Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy

A method for sensitively monitoring enzyme kinetics and activities by using dual-color fluorescence cross-correlation spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme’s action between two fluorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and fluorophores at each 5′ end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored on-line at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis–Menten equation with a K(M) of 14 ± 1 nM and a k(cat) of 4.6 ± 0.2 min(−1). In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color fluorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology

Comparison of Ventricular Inducibility with Late Gadolinium Enhancement and Myocardial Inflammation in Endomyocardial Biopsy in Patients with Dilated Cardiomyopathy.

Risk stratification of patients with non-ischemic dilated cardiomyopathy remains a matter of debate in the era of device implantation.We investigated associations between histopathological findings, contrast-enhanced cardiac MRI and the inducibility of ventricular tachycardia (VT) or fibrillation (VF) in programmed ventricular stimulation.56 patients with impaired left ventricular ejection fraction (LVEF≤50%, mean 36.6±10.5%) due to non-ischemic dilated cardiomyopathy underwent cardiac MRI, programmed ventricular stimulation, and endomyocardial biopsy and were retrospectively investigated. Inducibility was defined as sustained mono- or polymorphic VT or unstable VT/VF requiring cardioversion/defibrillation. Primary study endpoint was defined as the occurrence of hemodynamically relevant VT/VF and/or adequate ICD-therapy during follow-up.Endomyocardial biopsy detected cardiac fibrosis in 18 (32.1%) patients. Cardiac MRI revealed 35 (62.5%) patients with positive late gadolinium enhancement. VT/VF was induced in ten (17.9%) patients during programmed ventricular stimulation. Monomorphic VT was inducible in 70%, while 20% of patients showed polymorphic VT. One patient (10%) presented with VF. Inducibility correlated significantly with the presence of positive late gadolinium enhancement in cardiac MRI (p<0.01). We could not find a significant association between inducibility and the degree of cardiac inflammation and fibrosis in non-site directed routine right ventricular endomyocardial biopsy. During a mean follow-up of 2.6 years, nine (16.1%) patients reached the primary endpoint. Monomorphic VTs were found in 66.7% patients and were terminated by antitachycardia pacing therapy. One patient with polymorphic VT and two patients with VF received adequate therapy by an ICD-shock. However, inducibility did not correlate with the occurrence of endpoints.Inducibilty during programmed ventricular stimulation is associated with positive late gadolinium enhancement in cardiac MRI of patients with non-ischemic dilated cardiomyopathy. The presence of myocardial fibrosis or inflammation in undirected endomyocardial biopsy does not seem to be sufficient to predict future ventricular arrhythmias

Impact of phospholipids on plasmid packaging and toxicity of gemini nanoparticles

Street signs and buildings in the Admiralty area; Images taken in 2009 of Central and Admiralty areas, Hong Kong Island, highlighting the traffic conditions with cars and mass transit. Also views of the Central Elevated Walkway, an extensive footbridge network spanning Admiralty and Central, near Victoria Harbour in Hong Kong. The British colony of Hong Kong introduced its first double-decker buses in 1949 by Kowloon Motor Bus. By law, double-decker buses in Hong Kong are limited to a length of 12 metres (40 feet). Today, the majority of buses running in Hong Kong are double-decker buses. There are also double-deck trams; Hong Kong Tramways is the only tram company in the world that operates double-deck trams exclusively. Source: Wikipedia; http://en.wikipedia.org/wiki/Main_Page (accessed 7/22/2013

Association of ventricular inducibility with late gadonlinium enhancement in contrast-enhanced cardiac MRI.

<p>Fig 3 depicts the association of ventricular inducibility in patients with non-ischemic cardiomyopathy and the detection of positive late gadolinium enhancement in contrast-enhanced cardiac MRI. Ventricular inducibility correlates significantly with the presence of late gadolinium enhancement, p<0.01.</p

Patients’ demographics and baseline characteristics.

<p>Patients’ demographics and baseline characteristics.</p

Kaplan-Meier-Curves for the occurrence of primary endpoints stratified by inducibility in programmed ventricular stimulation in all patients (A) and in a subgroup of patients with low inflammatory activity (B).

<p><b>A.</b> Kaplan-Meier estimates illustrate the occurrence of the primary endpoints hemodynamically relevant sustained ventricular tachycardia and/or adequate ICD-therapy (antitachycardia pacing, shock) for all enrolled patients during follow-up. There is no significant difference between inducible and non-inducible patients regarding the occurrence of ventricular arrhythmia (LogRank 0.14, p<0.71). <b>B.</b> Kaplan-Meier estimates illustrate the occurrence of the primary endpoints hemodynamically relevant sustained ventricular tachycardia and/or adequate ICD-therapy (antitachycardia pacing, shock) during follow-up for a subgroup of patients with low inflammatory activity. There is no significant difference between inducible and non-inducible patients regarding the occurrence of ventricular arrhythmia (LogRank 0.63, p<0.43).</p

Association between ventricular inducibility and immunohistochemical markers of inflammation detected in endomyocardial biopsy.

<p>There was no significant association between the presence of the inflammatory marker CD3 and ventricular inducibility (p = 0.4), CD68 and ventricular inducibility (p = 0.2), MHCII and ventricular inducibility (p = 0.1), and the combination of all inflammatory markers (CD3, CD68, and MHCII) within the myocardium and ventricular inducibility (p = 0.2).</p