RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.

Transmission X-ray microscopy (TXM) has been well recognized as a powerful tool for non-destructive investigation of the three-dimensional inner structure of a sample with spatial resolution down to a few tens of nanometers, especially when combined with synchrotron radiation sources. Recent developments of this technique have presented a need for new tools for both system control and data analysis. Here a software package developed in MATLAB for script command generation and analysis of TXM data is presented. The first toolkit, the script generator, allows automating complex experimental tasks which involve up to several thousand motor movements. The second package was designed to accomplish computationally intense tasks such as data processing of mosaic and mosaic tomography datasets; dual-energy contrast imaging, where data are recorded above and below a specific X-ray absorption edge; and TXM X-ray absorption near-edge structure imaging datasets. Furthermore, analytical and iterative tomography reconstruction algorithms were implemented. The compiled software package is freely available.

These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin-or (6). However, the same techniques have not been used successfully to study pDNA nuclear transport because <1% of the cells expressed the cytoplasmically injected pDNA (7-10). The absence of expression cannot be explained by a problem with the microinjection technique, since 50-100% of the cells showed protein expression following injection of the pDNA directly into the nucleus. These results argue against the common assumption that the efficiency of pDNA expression could be enhanced by increased cytoplasmic delivery (4). In addition, they have precluded the use of current microinjection techniques to study the mechanisms of pDNA nuclear transport in these cell types.In the present study, up to two-thirds of primary rat myotubes that were cytoplasmically microinjected with 3-galactosidase expression plasmids had 13-galactosidase activity. The ability for cytoplasmically injected pDNA to be expressed with relatively high efficiency in myotubes provided a system for the study of pDNA nuclear transport.

We report on the characterization of nanometer-scale resonators. Each device incorporates one multiwalled carbon nanotube (MWNT) as a torsional spring. The devices are actuated electrostatically, and their deflections, both low frequency and on resonance, are detected optically. These are some of the smallest electromechanical devices ever created and are a demonstration of practical integrated MWNT-based oscillators. The results also show surprising intershell mechanical coupling behavior in the MWNTs.

We report on the characterization of torsional oscillators which use multiwalled carbon nanotubes as the spring elements. Through atomic-force-microscope force-distance measurements we are able to apply torsional strains to the nanotubes and measure their torsional spring constants, and estimate their effective shear moduli. The data show that the nanotubes are stiffened by repeated flexing. We speculate that changes in the intershell mechanical coupling are responsible for the stiffening.

Previously, we showed that rodent muscle has the ability to take up and express plasmid genes injected intramuscularly. This study now demonstrates that nonhuman primate muscle also has this ability to express injected plasmids. A scaled-up version of the standard large preparation of plasmid DNA allowed several tens of milligrams of CCC plasmid DNA to be relatively easily produced and administered to monkeys. After the injection of the E. coli beta-galactosidase reporter gene in pRSVLac-Z, foreign gene expression was localized to both type I and type II myofibers. The luciferase reporter gene in pRSVL was used to quantify the amount of expression. The multiple implantation of plasmid DNA pellets was more efficient in expressing luciferase than the injection of DNA in normal saline. Luciferase activity persisted for at least 4 months after injection. However, the luciferase expression was considerably less than that in rodents. Preliminary studies explored why expression was less in monkeys. Of particular interest was the increased thickness of the perimysium of monkeys as compared to that in rodents. This increased connective tissue may decrease delivery of the plasmid DNA to the myofibers. Anti-nuclear or anti-DNA antibodies were not observed, even after repetitive DNA administrations, and no adverse effects were observed in any of the monkeys.

The present data suggest that if promoter inactivation can be overcome, intravascular delivery of plasmid DNA could be a highly efficient, simple and non-toxic liver gene therapy approach. Intravascular delivery of pDNA allows for the rapid screening of novel expression vectors in vivo.

Mutations in some mismatch repair (MMR) genes are associated with Lynch syndrome (LS; also called hereditary nonpolyposis colorectal cancer [HNPCC]), an autosomal dominant cancer susceptibility syndrome. Colorectal cancer (CRC) is the most frequent cancer observed in LS. However, tumors occur at a variety of extracolonic sites and individuals may have multiple primary cancers. LS is the most common hereditary form of CRC, accounting for approximately 1% of all CRC. Since the first account of mutations in MSH2 causing this cancer susceptibility syndrome in 1993, mutations in three additional MMR genes, MLH1, MSH6, and PMS2, have been shown to cause LS. More than 1,500 different variants have been identified in these four genes and approximately 80% of the alterations have been identified in MLH1 and MSH2. There have been a few previous attempts to systematically record MMR variants associated with LS patients; however, they were not complete nor were they continuously updated. Thus, it was our goal to generate and maintain a comprehensive catalogue of MMR variants from genes known to be mutated in LS (http://www.med.mun.ca/MMRvariants; last accessed 8 February 2007). Providing such a resource should aid investigators in understanding the significance of the variants. Hum Mutat 28(7), 669–673, 2007. © 2007 Wiley‐Liss, Inc.