Improved Force Spectroscopy Using Focused-Ion-Beam-Modified Cantilevers.

Atomic force microscopy (AFM) is widely used in biophysics, including force-spectroscopy studies of protein folding and protein-ligand interactions. The precision of such studies increases with improvements in the underlying quality of the data. Currently, data quality is limited by the mechanical properties of the cantilever when using a modern commercial AFM. The key tradeoff is force stability vs short-term force precision and temporal resolution. Here, we present a method that avoids this compromise: efficient focused-ion-beam (FIB) modification of commercially available cantilevers. Force precision is improved by reducing the cantilever's hydrodynamic drag, and force stability is improved by reducing the cantilever stiffness and by retaining a cantilever's gold coating only at its free end. When applied to a commonly used short cantilever (L=40µm), we achieved sub-pN force precision over 5 decades of bandwidth (0.01-1000Hz) without significantly sacrificing temporal resolution (~75µs). Extending FIB modification to an ultrashort cantilever (L=9µm) also improved force precision and stability, while maintaining 1-µs-scale temporal resolution. Moreover, modifying ultrashort cantilevers also eliminated their inherent underdamped high-frequency motion and thereby avoided applying a rapidly oscillating force across the stretched molecule. Importantly, fabrication of FIB-modified cantilevers is accessible after an initial investment in training. Indeed, undergraduate researchers routinely modify 2-4 cantilevers per hour with the protocol detailed here. Furthermore, this protocol offers the individual user the ability to optimize a cantilever for a particular application. Hence, we expect FIB-modified cantilevers to improve AFM-based studies over broad areas of biophysical research.

Helicobacter pylori eradication in Western Australia using novel quadruple therapy combinations.

BACKGROUND: Helicobacter pylori eradication rates with standard triple therapy are declining worldwide. The optimal management of H. pylori is evolving and new treatment combinations for antibiotic resistant H. pylori strains are required, especially for patients with penicillin allergy. AIM: To review the effectiveness of alternative antibiotic combinations and necessity of pre-antibiotic sensitivity testing. METHODS: A total of 310 consecutive patients who had failed at least one course of standard 7-day triple therapy initially prescribed by their physicians were included in this study between year 2007 and 2011. Antibiotics were prescribed based on pre-antibiotic sensitivity tests and, if any, patient's allergy to penicillin. RESULTS: In 98.7% of the patients' samples, H. pylori was successfully cultured. The proportion resistant to clarithromycin and metronidazole was 94.1% and 67.6% respectively, with 65% resistant to both. For the in-house primary quadruple therapy, with Proton pump inhibitor, Amoxicillin, Rifabutin and Ciprofloxacin (PARC), H. pylori was successfully eradicated in 95.2% of patients. For patients allergic to amoxicillin, an alternative quadruple therapy using Proton pump inhibitor, Bismuth subcitrate, Rifabutin and Ciprofloxacin (PBRC) gave an eradication rate of 94.2%. Patients needing alternative salvage therapy were given novel personalised combinations consisting of bismuth, rifabutin, tetracycline or furazolidone; the eradication rate was 73.8%. CONCLUSIONS: Patients who present with antibiotic resistant H. pylori can be confidently treated with PARC, PBRC or other personalised salvage therapies. These regimens can be used when treatment options are limited by penicillin allergy. Pre-treatment H. pylori antibiotic sensitivity tests contributed to the high eradication rate in this study.

Single polymer dynamics in an elongational flow.

The stretching of individual polymers in a spatially homogeneous velocity gradient was observed through use of fluorescently labeled DNA molecules. The probability distribution of molecular extension was determined as a function of time and strain rate. Although some molecules reached steady state, the average extension did not, even after a approximately 300-fold distortion of the underlying fluid element. At the highest strain rates, distinct conformational shapes with differing dynamics were observed. There was considerable variation in the onset of stretching, and chains with a dumbbell shape stretched more rapidly than folded ones. As the strain rate was increased, chains did not deform with the fluid element. The steady-state extension can be described by a model consisting of two beads connected by a spring representing the entropic elasticity of a worm-like chain, but the average dynamics cannot.

Stretching of a single tethered polymer in a uniform flow.

The stretching of single, tethered DNA molecules by a flow was directly visualized with fluorescence microscopy. Molecules ranging in length (L) from 22 to 84 micrometers were held stationary against the flow by the optical trapping of a latex microsphere attached to one end. The fractional extension x/L is a universal function of eta vL 0.54 +/- 0.05, where eta and v are the viscosity and velocity of the flow, respectively. This relation shows that the DNA is not "free-draining" (that is, hydrodynamic coupling within the chain is not negligible) even near full extension (approximately 80 percent). This function has the same form over a long range as the fractional extension versus force applied at the ends of a worm-like chain. For small deformations (< 30 percent of full extension), the extension increases with velocity as x approximately v0.70 +/- 0.08. The relative size of fluctuations in extension decreases as sigma x/x approximately equal to 0.42 exp (-4.9 x/L). Video images of the fluctuating chain have a cone-like envelope and show a sharp increase in intensity at the free end.

Relaxation of a single DNA molecule observed by optical microscopy.

Single molecules of DNA, visualized in video fluorescence microscopy, were stretched to full extension in a flow, and their relaxation was measured when the flow stopped. The molecules, attached by one end to a 1-micrometer bead, were manipulated in an aqueous solution with optical tweezers. Inverse Laplace transformations of the relaxation data yielded spectra of decaying exponentials with distinct peaks, and the longest time component (tau) increased with length (L) as tau approximately L 1.68 +/- 0.10. A rescaling analysis showed that most of the relaxation curves had a universal shape and their characteristic times (lambda t) increased as lambda t approximately L 1.65 +/- 0.13. These results are in qualitative agreement with the theoretical prediction of dynamical scaling.

Direct observation of tube-like motion of a single polymer chain.

Tube-like motion of a single, fluorescently labeled molecule of DNA in an entangled solution of unlabeled lambda-phage DNA molecules was observed by fluorescence microscopy. One end of a 16- to 100-micrometer-long DNA was attached to a 1-micrometer bead and moved with optical tweezers. The molecule was stretched into various conformations having bends, kinks, and loops. As the polymer relaxed, it closely followed a path defined by its initial contour. The relaxation time of the disturbance caused by the bead was roughly 1 second, whereas tube-like motion in small loops persisted for longer than 2 minutes. Tube deformation, constraint release, and excess chain segment diffusion were also observed. These observations provide direct evidence for several key assumptions in the reptation model developed by de Gennes, Edwards, and Doi.