Crosstalk Between Ethylene and Abscisic Acid During Changes in Soil Water Content Reveals a New Role for 1-Aminocyclopropane-1- Carboxylate in Coffee Anthesis Regulation.

Coffee (Coffea arabica L.) presents an asynchronous flowering regulated by an endogenous and environmental stimulus, and anthesis occurs once plants are rehydrated after a period of water deficit. We evaluated the evolution of Abscisic Acid (ABA), ethylene, 1-aminocyclopropane-1-carboxylate (ACC) content, ACC oxidase (ACO) activity, and expression analysis of the Lysine Histidine Transporter 1 (LHT1) transporter, in the roots, leaves, and flower buds from three coffee genotypes (C. arabica L. cv Oeiras, Acauã, and Semperflorens) cultivated under field conditions with two experiments. In a third field experiment, the effect of the exogenous supply of ACC in coffee anthesis was evaluated. We found an increased ACC level, low ACO activity, decreased level of ethylene, and a decreased level of ABA in all tissues from the three coffee genotypes in the re-watering period just before anthesis, and a high expression of the LHT1 in flower buds and leaves. The ethylene content and ACO activity decreased from rainy to dry period whereas the ABA content increased. A higher number of opened and G6 stage flower buds were observed in the treatment with exogenous ACC. The results showed that the interaction of ABA-ACO-ethylene and intercellular ACC transport among the leaves, buds, and roots in coffee favors an increased level of ACC that is most likely, involved as a modulator in coffee anthesis. This study provides evidence that ACC can play an important role independently of ethylene in the anthesis process in a perennial crop.

A Comprehensive 2D-LC/MS Online Platform for Screening of Acetylcholinesterase Inhibitors.

The continuous interest in discovering new bioactive molecules derived from natural products (NP) has stimulated the development of improved screening assays to help overcome challenges in NP-based drug discovery. Here, we describe a unique platform for the online screening of acetylcholinesterase inhibitors without the need for pre-treating the sample. In the current study, we have demonstrated the ability to combine reversed-phase separation with a capillary immobilized enzyme reactor (cIMER) in two-dimensional liquid chromatography system coupled with mass spectrometry detection. We systematically investigated the effects of method parameters that are of practical significance and are known to affect the enzyme assay and interfere in the analysis such as: bioreactor dimensions, loop sizes, amount of immobilized enzyme, second dimension flow rates, reaction time, substrate concentration, presence of organic modifier, limit of detection and signal suppression. The performance of this new platform was evaluated using a mixture containing three known AChE inhibitors (tacrine, galanthamine and donepezil) and an ethanolic extract obtained from the dry bulbs of Hippeastrum calyptratum (Amaryllidaceae) was investigated to provide a proof of concept of the applicability of the platform for the analysis of complex mixtures such as those derived from NPs.

Micro- and nano-sized amine-terminated magnetic beads in a ligand fishing assay.

Functionalized micro- and nano-sized magnetic beads (MBs) have been widely used as versatile supports for proteins, enzymes, and drugs. Immobilized protein on MB surfaces has been successfully applied for ligand fishing assays allowing for direct identification of active ligands from complex mixtures, such as natural products and synthetic libraries. MBs with different properties such as different core compositions, sizes, coatings, and surface modifications are available commercially. Studies have been conducted to understand the role of these properties for ligand fishing assays. Here we evaluated, for the first time, the effect of MB size on the ligand fishing assay for acetylcholinesterase from Electrophorus electricus (AChE). For this purpose, four commercially available amine-terminated magnetic particles with diameters ranging from 4.5 nm to 106 µm were evaluated to fish out galantamine, a well-known AChE inhibitor, from an aqueous solution. All MBs were efficient at using glutaraldehyde to covalently immobilize AChE. The particles with diameters of about 1 µm (small microparticles) presented a higher protein mass capacity per milligram of particle than did those with diameters of about 4.5 nm (nanoparticles) and those with diameters of about 106 µm (large microparticles). The influence of these supports on the produced AChE-MBs with regards to hydrolysis turnover and ligand fishing was evaluated and is fully discussed.

A novel on-flow mass spectrometry-based dual enzyme assay.

This work describes a new simultaneous on-flow dual parallel enzyme assay based on immobilized enzyme reactors (ICERs) with mass spectrometry detection. The novelty of this work relies on the fact that two different enzymes can be screened at the same time with only one single sample injection and in less than 6 min. The system consisted of two immobilized capillary enzyme reactors (ICERs). More specifically, the ICERs comprised two different enzymes that were accommodated in parallel and were placed between a liquid chromatography (LC) system and a mass spectrometer (MS). The resulting system could be adapted to other types of enzyme reactors with different supports. All the elements in the system were interfaced by means of two 10-port/two-position switching valves. Different tubing dimensions allowed us to monitor the activity of each enzyme independently during the same analysis. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) bioreactors were chosen as proof of concept. Acetylcholine (ACh) was used as substrate; the area of its protonated enzymatic hydrolysis product ion, choline, [M+H]+m/z 104.0, was monitored in the presence and absence of the standard cholinesterase inhibitor galantamine. This method proved to be an interesting tool for fast, simultaneous, and independent label-free dual enzyme inhibitor assay.

An improved immobilized enzyme reactor-mass spectrometry-based label free assay for butyrylcholinesterase ligand screening.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are key cholinesterase enzymes responsible for the hydrolysis of acetylcholine into choline and acetic acid, an essential process for the restoration of the cholinergic neuron. The loss of cholinergic function in the central nervous system contributes to the cognitive decline associated with advanced age and Alzheimer's disease (AD). Inhibitions assays represent a significant role in the drug discovery process. Herein, we describe an improved label free method to screen and characterize new BChE ligands. The liquid chromatography system uses an immobilized capillary enzyme reactor (ICER) as a low affinity and high selectivity column coupled to a mass spectrometer (MS). The enzyme activity was evaluated by monitoring the choline's precursor ion [M + H]+m/z 104 for a brief period. The method was validated using two known cholinesterase inhibitors tacrine and galanthamine. The IC50 values were 0.03 ±â€¯0.006 µM and 0.88 ±â€¯0.2 for tacrine and galanthamine respectively, and Ki was 0.11 ±â€¯0.2 for galanthamine. The efficient combination of the huBChE-ICER with sensitive enzymatic assay detection such as MS, improved the reliable, fast identification of new ligands. Moreover, specific direct quantitation of the product contributes to the reduction of false positive and negative results.

Label-free offline versus online activity methods for nucleoside diphosphate kinase b using high performance liquid chromatography.

Nucleoside diphosphate kinase from Leishmania spp. (LmNDKb) has recently been described as a potential drug target to treat leishmaniasis disease. Therefore, screening of LmNDKb ligands requires methodologies that mimic the conditions under which LmNDKb acts in biological systems. Here, we compare two label-free methodologies that could help screen LmNDKb ligands and measure NDKb activity: an offline LC-UV assay for soluble LmNDKb and an online two-dimensional LC-UV system based on LmNDKb immobilised on a silica capillary. The target enzyme was immobilised on the silica capillary via Schiff base formation (to give LmNDKb-ICER-Schiff) or affinity attachment (to give LmNDKb-ICER-His). Several aspects of the ICERs resulting from these procedures were compared, namely kinetic parameters, stability, and procedure steps. Both the LmNDKb immobilisation routes minimised the conformational changes and preserved the substrate binding sites. However, considering the number of steps involved in the immobilisation procedure, the cost of reagents, and the stability of the immobilised enzyme, immobilisation via Schiff base formation proved to be the optimal procedure.

Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay.

The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the TcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave KM of 0.317 ± 0.044 mmol·L-1, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L-1, which is in accordance with the data for the enzyme in solution.

As Formas de Comunicação e de Inclusão da Criança Kaiowá Surda na Família e na Escola: um Estudo Etnográfico / Ways of Communication and Inclusion of the Kaiowá Deaf Children in Family and School: an Ethnographic Study

atualmente a etnografia está consolidada como método de pesquisa no campo das ciências sociais, contudo ganha gradativamente mais espaço no cenário educacional. Este estudo configurou-se pelo delineamento do objeto a partir do viver dentro, na tentativa de compreender a experiência cultural dos povos indígenas surdos Guarani-Kaiowá. Assim, tem como objetivo geral investigar as formas de comunicação e de inclusão da criança surda no contexto familiar e escolar das comunidades indígenas das Aldeias Bororó e Jaguapiru, em Dourados, MS. Os objetivos específicos foram: a) compreender como a criança indígena surda se relaciona e se comunica com a família e a escola; b) identificar as facilidades e as dificuldades encontradas na forma de comunicação e na inclusão; e c) descrever as ações e as estratégias utilizadas pela família e pela escola para a comunicação e a efetivação da inclusão da criança nesses sistemas. As análises têm bases conceituais dos estudos culturais, dos estudos surdos e das premissas do desenvolvimento humano ecológico, pautadas na interdependência entre as culturas familiares e os diferentes contextos de socialização, fatores estes determinantes para o desenvolvimento humano. Os resultados do estudo permitiram: conhecer um sistema incipiente de comunicação utilizado pelos familiares da criança; identificar os irmãos como mediadores da comunicação na família e na escola; e reconhecer, nas falas dos professores, o papel do intérprete de Libras como estratégia pedagógica e comunicativa para a inclusão da criança indígena surda. Por fim, a pesquisa etnográfica permitiu uma investigação a partir dos olhares de dentro, apontando indícios para o estabelecimento de um diálogo intercultural.

Currently, ethnographies are recognized as serious research methods in the field of social sciences, though they are still gaining recognition in the educational scene. This study is based on the delimitation of the object from an inside perspective, aiming to understand the cultural experience of the Guarani-Kaiowá deaf people. The general goal is to investigate the communication and inclusion process of deaf children in family and school contexts of the indigenous communities of Bororó and Jaguapiru Villages, in Dourados/Mato Grosso do Sul. The specific goals were: a) to comprehend how indigenous deaf children relate to and communicate with their family and in school; b) to identify what is comfortable and what is difficult for them regarding communication and inclusion; and c) to describe actions and strategies used by the family and by the school to achieve communication and effective inclusion of children in these systems. The analyses are conceptually based on cultural studies, deaf studies and assumptions of ecological human development, guided by the interdependence between family cultures and different contexts of socialization - determining factors for human development. The results of the study allowed us to: identify an emerging system of communication used by family and child; to identify siblings as mediators of communication in the family and at school; and to recognize, in the speech of teachers, the role of the sign language interpreter as a pedagogical and communicative strategy for the inclusion of indigenous deaf children. Finally, ethnographic research enabled us to investigate this situation with an insider view, highlighting the establishment of intercultural dialogue.