Passive Proton Research Articles

In Vitro Demonstration of Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

A new subfamily of rhodopsins was discovered in bacteria and proposed to function as dual function light-driven H+/Na+ pumps, ejecting Na+ from cells in the presence of Na+ and H+ in its absence (Inoue et al 2013. Nat. Commun. 4:1678). This proposal was based primarily on light-induced proton flux measurements in suspensions of E. coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the Na+ and H+ transport definitively. We expressed IAR from Indibacter alkaliphilus in E. coli cells and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta reported earlier. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na+ pumping activity in vitro, namely Na+-dependent light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na+-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore, showing H+ pumping activity. The direction of proton fluxes indicated that IAR was inserted inside-out into the sealed LUV system, which we confirmed by site-directed spin-label EPR spectroscopy. The in vitro LUV system proves that the dual light-driven H+/Na+ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo. We are currently investigating ion fluxes through natural anion channelrhodopsins with this system.

Spot Scanning and Passive Scattering Proton Therapy: Relative Biological Effectiveness and Oxygen Enhancement Ratio in Cultured Cells

To determine the relative biological effectiveness (RBE), oxygen enhancement ratio (OER), and contribution of the indirect effect of spot scanning proton beams, passive scattering proton beams, or both in cultured cells in comparison with clinically used photons. The RBE of passive scattering proton beams at the center of the spread-out Bragg peak (SOBP) was determined from dose-survival curves in 4 cell lines using 6-MV X rays as controls. Survival of 2 cell lines after spot scanning and passive scattering proton irradiation was then compared. Biological effects at the distal end region of the SOBP were also investigated. The OER of passive scattering proton beams and 6 MX X rays were investigated in 2 cell lines. The RBE and OER values were estimated at a 10% cell survival level. The maximum degree of protection of radiation effects by dimethyl sulfoxide was determined to estimate the contribution of the indirect effect against DNA damage. All experiments comparing protons and X rays were made under the same biological conditions. The RBE values of passive scattering proton beams in the 4 cell lines examined were 1.01 to 1.22 (average, 1.14) and were almost identical to those of spot scanning beams. Biological effects increased at the distal end of the SOBP. In the 2 cell lines examined, the OER was 2.74 (95% confidence interval, 2.56-2.80) and 3.08 (2.84-3.11), respectively, for X rays, and 2.39 (2.38-2.43) and 2.72 (2.69-2.75), respectively, for protons (P<.05 for both cells between X rays and protons). The maximum degree of protection was significantly higher for X rays than for proton beams (P<.05). The RBE values of spot scanning and passive scattering proton beams were almost identical. The OER was lower for protons than for X rays. The lower contribution of the indirect effect may partly account for the lower OER of protons.

Supramolecular tryptophan-zipper forms a tripeptide as a regular proton transporter

The diverse structure and assembly of two peptide analogues have been investigated. The tripeptide containing tryptophan, α-aminoisobutyric acid and valine self-assembles through various noncovalent interactions to form a tryptophan-zipper-like structure by intermolecular hydrogen bonding and π–π interactions. But the leucine analogue adopts a water-mediated hydrogen-bonded cage-like structure. X-ray crystallography shed some light on the structure of the tryptophan-zipper at the atomic level. Moreover, in U-tube experiments using pH gradient as the driving force across a liquid chloroform phase, the supramolecular tryptophan-zipper acts as a passive regular proton transporter.

Conversion of a light-driven proton pump into a light-gated ion channel.

Interest in microbial rhodopsins with ion pumping activity has been revitalized in the context of optogenetics, where light-driven ion pumps are used for cell hyperpolarization and voltage sensing. We identified an opsin-encoding gene (CsR) in the genome of the arctic alga Coccomyxa subellipsoidea C-169 that can produce large photocurrents in Xenopus oocytes. We used this property to analyze the function of individual residues in proton pumping. Modification of the highly conserved proton shuttling residue R83 or its interaction partner Y57 strongly reduced pumping power. Moreover, this mutation converted CsR at moderate electrochemical load into an operational proton channel with inward or outward rectification depending on the amino acid substitution. Together with molecular dynamics simulations, these data demonstrate that CsR-R83 and its interacting partner Y57 in conjunction with water molecules forms a proton shuttle that blocks passive proton flux during the dark-state but promotes proton movement uphill upon illumination.

A Novel Set of Lyman Kutcher Burman (LKB) Model Parameters in Predicting Radiation Esophagitis in Passive Scattering Proton Therapy

A Novel Set of Lyman Kutcher Burman (LKB) Model Parameters in Predicting Radiation Esophagitis in Passive Scattering Proton Therapy

Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector

The membrane sector (Vo) of the proton pumping vacuolar ATPase (V-ATPase, V1Vo-ATPase) from Saccharomyces cerevisiae was purified to homogeneity, and its structure was characterized by EM of single molecules and two-dimensional crystals. Projection images of negatively stained Vo two-dimensional crystals showed a ring-like structure with a large asymmetric mass at the periphery of the ring. A cryo-EM reconstruction of Vo from single-particle images showed subunits a and d in close contact on the cytoplasmic side of the proton channel. A comparison of three-dimensional reconstructions of free Vo and Vo as part of holo V1Vo revealed that the cytoplasmic N-terminal domain of subunit a (aNT) must undergo a large conformational change upon enzyme disassembly or (re)assembly from Vo, V1, and subunit C. Isothermal titration calorimetry using recombinant subunit d and aNT revealed that the two proteins bind each other with a Kd of ~5 μm. Treatment of the purified Vo sector with 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] resulted in selective release of subunit d, allowing purification of a VoΔd complex. Passive proton translocation assays revealed that both Vo and VoΔd are impermeable to protons. We speculate that the structural change in subunit a upon release of V1 from Vo during reversible enzyme dissociation plays a role in blocking passive proton translocation across free Vo and that the interaction between aNT and d seen in free Vo functions to stabilize the Vo sector for efficient reassembly of V1Vo.

Passive proton therapy vs. IMRT planning study with focal boost for prostate cancer.

BackgroundExploiting biologic imaging, studies have been performed to boost dose to gross intraprostatic tumor volumes (GTV) while reducing dose elsewhere in the prostate. Interest in proton beams has increased due to superior normal-tissue sparing they afford. Our goal was to dosimetrically compare 3D conformal proton boost plans with intensity-modulated radiation therapy (IMRT) plans with respect to target coverage and avoiding organs at risk.MethodsTreatment planning computer tomography scans of ten patients were selected. For each patient, two hypothetical but realistic GTVs each with a fixed volume were contoured in different anatomical locations of the prostate. IMRT and proton beam plans were created with a prescribed dose of 50.4 Gy to the initial planning target volume (PTV) including the PTV of the seminal vesicles (PSV), 70.2 Gy to the PTV of the prostate (PPS), and 90 Gy to the PTV of the gross tumor volumes (PGTVs). For proton plans, uncertainties of range and patient setup were accounted for; apertures were adjusted until the dose-volume coverage of PTVs matched that of the IMRT plan. For both plans, prescribed PTV doses were made identical to allow for comparing normal-tissue doses.ResultsProtons delivered more homogeneous but less conformal doses to PGTVs than IMRT did and comparable doses to PSV and PPS. Volumes of bladder and rectum receiving doses higher than 65 Gy were similar for both plans. However, volumes receiving less than 65 Gy were significantly reduced, i.e., protons reduced integral dose by 45.6 % and 26.5 % for rectum and bladder, respectively. This volume-sparing was also seen in femoral heads and penile bulb.ConclusionsProtons delivered comparable doses to targets in dose homogeneity and conformity and spared normal tissues from intermediate-to-low doses better than IMRT did. Further improvement of dose sparing and changes in homogeneity and conformity may be achieved by reducing proton range uncertainties and from implementing intensity modulation.

Designing a range modulator wheel to spread-out the Bragg peak for a passive proton therapy facility

In proton beam therapy, a Spread-Out Bragg peak (SOBP) is used to establish a uniform dose distribution in the target volume. In order to create a SOBP, several Bragg peaks of different ranges, corresponding to different entrance energies, with certain intensities (weights) should be combined each other. In a passive beam scattering system, the beam is usually extracted from a cyclotron at a constant energy throughout a treatment. Therefore, a SOBP is produced by a range modulator wheel, which is basically a rotating wheel with steps of variable thicknesses, or by using the ridge filters. In this study, we used the Geant4 toolkit to simulate a typical passive scattering beam line. In particular, the CATANA transport beam line of INFN Laboratori Nazionali del Sud (LNS) in Catania has been reproduced in this work. Some initial properties of the entrance beam have been checked by benchmarking simulations with experimental data. A class dedicated to the simulation of the wheel modulators has been implemented. It has been designed in order to be easily modified for simulating any desired modulator wheel and, hence, any suitable beam modulation. By using some auxiliary range-shifters, a set of pristine Bragg peaks was obtained from the simulations. A mathematical algorithm was developed, using the simulated pristine dose profiles as its input, to calculate the weight of each pristine peak, reproduce the SOBP, and finally generate a flat dose distribution. Therefore, once the designed modulator has been realized, it has been tested at CATANA facility, comparing the experimental data with the simulation results.

In Vitro Demonstration of Dual Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

A subfamily of rhodopsin pigments was recently discovered in bacteria and proposed to function as dual-function light-driven H+/Na+ pumps, ejecting sodium ions from cells in the presence of sodium and protons in its absence. This proposal was based primarily on light-induced proton flux measurements in suspensions of Escherichia coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the putative Na+ and H+ transport definitively. We expressed IAR, an uncharacterized member from Indibacter alkaliphilus in E. coli cell suspensions, and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na+ pumping activity in vitro, namely, light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na+-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore. These results meet the criteria for electrogenic Na+ transport and electrogenic H+ transport, respectively, in the presence and absence of Na+. The direction of proton fluxes indicated that IAR was inserted inside-out into our sealed LUV system, which we confirmed by site-directed spin-label electron paramagnetic resonance spectroscopy. We further demonstrate that Na+ transport by IAR requires Na+ only on the cytoplasmic side of the protein. The in vitro LUV system proves that the dual light-driven H+/Na+ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo.

Atomistic Characterization of the First Step of Calcium Pump Activation Associated with Proton Countertransport

The calcium pump [sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA)] transports Ca(2+) from the cytosol to the SR lumen at the expense of ATP hydrolysis and proton countertransport, thus playing a central role in Ca(2+) homeostasis and muscle contractility. Proton countertransport via deprotonation of transport site residue Glu309 is a critical first step in SERCA activation because it accelerates the E2-E1 structural transition. Previous studies have suggested that flipping of Glu309 toward the cytosol constitutes the primary mechanism for Glu309 deprotonation, but no conclusive data to support this hypothesis have been published. Therefore, we performed three independent 1 μs molecular dynamics simulations of the E2 state protonated at transport site residues Glu309, Glu771, and Glu908. Structural analysis and pKa calculations showed that Glu309 deprotonation occurs by an inward-to-outward side-chain transition. We also found that Glu309 deprotonation and proton countertransport occur through transient (~113 ps) water wires connecting Glu309 with the cytosol. Although both mechanisms are operational, we found that transient water wire formation, and not Glu309 flipping, is the primary mechanism for Glu309 deprotonation and translocation of protons to the cytosol. The outward-to-inward transition of protonated Glu309 and the presence of water wires suggest that protons from the cytosol might be passively transported to the lumen via Glu309. However, structural analysis indicates that passive SR proton leakage into the lumen unlikely occurs through Glu309 in the E2 state. These findings provide a time-resolved visualization of the first step in the molecular mechanism of SERCA activation and proton transport across the SR.

SU‐E‐T‐61: A Practical Process for Fabricating Passive Scatter Proton Beam Modulation Compensation Filters Using 3D Printing

Purpose:The purpose of this project was to devise a practical fabrication process for passive scatter proton beam compensation filters (CF) that is competitive in time, cost and effort using 3D printing.Methods:DICOM compensator filter files for a proton beam were generated by our Eclipse (Varian, Inc.) treatment planning system. The compensator thickness specifications were extracted with in‐house software written in Matlab (MathWorks, Inc.) code and written to a text file which could be read by the Rhinoceros 5, computer‐aided design (CAD) package (Robert McNeel and Associates), which subsequently generated a smoothed model in a STereoLithographic also known as a Standard Tesselation Language file (STL). The model in the STL file was subsequently refined using Netfabb software and then converted to printing instructions using Cura. version 15.02.1. for our 3D printer. The Airwolf3D, model HD2x, fused filament fabrication (FFF) 3D printer (Airwolf3D.com) was used for our fabrication system with a print speed of 150mm per second. It can print in over 22 different plastic filament materials in a build volume of 11” × 8” × 12”. We choose ABS plastic to print the 3D model of the imprint for our CFs.Results:Prints of the CF could be performed at a print speed of 70mm per second. The time to print the 3D topology for the CF for the 14 cm diameter snout of our Mevion 250 proton accelerator was less than 3 hours. The printed model is intended to subsequently be used as a mold to imprint a molten wax cylindrical to form the compensation after cooling. The whole process should be performed for a typical 3 beam treatment plan within a day.Conclusion:Use of 3D printing is practical and can be used to print a 3D model of a CF within a few hours.

SU‐E‐T‐369: Evaluating Intensity Modulated Proton Therapy Relative to Passive Scattering Proton Therapy for Increased Vertebral Column Sparing in CSI of Pediatric Patients

Purpose:To investigate the trade‐off between vertebral column sparing and thecal‐sac target coverage in craniospinal irradiation (CSI) of pediatric patients treated with passive‐scattering (PS) and intensity modulated (IMPT) proton therapy.Methods:We selected 2 pediatric patients treated with PS CSI for medulloblastoma. Spinal irradiation was re‐planned with IMPT. For all cases, we assumed prescription dose of 23.4 Gy(RBE), with the spinal canal receiving at least 95% of 23.4 Gy(RBE). PS planning was performed using the commercial system XiO. IMPT planning was done using the Astroid planning system. Beam arrangements consisted of (a) PS posterior‐anterior (PA) field, PS‐PA, (b) IMPT PA field, IMPT‐PA, and (c) two posterior oblique IMPT fields, IMPT2 (‐35°, 35°). Dose distributions were re‐calculated using TOPAS Monte Carlo, along with LET distributions, to investigate LET variations within the target and vertebra anatomy. Variable RBE‐weighed dose distributions were also calculated based on a dose and LET‐dependent biophysical model. Dosimetric data were compared among the plans for the target volume, spinal cord and adjacent critical organs (thecal‐sac and cauda equina).Results:IMPT2 resulted in better sparing of the posterior vertebral column (entrance region posterior to thecal‐sac), where planned dose was approximately 6–8Gy(RBE). For IMPT‐PA and PS‐PA the MC‐calculated dose to the posterior vertebral column was, on average, 20Gy and 18Gy respectively. For IMPT2 higher mean‐LET (5keV/µm/(g/cm3)) values were observed in anterior vertebral column (beyond the thecal‐sac) relative to IMPT‐PA and PS‐PA, where mean‐LET was 3.5keV/µm/(g/cm3) and 2.5keV/µm/(g/cm3) respectively. The higher LET region observed for both IMPT plans was in the distal end of treatment fields, where dose delivered was less 5Gy(RBE).Conclusion:The two‐oblique proton beams IMPT2 best spared the spinal column, while reducing the dose to the posterior spinal column from 18–20 to 6–8 Gy(RBE). The best LET distribution was obtained with the PS‐PA fields.

SU‐E‐T‐486: In Vivo Skin Dosimetry Using the Exradin W1 Plastic Scintillation Detector for Passively Scattered Proton Beam Therapy

Purpose:To evaluate the accuracy and usefulness of plastic scintillation detectors used for skin dosimetry of patients undergoing passive scatter proton therapy.Methods:Following an IRB approved protocol, six patients undergoing passively scattered proton beam therapy for prostate cancer were selected for in vivo skin dosimetry using the Exradin W1 plastic scintillator. The detector was calibrated on a Cobalt‐60 unit, and phantom measurements in the proton beam with the W1 and a calibrated parallel plate ion chamber were used to account for the under‐response due to high LET at energies used for treatment. Measurements made in a heated water tank were used to account for temperature dependence. For in vivo measurements, the W1 is fixed to the patient's skin with medical tape in the center of each of two laterally opposed treatment fields. Measurements will be performed once per week for each patient for the duration of treatment, for a total of thirty six measurements. The measured dose will be compared to the expected dose, extracted from the Eclipse treatment planning system. The average difference over all measurements and per‐patient will be computed, as well as standard deviations.Results:The calibrated detector exhibited a 7% under‐response in 225 and 250 MeV beams, and a 4% under‐response when used at 37 °C (relative to the response at the calibration temperature of 20 °C). Patient measurements are ongoing.Conclusion:The Exradin W1 plastic scintillator detector is a strong candidate for in vivo skin dosimetry in passively scattered proton beams as PSDs are water equivalent and very small (2mm in diameter), permitting accurate measurements that do not perturb the delivered dose.This project was supported in part by award number CA182450 from the National Cancer Institute.

SU‐E‐T‐439: Fundamental Verification of Respiratory‐Gated Spot Scanning Proton Beam Therapy

Purpose:The spot‐scanning proton beam irradiation with respiratory gating technique provides quite well dose distribution and requires both dosimetric and geometric verification prior to clinical implementation. The purpose of this study is to evaluate the impact of gating irradiation as a fundamental verification.Methods:We evaluated field width, flatness, symmetry, and penumbra in the gated and non‐gated proton beams. The respiration motion was distinguished into 3 patterns: 10, 20, and 30 mm. We compared these contents between the gated and non‐gated beams. A 200 MeV proton beam from PROBEAT‐III unit (Hitachi Co.Ltd) was used in this study. Respiratory gating irradiation was performed by Quasar phantom (MODUS medical devices) with a combination of dedicated respiratory gating system (ANZAI Medical Corporation). For radiochromic film dosimetry, the calibration curve was created with Gafchromic EBT3 film (Ashland) on FilmQA Pro 2014 (Ashland) as film analysis software.Results:The film was calibrated at the middle of spread out Bragg peak in passive proton beam. The field width, flatness and penumbra in non‐gated proton irradiation with respiratory motion were larger than those of reference beam without respiratory motion: the maximum errors of the field width, flatness and penumbra in respiratory motion of 30 mm were 1.75% and 40.3% and 39.7%, respectively. The errors of flatness and penumbra in gating beam (motion: 30 mm, gating rate: 25%) were 0.0% and 2.91%, respectively. The results of symmetry in all proton beams with gating technique were within 0.6%.Conclusion:The field width, flatness, symmetry and penumbra were improved with the gating technique in proton beam. The spot scanning proton beam with gating technique is feasible for the motioned target.

SU‐E‐T‐743: The Simple Monitor Unit Calculation for Irregular Field in Passive Proton Beam

Purpose:We try to apply the monitor unit (MU) calculation procedure for passively proton beam therapy. For estimation of field size factor (FSF) in the process, we used root A method for conversion from irregular field to equivalent field so far. In this time, we changed the conversion from root A method to Clarkson method. The purpose of this study is to ensure the calculation accuracy by comparative evaluation between root A and Clarkson methods.Methods:As fundamental study, we measured absolute dose with several circular field. Then we performed experimental fitting of conversion curve. With this method, the conversion from the equivalent circular field to equivalent square field is available by the assumed equation. As clinical study, we compared the measured MU with the calculated MU (root A and Clarkson method) in 28 prostate patients (76 fields) for the purpose of evaluating the feasibility of our procedure. To simplify the comparison of the calculation procedure, we built the dedicated MU calculation software.Results:As the Result of fundamental study, the assumed equation for conversion from circular field to square field was estimated. As the Result of clinical study, the differences between the measured MU and the calculated MU were within 2% in all cases. All calculated MU were calculated by our software. The differences of MU calculation between measurement MU and calculation MU (root A and Clarkson) were less than 2% for all cases. The average difference and standard deviation of MU calculation between the root A and Clarkson was 0.05±0.05%. However, the calculation accuracy of Clarkson method should be verified in the complicated cases.Conclusion:The MU calculation by use of Clarkson method was supposed and verified in this study. For prostate cases, the Clarkson method is feasible for MU calculation procedure.

Study of Morphological Variation on SiO2/Nafion Composite Membrane Under Different Hydration Condition

Passive air breathing proton exchange membranes fuel cells (AB-PEMFC) have a unique advantage such as remarkably reducing stack weight by minimizing balance of plant (BOP). However, the performance of the AB-PEMFC is worse than general PEMFC because it is difficult to controlling humidification and stack temperature. Thus, the proton exchange membranes (PEMs) which are used to AB-PEMFC have to stably work under various humidification and temperature. A lot of research groups are reported that inorganic fillers (SiO2, TiO2, ZrO2) and Nafion composite membrane show high performance under low relative humidity by increasing water retention on the membrane. In this study, we synthesize SiO2/Nafion composite membrane for compensating the weakness of AB-PEMFC. The performance of membrane electrode assembly (MEA) with SiO2/Nafion composite membrane is tested under different hydration and temperature conditions. For more detailed characterization, morphological variations of the composite membrane under hydration conditions are studied by using dynamic mode atomic force microscopy (DMAFM). DMAFM can map amplitude and phase distribution on the specimen surface. The phase lag, which is mechanical vibration changing of a cantilever, is resulting of tip-sample interaction, which is induced by microscopic mechanical property of sample. Thus, the DMAFM is best tool for characterizing heterogeneous materials such as composite membrane. The morphological structure variation on the composite membrane by water retention elevation is systematically studied by using phase lag changing under different humidification condition. References C. A. Hayden, and G. R. Goward, Proton Dynamics of Nafion and Nafion/SiO2 Composites by Solid tate NMR and Pulse Field Gradient NMR Macromolecules 40, (2007), 1529-1537 Bella Nicotera, Apostolos Enotiadis, Kristina Angjeli, Luigi Coppola, Giuseppe A. Ranieri, and imitrios Gournis, Effective Improvement of Water-Retention in Nanocomposite embranes Using Novel Organo-Modified Clays as Fillers or High Temperature PEMFCs, Journal of Physical Chemistry B 115, (2011), 9087–9097 Figure 1

M2 proton channel: toward a model of a primitive proton pump.

Transmembrane proton transfer was essential to early cellular systems in order to transduce energy for metabolic functions. The reliable, efficient and controlled generation of proton gradients became possible only with the emergence of active proton pumps. On the basis of features shared by most modern proton pumps we identify the essential mechanistic steps in active proton transport. Further, we discuss the mechanism of action of a small, transmembrane M2 proton channel from influenza A virus as a model for proton transport in protocells. The M2 channel is a 94-residue long, α-helical tetramer that is activated at low pH and exhibits high selectivity and directionality. A shorter construct, built of transmembrane fragments that are only 24 amino acids in length, exhibits very similar proton transport properties. Molecular dynamics simulations on the microsecond time-scale carried out for the M2 channel provided atomic level details on the activation of the channel in response to protonation of the histidine residue, His37. The pathway of proton conduction is mediated by His37, which accepts and donates protons at different interconverting conformation states when pH is lower than 6.5. The Val27 and Trp41 gates and the salt bridge between Asp44 and Arg45 further enhance the directionality of proton transport. It is argued that the architecture and the mechanism of action similar to that found in the M2 channel might have been the perfect starting point for evolution towards the earliest proton pumps, indicating that active proton transport could have readily emerged from simple, passive proton channels.

Biological Effects of Passive Versus Active Scanning Proton Beams on Human Lung Epithelial Cells

The goal was to characterize differences in cell response after exposure to active beam scanning (ABS) protons compared to a passive delivery system. Human lung epithelial (HLE) cells were evaluated at various locations along the proton depth dose profile. The dose delivered at the Bragg peak position was essentially identical (∼4 Gy) with the two techniques, but depth dose data showed that ABS resulted in lower doses at entry and more rapid drop-off after the peak. Average dose rates for the passive and ABS beams were 1.1 Gy/min and 5.1 Gy/min, respectively; instantaneous dose rates were 19.2 Gy/min and 2,300 Gy/min (to a 0.5 × 0.5 mm(2) voxel). Analysis of DNA synthesis was based on (3)H-TdR incorporation. Quantitative real-time polymerase chain reaction (RT-PCR) was done to determine expression of genes related to p53 signaling and DNA damage; a total of 152 genes were assessed. Spectral karyotyping and analyses of the Golgi apparatus and cytokines produced by the HLE cells were also performed. At or near the Bragg peak position, ABS protons resulted in a greater decrease in DNA synthesis compared to passively delivered protons. Genes with >2-fold change (P < 0.05 vs. 0 Gy) after passive proton irradiation at one or more locations within the Bragg curve were BTG2, CDKN1A, IFNB1 and SIAH1. In contrast, many more genes had >2-fold difference with ABS protons: BRCA1, BRCA2, CDC25A, CDC25C, CCNB2, CDK1, DMC1, DNMT1, E2F1, EXO1, FEN1, GADD45A, GTSE1, IL-6, JUN, KRAS, MDM4, PRC1, PTTG1, RAD51, RPA1, TNF, WT1, XRCC2, XRCC3 and XRCC6BP1. Spectral karyotyping revealed numerous differences in chromosomal abnormalities between the two delivery systems, especially at or near the Bragg peak. Percentage of cells staining for the Golgi apparatus was low after exposure to passive and active proton beams. Studies such as this are needed to ensure patient safety and make modifications in ABS delivery, if necessary.

Lipid-dependent pore formation by antimicrobial peptides arenicin-2 and melittin demonstrated by their proton transfer activity

This work presents a comparative study of proton transfer activity (PTA) of two cationic (+6) antimicrobial peptides, β-structural arenicin-2 and α-helical melittin. A new approach was proposed for the detection of passive proton transfer by using proteoliposomes containing bacteriorhodopsin, which creates a small light-induced electrochemical proton gradient ∆ΔpH. Addition of several nanomoles of the peptides lowers ∆ΔpH that is proximately indicative of the pore formation. The quantitative analysis of sigmoidal dependences of ∆pH on the peptides concentration was carried out using liposomes prepared from PC, PC/PE, PC/PE/PI and PC/PG. Substitution of PC-containing liposomes with PE-containing ones, having negative spontaneous curvature, reduced the PTA of α-helical melittin and increased that of β-structural arenicin-2. This result indicates an essential difference in the pore formation by these peptides. Further increase of PTA in response to arenicin-2 (in contrast to melittin) was observed in the liposomes prepared from PC/PE/PI. The data analysis leads to the conclusion that PTA is influenced by (i) efficiency of the pore assemblage, which depends on the structure of pore-forming peptides, and the spontaneous curvature of lipids and (ii) the presence of mobile protons in the polar head groups of phospholipids.

SU-E-T-591: Measurement and Monte Carlo Simulation of Stray Neutrons in Passive Scattering Proton Therapy: Needs and Challenges

Purpose: Measure stray radiation inside a passive scattering proton therapy facility, compare values to Monte Carlo (MC) simulations and identify the actual needs and challenges. Methods: Measurements and MC simulations were considered to acknowledge neutron exposure associated with 75 MeV ocular or 180 MeV intracranial passively scattered proton treatments. First, using a specifically-designed high sensitivity Bonner Sphere system, neutron spectra were measured at different positions inside the treatment rooms. Next, measurement-based mapping of neutron ambient dose equivalent was fulfilled using several TEPCs and rem-meters. Finally, photon and neutron organ doses were measured using TLDs, RPLs and PADCs set inside anthropomorphic phantoms (Rando, 1 and 5-years-old CIRS). All measurements were also simulated with MCNPX to investigate the efficiency of MC models in predicting stray neutrons considering different nuclear cross sections and models. Results: Knowledge of the neutron fluence and energy distribution inside a proton therapy room is critical for stray radiation dosimetry. However, as spectrometry unfolding is initiated using a MC guess spectrum and suffers from algorithmic limits a 20% spectrometry uncertainty is expected. H*(10) mapping with TEPCs and rem-meters showed a good agreement between the detectors. Differences within measurement uncertainty (10–15%) were observed and are inherent to the energy, fluence and directional response of each detector. For a typical ocular and intracranial treatment respectively, neutron doses outside the clinical target volume of 0.4 and 11 mGy were measured inside the Rando phantom. Photon doses were 2–10 times lower depending on organs position. High uncertainties (40%) are inherent to TLDs and PADCs measurements due to the need for neutron spectra at detector position. Finally, stray neutrons prediction with MC simulations proved to be extremely dependent on proton beam energy and the used nuclear models and cross sections. Conclusion: This work highlights measurement and simulation limits for ion therapy radiation protection applications.