ExCyto PCR Amplification

ExCyto PCR cells provide a novel and cost effective means to amplify DNA transformed into competent bacterial cells. ExCyto PCR uses host E. coli with a chromosomally integrated gene encoding a thermostable DNA polymerase to accomplish robust, hot-start PCR amplification of cloned sequences without addition of exogenous enzyme.Because the thermostable DNA polymerase is stably integrated into the bacterial chromosome, ExCyto cells can be transformed with a single plasmid or complex library, and then the expressed thermostable DNA polymerase can be used for PCR amplification. We demonstrate that ExCyto cells can be used to amplify DNA from different templates, plasmids with different copy numbers, and master mixes left on ice for up to two hours. Further, PCR amplification with ExCyto cells is comparable to amplification using commercial DNA polymerases. The ability to transform a bacterial strain and use the endogenously expressed protein for PCR has not previously been demonstrated.ExCyto PCR reduces pipetting and greatly increases throughput for screening EST, genomic, BAC, cDNA, or SNP libraries. This technique is also more economical than traditional PCR and thus broadly useful to scientists who utilize analysis of cloned DNAs in their research

Statins in the Treatment of Chronic Heart Failure: A Systematic Review

BACKGROUND: The efficacy of statin therapy in patients with established chronic heart failure (CHF) is a subject of much debate. METHODS AND FINDINGS: We conducted three systematic literature searches to assess the evidence supporting the prescription of statins in CHF. First, we investigated the participation of CHF patients in randomized placebo-controlled clinical trials designed to evaluate the efficacy of statins in reducing major cardiovascular events and mortality. Second, we assessed the association between serum cholesterol and outcome in CHF. Finally, we evaluated the ability of statin treatment to modify surrogate endpoint parameters in CHF. Using validated search strategies, we systematically searched PubMed for our three queries. In addition, we searched the reference lists from eligible studies, used the “see related articles” feature for key publications in PubMed, consulted the Cochrane Library, and searched the ISI Web of Knowledge for papers citing key publications. Search 1 resulted in the retrieval of 47 placebo-controlled clinical statin trials involving more than 100,000 patients. CHF patients had, however, been systematically excluded from these trials. Search 2 resulted in the retrieval of eight studies assessing the relationship between cholesterol levels and outcome in CHF patients. Lower serum cholesterol was consistently associated with increased mortality. Search 3 resulted in the retrieval of 18 studies on the efficacy of statin treatment in CHF. On the whole, these studies reported favorable outcomes for almost all surrogate endpoints. CONCLUSIONS: Since CHF patients have been systematically excluded from randomized, controlled clinical cholesterol-lowering trials, the effect of statin therapy in these patients remains to be established. Currently, two large, randomized, placebo-controlled statin trials are under way to evaluate the efficacy of statin treatment in terms of reducing clinical endpoints in CHF patients in particular

Changes in absolute gravity 2000–2015, South Island, New Zealand

<p>We quantify changes in gravity that have occurred over the past 15 years at 14 points between latitudes 42.5°S and 44°S in the South Island of New Zealand. Ten of the points form two transects across the Southern Alps and four lie in the epicentral region of the 2010 Canterbury earthquakes. At each location gravity was measured using an absolute gravimeter (FG5–111) with a nominal accuracy of 1 µGal. Observed changes in gravity varied from −53 to +43 µGal in the presence of surface elevation changes in the range −11 cm to +20 cm. Despite the difficulty in quantifying gravitational contributions from surface and subsurface water, uplift in both the Southern Alps and the Christchurch region is consistent with a Bouguer surface gradient of approximately 1.97 µGal cm^–1, appropriate for the mean density of crustal rocks.</p

Robustness of ExCyto PCR.

<p>Eight different clones from a cDNA library transformed into ExCyto cells were grown overnight, and 2 ul of the overnight cultures were added to reaction mix and left on ice for 0, 30, 60 and 120 minutes prior to ExCyto PCR amplification. One kb molecular weight markers are shown in the last lane.</p

ExCyto <i>versus</i> standard PCR.

<p>(A) Twenty-four different clones from a cDNA library transformed in ExCyto cells were grown overnight, and 2 µl of overnight cultures were used for ExCyto PCR amplification. (B, C, & D) Standard PCR on 2 µl of overnight cultures from the same 24 clones as in (A) above, except clones were transformed into parent cells without the integrated thermostable polymerase. The cDNA inserts were amplified using Invitrogen's Taq DNA Polymerase (B), Promega's GoTaq DNA Polymerase (C), or Stratagene's Taq DNA Polymerase (D). One kb molecular weight markers are shown in the last lane.</p