Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.
Septal penetration of high-energy photons affects quantitative results in imaging of 123 I-labeled tracers. We investigated acquisition protocols (collimator choice and energy window setting) and correction methods for estimating the heart-to-mediastinum (H/M) ratio in cardiac 123 I-metaiodobenzylguanidine (MIBG) imaging. Methods: Four hours after 123 I-MIBG injection, 40 patients successively underwent planar anterior chest imaging with the medium-energy (ME) (ME method) and low-energy high-resolution (LEHR) (LEHR method) collimators. A 20% energy window was used for both collimators. Another 40 patients were imaged successively with the ME collimator and a 20% window (ME method), the low-medium-energy (LME) collimator and a 20% window (LME20 method), and the LME collimator and a 15% window (LME15 method). The H/M ratios obtained by the LEHR, LME20, and LME15 methods were corrected using their correlations with the H/M ratio obtained by the ME method (empiric correction). The 123 I-dual-window (IDW) correction was also applied to remove the influence of high-energy photons. Results: Without correction, severe underestimation of the H/M ratio was shown for the LEHR method using the ME method as a standard, and this underestimation increased with increasing H/M ratios. Underestimation substantially decreased using the LME20 method and further using the LME15 method. Empiric correction reduced the error in the H/M ratio by the LEHR method, but the error was still evident. After empiric correction, the H/M ratios with the LME collimator were comparable to those with the ME collimator. The IDW correction only partially reduced underestimation by the LEHR method and caused a small overestimation for the LME15 method. Conclusion: The use of an LME collimator appears to be acceptable for cardiac 123 I-MIBG imaging as an alternative to an ME collimator, and the application of a 15% energy window is recommended when an LME collimator is used. Empiric correction is also expected to improve exchangeability between H/M ratios calculated with ME and LME collimators. Neither the use of an LEHR collimator nor the use of IDW correction is recommended.
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