responsivos positivos (DR4, F2, e TREpal) por ensaio de gene repórter luciferase e a interação com os correguladores pelo ensaio de interação proteína-proteína em solução (GST “pull down”). Além disso, utilizamos o mutante F451X, descrito na síndrome de resistência ao HT, pois esse receptor se interage avidamente aos correpressores. Nossos resultados demonstraram que os mutantes I280K e I280R foram transcricionalmente inativos em DR4, F2 e TREpal. Por outro lado, quando comparado ao TR1 selvagem, a mutação I280M não modificou a atividade transcricional em DR4, F2 e TREpal. Adicionalmente, a diferença observada nas capacidades transcricionais de I280M, I280R, e I280K em DR4, F2 e TREpal correlacionou-se com a capacidade de se interagir com os coativadores, pois, apenas o mutante I280M foi capaz de se ligar ao GRIP e ao SRC1 de forma semelhante ao TR selvagem. Além disso, I280M, I280K e I280R apresentaram uma diminuição na capacidade de se interagir o correpressor SMRT e a introdução da mutação I280R no F451X aboliu a alta capacidade de ligação deste mutante ao correpressores. Para completa compreensão das diferenças entre I280M, I280R, e I280K testamos a capacidade de ligação ao T3 pelo ensaio de ligação ao T3-I125. Observamos que a mutação I280M reduz discretamente a afinidade do T3 ao TR1, enquanto que as mutações I280R e I280K abole a ligação do T3 ao TR1. Assim, I280R e I280K além de impedir a ligação os correpressores impedem também à ligação ao hormônio, o que não ocorre com o I280M. Por outro lado, I280M, I280R e I280K se heterodimerizaram com RXR de forma semelhante ao TR selvagem. Diante dessas observações, para melhor compreensão do mecanismo molecular envolvido na repressão mediada por T3, avaliamos a atividade transcricional dos mutantes I280R, I280M, I280K, F451X e I280R/F451X sobre alguns promotores regulados negativamente como: Colagenase-1, Hormônio liberador de tirotropina (TRH) e Superóxido dismutase 1 (SOD-1). A soma dos nossos achados demonstra que, de forma geral, nesses promotores, na ausência de T3, TR estimula a transcrição e que essa ativação é de alguma forma dependente da interação com os correpressores. Por outro lado, a repressão da transcrição mediada por T3, depende da manutenção da superfície de interação com os coativadores. Em conclusão, as diferenças nos resultados observados em nossos mutantes parece ser secundária às diferenças existentes entre as cadeias laterais dos aminoácidos isoleucina, arginina, lisina e metionina (Isoleucina e metionina possuem cadeia lateral hidrofóbica, arginina e lisina possuem cadeias laterais polares e carregadas positivamente). O resíduo I280 está localizado na superfície de ligação ao correpressor, não faz contato direto com o co-ativador, em outros receptores nucleares seu correspondente é sempre de resíduos hidrofóbicos (isoleucina, Leucina, Valina, Metionina) e aparentemente tem a função de interagir com aminoácidos da face interna da H12. Dessa forma, as cadeias laterais carregadas positivamente pelos aminoácidos lisina e arginina poderiam impedir a sustentação da H12 em sua posição correta o que, conseqüentemente, impedir a formação da superfície de ligação aos co-ativadores e também diminuir a afinidade pelo ligante. Além disso, nos promotores regulados negativamente, na ausência de T3, a ativação da transcrição por TR depende da manutenção da superfície de interação com correpressores, enquanto que a repressão da transcrição mediada por T3 exige a preservação da interface de interação do TR com os coativadores. ___________________________________________________________________________________ ABSTRACTThe Thyroid hormones (THs) are necessary for differentiation, growth and metabolism of various mammals tissues. Its effects are mediated by thyroid hormone receptors (TRs) that belong to the nuclear receptors superfamily. The TRs are transcription factors that bind to DNA in the promoters of target genes, in regions called the TR-responsive elements (TRE). To modulate transcriptional activity (repression or activation) TR interacts with correpressores and co-activators, in the absence and presence of T3, respectively. To better to understand the relationship between structure and function of TR, we investigated the role of isoleucine 280 in the TR interaction with the correpressor SMRT and the coactivators SRC1 and GRIP. TRs mutants where the isoleucine 280 has been replaced by an arginine (I280R), methionine (I280M) or lysine (I280K) were created. The transcriptional activity of these mutants were assessed in positive responsive elements (DR4, F2, and TREpal) by luciferase reporter gene assay, and the interaction with the correguladores were assessed by GST pull down assays. Furthermore, we use the mutant F451X, described in the syndrome of resistance to TH, because this receptor interacts eagerly to correpressores. Our results showed that the mutants I280K and I280R were transcriptionally inactive in DR4, F2 and TREpal. Furthermore, when compared TR1 wild type, the mutation I280M not changed the transcriptional activity in DR4, F2 and TREpal. Additionally, the difference observed in the transcriptional capacity of I280M, I280R and I280K in DR4, F2 and TREpal correlated with the ability to interact with coactivators, therefore only the mutant I280M was able to connect to GRIP and the SRC1 in a manner similar to the TR wild type. Moreover, I280M, I280K and I280R showed a decrease in the ability to interact correpressor SMRT and the introduction of the mutation I280R in F451X abolished the high binding capacity of this mutant to corepressors. To complete understanding of the differences between I280M, I280R and I280K we tested the ability to bind to T3. We observed that the mutation I280M slightly reduces the affinity to T3, while the mutations I280R and I280K abolishing the bind to T3. So I280R and I280K addition to prevent the TR binding to corepressors, also prevent the binding to the hormone, which is not the case with the I280M. Moreover, I280M, I280R and I280K heterodimerization with RXR was similar to the TR wild type. Given these observations, for the better understanding of the molecular mechanism involved in the repression mediated by T3, we assessed the transcriptional activity of the mutant I280R, I280M, I280K, F451X and I280R/F451X in some promoters negatively regulated by T3 as: Collagenase-1, thyrotropin releasing hormone (TRH) and Superoxide dismutase-1 (SOD-1). The sum of our findings show that, in general, these promoters, in the absence of T3, TR stimulates transcription activation and this is somehow dependent on the interaction with correpressores. Moreover, the transcription repression mediated by T3, depends on the maintenance of the coactivator interaction surface. In conclusion, differences in the results seen in our mutants appears to be secondary to differences between the amino acids side chains isoleucine, arginine, lysine and methionine (Isoleucine and methionine have hydrophobic side chains, arginine and lysine have polar and positively charded side chains). The residue I280 is located on the corepressor binding surface, it makes direct contact with the corepressors, in other nuclear receptors its correspondent is always hydrophobic (isoleucine, Leucine, Valine, Methionine), and apparently has the task of interacting with amino acids from the inner surface of H12. Thus, positively charded side chain amino acids lysine and arginine might prevent support of H12 in its correct position, which, therefore, prevent the formation of coactivator binding surface and also decrease the affinity for the ligand. Moreover, the negatively regulated promoters, in the absence of T3, and the TR transcription activation depends on the maintenance of the corepressor interaction surface, while the T3 mediated transcription repression requires the maintenance of the coactivators interaction surface

Analytical Sensitivity and Specificity of Two RT-qPCR Protocols for SARS-CoV-2 Detection Performed in an Automated Workflow

WHO declared the novel coronavirus (COVID-19) outbreak a global pandemic on 11 March 2020. The establishment of standardized RT-qPCR protocols for respiratory secretions testing, as well as sharing of specimens, data, and information became critical. Here, we investigate the analytical performance of two interim RT-qPCR protocols (Charité and Centers for Disease Control (CDC)) for the qualitative detection of SARS-CoV-2 executed in a fully automated platform. Analytical specificity, PCR amplification efficiency, analytical sensitivity (limit of detection), and cross-reactivity were evaluated using contrived samples. The on-going accuracy was evaluated by retrospective analysis of our test results database (real clinical samples). N1, E, and a modified version of RdRP assays presented adequate analytical specificity, amplification efficiency, and analytical sensitivity using contrived samples. The three assays were applied to all individuals who requested the SARS-CoV-2 molecular test assay in our laboratory and it was observed that N1 gave more positive results than E, and E gave more positive results than RdRP (modified). The RdRP and E were removed from the test and its final version, based on N1 assay only, was applied to 30,699 Brazilian individuals (from 19 February 2020 to 8 May 2020). The aggregated test results available in the database were also presented