High-resolution fleezers reveal duplex opening and stepwise assembly by an oligomer of the DEAD-box helicase Ded1p.

DEAD-box RNA helicases are ubiquitous in all domains of life where they bind and remodel RNA and RNA-protein complexes. DEAD-box helicases unwind RNA duplexes by local opening of helical regions without directional movement through the duplexes and some of these enzymes, including Ded1p from Saccharomyces cerevisiae, oligomerize to effectively unwind RNA duplexes. Whether and how DEAD-box helicases coordinate oligomerization and unwinding is not known and it is unclear how many base pairs are actively opened.

An accurate probabilistic step finder for time-series analysis.

Noisy time-series data-from various experiments, including Förster resonance energy transfer, patch clamp, and force spectroscopy, among others-are commonly analyzed with either hidden Markov models or step-finding algorithms, both of which detect discrete transitions. Hidden Markov models, including their extensions to infinite state spaces, inherently assume exponential-or technically geometric-holding time distributions, biasing step locations toward steps with geometric holding times, especially in sparse and/or noisy data. In contrast, existing step-finding algorithms, while free of this restraint, often rely on ad hoc metrics to penalize steps recovered in time traces (by using various information criteria) and otherwise rely on approximate greedy algorithms to identify putative global optima.

An accurate probabilistic step finder for time-series analysis.

Noisy time-series data is commonly collected from sources including Förster Resonance Energy Transfer experiments, patch clamp and force spectroscopy setups, among many others. Two of the most common paradigms for the detection of discrete transitions in such time-series data include: hidden Markov models (HMMs) and step-finding algorithms. HMMs, including their extensions to infinite state-spaces, inherently assume in analysis that holding times in discrete states visited are geometrically-or, loosely speaking in common language, exponentially-distributed.

Few-body hydrodynamic interactions probed by optical trap pulling experiment.

We study the hydrodynamic coupling of neighboring micro-beads placed in a multiple optical trap setup allowing us to precisely control the degree of coupling and directly measure time-dependent trajectories of entrained beads. We performed measurements on configurations with increasing complexity starting with a pair of entrained beads moving in one dimension, then in two dimensions, and finally a triplet of beads moving in two dimensions. The average experimental trajectories of a probe bead compare well with the theoretical computation, illustrating the role of viscous coupling and setting timescales for probe bead relaxation.

High-Resolution Optical Tweezers Combined with Multicolor Single-Molecule Microscopy.

We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy. Biological macromolecules exhibit complex conformation and stoichiometry changes in coordination with their motion and activity. To further our understanding of the complex machinery of life, we need methods that can simultaneously probe more than one degree of freedom of single molecules and complexes.

Observation of processive telomerase catalysis using high-resolution optical tweezers.

Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled.

Combined High-Resolution Optical Tweezers and Multicolor Single-Molecule Fluorescence with an Automated Single-Molecule Assembly Line.

We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy along with automated single-molecule assembly. The multicolor allows the simultaneous observation of multiple molecules or multiple degrees of freedom, which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, with a reliable alignment scheme, which will allow three independent fluorescent probe or FRET measurements and also increases flexibility in the choice of fluorescent molecules.

Combined Force Ramp and Equilibrium High-Resolution Investigations Reveal Multipath Heterogeneous Unfolding of Protein G.

Over the past two decades, one of the standard models of protein folding has been the "two-state" model, in which a protein only resides in the folded or fully unfolded states with a single pathway between them. Recent advances in spatial and temporal resolution of biophysical measurements have revealed "beyond-two-state" complexity in protein folding, even for small, single-domain proteins. In this work, we used high-resolution optical tweezers to investigate the folding/unfolding kinetics of the B1 domain of immunoglobulin-binding protein G (GB1), a well-studied model system.

A force sensor that converts fluorescence signal into force measurement utilizing short looped DNA.

A force sensor concept is presented where fluorescence signal is converted into force information via single-molecule Förster resonance energy transfer (smFRET). The basic design of the sensor is a ~100 base pair (bp) long double stranded DNA (dsDNA) that is restricted to a looped conformation by a nucleic acid secondary structure (NAS) that bridges its ends. The looped dsDNA generates a tension across the NAS and unfolds it when the tension is high enough.

Single molecule force spectroscopy at high data acquisition: A Bayesian nonparametric analysis.

Bayesian nonparametrics (BNPs) are poised to have a deep impact in the analysis of single molecule data as they provide posterior probabilities over entire models consistent with the supplied data, not just model parameters of one preferred model. Thus they provide an elegant and rigorous solution to the difficult problem encountered when selecting an appropriate candidate model. Nevertheless, BNPs' flexibility to learn models and their associated parameters from experimental data is a double-edged sword.

Randomizing phase to remove acousto-optic device wiggle errors for high-resolution optical tweezers.

Acousto-optic (AO) devices have been used extensively in optical tweezers because of their flexibility and speed; however, these devices have trap positioning inaccuracies that limit their usefulness, especially for high-resolution applications. We show that these inaccuracies are due to interference patterns within the AO device sound fields. We have devised a method that removes these inaccuracies by reducing the coherence of the sound fields by directly controlling and randomizing the phase of the radio frequency voltage input signal.

Ultrashort Nucleic Acid Duplexes Exhibit Long Wormlike Chain Behavior with Force-Dependent Edge Effects.

Despite their importance in biology and use in nanotechnology, the elastic behavior of nucleic acids on "ultrashort" (<15  nt) length scales remains poorly understood. Here, we use optical tweezers combined with fluorescence imaging to observe directly the hybridization of oligonucleotides (7-12 nt) to a complementary strand under tension and to measure the difference in end-to-end extension between the single-stranded and duplex states. Data are consistent with long-polymer models at low forces (<8  pN) but smaller than predicted at higher forces (>8  pN), the result of the sequence-dependent duplex edge effects.

Elasticity of the transition state for oligonucleotide hybridization.

Despite its fundamental importance in cellular processes and abundant use in biotechnology, we lack a detailed understanding of the kinetics of nucleic acid hybridization. In particular, the identity of the transition state, which determines the kinetics of the two-state reaction, remains poorly characterized. Here, we used optical tweezers with single-molecule fluorescence to observe directly the binding and unbinding of short oligonucleotides (7-12 nt) to a complementary strand held under constant force.

The RNA helicase Mtr4p is a duplex-sensing translocase.

The conserved Saccharomyces cerevisiae Ski2-like RNA helicase Mtr4p plays essential roles in eukaryotic nuclear RNA processing. RNA helicase activity of Mtr4p is critical for biological functions of the enzyme, but the molecular basis for RNA unwinding is not understood. Here, single-molecule high-resolution optical trapping measurements reveal that Mtr4p unwinds RNA duplexes by 3'-to-5' translocation on the loading strand, that strand separation occurs in discrete steps of 6 base pairs and that a single Mtr4p molecule performs consecutive unwinding steps.

High-Resolution "Fleezers": Dual-Trap Optical Tweezers Combined with Single-Molecule Fluorescence Detection.

Recent advances in optical tweezers have greatly expanded their measurement capabilities. A new generation of hybrid instrument that combines nanomechanical manipulation with fluorescence detection-fluorescence optical tweezers, or "fleezers"-is providing a powerful approach to study complex macromolecular dynamics. Here, we describe a combined high-resolution optical trap/confocal fluorescence microscope that can simultaneously detect sub-nanometer displacements, sub-piconewton forces, and single-molecule fluorescence signals.

Protein structure. Direct observation of structure-function relationship in a nucleic acid-processing enzyme.

The relationship between protein three-dimensional structure and function is essential for mechanism determination. Unfortunately, most techniques do not provide a direct measurement of this relationship. Structural data are typically limited to static pictures, and function must be inferred.

Ultrahigh-resolution optical trap with single-fluorophore sensitivity.

We present a single-molecule instrument that combines a time-shared ultrahigh-resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, we observed individual single fluorophore-labeled DNA oligonucleotides to bind and unbind complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, we clearly observed coincident angstrom-scale changes in tether extension.

Surface anchoring and dynamics of thiolated azobenzene molecules on Au(111).

We have investigated the temperature-dependent behavior of thiolated azobenzene molecules on Au(111) using scanning tunneling microscopy. The addition of a thiol functional group to azobenzene molecules leads to increased surface anchoring of single azobenzene molecules to gold. Thiolated azobenzene shows diverse surface morphology and does not form well-ordered structures at low coverage.

Self-patterned molecular photoswitching in nanoscale surface assemblies.

Photomechanical switching (photoisomerization) of molecules at a surface is found to strongly depend on molecule-molecule interactions and molecule-surface orientation. Scanning tunneling microscopy was used to image photoswitching behavior in the single-molecule limit of tetra-tert-butyl-azobenzene molecules adsorbed onto Au(111) at 30 K. Photoswitching behavior varied strongly with surface molecular island structure, and self-patterned stripes of switching and nonswitching regions were observed having approximately 10 nm pitch.

Facility cost analysis in outpatient plastic surgery: implications for the academic health center.

Background: The authors examined the economic patterns of outpatient aesthetic and reconstructive plastic surgical procedures performed within an academic health center.

Methods: For fiscal years 2003 and 2004, the University of Michigan Health System's accounting database was queried to identify all outpatient plastic surgery cases (aesthetic and reconstructive) from four surgical facilities. Total facility charges, cost, revenue, and margin were calculated for each case.

Reversible photomechanical switching of individual engineered molecules at a metallic surface.

We have observed reversible light-induced mechanical switching for individual organic molecules bound to a metal surface. Scanning tunneling microscopy (STM) was used to image the features of individual azobenzene molecules on Au(111) before and after reversibly cycling their mechanical structure between trans and cis states using light. Azobenzene molecules were engineered to increase their surface photomechanical activity by attaching varying numbers of tert-butyl (TB) ligands ("legs") to the azobenzene phenyl rings.

Hospital volume outcome and discharge disposition of burn patients.

Background: The purpose of this investigation was to determine the impact of hospital clinical volume on patient outcomes (i.e., in-hospital mortality, length of stay) and discharge disposition of burn patients using a large nationally representative database.

Absolute orientation of molecules at interfaces.

A method to determine the absolute orientation of molecules at liquid interfaces by sum frequency generation (SFG) is reported. It is based on measurements of the orientations of two nonparallel vibrationally active chromophores in the molecule of interest combined with a rotation matrix formulation to obtain the absolute molecular orientation. We chose m-tolunitrile, a planar molecule adsorbed to the air/water interface, as a proof-of-method experiment.

Certificate-of-Need regulation in outpatient surgery and specialty care: implications for plastic surgeons.

For plastic surgeons, independent development of outpatient surgical centers and specialty facilities is becoming increasingly common. These facilities serve as important avenues not only for increasing access and efficiency but in maintaining a sustainable, competitive specialty advantage. Certificate of Need regulation represents a major hurdle to plastic surgeons who attempt to create autonomy in this fashion.