Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery.

Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.

Immunomodulatory actions of a kynurenine-derived endogenous electrophile.

The up-regulation of kynurenine metabolism induces immunomodulatory responses via incompletely understood mechanisms. We report that increases in cellular and systemic kynurenine levels yield the electrophilic derivative kynurenine-carboxyketoalkene (Kyn-CKA), as evidenced by the accumulation of thiol conjugates and saturated metabolites. Kyn-CKA induces NFE2 like bZIP transcription factor 2- and aryl hydrocarbon receptor-regulated genes and inhibits nuclear factor κB- and NLR family pyrin domain containing 3-dependent proinflammatory signaling. Sickle cell disease (SCD) is a hereditary hemolytic condition characterized by basal inflammation and recurrent vaso-occlusive crises. Both transgenic SCD mice and patients with SCD exhibit increased kynurenine and Kyn-CKA metabolite levels. Plasma hemin and kynurenine concentrations are positively correlated, indicating that Kyn-CKA synthesis in SCD is up-regulated during pathogenic vascular stress. Administration of Kyn-CKA abrogated pulmonary microvasculature occlusion in SCD mice, an important factor in lung injury development. These findings demonstrate that the up-regulation of kynurenine synthesis and its metabolism to Kyn-CKA is an adaptive response that attenuates inflammation and protects tissues.

Nrf2 activation protects against lithium-induced nephrogenic diabetes insipidus.

Lithium (Li) is the mainstay pharmacotherapeutic mood stabilizer in bipolar disorder. Its efficacious use is complicated by acute and chronic renal side effects, including nephrogenic diabetes insipidus (NDI) and progression to chronic kidney disease (CKD). The nuclear factor erythroid-derived 2-related factor 2 (Nrf2) pathway senses and coordinates cellular responses to oxidative and electrophilic stress. Here, we identify that graded genetic activation of Nrf2 protects against Li-induced NDI (Li-NDI) and volume wasting via an aquaporin 2-independent mechanism. Renal Nrf2 activity is differentially expressed on functional segments of the nephron, and its activation along the distal tubule and collecting duct directly modulates ion transporter expression, mimicking paradoxical effects of diuretics in mitigating Li-NDI. In addition, Nrf2 reduces cyclooxygenase expression and vasoactive prostaglandin biosynthesis. Pharmacologic activation of Nrf2 confers protective effects, confirming this pathway as a potentially novel druggable target for the prevention of acute and chronic renal sequelae of Li therapy.

Dual-purpose Guzerá cattle exhibit high dairy performance under heat stress.

The present study evaluated the heat stress response pattern of dual-purpose Guzerá cattle for test-day (TD) milk yield records of first lactation and estimated genetic parameters and trends related to heat stress. A total of 31,435 TD records from 4,486 first lactations of Guzerá cows, collected between 1986 and 2012, were analysed. Two random regression models considered days in milk (DIM) and/or temperature × humidity-dependent (THI) covariate. Impacts of -0.037, -0.019 and -0.006 kg/day/THI for initial and intermediate stages of lactation were observed when considering the mean maximum daily temperature and humidity to calculate THI. Heritability estimates ranged from 0.16 to 0.35 throughout lactation and THI values, suggesting the possibility to expect gains from selection for such trait. The variable trajectory of breeding values for dual-purpose Guzerá sires in response to changes in THI values confirms that the genotype × environment interaction due to heat stress can have some effect on TD milk yield. Despite the high dairy performance of Guzerá cattle under heat stress, estimated genetic trends showed a progressive reduction in heat tolerance. Therefore, new strategies should be adopted to prevent negative impacts of heat stress over milk production in Guzerá animals in future.

Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis.

Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO2-OA) is an electrophilic fatty acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis upon activation of Nrf2 signaling. The effects of NO2-OA on intracellular GSH homeostasis were evaluated. In addition to upregulation of GSH biosynthesis, we observed that NO2-OA increased intracellular GSSG in an oxidative stress-independent manner. NO2-OA directly inhibited GR in vitro by covalent modification of the catalytic Cys61, with kon of (3.45 ± 0.04) × 103 M-1 s-1, koff of (4.4 ± 0.4) × 10-4 s-1, and Keq of (1.3 ± 0.1) × 10-7 M. Akin to NO2-OA, the electrophilic Nrf2 activators bardoxolone-imidazole (CDDO-Im), bardoxolone-methyl (CDDO-Me) and dimethyl fumarate (DMF) also upregulated GSH biosynthesis while promoting GSSG accumulation, but without directly inhibiting GR activity. In vitro assays in which GR was treated with increasing GSH concentrations and GSH depletion experiments in cells revealed that GR activity is finely regulated via product inhibition, an observation further supported by theoretical (kinetic modeling of cellular GSSG:GSH levels) approaches. Together, these results describe two independent mechanisms by which electrophiles modulate the GSH/GSSG couple, and provide a novel conceptual framework to interpret experimentally determined values of GSH and GSSG.

Fuzzy system to predict physiological responses of Holstein cows in southeastern Brazil / Sistema difuso para predecir respuestas fisiológicas de vacas Holstein en el sureste de Brasil / Sistema fuzzy para predizer respostas fisiológicas de vacas Holstein na região sudeste do Brasil

Background:thermal environment exerts a direct influence on animal performance. Environmental factors, in different circumstances, may affect milk production and fertility of animals, compromising the profitability of the activity. Under heat stress conditions dairy cows reduce feed intake and, consequently, milk production. Sweating and panting are some of the mechanisms these animals use to relieve thermal stress. In addition, animals often suffer physiological and behavioral changes caused by heat stress.Objective: the goal of the present study was to develop and evaluate a model based on fuzzy set theory to predict rectal temperature (°C), and respiratory rate (breaths per minute) responses of Holstein cows exposed to different environmental thermal conditions. Methods: the proposed fuzzy model was based on data obtained experimentally (5,884 records) as well as from the literature (792 records) referring to the effect of environmental variables on both physiological responses. Input variables of each record were dry bulb air temperature and relative humidity. Output variables were rectal temperature and respiratory rate. Results: the adjusted model was evaluated for its ability to predict response variables as a function of input variables. The model was able to predict respiration rate with an average standard error of 7.73 and rectal temperature with an average standard error of 0.27. Conclusion: a fuzzy model was developed to predict physiological responses. The error (%) of model prediction for respiration rate and rectal temperature was +/- 12 and 0.5%, respectively.

Antecedentes: el ambiente termal ejerce una influencia directa en el desempeño animal. Factores ambientales, en diferentes circunstancias, pueden afectar la producción de leche y la fertilidad de los animales, comprometiendo la rentabilidad de la actividad. Bajo condiciones de estrés por calor, las vacas lecheras reducen su consumo de alimento y, consecuentemente su producción de leche. Sudar y jadear son algunos de los mecanismos que estos animales usan para aliviar el estrés térmico. Además de estas consecuencias, los animales a menudo sufren cambios fisiológicos y comportamentales causados por el estrés calórico, causando una reducción en la producción de leche. Objetivo: el objetivo del presente estudio fue desarrollar y evaluar un modelo basado en la teoría de los conjuntos fuzzy para predecir respuestas fisiológicas, temperatura rectal y frecuencia respiratoria, de vacas lecheras de raza holandesa blanco y negro expuestas a diferentes condiciones ambientales. Métodos: el modelo fuzzy propuesto fue basado en datos obtenidos experimentalmente (5.884 registros), también como de la literatura (792 registros), refiriéndose a la influencia de las variables ambientales sobre esas respuestas fisiológicas. Cada registro incluye valores de temperatura de bulbo seco del aire, humedad relativa (variables de entrada), temperatura rectal y frecuencia respiratoria (variables de salida). Resultados: el modelo ajustado fue evaluado para cada variable respuesta y predice estas en función de las variables de entrada. Este modelo fue capaz de predecir la frecuencia respiratoria con un error estándar medio de 7,73 y la temperatura rectal con un error estándar medio de 0,27. Conclusión: un modelo fuzzy fue exitosamente desarrollado para predecir respuestas fisiológicas. El modelo fue capaz de predecir frecuencia respiratoria y temperatura rectal con errores porcentuales de +/- 12 y 0,5%, respectivamente.

Antecedentes: o ambiente térmico exerce uma influencia direta no desempenho animal. Fatores ambientais, em diferentes circunstancias, podem afetar a produção de leite e a fertilidade dos animais, comprometendo assim a rentabilidade da atividade. Sobcondições de estresse por calor, as vacas leiteiras reduzem o seu consumo de alimento e, consequentemente, a sua produção de leite. Sudorese e respiração ofegante são alguns dos mecanismos que estes animais usam para aliviar o estresse térmico. Além destas consequências, os animais com frequência sofrem mudanças fisiológicas e comportamentais causados pelo estresse calórico, causando uma redução na produção de leite. Objetivo: o objetivo do presente estudo foi desenvolver e avaliar um modelo baseado na teoria dos conjuntos fuzzy para predizer respostas fisiológicas, temperatura retal e frequência respiratória, de vacas leiteiras de raça holandesa branca e preta, expostas a diferentes condições térmicas ambientais. Métodos: o modelo fuzzy proposto foi baseado em dados obtidos experimentalmente (5,884 registros) bem como da literatura (792 registros), referindo-se à influência das variáveis ambientais sobre essas respostas fisiológicas. Cada registro inclui valores de temperatura de bulbo seco do ar, umidade relativa (variáveis de entrada), temperatura retal e frequência respiratória (variáveis de saída). Resultados: o modelo ajustado foi avaliado para cada variável resposta e prediz estas em função das variáveis de entrada. Este modelo foi capaz de predizer a frequência respiratória com um erro padrão médio de 7,73 e a temperatura retal com um erro padrão médio de 0,27. Conclusão: o modelo fuzzy foi desenvolvido com sucesso para predizer respostas fisiológicas. O modelo foi capaz de predizer frequência respiratória e temperatura retal com erros percentuais de +/- 12 y 0,5%, respectivamente.

Constituents of Encyclia longifolia Schltr. (Orchidaceae) / Constituintes de Encyclia longifolia Schltr. (Orchidaceae)

A detailed chemical analysis of the non-volatile secondary metabolites of Encyclia longifolia Schltr. syn. Epidendrum longifolium Barb. Rodr. was carried out in an attempt to lend a chemotaxonomic support for the recent separation of the genus Encyclia into Encyclia Hook and Prosthechea Knowles & Westc.

Uma análise química detalhada dos metabólitos secundários não voláteis de Encyclia longifolia Schltr. Sin. Epidendrum longifolium Barb. Rodr. foi realizada na tentativa de prestar suporte quimiotaxonômico para a recente separação do gênero Encyclia em Encyclia Hook e Prosthechea Knowles & Westc.

Human parvovirus B19 infections among exanthematic diseases notified as measles

A total of 1397 sera collected from 1095 cases of exanthematic disease notified as measles in ES and RJ states during July 1992 to December 1994 were investigated. These sera were first tested for measles and rubella specific IgM. When they proved negative, they were tested for B19 specific IgM by an enzyme immunoassay. B19 infection was confirmed in 27 (2.5 per cent) of these cases. Sera from 194 negative cases for measles and rubella IgM received from other Brazilian states were also investigated and B19 infection was confirmed for 11 of them. Sera from these 38 IgM positive cases for B19, were tested for anti-B19 IgG by an enzyme immunoassay and for B19 DNA by dot blot hybridization. Anti-B19 IgG antibodies were detected in most of the acute sera. B19 DNA was detected in the acute serum of one patient that had been splenectomized before. As the exanthem caused by human parvovirus infection may be clinically diagnosed as rubella, it could be important to diagnose B19 infection in Brazil since it is becoming prevalent as the cause of rash in countries where rubella is controlled by vaccination.