Statistical breathing curve sampling to quantify interplay effects of moving lung tumors in a 4D Monte Carlo dose calculation framework.

PURPOSE: The interplay between respiratory tumor motion and dose application by intensity modulated radiotherapy (IMRT) techniques can potentially lead to undesirable and non-intuitive deviations from the planned dose distribution. We developed a 4D Monte Carlo (MC) dose recalculation framework featuring statistical breathing curve sampling, to precisely simulate the dose distribution for moving target volumes aiming at a comprehensive assessment of interplay effects. METHODS: We implemented a dose accumulation tool that enables dose recalculations of arbitrary breathing curves including the actual breathing curve of the patient. This MC dose recalculation framework is based on linac log-files, facilitating a high temporal resolution up to 0.1 s. By statistical analysis of 128 different breathing curves, interplay susceptibility of different treatment parameters was evaluated for an exemplary patient case. To facilitate prospective clinical application in the treatment planning stage, in which patient breathing curves or linac log-files are not available, we derived a log-file free version with breathing curves generated by a random walk approach. Interplay was quantified by standard deviations σ in D5%, D50% and D95%. RESULTS: Interplay induced dose deviations for single fractions were observed and evaluated for IMRT and volumetric arc therapy (σD95% up to 1.3 %) showing a decrease with higher fraction doses and an increase with higher MU rates. Interplay effects for conformal treatment techniques were negligible (σ<0.1%). The log-file free version and the random walk generated breathing curves yielded similar results (deviations in σ< 0.1 %) and can be used as substitutes for interplay assessment. CONCLUSION: It is feasible to combine statistically sampled breathing curves with MC dose calculations. The universality of the presented framework allows comprehensive assessment of interplay effects in retrospective and prospective clinically relevant scenarios.

Single-isocenter stereotactic radiosurgery for multiple brain metastases: Impact of patient misalignments on target coverage in non-coplanar treatments.

Frameless single-isocenter non-coplanar stereotactic radiosurgery (SRS) for patients with multiple brain metastases is a treatment at high geometrical complexity. The goal of this study is to analyze the dosimetric impact of non-coplanar image guidance with stereoscopic X-ray imaging. Such an analysis is meant to provide insights on the adequacy of safety margins, and to evaluate the benefit of imaging at non-coplanar configurations. The ExacTrac® (ET) system (Brainlab AG, Munich, Germany) was used for stereoscopic X-ray imaging in frameless single-isocenter non-coplanar SRS for multiple brain metastases. Sub-millimeter precision was found for the ET-based pre-treatment setup, whereas a degradation was noted for non-coplanar treatment angles. Misalignments without intra-fractional positioning corrections were reconstructed in 6 degrees of freedom (DoF) to resemble the situation without non-coplanar image guidance. Dose recalculation in 20 SRS patients with applied positioning corrections did not reveal any significant differences in D98% for 75 planning target volumes (PTVs) and gross tumor volumes (GTVs). For recalculation without applied positioning corrections, significant differences (p<0.05) were reported in D98% for both PTVs and GTVs, with stronger effects for small PTV volumes. A worst-case analysis at increasing translational and rotational misalignment revealed that dosimetric changes are a complex function of the combination thereof. This study highlighted the important role of positioning correction with ET at non-coplanar configurations in frameless single-isocenter non-coplanar SRS for patients with multiple brain metastases. Uncorrected patient misalignments at non-coplanar couch angles were linked to a significant loss of PTV coverage, with effects varying according to the combination of single DoF and PTV geometrical properties.

Planning Benchmark Study for Stereotactic Body Radiation Therapy of Liver Metastases: Results of the DEGRO/DGMP Working Group on Stereotactic Radiation Therapy and Radiosurgery.

PURPOSE: Our purpose was to investigate whether liver stereotactic body radiation therapy treatment planning can be harmonized across different treatment planning systems, delivery techniques, and institutions by using a specific prescription method and to minimize the knowledge gap concerning intersystem and interuser differences. We provide best practice guidelines for all used techniques. METHODS AND MATERIALS: A multiparametric specification of target dose (gross target volume [GTV]D50%, GTVD0.1cc, GTVV90%, planning target volume [PTV]V70%) with a prescription dose of GTVD50% = 3 × 20 Gy and organ-at-risk (OAR) limits were distributed with computed tomography and structure sets from 3 patients with liver metastases. Thirty-five institutions provided 132 treatment plans using different irradiation techniques. These plans were first analyzed for target and OAR doses. Four different renormalization methods were performed (PTVDmin, PTVD98%, PTVD2%, PTVDmax). The resulting 660 treatments plans were evaluated regarding target doses to study the effect of dose renormalization to different prescription methods. A relative scoring system was used for comparisons. RESULTS: GTVD50% prescription can be performed in all systems. Treatment plan harmonization was overall successful, with standard deviations for Dmax, PTVD98%, GTVD98%, and PTVDmean of 1.6, 3.3, 1.9, and 1.5 Gy, respectively. Primary analysis showed 55 major deviations from clinical goals in 132 plans, whereas in only <20% of deviations GTV/PTV dose was traded for meeting OAR limits. GTVD50% prescription produced the smallest deviation from target planning objectives and between techniques, followed by the PTVDmax, PTVD98%, PTVD2%, and PTVDmin prescription. Deviations were significant for all combinations but for the PTVDmax prescription compared with GTVD50% and PTVD98%. Based on the various dose prescription methods, all systems significantly differed from each other, whereas GTVD50% and PTVD98% prescription showed the least difference between the systems. CONCLUSIONS: This study showed the feasibility of harmonizing liver stereotactic body radiation therapy treatment plans across different treatment planning systems and delivery techniques when a sufficient set of clinical goals is given.

Stereotactic body radiotherapy in patients with hepatocellular carcinoma in a multimodal treatment setting.

PURPOSE: Retrospective evaluation of stereotactic body radiation therapy (SBRT) in patients with hepatocellular carcinoma (HCC). METHODS: We retrospectively analyzed 36 patients (45 lesions) treated between 2011 and 2017. Twenty-seven had previous treatments. Current treatment consisted of SBRT alone (n = 15) or selective transarterial chemoembolization (TACE) followed by SBRT to the same lesions (n = 21). Eight patients received additional local treatments to different lesions. Liver function was predominantly moderately restricted (Child A: 29, Child B: 6, Child C: 1). Treatment planning was based on 4D-computed tomography, dose/fractionation varied depending on location and size, most commonly 3 fractions of 12.5 Gy (65% isodose) and 5 fractions of 8 Gy (80% isodose). RESULTS: Median follow-up was 15 months. Local recurrence was observed in 3 lesions (7%), resulting in 1­and 2­year local control rates of 93%. The only significantly predicting factor was the use of abdominal compression. New hepatic lesions occurred in 19 patients (52%), 1­ and 2­year freedom-from-hepatic-failure (FFHF) was 39% and 32%, respectively. Only the number of treated lesions was predictive for FFHF. Sixteen patients have died, resulting in 1­ and 2­year overall survival (OS) of 64% and 41%, respectively, significantly impacted by the number of treated lesions and Child-Pugh class. Severe acute and late toxicity (≥grade 3) was observed in 3% and 8%, respectively. 6 patients (17%) received liver transplantation (OLT) after SBRT, of whom 5 showed pathological complete remission. CONCLUSION: SBRT (±TACE) in highly pretreated HCC is effective and associated with excellent LC and low toxicity. SBRT may be used as definitive or bridging treatment prior to OLT. Patients with multifocal lesions have significantly decreased 1­ and 2­year FFHF and OS.

Comparison of planned dose on different CT image sets to four-dimensional Monte Carlo dose recalculation using the patient's actual breathing trace for lung stereotactic body radiation therapy.

PURPOSE: The need for four-dimensional (4D) treatment planning becomes indispensable when it comes to radiation therapy for moving tumors in the thoracic and abdominal regions. The primary purpose of this study is to combine the actual breathing trace during each individual treatment fraction with the Linac's log file information and Monte Carlo 4D dose calculations. We investigated this workflow on multiple computed tomography (CT) datasets in a clinical environment for stereotactic body radiation therapy (SBRT) treatment planning. METHODS: We have developed a workflow, which allows us to recalculate absorbed dose to a 4DCT dataset using Monte Carlo calculation methods and accumulate all 4D doses in order to compare them to the planned dose using the Linac's log file, a 4DCT dataset, and the patient's actual breathing curve for each individual fraction. For five lung patients, three-dimensional-conformal radiation therapy (3D-CRT) and volumetric modulated arc treatment (VMAT) treatment plans were generated on four different CT image datasets: a native free-breathing 3DCT, an average intensity projection (AIP) and a maximum intensity projection (MIP) CT both obtained from a 4DCT, and a 3DCT with density overrides based on the 3DCT (DO). The Monte Carlo 4D dose has been calculated on each 4DCT phase using the Linac's log file and the patient's breathing trace as a surrogate for tumor motion and dose was accumulated to the gross tumor volume (GTV) at the 50% breathing phase (end of exhale) using deformable image registration. RESULTS: Δ D 98 % and Δ D 2 % between 4D dose and planned dose differed largely for 3DCT-based planning and also for DO in three patients. Least dose differences between planned and recalculated dose have been found for AIP and MIP treatment planning which both tend to be superior to DO, but the results indicate a dependency on the breathing variability, tumor motion, and size. An interplay effect has not been observed in the small patient cohort. CONCLUSIONS: We have developed a workflow which, to our best knowledge, is the first incorporation of the patient breathing trace over the course of all individual treatment fractions with the Linac's log file information and 4D Monte Carlo recalculations of the actual treated dose. Due to the small patient cohort, no clear recommendation on which CT can be used for SBRT treatment planning can be given, but the developed workflow, after adaption for clinical use, could be used to enhance a priori 4D Monte Carlo treatment planning in the future and help with the decision on which CT dataset treatment planning should be carried out.

Dose variability in different lymph node levels during locoregional breast cancer irradiation: the impact of deep-inspiration breath hold.

PURPOSE: Aim of the present analysis was to evaluate the movement and dose variability of the different lymph node levels of node-positive breast cancer patients during adjuvant radiotherapy (RT) with regional nodal irradiation (RNI) in deep-inspiration breath hold (DIBH). METHODS: Thirty-five consecutive node-positive breast cancer patients treated from October 2016 to February 2018 receiving postoperative RT of the breast or chest wall including RNI of the supra-/infraclavicular lymph node levels (corresponding to levels IV, III, Rotter LN (interpectoral), and some parts of level II) were analyzed. To evaluate the lymph node level movement, a center of volume (COV) was obtained for each lymph node level for free-breathing (FB) and DIBH plans. Geometric shifts and dose differences between FB and DIBH were analyzed. RESULTS: A significant movement of the COV in anterior (y) and cranial (z) dimensions was observed for lymph node levels I-II and Rotter lymph nodes (p < 0.001) due to DIBH. Only minor changes in the lateral dimension (x axis) were observed, without reaching significance for levels III, IV, and internal mammary. There was a significant difference in the mean dose of level I (DIBH vs. FB: 38.2 Gy/41.3 Gy, p < 0.001) and level II (DIBH vs. FB: 45.9 Gy/47.2 Gy, p < 0.001), while there was no significant difference in level III (p = 0.298), level IV (p = 0.476), or internal mammary nodes (p = 0.471). CONCLUSION: A significant movement of the axillary lymph node levels was observed during DIBH in anterior and cranial directions for node-positive breast cancer patients in comparison to FB. The movement leads to a significant dose reduction in level I and level II.

Stereotactic body radiation therapy (SBRT) in patients with hepatocellular carcinoma and oligometastatic liver disease.

BACKGROUND: To report our experience with SBRT in primary and secondary liver tumors. METHODS: We retrospectively analysed 55 patients (70 lesions) with a median follow-up of 10 months (range 1-57) treated from 2011 to 2016. All patients had not been eligible for other local treatment options. Median age was 64 years and 64% were male. 27 patients (36 lesions) suffered from hepatocellular carcinoma (HCC, Child A:78%, Child B:18%, Child C:4%), 28 patients (34 lesions) had oligometastatic liver disease (MD). Treatment planning was based on 4D-CT usually after placement of fiducials. Dose and fractionation varied depending on localization and size, most commonly 3 × 12.5 Gy (prescribed to the surrounding 65%-isodose) in 56% and 5x8Gy (80% isodose) in 20% of the treated lesions. RESULTS: Local recurrence was observed in 7 patients (13%) and 8 lesions (11%), resulting in estimated 1- and 2-year local control rates (LC) of 91 and 74%. Estimated 1- and 2-year rates of Freedom from hepatic failure (FFHF) were 42 and 28%. Number of lesions was predictive for LC and FFHF in the entire cohort. Estimated 1- and 2-year overall survival (OS) was 76 and 57%. OS was significantly affected by number of treated lesions and performance status. In the HCC subgroup, pretreatment liver function and gender were also predictive for OS. Maximum acute non-hepatic toxicity was grade 1 in 16% and grade 2 in 10% of the patients. Three HCC patients (11%) developed marked deterioration of liver function (grade 3/4). CONCLUSIONS: SBRT resulted in high local control and acceptable survival rates in patients with HCC or MD not amendable to other locally-ablative treatment options with limited toxicity. Care should be taken in HCC patients with Child B cirrhosis.

Clinical workflow optimization to improve 4DCT reconstruction for Toshiba Aquilion CT scanners.

Respiratory motion remains a source of major uncertainties in radiotherapy. Respiratory correlated computed tomography (referred to as 4DCT) serves as one way of reducing breathing artifacts in 3D-CTs and allows the investigation of tumor motion over time. The quality of the 4DCT images depends on the data acquisition scheme, which in turn is dependent on the vendor. Specifically, the only way Toshiba Aquilion LB CT scanners can reconstruct 4DCTs is a cycle-based reconstruction using triggers provided by an external surrogate signal. The accuracy is strongly dependent on the method of trigger generation. Two consecutive triggers are used to define a breathing cycle which is divided into respiratory phases of equal duration. The goal of this study is to identify if there are advantages in the usage of local-amplitude based sorting (LAS) of the respiration motion states, in order to reduce image artifacts and improve 4DCT quality. Furthermore, this study addresses the generation and optimization of a clinical workflow using as surrogate motion monitoring system the Sentinel™ (C-RAD AB, Sweden) optical surface scanner in combination with a Toshiba Aquilion LB CT scanner. For that purpose, a phantom study using 10 different breathing waveforms and a retrospective patient study using the 4DCT reconstructions of 10 different patients has been conducted. The error in tumor volume has been reduced from 2.9±3.7% to 2.7±2.6% using optimal cycle-based triggers (manipulated CBS) and to 2.7±2.2% using LAS in the phantom study. Moreover, it was possible to decrease the tumor volume variability from 5.0±3.6% using the original cycle-based triggers (original CBS) to 3.5±2.5% using the optimal triggers and to 3.7±2.7% using LAS in the patient data analysis. We therefore propose the usage of the manipulated CBS, also with regard to an accurate and safe clinical workflow.

Planning benchmark study for SBRT of early stage NSCLC : Results of the DEGRO Working Group Stereotactic Radiotherapy.

PURPOSE: The aim was to evaluate stereotactic body radiation therapy (SBRT) treatment planning variability for early stage nonsmall cell lung cancer (NSCLC) with respect to the published guidelines of the Stereotactic Radiotherapy Working Group of the German Society for Radiation Oncology (DEGRO). MATERIALS AND METHODS: Planning computed tomography (CT) scan and the structure sets (planning target volume, PTV; organs at risk, OARs) of 3 patients with early stage NSCLC were sent to 22 radiotherapy departments with SBRT experience: each department was asked to prepare a treatment plan according to the DEGRO guidelines. The prescription dose was 3 fractions of 15 Gy to the 65% isodose. RESULTS: In all, 87 plans were generated: 36 used intensity-modulated arc therapy (IMAT), 21 used three-dimensional conformal radiation therapy (3DCRT), 6 used static field intensity-modulated radiation therapy (SF-IMRT), 9 used helical radiotherapy and 15 used robotic radiosurgery. PTV dose coverage and simultaneously kept OARs doses were within the clinical limits published in the DEGRO guidelines. However, mean PTV dose (mean 58.0 Gy, range 52.8-66.4 Gy) and dose conformity indices (mean 0.75, range 0.60-1.00) varied between institutions and techniques (p ≤ 0.02). OARs doses varied substantially between institutions, but appeared to be technique independent (p = 0.21). CONCLUSION: All studied treatment techniques are well suited for SBRT of early stage NSCLC according to the DEGRO guidelines. Homogenization of SBRT practice in Germany is possible through the guidelines; however, detailed treatment plan characteristics varied between techniques and institutions and further homogenization is warranted in future studies and recommendations. Optimized treatment planning should always follow the ALARA (as low as reasonably achievable) principle.

Stereoscopic X-ray imaging, cone beam CT, and couch positioning in stereotactic radiotherapy of intracranial tumors: preliminary results from a cross-modality pilot installation.

BACKGROUND: To assess the accuracy and precision of a fully integrated pilot installation of stereoscopic X-ray imaging and kV-CBCT for automatic couch positioning in stereotactic radiotherapy of intracranial tumors. Positioning errors as detected by stereoscopic X-ray imaging are compared to those by kV-CBCT (i.e. the accuracy of the new method is verified by the established method), and repeated X-ray images are compared (i.e. the precision of new method is determined intra-modally). METHODS: Preliminary results are reported from a study with 32 patients with intracranial tumors. Patients were treated with stereotactic radiotherapy guided by stereoscopic X-ray imaging and kV-CBCT. Patient positioning was automatically corrected by a robotic couch. Cross-modal discrepancies in position detection were measured (N = 42). Intra-modal improvements after correction and re-verification by stereoscopic X-ray imaging were measured (N = 70). The accuracy and precision of stereoscopic X-ray imaging and the accuracy and precision of CBCT were confirmed in phantom measurements (N = 12 shifts of a ball bearing phantom, N = 24 shifts of a head phantom). RESULTS: After correction based on stereoscopic X-ray imaging 95% of residual mean errors were below 0.4, 0.4, 0.5, and 0.7 mm (lateral, longitudinal, vertical, radial, respectively). Stereoscopic X-ray imaging and CBCT were in close agreement with an average discrepancy of 0.1, 0.5, 0.3 and 0.8 mm, respectively. 95% of discrepancies were below 0.8, 1.2, 1.0, and 1.4 mm, respectively. After correction and re-verification by stereoscopic X-ray imaging, the remaining intra-modal residual error was consistent with zero (p = 0.31, p = 0.48, p = 0.81 in lateral, longitudinal, and vertical direction; p-values from two-tailed t-test). The inherent technical accuracy and precision of stereoscopic X-ray imaging and the accuracy and precision of CBCT were found to be of the order of 0.1 mm in controlled phantom settings. CONCLUSIONS: In a routine clinical setting, both stereoscopic X-ray imaging and CBCT were able to reduce positioning errors by an order of magnitude. The end-to-end precision of the system, measured from the discrepancy (mean) between ExacTrac and CBCT, in a clinical setting seems to be about 0.8 mm radially, including couch positioning. The precision (measured from repeatability of ExacTrac, intra-modal) was found to be about 0.7 mm radially in a clinical setting.

Feasibility study on image guided patient positioning for stereotactic body radiation therapy of liver malignancies guided by liver motion.

BACKGROUND: Fiducial markers are the superior method to compensate for interfractional motion in liver SBRT. However this method is invasive and thereby limits its application range. In this retrospective study, the compensation method for the interfractional motion using fiducial markers (gold standard) was compared to a new non-invasive approach, which does rely on the organ motion of the liver and the relative tumor position within this volume. METHODS: We analyzed six patients (3 m, 3f) treated with SBRT in 2014. After fiducial marker implantation, all patients received a treatment CT (free breathing, without abdominal compression) and a 4D-CT (consisting of 10 respiratory phases). For all patients the gross tumor volumes (GTVs), internal target volume (ITV), planning target volume (PTV), internal marker target volumes (IMTVs) and the internal liver target volume (ILTV) were delineated based on the CT and 4D-CT images. CBCT imaging was used for the standard treatment setup based on the fiducial markers. According to the patient coordinates the 3 translational compensation values (t x , t y , t z ) for the interfractional motion were calculated by matching the blurred fiducial markers with the corresponding IMTV structures. 4 observers were requested to recalculate the translational compensation values for each CBCT (31) based on the ILTV structures. The differences of the translational compensation values between the IMTV and ILTV approach were analyzed. RESULTS: The magnitude of the mean absolute 3D registration error with regard to the gold standard overall patients and observers was 0.50 cm ± 0.28 cm. Individual registration errors up to 1.3 cm were observed. There was no significant overall linear correlation between the respiratory motion and the registration error of the ILTV approach. CONCLUSIONS: Two different methods to calculate the translational compensation values for interfractional motion in stereotactic liver therapy were evaluated. The registration accuracy of the ILTV approach is mainly limited by the non-rigid behavior of the liver and the individual registration experience of the observer. The ILTV approach lacks the accuracy that would be desired for stereotactic radiotherapy of the liver.

Technical evaluation of different respiratory monitoring systems used for 4D CT acquisition under free breathing.

Respiratory monitoring systems are required to supply CT scanners with information on the patient's breathing during the acquisition of a respiration-correlated computer tomography (RCCT), also referred to as 4D CT. The information a respiratory monitoring system has to provide to the CT scanner depends on the specific scanner. The purpose of this study is to compare two different respiratory monitoring systems (Anzai Respiratory Gating System; C-RAD Sentinel) with respect to their applicability in combination with an Aquilion Large Bore CT scanner from Toshiba. The scanner used in our clinic does not make use of the full time dependent breathing signal, but only single trigger pulses indicating the beginning of a new breathing cycle. Hence the attached respiratory monitoring system is expected to deliver accurate online trigger pulse for each breathing cycle. The accuracy of the trigger pulses sent to the CT scanner has to be ensured by the selected respiratory monitoring system. Since a trigger pulse (output signal) of a respiratory monitoring system is a function of the measured breathing signal (input signal), the typical clinical range of the input signal is estimated for both examined respiratory monitoring systems. Both systems are analyzed based on the following parameters: time resolution, signal amplitude, noise, signal-to-noise ratio (SNR), signal linearity, trigger compatibility, and clinical examples. The Anzai system shows a better SNR (≥ 28 dB) than the Sentinel system (≥ 14.6 dB). In terms of compatibility with the cycle-based image sorting algorithm of the Toshiba CT scanner, the Anzai system benefits from the possibility to generate cycle-based triggers, whereas the Sentinel system is only able to generate amplitude-based triggers. In clinical practice, the combination of a Toshiba CT scanner and the Anzai system will provide better results due to the compatibility of the image sorting and trigger release methods.

Characteristics of gated treatment using an optical surface imaging and gating system on an Elekta linac.

BACKGROUND: Knowing the technical characteristics of gated radiotherapy equipment is crucial for ensuring precise and accurate treatment when using techniques such as Deep-Inspiration Breath-Hold and gating under free breathing. With one of the first installations of the novel surface imaging system Catalyst™ (C-RAD AB, Sweden) in connection with an Elekta Synergy linear accelerator (Elekta AB, Sweden) via the Elekta Response Interface, characteristics like dose delivery accuracy and time delay were investigated prior to clinical implementation of gated treatments in our institution. METHODS: In this study a moving phantom was used to simulate respiratory motion which was registered by the Catalyst™ system. The gating level was set manually. Within this gating window a trigger signal is automatically sent to the linac initiating treatment delivery. Dose measurements of gated linac treatment beams with different gating levels were recorded with a static 2D-Diode Array (MapCheck2, Sun Nuclear Co., USA) and compared to ungated reference measurements for different field sizes. In addition, the time delay of gated treatment beams was measured using radiographic film. RESULTS: The difference in dose delivery between gated and ungated treatment decreases with the size of the chosen gating level. For clinically relevant gating levels of about 30%, the differences in dose delivery accuracy remain below 1%. In comparison with other system configurations in literature, the beam-on time delay shows a large deviation of 851 ms ± 100 ms. CONCLUSIONS: When performing gated treatment, especially for free-breathing gating, factors as time delay and dose delivery have to be evaluated regularly in terms of a quality assurance process. Once these parameters are known they can be accounted and compensated for, e.g. by adjusting the pre-selected gating level or the internal target volume margins and by using prediction algorithms for breathing curves. The usage of prediction algorithms becomes inevitable with the high beam-on time delay which is reported here.

Identification and characterization of a novel porin family highlights a major difference in the outer membrane of chlamydial symbionts and pathogens.

The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P. amoebophila by in silico analysis. Two of these Protochlamydiaouter membrane proteins, PomS (pc1489) and PomT (pc1077), are highly abundant in outer membrane preparations of this organism. Here we show that all four members of this putative porin family are toxic when expressed in the heterologous host Escherichia coli. Immunofluorescence analysis using antibodies against heterologously expressed PomT and PomS purified directly from elementary bodies, respectively, demonstrated the location of both proteins in the outer membrane of P. amoebophila. The location of the most abundant protein PomS was further confirmed by immuno-transmission electron microscopy. We could show that pomS is transcribed, and the corresponding protein is present in the outer membrane throughout the complete developmental cycle, suggesting an essential role for P. amoebophila. Lipid bilayer measurements demonstrated that PomS functions as a porin with anion-selectivity and a pore size similar to the Chlamydiaceae MOMP. Taken together, our results suggest that PomS, possibly in concert with PomT and other members of this porin family, is the functional equivalent of MOMP in P. amoebophila. This work contributes to our understanding of the adaptations of symbiotic and pathogenic chlamydiae to their different eukaryotic hosts.

Proteomic analysis reveals a virtually complete set of proteins for translation and energy generation in elementary bodies of the amoeba symbiont Protochlamydia amoebophila.

Chlamydiae belong to the most successful intracellular bacterial pathogens. They display a complex developmental cycle and an extremely broad host spectrum ranging from vertebrates to protozoa. The family Chlamydiaceae comprises exclusively well-known pathogens of humans and animals, whereas the members of its sister group, the Parachlamydiaceae, naturally occur as symbionts of free-living amoebae. Comparative analysis of these two groups provides valuable insights into chlamydial evolution and mechanisms for microbe-host interaction. Based on the complete genome sequence of the Acanthamoeba spp. symbiont Protochlamydia amoebophila UWE25, we performed the first detailed proteome analysis of the infectious stage of a symbiotic chlamydia. A 2-D reference proteome map was established and the analysis was extensively complemented by shotgun proteomics. In total, 472 proteins were identified, which represent 23.2% of all encoded proteins. These cover a wide range of functional categories, including typical house-keeping proteins, but also putative virulence-associated proteins. A number of proteins that are not encoded in genomes of Chlamydiaceae were observed and the expression of 162 proteins classified as hypothetical or unknown proteins could be demonstrated. Our findings indicate that P. amoebophila exploits its additional genetic repertoire (compared with the Chlamydiaceae), and that its elementary bodies are remarkably well equipped with proteins involved in transcription, translation, and energy generation.

Proteomic analysis of the outer membrane of Protochlamydia amoebophila elementary bodies.

Chlamydiae are obligate intracellular bacteria, comprising some of the most important bacterial pathogens of animals and humans. During their unique developmental cycle they have to attach to and enter their eukaryotic host cells, a process mediated by proteins in the chlamydial outer membrane. So far the only experimental data for chlamydial outer membrane proteins are available from members of the Chlamydiaceae, a family comprising exclusively human and animal pathogens. To get further insights into the evolution of the protein composition of the chlamydial outer membrane and into host-dependent differences, we performed an extensive experimental analysis of outer membrane fractions of Protochlamydia amoebophila elementary bodies, which constitute the infectious form of this non-pathogenic member of the Chlamydiae that thrives as a symbiont in Acanthamoeba spp. We used 1-D and 2-DE in combination with MALDI-TOF, MALDI-TOF/TOF and nanoLC-ESI-MS/MS, and compared our experimental results with a previously published in silico analysis of chlamydial outer membrane proteins. This resulted in the identification of 38 proteins supported by both studies and therefore very likely to be located in the P. amoebophila outer membrane. The obtained experimental data provide the first comprehensive overview of outer membrane proteins of a chlamydial organism outside the Chlamydiaceae. They reveal both fundamental differences and convergent evolution between pathogenic and symbiotic chlamydiae.

Quantification of adipocere degradation with and without access to oxygen and to the living soil.

Adipocere is formed from body fat in moist and oxygen-deficient decay conditions. The persistence of adipocere may cause problems for the reuse of graves after the expiration of statutory resting times in some countries. Up to now, no quantitative data existed on the persistence of adipocere in either aerated or anoxic conditions. We investigated the rate of degradation (disappearance) of adipocere in five different samples from human corpses. The experimental incubation was (a) in water without air contact, (b) in water with access to air, (c) in physiological saline with access to air, (d) on sterilized quartz sand, (e) in vitro on living soil, and (f) buried 15 cm deep in field soil. The weight loss of the samples was determined after 215 (293) days and half-lives were calculated under the assumption of simple first-order kinetics. Furthermore, the nitrogen content and the fatty acid composition of the adipocere samples were analyzed. The results revealed half-lives that differ between the adipocere samples from 11 to 82 years under anaerobic conditions (mean of all samples, 37 years). In air, the half-life of adipocere was reduced to about one tenth, ranging from 0.7 to 10 years (mean of 2.8 years for all samples incubated in aerated physiological saline, mean of 4.0 years for all samples incubated on living soil in the laboratory). Burying adipocere in a biologically active field soil resulted in half-lives of disappearance from 1.2 years to 2.1 years (mean, 1.5 years). The N content of the adipocere samples ranged between 1.9 and 6.7 mg N g(-1). The sample with the highest N content was also that with the lowest half-life of disappearance in all types of incubation. The fatty acid analysis of the samples revealed a composition typical of adipocere, with a clear dominance of saturated acids (palmitic, myristic and stearic acid) over unsaturated ones. The variation of fatty acid composition between the different adipocere samples could only be attributed partly to their age and the burial conditions. It can be concluded that the aeration of adipocere-laden corpses will lead to a disappearance of adipocere (and hence restitution of the decay process) within a time span of several years.

Rv1698 of Mycobacterium tuberculosis represents a new class of channel-forming outer membrane proteins.

Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 +/- 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes.

Characterization of nanostructured surfaces generated by reconstitution of the porin MspA from Mycobacterium smegmatis.

Nanostructures with long-term stability at the surface of gold electrodes are generated by reconstituting the porin MspA from Mycobacterium smegmatis into a specially designed monolayer of long-chain lipid surfactant on gold. Tailored surface coverage of gold electrodes with long-chain surfactants is achieved by electrochemically assisted deposition of organic thiosulfates (Bunte salts). The subsequent reconstitution of the octameric-pore MspA is guided by its extraordinary self-assembling properties. Importantly, electrochemical reduction of copper(II) yields copper nanoparticles within the MspA nanopores. Electrochemical impedance spectroscopy, reflection electron microscopy, and atomic force microscopy (AFM) show that: 1) the MspA pores within the self-assembled monolayer (SAM) are monodisperse and electrochemically active, 2) MspA reconstitutes in SAMs and with a 10-nm thickness, 3) AFM is a suitable method to detect pores within SAMs, and 4) the electrochemical reduction of Cu2+ to Cu0 under overpotential conditions starts within the MspA pores.