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At least 1,099 records · Page 61
All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

SU-E-T-799: Verification of a Simultaneous Treatment of Multiple Brain Metastases Using VMAT Technique by a Composite Alanine-Gel Dosimeter Phantom

Purpose: This work presents an end-to-end test using a Gel-Alanine phantom to validate the three-dimensional (3D) dose distribution (DD) delivered by a single isocenter VMAT technique on the simultaneous treatment of multiple brain metastases. Methods: Three cylindrical phantons containing MAGIC-f gel dosimeter were used to measure the 3D DD of a VMAT treatment, the first two were filled with the gel dosimeter (Gel 1 and 2) and the third one was filled with gel and 12 alanine dosimeters distributed along it (Gel 3). Gels 1 and 3 were irradiated and gel 2 was used to map the magnetic resonance image (MRI) scanner field inomogeneities. A CT scan of gel 3 was used for the VMAT treatment planning and 5 alanine pellets were chosen as lesions, around them a PTV was grown and different dose prescriptions were assigned for each one, varying from 5 to 9Gy. Before treatment, the plan was approved in a QA based on an ionization chamber absolute dose measurement, a radiochromic film planar dose measurement and a portal dosimetry per field verification; and also the phantons positioning were verified by ExacTrac 6D correction and OBI kV Cone Beam CT. The gels were irradiated, the MRIs were acquired 24 hours after irradiation and finally, the alanine dosimeters were analysed in a X-band Electron Spin Resonance spectrometer. Results: The association of the two detectors enabled the 3D dose evaluation by gel and punctually inside target volumes by alanine. In the gamma analyses (3%/3mm) comparing the 5 PTVs’ central images DD with TPS expected DD more than 95% of the points were approved. The alanine absolute dose measurements were in agreement with TPS by less than 5%. Conclusion: The gel-alanine phantom enabled the dosimetric validation of multiple brain metastases treatment using VMAT, being an almost ideal tool for this application. This work is partially supported by FAPESP.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

MO-F-CAMPUS-T-01: Radiosurgery of Multiple Brain Metastases with Single-Isocenter VMAT: Optimizing Treatment Geometry to Reduce Normal Brain Dose

Purpose: To develop an optimization algorithm to reduce normal brain dose by optimizing couch and collimator angles for single isocenter multiple targets treatment of stereotactic radiosurgery. Methods: Three metastatic brain lesions were retrospectively planned using single-isocenter volumetric modulated arc therapy (VMAT). Three matrices were developed to calculate the projection of each lesion on Beam’s Eye View (BEV) by the rotating couch, collimator and gantry respectively. The island blocking problem was addressed by computing the total area of open space between any two lesions with shared MLC leaf pairs. The couch and collimator angles resulting in the smallest open areas were the optimized angles for each treatment arc. Two treatment plans with and without couch and collimator angle optimization were developed using the same objective functions and to achieve 99% of each target volume receiving full prescription dose of 18Gy. Plan quality was evaluated by calculating each target’s Conformity Index (CI), Gradient Index (GI), and Homogeneity index (HI), and absolute volume of normal brain V8Gy, V10Gy, V12Gy, and V14Gy. Results: Using the new couch/collimator optimization strategy, dose to normal brain tissue was reduced substantially. V8, V10, V12, and V14 decreased by 2.3%, 3.6%, 3.5%, and 6%, respectively. There were no significant differences in the conformity index, gradient index, and homogeneity index between two treatment plans with and without the new optimization algorithm. Conclusion: We have developed a solution to the island blocking problem in delivering radiation to multiple brain metastases with shared isocenter. Significant reduction in dose to normal brain was achieved by using optimal couch and collimator angles that minimize total area of open space between any of the two lesions with shared MLC leaf pairs. This technique has been integrated into Eclipse treatment system using scripting API.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SU-C-207A-06: On-Line Beam Range Verification with Multiple Scanning Particle Beams: Initial Feasibility Study with Simulations

Purpose: To investigate the feasibility and requirement for intra-fraction on-line multiple scanning particle beam range verifications (BRVs) with in-situ PET imaging, which is beyond the current single-beam BRV with extra factors that will affect the BR measurement accuracy, such as beam diameter, separation between beams, and different image counts at different BRV positions. Methods: We simulated a 110-MeV proton beam with 5-mm diameter irradiating a uniform PMMA phantom by GATE simulation, which generated nuclear interaction-induced positrons. In this preliminary study, we simply duplicated these positrons and placed them next to the initial protons to approximately mimic the two spatially separated positron distributions produced by two beams parallel to each other but with different beam ranges. These positrons were then imaged by a PET (∼2-mm resolution, 10% sensitivity, 320×320×128 mm^3 FOV) with different acquisition times. We calculated the positron activity ranges (ARs) from reconstructed PET images and compared them with the corresponding ARs of original positron distributions. Results: Without further image data processing and correction, the preliminary study show the errors between the measured and original ARs varied from 0.2 mm to 2.3 mm as center-to-center separations and range differences were in the range of 8–12 mm and 2–8 mm respectively, indicating the accuracy of AR measurement strongly depends on the beam separations and range differences. In addition, it is feasible to achieve ≤ 1.0-mm accuracy for both beams with 1-min PET acquisition and 12 mm beam separation. Conclusion: This study shows that the overlap between the positron distributions from multiple scanning beams can significantly impact the accuracy of BRVs of distributed particle beams and need to be further addressed beyond the established method of single-beam BRV, but it also indicates the feasibility to achieve accurate on-line multi-beam BRV with further improved method.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SU-F-T-495: Method for Simultaneous Commissioning of Multiple Truebeam (TB) Linacs: Small Field Data and the Choice of Dosimetric Lead Gap (DLG)

Purpose: Many times a set of multiple Varian-Truebeam (TB) linacs are acquired by an institution. Since “beam matching” is an important requirement for many facilities, we developed a strategy to perform a “simultaneous” commissioning between multiple linacs.Methods and Materials: We first commissioned the required photon beam data for eclipse on the 1st TB for all the energy modalities with a beam scanning system, while integrated measurements for output factors, of all field sizes (from 1×1 to 40×40cm{sup 2}) were conducted on the 2nd TB. Care was exercised during small field dosimetry so the appropriate detectors were used with data taken between two detectors be “linked” to a larger field size (4×4cm{sup 2}) with the “daisy-chaining” technique via: OF=[M-PTW(fs)×(M-PTW(4×4))-1]×[MA12s(4×4)×(M-A12S(10×10))−1]. For all energy modalities, data that span the entire range of field size, was repeated on the next TB linac, for verification. The primarily energy-dependent dosimetric leaf gap (DLG) which was measured separately on each TB. The modeled data was validated with special measurements conducted on both linacs during commissioning. Results: Our data agreed with the “TB representative beam data” to within 0.5% for all energy modalities and field sizes ≥3×3cm2. Sample depth-doses and cross-profiles of a 3×3cm2 between the linacs agreeing to within 1% between linacs. The measured DLGs were quite different with a uniform difference of 1.3% between the two linacs. The measured DLG values are independent of the average dose rate and medium used for the measurements. Conclusion: A comprehensive method of commissioning identical Varian-TB linacs, outlining the critical issues, especially small field dosimetry and DLG. The dosimetric effect of different DLG values, when it comes to, dynamic delivery and data comparisons will be presented. The dependence of DLG value on the measurement medium (in-air vs. water) or dose rate used will also be discussed. This work was supported by CAMC Cancer Center and Alliance Oncology.

61 RADIATION PROTECTION AND DOSIMETRY↗
DOE OSTI2016

SU-F-T-510: Single Isocenter Radiosurgery for the Simultaneous Treatment of Multiple Brain Metastases: Volumetric Modulated Arc Therapy Or Dynamic Conformal Arc?

Purpose: This study evaluates a novel algorithm that can be used with any treatment planning system for simple and rapid generation of stereotactic radiosurgery (SRS) plans for treating multiple brain metastases using a single isocenter dynamic conformal arc (DCA) approach. This technique is compared with a single isocenter volumetric modulated arc therapy (VMAT) technique in terms of delivery time, conformity, low dose spread and delivery accuracy. Methods: Five patients, with a total of 37 (5 – 11) targets were planned using a previously published method for generating optimal VMAT plans and using the proposed DCA algorithm. All planning target volumes (PTVs) were planned to 20 Gy, meeting a minimum 99% coverage and maximum 135 % hot spot for both techniques. Quality assurance was performed using radiochromic film, with films placed in the high dose regions of each PTV. Normal tissue volumes receiving 12 Gy and 6 Gy (V12 and V6) were computed for each plan. Conformity index (CI) and gamma evaluations (95% of points passing 4%/0.5mm) were computed for each PTV. Results: Delivery times, including beam on and table rotation times, were comparable: 17 – 22 minutes for all deliveries. V12s for DCA plans were (18.5±15.2 cc) vs. VMAT (19.7±14.4 cc). V6s were significantly lower for DCA (69.0±52.0 cc) compared with VMAT (154.0±91.0 cc) (p <<0.05). CIs for VMAT targets were (1.38±0.50) vs. DCA (1.61±0.41). 36 of 37 DCA planned targets passed gamma tests, while 29 of 37 VMAT planned targets passed. Conclusion: Single isocenter DCA plans were easily achieved. The evaluation suggests that DCA may represent a favorable technique compared with VMAT for multiple target SRS by reducing dose to normal tissue and more accurately depicting deliverable dose.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SU-G-201-17: Verification of Dose Distributions From High-Dose-Rate Brachytherapy Ir-192 Source Using a Multiple-Array-Diode-Detector (MapCheck2)

Purpose: To investigate quantitatively the accuracy of dose distributions for the Ir-192 high-dose-rate (HDR) brachytherapy source calculated by the Brachytherapy-Planning system (BPS) and measured using a multiple-array-diode-detector in a heterogeneous medium. Methods: A two-dimensional diode-array-detector system (MapCheck2) was scanned with a catheter and the CT-images were loaded into the Varian-Brachytherapy-Planning which uses TG-43-formalism for dose calculation. Treatment plans were calculated for different combinations of one dwell-position and varying irradiation times and different-dwell positions and fixed irradiation time with the source placed 12mm from the diode-array plane. The calculated dose distributions were compared to the measured doses with MapCheck2 delivered by an Ir-192-source from a Nucletron-Microselectron-V2-remote-after-loader. The linearity of MapCheck2 was tested for a range of dwell-times (2–600 seconds). The angular effect was tested with 30 seconds irradiation delivered to the central-diode and then moving the source away in increments of 10mm. Results: Large differences were found between calculated and measured dose distributions. These differences are mainly due to absence of heterogeneity in the dose calculation and diode-artifacts in the measurements. The dose differences between measured and calculated due to heterogeneity ranged from 5%–12% depending on the position of the source relative to the diodes in MapCheck2 and different heterogeneities in the beam path. The linearity test of the diode-detector showed 3.98%, 2.61%, and 2.27% over-response at short irradiation times of 2, 5, and 10 seconds, respectively, and within 2% for 20 to 600 seconds (p-value=0.05) which depends strongly on MapCheck2 noise. The angular dependency was more pronounced at acute angles ranging up to 34% at 5.7 degrees. Conclusion: Large deviations between measured and calculated dose distributions for HDR-brachytherapy with Ir-192 may be improved when considering medium heterogeneity and dose-artifact of the diodes. This study demonstrates that multiple-array-diode-detectors provide practical and accurate dosimeter to verify doses delivered from the brachytherapy Ir-192-source.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SU-G-BRC-05: Conundrum for VMAT Cranial Multiple Lesions Treated with HD120 MLC

Purpose: To commission a custom 6MV-SRS-AAA Eclipse beam model for VMAT multiple lesions cranial SRS treatment on a Varian TrueBeam STx. Methods: Six clinical plans were created using a customized beam model with dosimetric-leaf-gap(DLG) optimized for clinical treatments. Each plan had 4–6 non-isocentric targets with size from 0.2 to 7.1cc. All fields were measured with EBT3 film in the coronal plane in a solid water phantom and with an AS1000 EPID using gantry rotation. In addition, an end-to-end test was performed with coronal and sagittal films in an anthropomorphic phantom verifying dosimetry and localization accuracy. Portal dose distributions were generated with a custom portal dosimetry algorithm(PDIP). Measured dose distributions were compared with calculations using average dose difference (DD), and gamma function, γ. Using a 1.25mm grid, the γ criteria, local DD ≤ 3% and 2mm distance-to-agreement, were applied in regions with dose 50% of maximum. Results: The respective DD and γ for all films were <±2% and >94.2%. The portal dose γ scores for all the plans were >94.9%. However, local regions with underdose >10%, were observed when targets were treated with the 5mm leaves. The same plans re-optimized with two isocenters such that all lesions were under the 2.5mm leaves did not show this effect. The DD and localization error of the end-to-end test were within 3.4% and 1.0mm respectively. Conclusion: The custom AAA beam model is capable of calculating acceptable dosimetry for targets using only the 2.5 mm leaves. This restricts lesions to within ±4cm of isocenter. The observed underdose beneath the 5mm leaves is attributed to a limitation in Eclipse that uses a single DLG representing the DLG’s of both 2.5mm and 5mm leaves. If lesions are >4cm from isocenter, a multiple isocenter technique should be considered to allow the use of only the 2.5mm leaves.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SU-G-BRC-06: Evaluation of a Novel Radiosurgery Software for Treating Multiple Brain Metastases Simultaneously in a Single Fraction

Purpose: To evaluate a new, automated brain metastases planning software designed to treat up to ten brain metastases simultaneously. Methods: We treated 61 patients with multiple brain metastases using the Elements software by BrainLab (Munich, Germany). Patients had between 2–10 metastases ranging from 0.01–8.64 cc. Dose prescription was 18–24 Gy. Plans use up to 5 non-coplanar arcs with a single isocenter at the metastases’ center of mass. The high degree of automation shortens the planning time to 15–20 minutes per patient.For comparison we planned 21 of the patients using Rapid Arc (Varian, Palo Alto CA) (RA). We used two coplanar arcs so as to keep planning times comparable to the Elements. We also planned 8 patients using iPlan software (BrainLab). We compared conformity index (CI), volume of brain receiving over 12 Gy (V{sub 12}) and mean brain dose (MBD) for the three different planning systems (TPSs). Results: Plans from all TPSs were judged clinically acceptable. V{sub 12} and MBD were not statistically significantly different between TPSs.CI between RA and Elements was similar, however for iPlan CI was significantly worse compared to both RA and Elements (p<0.001). RA plans took approximately 40 minutes to plan (despite fusion and contouring being done in the Elements), and iPlan plans over an hour each. Delivery times were approximately 30 minutes for Elements, 10 minutes for RA, and up to 300 minutes for iPlan. Conclusion: Elements plans had good CI values and low brain doses. While treatment times for Elements were longer than for RA, 30 minutes is a significant improvement over conventional radiosurgery techniques where each metastasis is treated individually and delivery times to 10 metastases are close to 300 minutes.BrainLab Elements is a novel software allowing fast, automated planning and efficient irradiation of multiple brain metastases with minimal dose to healthy brain.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SU-G-IeP1-12: Size Selective Arterial Cerebral Blood Volume Mapping Using Multiple Inversion Time Arterial Spin Labeling

Purpose: To demonstrate the feasibility of a novel method for size specific arterial cerebral blood volume (aCBV) mapping using pseudo-continuous arterial spin labeling (PCASL), with multiple TI. Methods: Multiple PCASL images were obtained from a subject with TI of [300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000] ms. Each TI pair was averaged six times. Two scans were performed: one without a flow crusher gradient and the other with a crusher gradient (10cm/s in three directions) to remove signals from large arteries. Scan times were 5min. without a crusher gradient and 5.5 min with a crusher gradient. Non-linear fitting algorithm finds the minimum mean squared solution of per-voxel based aCBV, cerebral blood flow, and arterial transit time, and fits the data into a hemodynamic model that represents superposition of blood volume and flow components within a single voxel. Results: aCBV maps with a crusher gradient represent signals from medium and small sized arteries, while those without a crusher gradient represent signals from all sized arteries, indicating that flow crusher gradients can be effectively employed to achieve size-specific aCBV mapping. Regardless of flow crusher, the CBF and ATT maps are very similar in appearance. Conclusion: Quantitative size selective blood volume mapping controlled by a flow crusher is feasible without additional information because the ASL quantification process doesn’t require an arterial input function measured from a large artery. The size specific blood volume mapping is not interfered by sSignals from large arteries do not interfere with size specific aCBV mapping in the applications of interest in for applications in which only medium or small arteries are of interest.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SU-G-JeP3-15: Is the Reproducibility with Respect to Bone of Tumor Position at Simulation for Breath Hold CT Scans Correlated to the Reproducibility for Multiple Breath Hold CBCTs at Treatment in SBRT Thoracic Patients?

Purpose: To evaluate correlation between the reproducibility of tumor position under feedback guided voluntary deep inspiration breath hold gating at simulation and at treatment. Methods: All patients treated with breath hold (BH) have 3-6 BH CTs taken at simulation (sim). In addition, if the relationship between the tumor and nearby bony anatomy on treatment BH CT(or CBCT) is found to be greater than 5 mm different at treatment than it was at sim, a repeat BH CT is taken before treatment. We retrospectively analyzed the sim CTs for 19 patients who received BH SBRT lung treatments and had repeat BH CT on treatment. We evaluated the reproducibility of the tumor position during the simulation CTs and compared this to the reproducibility of the tumor position on the repeat treatment CT with our in-house CT alignment software (CT-Assisted Targeting for Radiotherapy). Results: Comparing the tumor position for multiple simulation BH CTs, we calculated: maximum difference (max) = 0.69cm; average difference (x) = 0.28cm; standard deviation (σ) = 0.18cm. Comparing the repeat BH CBCTs on treatment days we calculated: max = 0.44cm; x = 0.16cm; σ = 0.22cm. We also found that for 95% of our BH cases, the absolute variation in tumor position within the same imaging day was within 5mm of the range at the time of simulation and treatment. We found that 75% of the BH cases had less residual tumor motion on treatment days than at simulation. Conclusion: This suggests that a GTV contour based upon the residual tumor motion in multiple BH datasets plus 2 mm margin should be sufficient to cover the full range of residual tumor motion on treatment days.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

MO-FG-CAMPUS-TeP1-04: Pseudo-In-Vivo Dose Verification of a New Mono-Isocentric Technique for the Treatment of Multiple Brain Metastases

Purpose: To validate dose calculation and delivery accuracy of a recently introduced mono-isocentric technique for the treatment of multiple brain metastases in a realistic clinical case. Methods: Anonymized CT scans of a patient were used to model a hollow phantom that duplicates anatomy of the skull. A 3D printer was used to construct the phantom of a radiologically bone-equivalent material. The hollow phantom was subsequently filled with a polymer gel 3D dosimeter which also acted as a water-equivalent material. Irradiation plan consisted of 5 targets and was identical to the one delivered to the specific patient except for the prescription dose which was optimized to match the gel dose-response characteristics. Dose delivery was performed using a single setup isocenter dynamic conformal arcs technique. Gel dose read-out was carried out by a 1.5 T MRI scanner. All steps of the corresponding patient’s treatment protocol were strictly followed providing an end-to-end quality assurance test. Pseudo-in-vivo measured 3D dose distribution and calculated one were compared in terms of spatial agreement, dose profiles, 3D gamma indices (5%/2mm, 20% dose threshold), DVHs and DVH metrics. Results: MR-identified polymerized areas and calculated high dose regions were found to agree within 1.5 mm for all targets, taking into account all sources of spatial uncertainties involved (i.e., set-up errors, MR-related geometric distortions and registration inaccuracies). Good dosimetric agreement was observed in the vast majority of the examined profiles. 3D gamma index passing rate reached 91%. DVH and corresponding metrics comparison resulted in a satisfying agreement between measured and calculated datasets within targets and selected organs-at-risk. Conclusion: A novel, pseudo-in-vivo QA test was implemented to validate spatial and dosimetric accuracy in treatment of multiple metastases. End-to-end testing demonstrated that our gel dosimetry phantom is suited for such QA procedures, allowing for 3D analysis of both targeting placement and dose.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

MO-G-201-04: Knowledge-Based Planning for Single-Isocenter Stereotactic Radiosurgery to Multiple Brain Metastases

Purpose: Single-isocenter, linac-based SRS for multiple brain metastases (multi-mets) can deliver highly conformal radiation doses and reduce overall patient treatment time compared to other therapy techniques. This study aims to quantify the dosimetric benefits of knowledge-based planning (KBP) for multi-met treatments. Methods: Using a previously-published KBP methodology (an artificial neural network (ANN) trained on single-target linac-based SRS plans), 3D dose distribution predictions for multi-met patients were obtained by treating each brain lesion as a solitary target and subsequently combining individual predictions into a single distribution using a dose-weighted geometric averaging to obtain the best results in the inter-target space. 17 previously-treated multi-met plans, with target numbers ranging from N=2–5, were used to validate the ANN predictions and subsequent KBP auto-planning routine. The fully-deliverable KBP plans were developed by converting dose distribution predictions into patient-specific optimization objectives while maintaining identical target normalizations (typically PTV V100%=D98%). Plan quality improvements were quantified by the difference between SRS quality metrics (QMs): δdQM=QM(clinical)-QM(KBP). QMs of interest were: gradient measure (GM), conformity index (CI), brain V10 and V5, brainstem D0.1cc and heterogeneity index (HI). Finally, overall plan quality was judged via blinded plan comparison by SRS-specializing physicians. Results: Two clinical plans were found to be significant outliers wherein plan quality was dramatically worse than KBP. Despite indicating KBP superiority, these were removed from the QM analysis to prevent skewing the results. In the remaining cases, clinical and KBP QMs were nearly identical with modest improvements in the KBP sample: δGM=0.12±0.56mm, δCI=−0.01±0.04, Brain δV10=0.8±2.6cc, brain δV5=6.3 ±10.7cc, brainstem δD0.1cc=0.06±1.19Gy and δHI= −0.04±0.05. Ultimately, 13/17 KBP plans were deemed superior to the manual plans in blinded physician review. Conclusion: The results demonstrate that KBP-driven automated planning in linac-based single-isocenter treatments for multiple brain metastases is indistinguishable from, or even better than, traditional manual planning. J. Hattangadi: Research Grant; Varian Medical Systems; K.L. Moore: Research Grant: Varian Medical Systems.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Multiplicity counting using organic scintillators to distinguish neutron sources: An advanced teaching laboratory

In this advanced instructional laboratory, students explore complex detection systems and nondestructive assay techniques used in the field of nuclear physics. After setting up and calibrating a neutron detection system, students carry out timing and energy deposition analyses of radiation signals. Through the timing of prompt fission neutron signals, multiplicity counting is used to carry out a special nuclear material (SNM) nondestructive assay. Our experimental setup is comprised of eight trans-stilbene organic scintillation detectors in a well-counter configuration, and measurements are taken on a spontaneous fission source as well as two (α,n) sources. By comparing each source's measured multiplicity distribution, the resulting measurements of the (α,n) sources can be distinguished from that of the spontaneous fission source. Such comparisons prevent the spoofing, i.e., intentional imitation, of a fission source by an (α,n) neutron source. This instructional laboratory is designed for nuclear engineering and physics students interested in organic scintillators, neutron sources, and nonproliferation radiation measurement techniques.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2025

Generalizing multiple memories from a single drive: The hysteron latch

Far-from-equilibrium systems can form memories of previous deformations or driving. In systems from sheared glassy materials to buckling beams to crumpled sheets, this behavior is dominated by return-point memory, in which revisiting a past extremum of driving restores the system to a previous state. Cyclic driving with both positive and negative strains forms multiple nested memories, as in a single-dial combination lock, while asymmetric driving (only positive strain) cannot. We study this case in a general model of hysteresis that considers discrete elements called hysterons. We show how two hysterons with a frustrated interaction can violate return-point memory, realizing multiple memories of asymmetric driving. This reveals a general principle for designing systems that store sequences of cyclic driving, whether symmetric or asymmetric. In disordered systems, asymmetric driving is a sensitive tool for the direct measurement of frustration.

Science & Technology - Other Topics↗
DOE OSTI2019

Nitrate-Utilizing Microorganisms Resistant to Multiple Metals from the Heavily Contaminated Oak Ridge Reservation.

Contamination of environments with nitrate generated by industrial processes and the use of nitrogen-containing fertilizers is a growing problem worldwide. While nitrate can be removed from contaminated areas by microbial denitrification, nitrate frequently occurs with other contaminants, such as heavy metals, that have the potential to impede the process. In this work, nitrate-reducing microorganisms were enriched and isolated from both groundwater and sediments at the Oak Ridge Reservation (ORR) using concentrations of nitrate and metals (Al, Mn, Fe, Co, Ni, Cu, Cd, and U) similar to those observed in a contaminated environment at ORR. Seven new metal-resistant, nitrate-reducing strains were characterized, and their distribution across both noncontaminated and contaminated areas at ORR was examined. Additionally, while the seven strains have various pH ranges for growth, carbon source preferences, and degrees of resistance to individual and combinations of metals, all were able to reduce nitrate at similar rates both in the presence and absence of the mixture of metals found in the contaminated ORR environment. Four strains were identified in groundwater samples at different ORR locations by exact 16S RNA sequence variant analysis, and all four were found in both noncontaminated and contaminated areas. By using environmentally relevant metal concentrations, we successfully isolated multiple organisms from both ORR noncontaminated and contaminated environments that are capable of reducing nitrate in the presence of extreme mixed-metal contamination.IMPORTANCE Nitrate contamination is a global issue that affects groundwater quality. In some cases, cocontamination of groundwater with nitrate and mixtures of heavy metals could decrease microbially mediated nitrate removal, thereby increasing the duration of nitrate contamination. In this study, we used metal and nitrate concentrations that are present in a contaminated site at the Oak Ridge Reservation to isolate seven metal-resistant strains. All were able to reduce nitrate in the presence of high concentrations of a mixture of heavy metals. Four of seven strains were located in pristine as well as contaminated sites at the Oak Ridge Reservation. Further study of these nitrate-reducing strains will uncover mechanisms of resistance to multiple metals that will increase our understanding of the effect of nitrate and metal contamination on groundwater microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

The Lipid A 1-Phosphatase, LpxE, Functionally Connects Multiple Layers of Bacterial Envelope Biogenesis

Although distinct lipid phosphatases are thought to be required for processing lipid A (component of the outer leaflet of the outer membrane), glycerophospholipid (component of the inner membrane and the inner leaflet of the outer membrane), and undecaprenyl pyrophosphate (C 55 -PP; precursors of peptidoglycan and O antigens of lipopolysaccharide) in Gram-negative bacteria, we report that the lipid A 1-phosphatases, LpxEs, functionally connect multiple layers of cell envelope biogenesis in Gram-negative bacteria. We found that Aquifex aeolicus LpxE structurally resembles YodM in Bacillus subtilis, a phosphatase for phosphatidylglycerol phosphate (PGP) with a weak in vitro activity on C 55 -PP, and rescues Escherichia coli deficient in PGP and C 55 -PP phosphatase activities; deletion of lpxE in Francisella novicida reduces the MIC value of bacitracin, indicating a significant contribution of LpxE to the native bacterial C 55 -PP phosphatase activity. Suppression of plasmid-borne lpxE in F. novicida deficient in chromosomally encoded C 55 -PP phosphatase activities results in cell enlargement, loss of O-antigen repeats of lipopolysaccharide, and ultimately cell death. These discoveries implicate LpxE as the first example of a multifunctional regulatory enzyme that orchestrates lipid A modification, O-antigen production, and peptidoglycan biogenesis to remodel multiple layers of the Gram-negative bacterial envelope.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Pseudomonas putida Lysine Metabolism

Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged r andom b arcode t ra n sposon seq uencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both L - and D -lysine metabolism. We first describe three pathway enzymes that catabolize L -2-aminoadipate ( L -2AA) to 2-ketoglutarate (2KG), connecting D -lysine to the TCA cycle. One of these enzymes, P. putida 5260 (PP_5260), contains a DUF1338 domain, representing a family with no previously described biological function. Our work also identified the recently described coenzyme A (CoA)-independent route of L -lysine degradation that results in metabolization to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of selected pathway enzymes revealed that expression of catabolic genes is highly sensitive to the presence of particular pathway metabolites, implying intensive local and global regulation. This work demonstrated the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as its utility a powerful tool for validating previous research. IMPORTANCE P. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to D -2-hydroxyglutarate ( D -2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Novosphingobium aromaticivorans LigR coordinates transcription of genes involved in metabolism of multiple types of aromatics

Aromatic compounds are a ubiquitous and diverse family of chemicals with functions as biomolecules, natural products, industrial chemicals, and pollutants. Novosphingobium aromaticivorans DSM 12444 uses multiple inducible pathways to catabolize H-, G-, and S-type aromatics that contain zero, one, or two methoxy groups, respectively. Here, we obtain a systems-level view of the transcriptional control of its aromatic metabolic pathways. Several in vitro analyses found that a N. aromaticivorans homolog of the Sphingobium lignivorans SYK-6 transcription factor LigR bound genomic DNA upstream of genes involved in metabolism of multiple aromatic types. We found that a ΔLigR mutant had growth defects on all three types of aromatics as sole carbon sources. Transcriptomic analysis revealed that LigR was required to increase expression of gene products that function in metabolism of all three aromatic types. We also found that, in media containing both glucose and an aromatic carbon source, the ΔLigR mutant directed intermediates through alternative aromatic metabolic pathways. Protein-DNA binding assays showed that N. aromaticivorans LigR binds immediately upstream of promoters of genes involved in aromatic metabolism. We found that N. aromaticivorans LigR coordinates the expression of enzymes that function in the catabolism of H-, G-, and S-type aromatics, and that there are differences in the role of LigR in N. aromaticivorans and S. lignivorans. A comparative genomic analysis predicted that LigR homologs and the aromatic-metabolizing genes that it directly regulates are often co-localized in the genomes of Sphingomonadales, but often not found in this arrangement in many other known aromatic metabolizing bacteria.

Aromatic Compound Degradation↗