Single particle tracking has seen numerous applications in biophysics, ranging from the diffusion of proteins in cell membranes to the movement of molecular motors. A plethora of computer algorithms have been developed to monitor the sub-pixel displacement of fluorescent objects between successive video frames, and some have been claimed to have "nanometer" resolution. To date, there has been no rigorous comparison of these algorithms under realistic conditions. In this paper, we quantitatively compare specific implementations of four commonly used tracking algorithms: cross-correlation, sum-absolute difference, centroid, and direct Gaussian fit. Images of fluorescent objects ranging in size from point sources to 5 microm were computer generated with known sub-pixel displacements. Realistic noise was added and the above four algorithms were compared for accuracy and precision. We found that cross-correlation is the most accurate algorithm for large particles. However, for point sources, direct Gaussian fit to the intensity distribution is the superior algorithm in terms of both accuracy and precision, and is the most robust at low signal-to-noise. Most significantly, all four algorithms fail as the signal-to-noise ratio approaches 4. We judge direct Gaussian fit to be the best algorithm when tracking single fluorophores, where the signal-to-noise is frequently near 4.

Muscle contraction and many other cell movements are driven by cyclic interactions between actin filaments and the motor enzyme myosin. Conformational changes in the actin-myosin binding interface occur in concert with the binding of ATP, binding to actin, and loss of hydrolytic by-products, but the effects of these conformational changes on the strength of the actomyosin bond are unknown. The force-dependent kinetics of the actomyosin bond may be particularly important at high loads, where myosin may detach from actin before achieving its full power stroke. Here we show that over a physiological range of rapidly applied loads, actomyosin behaves as a ''catch'' bond, characterized by increasing lifetimes with increasing loads up to a maximum at Ϸ6 pN. Surprisingly, we found that the myosin-ADP bond is possessed of longer lifetimes under load than rigor bonds, although the load at which bond lifetime is maximal remains unchanged. We also found that actomyosin bond lifetime is ultimately dependent not only on load, but loading history as well. These data suggest a complex relationship between the rate of actomyosin dissociation and muscle force and shortening velocity. The 6-pN load for maximum bond lifetime is near the force generated by a single myosin molecule during isometric contraction. This raises the possibility that all catch bonds between load-bearing molecules are ''mechanokinetically'' tuned to their physiological environment. catch bonds ͉ dynamic force spectroscopy ͉ laser trap ͉ myosin T he crystal structure of the myosin head (S1) (1) reveals how small-scale conformational changes within the hydrolytic site are converted into relatively large-scale changes producing movement. A globular ''motor domain'' occupies the bulk of the structure and contains both the nucleotide-and actin-binding sites (see Fig. 1). Of particular note is an ␣-helical extension of the heavy chain, or ''neck,'' protruding from the globular motor domain that acts as a rigid ''lever arm'' to amplify small movements arising in the nucleotide-binding pocket (2-5). Concomitant with phosphate release, rotation of the neck causes a step of an Ϸ5.5-nm ''working stroke'' and an isometric force of 0.7-9 pN (3, 6-10) that has been the subject of numerous single-molecule mechanics studies.Another feature of particular note within the motor domain is a cleft that divides the actin-binding site (2). This cleft is thought to close upon binding to actin (1, 2, 11-13), bringing into position residues on both sides of the cleft that are involved in strong binding to actin: the so-called R-site and A-site. Evidence suggests that there may be additional conformational changes in the actin-binding interface that accompany ADP release. The cleft at the actin-binding interface of smooth muscle myosin may close further upon ADP release (14, 15) and is accompanied by an increase in R-site flexibility (14, 16). Thus, although both the R-site and A-site are tightly bound to actin in the presence of ADP, upon ADP release from smooth muscle myosin the R-...

Purified smooth muscle myosin in the in vitro motility assay propels actin filaments at 1/10 the velocity, yet produces 3-4 times more force than skeletal muscle myosin. At the level of a single myosin molecule, these differences in force and actin filament velocity may be reflected in the size and duration of single motion and force-generating events, or in the kinetics of the cross-bridge cycle. Specifically, an increase in either unitary force or duty cycle may explain the enhanced force-generating capacity of smooth muscle myosin. Similarly, an increase in attached time or decrease in unitary displacement may explain the reduced actin filament velocity of smooth muscle myosin. To discriminate between these possibilities, we used a laser trap to measure unitary forces and displacements from single smooth and skeletal muscle myosin molecules. We analyzed our data using mean-variance analysis, which does not rely on scoring individual events by eye, and emphasizes periods in the data with constant properties. Both myosins demonstrated multiple but similar event populations with discrete peaks at approximately +11 and -11 nm in displacement, and 1.5 and 3.5 pN in force. Mean attached times for smooth muscle myosin were longer than for skeletal-muscle myosin. These results explain much of the difference in actin filament velocity between these myosins, and suggest that an increased duty cycle is responsible for the enhanced force-generating capacity of smooth over skeletal-muscle myosin.

The anti-inflammatory eicosanoid lipoxin A4 (LXA4), aspirin-triggered 15-epi-LXA4, and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal epithelia. In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays. LXA4 analog alone did not significantly affect expression of any of the >7000 genes analyzed. However, LXA4 analog pretreatment attenuated induction of ∼50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium. A major subset of genes whose induction was reduced by LXA4 analog pretreatment is regulated by NF-κB, suggesting that LXA4 analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA4 analog reduced NF-κB-mediated transcriptional activation in a LXA4 receptor-dependent manner and inhibited induced degradation of IκBα. LXA4 analog did not affect earlier stimulus-induced signaling events that lead to IκBα degradation, such as S. typhimurium-induced epithelial Ca2+ mobilization or TNF-α-induced phosphorylation of IκBα. To establish the in vivo relevance of these findings, we examined whether LXA4 analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis. Oral administration of LXA4 analog (15-epi-16-para-fluoro-phenoxy-LXA4, 10 μg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis. Thus, LXA4 analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-κB pathway can be therapeutic for diseases characterized by mucosal inflammation.

There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients.