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A controllable cleavage activity of inteins to release target proteins from affinity tags is an attractive approach for the production of heterologous proteins. Expression of CellBD-GyrA-bfgf (CGF), a fusion precursor comprising the cellulose binding domain (CellBD) of an endoglucanase (Eng) encoded by the cenA gene of Cellulomonas fimi at the N-terminus, Mxe GyrA intein (GyrA) in the middle, and human basic fibroblast growth factor (bfgf) at the C-terminus, resulted in incomplete auto-catalytic cleavages and a partial release of free bFGF in the cytosol of Escherichia coli. Amino acid substitutions involving Cys1Ala (C1A) and His197Gln (H197Q) performed simultaneously at both termini of GyrA on CellBD-GyrA-bfgf, thus forming the 49-kDa full-length CGF (H197Q; C1A) mutant precursor (49-kDa MP), were found to exhibit an inhibitory effect on the cleavage detected between CellBD and GyrA, thereby enhancing the reservation of intact 49- kDa MP, which was shown to bind well to Avicel. Sequencing analysis showed that 49-kDa MP underwent complex auto-processing activities involving self-cleavages, proteolysis and fusion of degraded fragments to give rise to a variety of smaller intermediates. One such intermediate, 39-kDa (SI), was also found to be able to bind to Avicel. Free bFGF was effectively cleaved from 49-kDa MP and 39-kDa SI bound on Avicel at pH 8.5 or in the presence of succinic acid. Released bFGF was substantiated to possess the authentic structure and potent bioactivity. The findings may pave way for the development of a facile scalable approach for enhancing production of a heterologous protein. Keywords: Mxe GyrA Intein; Site-Specific Mutagenesis; Cellulose Binding Domain; Avicel; bFGF ; in vitro Cleavages; pH 8.5 ; Succinic Acid

Purpose Tomotherapy delivers an intensity-modulated radiation therapy (IMRT) treatment by the synchronization of gantry rotation, multileaf collimator (MLC), and couch movement. This dynamic nature makes the quality assurance (QA) important and challenging. The purpose of this study is to develop some methodologies using an ArcCHECK for accurate QA measurements of the gantry angle and speed, MLC synchronization and leaf open time, couch translation per gantry rotation, couch speed and uniformity, and constancy of longitudinal beam profile for a Tomotherapy unit. Methods Four test plans recommended by AAPM Task Group 148 (TG148) and the manufacturer were chosen for this study. Helical and static star shot tests are used for checking the leaves opened at the expected gantry angles. Another helical test is to verify the couch traveled the expected distance per gantry rotation. The final test is for checking the couch speed constancy with a static gantry. ArcCHECK can record the detector signal every 50 ms as a movie file, and has a virtual inclinometer for gantry angle measurement. These features made the measurement of gantry angle and speed, MLC synchronization and leaf open time, and longitudinal beam profile possible. A shaping parameter was defined for facilitating the location of the beam center during the plan delivery, which was thereafter used to calculate the couch translation per gantry rotation and couch speed. The full width at half maximum (FWHM) was calculated for each measured longitudinal beam profile and then used to evaluate the couch speed uniformity. Furthermore, a mean longitudinal profile was obtained for constancy check of field width. The machine trajectory log data were also collected for comparison. Inhouse programs were developed in MATLAB to process both the ArcCHECK and machine log data. Results The deviation of our measurement results from the log data for gantry angle was calculated to be less than 0.4°. The percentage differences between measured and planned leaf open time were found to be within 0.5% in all the tests. Our results showed mean values of MLC synchronization of 0.982, 0.983, and 0.995 at static gantry angle 0°, 45°, and 135°, respectively. The mean value of measured couch translation and couch speed by ArcCHECK had less than 0.1% deviation from the planned values. The variation in the value of FWHM suggested the couch speed uniformity was better than 1%. The mean of measured longitudinal profiles was suitable for constancy check of field width. Conclusion Precise and efficient methods for measuring the gantry angle and speed, leaf open time, couch translation per gantry rotation, couch speed and uniformity, and constancy of longitudinal beam profile of Tomotherapy using ArcCHECK have been developed and proven to be accurate compared with machine log data. Estimation of the Tomotherapy binary MLC leaf open time is proven to be precise enough to verify the leaf open time as small as 277.8 ms. Our method also makes the observation and quantification of the synchronization of leaves possible.

Although evaporimetry (the measurement of water vapour flux density from the skin) has often been used to study the impact on skin hydration of using products such as baby diapers and incontinence pads, it is difficult to interpret results and to compare data from different studies because of the diversity of unvalidated methodologies used. The aim of this work was to develop a robust methodology for measuring the excess water in over-hydrated skin and test it on volar forearm and hip skin which had been occluded with saline soaked patches. Three repeat measurements were made on the volar forearm and the hip of five young (31-44 years) and six older (67-85 years) women and moderately good within-subject repeatability was found for both skin sites for both subject groups. Measurements taken from the hip were significantly higher (P = 0.001) than those from the arm and had larger coefficients of variation (3.5-22.1%) compared to arms (3.0-14.0%). There were no significant differences between young and older skin, implying that women for future studies could be recruited without regard to age. This is the first time that a robust evaporimetric methodology for quantifying excess water in over-hydrated skin has been described and validated, and it will form a solid basis for future work.

A new method for measuring the coefficient of friction between nonwoven materials and the curved surface of the volar forearm has been developed and validated. The method was used to measure the coefficient of static friction for three different nonwoven materials on the normal (dry) and over-hydrated volar forearms of five female volunteers (ages 18–44). The method proved simple to run and had good repeatability: the coefficient of variation (standard deviation expressed as a percentage of the mean) for triplets of repeat measurements was usually (80 per cent of the time) less than 10 per cent. Measurements involving the geometrically simpler configuration of pulling a weighted fabric sample horizontally across a quasi-planar area of volar forearm skin proved experimentally more difficult and had poorer repeatability. However, correlations between values of coefficient of static friction derived using the two methods were good ( R = 0.81 for normal (dry) skin, and 0.91 for over-hydrated skin). Measurements of the coefficient of static friction for the three nonwovens for normal (dry) and for over-hydrated skin varied in the ranges of about 0.3–0.5 and 0.9–1.3, respectively. In agreement with Amontons' law, coefficients of friction were invariant with normal pressure over the entire experimental range (0.1–8.2 kPa).

Production of recombinant proteins by excretory expression has many advantages over intracellular expression in Escherichia coli. Hyperexpression of a secretory exoglucanase, Exg, of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of E. coli. In this study, we demonstrated that overexpression of the PspA in the JM101(pM1VegGcexL-pspA) strain enhanced excretion of Exg to 1.65 U/ml using shake-flask cultivation, which was 80% higher than the highest yield previously obtained from the optimized JM101(pM1VegGcexL) strain. A much higher excreted Exg activity of 4.5 U/ml was further achieved with high cell density cultivation using rich media. Furthermore, we showed that the PspA overexpression strain enjoyed an elevated critical value (CV), which was defined as the largest quotient between the intracellular unprocessed precursor and its secreted mature counterpart that was still tolerable by the host cells prior to the onset of cell death, improving from the previously determined CV of 20/80 to the currently achieved CV of 45/55 for Exg. The results suggested that the PspA overexpression strain might tolerate a higher level of precursor Exg making use of the SecYEG pathway for secretion. The reduced lethal effect might be attributable to the overexpressed PspA, which was postulated to be able to reduce membrane depolarization and damage. Our findings introduce a novel strategy of the combined application of metabolic engineering and construct optimization to the attainment of the best possible E. coli producers for secretory/excretory production of recombinant proteins, using Exg as the model protein.

An analytical mathematical model for friction between a fabric strip and the volar forearm has been developed and validated experimentally. The model generalizes the common assumption of a cylindrical arm to any convex prism, and makes predictions for pressure and tension based on Amontons' law. This includes a relationship between the coefficient of static friction ( μ) and forces on either end of a fabric strip in contact with part of the surface of the arm and perpendicular to its axis. Coefficients of friction were determined from experiments between arm phantoms of circular and elliptical cross-section (made from Plaster of Paris covered in Neoprene) and a nonwoven fabric. As predicted by the model, all values of μ calculated from experimental results agreed within ±8 per cent, and showed very little systematic variation with the deadweight, geometry, or arc of contact used. With an appropriate choice of coordinates the relationship predicted by this model for forces on either end of a fabric strip reduces to the prediction from the common model for circular arms. This helps to explain the surprisingly accurate values of μ obtained by applying the cylindrical model to experimental data on real arms.