Real-time monitoring of cell surface protein arrival with split luciferases

Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as a tag on the protein of interest and the second fragment as a supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells

Dihydroisoxazole inhibitors of Anopheles gambiae seminal transglutaminase AgTG3

Background: Current vector-based malaria control strategies are threatened by the rise of biochemical and behavioural resistance in mosquitoes. Researching mosquito traits of immunity and fertility is required to find potential targets for new vector control strategies. The seminal transglutaminase AgTG3 coagulates male Anopheles gambiae seminal fluids, forming a ‘mating plug’ that is required for male reproductive success. Inhibitors of AgTG3 can be useful both as chemical probes of A. gambiae reproductive biology and may further the development of new chemosterilants for mosquito population control. Methods: A targeted library of 3-bromo-4,5-dihydroxoisoxazole inhibitors were synthesized and screened for inhibition of AgTG3 in a fluorescent, plate-based assay. Positive hits were tested for in vitro activity using cross-linking and mass spectrometry, and in vivo efficacy in laboratory mating assays. Results: A targeted chemical library was screened for inhibition of AgTG3 in a fluorescent plate-based assay using its native substrate, plugin. Several inhibitors were identified with IC50 < 10 μM. Preliminary structure-activity relationships within the library support the stereo-specificity and preference for aromatic substituents in the chemical scaffold. Both inhibition of plugin cross-linking and covalent modification of the active site cysteine of AgTG3 were verified. Administration of an AgTG3 inhibitor to A. gambiae males by intrathoracic injection led to a 15% reduction in mating plug transfer in laboratory mating assays. Conclusions: A targeted screen has identified chemical inhibitors of A. gambiae transglutaminase 3 (AgTG3). The most potent inhibitors are known inhibitors of human transglutaminase 2, suggesting a common binding pose may exist within the active site of both enzymes. Future efforts to develop additional inhibitors will provide chemical tools to address important biological questions regarding the role of the A. gambiae mating plug. A second use for transglutaminase inhibitors exists for the study of haemolymph coagulation and immune responses to wound healing in insects

Efeito da estratificação em substrato esterilizado na qualidade sanitária de sementes de Ilex paraguariensis

The stratification of Ilex paraguariensis seeds (yerba mate) in wet sand for periods of four to six months is a common practice in nurseries, used to overcome their dormancy. However, this process may present difficult phytosanitary control due to the high moisture content and the high concentration of seeds in the same place. Thus, the present work had the objective of verifying the influence of the stratification in autoclaved sand on the sanitary quality of yerba mate seeds from different lots. Seeds of four lots were submitted to wet stratification for three and six months, and their sanity was evaluated at the end of these periods, as well as seeds that did not undergo stratification. At the end of the stratification period, the seeds had a darkened surface appearance, which may be related to the action of the fungi in the degradation of the endocarp. These ones were identified: Fusarium graminearum, Fusarium oxysporum, Fusarium guttiforme, Paecilomyces sp., Aspergillus sp., Penicillium sp. Already Aureobasidium melanogenum, Trichoderma sp., Curvularia sp., Rhizopus sp., Alternaria sp., Cladosporium sp. and Bipolaris sp. had fewer occurrences. Observed reduction of Fusarium graminearum, Fusarium oxysporum, Fusarium guttiforme, Aspergillus sp. and Penicillium sp. after stratification and the genera Paecilomyces was only observed at 180 days of stratification, and may be related to the degradation of the endocarp of Ilex paraguariensis seeds.A estratificação das sementes de Ilex paraguariensis A. St.-Hill. (erva-mate) em areia úmida por períodos de quatro a seis meses é uma prática comum em viveiros, utilizada para a superação de dormência das mesmas. Entretanto, este processo pode apresentar difícil controle fitossanitário, devido ao elevado grau de umidade e a grande concentração de sementes no mesmo local. Assim, objetivou-se verificar a influência da estratificação em areia autoclavada na qualidade sanitária de sementes de erva-mate. Sementes de quatro lotes foram submetidas à estratificação em areia úmida autoclavada, durante três e seis meses, tendo sua sanidade avaliada em meio BDA ao final destes períodos, assim como em sementes que não passaram por estratificação. Ao final do período de estratificação, as sementes apresentaram aspecto superficial escurecido, o que pode estar ligado à ação dos fungos na degradação do endocarpo das mesmas. Foram identificados: Fusarium graminearum, F. oxysporum, F. guttiforme, Paecilomyces sp., Aspergillus sp., Penicillium sp. Já Aureobasidium melanogenum, Trichoderma sp., Curvularia  sp., Rhizopus sp., Alternaria sp., Cladosporium sp. e Bipolaris sp. tiveram menores ocorrências. Observou-se a redução de Fusarium graminearum, F. oxysporum, F. guttiforme, Aspergillus sp. e Penicillium sp. após a estratificação em areia autoclavada e o gênero Paecilomyces somente foi observado aos 180 dias de estratificação, podendo estar relacionado com a degradação do endocarpo de sementes de Ilex paraguariensis

Light-emitting diodes (LEDs) in development and response to water stress in Eucalyptus benthamii seedlings (Myrtaceae)

ABSTRACT: Plants use light as a source of energy in the process of photosynthesis. Different levels of luminosity cause physiological and morphological changes in the plant, and its success depends on its adaptation to these different levels. Light emitting diodes (LED) have been proposed as a light source in controlled environments. The present research evaluated physiological and morphological aspects in Eucalyptus benthamii (Myrtaceae) seedlings kept under different colors of LED lamps and submitted to water stress. The experiment was carried out in a completely randomized design, in which the treatments were defined as: T1 (control, with white light); T2 (blue light); T3 (red light) and T4 (mixed blue and red light). Red light resulted in the best response to most morphological variables in plant growth. In response to water stress, blue light LEDs resulted in a better seedling response, with slower reduction of photosynthetic rate and other variables. This may indicate the possibility of reducing water deficit damage in seedlings acclimated to blue light prior to field planting

Aspectos das fenofases reprodutiva e vegetativa de Campomanesia guazumifolia (Myrtaceae), na Região de Lages, Santa Catarina

Our objective was to understand the C. guazumifolia reproductive and vegetative cycles , monitoring its phenological events and correlating them to the climatic data of the studied region. Two evaluation methods were used, the activity index and the intensity index (Fournier index). The flowering phenophase occurred between the months of October and December, and from March to April, with Fournier peaks of intensity in November. The fruiting occurred from December to April, being December the most intense. The species presented the biggest foliar fall period between March and September, with intensity peak in August, and the budding presented a peak in September. It was observed a positive correlation between the climatic variables of maximum and minimum monthly temperatures to fruiting, and negative correlation between the climatic variables of maximum and medium monthly temperatures to foliar fall.Objetivou-se compreender os ciclos reprodutivo e vegetativo de Campomanesia guazumifolia (Cambess.) O. Berg., monitorando os eventos fenológicos e correlacionando com dados climáticos da região de estudo. Foram utilizados dois métodos de avaliação, o Índice de Atividade e o Índice de Intensidade (Índice de Fournier). A fenofase de floração ocorreu entre os meses de outubro a dezembro, e março a abril, com picos de intensidade de Fournier em novembro. A frutificação ocorreu de dezembro a abril, sendo que dezembro foi o mês de maior intensidade de frutos. A espécie apresentou período de maior queda foliar entre março e setembro, com pico de intensidade em agosto, e o brotamento com pico em setembro. Foi observada correlação positiva entre as variáveis climáticas de temperaturas máximas e mínimas mensais e a frutificação, e correlação negativa entre temperaturas máximas e médias mensais e a queda folia

Sementes de Zanthoxylum rhoifolium Lam.: tolerância à secagem, ao armazenamento e ao descongelamento

O armazenamento apropriado de sementes florestais consiste como um importante quesito para preservar a sua qualidade fisiológica. Desta forma, objetivou-se realizar a classificação fisiológica de sementes de Zanthoxylum rhoifolium de acordo com sua tolerância à dessecação e ao armazenamento e avaliar diferentes tratamentos para descongelamento. As sementes foram expostas à dessecação até 5% de teor de água e armazenadas em –20 ºC durante três meses. Em seguida, foram submetidas ao descongelamento em: B.O.D. a 25 ºC, estufa a 40 ºC, banho-maria a 50 ºC, e em temperatura ambiente de 18 a 25 ºC. A viabilidade das sementes foi avaliada pelo teste de tetrazólio, em todas as etapas. A viabilidade inicial das sementes foi de 74% e, após secagem, de 80%. As sementes da espécie Zanthoxylum rhoifolium são tolerantes à dessecação até 5% de umidade e ao armazenamento em -20º C, sendo classificadas como ortodoxas. Recomenda-se o descongelamento em B.O.D a 25 ºC. Recomenda-se o descongelamento em B.O.D a 25 ºC

Nucleocytosolic depletion of the energy metabolite acetyl-coenzyme a stimulates autophagy and prolongs lifespan.

Healthy aging depends on removal of damaged cellular material that is in part mediated by autophagy. The nutritional status of cells affects both aging and autophagy through as-yet-elusive metabolic circuitries. Here, we show that nucleocytosolic acetyl-coenzyme A (AcCoA) production is a metabolic repressor of autophagy during aging in yeast. Blocking the mitochondrial route to AcCoA by deletion of the CoA-transferase ACH1 caused cytosolic accumulation of the AcCoA precursor acetate. This led to hyperactivation of nucleocytosolic AcCoA-synthetase Acs2p, triggering histone acetylation, repression of autophagy genes, and an age-dependent defect in autophagic flux, culminating in a reduced lifespan. Inhibition of nutrient signaling failed to restore, while simultaneous knockdown of ACS2 reinstated, autophagy and survival of ach1 mutant. Brain-specific knockdown of Drosophila AcCoA synthetase was sufficient to enhance autophagic protein clearance and prolong lifespan. Since AcCoA integrates various nutrition pathways, our findings may explain diet-dependent lifespan and autophagy regulation

O protagonismo das mulheres em viveiros florestais / The protagonism of women in forest nurseries

A divisão de gênero do trabalho é uma prática existente desde os povos indígenas, que distinguiam as atividades de acordo com o gênero. Em algumas tribos as mulheres tinham o papel principal de plantar, colher, preparar a comida, cuidar dos filhos e fazer artesanatos, pois na visão indígena, as mulheres possuíam aptidão para realizar estes tipos de tarefas. Em muitos setores da economia, essa situação tem sido revertida, graças às lutas pelos direitos femininos. Todavia este conceito persiste até os dias de hoje, ainda se observa uma distinção das tarefas realizadas. Este artigo se ocupa de uma análise do trabalho feminino na produção de mudas em viveiros florestais. Com o objetivo de atestar a teoria de que as mulheres protagonizam a produção de mudas por tradicionalmente serem reconhecidas como mais atenciosas e delicadas nas atividades que realizam, e caracterizar suas condições de trabalho no setor a fim de melhor avaliar sua participação. Foram aplicados questionários em três viveiros produtores de mudas florestais, localizados no município de Marituba, São Francisco e Redenção, ambos no estado do Pará. Os questionários continham oito perguntas a respeito do setor produtivo de mudas no viveiro. Podemos concluir que apesar dos avanços dos direitos do trabalho feminino, ainda há desigualdade nas condições de trabalho e remuneração das mulheres que trabalham em viveiros florestais.