GC-MS with Headspace Extraction for Non-Invasive Diagnostics of IBD Dynamics in a Model of DSS-Induced Colitis in Rats.

Inflammatory bowel diseases are extremely common throughout the world. However, in most cases, it is asymptomatic at the initial stage. Therefore, it is important to develop non-invasive diagnostic methods that allow identification of the IBD risks in a timely manner. It is well known that gastrointestinal microbiota secrete volatile compounds (VOCs) and their composition may change in IBD. We propose a non-invasive method to identify the dynamics of IBD development in the acute and remission stage at the level of VOCs in model of dextran sulfate sodium (DSS) with chemically induced colitis measured by headspace GC/MS (HS GC/MS). Methods: VOCs profile was identified using a headspace GC/MS (HS GC/MS). GC/MS data were processed using MetaboAnalyst 5.0 and GraphPad Prism 8.0.1 software. The disease activity index (DAI) and histological method were used to assess intestinal inflammation. The peak of intestinal inflammation activity was reached on day 7, according to the disease activity index. Histological examination data showed changes in the intestine due to different stages of inflammation. As the acute inflammation stage was reached, the metabolomic profile also underwent changes, especially at the short-fatty acids level. A higher relative amounts of acetic acid (p value < 0.025) and lower relative amounts of propanoic acid (p value < 0.0005), butanoic acid (p value < 0.005) and phenol 4-methyl- (p value = 0.053) were observed in DSS7 group on day 7 compared to the control group. In remission stage, disease activity indexes decreased, and the histological picture also improved. But metabolome changes continued despite the withdrawal of the DSS examination. A lower relative amounts of propanoic acid (p value < 0.025), butanoic acid (p value < 0.0005), pentanoic acid (p value < 0.0005), and a significant de-crease of hexanoic acid (p value < 0.0005) relative amounts were observed in the DSS14 group compared to the control group on day 14. A model of DSS-induced colitis in rats was successfully implemented for metabolomic assessment of different stages of inflammation. We demonstrated that the ratios of volatile compounds change in response to DSS before the appearance of standard signs of inflammation, determined by DAI and histological examination. Changes in the volatile metabolome persisted even after visual intestine repair and it confirms the high sensitivity of the microbiota to the damaging effects of DSS. The use of HS GC/MS may be an important addition to existing methods for assessing inflammation at early stages.

Incorporation of Pectin into Vaterite Microparticles Prevented Effects of Adsorbed Mucin on Neutrophil Activation.

The application of vaterite microparticles for mucosal delivery depends on their interaction with mucin and immune cells. As we have shown previously, the binding of mucin onto particles enhances the generation of reactive oxygen species by neutrophils. The attenuation of the pro-oxidant effect of the bound mucin through the modification of vaterite could improve its biocompatibility. Hybrid microparticles composed of vaterite and pectin (CCP) were prepared using co-precipitation. In comparison with vaterite (CC), they had a smaller diameter and pores, a greater surface area, and a negative zeta-potential. We aimed to study the cytotoxicity and mucin-dependent neutrophil-activating effect of CCP microparticles. The incorporated pectin did not influence the neutrophil damage according to a lactate dehydrogenase test. The difference in the CC- and CCP-elicited luminol or lucigenin chemiluminescence of neutrophils was insignificant, with no direct pro- or antioxidant effects from the incorporated pectin. Unlike soluble pectin, the CCP particles were ineffective at scavenging radicals in an ABAP-luminol test. The fluorescence of SYTOX Green demonstrated a CCP-stimulated formation of neutrophil extracellular traps (NETs). The pre-treatment of CC and CCP with mucin resulted in a 2.5-times-higher CL response of neutrophils to the CC-mucin than to the CCP-mucin. Thus, the incorporation of pectin into vaterite microspheres enabled an antioxidant effect to be reached when the neutrophils were activated by mucin-treated microparticles, presumably via exposed ligands.

Methylglyoxal-Modified Human Serum Albumin Binds to Leukocyte Myeloperoxidase and Inhibits its Enzymatic Activity.

Hyperglycemia in diabetes mellitus induces modification of proteins by glucose and its derivative methylglyoxal (MG). Neutrophils perform their bactericidal activity mainly via reactive halogen (RHS) and oxygen (ROS) species generation catalyzed by myeloperoxidase (MPO) stored in neutrophil azurophilic granules (AGs) and membrane NADPH oxidase, respectively. Herein, we study the binding of human serum albumin (HSA) modified with MG (HSA-MG) to MPO and its effects on MPO activity and release by neutrophils. Peroxidase activity of MPO was registered by oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and chlorinating activity by decolorization of Celestine blue B dye. Binding of HSA-MG to MPO was studied by affinity chromatography, disc-electrophoresis, ligand Western blotting and enzyme-linked solid phase immunoassay using monoclonal antibodies (mAbs) to MPO. ROS and RHS generation were detected by lucigenin (Luc) and luminol (Lum) chemiluminescence (CL), respectively. Neutrophil degranulation was assessed by flow cytometry using fluorescent labeled antibodies to the marker proteins CD63 from AGs and CD11b from peroxidase-negative granules (PNGs). NETosis was assayed by quantifying DNA network-like structures (NET-like structures) in blood smears stained by Romanowsky. HSA-MG bound to MPO, giving a stable complex (Kd = 1.5 nM) and competing with mAbs, and non-competitively inhibited peroxidase and chlorinating MPO activity and induced degranulation of PNGs but not of AGs. HSA-MG enhanced Luc-CL per se or following PMA, unlike Lum-CL, and did not affect spontaneous or PMA-stimulated NETosis. Thus, HSA modified under hyperglycemia-like conditions stimulated NADPH oxidase of neutrophils but dampened their functions dependent on activity of MPO, with no effect on its release via degranulation or NETosis. This phenomenon could underlie the downregulation of bactericidal activity of MPO and neutrophils, and hence of innate immunity, giving rise to wound healing impairment and susceptibility to infection in patients with hyperglycemia.

Crystallization of semiflexible polymers in melts and solutions.

We studied the crystallization of semiflexible polymer chains in melts and poor-solvent solutions with different concentrations using dissipative particle dynamics (DPD) computer simulation techniques. We used the coarse-grained polymer model to reveal the general principles and microscopic scenario of crystallization in such systems at large time and length scales. It covers both primary and secondary nucleation as well as crystallites' merging. The parameters of the DPD model were chosen appropriately to reproduce the entanglements of polymer chains. We started from an initial homogeneous disordered solution of Gaussian chains and observed the initial stages of crystallization process caused in our model by orientational ordering of polymer chains and polymer-solvent phase separation. We found that the overall crystalline fraction at the end of the crystallization process decreases with the increasing polymer volume fraction while the steady-state crystallization speed at later stages does not depend on the polymer volume fraction. The average crystallite size has a maximal value in the systems with a polymer volume fraction from 0.7 to 0.95. In our model, these polymer concentrations represent an optimal value in the sense of balance between the amount of polymer material available to increase the crystallite size and chain entanglements, that prevent crystallites' growth and merging. On large time scales, our model allows us to observe lamellar thickening linear in logarithmic time scale.

Additive-induced ordered structures formed by PC71BM fullerene derivatives.

We report the results of an experimental and theoretical study of structure formation in mixtures of phenyl-C71-butyric acid methyl ester (PC71BM) with high boiling octane based solvent additives 1,8-octanedithiol (ODT), 1,8-dibromooctane, and 1,8-diiodooctane obtained by evaporation of a host-solvent (chlorobenzene). Experimental studies by DSC, SAXS and WAXS methods found evidence of crystallization of fullerenes in the presence of the high boiling additives in the mixtures. A molecular dynamics simulation of a PC71BM/ODT mixture revealed the self-assembly of fullerenes into sponge-like network structures.

Self-Assembly of Lecithin and Bile Salt in the Presence of Inorganic Salt in Water: Mesoscale Computer Simulation.

The influence of inorganic salt on the structure of lecithin/bile salt mixtures in aqueous solution is studied by means of dissipative particle dynamics simulations. We propose a coarse-grained model of phosphatidylcholine and two types of bile salts (sodium cholate and sodium deoxycholate) and also take into account the presence of low molecular weight salt. This model allows us to study the system on rather large time and length scales (up to about ∼20 µs and 50 nm) and to reveal mechanisms of experimentally observed increasing viscosity upon increasing the low molecular weight salt concentration in this system. We show that increasing the low molecular weight salt concentration induces the growth of cylinder-like micelles formed in lecithin/bile salt mixtures in water. These wormlike micelles can entangle into transient networks displaying perceptible viscoelastic properties. Computer simulation results are in good qualitative agreement with experimental observations.

Effects of Alkali Cations and Halide Anions on the Self-Assembly of Phosphatidylcholine in Oils.

The interactions between ions and phospholipids are closely associated with the structures and functions of cell membrane. Instead of conventional aqueous systems, we systematically investigated the effects of inorganic ions on the self-assembly of lecithin, a zwitterionic phosphatidylcholine, in cyclohexane. Previous studies have shown that addition of inorganic salts with specific divalent and trivalent cations can transform lecithin organosols into organogels. In this study, we focused on the effect of monovalent alkali halides. Fourier transform infrared spectroscopy was used to demonstrate that the binding strength of the alkali cations with the phosphate of lecithin is in the order Li+ > Na+ > K+. More importantly, the cation-phosphate interaction is affected by the paired halide anions, and the effect follows the series I- > Br- > Cl-. The salts of stronger interactions with lecithin, including LiCl, LiBr, LiI, and NaI, were found to induce cylindrical micelles sufficiently long to form organogels, while others remain organosols. A mechanism based on the charge density of ions and the enthalpy change of the ion exchange between alkali halides and lecithin headgroup is provided to explain the contrasting interactions and the effectiveness of the salts to induce organogelation.

Diagram of states and morphologies of flexible-semiflexible copolymer chains: A Monte Carlo simulation.

A single copolymer chain consisting of multiple flexible (F) and semiflexible (S) blocks has been studied using a continuum bead-spring model by Stochastic Approximation Monte Carlo simulations, which determine the density of states of the model. The only difference between F and S blocks is the intramolecular bending potential, all non-bonded interactions are equal. The state diagrams for this class of models display multiple nematic phases in the collapsed state, characterized through a demixing of the blocks of different stiffness and orientational ordering of the stiff blocks. We observe dumbbell-like morphologies, lamellar phases, and for the larger block lengths also Saturn-like structures with a core of flexible segments and the stiff segments forming a ring around the core.

Fractal globules: a new approach to artificial molecular machines.

The over-damped relaxation of elastic networks constructed by contact maps of hierarchically folded fractal (crumpled) polymer globules was investigated in detail. It was found that the relaxation dynamics of an anisotropic fractal globule is very similar to the behavior of biological molecular machines like motor proteins. When it is perturbed, the system quickly relaxes to a low-dimensional manifold, M, with a large basin of attraction and then slowly approaches equilibrium, not escaping M. Taking these properties into account, it is suggested that fractal globules, even those made by synthetic polymers, are artificial molecular machines that can transform perturbations into directed quasimechanical motion along a defined path.

Molecular interactions between lecithin and bile salts/acids in oils and their effects on reverse micellization.

It has been known that the addition of bile salts to lecithin organosols induces the formation of reverse wormlike micelles and that the worms are similar to long polymer chains that entangle each other to form viscoelastic solutions. In this study, we further investigated the effects of different bile salts and bile acids on the growth of lecithin reverse worms in cyclohexane and n-decane. We utilized rheological and small-angle scattering techniques to analyze the properties and structures of the reverse micelles. All of the bile salts can transform the originally spherical lecithin reverse micelles into wormlike micelles and their rheological behaviors can be described by the single-relaxation-time Maxwell model. However, their efficiencies to induce the worms are different. In contrast, before phase separation, bile acids can induce only short cylindrical micelles that are not long enough to impart viscoelasticity. We used Fourier transform infrared spectroscopy to investigate the interactions between lecithin and bile salts/acids and found that different bile salts/acids employ different functional groups to form hydrogen bonds with lecithin. Such effects determine the relative positions of the bile salts/acids in the headgroups of lecithin, thus resulting in varying efficiencies to alter the effective critical packing parameter for the formation of wormlike micelles. This work highlights the importance of intermolecular interactions in molecular self-assembly.

Scattering study of the conformational structure and aggregation behavior of a conjugated polymer solution.

The conformational structure and the interchain aggregation behavior of a semirigid conjugated polymer bearing a decyl side chain, poly(2,3-diphenyl-5-decyl-1,4-phenylenevinylene) (DP10-PPV), in solutions with chloroform and toluene have been investigated by means of small-angle neutron scattering (SANS), static light scattering (SLS) and dynamic light scattering (DLS). The radius of gyration, persistence length, and the second virial coefficient of the polymer in dilute solution as determined by SLS were higher in chloroform than in toluene; consequently, the polymer assumed a more extended wormlike chain conformation in the former. The difference in the strength of interaction in the two solvents gave rise to contrasting aggregation behavior of the polymer in the semidilute regime. While only a minor fraction of the polymer underwent segmental association in chloroform, a considerable fraction of it formed clusters (microgels) with several micrometers in size in toluene. These clusters were further found to consist of sheetlike nanodomains. Compared with the DP-PPV bearing a shorter hexyl side chain, DP6-PPV, the aggregates of DP10-PPV in toluene were weaker as they could be easily disrupted by moderate heating. This was attributed to a lack of strong pi-pi interaction between the DP10-PPV segments due to the greater steric hindrance imposed by the longer decyl side chains.

Columnar mesophases of the complexes of DNA with low-generation poly(amido amine) dendrimers.

The electrostatic complexes of polyanionic DNA with dendrimers have been considered as a class of nonviral vector for gene therapy. The gene transfection efficiency has been believed to be influenced by the structure of the complex. In this study, we have systematically characterized the supramolecular structures of the complexes of DNA duplexes with poly(amido amine) (PAMAM) dendrimers with generation two (G2) and three (G3) in pure water using small-angle X-ray scattering. The structures were examined as a function of the charge density of the dendrimer expressed by its degree of protonation (dp) and the molar ratio of the amine groups of dendrimer to the phosphate groups of DNA (N/P). The DNA chains in all complexes under study were found to self-organize into two-dimensional hexagonal or square lattice. In general, hexagonal phase was the favorable structure for G2 complexes, while the DNA in G3 complexes tended to organize into a square lattice. Interesting transitions between the columnar mesophases with respect to the changes of N/P ratio and dp have been identified. The geometric features of the dendrimer molecules accommodated within the interstitial tunnels of the DNA lattices have also been revealed. The B conformation of DNA was effectively retained in the complexes in spite of the influence of the electrostatic interaction with the dendrimers.

Aggregation of conjugated polymers in aromatic solvent.

Segments of conjugated polymers display the propensity to aggregate in solutions with common organic solvents. Here we revealed that the segmental aggregation of a conjugated polymer, poly(9,9-bis(2-ethylhexyl)fluorene-2,7-diyl), (PF2/6), in toluene was stabilized by the polymer-solvent complex formation through pi-pi stacking induction of solvent molecules and polymer segments. In this case, a portion of the solvent was trapped inside the aggregate domains upon bringing the system to the subambient temperatures. The residence time of these associated solvent molecules became long enough to yield a separate upfield-shifted NMR resonance. The line-shape of this resonance revealed alignment of the polymer segments in the aggregates. A portion of the solvent was frozen in the compact structure due to the formation of strong polymer-solvent complex.

Stability of toroid and rodlike globular structures of a single stiff-chain macromolecule for different bending potentials.

We study the effect of the bending potential on the stability of toroidal and rodlike globules which are typical collapsed conformations of a single stiff-chain macromolecule. We perform numerical calculations in the framework of the bead-stick model of a polymer chain. The intrinsic structure of globules is also analyzed. It was shown that the bending potential affects the packing geometry of bundles in a toroidal globule in the ground state. This potential also influences the bends at the ends of a rodlike globule: both the shape of the loops and the number of bonds in each loop have been investigated numerically as well as by Monte Carlo computer simulations performed for a separate loop. Our main results are (1) the shape of the bending potential could be possibly seen from the geometry of a globule; (2) toroidal globules are always more favorable than the rodlike ones.