Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.

Effect of endogenous hydrogen sulfide inhibition on structural and functional renal disturbances induced by gentamicin

Animal models of gentamicin nephrotoxicity present acute tubular necrosis associated with inflammation, which can contribute to intensify the renal damage. Hydrogen sulfide (H2S) is a signaling molecule involved in inflammation. We evaluated the effect of DL-propargylglycine (PAG), an inhibitor of endogenous H2S formation, on the renal damage induced by gentamicin. Male Wistar rats (N = 8) were injected with 40 mg/kg gentamicin (im) twice a day for 9 days, some of them also received PAG (N = 8, 10 mg·kg-1·day-1, ip). Control rats (N = 6) were treated with saline or PAG only (N = 4). Twenty-four-hour urine samples were collected one day after the end of these treatments, blood samples were collected, the animals were sacrificed, and the kidneys were removed for quantification of H2S formation and histological and immunohistochemical studies. Gentamicin-treated rats presented higher sodium and potassium fractional excretion, increased plasma creatinine [4.06 (3.00; 5.87) mg%] and urea levels, a greater number of macrophages/monocytes, and a higher score for tubular interstitial lesions [3.50 (3.00; 4.00)] in the renal cortex. These changes were associated with increased H2S formation in the kidneys from gentamicin-treated rats (230.60 ± 38.62 µg·mg protein-1·h-1) compared to control (21.12 ± 1.63) and PAG (11.44 ± 3.08). Treatment with PAG reduced this increase (171.60 ± 18.34), the disturbances in plasma creatinine levels [2.20 (1.92; 4.60) mg%], macrophage infiltration, and score for tubular interstitial lesions [2.00 (2.00; 3.00)]. However, PAG did not interfere with the increase in fractional sodium excretion provoked by gentamicin. The protective effect of PAG on gentamicin nephrotoxicity was related, at least in part, to decreased H2S formation.

Panic-like defensive behavior but not fear-induced antinociception is differently organized by dorsomedial and posterior hypothalamic nuclei of Rattus norvegicus (Rodentia, Muridae)

The hypothalamus is a forebrain structure critically involved in the organization of defensive responses to aversive stimuli. Gamma-aminobutyric acid (GABA)ergic dysfunction in dorsomedial and posterior hypothalamic nuclei is implicated in the origin of panic-like defensive behavior, as well as in pain modulation. The present study was conducted to test the difference between these two hypothalamic nuclei regarding defensive and antinociceptive mechanisms. Thus, the GABA A antagonist bicuculline (40 ng/0.2 µL) or saline (0.9% NaCl) was microinjected into the dorsomedial or posterior hypothalamus in independent groups. Innate fear-induced responses characterized by defensive attention, defensive immobility and elaborate escape behavior were evoked by hypothalamic blockade of GABA A receptors. Fear-induced defensive behavior organized by the posterior hypothalamus was more intense than that organized by dorsomedial hypothalamic nuclei. Escape behavior elicited by GABA A receptor blockade in both the dorsomedial and posterior hypothalamus was followed by an increase in nociceptive threshold. Interestingly, there was no difference in the intensity or in the duration of fear-induced antinociception shown by each hypothalamic division presently investigated. The present study showed that GABAergic dysfunction in nuclei of both the dorsomedial and posterior hypothalamus elicit panic attack-like defensive responses followed by fear-induced antinociception, although the innate fear-induced behavior originates differently in the posterior hypothalamus in comparison to the activity of medial hypothalamic subdivisions.

Whole-body vibration decreases the proliferativeb response of TCD4+ cells in elderly individuals with knee osteoarthritis

The aim of this study was to investigate the effect of adding whole-body vibration (WBV; frequency = 35 to 40 Hz; amplitude = 4 mm) to squat training on the T-cell proliferative response of elderly patients with osteoarthritis (OA) of the knee. This study was a randomized controlled trial in which the selected variables were assessed before and after 12 weeks of training. Twenty-six subjects (72 ± 5 years of age) were divided into three groups: 1) squat training with WBV (WBV, N = 8); 2) squat training without WBV (N = 10), and 3) a control group (N = 8). Women who were ≥60 years of age and had been diagnosed with OA in at least one knee were eligible. The intervention consisted of 12 uninterrupted weeks of squatting exercise training performed 3 times/week. Peripheral blood mononuclear cells were obtained from peripheral blood collected before and after training. The proliferation of TCD4+ and TCD8+ cells was evaluated by flow cytometry measuring the carboxyfluorescein succinimidyl ester fluorescence decay before and after the intervention (∆). The proliferative response of TCD4+ cells (P = 0.02, effect size = 1.0) showed a significant decrease (23%) in the WBV group compared to the control group, while there was no difference between groups regarding the proliferative response of TCD8+ cells (P = 0.12, effect size = 2.23). The data suggest that the addition of WBV to squat exercise training might modulate T-cell-mediated immunity, minimizing or slowing disease progression in elderly patients with OA of the knee.

Coronary artery plaque burden and calcium scores in healthy men adhering to long-term wine drinking or alcohol abstinence

Observational studies suggest there are clinical benefits to moderate red wine (RW) consumption. However, the effects on coronary vasculature and overall lifestyle are unclear. We investigated whether a lifestyle of regular long-term RW consumption is associated with changes in coronary plaque burden, calcium score, carotid intima/media thickness, endothelial function, and metabolic variables, compared with alcohol abstinence. Healthy volunteers were evaluated by coronary computed tomography angiography (CTA) as well as carotid and brachial artery ultrasound. Nutritional status, psychological status, and metabolic variables were assessed. The study included 101 drinkers [aged 58.9±7.3 years (means±SD)], from wine brotherhoods, and 104 abstainers, from Anglican, Evangelical and Catholic churches both in the city of São Paulo, Brazil. No significant differences in demographics were noted. Lesion prevalence per patient assessed by coronary CTA and classified as absent (0), 1-25, 26-49, and ≥50% stenosis was similar between groups. When analyzed by individual arteries, i.e., left anterior descending, circumflex, and right coronary, prevalence was also not different. On the other hand, calcium scores were higher among drinkers than abstainers (144.4±362.2 vs 122.0±370.3; P<0.01). However, drinkers reported less history of diabetes and exercised more. RW drinkers consumed 2127.9±387.7 kcal/day while abstainers consumed 1836.0±305.0 (P<0.0001). HDL cholesterol was significantly higher among drinkers compared to abstainers (46.9±10.9 vs 39.5±9.0 mg/dL; P<0.001), while fasting plasma glucose was lower (97.6±18.2 vs 118.4±29.6 mg/dL; P<0.02). Liver enzymes were normal in both groups. In conclusion, long-term wine drinkers displayed a similar plaque burden but greater calcium score than abstainers, despite a more atherogenic diet, and the mechanisms for the increased calcium scores in the former remain speculative.

Hypertension is associated with greater heat exchange during exercise recovery in a hot environment

Individuals with systemic arterial hypertension have a higher risk of heat-related complications. Thus, the aim of this study was to examine the thermoregulatory responses of hypertensive subjects during recovery from moderate-intensity exercise performed in the heat. A total of eight essential hypertensive (H) and eight normotensive (N) male subjects (age=46.5±1.3 and 45.6±1.4 years, body mass index=25.8±0.8 and 25.6±0.6 kg/m2, mean arterial pressure=98.0±2.8 and 86.0±2.3 mmHg, respectively) rested for 30 min, performed 1 h of treadmill exercise at 50% of maximal oxygen consumption, and rested for 1 h after exercise in an environmental chamber at 38°C and 60% relative humidity. Skin and core temperatures were measured to calculate heat exchange parameters. Mean arterial pressure was higher in the hypertensive than in the normotensive subjects throughout the experiment (P<0.05, unpaired t-test). The hypertensive subjects stored less heat (H=-24.23±3.99 W·m−2vs N=-13.63±2.24 W·m−2, P=0.03, unpaired t-test), experienced greater variations in body temperature (H=-0.62±0.05°C vsN=-0.35±0.12°C, P=0.03, unpaired t-test), and had more evaporated sweat (H=-106.1±4.59 W·m−2vs N=-91.15±3.24 W·m−2, P=0.01, unpaired t-test) than the normotensive subjects during the period of recovery from exercise. In conclusion, essential hypertensive subjects showed greater sweat evaporation and increased heat dissipation and body cooling relative to normotensive subjects during recovery from moderate-intensity exercise performed in hot conditions.

Inflammatory cytokines and plasma redox status responses in hypertensive subjects after heat exposure

Hypertension is characterized by a pro-inflammatory status, including redox imbalance and increased levels of pro-inflammatory cytokines, which may be exacerbated after heat exposure. However, the effects of heat exposure, specifically in individuals with inflammatory chronic diseases such as hypertension, are complex and not well understood. This study compared the effects of heat exposure on plasma cytokine levels and redox status parameters in 8 hypertensive (H) and 8 normotensive (N) subjects (age: 46.5±1.3 and 45.6±1.4 years old, body mass index: 25.8±0.8 and 25.6±0.6 kg/m2, mean arterial pressure: 98.0±2.8 and 86.0±2.3 mmHg, respectively). They remained at rest in a sitting position for 10 min in a thermoneutral environment (22°C) followed by 30 min in a heated environmental chamber (38°C and 60% relative humidity). Blood samples were collected before and after heat exposure. Plasma cytokine levels were measured using sandwich ELISA kits. Plasma redox status was determined by thiobarbituric acid reactive substances (TBARS) levels and ferric reducing ability of plasma (FRAP). Hypertensive subjects showed higher plasma levels of IL-10 at baseline (P<0.05), although levels of this cytokine were similar between groups after heat exposure. Moreover, after heat exposure, hypertensive individuals showed higher plasma levels of soluble TNF receptor (sTNFR1) and lower TBARS (P<0.01) and FRAP (P<0.05) levels. Controlled hypertensive subjects, who use angiotensin-converting-enzyme inhibitor (ACE inhibitors), present an anti-inflammatory status and balanced redox status. Nevertheless, exposure to a heat stress condition seems to cause an imbalance in the redox status and an unregulated inflammatory response.

Avaliação da influência dos milhos QPM nas características sensoriais de bolo

Atualmente já não existem mais dúvidas sobre o potencial nutricional dos milhos QPM na alimentação humana. No entanto pouco se conhece sobre os milhos QPM (Quality Protein Maize) na produção de alimentos. O presente trabalho teve como objetivo avaliar o efeito da adição de diferentes variedades de milho nas características de aparência, aroma, sabor e textura de bolo de fubá utilizando análise descritiva quantitativa. Sete formulações foram avaliadas por uma equipe de provadores treinados, onze atributos sensoriais foram definidos. Após um período de treinamento, os provadores avaliaram as amostras através de delineamento de blocos incompletos balanceados usando escala não estruturada de 9cm. As mesmas foram também avaliadas em relação a aceitabilidade através da escala hedônica de 9 pontos, por 70 consumidores de bolo de fubá. Os resultados foram analisados através da ANOVA seguida pelo teste de Tukey. As amostras apresentaram diferença significativa (p<0,05) em todos os atributos avaliados. As formulações diferiram quanto à aceitabilidade (p<0,05). Estas se situaram entre os termos hedônicos "gostei ligeiramente" e "gostei moderadamente"

Concentrações salinas combinadas com tempos de hidratação: efeito no tempo de cocção em feijão

Este trabalho teve como objetivo investigar o efeito da adição de diferentes doses de NaCl na água de hidratação, durante diferentes tempos, buscando uma redução no tempo de cocção. O Brasil, embora seja considerado um grande produtor e um dos maiores consumidores de feijão, está reduzindo gradativamente o consumo. Sendo assim, é necessário dispor de técnicas que facilitem o preparo do feijão, podendo dessa forma reverter a tendência de consumo e como consequência incentivar a produção. O experimento foi conduzido na área experimental do Instituto de Melhoramento e Genética Molecular da UDESC - IMEGEM, em Lages - SC. Foram avaliados os efeitos de três concentrações de NaCL (0, 50 e 125 g.L-1) em dois genótipos de feijão (Pérola e IPR Uirapuru) hidratados durante três tempos (0,5, 9 e 18 horas). Os resultados revelaram um efeito significativo das doses de NaCl na redução do tempo de cocção. A adição de 56,50 g.L-1 de NaCl foi suficiente para reduzir significativamente o tempo de cocção. A hidratação dos grãos de feijão propiciou a redução no tempo de cocção, sendo necessárias 12,5 horas para a máxima hidratação, independente dos genótipos avaliados.

Environment is crucial to the cooking time of beans

The objectives of this study were to understand how genotype, storage time, and storage conditions affect cooking time of beans and to indicate storage techniques that do not affect the cooking time. The grains were subjected to five different storage periods and six different storage conditions. The cooking time was estimated using the Mattson Cooker. The data were subjected to analysis of variance and a subsequent adjustment of simple linear regression for deployment of the interactions between the factors. Contrasts were used to determine the best levels of the factor storage condition. Genotype did not impact cooking time when the storage time and storage conditions were considered. Time and storage conditions affect the cooking time of beans in a dependent manner, but time of storage had the biggest influence. The best conditions for long-term storage of beans ensuring a smaller increase in cooking time is plastic storage at low temperatures. Thus, plastic freezer storage is a practical alternative for consumers.

Thermophysical and rheological properties of dulce de leche with and without coconut flakes as a function of temperature

Dulce de leche (DL), a dairy dessert highly appreciated in Brazil, is a concentrated product containing about 70% m/m of total solids. Thermophysical and rheological properties of two industrial Brazilian Dulce de leche formulations (classic Dulce de leche and Dulce de leche added with coconut flakes 1.5% m/m) were determined at temperatures comprised between 28.4 and 76.4 °C. In general, no significant differences (p < 0.05) were observed in the presence of coconut flakes in the two formulations. Heat capacity varied from 2633.2 to 3101.8 J/kg.°C; thermal conductivity from 0.383 to 0.452 W/m.°C; specific mass from 1350.7 to 1310.7 kg/m³; and, thermal diffusivity from (1.082 × 10-7 to 1.130 × 10-7) m²/s. The Bingham model was used to properly describe the non-Newtonian behavior of both formulations, with yielding stress values varying from 27.3 to 17.6 Pa and plastic viscosity from 19.9 to 5.9 Pa.s.

Nefrolitíase em pacientes pediátricos: investigação metabólica e anatômica

Desordens metabólicas são frequentes em pacientes com nefrolitíase pediátrica. OBJETIVOS: Estudar as alterações metabólicas e anatômicas e a análise química dos cálculos encontrados em pacientes da nossa região. MÉTODOS: Este é um estudo retrospectivo em 158 crianças com evidência de formação recente de cálculos, destes apenas 109 concluíram a investigação metabólica. A investigação laboratorial consistiu de duas amostras de urina de 24 horas com dosagem de cálcio, ácido úrico, citrato, oxalato, sódio e creatinina, cistinúria qualitativa, pH urinário seguido de 12 horas de jejum e restrição hídrica, cultura da urina e análise química quando os cálculos foram disponíveis. As técnicas de imagem incluíram ultrassonografia do trato urinário e urografia excretora. RESULTADOS: Em 96,3% das crianças alguma causa foi detectada. A principal alteração metabólica encontrada foi a hipercalciúria (73,4%). Análise química dos cálculos mostrou oxalato de cálcio em 90,9% dos casos. Alterações anatômicas foram encontradas em 18,0% dos pacientes investigados, e a mais frequente foi a duplicação pieloureteral (28,6%). CONCLUSÕES:Hipercalciúria foi a desordem mais encontrada, a alteração anatômica mais comum foi a duplicação pieloureteral e oxalato de cálcio foi o constituinte químico mais frequente. Este trabalho serviu para o conhecimento das características dos pacientes pediátricos portadores de nefrolitíase em nossa região.

Época de semeadura, produção e qualidade fisiológica de sementes de milheto

O milheto (Pennisetum americanum (L.) Leeke) é uma gramínea anual, de clima tropical, utilizado no pastejo, na produção de massa e cobertura de solo no sistema de plantio direto. Para estudar o comportamento do milheto, semeado em diferentes épocas do ano em condições de Botucatu, sob túnel plástico, com a finalidade de produção de sementes, foram realizados dois experimentos. No experimento 1, estudou-se nove épocas de semeadura (abril a dezembro), empregando-se a cultivar BN2 e no experimento 2, três épocas (janeiro a março), empregando-se a cultivar Comum. O delineamento experimental foi o de blocos casualizados, com cinco repetições. Foram avaliados componentes da produção, rendimento, germinação e vigor de sementes (primeira contagem do teste de germinação, condutividade elétrica e emergência de plântulas em solo). A época de semeadura afetou a produção e a qualidade das sementes de milheto. A massa de mil sementes e o número de sementes por panícula contribuíram para os melhores resultados da produção. As épocas de semeadura promissoras para produção de sementes milheto em Botucatu são dezembro e janeiro.

Época de semeadura, regimes de corte, produção e qualidade de sementes de milheto

O milheto é uma gramínea anual, de clima tropical, empregada na alimentação animal como forrageira, em pastejo direto, para corte, feno, silagem e colheita de grãos para ração. O presente trabalho teve o objetivo de estudar o comportamento do milheto, após diferentes regimes de corte, semeado em duas épocas, para verificar a possibilidade de cultivá-lo em condições de Botucatu, 815 metros de altitude, 22&deg;50'59''S e 48&deg;25'54''O, visando a produção de sementes, como cultura em sucessão/rotação com outra de cultivo das águas. O experimento foi conduzido em vaso, sob túnel plástico, em delineamento de blocos ao acaso, com duas épocas de semeadura (abril e setembro), cada uma com cinco tratamentos: T1) crescimento livre, sem cortes; T2) um corte quando a planta atingiu 50 a 70cm de altura; T3) um corte quando a planta atingiu o florescimento; T4) seqüência de três cortes quando a planta atingiu 50 a 70cm de altura; T5) seqüência de dois cortes quando a planta atingiu o florescimento, e cinco repetições. Foram avaliados a produção de sementes, os componentes de produção de sementes por planta, e a qualidade física e fisiológica das sementes. Os dados foram submetidos à análise estatística através da análise conjunta das épocas de semeadura e as médias comparadas pelo teste de Tukey a 5% de significância. Os resultados apontam que a época de semeadura e o regime de corte afetam a produção e a qualidade das sementes de milheto. A semeadura em abril com no máximo um corte na fase vegetativa mostrou ser a melhor combinação para a produção de sementes de milheto em Botucatu.

Teste de germinação com acondicionamento dos rolos de papel em sacos plásticos

O teste de germinação é realizado em laboratório, sob condições de ambiente controlado e favorável, visando a obtenção da mais completa e rápida germinação dos lotes de sementes. O substrato utilizado deve manter umidade suficiente para o processo de germinação, sendo que, muitas vezes os rolos de papel umedecidos necessitam ser acondicionados em sacos plásticos. O excesso de umidade também pode ser prejudicial à germinação, provocando atraso ou paralisação do desenvolvimento das plântulas. Essas alterações podem tornar o teste não representativo da verdadeira qualidade do lote. O objetivo do trabalho foi avaliar o efeito de embalagens plásticas, no acondicionamento dos conjuntos de rolo de papel mais sementes, durante o teste de germinação conduzido em germinadores de câmara vertical tipo B.O.D., visando a maximização dos resultados. Foram avaliadas duas espessuras (0,033 mm e 0,050 mm) e a presença ou a ausência de perfurações (128 furos de 5mm de diâmetro por face de 60 cm x 40 cm), nos sacos plásticos transparentes utilizados durante a realização do teste de germinação, para as seguintes espécies: milho doce (cv. 'Doce Cristal' e cv. 'Super Doce'), feijão (cv. 'Pérola' e cv. 'IAC-Carioca Tybatã') e soja (cv. 'Embrapa-48' dois lotes). Para sementes de milho doce e feijão, os tratamentos plástico grosso ou fino perfurados e plástico fino inteiro promoveram os melhores resultados do teste de germinação. Concluiu-se que, a espessura do plástico e a presença ou ausência de perfurações são fatores que interferem nos resultados do teste de germinação conduzido em germinadores de câmara vertical tipo B.O.D.