Comprehensive quality assurance phantom for the small animal radiation research platform (SARRP)

PurposeTo develop and test the suitability and performance of a comprehensive quality assurance (QA) phantom for the Small Animal Radiation Research Platform (SARRP).Methods and materialsA QA phantom was developed for carrying out daily, monthly and annual QA tasks including: imaging, dosimetry and treatment planning system (TPS) performance evaluation of the SARRP. The QA phantom consists of 15 (60 × 60 × 5 mm³) kV-energy tissue equivalent solid water slabs. The phantom can incorporate optically stimulated luminescence dosimeters (OSLD), Mosfet or film. One slab, with inserts and another slab with hole patterns are particularly designed for image QA.ResultsOutput constancy measurement results showed daily variations within 3%. Using the Mosfet in phantom as target, results showed that the difference between TPS calculations and measurements was within 5%. Annual QA results for the Percentage depth dose (PDD) curves, lateral beam profiles, beam flatness and beam profile symmetry were found consistent with results obtained at commissioning. PDD curves obtained using film and OSLDs showed good agreement. Image QA was performed monthly, with image-quality parameters assessed in terms of CBCT image geometric accuracy, CT number accuracy, image spatial resolution, noise and image uniformity.ConclusionsThe results show that the developed QA phantom can be employed as a tool for comprehensive performance evaluation of the SARRP. The study provides a useful reference for development of a comprehensive quality assurance program for the SARRP and other similar small animal irradiators, with proposed tolerances and frequency of required tests.     

Estimation of extremely small field radiation dose for brain stereotactic radiotherapy using the Vero4DRT system

The purpose of this study was a dosimetric validation of the Vero4DRT for brain stereotactic radiotherapy (SRT) with extremely small fields calculated by the treatment planning system (TPS) iPlan (Ver.4.5.1; algorithm XVMC). Measured and calculated data (e.g. percentage depth dose [PDD], dose profile, and point dose) were compared for small square fields of 30 × 30, 20 × 20, 10 × 10 and 5 × 5 mm² using ionization chambers of 0.01 or 0.04 cm³ and a diamond detector. Dose verifications were performed using an ionization chamber and radiochromic film (EBT3; the equivalent field sizes used were 8.2, 8.7, 8.9, 9.5, and 12.9 mm²) for five brain SRT cases irradiated with dynamic conformal arcs.The PDDs and dose profiles for the measured and calculated data were in good agreement for fields larger than or equal to 10 × 10 mm² when an appropriate detector was chosen. The dose differences for point doses in fields of 30 × 30, 20 × 20, 10 × 10 and 5 × 5 mm² were +0.48%, +0.56%, −0.52%, and +11.2% respectively. In the dose verifications for the brain SRT plans, the mean dose difference between the calculated and measured doses were −0.35% (range, −0.94% to +0.47%), with the average pass rates for the gamma index under the 3%/2 mm criterion being 96.71%, 93.37%, and 97.58% for coronal, sagittal, and axial planes respectively.The Vero4DRT system provides accurate delivery of radiation dose for small fields larger than or equal to 10 × 10 mm².     

An integrated database for managing animal study proposals and animal inventory for the small animal facility

The authors describe how they created a user-friendly, multifunctional database for use by a variety of vivarium staff.     

Feasibility study of the Fricke chemical dosimeter as an independent dosimetric system for the small animal radiation research platform (SARRP)

For the small animal radiation research platform (SARRP) with X-ray beams in the medium energy range (tube operating voltage at 220 kVp), reference dosimetry is based on the AAPM TG-61 recommendations following the in-phantom method. The objective of this study was to evaluate the feasibility of the Fricke solution as a dosimeter to determine the absorbed dose to water. Feasibility studies at this X-ray energy range are not widely available. We evaluated the accuracy, dose linearity and dose rate dependence in a comparison with an NE 2571 Farmer ionization chamber (IC) and measurements in water. The G(Fe³⁺) factor was calculated from the curve fitting of the chemical yields for two radioactive sources (¹⁹²Ir and ⁶⁰Co) and one X-ray system with a tube operating at 150 and 250 kVp. The same methodology was followed for the dependence of the G(Fe³⁺) value on the energy and the dose agreement assessment for 180 and 200 kVp in the SARRP. The Fricke system exhibits a good linear response over the range of 5–70 Gy and an accuracy better than 2% for a 2 Gy/min dose rate. The dose rate dependence is smaller than 1% for dose rates greater than 1 Gy/min. The dependence of the G(Fe³⁺) value on the energy is smaller than 0.41%, with dose agreements better than 2%. The feasibility of the dosimeter for measurements at high doses and high dose rates makes it a suitable tool for dosimetric verifications in several preclinical irradiation configurations.     

An accurate and reproducible method for proteome profiling of the effects of salt stress in the rice leaf lamina

Proteomic analysis of any biological system by two-dimensional gel electrophoresis (2-DE) requires high resolution and high reproducibility. The results presented here demonstrate the reproducible and accurate separation of rice (Oryza sativa L.) proteins using improved procedures for high resolution 2-DE, which were adapted for the separation of rice lamina proteins. Validation of this system was achieved by measuring the effects of sample preparation and biological variation on the coefficient of variation (CV) for replicate spots. The majority of experimental variation was shown to be introduced by the 2-DE technique (CV 0.26). Analysis of biological variation indicated that approximately 93-95% of spots were within a CV of 0.7. This provided a threshold value from which valid differences in expression between experimental groups could be screened. This system was then utilized for the proteomic analysis of short- and long-term salt-stress-responsive proteins in the rice leaf lamina. Analysis resulted in the separation of approximately 2500 protein species of which 32 were observed to be significantly regulated by salinity; so far 11 of these proteins have been identified by tandem mass spectrometry. An increase in eight proteins, including RuBisCO activase and ferritin, occurred by 24 h of exposure to sodium chloride (50 mM) and continued to increase during the following 6 d. Only one protein, a putative phosphoglycerate kinase, was found to increase in expression within 24 h and did not increase over a longer period of exposure to salt. There were also proteins that showed no change 24 h after exposure to salt, but had increased (superoxide dismutase) or decreased (S-adenosyl-L-methionine synthetase) after 7 d salt treatment.     

An efficient and reproducible method for transformation of genetically recalcitrant bifidobacteria

The epidemic community-associated methicillin-resistant clone Staphylococcus aureus USA300 is a major source of skin and soft tissue infections and involves strains with a diverse set of resistance genes. In this study, we report efficient transduction of penicillinase and tetracycline resistance plasmids by bacteriophages φ80α and φJB between clinical isolates belonging to the USA300 clone. High transduction frequencies (10(-5) - 10(-6) CFU/PFU) were observed using phages propagated on donor strains as well as prophages induced from donors by ultraviolet light. Quantitative real-time PCR was employed to detect penicillinase plasmids in transducing phage particles and determine the ratio of transducing particles in phage lysates to infectious phage particles (determined as approximately 1 : 1700). Successful transfer of plasmids between strains in USA300 clone proves transduction is an effective mechanism for spreading plasmids within the clone. Such events contribute to its evolution and to emergence of new multiple drug-resistant strains of this successful clone.     

An accurate method for estimating sizes of small and large plasmids and DNA fragments by gel electrophoresis

Several regression methods were tested for estimating the sizes of a wide range of plasmids (1.37-312 MDa) and restriction fragments (2.2-14.2 MDa) by agarose gel electrophoresis. The most accurate and least variable method was the multiple regression of log10 molecular size against log10 relative mobility and the reciprocal square root of the relative mobility. This method gave a good fit to all the data with low percentage errors of the molecular size estimates (less than or equal to 3.0 +/- 1.5%). It is suggested that with this method the molecular size of unknown plasmids can be accurately estimated using the plasmids from Escherichia coli V517 and E. coli IR713 as standards.     

An animal model for tumors of the large intestine]

Dimethylhydrazine (DMH) and its derivatives induced in many animal species tumors of the large intestine with a great specificity and reproductibility. Oour personal observations using rats BD IX injected weekly with DMH are summarized in this presentation. The induced tumors appear as invaded tumors already at the microscopic stade. Their growth is best described by a Gompertz function. The number of induced tumors increases with time as a non linear function. This model is very useful for the study of the biology of human tumors with a low development.     

An integrated biosensor platform for extraction and detection of nucleic acids

The development of portable systems for analysis of nucleic acids (NAs) is crucial for the evolution of biosensing in the context of future healthcare technologies. The integration of NA extraction, purification, and detection modules, properly actuated by microfluidics technologies, is a key point for the development of portable diagnostic systems. In this paper, we describe an integrated biosensor platform based on a silicon–plastic hybrid lab-on-disk technology capable of managing NA extraction, purification, and detection processes in an integrated format. The sample preparation process is performed by solid-phase extraction technology using magnetic beads on a plastic disk, while detection is done through quantitative real-time polymerase chain reaction (qRT-PCR) on a miniaturized silicon device. The movement of sample and reagents is actuated by a centrifugal force induced by a disk actuator instrument. The assessment of the NA extraction and detection performance has been carried out by using hepatitis B virus (HBV) DNA genome as a biological target. The quantification of the qRT-PCR chip in the hybrid disk showed an improvement in sensitivity with respect to the qRT-PCR commercial platforms, which means an optimization of time and cost. Limit of detection and limit of quantification values of about 8 cps/reaction and 26 cps/reaction, respectively, were found by using analytical samples (synthetic clone), while the results with real samples (serum with spiked HBV genome) indicate that the system performs as well as the standard methods.     

An accurate method for estimating 5-phosphoribosyl 1-pyrophosphate in animal tissues with the use of acid extraction.

In determination of the tissue content of 5-phosphoribosyl 1-pyrophosphate (PRPP), heat treatment has been generally used for the extraction. However, the procedure appeared to need well-controlled conditions which should be determined for each different tissue by preliminary examinations. Thus, extraction with perchloric acid was examined and found to give satisfactory results if combined with rapid processing of the tissues. The decomposition of the compound, though less than 20% by the procedure used, was corrected based on the recovery of ³H-labeled PRPP which was added at the step of homogenization. The acid extraction eliminated interfering enzyme activities in the extract and assured, together with the use of the internal standard, a high degree of reproducibility. It was also shown that the duration of anoxia that inevitably happens when animal tissues are excised must be minimized to the order of a few seconds.     

Availability of applying diaphragm matching with the breath-holding technique in stereotactic body radiation therapy for liver tumors

PurposeImage-guided radiotherapy (IGRT) based on bone matching can produce large target-positioning errors because of expiration breath-hold reproducibility during stereotactic body radiation therapy (SBRT) for liver tumors. Therefore, the feasibility of diaphragm-based 3D image matching between planning computed tomography (CT) and pretreatment cone-beam CT was investigated.MethodsIn 59 liver SBRT cases, Lipiodol uptake after transarterial chemoembolization was defined as a tumor marker. Further, the relative isocenter coordinate that was obtained by Lipiodol matching was defined as the reference coordinate. The distance between the relative isocenter coordinate and reference coordinate, which was obtained from diaphragm matching and bone matching techniques, was defined as the target positioning error. Furthermore, the target positioning error between liver matching and Lipiodol matching was evaluated.ResultsThe positioning errors in all directions by the diaphragm matching were significantly smaller than those obtained by using by the bone matching technique (p < 0.05). Further, the positioning errors in the A-P and C-C directions that were obtained by using liver matching were significantly smaller than those obtained by using bone matching (p < 0.05). The estimated PTV margins calculated by the formula proposed by van Herk for diaphragm matching, liver matching, and bone matching were 5.0 mm, 5.0 mm, and 11.6 mm in the C-C direction; 3.6 mm, 2.4 mm, and 6.9 mm in the A-P direction; and 2.6 mm, 4.1 mm, and 4.6 mm in the L-R direction, respectively.ConclusionsDiaphragm matching-based IGRT may be an alternative image matching technique for determining liver tumor positions in patients.     

Distributed parameters deterministic model for treatment of brain tumors using Galerkin finite element method

In this paper, we present a distributed parameters deterministic model for treatment of brain tumors using Galerkin finite element method. The dynamic model comprises system of three coupled reaction-diffusion models, involving the tumor cells, the normal tissues and the drug concentration. An optimal control problem is formulated with the goal of minimizing the tumor cell density and reducing the side effects of the drug. A distributed parameters method based on the application of variational calculus is used on an integral-Hamiltonian, which is then used to obtain an optimal coupled system of forward state equations and backward co-state equations. The Galerkin finite element method is used to realistically represent the brain structure as well as to facilitate computation. Finally a three-dimensional test case is considered and partitioned into a set of spherical finite elements, using tri-linear basis functions, except for the elements affected by singularities of polar and azimuthal angles, as well as the origin.     

An accurate method for the quantitation of Fmoc-derivatized solid phase supports

Summary By performing the Fmoc resin loading determination with DBU instead of piperidine, highly reproducible results were obtained that showed excellent correlation with data obtained by independent analytical methods.     

An accurate method for the quantitation of Fmoc-derivatized solid phase supports

By performing the Fmoc resin loading determination with DBU instead of piperidine, highly reproducible results were obtained that showed excellent correlation with data obtained by independent analytical methods.     

Tissue Raman spectroscopy for the study of radiation damage: brain irradiation of mice

Radiotherapy is routinely employed in the treatment of head and neck cancers. Acute cell death, radiation-activated chemical cascades, and the induction of genes coding for protective factors like cytokines are considered to be the major processes involved in radiation damage and repair. It should be possible to follow these processes by monitoring the biochemical interactions initiated by radiation. We have carried out Raman spectroscopy studies on tissue from mice subjected to brain irradiation to identify the biochemical changes occurring in tissue and brain as a result of radiation injury. These studies show that brain irradiation produces drastic spectral changes even in tissue far removed from the irradiation site. The changes are very similar to those produced by the stress of inoculation and restraint and the administration of an anesthetic drug. While the changes produced by stress or anesthetics last for only a short time (a few hours to 1 or 2 days), radiation-induced changes persist even after 1 week. The spectral changes can be interpreted in terms of the observation of new spectra that are dominated by bands due to proteins. The results thus support the hypothesis that various protective factors are released throughout the body when the central nervous system (CNS) is exposed to radiation.     

A simple epigenetic method for the diagnosis and classification of brain tumors

The new, simple, and reliable method for the diagnosis of brain tumors is described. It is based on a TLC quantitative determination of 5-methylcytosine (m(5)C) in relation to its damage products of DNA from tumor tissue. Currently, there is evidence that oxidative stress through reactive oxygen species (ROS) plays an important role in the etiology and progression of several human diseases. Oxidative damage of DNA, lipids, and proteins is deleterious for the cell. m(5)C, along with other basic components of DNA, is the target for ROS, which results in the appearance of new modified nucleic acid bases. If so, m(5)C residue constitutes a mutational hotspot position, whether it occurs within a nucleotide sequence of a structural gene or a regulatory region. Here, we show the results of the analysis of 82 DNA samples taken from brain tumor tissues. DNA was isolated and hydrolyzed into nucleotides, which, after labeling with [gamma-(32)P]ATP, were separated on TLC. Chromatograms were evaluated using PhosphorImager and the amounts of 5-methyldeoxycytosine (m(5)dC) were calculated as a ratio (R) of m(5)dC to m(5)dC + deoxycytosine + deoxythymidine spot intensities. The R value could not only be a good diagnostic marker for brain tumors but also a factor differentiating low-grade and high-grade gliomas. Therefore, DNA methylation pattern might be a useful tool to give a primary diagnosis of a brain tumor or as a marker for the early detection of the relapse of the disease. This method has several advantages over those existing nowadays.     

An improved LC-MS/MS procedure for brain prostanoid analysis using brain fixation with head-focused microwave irradiation and liquid-liquid extraction

High-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) allows a highly selective, sensitive, simultaneous analysis for prostanoids (PG) without derivatization. However, high chemical background noise reduces LC-MS/MS selectivity and sensitivity for brain PG analysis. Four common methods using different solvent systems for PG extraction were tested. Although these methods had the same recovery of PG, the modified acetone extraction followed by liquid/liquid purification had the greatest sensitivity. This method combined with hexane/2-propanol extraction permits the simultaneous analysis of other lipid molecules and PG in the same extract. We also determined that PG mass in brain powder stored at -80 degrees C was reduced 2- to 4- fold in 4 weeks; however, PG were stable for long periods (>3 months) in hexane/2-propanol extracts. PG mass was increased significantly when mice were euthanized by decapitation and the brains rapidly flash-frozen rather than euthanized using head-focused microwave irradiation. This reduction is not the result of PG trapping or destruction in microwave-irradiated brains, demonstrating its importance in limiting mass artifacts during brain PG analysis. Our improved procedure for brain PG analysis provides a reliable, rapid means to detect changes in brain PG mass under both basal and pathological conditions and demonstrates the importance of sample preparation in this process.     

An effective method for accurate nymphal-stage delimitation of the cicada Hyalessa fuscata

Subterranean nymphal development in cicadas presents challenges to researchers in accurately estimating the number of their developmental stages, although such information is crucial to understanding and predicting their population dynamics. While most studies have relied on head width as an attribute for life-stage determination to date, such character in cicadas can be highly variable and thus differentiation solely based on such morphology is prone to subjectivity in practice. Here, we propose a reliable method for instar estimation that is applicable to Hyalessa fuscata nymphs. We first obtained morphometrics of nymphs in all stages. Second, we computed logarithm-transformation and principal component analysis to extract a transformed variable that captures most of the variance of morphological characteristics. Third, k-means were computed to divide the dataset into distinct clusters assuming four-, five- and six life-stage scenarios for the best interferences of life stages. Finally, simple linear regression analysis was conducted to compare and select the best fit model. Our result shows that five nymphal stages best fit for H. fuscata nymphs. This method is expected to provide an easy-to-handle ecological tool for the study of life history of cicadas as well as other insects that have long life cycles and multiple developmental stages.