The architecture of the Gram-positive bacterial cell wall.

The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics1,2. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor1,3. In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength4-6. Here we applied atomic force microscopy7-12 to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface13,14, providing information complementary to traditional structural biology approaches.

High-speed large area atomic force microscopy using a quartz resonator.

A high-speed atomic force microscope for scanning large areas, utilizing a quartz bar driven close to resonance to provide the motion in the fast scan axis is presented. Images up to 170 × 170 µm2 have been obtained on a polydimethylsiloxane (PDMS) grating in 1 s. This is provided through an average tip-sample velocity of 28 cm s-1 at a line rate of 830 Hz. Scan areas up to 80 × 80 µm2 have been obtained in 0.42 s with a line rate of 1410 Hz. To demonstrate the capability of the scanner the spherulitic crystallization of a semicrystalline polymer was imaged in situ at high speed.

Immobilizing live bacteria for AFM imaging of cellular processes.

Coccoid cells of the bacterial species Staphylococcus aureus have been mechanically trapped in lithographically patterned substrates and imaged under growth media using atomic force microscopy (AFM) in order to follow cellular processes. The cells are not perturbed as there is no chemical linkage to the surface. Confinement effects are minimized compared to trapping the cells in porous membranes or soft gels. S. aureus cells have been imaged undergoing cell division whilst trapped in the patterned substrates. Entrapment in lithographically patterned substrates provides a novel way for anchoring bacterial cells so that the AFM tip will not push the cells off during imaging, whilst allowing the bacteria to continue with cellular processes.

Dietary fat type influences protein consumption in rats given 8-hydroxy-2-(Di-n-propylamino)tetralin.

Our previous work indicates that type and level of dietary fat influences selection of protein and carbohydrate diets. Serotonin (5HT) appears to be involved in this feeding behavior. In the present study, we examined the effects of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) on feeding behavior, and fenfluramine on in vitro 5HT and 5-hydroxyindoleacetic acid (5HIAA) release from the brain in rats fed tallow or corn oil. Male, Sprague-Dawley rats were given a diet containing corn oil or tallow for 2 days. In Experiment 1, rats received an injection of 8-OH-DPAT (a 5HT1A agonist) or saline and were then allowed to select from two diets: low protein/high carbohydrate or high protein/low carbohydrate. Prior exposure to tallow caused an increased intake of protein and 8-OH-DPAT blunted this effect. In Experiment 2, the dorsal raphe from rats fed tallow or corn oil was superfused with fenfluramine or vehicle. Superfusates were collected for analysis of 5HT and 5HIAA. Fenfluramine increased serotonin release in tallow-fed animals as compared to basal. These results suggest that serotonin may be involved in mediating tallow's effect on macronutrient selection.

Large scan area high-speed atomic force microscopy using a resonant scanner.

A large scan area high-speed scan stage for atomic force microscopy using the resonant oscillation of a quartz bar has been constructed. The sample scanner can be used for high-speed imaging in both air and liquid environments. The well-defined time-position response of the scan stage due to the use of resonance allows highly linearized images to be obtained with a scan size up to 37.5 mum in 0.7 s. The scanner is demonstrated for imaging highly topographic silicon test samples and a semicrystalline polymer undergoing crystallization in air, while images of a polymer and a living bacteria, S. aureus, are obtained in liquid.

In situ annealing and thickening of single crystals of C294H590 observed by atomic force microscopy.

The annealing behavior of twice-folded crystals of the long-chain alkane C294H590 is examined in situ, in real time, by atomic force microscopy (AFM). AFM is capable of following processes in real time provided that the time scale is sufficiently long for several images to be collected during the process. In this paper, we focus on the temperature dependence and the thickened morphology. We are able to investigate where the thickening starts and how this depends on temperature and how melting is influenced by morphology. By following the motion of holes within the crystal, a lower limit for the rate of diffusion of crystalline polyethylene is estimated. We also focus on the substrate effect on the crystal morphology and thickening, using mica, glass, and graphite.

Effects of a fat substitute, sucrose polyester, on food intake, body composition, and serum factors in lean and obese Zucker rats.

Sucrose polyester, a fat substitute, has shown promise in reducing blood cholesterol and body weight of obese individuals. Effects of this compound in the Zucker rat, a genetic model of obesity, are unknown. Thus, we examined food intake, body weight, body composition, and several metabolic parameters in sera of lean and obese female Zucker rats. Eight-week-old lean and obese animals were given a choice between a control diet (15% corn oil) and fat substitute diet (5% corn oil and 10% sucrose polyester) for 2 days. Next, one-half of the lean and obese groups received control diet; the remaining lean and obese rats received fat substitute diet for 18 days. Cumulative food intake was depressed in fat substitute groups relative to control-fed animals; however, this effect was more predominant in obese animals. Obese rats consuming fat substitute diet (O-FS) gained less weight as compared to obese control-fed animals (O-C). Lean rats given fat substitute (L-FS) did not have significantly different body weights as compared to the L-C group. Fat substitute groups, combined, had lower body fat and higher body water as compared to controls. The O-FS group had lower serum glucose and insulin and higher fatty acid levels compared to the O-C group. There were no differences in serum cholesterol, HDL, or triglyceride levels due to fat substitute diet. These data suggest that the obese Zucker rat is unable to defend its body weight when dietary fat is replaced with sucrose polyester.