Toggle switch residues control allosteric transitions in bacterial adhesins by participating in a concerted repacking of the protein core.

Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an 'inactive' low-affinity state (LAS) to an 'active' high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH's ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function.

Optimization of detergents in solubilization and reconstitution of Aquaporin Z: A structural approach.

BACKGROUND: The exceptional capacities of aquaporins in terms of water permeation and selectivity have made them an interesting system for membrane applications. Despite the multiple attempts for immobilizing the aquaporins over a porous substrate, there is a lack of studies related to the purification and reconstitution steps, principally associated with the use of detergents in solubilization and destabilization steps. This study analyzed the effect of detergents in Aquaporin Z solubilization, considering the purity and structural homogeneity of the protein. METHODS: The extraction process was optimized by the addition of detergent at the sonication step, which enabled the omission of the ultracentrifugation and resuspension steps. Two detergents, Triton X-100, and octyl-glucoside were also evaluated. Destabilization mediated by detergents was used as reconstitution method. Saturation and solubilization points were defined by detergent concentration and both, liposomes and proteoliposomes, were analyzed by size distribution and permeability assays. Detergent removal with Bio-beads was also analyzed. RESULTS: Octyl glucoside ensures structural stability and homogeneity of Aquaporin Z. However, high concentrations of detergents induce the presence of defects in proteoliposomes. While saturated liposomes create homogeneous and functional structures, solubilized liposomes get affected by a reassembly process, creating vesicle defects with anomalous permeability profiles. CONCLUSIONS: Detergent concentration affects the structural conformation of proteoliposomes in the reconstitution process. GENERAL SIGNIFICANCE: Since the destabilization process is dependent on vesicle, detergent, and buffer composition, optimization of this process should be mandatory for further studies. All these considerations will allow achieving the potential of Aquaporins and any other integral membrane protein in their applications for industrial purposes.

In-Situ Modification of Nanofiltration Membranes Using Carbon Nanotubes for Water Treatment.

Modification of thin-film composite (TFC) nanofiltration (NF) membranes to increase permeability and improve separation performance remains a significant challenge for water scarcity. This study aimed to enhance the permeability and selectivity of two commercial polyamide (PA) NF membranes, NF90 and NF270, by modifying them with carbon nanotubes (CNTs) using microwave (MW)-assisted in-situ growth. The conducting polymer, polypyrrole (Ppy), and a ferrocene catalyst were used to facilitate the growth process. Chemical and morphological analyses confirmed that the surface of both membranes was modified. The NF270-Ppy-CNT membrane was selected for ion rejection testing due to its superior permeability compared to the NF90-Ppy-CNT. The modified NF270 membrane showed a 14% increase in ion rejection while maintaining constant water permeability. The results demonstrated that it is feasible to attach CNTs to a polymeric surface without compromising its functional properties. The Spliegler-Kedem model was employed to model the rejection and permeate flux of NF270-Ppy-CNT and NF270 membranes, which indicated that diffusive transport contributes to the modification to increase NaCl rejection. The present study provides a promising approach for modifying membranes by in-situ CNT growth to improve their performance in water treatment applications, such as desalination.

Fourier-transform infrared imaging and multivariate analysis for direct identification of principal polysaccharides in brown seaweeds.

The current hydrocolloid industry requires new techniques for biomass characterization, which can quickly and ecologically characterize contained sugars. This work proposes the use of Fourier Transform Infrared microspectroscopy in combination with multivariate methods, to localize and identify the main carbohydrates and other components present in fresh brown seaweeds, avoiding time-consuming samples pre-treatments. Infrared images of Macrocystis pyrifera samples were analyzed by Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) and Principal Component Analysis (PCA) as chemometrics techniques to identify the compounds. MCR-ALS was the best strategy, delivering pure spectra of chemical compound that PCA did not. The carbohydrates identified by this method were 1-3-ß-glucans divided into endofibers and laminarin; two types of fucoidans (rich in fucose or mannuronic acid), alginate and mannitol, besides other compounds such as proteins. This technique represents an opportunity for the hydrocolloid industry for a modern, rapid and environmentally-friendly characterization of macroalgal biomass to enhance its use.

Efecto en la rata de los corticoides tópicos sobre los linfocitos circulantes / Effect in rats of the topical steroids on circulating lymphocytes

Los glucocorticoides tópicos se usan en diversas enfermedades inflamatorias dermatológicas, respiratorias, etc. Al igual que los glucocorticoides sistémicos pueden suprimir el eje hipotálamo-hipófisis-suprarrenal y producir efectos locales adversos. Por su uso en afecciones crónicas interesa conocer si los glucocorticoides tópicos tienen influencia sobre el número de linfocitos periféricos, lo que repercutiría en el sistema inmune. Se estudió el efecto de dos formas de corticoide de acción porlongada, dipropionato de betametasona, sobre los linfocitos circulantes y algunos órgano como la glándula suprarrenal y piel, en 46 ratas Wistar, aplicándose corticoide tópico en el 25 por ciento de la superficie corporal 2 veces al día a un grupo y sistémico por vía intramuscular por una sola vez en distintas dosis a otros 3 grupos. Después de realizar recuento de linfocitos los días 0,3 y 21, se observaron diferencias estadísticamente significativas en el número de linfocitos al día 3, alteración que revirtió en el análisis del recuento del día 21. En relación al uso de glucocorticoides sistémicos se observaron variaciones características de linfopenia y neutrofilia, que fueron más intensas a mayor dosis empleada. En análisis histopatológico se ralizó en el grupo de ratas con glucocorticoide tópico y sistémico a dosis baja y en las controles. Hubo cambios histopatológicos sólo a nivel de la corteza suprarrenal de las ratas tratadas con glucocorticoide sistémico a dosis baja y a nivel de piel para las ratas sometidasa tratamiento tópico