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This work presents a magnetic purification method of human erythrocyte Acetylcholinesterase (EC 3.1.1.7; AChE) based on affinity binding to procainamide (Proca) as ligand. Acetylcholinesterase is an acetylcholine-regulating enzyme found in different areas of the body and associated with various neurological disorders, such as Parkinson, Alzheymer and Amyotrophic Lateral Sclerosis. AChE from human erythrocyte purification has been attempted in recent years with low degree of purity. Here, magnetic nanoparticles (MNP) were synthesized and coated with polyaniline (PANI) and procainamide (PROCA) was covalently linked to the PANI. The extracted human erythrocyte AChE formed a complex with the MNP@PANI-PROCA and an external magnet separated it from the undesired proteins. Finally, the enzyme was collected by increasing the ionic strength. Experimental Box-Behnken design was developed to optimize this process of human erythrocyte AChE purification protocol. The enzyme was purified in all fifteen experiments. However, the best AChE purification result was achieved, about 2000 times purified, when 100 mg of MNP@PANI-PROCA was incubated for one hour with 4 ml hemolysate extract. The SDS-PAGE of this preparation presented a molecular weight of approximately 70 kDa, corroborating with few previous studies of AChE from erythrocyte purification.

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Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology.

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The ability to reversibly bind carbohydrates is an incredible property from lectins. Such characteristic has led these molecules to be employed in several applications involving medical research and biotechnology. Generally, these proteins follow several steps towards purification. Here, the synthesis, physical characterization, and use of levan-coated magnetite nanoparticles (MNPs-levan) for lectin isolation is described. Canavalia ensiformis and Cratylia mollis were used as sources of Concanavalin A and Cramoll, respectively, that were purified by using MNPs-levan. Mass spectrometry, SDS-PAGE, and hemagglutinating activity were employed to assess the efficiency of the process. Moreover, by using mass spectrometry approaches, a novel lectin, similar to Canavalin, was also identified for C. mollis, corroborating the advantages of using nanoparticles over microparticles. MNPs-levan could also be recycled, making this a low-cost, scalable process that can be efficiently employed over crude samples.

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Carbohydrates perform important physiological functions in eukaryotic and prokaryotic cells. Indeed, alterations in glycan patterns may be associated with disorders. The analysis of these sugars can be reached using nanoprobes composed by lectins associated with fluorescent nanoparticles. This study reports the conjugation of a galactose-binding lectin (BmoLL) isolated from Bauhinia monandra leaves with quantum dots (QDs) by adsorption. QDs-BmoLL conjugates showed bright fluorescence and the hemagglutination assay revealed that the lectin preserved its carbohydrate-binding ability after the conjugation. To evaluate the efficiency/specificity of the bioconjugate, ABO human red blood cells (RBCs) were used as biological models and the labeling was analyzed by flow cytometry. Among ABO blood groups, higher labeling (71.7 ± 5.9%) was detected for B-type RBCs, whose antigens have galactose in their structure. The specificity of labeling was confirmed since A- and O-types RBCs incubated with QDs-BmoLL, as well as B-type cells incubated with previously galactose-inhibited conjugates, were labeled below 6%. In AB-type RBCs, which simultaneously have B and A (N-acetylgalactosamine) antigens on their membrane, the labeling was ca. 14.1 ± 4.8%. Therefore, a successful conjugation was reached and QDs-BmoLL conjugates can be considered promising fluorescent nanoprobes for biological investigations.

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Glycoconjugates found on cell walls of Candida species are fundamental for their pathogenicity. Laborious techniques have been employed to investigate the sugar composition of these microorganisms. Herein, we prepared a nanotool, based on the fluorescence of quantum dots (QDs) combined with the specificity of Cramoll lectin, to evaluate glucose/mannose profiles on three Candida species. The QDs-Cramoll conjugates presented specificity and bright fluorescence emission. The lectin preserved its biological activity after the conjugation process mediated by adsorption interactions. The labeling of Candida species was analyzed by fluorescence microscopy and quantified by flow cytometry. Morphological analyses of yeasts labeled with QDs-Cramoll conjugates indicated that C. glabrata (2.7 µm) was smaller when compared to C. albicans (4.0 µm) and C. parapsilosis sensu stricto (3.8 µm). Also, C. parapsilosis population was heterogeneous, presenting rod-shaped blastoconidia. More than 90% of cells of the three species were labeled by conjugates. Inhibition and saturation assays indicated that C. parapsilosis had a higher content of exposed glucose/mannose than the other two species. Therefore, QDs-Cramoll conjugates demonstrated to be effective fluorescent nanoprobes for evaluation of glucose/mannose constitution on the cell walls of fungal species frequently involved in candidiasis.

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Abstract Objective: To determine the association between occupational ionizing radiation exposure in the cardiovascular catheterization laboratories staff and development of lens changes. Methods: A cross-sectional study was made. Participants were recruited at the XIII Colombian Conference of Interventional Cardiology and SOLACI conferences in Bogotá - Colombia 2017. Informed consent was obtained from all study participants. Demographics and clinical data were collected. Associated lens changes were determined according to the ionizing radiation exposure status. Results: A total of 89 subjects (178 eyes) participated in the study. In general, the mean age was 40 years old, ranging from 19 to 78 years old. Distribution according gender was 55.3% male and 44.7% female. 31 subjects (62 eyes) were deemed to have not occupational exposure to radiation and 58 subjects (116 eyes) with a history of occupational radiation exposure. Exposed eyes had twice risk for present subcapsular posterior lens opacity compared to non-exposed eyes. The subjects with older age and physician role were associated to the presence of lens opacity, with a p value 0.001 in both cases. Likewise, exposed subjects with any lens opacity had a high mean occupational lifetime, with a p 0.001. Conclusions: This study confirms a statistically significant increase in radiation-associated subcapsular lens changes in the eyes of interventional cardiology staff compared to unexposed controls.

Resumen Objetivo: Determinar la asociación entre la exposición a radiación ionizante en el personal de las salas de cateterismo cardiovascular y el desarrollo de cambios en el cristalino. Métodos: Se hizo un estudio transversal. Los participantes fueron reclutados en el XIII Congreso Colombiano de Cardiología Intervencionista y las Conferencias SOLACI en Bogotá - Colombia 2017. Se obtuvo el consentimiento informado de todos los participantes. Se recolectaron datos demográficos y clínicos. Se determinó la asociación entre los cambios en el cristalino y la exposición a la radiación ionizante. Resultados: Un total de 89 sujetos (178 ojos) participaron en el estudio. En general, la edad promedio fue de 40 años, con edades comprendidas entre los 19 años y 78 años. La distribución según el género fue del 55,3% en hombres y el 44,7% en mujeres. Se consideró que 31 sujetos (62 ojos) no tenían exposición ocupacional a la radiación y 58 sujetos (116 ojos) presentaban exposición ocupacional a esta. Los ojos expuestos tenían dos veces el riesgo de presentar opacidad subcapsular posterior del cristalino en comparación con los ojos no expuestos. Los sujetos de mayor edad y con rol de médico se asociaron a la presencia de opacidad del cristalino, con un valor p de 0.001 en ambos casos. Del mismo modo, los sujetos expuestos con cualquier opacidad del cristalino tenían una vida laboral más larga, con una p 0.001. Conclusiones: Este estudio confirma un aumento estadísticamente significativo en la presencia de cambios en el cristalino en el personal de las salas de cateterismo cardiovascular en comparación con los controles no expuestos.

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[This corrects the article DOI: 10.1155/2010/834060.].

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BACKGROUND: Site-specific multimodal nanoplatforms with fluorescent-magnetic properties have great potential for biological sciences. For this reason, we developed a multimodal nanoprobe (BNPs-Tf), by covalently conjugating an optical-magnetically active bimodal nanosystem, based on quantum dots and iron oxide nanoparticles, with the human holo-transferrin (Tf). METHODS: The Tf bioconjugation efficiency was evaluated by the fluorescence microplate assay (FMA) and the amount of Tf immobilized on BNPs was quantified by fluorescence spectroscopy. Moreover, relaxometric and fluorescent properties of the BNPs-Tf were evaluated, as well as its ability to label specifically HeLa cells. Cytotoxicity was also performed by Alamar Blue assay. RESULTS: The FMA confirmed an efficient bioconjugation and the fluorescence spectroscopy analysis indicated that 98% of Tf was immobilized on BNPs. BNPs-Tf also presented a bright fluorescence and a transversal/longitudinal relaxivities ratio (r2/r1) of 65. Importantly, the developed BNPs-Tf were able to label, efficiently and specifically, the Tf receptors in HeLa cells, as shown by fluorescence and magnetic resonance imaging assays. Moreover, this multimodal system did not cause noteworthy cytotoxicity. CONCLUSIONS: The prepared BNPs-Tf hold great promise as an effective and specific multimodal, highly fluorescent-magnetic, nanoplatform for fluorescence analyses and T2-weighted images. GENERAL SIGNIFICANCE: This study developed an attractive and versatile multimodal nanoplatform that has potential to be applied in a variety of in vitro and in vivo studies, addressing biological processes, diagnostic, and therapeutics. Moreover, this work opens new possibilities for designing other efficient multimodal nanosystems, considering other biomolecules in their composition able to provide them important functional properties.

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Tannase (tannin acyl hydrolase, E.C. 3.1.1.20) is an enzyme that catalyzes the hydrolysis of ester and depside linkages in hydrolysable tannins such as tannic acid, releasing gallic acid and glucose. It has several commercial applications in food industry, among which are gallic acid production, reduction of tannin content in fruit juices, and preparation of instantaneous tea. In this study we immobilized Aspergillus ficuum tannase in calcium alginate beads and then used it to treat boldo (Peumus boldus) tea. Such a technique allowed entrapping tannase with a 75% efficiency and appreciably increasing its thermal and pH stability compared with the free enzyme. Storage stability and reuse of the immobilized enzyme were very promising, in that about 60% of starting enzyme activity was retained after bead storage for 90 days at 4 °C or after six cycles of use. Boldo tea treatment with immobilized tannase for 120 min at 40 °C led to 31 and 60% removals of tannins and epigallocatechin gallate, an increase of about two orders of magnitude in gallic acid content, 56 and 109% increases in total flavonoids and epigallocatechin contents, a 42.8% increase in antioxidant activity and significant enhancements of tea color, clarity and pH.

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Acetylcholinesterase (AChE) acts on the hydrolysis of acetylcholine, rapidly removing this neurotransmitter at cholinergic synapses and neuromuscular junctions as well as in neuronal growth and differentiation, modulation of cell adhesion ("electrotactins") and aryl-acylamidase activity (AAA). This enzyme is also found in erythrocyte, as 160 kDa dimer that anchors to the plasma membrane via glycophosphatidylinositol. The function of this enzyme in erythrocytes has not yet been elucidated; however, it is suspected to participate in cell-to-cell interactions. Here, a review on erythrocyte AChE characteristics and use as biomarker for organophosphorus and carbamate insecticides is presented since it is the first specific target/barrier of the action of these pesticides, besides plasma butyrylcholinesterase (BChE). However, some past and current methods have disadvantages: (a) not discriminating the activities of AChE and BChE; (b) low accuracy due to interference of hemoglobin in whole blood samples. On the other hand, extraction methods of hemoglobin-free erythrocyte AChE allows: (a) the freezing and transporting of samples; (b) samples free of colorimetric interference; (c) data from only erythrocyte AChE activity; (d) erythrocyte AChE specific activity presents higher correlation with the central nervous system AChE than other peripheral ChEs; (e) slow spontaneous regeneration against anti-ChEs agents of AChE in comparison to BChE, thus increasing the chances of detecting such compounds following longer interval after exposure. As monitoring perspectives, hemoglobin-free methodologies may be promising alternatives to assess the degree of exposure since they are not influenced by this interfering agent.

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