The preponderance of P450s in the Mycobacterium tuberculosis genome.

The genome of Mycobacterium tuberculosis (Mtb) encodes 20 different cytochrome P450 enzymes (P450s). P450s are mono-oxygenases, which are historically considered to facilitate prokaryotic usage of unusual carbon sources. However, their preponderance in Mtb strongly indicates crucial physiological functions, as does the fact that polycyclic azoles (known P450 inhibitors) have potent anti-mycobacterial effects. Recent structural and enzyme characterization data reveal novel features for at least two Mtb P450s (CYP121 and CYP51). Genome analysis, knockout studies and structural comparisons signify important roles in cell biology and pathogenesis for various P450s and redox partner enzymes in Mtb. Elucidation of structure, function and metabolic roles will be essential in targeting the P450s as an 'Achilles heel' in this major human pathogen.

Gamma histograms for radiotherapy plan evaluation.

BACKGROUND AND PURPOSE: The technique known as the 'gamma evaluation method' incorporates pass-fail criteria for both distance-to-agreement and dose difference analysis of 3D dose distributions and provides a numerical index (gamma) as a measure of the agreement between two datasets. As the gamma evaluation index is being adopted in more centres as part of treatment plan verification procedures for 2D and 3D dose maps, the development of methods capable of encapsulating the information provided by this technique is recommended. PATIENTS AND METHODS: In this work the concept of gamma index was extended to create gamma histograms (GH) in order to provide a measure of the agreement between two datasets in two or three dimensions. Gamma area histogram (GAH) and gamma volume histogram (GVH) graphs were produced using one or more 2D gamma maps generated for each slice of the irradiated volume. GHs were calculated for IMRT plans, evaluating the 3D dose distribution from a commercial treatment planning system (TPS) compared to a Monte Carlo (MC) calculation used as reference dataset. RESULTS: The extent of local anatomical inhomogenities in the plans under consideration was strongly correlated with the level of difference between reference and evaluated calculations. GHs provided an immediate visual representation of the proportion of the treated volume that fulfilled the gamma criterion and offered a concise method for comparative numerical evaluation of dose distributions. CONCLUSIONS: We have introduced the concept of GHs and investigated its applications to the evaluation and verification of IMRT plans. The gamma histogram concept set out in this paper can provide a valuable technique for quantitative comparison of dose distributions and could be applied as a tool for the quality assurance of treatment planning systems.

A comparison of electronic portal dosimetry verification methods for use in stereotactic radiotherapy.

Three methods of transit dosimetry using Electronic Portal Imaging Devices (EPIDs) were investigated for use in routine in-vivo dosimetry for cranial stereotactic radiosurgery and radiotherapy. The approaches examined were (a) A full Monte Carlo (MC) simulation of radiation transport through the linear accelerator and patient; (b) Calculation of the expected fluence by a treatment planning system (TPS); (c) Point doses calculated along the central axis compared to doses calculated using parameters acquired using the EPID. A dosimetric comparison of each of the three methods predicted doses at the imager plane to within ±5% and a gamma comparison for the MC and TPS based approaches showed good agreement for a range of dose and distance to agreement criteria. The MC technique was most time consuming, followed by the TPS calculation with the point dose calculation significantly quicker than the other methods.

Observer variability when evaluating patient movement from electronic portal images of pelvic radiotherapy fields.

BACKGROUND AND PURPOSE: A study has been performed to evaluate inter-observer variability when assessing pelvic patient movement using an electronic portal imaging device (EPID). MATERIALS AND METHODS: Four patient image sets were used with 3-6 portal images per set. The observer group consisted of nine radiographers with 3-18 months clinical EPID experience. The observers outlined bony landmarks on a digital simulator image and used matching software to evaluate field placement errors (FPEs) on each portal image relative to the reference simulator image. Data were evaluated statistically, using a two-component analysis of variance technique, to quantify both the inter-observer variability in evaluating FPEs and inter-fraction variability in patient position relative to the residuals of the analysis. Intra-observer variability was also estimated using four of the observers carrying out three sets of repeat readings. RESULTS: Eight sets of variance data were analysed, based on FPEs in two orthogonal directions for each of the four patient image sets studied. Initial analysis showed that both inter-observer variation and inter-fraction-patient position variation were statistically significant (P<0.05) in seven of the eight cases evaluated. The averaged root-mean-square (RMS) deviation of the observers from the group mean was 1.1 mm, with a maximum deviation of 5.0 mm recorded for an individual observer. After additional training and re-testing of two of the observers who recorded the largest deviations from the group mean, a subsequent analysis showed the inter-observer variability for the group to be significant in only three of the eight cases, with averaged RMS deviation reduced to 0.5 mm, with a maximum deviation of 2.7 mm. The intra-observer variability was 0.5 mm, averaged over the four observers tested. CONCLUSIONS: We have developed a quantitative approach to evaluate inter-observer variability in terms of its statistical significance compared to inter-fraction patient movement. This will assist us in training and assessing observers required to perform this task on a routine basis.

Characterization of active site structure in CYP121. A cytochrome P450 essential for viability of Mycobacterium tuberculosis H37Rv.

Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.