A Single-Molecule Surface-Based Platform to Detect the Assembly and Function of the Human RNA Polymerase II Transcription Machinery.

Single-molecule detection and manipulation is a powerful tool for unraveling dynamic biological processes. Unfortunately, success in such experiments is often challenged by tethering the biomolecule(s) of interest to a biocompatible surface. Here, we describe a robust surface passivation method by dense polymer brush grafting, based on optimized polyethylene glycol (PEG) deposition conditions, exactly at the lower critical point of an aqueous biphasic PEG-salt system.

Structural mechanism of transcription inhibition by lasso peptides microcin J25 and capistruin.

We report crystal structures of the antibacterial lasso peptides microcin J25 (MccJ25) and capistruin (Cap) bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). Both peptides bind within the RNAP secondary channel, through which NTP substrates enter the RNAP active site, and sterically block trigger-loop folding, which is essential for efficient catalysis by the RNAP. MccJ25 binds deep within the secondary channel in a manner expected to interfere with NTP substrate binding, explaining the partial competitive mechanism of inhibition with respect to NTPs found previously [Mukhopadhyay J, Sineva E, Knight J, Levy RM, Ebright RH (2004) 14:739-751].

RNA polymerase motions during promoter melting.

All cellular RNA polymerases (RNAPs), from those of bacteria to those of man, possess a clamp that can open and close, and it has been assumed that the open RNAP separates promoter DNA strands and then closes to establish a tight grip on the DNA template. Here, we resolve successive motions of the initiating bacterial RNAP by studying real-time signatures of fluorescent reporters placed on RNAP and DNA in the presence of ligands locking the clamp in distinct conformations. We report evidence for an unexpected and obligatory step early in the initiation involving a transient clamp closure as a prerequisite for DNA melting.

Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry.

Many essential cellular processes, such as gene control, employ elaborate mechanisms involving the coordination of large, multi-component molecular assemblies. Few structural biology tools presently have the combined spatial-temporal resolution and molecular specificity required to capture the movement, conformational changes, and subunit association-dissociation kinetics, three fundamental elements of how such intricate molecular machines work. Here, we report a 3D single-molecule super-resolution imaging study using modulation interferometry and phase-sensitive detection that achieves <2 nm axial localization precision, well below the few-nanometer-sized individual protein components.

Maintaining competency for float nursing staff.

Orienting staff to multiple areas is challenging, as is maintaining multiple competencies, which might be used infrequently. Creating a strategy to regularly assess needed competencies to maintain a highly skilled pool of nurses, prepared to float to multiple areas, is critical to supporting flexible staffing. A plan for how to achieve this complex analysis is described and can be translated to other complex environments.

Arabidopsis Argonaute MID domains use their nucleotide specificity loop to sort small RNAs.

The 5'-nucleotide of small RNAs associates directly with the MID domain of Argonaute (AGO) proteins. In humans, the identity of the 5'-base is sensed by the MID domain nucleotide specificity loop and regulates the integrity of miRNAs. In Arabidopsis thaliana, the 5'-nucleotide also controls sorting of small RNAs into the appropriate member of the AGO family; however, the structural basis for this mechanism is unknown.

Low-dose inhaled nitric oxide improves the oxygenation and ventilation of infants and children with acute, hypoxemic respiratory failure.

Objective: To describe the effects of inhaled nitric oxide on oxygenation and ventilation in patients with acute, hypoxic respiratory failure and to characterize those who respond to low doses with a significant improvement in PaO2.

Design: Prospective dose response trial of inhaled nitric oxide. Patients who demonstrated a > or =15% improvement in PaO2 were randomized to receive conventional mechanical ventilation with or without prolonged inhaled nitric oxide.

The evaluation of performance and cost in a hypertension control program.

This article examines the Performance Measurement System (PMS), a new approach to evaluating performance and cost of public health departments, developed under the sponsorship of the National Science Foundation. Four weaknesses characterizing presently used evaluation designs are described. The PMS method is briefly summarized and one practical application of the method, to hypertension control, is discussed in detail.