Simulation approach for the evaluation of tracking accuracy in radiotherapy: A preliminary study

Real-time tumor tracking in external radiotherapy can be achieved by diagnostic (kV) X-ray imaging with a dynamic flat-panel detector (FPD). It is important to keep the patient dose as low as possible while maintaining tracking accuracy. A simulation approach would be helpful to optimize the imaging conditions. This study was performed to develop a computer simulation platform based on a noise property of the imaging system for the evaluation of tracking accuracy at any noise level. Flat-field images were obtained using a direct-type dynamic FPD, and noise power spectrum (NPS) analysis was performed. The relationship between incident quantum number and pixel value was addressed, and a conversion function was created. The pixel values were converted into a map of quantum number using the conversion function, and the map was then input into the random number generator to simulate image noise. Simulation images were provided at different noise levels by changing the incident quantum numbers. Subsequently, an implanted marker was tracked automatically and the maximum tracking errors were calculated at different noise levels. The results indicated that the maximum tracking error increased with decreasing incident quantum number in flat-field images with an implanted marker. In addition, the range of errors increased with decreasing incident quantum number. The present method could be used to determine the relationship between image noise and tracking accuracy. The results indicated that the simulation approach would aid in determining exposure dose conditions according to the necessary tracking accuracy. © The Author 2012. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Therapeutic Radiology and Oncology

Double knockdown of α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC

<p>Abstract</p> <p>Background</p> <p>Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans <it>in vivo</it>. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies.</p> <p>Results</p> <p>Firstly, loss-of-function analyses using small interfering RNAs (siRNAs) against the three key genes involved in oligosaccharide fucose modification, i.e. α1,6-fucosyltransferase (<it>FUT8</it>), GDP-mannose 4,6-dehydratase (<it>GMD</it>), and GDP-fucose transporter (<it>GFT</it>), revealed that single-gene knockdown of each target was insufficient to completely defucosylate the products in antibody-producing cells, even though the most effective siRNA (>90% depression of the target mRNA) was employed. Interestingly, beyond our expectations, synergistic effects of <it>FUT8 </it>and <it>GMD </it>siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with <it>GFT </it>siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of <it>FUT8 </it>and <it>GMD</it>, and we converted antibody-producing Chinese hamster ovary (CHO) cells to fully non-fucosylated antibody producers within two months, and with high converting frequency. Finally, the stable manufacture of fully non-fucosylated antibodies with enhanced ADCC was confirmed using the converted cells in serum-free fed-batch culture.</p> <p>Conclusion</p> <p>Our results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of <it>FUT8 </it>and <it>GMD </it>in antibody-producing cells could serve as a new strategy for producing next-generation therapeutic antibodies fully lacking core fucosylation and with enhanced ADCC. This approach offers tremendous cost- and time-sparing advantages for the development of next-generation therapeutic antibodies.</p

A Proteomic Approach for Comprehensively Screening Substrates of Protein Kinases Such as Rho-Kinase

BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity

Effects of image lag on real-time target tracking in radiotherapy

There is a concern that image lag may reduce accuracy of real-time target tracking in radiotherapy. This study was performed to investigate influence of image lag on the accuracy of target tracking in radiotherapy. Fluoroscopic image: were obtained using a direct type of dynamic flat-panel detector (FPD) system under conditions of target tracking during radiotherapy. The images continued to be read out after X-irradiations and cutoff, and image lag properties in the system were then determined. Subsequently, a tungsten materials plate with a precision edge was mounted on to a motor control device, which provided a constant velocity. The plate was moved into the center of the detector at movement rate of 1 and 20 mm/s, covering lung tumor movement of normal breathing, and MTF and profile curves were measured on the edges covering and uncovering the detector. A lung tumor with blurred edge due to image lag was simulated using the results and then superimposed on breathing chest radiographs of a patient. The moving target with and without image lag was traced using a template-matching technique. In the results, the target could be traced within a margin for error ii external radiotherapy. The results indicated that there was no effect of image lag on target tracking in usual breathing speed in a radiotherapy situation. Further studies are required to investigate influence by the other factors, such a: exposure dose, target size and shape, imaging rate, and thickness of a patient\u27s body. © 2010 SPIE