A comparison of clinic based dosimeters based on silica optical fibre and plastic optical fibre for in-vivo dosimetry

Four sensors based on silica optical fibre and plastic optical fibre for clinical in-vivo dosimetry have been fabricated and tested on site at Galway Clinic. The initial comparison results have been attained for the four sensors when they have been irradiated with beam energies of 6 MV and 15 MV at different dose rates using a modern clinical linear accelerator (Linac) as the radiation source. According to the experimental test results, the sensors based on silica optical fibre exhibit greater sensitivity to the incident radiation beam than the sensors based on plastic optical fibre when they are exposed to identical irradiation conditions. The output intensity from the sensor based on silica fibre is 5 times higher than the sensor based on plastic optical fibre

Highly sensitive extrinsic X-ray polymer optical fiber sensors based on fiber tip modification

An extrinsic X-ray fiber sensor with improved sensitivity is presented. The developed device is based on an inorganic scintillator (Terbium doped Gadolinium Oxysulfide) attached to a polymer fiber tip. Sensitivity improvement has been accomplished by modifying the fiber tip in two ways, either by thermomechanical tapering of chemical etching. Thus, the useful surface in contact with the scintillator increases, and so the fluorescence light gathering capability. All the fabricated devices have been tested under X-ray irradiation from a Linac at a dose rate of 300 monitor units/min, with a readout every 100 ms. The obtained results shown a signal improvement of up to 43 times when compared with previous reported proposals using inorganic scintillators and polymer fibers. Moreover, the recorded signal under the same measurement conditions can be further improved if the inorganic scintillator is used in powder format instead into an epoxy matrix

Advanced characterization of an optical fibre sensor system based on an MPPC detector for measurement of X-ray radiation in clinical linacs

A reliable, accurate and in-vivo dosimetry system for measuring the radiation dose and profiling the X-ray beam during radiotherapy is reported. Its dynamic range is investigated using an accurately controlled pulsed light emitting diode (LED) system. Highly resolved temporal analog and digital signals were captured from the analog and digital outputs of a multi-pixel photon counter (MPPC) detector when exposed to the LED system. The photon distribution of a low intensity pulsed LED light source was observed and is found to obey a Poisson distribution with changing light intensity. The average number of photons was obtained using the digital MPPC output signals which in turn allowed the appropriate intensity of the light source to be determined for the correct light exposure conditions for the detector. The average analog output voltage over a single 3 μs pulse is determined to indicate the intensity of the detected light. The MPPC detector output analog signal is limited to a narrow range (0.6 V to 1.4 V) to ensure adequate signal detection level (the lower limit) and prevention of entry into saturation (the upper limit) which also corresponds to a digital output signal range (in counts). An average photon number range of 3 to 7 for the digital output signal is established, which leads to the establishment of a unique and constant photon number to average output voltage ratio of 4.64 ± 0.10. Experimental results show that the establishment of this ratio is significant as adherence to it ensures the correct exposure conditions of the MPPC and speeds up the measurement cycle in the clinical setting

Proteomics Analysis of Co-Purifying Cellular Proteins Associated with rAAV Vectors

<div><p>Recombinant adeno-associated vectors (rAAV) are commonly purified by either chromatography or equilibrium CsCl gradient. Nevertheless, even after purification various cellular proteins often associate with rAAV vector capsids. Such co-purifying cellular proteins may raise concern about safety of gene therapy. Here we report identification and characterization of the co-purifying cellular protein in the vector preparations by using a combination of two proteomics approaches, GeLC-MS (gel electrophoresis liquid chromatography-mass spectrometry) and 2DE (two-dimensional gel electrophoresis). Most prominent bands revealed by Coomassie Blue staining were mostly similar to the AAV capsid proteins. Posttranslational modifications of capsid proteins were detected by the proteomics analysis. A total of 13 cellular proteins were identified in the rAAV vectors purified by two rounds of cesium chloride gradient centrifugation, including 9 by the GeLC-MS analysis and 4 by the 2DE analysis. Selected cellular proteins were verified by western blot. Furthermore, the cellular proteins could be consistently found associated with different AAV serotypes and carrying different transgenes. Yet, the proteins were not integral components of the viral capsis since a stringent washing procedure by column purification could remove them. These co-purified proteins in AAV vector preparations may have a role in various stages of the AAV life cycle.</p></div

Detection of protein SET in different fractions of the AAV2-dsEGFP preparation.

<p>The vector was produced by triple plasmid transfection and purified by two rounds of cesium chloride ultra-centrifugation. The fractions with different densities were collected after the second round of ultra-centrifugation. Protein form 1×10¹⁰ viral particles was resolved on 10% SDS/PAGE. The full AAV vector particles have buoyant densities in CsCl from 1.41 to 1.45 g/cm³ while empty particles have the density of 1.32 g/cm³. A, silver staining; and B, western blot.</p

2DE map of recombinant AAV vector.

<p>The top 2DE map was obtained using a wide pH range of IPG strip pH–10, and the bottom one using a narrow pH range of IPG strip pH 4–7. The circled and numbered spots were identified as human proteins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086453#pone-0086453-t002" target="_blank">Table 2</a>), the spots only circled were identified as AAV capsid proteins.</p

List of human cellular proteins identified from 2DE analysis via nano-LC IT MS.

<p>List of human cellular proteins identified from 2DE analysis via nano-LC IT MS.</p

List of cellular proteins identified using GeLC-MS analysis of adeno-associated virus vectors.

<p>List of cellular proteins identified using GeLC-MS analysis of adeno-associated virus vectors.</p

Identification of celluar proteins co-purifed with recombinant AAV vector.

<p>A, 1DE-SDS PAGE of recombinant AAV vector. The seven marked bands were excised and processed for MALDI-TOF analysis. B, All seven bands were identified as capsid-related proteins by the sequence homology. The Mowse score and the number of peptides matched were from the matching results against capsid protein VP1.</p

The effect of AAV serotypes, transgenes and purification methods on the presence of SET proteins in the rAAV vector preparations.

<p>Protein from 1×1010 viral particles was resolved on 10% SDS/PAGE. AAV2-EGFP/C and AAV2-FIX/C were purified by ion exchange chromatography. A, silver staining; and B, western blot.</p