Rama Bansil

Helicobacter pylori moves through mucus by reducing mucin viscoelasticity

The ulcer-causing gastric pathogen Helicobacter pylori is the only bacterium known to colonize the harsh acidic environment of the human stomach. H. pylori survives in acidic conditions by producing urease, which catalyzes hydrolysis of urea to yield ammonia thus elevating the pH of its environment. However, the manner in which H. pylori is able to swim through the viscoelastic mucus gel that coats the stomach wall remains poorly understood. Previous rheology studies on gastric mucin, the key viscoelastic component of gastric mucus, indicate that the rheology of this material is pH dependent, transitioning from a viscous solution at neutral pH to a gel in acidic conditions. Bulk rheology measurements on porcine gastric mucin (PGM) show that pH elevation by H. pylori induces a dramatic decrease in viscoelastic moduli. Microscopy studies of the motility of H. pylori in gastric mucin at acidic and neutral pH in the absence of urea show that the bacteria swim freely at high pH, and are strongly constrained at low pH. By using two-photon fluorescence microscopy to image the bacterial motility in an initially low pH mucin gel with urea present we show that the gain of translational motility by bacteria is directly correlated with a rise in pH indicated by 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH sensitive fluorescent dye. This study indicates that the helicoidal-shaped H. pylori does not bore its way through the mucus gel like a screw through a cork as has previously been suggested, but instead achieves motility by altering the rheological properties of its environment.

pH-dependent conformational change of gastric mucin leads to sol-gel transition.

We present dynamic light scattering (DLS) and hydrophobic dye-binding data in an effort to elucidate a molecular mechanism for the ability of gastric mucin to form a gel at low pH, which is crucial to the barrier function of gastric mucus. DLS measurements of dilute mucin solutions were not indicative of intermolecular association, yet there was a steady fall in the measured diffusion coefficient with decreasing pH, suggesting an apparent increase in size. Taken together with the observed rise in depolarized scattering ratio with decreasing pH, these results suggest that gastric mucin undergoes a conformational change from a random coil at pH >/= 4 to an anisotropic, extended conformation at pH < 4. The increased binding of mucin to hydrophobic fluorescent with decreasing pH indicates that the change to an extended conformation is accompanied by exposure of hydrophobic binding sites. In concentrated mucin solutions, the structure factor S(q, t) derived from DLS measurements changed from a stretched exponential decay at pH 7 to a power-law decay at pH 2, which is characteristic of a sol-gel transition. We propose that the conformational change facilitates cross-links among mucin macromolecules through hydrophobic interactions at low pH, which in turn leads to a sol-gel transition when the mucin solution is sufficiently concentrated.

Low‐frequency Raman spectrum of supercooled water

We report measurements of the Raman spectrum of supercooled water in the hindered translational region (20–400 cm−1) down to a temperature of −20 °C. The spectra are analyzed after correcting for the effects of Boltzmann factor and harmonic oscillator coupling, i.e., in the reduced R(ν) representation of Shuker and Gammon. Spectral deconvolution shows that in addition to the previously observed 0‐0‐0 bending mode (≂60 cm−1) and the 0‐0 stretching mode (≂190 cm−1), there is a weak feature at 260 cm−1 whose intensity increases by almost an order of magnitude as temperature decreases from 40 to −20 °C. A plausible interpretation of the 260 cm−1 band is that it is analogous to the 310 cm−1 band seen in ice I and probably arises because of differing electrostatic interactions in different configurations of coupled H bonds of neighboring H2O molecules. The 0‐0 stretching band at 190 cm−1 changes in many respects as temperature decreases from 40 to −20 °C: (i) Its peak intensity increases almost four times; (ii...

Confocal light absorption and scattering spectroscopic microscopy

We have developed a novel optical method for observing submicrometer intracellular structures in living cells, which is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. It combines confocal microscopy, a well-established high-resolution microscopic technique, with light-scattering spectroscopy. CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales of the order of 100 nm.

The Influence of Mucus Microstructure and Rheology in Helicobacter pylori Infection

The bacterium Helicobacter pylori (H. pylori), has evolved to survive in the highly acidic environment of the stomach and colonize on the epithelial surface of the gastric mucosa. Its pathogenic effects are well known to cause gastritis, peptic ulcers, and gastric cancer. In order to infect the stomach and establish colonies on the mucus epithelial surface, the bacterium has to move across the gel-like gastric mucus lining of the stomach under acidic conditions. In this review we address the question of how the bacterium gets past the protective mucus barrier from a biophysical perspective. We begin by reviewing the molecular structure of gastric mucin and discuss the current state of understanding concerning mucin polymerization and low pH induced gelation. We then focus on the viscoelasticity of mucin in view of its relevance to the transport of particles and bacteria across mucus, the key first step in H. pylori infection. The second part of the review focuses on the motility of H. pylori in mucin solutions and gels, and how infection with H. pylori in turn impacts the viscoelastic properties of mucin. We present recent microscopic results tracking the motion of H. pylori in mucin solutions and gels. We then discuss how the biochemical strategy of urea hydrolysis required for survival in the acid is also relevant to the mechanism that enables flagella-driven swimming across the mucus gel layer. Other aspects of the influence of H. pylori infection such as, altering gastric mucin expression, its rate of production and its composition, and the influence of mucin on factors controlling H. pylori virulence and proliferation are briefly discussed with references to relevant literature.

Effect of varying crosslinking density on polyacrylamide gels

Abstract The copolymerization of acrylamide and N, N′-methylene bisacrylamide has been investigated over a wide range of total monomer concentration (T) and with the proportion of the crosslinking monomer (C) varying from 0 to 100%. The threshold value of the total monomer concentration above which gel formation becomes possible decreases as C increases. Gels with high C (>4%) become turbid and eventually opaque. An analogous phase separation is observed in the sol phase, where actual precipitation of a branched polymer takes place. The gelation curve continues into the phase separated region. Raman spectroscopy of gels with different values of C indicates that the opacity is related to the formation of cluster of varying sizes, with varying amounts of the monomers. The spectral data also shows that the BIS molecules may aggregate and that some unreacted vinyl groups of the BIS molecules are incorporated into the polymer network.

Effects of gelation on spinodal decomposition kinetics in gelatin

Abstract We have studied the kinetics of spinodal decomposition in a gelatin-water-methanol mixture, which undergoes a sol-gel transition simultaneously with phase separation, using light scattering techniques. The kinetics of the sol-gel transition was investigated using falling-ball microviscometry techniques. We found that the kinetics of phase separation depend very strongly on temperature; and for deep quenches, where the rates of phase separation and gelation are comparable, the phase-separation process does not go to completion. Prior to the onset of gelation the initial growth rate of the structure factor can be described according to the Cahn-Hilliard model, even though the wavevector at maximum scattered intensity decreases as k m ∼ t −0.6 . For deep quenches after the onset of gelation, k m remains unchanged with time, while the peak intensity exhibits a slow growth. The final domain size, k −1 final , at which the phase-separating morphology becomes pinned decreases with increasing quench depth. Micrographs of the phase-separated domains reveal a connected structure on length scales of a hundred micrometres superimposed on a finer network structure due to gelation.

Raman spectroscopy of supercooled water

We report Raman spectroscopic measurements on supercooled water down to a temperature of −20 °C. The spectral region investigated is the uncoupled OD stretching vibration (2000–3000 cm−1) from a 5% solution of D2O in H2O. The results are consistent with an interpretation in terms of two categories of OD bonds, those that are hydrogen bonded and those that are essentially free. The fraction of H‐bonded OD bonds varies from 0.9 to 0.95 as the temperature decreases from 0 to −20 °C. We observe that the frequency of the bonded OD component decreases with temperature at a slightly slower rate in the supercooled region than in the normal liquid region. With decreasing temperature, a decrease in width of this component band is observed, and this decrease appears to have a much larger slope in the supercooled region as compared to that observed previously in the normal temperature region.

Swelling equilibria of ionized poly(methacrylic acid) gels in the absence of salt

Abstract The kinetic theory of rubber elasticity for polyelectrolytes has been modified by including an effect of the electrostatic persistence length. The relations for swelling equilibria as a function of the degree of neutralization in polyelectrolyte gels are derived. We find that the volume fraction of the polymer in the swollen gel does not decrease monotonically with increasing α, but instead exhibits a minimum near α ∼ 0.1. Beyond this value of α the extent of swelling decreases with increasing degree of neutralization. The theory has been verified by measurements of the swelling behaviour of weakly crosslinked polymethacrylic acid gels as a function of varying degree of neutralization.

Intermolecular coupling in HOD solutions

Intermolecular coupling of OD oscillators from HOD in H2O has been investigated by Raman spectroscopy. The data indicate that at concentrations of HOD greater than 10 mol % the effects of intermolecular coupling of OD ⋅⋅⋅ OD pairs become noticeable. Difference spectra show a characteristic derivativelike feature with an increase of intensity around 2400 cm−1 as intermolecular coupling increases. The peak frequency of the OD stretching vibration in HOD decreases from 2525 cm−1 at infinite dilution to 2500 cm−1 in 50 mol % HOD, while the width of the OD stretching band increases from 150 cm−1 (infinite dilution) to 178 cm−1 (50 mol % HOD). Depolarization measurement indicates that the band at ∼2500 cm−1 is polarized. By comparing these difference spectra with the spectrum of OD oscillators from D2O we suggest that the major features of the D2O spectrum in the liquid state can be obtained by considering intermolecular coupling of OD oscillators.