Monomeric PcrA helicase processively unwinds plasmid lengths of DNA in the presence of the initiator protein RepD

The helicase PcrA unwinds DNA during asymmetric replication of plasmids, acting with an initiator protein, in our case RepD. Detailed kinetics of PcrA activity were measured using bulk solution and a single-molecule imaging technique to investigate the oligomeric state of the active helicase complex, its processivity and the mechanism of unwinding. By tethering either DNA or PcrA to a microscope coverslip surface, unwinding of both linear and natural circular plasmid DNA by PcrA/RepD was followed in real-time using total internal reflection fluorescence microscopy. Visualization was achieved using a fluorescent single-stranded DNA-binding protein. The single-molecule data show that PcrA, in combination with RepD, can unwind plasmid lengths of DNA in a single run, and that PcrA is active as a monomer. Although the average rate of unwinding was similar in single-molecule and bulk solution assays, the single-molecule experiments revealed a wide distribution of unwinding speeds by different molecules. The average rate of unwinding was several-fold slower than the PcrA translocation rate on single-stranded DNA, suggesting that DNA unwinding may proceed via a partially passive mechanism. However, the fastest dsDNA unwinding rates measured in the single-molecule unwinding assays approached the PcrA translocation speed measured on ssDNA

A Microcantilever Sensor Array for the Detection and Inventory of Desert Tortoises

We have designed and tested a portable instrument consisting of a small infrared camera coupled with an array of piezoresistive microcantilever sensors that is used to provide real‐time, non-invasive data on desert tortoise den occupancy. The piezoresistive microcantilever (PMC) sensors are used to obtain a chemical “signature” of tortoise presence from the air deep within the dens, and provide data in cases where the camera cannot extend deep enough into the den to provide visual evidence of tortoise presence. The infrared camera was used to verify the PMC data during testing, and in many cases, such as shallower dens, may be used to provide exact numbers on den populations

X-Ray photoelectron spectroscopy and mass spectrometry studies of X-ray-processed solid CO2

Solid CO2 films have been grown on a stainless steel substrate and processed by X-ray bombardment for up to 6 hr.. The reactions induced were monitored using X-ray photoelectron spectroscopy (XPS) and mass spectrometry. The XPS results are twofold: direct X-ray photolysis of the CO2 ice produced CO and an unidentified O product, possibly atomic O; secondary effects resulting from surface reactions between CO, O, and residual H from the vacuum environment produced H2CO, CH3OH, and a water ice cap on the CO2 film. The rate of production of CO from direct X-ray photolysis of CO2 is measured to be 5.4 × 102 molecule photon-1, corresponding to a formation cross section of 4.7 × 10-20 cm2. The growth rate for the water cap is calculated to be 2.6 × 10-4 monolayers s-1 for a partial pressure of H equal to 2 × 10-10 Torr. The appearance of gas-phase products from the film showed a time lag which indicates that the diffusion of the product species in the bulk CO2 is affected by some time-dependent process, possibly the creation of defects in the film. A model for the observed time dependence of the dissociation products in the gas phase yields diffusion coefficients in the CO2 of 5 × 10-12 and 1 × 10-12 cm2 s-1, for O and CO, respectively

Implications of Arm Restraint on Lower Extremity Kinetics During Gait

Background Literature indicates the importance of the upper extremities in providing stability and propulsion for the body during ambulation. However, the kinetic implications of upper extremity restraint during gait are not as well documented. Aim The objective of this study was to examine the effect of arm restraint (unilateral and bilateral) on lower extremity joint kinetics during walking. Methods Twenty-three healthy young participants were instrumented for three dimensional motion analysis, and tested in four randomly ordered upper extremity restraint conditions (unrestrained, bilateral restraint, right side restraint, and left side restraint). Temporal spatial parameters and gait/phase-specific lower extremity kinetics and kinematics were measured. For each restraint condition, pointwise differences from the unrestrained condition were compared using a two-way ANOVA model of restraint condition (“Condition”) and gait cycle phase (“Timing”). Results Decreases in walking speed and stride length were observed for all restraint conditions. Differences in kinetic demands were also noted, primarily at the hip and knee. Conclusion Upper extremity restraint in healthy young adults leads to significant changes in temporal-spatial parameters and proximal joint kinetics, most prominently during periods of load accommodation and balance

Visualizing helicases unwinding DNA at the single molecule level

DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes

Developing core sets for persons following amputation based on the International Classification of Functioning, Disability and Health as a way to specify functioning

Amputation is a common late stage sequel of peripheral vascular disease and diabetes or a sequel of accidental trauma, civil unrest and landmines. The functional impairments affect many facets of life including but not limited to: Mobility; activities of daily living; body image and sexuality. Classification, measurement and comparison of the consequences of amputations has been impeded by the limited availability of internationally, multiculturally standardized instruments in the amputee setting. The introduction of the International Classification of Functioning, Disability and Health (ICF) by the World Health Assembly in May 2001 provides a globally accepted framework and classification system to describe, assess and compare function and disability. In order to facilitate the use of the ICF in everyday clinical practice and research, ICF core sets have been developed that focus on specific aspects of function typically associated with a particular disability. The objective of this paper is to outline the development process for the ICF core sets for persons following amputation. The ICF core sets are designed to translate the benefits of the ICF into clinical routine. The ICF core sets will be defined at a Consensus conference which will integrate evidence from preparatory studies, namely: (a) a systematic literature review regarding the outcome measures of clinical trails and observational studies, (b) semi-structured patient interviews, (c) international experts participating in an internet-based survey, and (d) cross-sectional, multi-center studies for clinical applicability. To validate the ICF core sets field-testing will follow. Invitation for participation: The development of ICF Core Sets is an inclusive and open process. Anyone who wishes to actively participate in this process is invited to do so