Stability of ß-lactam antibiotics in bacterial growth media.

Laboratory assays such as MIC tests assume that antibiotic molecules are stable in the chosen growth medium-but rapid degradation has been observed for antibiotics including ß-lactams under some conditions in aqueous solution. Degradation rates in bacterial growth medium are less well known. Here, we develop a 'delay time bioassay' that provides a simple way to estimate antibiotic stability in bacterial growth media, using only a plate reader and without the need to measure the antibiotic concentration directly. We use the bioassay to measure degradation half-lives of the ß-lactam antibiotics mecillinam, aztreonam and cefotaxime in widely-used bacterial growth media based on MOPS and Luria-Bertani (LB) broth. We find that mecillinam degradation can occur rapidly, with a half-life as short as 2 hours in MOPS medium at 37°C and pH 7.4, and 4-5 hours in LB, but that adjusting the pH and temperature can increase its stability to a half-life around 6 hours without excessively perturbing growth. Aztreonam and cefotaxime were found to have half-lives longer than 6 hours in MOPS medium at 37°C and pH 7.4, but still shorter than the timescale of a typical minimum inhibitory concentration (MIC) assay. Taken together, our results suggest that care is needed in interpreting MIC tests and other laboratory growth assays for ß-lactam antibiotics, since there may be significant degradation of the antibiotic during the assay.

Molecular Interactions behind the Self-Assembly and Microstructure of Mixed Sterol Organogels.

In this work, we have employed docking and atomistic molecular dynamics (MD) simulations supported by complementary experiments using atomic force microscopy, rheology, and spectroscopy to investigate the self-assembled structure of ß-sitosterol and γ-oryzanol molecules into cylindrical tubules in a nonaqueous solvent. Docking models of several phytosterols, including sitosterol, with oryzanol and other sterol esters demonstrate that for systems to form tubules, the phytosterol sterane group must be stacked in a wedge shape with the ester sterane group and a hydrogen bond must form between the hydroxyl group of the phytosterol and the carbonyl group of the ester. MD of the self-assembled structure were initiated with the molecules in a roughly cylindrical configuration, as suggested from previous experimental studies, and the configurations were found to be stable during 50 ns simulations. We performed MD simulations of two tubules in proximity to better understand the aggregation of these fibrils and how the fibrils interact in order to stick together. We found that an interfibril network of noncovalent bonds, in particular van der Waals and π-π contacts, which is formed between the ferulic acid groups of oryzanol through the hydroxyl, methoxy, and aromatic groups, is responsible for the surface-to-surface interactions between fibrils; an observation supported by molecular spectroscopy. We believe that these interactions are of primary importance in creating a strong organogel network.

A multipurpose modular system for high-resolution microscopy at high hydrostatic pressure.

We have developed a modular system for high-resolution microscopy at high hydrostatic pressure. The system consists of a pressurized cell of volume approximately 100 microl, a temperature controlled holder, a ram, and a piston. We have made each of these components in several versions which can be interchanged to allow a wide range of applications. Here, we report two pressure cells with pressure ranges 0.1-700 MPa and 0.1-100 MPa, which can be combined with hollow or solid rams and pistons. Our system is designed to work with fluorescent samples (using a confocal or epifluorescence microscope), but also allows for transmitted light microscopy via the hollow ram and piston. The system allows precise control of pressure and temperature (-20 to 70 degrees C), as well as rapid pressure quenching. We demonstrate its performance and versatility with two applications: time-resolved imaging of colloidal phase transitions caused by pressure changes between 0.1 and 100 MPa, and imaging the growth of Escherichia coli bacteria at 50 MPa. We also show that the isotropic-nematic phase transition of pentyl-cyanobiphenyl (5CB) liquid crystal provides a simple, convenient, and accurate method for calibrating pressure in the range 0.1-200 MPa.

Probing the liquid-state structure and dynamics of aqueous solutions by fluorescence spectroscopy.

Time-resolved fluorescence spectroscopy of the solvent-sensitive molecule 1,8-anilinonaphthalene sulfonate (ANS) is used to probe the structure and dynamics of an aqueous methanol solution (mole fraction = 0.5). The intensity decay of ANS in the mixed solvent displays single exponential kinetics under ambient conditions. At low temperature, a simple two-state solvent relaxation model describes the fluorescence decay for ANS in both methanol and the mixed solvent. The temperature dependence of ANS fluorescence in the mixed solvent is attributed to the onset of glassy dynamics in the aqueous component at higher temperature, implying a partial demixing of the water and methanol due to self-association. We discuss the absence of more complicated fluorescence decays in such a heterogeneous solvent system.