γ-SNAP stimulates disassembly of endosomal SNARE complexes and regulates endocytic trafficking pathways.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that reside in the target membranes and transport vesicles assemble into specific SNARE complexes to drive membrane fusion. N-ethylmaleimide-sensitive factor (NSF) and its attachment protein, α-SNAP (encoded by NAPA), catalyze disassembly of the SNARE complexes in the secretory and endocytic pathways to recycle them for the next round of fusion events. γ-SNAP (encoded by NAPG) is a SNAP isoform, but its function in SNARE-mediated membrane trafficking remains unknown. Here, we show that γ-SNAP regulates the endosomal trafficking of epidermal growth factor (EGF) receptor (EGFR) and transferrin. Immunoprecipitation and mass spectrometry analyses revealed that γ-SNAP interacts with a limited range of SNAREs, including endosomal ones. γ-SNAP, as well as α-SNAP, mediated the disassembly of endosomal syntaxin-7-containing SNARE complexes. Overexpression and small interfering (si)RNA-mediated depletion of γ-SNAP changed the morphologies and intracellular distributions of endosomes. Moreover, the depletion partially suppressed the exit of EGFR and transferrin from EEA1-positive early endosomes to delay their degradation and uptake. Taken together, our findings suggest that γ-SNAP is a unique SNAP that functions in a limited range of organelles - including endosomes - and their trafficking pathways.

A novel analytical procedure for assaying lysozyme activity using an end-blocked chitotetraose derivative as substrate.

An end-modified ß-d-galactosyl chitotetraose derivative [44-O-ß-d-galactosyl-ß-tri-N-acetylchitotriosyl 2-acetamide-2,3-dideoxy-glucopyranose; Gal(GlcN)3D] was designed and synthesized from chitin tetrasaccharide. The derivative was chemically modified by dehydration of the reducing end GlcN and enzymatic addition of a Gal group to the non-reducing end GlcN. Hydrolysis of Gal(GlcN)3D and related compounds using hen egg-white lysozyme was then examined. Gal(GlcN)3D was specifically cleaved to Gal(GlcN)2 and GlcND. Kinetic studies and docking simulations were further conducted to elucidate its mode of binding to lysozyme. These analyses revealed the binding of Gal(GlcN)3D to lysozyme is more favorable than that of (GlcN)4D. We conclude the 4-O-substituted Gal group at the non-reducing end of Gal(GlcN)3D does not prohibit the action of lysozyme, but gives some affinity to the subsite (i.e. equivalent to GlcN). From these results, a new assay method for quantifying lysozyme was established by utilizing the Morgan-Elson reaction based on the generation of product D (2-acetamide-2,3-dideoxy-glucopyranose), which serves as a chromophore, formed from Gal(GlcN)3D by lysozyme through a conjugated reaction involving ß-N-acetylhexosaminidase. The assay system gave a linear dose-response curve in the range of 2-31 µg of lysozyme during a 15 min incubation. This novel assay method for the quantification of lysozyme is highly specific, sensitive, accurate and reproducible.

Patient handling system for carbon ion beam scanning therapy.

Our institution established a new treatment facility for carbon ion beam scanning therapy in 2010. The major advantages of scanning beam treatment compared to the passive beam treatment are the following: high dose conformation with less excessive dose to the normal tissues, no bolus compensator and patient collimator/multi-leaf collimator, better dose efficiency by reducing the number of scatters. The new facility was designed to solve several problems encountered in the existing facility, at which several thousand patients were treated over more than 15 years. Here, we introduce the patient handling system in the new treatment facility. The new facility incorporates three main systems, a scanning irradiation system (S-IR), treatment planning system (TPS), and patient handling system (PTH). The PTH covers a wide range of functions including imaging, geometrical/position accuracy including motion management (immobilization, robotic arm treatment bed), layout of the treatment room, treatment workflow, software, and others. The first clinical trials without respiratory gating have been successfully started. The PTH allows a reduction in patient stay in the treatment room to as few as 7 min. The PTH plays an important role in carbon ion beam scanning therapy at the new institution, particularly in the management of patient handling, application of image-guided therapy, and improvement of treatment workflow, and thereby allows substantially better treatment at minimum cost.

Development of digital reconstructed radiography software at new treatment facility for carbon-ion beam scanning of National Institute of Radiological Sciences.

To increase the accuracy of carbon ion beam scanning therapy, we have developed a graphical user interface-based digitally-reconstructed radiograph (DRR) software system for use in routine clinical practice at our center. The DRR software is used in particular scenarios in the new treatment facility to achieve the same level of geometrical accuracy at the treatment as at the imaging session. DRR calculation is implemented simply as the summation of CT image voxel values along the X-ray projection ray. Since we implemented graphics processing unit-based computation, the DRR images are calculated with a speed sufficient for the particular clinical practice requirements. Since high spatial resolution flat panel detector (FPD) images should be registered to the reference DRR images in patient setup process in any scenarios, the DRR images also needs higher spatial resolution close to that of FPD images. To overcome the limitation of the CT spatial resolution imposed by the CT voxel size, we applied image processing to improve the calculated DRR spatial resolution. The DRR software introduced here enabled patient positioning with sufficient accuracy for the implementation of carbon-ion beam scanning therapy at our center.

Mid-term outcome of transarterial embolization of renal artery pseudoaneurysm and arteriovenous fistula after partial nephrectomy screened by early postoperative contrast-enhanced CT.

PURPOSE: To retrospectively evaluate the mid-term outcome of transarterial embolization (TAE) of renal artery pseudoaneurysm (RAP) including arteriovenous fistula (AVF) after partial nephrectomy screened by early postoperative contrast-enhanced CT (CE-CT). MATERIALS AND METHODS: Eighty-two patients (7.0%) who underwent TAE after partial nephrectomy were reviewed, from 1166 partial nephrectomies performed over 6 years. In 18 patients (22.0%), TAE was performed emergently on the median postoperative day (POD) seven. In the remaining patients, elective TAE was performed on the median POD six for RAP detected by early postoperative CE-CT or that emerged on follow-up CE-CT. RESULTS: In one patient (1.2%), TAE was performed twice because one of two RAPs could not be embolized during the first TAE, being successfully embolized at the second TAE after readmission with hematuria. Otherwise, no bleeding recurrence or RAPs were observed during the median 1354 follow-up days. Thus, the primary and secondary success rates of TAE were 98.8% (81 of 82 patients) and 100% (82 of 82 patients), respectively. On angiography, the average number of lesions was 1.7 ± 0.9 and the average RAP size was 12.8 ± 6.0 mm. The shapes of the lesions varied: oval-round 60, oval-round + AVF 36, irregular + AVF 14, AVF 12, irregular 10, disruption 4, and extravasation 3. No major complications were observed. The median inpatient days after TAE were two. No estimated glomerular filtration rate deterioration was observed (64.6 ± 18.6 vs. 64.2 ± 18.4 mL/min/1.73 m2, p = 0.902). CONCLUSION: TAE is largely effective and safe for treating bleedings or RAPs, including AVFs, after partial nephrectomy, as screened by early postoperative CE-CT.

Sustained Neurotrophin Release from Protein Nanoparticles Mediated by Matrix Metalloproteinases Induces the Alignment and Differentiation of Nerve Cells.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.

A simulation study on the dosimetric benefit of real-time motion compensation in spot-scanning proton therapy for prostate.

For proton spot scanning, use of a real-time-image gating technique incorporating an implanted marker and dual fluoroscopy facilitates mitigation of the dose distribution deterioration caused by interplay effects. This study explored the advantages of using a real-time-image gating technique, with a focus on prostate cancer. Two patient-positioning methods using fiducial markers were compared: (i) patient positioning only before beam delivery, and (ii) patient positioning both before and during beam delivery using a real-time-gating technique. For each scenario, dose distributions were simulated using the CT images of nine prostate cancer patients. Treatment plans were generated using a single-field proton beam with 3-mm and 6-mm lateral margins. During beam delivery, the prostate was assumed to move by 5 mm in four directions that were perpendicular to the beam direction at one of three separate timings (i.e. after the completion of the first, second and third quartiles of the total delivery of spot irradiation). Using a 3-mm margin and second quartile motion timing, the averaged values for ΔD99, ΔD95, ΔD5 and D5-95 were 5.1%, 3.3%, 3.6% and 9.0%, respectively, for Scenario (i) and 2.1%, 1.5%, 0.5% and 4.1%, respectively, for Scenario (ii). The margin expansion from 3 mm to 6 mm reduced the size of ΔD99, ΔD95, ΔD5 and D5-95 only with Scenario (i). These results indicate that patient positioning during beam delivery is an effective way to obtain better target coverage and uniformity while reducing the target margin when the prostate moves during irradiation.

Development of Continuous Line Scanning System Prototype for Proton Beam Therapy.

PURPOSE: Taking advantage of the continuous, high-intensity beam of the cyclotron at the National Cancer Center Hospital East, we developed a continuous line scanning system (CLSS) prototype for prostate cancer in collaboration with Sumitomo Heavy Industries, Ltd (Tokyo, Japan). MATERIALS AND METHODS: The CLSS modulates dose distribution at each beam energy level by varying scanning speed while keeping the beam intensity constant through a beam-intensity control system and a rapid on/off beam-switching system. In addition, we developed a beam alignment system to improve the precision of the beam position. The scanning control system is used to control the scanning pattern and set the value of the nozzle apparatus. It also collects data for monitoring and for cyclotron parameters and transmits information to the scanning power supplies and monitor amplifiers, which serve as the measurement system, and to the nozzle-control and beam-transfer systems. The specifications of the line scanning beam were determined in performance tests. Finally, a patient-specific dosimetric measurement for prostate cancer was also performed. RESULTS: The beam size, position, intensity, and scanning speed of our CLSS were found to be well within clinical requirements. The CLSS produced an accurate 3-dimensional dose distribution for clinical treatment planning. CONCLUSION: The performance of our new CLSS was confirmed to comply with clinical requirements. We have been employing it in prostate cancer treatments since October 23, 2015.

Impact of Real-Time Image Gating on Spot Scanning Proton Therapy for Lung Tumors: A Simulation Study.

PURPOSE: To investigate the effectiveness of real-time-image gated proton beam therapy for lung tumors and to establish a suitable size for the gating window (GW). METHODS AND MATERIALS: A proton beam gated by a fiducial marker entering a preassigned GW (as monitored by 2 fluoroscopy units) was used with 7 lung cancer patients. Seven treatment plans were generated: real-time-image gated proton beam therapy with GW sizes of ±1, 2, 3, 4, 5, and 8 mm and free-breathing proton therapy. The prescribed dose was 70 Gy (relative biological effectiveness)/10 fractions to 99% of the target. Each of the 3-dimensional marker positions in the time series was associated with the appropriate 4-dimensional computed tomography phase. The 4-dimensional dose calculations were performed. The dose distribution in each respiratory phase was deformed into the end-exhale computed tomography image. The D99 and D5 to D95 of the clinical target volume scaled by the prescribed dose with criteria of D99 >95% and D5 to D95 <5%, V20 for the normal lung, and treatment times were evaluated. RESULTS: Gating windows ≤ ±2 mm fulfilled the CTV criteria for all patients (whereas the criteria were not always met for GWs ≥ ±3 mm) and gave an average reduction in V20 of more than 17.2% relative to free-breathing proton therapy (whereas GWs ≥ ±4 mm resulted in similar or increased V20). The average (maximum) irradiation times were 384 seconds (818 seconds) for the ±1-mm GW, but less than 226 seconds (292 seconds) for the ±2-mm GW. The maximum increased considerably at ±1-mm GW. CONCLUSION: Real-time-image gated proton beam therapy with a GW of ±2 mm was demonstrated to be suitable, providing good dose distribution without greatly extending treatment time.

NTCP modeling analysis of acute hematologic toxicity in whole pelvic radiation therapy for gynecologic malignancies - A dosimetric comparison of IMRT and spot-scanning proton therapy (SSPT).

PURPOSE: This treatment planning study was conducted to determine whether spot scanning proton beam therapy (SSPT) reduces the risk of grade ⩾3 hematologic toxicity (HT3+) compared with intensity modulated radiation therapy (IMRT) for postoperative whole pelvic radiation therapy (WPRT). METHODS AND MATERIALS: The normal tissue complication probability (NTCP) of the risk of HT3+ was used as an in silico surrogate marker in this analysis. IMRT and SSPT plans were created for 13 gynecologic malignancy patients who had received hysterectomies. The IMRT plans were generated using the 7-fields step and shoot technique. The SSPT plans were generated using anterior-posterior field with single field optimization. Using the relative biological effectives (RBE) value of 1.0 for IMRT and 1.1 for SSPT, the prescribed dose was 45Gy(RBE) in 1.8Gy(RBE) per fractions for 95% of the planning target volume (PTV). The homogeneity index (HI) and the conformity index (CI) of the PTV were also compared. RESULTS: The bone marrow (BM) and femoral head doses using SSPT were significantly lower than with IMRT. The NTCP modeling analysis showed that the risk of HT3+ using SSPT was significantly lower than with IMRT (NTCP=0.04±0.01 and 0.19±0.03, p=0.0002, respectively). There were no significant differences in the CI and HI of the PTV between IMRT and SSPT (CI=0.97±0.01 and 0.96±0.02, p=0.3177, and HI=1.24±0.11 and 1.27±0.05, p=0.8473, respectively). CONCLUSION: The SSPT achieves significant reductions in the dose to BM without compromising target coverage, compared with IMRT. The NTCP value for HT3+ in SSPT was significantly lower than in IMRT.

Optimization and evaluation of multiple gating beam delivery in a synchrotron-based proton beam scanning system using a real-time imaging technique.

PURPOSE: To find the optimum parameter of a new beam control function installed in a synchrotron-based proton therapy system. METHODS: A function enabling multiple gated irradiation in the flat top phase has been installed in a real-time-image gated proton beam therapy (RGPT) system. This function is realized by a waiting timer that monitors the elapsed time from the last gate-off signal in the flat top phase. The gated irradiation efficiency depends on the timer value, Tw. To find the optimum Tw value, gated irradiation efficiency was evaluated for each configurable Tw value. 271 gate signal data sets from 58 patients were used for the simulation. RESULTS: The highest mean efficiency 0.52 was obtained in TW=0.2s. The irradiation efficiency was approximately 21% higher than at TW=0s, which corresponds to ordinary synchrotron operation. The irradiation efficiency was improved in 154 (57%) of the 271 cases. The irradiation efficiency was reduced in 117 cases because the TW value was insufficient or the function introduced an unutilized wait time for the next gate-on signal in the flat top phase. In the actual treatment of a patient with a hepatic tumor at Tw=0.2s, 4.48GyE irradiation was completed within 250s. In contrast, the treatment time of ordinary synchrotron operation was estimated to be 420s. CONCLUSIONS: The results suggest that the multiple gated-irradiation function has potential to improve the gated irradiation efficiency and to reduce the treatment time.

Development and evaluation of a short-range applicator for treating superficial moving tumors with respiratory-gated spot-scanning proton therapy using real-time image guidance.

Treatment of superficial tumors that move with respiration (e.g. lung tumors) using spot-scanning proton therapy (SSPT) is a high-priority research area. The recently developed real-time image-gated proton beam therapy (RGPT) system has proven to be useful for treating moving tumors deep inside the liver. However, when treating superficial tumors, the proton's range is small and so is the sizes of range straggling, making the Bragg-peaks extremely sharp compared to those located in deep-seated tumors. The extreme sharpness of Bragg-peaks is not always beneficial because it necessitates a large number of energy layers to make a spread-out Bragg-peak, resulting in long treatment times, and is vulnerable to motion-induced dose deterioration. We have investigated a method to treat superficial moving tumors in the lung by the development of an applicator compatible with the RGPT system. A mini-ridge filter (MRF) was developed to broaden the pristine Bragg-peak and, accordingly, decrease the number of required energy layers to obtain homogeneous irradiation. The applicator position was designed so that the fiducial marker's trajectory can be monitored by fluoroscopy during proton beam-delivery. The treatment plans for three lung cancer patients were made using the applicator, and four-dimensional (4D) dose calculations for the RGPT were performed using patient respiratory motion data. The effect of the MRF on the dose distributions and treatment time was evaluated. With the MRF, the number of energy layers was decreased to less than half of that needed without it, whereas the target volume coverage values (D99%, D95%, D50%, D2%) changed by less than 1% of the prescribed dose. Almost no dose distortion was observed after the 4D dose calculation, whereas the treatment time decreased by 26%-37%. Therefore, we conclude that the developed applicator compatible with RGPT is useful to solve the issue in the treatment of superficial moving tumors with SSPT.

Effective and organ doses using helical 4DCT for thoracic and abdominal therapies.

The capacity of 4DCT to quantify organ motion is beyond conventional 3DCT capability. Local control could be improved. However we are unaware of any reports of organ dose measurements for helical 4DCT imaging. We therefore quantified the radiation doses for helical 4DCT imaging. Organ and tissue dose was measured for thoracic and abdominal 4DCT in helical mode using an adult anthropomorphic phantom. Radiation doses were measured with thermoluminescence dosimeter chips inserted at various anatomical sites on the phantom. For the helical thoracic 4DCT, organ doses were 57.2 mGy for the lung, 76.7 mGy for the thyroids, 48.1 mGy for the breasts, and 10.86 mGy for the colon. The effective doses for male and female phantoms were very similar, with a mean value of 33.1 mSv. For abdominal 4DCT imaging, organ doses were 14.4 mGy for the lung, 0.78 mGy for the thyroids, 9.83 mGy for breasts, and 58.2 mGy for the colon (all obtained by using ICRP 103). We quantified the radiation exposure for thoracic and abdominal helical 4DCT. The doses for helical 4DCT were approximately 1.5 times higher than those for cine 4DCT, however the stepwise image artifact was reduced. 4DCT imaging should be performed with care in order to minimize radiation exposure, but the advantages of 4DCT imaging mandates its incorporation into routine treatment protocols.

First clinical experience in carbon ion scanning beam therapy: retrospective analysis of patient positional accuracy.

Our institute has constructed a new treatment facility for carbon ion scanning beam therapy. The first clinical trials were successfully completed at the end of November 2011. To evaluate patient setup accuracy, positional errors between the reference Computed Tomography (CT) scan and final patient setup images were calculated using 2D-3D registration software. Eleven patients with tumors of the head and neck, prostate and pelvis receiving carbon ion scanning beam treatment participated. The patient setup process takes orthogonal X-ray flat panel detector (FPD) images and the therapists adjust the patient table position in six degrees of freedom to register the reference position by manual or auto- (or both) registration functions. We calculated residual positional errors with the 2D-3D auto-registration function using the final patient setup orthogonal FPD images and treatment planning CT data. Residual error averaged over all patients in each fraction decreased from the initial to the last treatment fraction [1.09 mm/0.76° (averaged in the 1st and 2nd fractions) to 0.77 mm/0.61° (averaged in the 15th and 16th fractions)]. 2D-3D registration calculation time was 8.0 s on average throughout the treatment course. Residual errors in translation and rotation averaged over all patients as a function of date decreased with the passage of time (1.6 mm/1.2° in May 2011 to 0.4 mm/0.2° in December 2011). This retrospective residual positional error analysis shows that the accuracy of patient setup during the first clinical trials of carbon ion beam scanning therapy was good and improved with increasing therapist experience.

Nuclear collision processes around the Bragg peak in proton therapy.

In the physical processes of proton interaction in bio-materials, most of the proton energy is transferred to electrons. Ionization and excitation occur most frequently around the Bragg peak region, where nuclear reactions also exist. In this study, we investigated the processes of energy deposition by considering interactions including the nuclear reactions between protons and water molecules by a Monte Carlo simulation for proton therapy. We estimated the number of particles produced by a variety of nuclear reactions, and we focused on the interaction in the low-energy region (below 1 MeV). Furthermore, we considered the charge-changing processes in the low-energy region (less than a few hundred keV). Finally, we evaluated the total dose and the contribution of primary protons and secondary particles through nuclear reactions to the absorbed dose. The results showed that the protons generate numerous neutrons via nuclear reactions. Particularly, neutrons with relatively low energies produce recoil protons by elastic collisions with the hydrogen atoms. Around the Bragg peak, low-energy primary protons (slowed-down protons) are prevalent, whereas recoil (secondary) protons gradually become dominant behind the distal falloff region of the Bragg peak. Therefore, around the Bragg peak, the main contribution to the absorbed dose is that of the primary protons (from 80 to 90%), whereas secondary protons created by primary proton-induced reactions contribute to the dose from 20 to 5%. Behind the distal endpoint of the Bragg peak, the absorbed dose is mainly due to the protons produced by (1)H(n, p), and the contribution of these is about 70%.