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At least 955 records · Page 53

SU-F-T-558: ArcCheck for Patient Specific QA in Stereotactic Ablative Radiotherapy

Purpose: Stereotactic Ablative Radiotherapy (SABR) is one of the most preferred treatment techniques for early stage lung cancer. This technique has been extended to other treatment sites like Spine, Liver, Scapula, Sternum etc., This has resulted in increased physics QA time on machine. In this study, we’ve tested the feasibility of using ArcCheck as an alternative method to replace film dosimetry. Methods: Twelve patients with varied diagnosis of Lung, Liver, scapula, sternum and Spine undergoing SABR were selected for this study. Pre-treatment QA was performed for all the patients which include ionization chamber and film dosimetry. The required gamma criteria for each SABR plan to pass QA and proceed to treatment is 95% (3%,1mm). In addition to this routine process, the treatment plans were exported on to an ArcCheck phantom. The planned and measured dose from the ArcCheck device were compared using four different gamma criteria: 2%,2 mm, 3%,2 mm, 3%,1 mm and 3%, 3 mm. In addition to this, we’ve also introduced errors to gantry, collimator and couch angle to assess sensitivity of the ArcCheck with potential delivery errors. Results: The ArcCheck mean passing rates for all twelve cases were 76.1%±9.7% for gamma criteria 3%,1 mm, 89.5%±5.3% for 2%,2 mm, 92.6%±4.2% for 3%,2 mm, and 97.6%±2.4% for 3%,3 mm gamma criteria. When SABR spine cases are excluded, we observe ArcCheck passing rates higher than 95% for all the studied cases with 3%, 3mm, and ArcCheck results in acceptable agreement with the film gamma results. Conclusion: Our ArcCheck results at 3%, 3 mm were found to correlate well with our non-SABR spine routine patient specific QA results (3%,1 mm). We observed significant reduction in QA time on using ArcCheck for SABR QA. This study shows that ArcCheck could replace film dosimetry for all sites except SABR spine.

60 APPLIED LIFE SCIENCES↗

SU-F-T-586: Pre-Treatment QA of InCise2 MLC Plans On a Cyberknife-M6 Using the Delta4 System in SBRT

Purpose: Performing pre-treatment quality assurance (QA) with the Delta4 system (ScandiDos Inc., Madison, WI) is well established for linac-based radiotherapy. This is not true when using a Cyberknife (Accuray Inc., Sunnyvale, CA) where, typically film-based QA is applied. The goal of this work was to test the feasibility to use the Delta4 system for pre-treatment QA for stereotactic body radiation therapy (SBRT) using a Cyberknife-M6 equipped with the InCise2 multileaf collimator (MLC). Methods: In order to perform measurements without accelerator pulse signal, the Tomotherapy option within the Delta4 software was used. Absolute calibration of the Delta4 phantom was performed using a 10×10 cm{sup 2} field shaped by the InCise2 MLC of the Cyberknife-M6. Five fiducials were attached to the Delta4 phantom in order to be able to track the phantom before and during measurements. For eight SBRT treatment plans (two liver, two prostate, one lung, three bone metastases) additional verification plans were recalculated on the Delta4 phantom using MultiPlan. Dicom data was exported from MultiPlan and was adapted in order to be compatible with the Delta4 software. The measured and calculated dose distributions were compared using the gamma analysis of the Delta4 system. Results: All eight SBRT plans were successfully measured with the aid of the Delta4 system. In the mean, 98.0±1.9%, 95.8±4.1% and 88.40±11.4% of measured dose points passed the gamma analysis using a global dose deviation criterion of 3% (100% corresponds to the dose maximum) and a distance-to-agreement criterion of 3 mm, 2 mm and 1 mm, respectively, and a threshold of 20%. Conclusion: Pre-treatment QA of SBRT plans using the Delta4 system on a Cyberknife-M6 is feasible. Measured dose distributions of SBRT plans showed clinically acceptable agreement with the corresponding calculated dose distributions.

60 APPLIED LIFE SCIENCES↗

SU-F-T-601: Equivalence of CyberKnife and TrueBeam STx for Spine Irradiation

Purpose: To evaluate the equivalence of spine SBRT treatment plans created in Eclipse for the TrueBeam STx (Varian Medical System, Palo Alto, CA) compared to plans using CyberKnife and MultiPlan (Accuray, Sunnyvale, CA). Methods: CT data and contours for 23 spine SBRT patients previously treated using CyberKnife (CK) were exported from MultiPlan treatment planning system into Eclipse where they were planned using static IMRT 6MV coplanar beams. Plans were created according to the original prescription dose and fractionation schedule while limiting spinal dose according to the RTOG 0631 protocol and maintaining target coverage comparable to the original CK plans. Plans were evaluated using new conformity index (nCI), homogeneity index (HI), dose-volume histogram data, number of MU, and estimated treatment time. To ensure all Eclipse plans were deliverable, standard clinical IMRT QA was performed. The plan results were matched with their complimentary CK plans for paired statistical analysis. Results: Plans generated in Eclipse demonstrated statistically significant (p<0.01) improvements compared to complimentary CK plans in median values of maximum spinal cord dose (17.39 vs. 18.12 Gy), RTOG spinal cord constraint dose (14.50 vs. 16.93 Gy), nCI (1.28 vs. 1.54), HI (1.13 vs. 1.27), MU (3918 vs. 36416), and estimated treatment time (8 vs. 48 min). All Eclipse generated plans passed our clinically used protocols for IMRT QA. Conclusion: CK spine SBRT replanned utilizing Eclipse for LINAC delivery demonstrated dosimetric advantages. We propose improvements in plan quality metrics reviewed in this study may be attributed to dynamic MLCs that facilitate treatment of complicated geometries as well as posterior beams ideal for centrally located and/or posterior targets afforded by gantry-based RT delivery.

60 APPLIED LIFE SCIENCES↗

SU-F-T-634: Feasibility Study of Respiratory Gated RapidArc SBRT Using a 6MV FFF Photon Beam

Purpose: To conduct a feasibility study on retrospective respiratory gating and marker tracking for lung stereotactic body radiotherapy (SBRT) with a gated RapidArc delivery using a 6MV flattened filter free photon mode. Methods: A CIRS dynamic thorax phantom Model 008A with different inserts was used for treatment planning and respiratory gating. 4D CT had a free breathing simulation followed by a respiration gated, ten phased CT using a Philips Brilliance CT with a Varian RPM respiratory gating system. The internal target volume was created from the ten phase gated CT images, followed by exporting to a Varian Eclipse TPS v11 for treatment planning on the free breath images. Both MIP and AIP were also generated for comparison of planning and target motion tracking. The planned dose was delivered with a 6MV FFF photon beam from a Varian TrueBeam accelerator. Gated target motion was also verified by tracking the implanted makers during delivery using continuous kV imaging in addition to CBCT, kV and MV localization and verification. Results: Gating was studied in three situations of lower, normal, and faster breathing at a respiratory cycle of 5, 15 and 25 breaths per minute, respectively. 4D treatment planning was performed at a normal breathing of 15 breaths per minute. The gated patterns obtained using the TrueBeam IR camera were compared with the planned ones while gating operation was added prior to delivery . Gating was realized only when the measured respiratory patterns matched to the planned ones. The gated target motion was verified within the tolerance by kV and MV imaging. Either free breathing CT or averaged CT images were studied to be good for image guidance to align the target. Conclusion: Gated RapidArc SBRT delivered with a 6MV FFF photon beam is realized using a dynamic lung phantom.

60 APPLIED LIFE SCIENCES↗

SU-F-T-680: Radiobiological Analysis of the Impact of Daily Patient Deformation and Setup Variations Through the Use of the Cone Beam CT and Deformable Image Registration in Lung Cancer IMRT

Purpose: To estimate the dose distributions delivered to the patient in each treatment fraction using deformable image registration (DIR) and assess the radiobiological impact of the inter-fraction variations due to patient deformation and setup. Methods: The work is based on the cone beam CT (CBCT) images and treatment plans of two lung cancer patients. Both patients were treated with intensity modulated radiation therapy (IMRT) to 66Gy in 2Gy/fraction. The treatment plans were exported from the treatment planning system (TPS) to the Velocity AI where DIR was performed and the same deformation matrix was used for the deformation of the planned dose distribution and organ contours to each CBCT dataset. A radiobiological analysis was performed based on the radiobiological parameters of the involved organs at risk (OARs) and planning target volume (PTV). Using the complication free tumor control probability (P+) index, differences in P+ were observed between each CBCT as well as between CBCT and planning dose distributions. Results: The optimal CBCT P? values ranged from 91.6 % to 94.8 % for patient #1 and from 88.8 % to 90.6 % for patient #2. At the dose level of the clinical prescription, the CBCT P+ values ranged from 80.3% to 80.7% for patient #1 and from 80.7% to 81.0% for the patient #2. The planning CT P+ values were 81.0% and 80.7% for the two patients, respectively. These differences emphasize the significance of using the radiobiological analysis when assessing changes in the dose distribution due to the tumor motion and lung deformations. Conclusion: Daily setup variations yield to differences in the actual dose delivered versus the planned one. The observed differences were rather small when only looking at the dosimetric comparison of the dose distributions, however the radiobiology analysis was able to detect clinically relevant differences among the studied dose distributions.

60 APPLIED LIFE SCIENCES↗

SU-G-BRC-04: Collimator Angle Optimization in Volumetric Modulated Arc Therapy

Purpose: Volumetric modulated arc therapy (VMAT) has revolutionized radiation treatment by decreasing treatment time and monitor units, thus reducing scattered and whole body radiation dose. As the collimator angle changes the apparent leaf gap becomes larger which can impact plan quality, organ at risk (OAR) sparing as well as IMRT QA passing rate which is investigated. Methods: Two sites (prostate and head and neck) that have maximum utilization of VMAT were investigated. Two previously treated VMAT patients were chosen. For each patient 10 plans were created by maintaining constant optimization constraints while varying collimator angles from 0-90 deg at an interval of 10 degrees for the first arc and the appropriate complimentary angle for the second arc. Plans were created with AAA algorithm using 6 MV beam on a Varian IX machine with Millennium 120 MLC. The dose-volume histogram (DVH) for each plan was exported and dosimetric parameters (D98, D95, D50, D2) as well homogeneity index (HI) and conformity index (CI) were computed. Each plan was validated for QA using ArcCheck with gamma index passing criteria of 2%/2 mm and 3%/3 mm. Additionally, normal tissue complication probability (NTCP) for each OAR was computed using Uzan-Nahum software. Results: The CI values for both sites had no impact as target volume coverage in every collimator angle were the same since it was optimized for adequate coverage. The HI which is representative of DVH gradient or dose uniformity in PTV showed a clear trend in both sites. The NTCP for OAR (brain and cochlea) in H&N plan and (bladder and rectum) in prostate plan showed a distinct superiority for collimator angles between 15-30 deg. The gamma passing rates were not correlated with angle. Conclusion: Based on CI, HI, NTCP and gamma passing index, it can be concluded that collimator angles should be maintained within 15–30 deg.

60 APPLIED LIFE SCIENCES↗

SU-G-BRC-08: Evaluation of Dose Mass Histogram as a More Representative Dose Description Method Than Dose Volume Histogram in Lung Cancer Patients

Purpose: Dose-volume-histogram (DVH) is widely used for plan evaluation in radiation treatment. The concept of dose-mass-histogram (DMH) is expected to provide a more representative description as it accounts for heterogeneity in tissue density. This study is intended to assess the difference between DVH and DMH for evaluating treatment planning quality. Methods: 12 lung cancer treatment plans were exported from the treatment planning system. DVHs for the planning target volume (PTV), the normal lung and other structures of interest were calculated. DMHs were calculated in a similar way as DVHs expect that the voxel density converted from the CT number was used in tallying the dose histogram bins. The equivalent uniform dose (EUD) was calculated based on voxel volume and mass, respectively. The normal tissue complication probability (NTCP) in relation to the EUD was calculated for the normal lung to provide quantitative comparison of DVHs and DMHs for evaluating the radiobiological effect. Results: Large differences were observed between DVHs and DMHs for lungs and PTVs. For PTVs with dense tumor cores, DMHs are higher than DVHs due to larger mass weighing in the high dose conformal core regions. For the normal lungs, DMHs can either be higher or lower than DVHs depending on the target location within the lung. When the target is close to the lower lung, DMHs show higher values than DVHs because the lower lung has higher density than the central portion or the upper lung. DMHs are lower than DVHs for targets in the upper lung. The calculated NTCPs showed a large range of difference between DVHs and DMHs. Conclusion: The heterogeneity of lung can be well considered using DMH for evaluating target coverage and normal lung pneumonitis. Further studies are warranted to quantify the benefits of DMH over DVH for plan quality evaluation.

60 APPLIED LIFE SCIENCES↗

SU-G-TeP1-14: SRS Dose Calculation Accuracy Comparison Between Pencil Beam and Monte Carlo Algorithms

Purpose: Stereotactic radiosurgery (SRS) outcomes are related to the delivered dose to the target and to surrounding tissue. We have commissioned a Monte Carlo based dose calculation algorithm to recalculated the delivered dose planned using pencil beam calculation dose engine. Methods: Twenty consecutive previously treated patients have been selected for this study. All plans were generated using the iPlan treatment planning system (TPS) and calculated using the pencil beam algorithm. Each patient plan consisted of 1 to 3 targets and treated using dynamically conformal arcs or intensity modulated beams. Multi-target treatments were delivered using multiple isocenters, one for each target. These plans were recalculated for the purpose of this study using a single isocenter. The CT image sets along with the plan, doses and structures were DICOM exported to Monaco TPS and the dose was recalculated using the same voxel resolution and monitor units. Benchmark data was also generated prior to patient calculations to assess the accuracy of the two TPS against measurements using a micro ionization chamber in solid water. Results: Good agreement, within −0.4% for Monaco and +2.2% for iPlan were observed for measurements in water phantom. Doses in patient geometry revealed up to 9.6% differences for single target plans and 9.3% for multiple-target-multiple-isocenter plans. The average dose differences for multi-target-single-isocenter plans were approximately 1.4%. Similar differences were observed for the OARs and integral dose. Conclusion: Accuracy of the beam is crucial for the dose calculation especially in the case of small fields such as those used in SRS treatments. A superior dose calculation algorithm such as Monte Carlo, with properly commissioned beam models, which is unaffected by the lack of electronic equilibrium should be preferred for the calculation of small fields to improve accuracy.

60 APPLIED LIFE SCIENCES↗

SU-G-IeP3-12: Preliminary Report On the Experience of Patient Radiation Dose Monitoring and Tracking Systems; PEMNET, Radimetrics and DoseWatch

Purpose: Three patient radiation dose monitoring and tracking (PRDMT) systems have been in operation at this institution for the past 6 months. There are useful information that should be disseminated to those who are considering installation of PRDMT programs. In addition, there are “problems” uncovered in the process of estimating fluoroscopic “peak” skin dose (PSD), especially, for those patients who received interventional angiographic studies and in conjunction with surgical procedures. Methods: Upon exporting the PRDMT data to Microsoft Excel program, the peak skin dose can be estimated by applying various correction factors including; attenuation due to the tabletop and examination mattress, table height, tabletop translation, backscatter, etc. A procedure was established to screen and divide the PRDMT reported radiation dose and estimated PSD to three different levels of threshold to assess the potential skin injuries, to assist patient follow-up, risk management and provide radiation dosimetry information in case of “Sentinel Event”. Results: The Radiation Dose Structured Report (RDSR) was found to be the prerequisite for the PRDMT systems to work seamlessly. And, the geometrical parameters (gantry and table orientation) displayed by the equipment are not necessarily implemented in the “patient centric” manner which could result in a large error in the PSD estimation. Since, the PRDMT systems obtain their pertinent data from the DICOM tags including the polarity (+ and − signs), the geometrical parameters need to be verified. Conclusion: PRDMT systems provide a more accurate PSD estimation than previously possible as the air-kerma-area dose meter become widely implemented. However, care should be exercised to correctly apply the geometrical parameters in estimating the patient dose. In addition, further refinement is necessary for these software programs to account for all geometrical parameters such as the tabletop translation in the z-direction in particular.

60 APPLIED LIFE SCIENCES↗

TU-AB-BRC-09: Fast Dose-Averaged LET and Biological Dose Calculations for Proton Therapy Using Graphics Cards

Purpose: To demonstrate fast and accurate Monte Carlo (MC) calculations of proton dose-averaged linear energy transfer (LETd) and biological dose (BD) on a Graphics Processing Unit (GPU) card. Methods: A previously validated GPU-based MC simulation of proton transport was used to rapidly generate LETd distributions for proton treatment plans. Since this MC handles proton-nuclei interactions on an event-by-event using a Bertini intranuclear cascade-evaporation model, secondary protons were taken into account. The smaller contributions of secondary neutrons and recoil nuclei were ignored. Recent work has shown that LETd values are sensitive to the scoring method. The GPU-based LETd calculations were verified by comparing with a TOPAS custom scorer that uses tabulated stopping powers, following recommendations by other authors. Comparisons were made for prostate and head-and-neck patients. A python script is used to convert the MC-generated LETd distributions to BD using a variety of published linear quadratic models, and to export the BD in DICOM format for subsequent evaluation. Results: Very good agreement is obtained between TOPAS and our GPU MC. Given a complex head-and-neck plan with 1 mm voxel spacing, the physical dose, LETd and BD calculations for 10{sup 8} proton histories can be completed in ∼5 minutes using a NVIDIA Titan X card. The rapid turnover means that MC feedback can be obtained on dosimetric plan accuracy as well as BD hotspot locations, particularly in regards to their proximity to critical structures. In our institution the GPU MC-generated dose, LETd and BD maps are used to assess plan quality for all patients undergoing treatment. Conclusion: Fast and accurate MC-based LETd calculations can be performed on the GPU. The resulting BD maps provide valuable feedback during treatment plan review. Partially funded by Varian Medical Systems.

60 APPLIED LIFE SCIENCES↗

TH-AB-201-05: Determining the Direction Distribution of the Primary Radiation for a Cyberknife-M6

Purpose: Radiation protection regulatory differentiates between primary and scatter radiation. Whereas for conventional clinical linear accelerators the solid angle for primary radiation is planar, the Cyberknife (Accuray Inc., Sunnyvale, CA) may point its beam in all spatial directions. In order to be able to judge on radiation protection calculations for a Cyberknife-M6 vault, the direction distribution for delivered plans was evaluated based on clinical experiences. Methods: The log-files of 121 delivered patient treatment plans were exported, divided into cranial and extra-cranial treatments and the delivered monitor units (MU) together with the corresponding beam directions were analyzed. This MU-weighted spatial distribution was then projected to a 9.5 × 5.9 × 3.9 m{sup 3} vault, generating an “intensity map” using a binning of 50 × 50 cm{sup 2}. The factor of direction (FOD) is reported as a fraction of the total applied MUs to the walls, ceiling and floor in the perspective of a patient lying in head-first-supine position on the couch. In this study, the term intFOD refers to the integral FOD and maxFOD refers to the maximal FOD for a single bin. Results: For all kind of treatments and collimators, intFOD and maxFOD for the wall behind the patient’s head is 0.0. The intFOD for the floor varies between 0.65 and 0.74. For the ceiling, maxFOD is 0.002 and 0.0 for cranial and extra-cranial cases, respectively. The intFOD for the wall at the patient’s feet, is 0.094 for cranial and 0.005 for extra-cranial cases. There is nearly no difference between the maxFOD of the right and left wall for cranial cases, whereas for extra-cranial cases these numbers differ by a factor of 1.75. Conclusion: The varying FODs for the Cyberknife were quantified based on delivered treatment plans. These findings are helpful regarding the design of Cyberknife vaults.

60 APPLIED LIFE SCIENCES↗

TH-EF-BRB-10: Dosimetric Validation of a Trajectory Based Cranial SRS Treatment Technique On a Varian TrueBeam Linac

Purpose: This work investigates the dosimetric accuracy of a trajectory based delivery technique in which an optimized radiation beam is delivered along a Couch-Gantry trajectory that is formed by simultaneous rotation of the linac gantry and the treatment couch. Methods: Nine trajectory based cranial SRS treatment plans were created using in-house optimization software. The plans were calculated for delivery on the TrueBeam STx linac with 6MV photon beam. Dose optimization was performed along a user-defined trajectory using MLC modulation, dose rate modulation and jaw tracking. The pre-defined trajectory chosen for this study is formed by a couch rotation through its full range of 180 degrees while the gantry makes four partial arc sweeps which are 170 degrees each. For final dose calculation, the trajectory based plans were exported to the Varian Eclipse Treatment Planning System. The plans were calculated on a homogeneous cube phantom measuring 18.2×18.2×18.2 cm3 with the analytical anisotropic algorithm (AAA) using a 1mm3 calculation voxel. The plans were delivered on the TrueBeam linac via the developer’s mode. Point dose measurements were performed on 9 patients with the IBA CC01 mini-chamber with a sensitive volume of 0.01 cc. Gafchromic film measurements along the sagittal and coronal planes were performed on three of the 9 treatment plans. Point dose values were compared with ion chamber measurements. Gamma analysis comparing film measurement and AAA calculations was performed using FilmQA Pro. Results: The AAA calculations and measurements were in good agreement. The point dose difference between AAA and ion chamber measurements were within 2.2%. Gamma analysis test pass rates (2%, 2mm passing criteria) for the Gafchromic film measurements were >95%. Conclusion: We have successfully tested TrueBeam’s ability to deliver accurate trajectory based treatments involving simultaneous gantry and couch rotation with MLC and dose rate modulation along the trajectory.

60 APPLIED LIFE SCIENCES↗

Poster - 47: A parametrized prediction model of rectal toxicity in focal SBRT of low risk prostate cancer

There has been a recent trend towards watchful waiting in place of intervention for early stage prostate cancer (CaP). However, this approach can allow for disease progression, and subsequent whole-gland therapies such as prostatectomy and whole gland irradiation can result in functional deficits or rectal toxicities or both. A controversial alternative approach for this patient cohort is the use of focal therapy, where the treatment is focussed on an identified dominant index lesion (DIL). This work aims to investigate the treatment parameters for focal SBRT of the prostate under which clinically acceptable rectal NTCP levels can be achieved. For each of 25 low risk CaP patients, a hypothetical 2 cc DIL was modeled in the right-posterior quadrant of the prostate, and was used to build a PTV as the target for SBRT simulation. An SBRT prescriptions of 41 Gy and 37 Gy in 5 fractions were chosen, corresponding to the boost levels used in previous CaP dose escalation studies. DVH data were exported and used to calculate rectal NTCP values based on the Lyman-Kutcher-Burman (LKB) model using the QUANTEC reccommended model parameters. Rectal NTCP dependence on DIL-to-rectum separation, dose level, and DIL volume were investigated. The final goal of this ongoing work is to create a map of the maximum allowable prescription dose for a given patient geometry that achieves a clinically acceptable rectal NTCP level.

60 APPLIED LIFE SCIENCES↗

High-latitude warming initiated the onset of the last deglaciation in the tropics

Atmospheric greenhouse gas concentrations are thought to have synchronized global temperatures during Pleistocene glacial–interglacial cycles, yet their impact relative to changes in high-latitude insolation and ice-sheet extent remains poorly constrained. Here, we use tropical glacial fluctuations to assess the timing of low-latitude temperature changes relative to global climate forcings. We report 10 Be ages of moraines in tropical East Africa and South America and show that glaciers reached their maxima at ~29 to 20 ka, during the global Last Glacial Maximum. Tropical glacial recession was underway by 20 ka, before the rapid CO 2 rise at ~18.2 ka. This “early” tropical warming was influenced by rising high-latitude insolation and coincident ice-sheet recession in both polar regions, which lowered the meridional thermal gradient and reduced tropical heat export to the high latitudes.

54 ENVIRONMENTAL SCIENCES↗

Human perturbations to mercury in global rivers

Mercury compounds are potent neurotoxins that pose threats to human health, primarily through fish consumption. Rivers, critical for drinking water and food supply, have seen rapid increases in mercury concentrations and export to coastal margins since the Industrial Revolution (~1850). However, patterns of these changes remain understudied, limiting assessments of environmental policies. Here, we develop a global model to simulate preindustrial riverine total mercury and assess human perturbations by comparing it to present-day conditions. We find that global rivers transported ~390 megagrams annually of mercury to the oceans in the preindustrial era, with spatial variability. Human activities have elevated riverine mercury budgets by two to three times in the present day. Establishing a baseline riverine mercury level, our findings reveal rapid responses of riverine mercury to human perturbations and could be used to inform targets for global riverine mercury restoration. Total riverine mercury concentrations could also be used as indicators to comprehensively understand the effectiveness of mercury pollution governance.

Science & Technology - Other Topics↗

Novel Metal Cation Resistance Systems from Mutant Fitness Analysis of Denitrifying Pseudomonas stutzeri

Metal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu 2+ and Zn 2+ resistance systems in Pseudomonas stutzeri strain RCH2 are compared with those experimentally implicated in Cu 2+ and Zn 2+ resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu 2+ and Zn 2+ resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu 2+ resistance systems do not show decreased fitness relative to the wild type when exposed to Cu 2+ . In addition, new genes are identified that have no known connection to Zn 2+ ( corB , corC , Psest_3226, Psest_3322, and Psest_0618) or Cu 2+ resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lacking corB , corC , Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsible for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu 2+ - and Zn 2+ -based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu 2+ and Zn 2+ efflux and resistance and their effects on denitrifying metabolism. Here in this study, genome-wide mutant fitness data in P. stutzeri RCH2 combined with regulon predictions identify several proteins of unknown function that are involved in resisting zinc and copper toxicity. For zinc, these include a member of the UPF0016 protein family that was previously implicated in Ca 2+ /H + antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg 2+ transport, and Psest_3322 and Psest_0618, two proteins with no characterized homologs. Experiments using mutants lacking Psest_3226, Psest_3322, corB , corC , or czcI verified their proposed functions, which will enable future studies of these little-characterized zinc resistance determinants. Likewise, Psest_2850, annotated as an ion antiporter subunit, and the conserved hypothetical protein Psest_0584 are implicated in copper resistance. Physiological connections between previous studies and phenotypes presented here are discussed. Functional and mechanistic understanding of transport proteins improves the understanding of systems in which members of the same protein family, including those in humans, can have different functions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Herbivorous Fish Microbiome Adaptations to Sulfated Dietary Polysaccharides

Marine herbivorous fish that feed primarily on macroalgae, such as those from the genus Kyphosus, are essential for maintaining coral health and abundance on tropical reefs. Here, deep metagenomic sequencing and assembly of gut compartment-specific samples from three sympatric, macroalgivorous Hawaiian kyphosid species have been used to connect host gut microbial taxa with predicted protein functional capacities likely to contribute to efficient macroalgal digestion. Bacterial community compositions, algal dietary sources, and predicted enzyme functionalities were analyzed in parallel for 16 metagenomes spanning the mid- and hindgut digestive regions of wild-caught fishes. Gene colocalization patterns of expanded carbohydrate (CAZy) and sulfatase (SulfAtlas) digestive enzyme families on assembled contigs were used to identify likely polysaccharide utilization locus associations and to visualize potential cooperative networks of extracellularly exported proteins targeting complex sulfated polysaccharides. These insights into the gut microbiota of herbivorous marine fish and their functional capabilities improve our understanding of the enzymes and microorganisms involved in digesting complex macroalgal sulfated polysaccharides.

09 BIOMASS FUELS↗

A Rhizobiales-Specific Unipolar Polysaccharide Adhesin Contributes to Rhodopseudomonas palustris Biofilm Formation across Diverse Photoheterotrophic Conditions

Bacteria predominantly exist as members of surfaced-attached communities known as biofilms. Many bacterial species initiate biofilms and adhere to each other using cell surface adhesins. This is the case for numerous ecologically diverse Alphaprotebacteria , which use polar exopolysaccharide adhesins for cell-cell adhesion and surface attachment. Here, we show that Rhodopseudomonas palustris , a metabolically versatile member of the alphaproteobacterial order Rhizobiales , contains a functional unipolar polysaccharide (UPP) biosynthesis gene cluster. Deletion of genes predicted to be critical for UPP biosynthesis and export abolished UPP production. We also found that R. palustris uses UPP to mediate biofilm formation across diverse photoheterotrophic growth conditions, wherein light and organic substrates are used to support growth. However, UPP was less important for biofilm formation during photoautotrophy, where light and CO 2 support growth, and during aerobic respiration with organic compounds. Expanding our analysis beyond R. palustris , we examined the phylogenetic distribution and genomic organization of UPP gene clusters among Rhizobiales species that inhabit diverse niches. Our analysis suggests that UPP is a conserved ancestral trait of the Rhizobiales but that it has been independently lost multiple times during the evolution of this clade, twice coinciding with adaptation to intracellular lifestyles within animal hosts. Bacteria are ubiquitously found as surface-attached communities and cellular aggregates in nature. Here, we address how bacterial adhesion is coordinated in response to diverse environments using two complementary approaches. First, we examined how Rhodopseudomonas palustris , one of the most metabolically versatile organisms ever described, varies its adhesion to surfaces in response to different environmental conditions. We identified critical genes for the production of a unipolar polysaccharide (UPP) and showed that UPP is important for adhesion when light and organic substrates are used for growth. Looking beyond R. palustris , we performed the most comprehensive survey to date on the conservation of UPP biosynthesis genes among a group of closely related bacteria that occupy diverse niches. Our findings suggest that UPP is important for free-living and plant-associated lifestyles but dispensable for animal pathogens. Additionally, we propose guidelines for classifying the adhesins produced by various Alphaprotebacteria , facilitating future functional and comparative studies.

59 BASIC BIOLOGICAL SCIENCES↗