Biao Lu

Identification and quantification of coronary atherosclerotic plaques: a comparison of 64-MDCT and intravascular ultrasound.

OBJECTIVE The purpose of this study was to compare the accuracy of 64-MDCT with that of intravascular ultrasound (IVUS) for the identification and quantitative analysis of coronary atherosclerotic plaques. MATERIALS AND METHODS Twenty-six patients (17 men, nine women; mean age, 56 years) with suspected coronary atherosclerotic disease were studied using contrast-enhanced 64-MDCT and IVUS. The coronary arteries were divided into 10-mm segments and vascular cross-sectional area (CSA), luminal CSA, and plaque burden were measured in each segment. Plaque analysis software was used to automatically detect both plaques and vessel walls on CT images. Two investigators who were blinded to IVUS results independently determined the presence, classification, and quantitative measurement of atherosclerotic plaques on the CT images, which were then compared with the IVUS images. RESULTS Of 40 coronary arteries, 247 of 263 segments were imaged and analyzed by both contrast-enhanced 64-MDCT and IVUS. Sixteen segments were ruled out because of poor CT image quality. Compared with IVUS, 64-MDCT enabled correct detection in 86 of 89 (96.6%) segments containing noncalcified plaques, 25 of 27 (92.6%) segments containing calcified plaques, and 118 of 131 (90.1%) segments without atherosclerotic plaques. Sensitivity, specificity, positive predictive value, and negative predictive value for the detection of plaques by 64-MDCT were 97.4%, 90.1%, 89.7%, and 97.5%, respectively. Plaque analysis software using predetermined Hounsfield unit ranges for different components of plaque was able to distinguish between fibrous, fibrous-soft, and calcified plaques to a significant degree, but was less able to distinguish between soft and fibrous, and between soft and fibrous-soft plaque. Cohens kappa coefficient for the sole detection of atherosclerotic segments by observers was 0.91. The correlation coefficients to determine vascular CSA, luminal CSA, and plaque burden were r = 0.85, 0.82, and 0.77, respectively (p < 0.01). CONCLUSION Compared with IVUS, contrast-enhanced 64-MDCT has a good ability to identify and quantify coronary atherosclerotic plaques. However, the reliable differentiation of the composition of noncalcified plaques is still limited.

A do-it-yourself protocol for simple transcription activator-like effector assembly

BackgroundTALEs (transcription activator-like effectors) are powerful molecules that have broad applications in genetic and epigenetic manipulations. The simple design of TALEs, coupled with high binding predictability and specificity, is bringing genome engineering power to the standard molecular laboratory. Currently, however, custom TALE assembly is either costly or limited to few research centers, due to complicated assembly protocols, long set-up time and specific training requirements.ResultsWe streamlined a Golden Gate-based method for custom TALE assembly. First, by providing ready-made, quality-controlled monomers, we eliminated the procedures for error-prone and time-consuming set-up. Second, we optimized the protocol toward a fast, two-day assembly of custom TALEs, based on four thermocycling reactions. Third, we increased the versatility for diverse downstream applications by providing series of vector sets to generate both TALENs (TALE nucleases) and TALE-TFs (TALE-transcription factors) under the control of different promoters. Finally, we validated our system by assembling a number of TALENs and TALE-TFs with DNA sequencing confirmation. We further demonstrated that an assembled TALE-TF was able to transactivate a luciferase reporter gene and a TALEN pair was able to cut its target.ConclusionsWe established and validated a do-it-yourself system that enables individual researchers to assemble TALENs and TALE-TFs within 2 days. The simplified TALE assembly combined with multiple choices of vectors will facilitate the broad use of TALE technology.

Gadofluorine-Enhanced Magnetic Resonance Imaging of Carotid Atherosclerosis in Yucatan Miniswine

Objective:The aim of this study was to determine whether gadofluorine, a paramagnetic magnetic resonance imaging (MRI) contrast agent, selectively enhances carotid atherosclerotic plaques in Yucatan miniswine. Methods:Atherosclerotic plaques were induced in the left carotid arteries (LCA) of Yucatan miniswine (n = 3) by balloon denudation and high cholesterol diet. T1-weighted MRI was performed before and 24 hours after gadofluorine injection (at a dose of 100 &mgr;mol/kg) to assess the enhancement of the balloon-injured LCA wall relative to healthy, uninjured right carotid artery (RCA) wall. Histopathology was performed to verify the presence and composition of the atherosclerotic plaques imaged with MRI. Results:Gadofluorine was found to enhance LCA atherosclerotic lesions relative to RCA wall by 21% (P < 0.025) 24 hours after contrast injection. Enhancement of healthy LCA wall relative to healthy RCA wall was not observed. Conclusion:Gadofluorine selectively enhances carotid atherosclerotic plaques in Yucatan miniswine. Gadofluorine appears to be a promising MR contrast agent for detection of atherosclerotic plaques in vivo.

TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells

Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells.

A TALEN-based strategy for efficient bi-allelic miRNA ablation in human cells.

Significant progress in the functional understanding of microRNAs (miRNAs) has been made in mice, but a need remains to develop efficient tools for bi-allelic knockouts of miRNA in the human genome. Transcription activator-like effector nucleases (TALENs) provide an exciting platform for targeted gene ablation in cultured human cells, but bi-allelic modifications induced by TALENs alone occur at low frequency, making screening for double knockouts a tedious task. Here, we present an approach that is highly efficient in bi-allelic miRNA ablation in the human genome by combining TALENs targeting to the miRNA seed region with a homologous recombination donor vector and a positive selection strategy. A pilot test of this approach demonstrates bi-allelic miR-21 gene disruption at high frequency (∼87%) in cultured HEK293 cells. Analysis of three independent clones showed a total loss of miR-21 expression. Phenotypical analysis revealed increased miR-21 target gene expression, reduced cell proliferation, and alterations of global miRNA expression profiles. Taken together, our study reveals a feasible and efficient approach for bi-allelic miRNA ablation in cultured human cells and demonstrates its usefulness in elucidating miRNA function in human cells.

Black-blood steady-state free precession (SSFP) coronary wall MRI for cardiac allografts: a feasibility study.

To assess the hypothesis that steady‐state free procession (SSFP) allows for imaging of the coronary wall under the conditions of fast heart rate in heart transplantation (HTx) patients.

Comparison of X‐ray fluoroscopy and interventional magnetic resonance imaging for the assessment of coronary artery stenoses in swine

The accuracy of a two‐step interventional MRI protocol to quantify coronary artery disease was compared to the clinical gold standard, X‐ray angiography. Studies were conducted in nine swine with a surgically induced stenosis in the proximal left circumflex coronary artery. The two‐step protocol consisted of catheter‐directed magnetic resonance angiography (MRA), which was first used to localize the stenosis, followed by MRI cross‐sectional images to quantify the degree of stenosis without the use of contrast agent. Line signal intensity profiles were drawn across the vessel diameter at the stenosis site and proximal to the stenosis for each data set to measure percentage stenosis for each animal. Catheter‐directed MRA successfully detected eight of nine stenoses. Cross‐sectional MRI accurately quantified each stenosis, with strong agreement to the measurements made using X‐ray fluoroscopy (intraclass correlation coefficient = 0.955; P < 0.05). This study demonstrates that in the future interventional MRI may be an alternative to X‐ray angiography for the detection and quantification of coronary artery disease. Magn Reson Med, 2005.

A robust dual reporter system to visualize and quantify gene expression mediated by transcription activator-like effectors

BackgroundTranscription activator-like effectors (TALEs) are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The modularity of TALEs DNA binding domain enables sequence-specific perturbation and offers broad applications in genetic and epigenetic studies. Although the efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking.ResultsWe established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP) applications.ConclusionWe have designed, constructed and validated a novel dual reporter system for assessing TALE mediated gene regulations. This system offers a robust and easy-to- use tool for real-time monitoring and quantifying gene expression in mammalian cells.

A cooled CCD camera-based protocol provides an effective solution for in vitro monitoring of luciferase

Luciferase assay has become an increasingly important technique to monitor a wide range of biological processes. However, the mainstay protocols require a luminometer to acquire and process the data, therefore limiting its application to specialized research labs. To overcome this limitation, we have developed an alternative protocol that utilizes a commonly available cooled charge-coupled device (CCCD), instead of a luminometer for data acquiring and processing. By measuring activities of different luciferases, we characterized their substrate specificity, assay linearity, signal-to-noise levels, and fold-changes via CCCD. Next, we defined the assay parameters that are critical for appropriate use of CCCD for different luciferases. To demonstrate the usefulness in cultured mammalian cells, we conducted a case study to examine NFκB gene activation in response to inflammatory signals in human embryonic kidney cells (HEK293 cells). We found that data collected by CCCD camera was equivalent to those acquired by luminometer, thus validating the assay protocol. In comparison, The CCCD-based protocol is readily amenable to live-cell and high-throughput applications, offering fast simultaneous data acquisition and visual and quantitative data presentation. In conclusion, the CCCD-based protocol provides a useful alternative for monitoring luciferase reporters. The wide availability of CCCD will enable more researchers to use luciferases to monitor and quantify biological processes.

Live visualization and quantification of pathway signaling with dual fluorescent and bioluminescent reporters.

Despite their fundamental importance, the dynamics of signaling pathways in living cells remain challenging to study, due to a lack of non-invasive tools for temporal assessment of signal transduction in desired cell models. Here we report a dual-reporter strategy that enables researchers to monitor signal transduction in mammalian cells in real-time, both temporally and quantitatively. This is achieved by co-expressing green fluorescent protein and firefly luciferase in response to signaling stimuli. To display the versatility of this approach, we constructed and assessed eight unique signaling pathway reporters. We further validated the system by establishing stable NF-κB pathway reporter cell lines. Using these stable cell lines, we monitored the activity of NF-κB-mediated inflammatory pathway in real-time, both visually and quantitatively. Live visualization has the power to reveal individual cell responses and is compatible with single cell analysis, In addition, we provide evidence that this system is readily amenable to a high-throughput format. Together, our findings demonstrate the potential of the dual reporter system, which significantly improves the capacity to study signal transduction pathways in mammalian cells.