MRI of Lymphedema.

Lymphedema is a devastating disease that has no cure. Management of lymphedema has evolved rapidly over the past two decades with the advent of surgeries that can ameliorate symptoms. MRI has played an increasingly important role in the diagnosis and evaluation of lymphedema, as it provides high spatial resolution of the distribution and severity of soft tissue edema, characterizes diseased lymphatic channels, and assesses secondary effects such as fat hypertrophy. Many different MR techniques have been developed for the evaluation of lymphedema, and the modality can be tailored to suit the needs of a lymphatic clinic. In this review article we provide an overview of lymphedema, current management options, and the current role of MRI in lymphedema diagnosis and management. EVIDENCE LEVEL: 5 TECHNICAL EFFICACY: Stage 5.

Comparison of End-Expiration Versus End-Inspiration Breath-Holds With Respect to Respiratory Motion Artifacts on T1-Weighted Abdominal MRI.

OBJECTIVE. The purpose of this study was to compare respiratory motion artifact and diagnostic image quality between end-inspiration and end-expiration breath-holding techniques on unenhanced and contrast-enhanced axial T1-weighted MRI of the liver. MATERIALS AND METHODS. This retrospective observational study included 50 consecutive subjects undergoing axial T1-weighted liver MRI, with unenhanced images acquired with both end-inspiration and end-expiration breath-holding techniques, and with contrast-enhanced images acquired for 47 of the subjects with either the end-inspiration or the end-expiration breath-holding technique. Three radiologists performed blinded independent evaluations of each unenhanced sequence, contrast-enhanced sequence, and subtraction (contrast-enhanced minus unenhanced) image, using a scale ranging from 1 point (denoting nondiagnostic imaging) to 5 points (denoting no artifacts). Blinded side-by-side assessment of each pair of unenhanced sequences was also performed. Two-tailed Wilcoxon signed rank and Wilcoxon rank sum tests were used to assess statistical significance. RESULTS. A significant improvement in motion scores was noted for sequences acquired in end-expiration, compared with those acquired in end-inspiration, for unenhanced sequences (mean, 3.35 vs 2.80; p < 0.00001), contrast-enhanced sequences (mean, 4.02 vs 3.46; p = 0.0003), and subtraction images (mean, 3.67 vs 2.41; p < 0.00001). Severe degradation of image quality or nondiagnostic image quality was noted for 15% of unenhanced images (23/150), 0% of contrast-enhanced images, and 8% (5/63) of subtraction images acquired on end-expiration, whereas it was noted for 36% (54/150) of unenhanced images, 13% (10/78) of contrast-enhanced images, and 59% (46/78) of subtraction images acquired on end-inspiration. When side-by-side assessment of paired unenhanced sequences was performed, images acquired in end-expiration were significantly favored in 59% of paired sequences (88/150) (p < 0.00001), and no difference between images acquired with both breath-hold techniques was noted for 21% (32/150) of paired sequences. CONCLUSION. The end-expiration breath-holding technique leads to significant decreases in respiratory motion artifacts, compared with the end-inspiration technique, on unenhanced and contrast-enhanced T1-weighted liver MRI.

BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals.

Taking advantage of the bioluminescence resonance energy transfer (BRET) phenomenon, we report the development of a highly photon-efficient, self-illuminating fusion protein combining a mutant red fluorescent protein (mOrange) and a mutant Renilla reniformis luciferase (RLuc8). This new BRET fusion protein (BRET3) exhibits severalfold improvement in light intensity in comparison with existing BRET fusion proteins. BRET3 also exhibits the most red-shifted light output (564-nm peak wavelength) of any reported bioluminescent protein that utilizes its natural substrate coelenterazine, a benefit of which is demonstrated at various tissue depths in small animals. The imaging utility of BRET3 at the single-cell level is demonstrated using an intramolecular sensor incorporating two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. With its increased photon intensity, red-shifted light output, and good spectral resolution (approximately 85 nm), BRET3 shows improved spatial and temporal resolution for measuring intracellular events in single cells and in living small animal models. The development of further BRET3-based assays will allow imaging of protein-protein interactions using a single assay directly scalable from intact living cells to small living subjects, allowing accelerated drug discovery.

An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects.

Bioluminescence resonance energy transfer (BRET) is currently used for monitoring various intracellular events, including protein-protein interactions, in normal and aberrant signal transduction pathways. However, the BRET vectors currently used lack adequate sensitivity for imaging events of interest from both single living cells and small living subjects. Taking advantage of the critical relationship of BRET efficiency and donor quantum efficiency, we report generation of a novel BRET vector by fusing a GFP(2) acceptor protein with a novel mutant Renilla luciferase donor selected for higher quantum yield. This new BRET vector shows an overall 5.5-fold improvement in the BRET ratio, thereby greatly enhancing the dynamic range of the BRET signal. This new BRET strategy provides a unique platform to assay protein functions from both single live cells and cells located deep within small living subjects. The imaging utility of the new BRET vector is shown by constructing a sensor using two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. This new BRET vector should facilitate high-throughput sensitive BRET assays, including studies in single live cells and small living subjects. Applications will include anticancer therapy screening in cell culture and in small living animals.

Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output.

Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational analysis were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output. These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.

Red-shifted Renilla reniformis luciferase variants for imaging in living subjects.

The use of R. reniformis luciferase (RLuc) as a reporter gene in small-animal imaging has been hampered by its 481 nm peaked emission spectrum, as blue wavelengths are strongly attenuated in biological tissues. To overcome this, we generated variants of RLuc with bathochromic (red) shifts of up to 66 nm (547 nm peak) that also had greater stability and higher light emission than native RLuc.

Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis.

Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 A and demonstrate a classic alpha/beta-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.

AMIDE: a free software tool for multimodality medical image analysis.

Amide's a Medical Image Data Examiner (AMIDE) has been developed as a user-friendly, open-source software tool for displaying and analyzing multimodality volumetric medical images. Central to the package's abilities to simultaneously display multiple data sets (e.g., PET, CT, MRI) and regions of interest is the on-demand data reslicing implemented within the program. Data sets can be freely shifted, rotated, viewed, and analyzed with the program automatically handling interpolation as needed from the original data. Validation has been performed by comparing the output of AMIDE with that of several existing software packages. AMIDE runs on UNIX, Macintosh OS X, and Microsoft Windows platforms, and it is freely available with source code under the terms of the GNU General Public License.