DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force

Proteins interacting at multiple sites on DNA via looping play an important role in many fundamental biochemical processes. Restriction endonucleases that must bind at two recognition sites for efficient activity are a useful model system for studying such interactions. Here we used single DNA manipulation to study sixteen known or suspected two-site endonucleases. In eleven cases (BpmI, BsgI, BspMI, Cfr10I, Eco57I, EcoRII, FokI, HpaII, NarI, Sau3AI and SgrAI) we found that substitution of Ca(2+) for Mg(2+) blocked cleavage and enabled us to observe stable DNA looping. Forced disruption of these loops allowed us to measure the frequency of looping and probability distributions for loop size and unbinding force for each enzyme. In four cases we observed bimodal unbinding force distributions, indicating conformational heterogeneity and/or complex binding energy landscapes. Measured unlooping events ranged in size from 7 to 7500 bp and the most probable size ranged from less than 75 bp to nearly 500 bp, depending on the enzyme. In most cases the size distributions were in much closer agreement with theoretical models that postulate sharp DNA kinking than with classical models of DNA elasticity. Our findings indicate that DNA looping is highly variable depending on the specific protein and does not depend solely on the mechanical properties of DNA

Validation of an abbreviated Treatment Satisfaction Questionnaire for Medication (TSQM-9) among patients on antihypertensive medications

<p>Abstract</p> <p>Background</p> <p>The 14-item Treatment Satisfaction Questionnaire for Medication (TSQM) Version 1.4 is a reliable and valid instrument to assess patients' satisfaction with medication, providing scores on four scales – side effects, effectiveness, convenience and global satisfaction. In naturalistic studies, administering the TSQM with the side effects domain could provoke the physician to assess the presence or absence of adverse events in a way that is clinically atypical, carrying the potential to interfere with routine medical care. As a result, an abbreviated 9-item TSQM (TSQM-9), derived from the TSQM Version 1.4 but without the five items of the side effects domain was created. In this study, an interactive voice response system (IVRS)-administered TSQM-9 was psychometrically evaluated among patients taking antihypertensive medication.</p> <p>Methods</p> <p>A total of 3,387 subjects were invited to participate in the study from an online panel who self-reported taking a prescribed antihypertensive medication. The subjects were asked to complete the IVRS-administered TSQM-9 at the start of the study, along with the modified Morisky scale, and again within 7 to 14 days. Standard psychometric analyses were conducted; including Cronbach's alpha, intraclass correlation coefficients, structural equation modeling, Spearman correlation coefficients and analysis of covariance (ANCOVA).</p> <p>Results</p> <p>A total of 396 subjects completed all the study procedures. Approximately 50% subjects were male with a good racial/ethnic mix: 58.3% white, 18.9% black, 17.7% Hispanic and 5.1% either Asian or other. There was evidence of construct validity of the TSQM-9 based on the structural equation modeling findings of the observed data fitting the Decisional Balance Model of Treatment Satisfaction even without the side effects domain. TSQM-9 domains had high internal consistency as evident from Cronbach's alpha values of 0.84 and greater. TSQM-9 domains also demonstrated good test-retest reliability with high intraclass correlation coefficients exceeding 0.70. As expected, the TSQM-9 domains were able to differentiate between individuals who were low, medium and high compliers of medication, with moderate to high effect sizes. There was evidence of convergent validity with significant correlations with the medication adherence scale.</p> <p>Conclusion</p> <p>The IVRS-administered TSQM-9 was found to be a reliable and valid measure to assess treatment satisfaction in naturalistic study designs, in which there is potential that the administration of the side effects domain of the TSQM would interfere with routine clinical care.</p

Calculating the Energy Cost of CO2 Removal in a Coal Based Gas Turbine Fuel Cell Hybrid Power Generation System with an Isolated Anode Stream

In recent years there has been significant interest in identifying carbon capturing technologies that can be applied to fossil fuel power generation plants.CO2 capture technologies seek to reduce the amount of CO2 that would normally be emitted into the atmosphere from the daily operation of these plants. In terms of system efficiency and operating costs, this carbon capture is expensive. Further, the additional equipment that would be used to capture CO2 emissions greatly adds to the complexity of the system. There has also been significant interest in coal based gas turbine fuel cell hybrid power plants. A hybrid power plant can have much greater system efficiency than a normal gas turbine power plant because the heat that is normally unused in a standalone solid oxide fuel cell (SOFC) is recovered and used to drive a power producing turbine. It is thought that the increased system efficiency of the hybrid system might compensate for the increased expense of performing carbon capture. In order to provide some analytical insight on this tradeoff we present a 100 MW class coal fired gas turbine SOFC hybrid power generation system. The hybrid system operates at a pressure ratio of 6, and uses heat recuperation and cathode air recirculation to control the SOFC inlet temperature and the temperature change across the SOFC. A carbon capture scheme is added to this system in order to calculate the relative energy cost in terms of system efficiency due to CO2 ompression. The carbon capture is performed by burning the unused fuel from the SOFC in an isolated anode stream using pure O2 injection. The resulting heat that is generated from this process is then used to drive a secondary turbine that is placed in the anode exhaust stream where more work is extracted. With an isolated anode stream, the products of combustion from this secondary combustion process are mostly water and carbon dioxide. The water by-product is then condensed out of the stream leaving a relatively high concentration of CO2. This is then compressed, and removed from the system. In this study we present power plant efficiency calculations for the performance of the hybrid system with the carbon capturing loop. Our results show the effects on system performance that result from a changing fuel utilization factor

A general method for manipulating DNA sequences from any organism with optical tweezers

Mechanical manipulation of single DNA molecules can provide novel information about DNA properties and protein–DNA interactions. Here we describe and characterize a useful method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and are tethered between two optically trapped microspheres. We demonstrate that human, insect, plant, bacterial and viral sequences ranging from ∼10 to 40 kilobasepairs can be manipulated. Force-extension measurements show that these constructs exhibit uniform elastic properties in accord with the expected contour lengths for the targeted sequences. Detailed protocols for preparing and manipulating these molecules are presented, and tethering efficiency is characterized as a function of DNA concentration, ionic strength and pH. Attachment strength is characterized by measuring the unbinding time as a function of applied force. An alternative stronger attachment method using an amino–carboxyl linkage, which allows for reliable DNA overstretching, is also described

Combustor oscillation pressure stabilizer

In accordance with the objective of the present invention, the active control of unsteady combustion induced oscillations in a combustion chamber fired by a suitable fuel and oxidizer mixture, such as a hydrocarbon fuel and air mixture, is provided by restructuring and moving the position of the main flame front and thereby increasing the transport time and displacing the pressure wave further away from the in-phase relationship with the periodic heat release. The restructuring and repositioning of the main flame are achieved by utilizing a pilot flame which is pulsed at a predetermined frequency corresponding to less than about one-half the frequency of the combustion oscillation frequency with the duration of each pulse being sufficient to produce adequate secondary thermal energy to restructure the main flame and thereby decouple the heat release from the acoustic coupling so as to lead to a reduction in the dynamic pressure amplitude. The pulsating pilot flame produces a relatively small and intermittently existing flame front in the combustion zone that is separate from the oscillating main flame front but which provides the thermal energy necessary to effectively reposition the location of the oscillating main flame front out of the region in the combustion zone where the acoustic coupling can occur with the main flame and thereby effectively altering the oscillation-causing phase relationship with the heat of combustion

The burden of selected digestive diseases in the United States

AbstractBackground & Aims: Gastrointestinal (GI) and liver diseases inflict a heavy economic burden. Although the burden is considerable, current and accessible information on the prevalence, morbidity, and cost is sparse. This study was undertaken to estimate the economic burden of GI and liver disease in the United States for use by policy makers, health care providers, and the public. Methods: Data were extracted from a number of publicly available and proprietary national databases to determine the prevalence, direct costs, and indirect costs for 17 selected GI and liver diseases. Indirect cost calculations were purposefully very conservative. These costs were compared with National Institutes of Health (NIH) research expenditures for selected GI and liver diseases. Results: The most prevalent diseases were non–food-borne gastroenteritis (135 million cases/year), food-borne illness (76 million), gastroesophageal reflux disease (GERD; 19 million), and irritable bowel syndrome (IBS; 15 million). The disease with the highest annual direct costs in the United States was GERD ($9.3 billion), followed by gallbladder disease ($5.8 billion), colorectal cancer ($4.8 billion), and peptic ulcer disease ($3.1 billion). The estimated direct costs for these 17 diseases in 1998 dollars were $36.0 billion, with estimated indirect costs of$22.8 billion. The estimated direct costs for all digestive diseases were $85.5 billion. Total NIH research expenditures were$676 million in 2000. Conclusions: GI and liver diseases exact heavy economic and social costs in the United States. Understanding the prevalence and costs of these diseases is important to help set priorities to reduce the burden of illness.GASTROENTEROLOGY 2002;122:1500-151

Fused eco29kIR- and M genes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems

<p>Abstract</p> <p>Background</p> <p>The discovery of restriction endonucleases and modification DNA methyltransferases, key instruments of genetic engineering, opened a new era of molecular biology through development of the recombinant DNA technology. Today, the number of potential proteins assigned to type II restriction enzymes alone is beyond 6000, which probably reflects the high diversity of evolutionary pathways. Here we present experimental evidence that a new type IIC restriction and modification enzymes carrying both activities in a single polypeptide could result from fusion of the appropriate genes from preexisting bipartite restriction-modification systems.</p> <p>Results</p> <p>Fusion of <it>eco29kIR </it>and <it>M </it>ORFs gave a novel gene encoding for a fully functional hybrid polypeptide that carried both restriction endonuclease and DNA methyltransferase activities. It has been placed into a subclass of type II restriction and modification enzymes - type IIC. Its MTase activity, 80% that of the M.Eco29kI enzyme, remained almost unchanged, while its REase activity decreased by three times, concurrently with changed reaction optima, which presumably can be caused by increased steric hindrance in interaction with the substrate. <it>In vitro </it>the enzyme preferentially cuts DNA, with only a low level of DNA modification detected. <it>In vivo </it>new RMS can provide a 10²-fold less protection of host cells against phage invasion.</p> <p>Conclusions</p> <p>We propose a molecular mechanism of appearing of type IIC restriction-modification and M.SsoII-related enzymes, as well as other multifunctional proteins. As shown, gene fusion could play an important role in evolution of restriction-modification systems and be responsible for the enzyme subclass interconversion. Based on the proposed approach, hundreds of new type IIC enzymes can be generated using head-to-tail oriented type I, II, and III restriction and modification genes. These bifunctional polypeptides can serve a basis for enzymes with altered recognition specificities. Lastly, this study demonstrates that protein fusion may change biochemical properties of the involved enzymes, thus giving a starting point for their further evolutionary divergence.</p