Electrochemical Study of Butyl-Pyrene Nitrobenzoate Derivatives Trapped on MWCNT Nanostructured Electrodes

In the scope of our studies tending to find new nanostructured electrodic platforms containing nitroaromatic compounds (NACs) capable of generating in situ electrocatalytic redox couples, we synthesized and electrochemically studied three related 4-(pyren-1-yl)-butyl-substituted nitrobenzoates (2-NBPy, 3-NBPy and 4-NBPy). The design of the compounds is based on a combination of a) an adsorptive tail (-butyl-pyrene) capable of interacting via π-π stacking with the MWCNT nanostructured electrodes and b) nitroaromatic compounds (NACs) capable of electrochemically activating to form a RNHOH/NO redox couple trapped on the nanostructured electrodic platform. Morphological and structural analyses of the nanostructured interfaces were performed by SEM and WAXS/SAXS analysis. All of the NBPy compounds trapped on the nanostructured electrodic platform were susceptible to reduction, generating the corresponding hydroxylamine derivative. The order of ease of reduction for the nitrocompounds is 4-NBPy > 2-NBPy > 3-NBPy. After electrochemical activation, all compounds generated an RNHOH/NO redox mediator couple with the following order of stability of the mediator couple: 2-NBPy > 3-NBPy > 4-NBPy. For the 2-NBPy and 3-NBPy derivatives, excellent stability of the couple was observed, and a decrease in the peak current of 6% was observed after 60 minutes.

Stabilization by Nano Spray Dryer of Pioglitazone Polymeric Nanosystems: Development, In Vivo, Ex Vivo and Synchrotron Analysis

Pioglitazone-loaded PLGA-PEG nanoparticles (NPs) were stabilized by the spray drying technique as an alternative to the treatment of ocular inflammatory disorders. Pioglitazone-NPs were developed and characterized physiochemically. Interaction studies, biopharmaceutical behavior, ex vivo corneal and scleral permeation, and in vivo bioavailability evaluations were conducted. Fibrillar diameter and interfibrillar corneal spacing of collagen was analyzed by synchrotron X-ray scattering techniques and stability studies at 4 °C and was carried out before and after the spray drying process. NPs showed physicochemical characteristics suitable for ocular administration. The release was sustained up to 46 h after drying; ex vivo corneal and scleral permeation profiles of pioglitazone-NPs before and after drying demonstrated higher retention and permeation through cornea than sclera. These results were correlated with an in vivo bioavailability study. Small-angle X-ray scattering (SAXS) analysis did not show a significant difference in the organization of the corneal collagen after the treatment with pioglitazone-NPs before and after the drying process, regarding the negative control. The stabilization process by Nano Spray Dryer B-90 was shown to be useful in preserving the activity of pioglitazone inside the NPs, maintaining their physicochemical characteristics, in vivo bioavailability, and non-damage to corneal collagen function after SAXS analysis was observed.

A Study of the Interaction, Morphology, and Structure in Trypsin-Epigallocatechin-3-Gallate Complexes

Understanding the interaction between proteins and polyphenols is of significance to food industries. The aim of this research was to investigate the mode of aggregation for trypsin-EGCG (Epigallocatechin-3-gallate) complexes. For this, the complex was characterized by fluorescence spectroscopy, circular dichroism (CD) spectra, small-angel X-ray scattering (SAXS), and atomic force microscope (AFM) techniques. The results showed that the fluorescence intensity of trypsin-EGCG complexes decreased with increasing the concentration of EGCG, indicating that the interaction between trypsin and EGCG resulted in changes in the microenvironment around fluorescent amino acid residues. The results of CD analysis showed conformational changes in trypsin after binding with EGCG. The results from SAXS analysis showed that the addition of EGCG results in the formation of aggregates of trypsin-EGCG complexes, and increasing the concentration of EGCG resulted in larger aggregates. AFM images showed that the trypsin-EGCG complex formed aggregates of irregular ellipsoidal shapes with the size of about 200 × 400 × 200 nm, with EGCG interconnecting the trypsin particles. Overall, according to these results, it was concluded that the large aggregates of trypsin-EGCG complexes are formed from several small aggregates that are interconnected. The results of this study shed some light on the interaction between digestive enzymes and EGCG.

Structural insights into the substrate-binding proteins Mce1A and Mce4A from Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), which is responsible for more than a million deaths annually, uses lipids as the source of carbon and energy for its survival in the latent phase of infection. Mtb cannot synthesize all of the lipid molecules required for its growth and pathogenicity. Therefore, it relies on transporters such as the mammalian cell entry (Mce) complexes to import lipids from the host across the cell wall. Despite their importance for the survival and pathogenicity of Mtb, information on the structural properties of these proteins is not yet available. Each of the four Mce complexes in Mtb (Mce1–4) comprises six substrate-binding proteins (SBPs; MceA–F), each of which contains four conserved domains (N-terminal transmembrane, MCE, helical and C-terminal unstructured tail domains). Here, the properties of the various domains of Mtb Mce1A and Mce4A, which are involved in the import of mycolic/fatty acids and cholesterol, respectively, are reported. In the crystal structure of the MCE domain of Mce4A (MtMce4A39–140) a domain-swapped conformation is observed, whereas solution studies, including small-angle X-ray scattering (SAXS), indicate that all Mce1A and Mce4A domains are predominantly monomeric. Further, structural comparisons show interesting differences from the bacterial homologs MlaD, PqiB and LetB, which form homohexamers when assembled as functional transporter complexes. These data, and the fact that there are six SBPs in each Mtb mce operon, suggest that the MceA–F SBPs from Mce1–4 may form heterohexamers. Also, interestingly, the purification and SAXS analysis showed that the helical domains interact with the detergent micelle, suggesting that when assembled the helical domains of MceA–F may form a hydrophobic pore for lipid transport, as observed in EcPqiB. Overall, these data highlight the unique structural properties of the Mtb Mce SBPs.

PlzA is a bifunctional c-di-GMP biosensor that promotes tick and mammalian host-adaptation of Borrelia burgdorferi

In this study, we examined the relationship between c-di-GMP and its only known effector protein, PlzA, in Borrelia burgdorferi during the arthropod and mammalian phases of the enzootic cycle. Using a B. burgdorferi strain expressing a plzA point mutant (plzA-R145D) unable to bind c-di-GMP, we confirmed that the protective function of PlzA in ticks is c-di-GMP-dependent. Unlike ΔplzA spirochetes, which are severely attenuated in mice, the plzA-R145D strain was fully infectious, firmly establishing that PlzA serves a c-di-GMP-independent function in mammals. Contrary to prior reports, loss of PlzA did not affect expression of RpoS or RpoS-dependent genes, which are essential for transmission, mammalian host-adaptation and murine infection. To ascertain the nature of PlzA’s c-di-GMP-independent function(s), we employed infection models using (i) host-adapted mutant spirochetes for needle inoculation of immunocompetent mice and (ii) infection of scid mice with in vitro-grown organisms. Both approaches substantially restored ΔplzA infectivity, suggesting that PlzA enables B. burgdorferi to overcome an early bottleneck to infection. Furthermore, using a Borrelia strain expressing a heterologous, constitutively active diguanylate cyclase, we demonstrate that ‘ectopic’ production of c-di-GMP in mammals abrogates spirochete virulence and interferes with RpoS function at the post-translational level in a PlzA-dependent manner. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA revealed marked conformational changes that underlie its biphasic functionality. This structural plasticity likely enables PlzA to serve as a c-di-GMP biosensor that in its respective liganded and unliganded states promote vector- and host-adaptation by the Lyme disease spirochete.

SAXS analysis of the intrinsic tenase complex bound to lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

The intrinsic tenase (Xase) complex, formed by factors (f)VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI) and small angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.

Crystal structure of the human NKR-P1 bound to its lymphocyte ligand LLT1 reveals receptor clustering in the immune synapse

Human NKR-P1 (CD161, KLRB1) and its ligand LLT1 (CLEC2D) are a prototypical inhibitory C-type lectin-like receptor:ligand pair of NK cells with a critical role in homing lymphocytes to immune-privileged sites, particularly in multiple sclerosis, rheumatoid arthritis, and Crohn's disease. Furthermore, NKR-P1:LLT1 inhibitory signaling is associated with glioblastoma, non-Hodgkin's lymphoma, breast, and prostate cancer. However, the lack of structural data on the formation of the NKR-P1:LLT1 complex limits our understanding of this signaling. We thus solved the crystal structures of NKR-P1 and the NKR-P1:LLT1 complex. NKR-P1 forms a homodimer with an unexpected arrangement that enables LLT1 binding in two modes, bridging two LLT1 molecules, thereby forming interaction clusters suggestive of an inhibitory immune synapse. Moreover, observing the formation of these clusters by SEC-SAXS analysis in solution and by dSTORM super-resolution microscopy on the cell surface, and following their role in receptor signaling using in vitro cytotoxicity assay with freshly isolated NK cells, we show how NKR-P1:LLT1 clustering allows these proteins to overcome the weak affinity of C-type lectin-like receptors to their ligands. Furthermore, only the ligation of both primary and secondary LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. Therefore, our findings show how inhibitory receptor cross-linking and clustering work together to trigger signal transduction upon cellular contact in the immune synapse.

Physicochemical and Adsorption Characteristics of Divinylbenzene-co-Triethoxyvinylsilane Microspheres as Materials for the Removal of Organic Compounds

In this work, organic-inorganic materials with spherical shape consisting of divinylbenzene (DVB) and triethoxyvinylsilane (TEVS) were synthesized and investigated by different complementary techniques. The obtained microspheres may be applied as sorbent systems for the purification of organic compounds from water. The hybrid microspheres combine the properties of the constituents depending on the morphologies and interfacial bonding. In this work, the influence of the molar ratio composition of crosslinked monomer (DVB) and silane coupling agent (TEVS) (DVB:TEVS molar ratios: 1:2, 1:1 and 2:1) on the morphology and quality of organic-inorganic materials have been examined. The materials were analysed using small angle X-ray scattering (SAXS) analysis, low-temperature nitrogen sorption, scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) to provide information on their structural and surface properties. Moreover, thermal analysis was performed to characterize the thermal stability of the studied materials and the adsorbent-adsorbate interactions, while adsorption kinetic studies proved the utility of the synthesized adsorbents for water and wastewater treatment.

Pilot study on the effects of preservatives on corneal collagen parameters measured by small angle X-ray scattering analysis

Objective Small angle X-ray scattering (SAXS) analysis is a sensitive way of determining the ultrastructure of collagen in tissues. Little is known about how parameters measured by SAXS are affected by preservatives commonly used to prevent autolysis. We determined the effects of formalin, glutaraldehyde, Triton X and saline on measurements of fibril diameter, fibril diameter distribution, and D-spacing of corneal collagen using SAXS analysis. Results Compared to sections of sheep and cats’ corneas stored frozen as controls, those preserved in 5% glutaraldehyde and 10% formalin had significantly larger mean collagen fibril diameters, increased fibril diameter distribution and decreased D-spacing. Sections of corneas preserved in Triton X had significantly increased collagen fibril diameters and decreased fibril diameter distribution. Those preserved in 0.9% saline had significantly increased mean collagen fibril diameters and decreased diameter distributions. Subjectively, the corneas preserved in 5% glutaraldehyde and 10% formalin maintained their transparency but those in Triton X and 0.9% saline became opaque. Subjective morphological assessment of transmission electron microscope images of corneas supported the SAXS data. Workers using SAXS analysis to characterize collagen should be alerted to changes that can be introduced by common preservatives in which their samples may have been stored.

REGALS: a general method to deconvolve X-ray scattering data from evolving mixtures

Mixtures of biological macromolecules are inherently difficult to study using structural methods, as increasing complexity presents new challenges for data analysis. Recently, there has been growing interest in studying evolving mixtures using small-angle X-ray scattering (SAXS) in conjunction with time-resolved, high-throughput or chromatography-coupled setups. Deconvolution and interpretation of the resulting datasets, however, are nontrivial when neither the scattering components nor the way in which they evolve are known a priori. To address this issue, the REGALS method (regularized alternating least squares) is introduced, which incorporates simple expectations about the data as prior knowledge, and utilizes parameterization and regularization to provide robust deconvolution solutions. The restraints used by REGALS are general properties such as smoothness of profiles and maximum dimensions of species, making it well suited for exploring datasets with unknown species. Here, REGALS is applied to the analysis of experimental data from four types of SAXS experiment: anion-exchange (AEX) coupled SAXS, ligand titration, time-resolved mixing and time-resolved temperature jump. Based on its performance with these challenging datasets, it is anticipated that REGALS will be a valuable addition to the SAXS analysis toolkit and enable new experiments. The software is implemented in both MATLAB and Python and is available freely as an open-source software package.