DNA O-MAP uncovers the molecular neighborhoods associated with specific genomic loci.

The accuracy of crucial nuclear processes such as transcription, replication, and repair, depends on the local composition of chromatin and the regulatory proteins that reside there. Understanding these DNA-protein interactions at the level of specific genomic loci has remained challenging due to technical limitations. Here, we introduce a method termed "DNA O-MAP", which uses programmable peroxidase-conjugated oligonucleotide probes to biotinylate nearby proteins.

Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy.

Ligand-induced conformational changes are critical to the function of many membrane proteins and arise from numerous intramolecular interactions. In the photocycle of the model membrane protein bacteriorhodopsin (bR), absorption of a photon by retinal triggers a conformational cascade that results in pumping a proton across the cell membrane. While decades of spectroscopy and structural studies have probed this photocycle in intricate detail, changes in intramolecular energetics that underlie protein motions have remained elusive to experimental quantification.

Force-Activated DNA Substrates for In Situ Generation of ssDNA and Designed ssDNA/dsDNA Structures in an Optical-Trapping Assay.

Single-molecule force spectroscopy can precisely probe the biomechanical interactions of proteins that unwind duplex DNA and bind to and wrap around single-stranded (ss)DNA. Yet assembly of the required substrates, which often contain a ssDNA segment embedded within a larger double-stranded (ds)DNA construct, can be time-consuming and inefficient, particularly when using a standard three-way hybridization protocol. In this chapter, we detail how to construct a variety of force-activated DNA substrates more efficiently.

Feeding a high-energy finishing diet upon arrival to high-risk feedlot calves: effects on health, performance, ruminal pH, rumination, serum metabolites, and carcass traits.

This study evaluated the impacts of feeding a high-energy finishing diet during both the receiving and finishing period compared with a lower-energy receiving diet with adaptation to the finishing diet on health, performance, serum chemistry, ruminal pH, rumination, and carcass characteristics of high-risk feedlot cattle. Five truck-load blocks of steers (n = 101) and bulls (n = 299) were used in a generalized complete block design and randomly assigned to receive: 1) finishing diet for the entire feeding period (FIN) or 2) receiving diet for the first 56 d, followed by a transition to the finishing diet (REC). All cattle were fed ad libitum and consumed the same diet by day 74.

Insulin and IGF-2 support rat corneal endothelial cell growth and wound repair in the organ cultured tissue.

The ability of insulin and IGF-2 to support wound repair in the organ-cultured rat corneal endothelium was investigated. Corneas given a circular transcorneal freeze injury, were explanted into organ cultures containing either insulin or IGF-2 and cultured up to72 h. Both factors increased [H]-thymidine incorporation and mitotic levels compared to controls.

Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy.

Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔ). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein.

Type III secretion system effector proteins are mechanically labile.

Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins-such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)-impairs secretion and results in obstruction of the T3SS.

Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations.

Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to μm-scale force probes via flexible linkers? Here, we studied the folding/unfolding of αD using atomic force microscopy (AFM)-based force spectroscopy. αD offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed αD's configurational diffusion constant within the context of Kramers theory varies with pH.

Correcting molecular transition rates measured by single-molecule force spectroscopy for limited temporal resolution.

Equilibrium free-energy-landscape parameters governing biomolecular folding can be determined from nonequilibrium force-induced unfolding by measuring the rates k for transitioning back and forth between states as a function of force F. However, bias in the observed forward and reverse rates is introduced by limited effective temporal resolution, which includes the mechanical response time of the force probe and any smoothing used to improve the signal-to-noise ratio. Here we use simulations to characterize this bias, which is most prevalent when the ratio of forward and reverse rates is far from unity.

Quantifying the Native Energetics Stabilizing Bacteriorhodopsin by Single-Molecule Force Spectroscopy.

We quantified the equilibrium (un)folding free energy ΔG_{0} of an eight-amino-acid region starting from the fully folded state of the model membrane-protein bacteriorhodopsin using single-molecule force spectroscopy. Analysis of equilibrium and nonequilibrium data yielded consistent, high-precision determinations of ΔG_{0} via multiple techniques (force-dependent kinetics, Crooks fluctuation theorem, and inverse Boltzmann analysis). We also deduced the full 1D projection of the free-energy landscape in this region.

Bending and looping of long DNA by Polycomb repressive complex 2 revealed by AFM imaging in liquid.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that methylates histone H3 at Lysine 27. PRC2 is critical for epigenetic gene silencing, cellular differentiation and the formation of facultative heterochromatin. It can also promote or inhibit oncogenesis.

Alzheimer's Treatment: Real-World Physician Behavior Across Countries.

Objective: Timely initiation of Alzheimer's disease (AD)-specific treatment may postpone cognitive deterioration and preserve patient independence. We explored real-world physician behavior in the treatment of AD.

Methods: Online questionnaires and patient record forms (PRFs) were completed by participating physicians.

Alzheimer's Diagnosis: Real-World Physician Behavior Across Countries.

Introduction: Appropriate management of patients with Alzheimer's disease (AD) helps preserve their independence and time at home. We explored physician behavior in the management of AD, focusing on diagnosis.

Methods: Online questionnaires and patient record forms (PRFs) were created by an independent market research agency and completed by participating physicians.

Membrane-Protein Unfolding Intermediates Detected with Enhanced Precision Using a Zigzag Force Ramp.

Precise quantification of the energetics and interactions that stabilize membrane proteins in a lipid bilayer is a long-sought goal. Toward this end, atomic force microscopy has been used to unfold individual membrane proteins embedded in their native lipid bilayer, typically by retracting the cantilever at a constant velocity. Recently, unfolding intermediates separated by as few as two amino acids were detected using focused-ion-beam-modified ultrashort cantilevers.

Analysis of magnetic resonance spectroscopy relative metabolite ratios in mild traumatic brain injury and normative controls.

Introduction: We evaluated magnetic resonance spectroscopy (MRS) in United States military personnel with persistent symptoms after mild traumatic brain injury (mTBI), comparing over time two groups randomized to receive hyperbaric oxygen or sham chamber sessions and a third group of normative controls.

Methods: Active-duty or veteran military personnel and normative controls underwent MRS outcome measures at baseline, 13 weeks (mTBI group only), and six months. Participants received 3.

Quantitative analysis tool for clinical functional MRI in mild traumatic brain injury.

Functional magnetic resonance imaging (fMRI) has been available commercially for clinical diagnostic use for many years. However, both clinical interpretation of fMRI by a neuroradiologist and quantitative analysis of fMRI data can require significant personnel resources that exceed reimbursement. In this report, a fully automated computer-based quantification methodology (Enumerated Auditory Response, EAR) has been developed to provide an auditory fMRI assessment of patients who have suffered a mild traumatic brain injury.

Imaging DNA Equilibrated onto Mica in Liquid Using Biochemically Relevant Deposition Conditions.

For over 25 years, imaging of DNA by atomic force microscopy has been intensely pursued. Ideally, such images are then used to probe the physical properties of DNA and characterize protein-DNA interactions. The atomic flatness of mica makes it the preferred substrate for high signal-to-noise ratio (SNR) imaging, but the negative charge of mica and DNA hinders deposition.

Quantifying the Initial Unfolding of Bacteriorhodopsin Reveals Retinal Stabilization.

The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important, but poorly resolved, are the forces present during the initial unfolding of a membrane protein, where the most native set of interactions is present. A high-precision, atomic force microscopy assay was developed to study the initial unfolding of bacteriorhodopsin.

High-Precision Single-Molecule Characterization of the Folding of an HIV RNA Hairpin by Atomic Force Microscopy.

The folding of RNA into a wide range of structures is essential for its diverse biological functions from enzymatic catalysis to ligand binding and gene regulation. The unfolding and refolding of individual RNA molecules can be probed by single-molecule force spectroscopy (SMFS), enabling detailed characterization of the conformational dynamics of the molecule as well as the free-energy landscape underlying folding. Historically, high-precision SMFS studies of RNA have been limited to custom-built optical traps.

FEATHER: Automated Analysis of Force Spectroscopy Unbinding and Unfolding Data via a Bayesian Algorithm.

Single-molecule force spectroscopy (SMFS) provides a powerful tool to explore the dynamics and energetics of individual proteins, protein-ligand interactions, and nucleic acid structures. In the canonical assay, a force probe is retracted at constant velocity to induce a mechanical unfolding/unbinding event. Next, two energy landscape parameters, the zero-force dissociation rate constant (k) and the distance to the transition state (Δx), are deduced by analyzing the most probable rupture force as a function of the loading rate, the rate of change in force.

Improved free-energy landscape reconstruction of bacteriorhodopsin highlights local variations in unfolding energy.

Precisely quantifying the energetics that drive the folding of membrane proteins into a lipid bilayer remains challenging. More than 15 years ago, atomic force microscopy (AFM) emerged as a powerful tool to mechanically extract individual membrane proteins from a lipid bilayer. Concurrently, fluctuation theorems, such as the Jarzynski equality, were applied to deduce equilibrium free energies (ΔG) from non-equilibrium single-molecule force spectroscopy records.

Going Vertical To Improve the Accuracy of Atomic Force Microscopy Based Single-Molecule Force Spectroscopy.

Single-molecule force spectroscopy (SMFS) is a powerful technique to characterize the energy landscape of individual proteins, the mechanical properties of nucleic acids, and the strength of receptor-ligand interactions. Atomic force microscopy (AFM)-based SMFS benefits from ongoing progress in improving the precision and stability of cantilevers and the AFM itself. Underappreciated is that the accuracy of such AFM studies remains hindered by inadvertently stretching molecules at an angle while measuring only the vertical component of the force and extension, degrading both measurements.

Force Spectroscopy with 9-μs Resolution and Sub-pN Stability by Tailoring AFM Cantilever Geometry.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize the unfolding/refolding dynamics of individual molecules and resolve closely spaced, transiently occupied folding intermediates. On a modern commercial AFM, these applications and others are now limited by the mechanical properties of the cantilever. Specifically, AFM-based SMFS data quality is degraded by a commercial cantilever's limited combination of temporal resolution, force precision, and force stability.

Improved Free-Energy Landscape Quantification Illustrated with a Computationally Designed Protein-Ligand Interaction.

Quantifying the energy landscape underlying protein-ligand interactions leads to an enhanced understanding of molecular recognition. A powerful yet accessible single-molecule technique is atomic force microscopy (AFM)-based force spectroscopy, which generally yields the zero-force dissociation rate constant (k ) and the distance to the transition state (Δx ). Here, we introduce an enhanced AFM assay and apply it to probe the computationally designed protein DIG10.