Clustered DNA Damage Patterns after Proton Therapy Beam Irradiation Using Plasmid DNA.

Modeling ionizing radiation interaction with biological matter is a major scientific challenge, especially for protons that are nowadays widely used in cancer treatment. That presupposes a sound understanding of the mechanisms that take place from the early events of the induction of DNA damage. Herein, we present results of irradiation-induced complex DNA damage measurements using plasmid pBR322 along a typical Proton Treatment Plan at the MedAustron proton and carbon beam therapy facility (energy 137-198 MeV and Linear Energy Transfer (LET) range 1-9 keV/µm), by means of Agarose Gel Electrophoresis and DNA fragmentation using Atomic Force Microscopy (AFM). The induction rate Mbp-1 Gy-1 for each type of damage, single strand breaks (SSBs), double-strand breaks (DSBs), base lesions and non-DSB clusters was measured after irradiations in solutions with varying scavenging capacity containing 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris) and coumarin-3-carboxylic acid (C3CA) as scavengers. Our combined results reveal the determining role of LET and Reactive Oxygen Species (ROS) in DNA fragmentation. Furthermore, AFM used to measure apparent DNA lengths provided us with insights into the role of increasing LET in the induction of highly complex DNA damage.

On the measurement uncertainty of microdosimetric quantities using diamond and silicon microdosimeters in carbon-ion beams.

PURPOSE: The purpose of this paper is to compare the response of two different types of solid-state microdosimeters, that is, silicon and diamond, and their uncertainties. A study of the conversion of silicon microdosimetric spectra to the diamond equivalent for microdosimeters with different geometry of the sensitive volumes is performed, including the use of different stopping power databases. METHOD: Diamond and silicon microdosimeters were irradiated under the same conditions, aligned at the same depth in a carbon-ion beam at the MedAustron ion therapy center. In order to estimate the microdosimetric quantities, the readout electronic linearity was investigated with three different methods, that is, the first being a single linear regression, the second consisting of a double linear regression with a channel transition and last a multiple linear regression by splitting the data into odd and even groups. The uncertainty related to each of these methods was estimated as well. The edge calibration was performed using the intercept with the horizontal axis of the tangent through the inflection point of the Fermi function approximation multi-channel analyzer spectrum. It was assumed that this point corresponds to the maximum energy difference of particle traversing the sensitive volume (SV) for which the residual range difference in the continuous slowing down approximation is equal to the thickness of the SV of the microdosimeter. Four material conversion methods were explored, the edge method, the density method, the maximum-deposition energy method and the bin-by-bin transformation method. The uncertainties of the microdosimetric quantities resulting from the linearization, the edge calibration and the detectors thickness were also estimated. RESULTS: It was found that the double linear regression had the lowest uncertainty for both microdosimeters. The propagated standard (k = 1) uncertainties on the frequency-mean lineal energy y ¯ F ${\bar{y}}_{\rm{F}}$ and the dose-mean lineal energy y ¯ D ${\bar{y}}_{\rm{D}}$ values from the marker point, in the spectra, in the plateau were 0.1% and 0.2%, respectively, for the diamond microdosimeter, whilst for the silicon microdosimeter data converted to diamond, the uncertainty was estimated to be 0.1%. In the range corresponding to the 90% of the amplitude of the Bragg Peak at the distal part of the Bragg curve (R90 ) the uncertainty was found to be 0.1%. The uncertainty propagation from the stopping power tables was estimated to be between 5% and 7% depending on the method. The uncertainty on the y ¯ F ${\bar{y}}_{\rm{F}}$ and y ¯ D ${\bar{y}}_{\rm{D}}$ coming from the thickness of the detectors varied between 0.3% and 0.5%. CONCLUSION: This article demonstrate that the linearity of the readout electronics affects the microdosimetric spectra with a difference in y ¯ F ${\bar{y}}_{\rm{F}}$ values between the different linearization methods of up to 17.5%. The combined uncertainty was dominated by the uncertainty of stopping power on the edge.

Technical note: Experimental determination of the effective point of measurement of the PTW-31010 ionization chamber in proton and carbon ion beams.

PURPOSE: The accurate knowledge of the effective point of measurement (Peff ) is particularly important for measurements in proximity to high dose gradients such as in the distal fall-off of particle beams. For plane-parallel ionization chambers (ICs), Peff is well known and located at the center of the inner surface of the entrance window. For cylindrical ICs, Peff is shifted from the chamber's center toward the beam source. According to IAEA TRS-398, this shift can be calculated as 0.75·rcyl for light ions with rcyl being the radius of the cavity. For proton beams and in absence of a dose gradient, no shift is recommended. We have experimentally determined Peff for the 0.125 cc Semiflex IC in both proton and carbon ion beams. METHODS: The first method consisted of simultaneous irradiation of a plane-parallel IC and the Semiflex in a 4-cm wide spread-out Bragg peak. In the second method, a single-energy beam was used, and both ICs were positioned successively at the same measurement depths. For both approaches, the shift of the distal edge of the depth ionization distributions recorded by the two chambers at different reference points was used to calculate Peff of the Semiflex. Both methods were applied in carbon ion beams, and only the latter was applied in proton beams. RESULTS: Both methods yielded a similar Peff for carbon ions, 0.88·rcyl , and 0.84·rcyl , which results in a difference of only 0.1 mm. The difference to the recommended value of 0.75·rcyl is 0.4 and 0.3 mm, respectively, which is larger than the positioning uncertainty. In the proton beam, a Peff of 0.92·rcyl was obtained. CONCLUSIONS: The Peff for the 0.125 cc Semiflex IC is shifted further from the cavity center as recommended by IAEA TRS-398 for light ions, with the shift for proton beams being even larger than for carbon ion beams.

The Positive Impact of Vitamin D on Glucocorticoid-Dependent Skeletal Muscle Atrophy.

(1) The study aimed to investigate whether vitamin D3 supplementation would positively affect rats with glucocorticoids-induced muscle atrophy as measured by skeletal muscle mass in two experimental conditions: chronic dexamethasone (DEX) administration and a model of the chronic stress response. (2) The study lasted 28 consecutive days and was performed on 45 male Wistar rats randomly divided into six groups. These included two groups treated by abdominal injection of DEX at a dose of 2 mg/kg/day supplemented with vegetable oil (DEX PL; n = 7) or with vitamin D3 600 IU/kg/day (DEX SUP; n = 8), respectively, and a control group treated with an abdominal injection of saline (CON; n = 6). In addition, there were two groups of rats chronically stressed by cold water immersion (1 hour/day in a glass box with 1-cm-deep ice/water mixture; temperature ~4 °C), which were supplemented with vegetable oil as a placebo (STR PL; n = 9) or vitamin D3 at 600 IU/kg/day (STR SUP; n = 9). The last group was of sham-stressed rats (SHM; n = 6). Blood, soleus, extensor digitorum longus, gastrocnemius, tibialis anterior, and quadriceps femoris muscles were collected and weighed. The heart, liver, spleen, and thymus were removed and weighed immediately after sacrifice. The plasma corticosterone (CORT) and vitamin D3 metabolites were measured. (3) We found elevated CORT levels in both cold water-immersed groups; however, they did not alter body and muscle weight. Body weight and muscle loss occurred in groups with exogenously administered DEX, with the exception of the soleus muscle in rats supplemented with vitamin D3. Decreased serum 25(OH)D3 concentrations in DEX-treated rats were observed, and the cold water immersion did not affect vitamin D3 levels. (4) Our results indicate that DEX-induced muscle loss was abolished in rats supplemented with vitamin D3, especially in the soleus muscle.

Modelling and measurements of distributions in an adult human phantom undergoing proton scanning beam radiotherapy: lung- and prostate-located tumours.

Proton radiotherapy has been shown to offer a significant dosimetric advantage in cancer patients, in comparison to conventional radiotherapy, with a decrease in dose to healthy tissue and organs at risk, because the bulk of the beam energy is deposited in the Bragg peak to be located within a tumour. However, it should be kept in mind that radiotherapy of cancer is still accompanied by adverse side effects, and a better understanding and improvement of radiotherapy can extend the life expectancy of patients following the treatment of malignant tumours. In this study, the dose distributions measured with thermoluminescent detectors (TLDs) inside a tissue-equivalent adult human phantom exposed for lung and prostate cancer using the modern proton beam scanning radiotherapy technique were compared. Since the TLD detection efficiency depends on the ionization density of the radiation to be detected, and since this efficiency is detector specific, four different types of TLDs were used to compare their response in the mixed radiation fields. Additionally, the dose distributions from two different cancer treatment modalities were compared using the selected detectors. The measured dose values were benchmarked against Monte Carlo simulations and available literature data. The results indicate an increase in the lateral dose with an increase of the primary proton energy. However, the radiation quality factor of the mixed radiation increases by 20% in the vicinity to the target for the lower initial proton energy, due to the production of secondary charged particles of low-energy and short range. For the cases presented here the MTS-N TLD detector seems to be the most optimal tool for dose measurements within the target volume, while the MCP-N TLD detector, due to an interplay of its enhanced thermal neutron response and decreased detection efficiency to highly ionising radiation, is a better choice for the out-of-field measurements. The pairs of MTS-6 and MTS-7 TLDs used also in this study allowed for a direct measurement of the neutron dose equivalent. Before it can be concluded that they offer an alternative to the time-consuming nuclear track detectors, however, more research is needed to unambiguously confirm whether this observation was just accidental or whether it only applies to certain cases. Since there is no universal detector, which would allow the determination of the dosimetric quantities relevant for risk estimation, this work expands the knowledge necessary to improve the quality of dosimetry data and might help scientists and clinicians in choosing the right tools to measure radiation doses in mixed radiation fields.

ELAV and FNE Determine Neuronal Transcript Signatures through EXon-Activated Rescue.

The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. Here, we report that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. We uncover an endogenous strategy of functional gene rescue that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. We propose that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors.

Benchmarking of PHITS for Carbon Ion Therapy.

PURPOSE: Up to now, carbon ions have shown the most favorable physical and radiobiological properties for radiation therapy of, for example, deep-seated radioresistant tumors. However, when carbon ions penetrate matter, they undergo inelastic nuclear reactions that give rise to secondary fragments contributing to the dose in the healthy tissue. This can cause damage to radiosensitive organs at risk when they are located in the vicinity of the tumor. Therefore, predictions of the yields and angular distributions of the secondary fragments are needed to be able to estimate the resulting biological effects in both the tumor region and the healthy tissues. This study presents the accuracy of simulations of therapeutic carbon ion beams with water, with the 3D MC (Monte Carlo) general purpose particle and ion transport code PHITS. MATERIALS AND METHODS: Simulations with PHITS of depth-dose distributions, beam attenuation, fragment yields, and fragment angular distributions from interactions of therapeutic carbon ion beams with water are compared to published measurements performed at Gesellschaft für Schwerionen Forschung (GSI). RESULTS: The results presented in this study demonstrate that PHITS simulations of therapeutic carbon ion beams in water show overall a good agreement with measurements performed at GSI; for example, for light ions like H and He, simulations agree within about 10%. However, there is still a need to further improve the calculations of fragment yields, especially for underproduction of Li of up to 50%, by improving the nucleus-nucleus cross-section models. CONCLUSION: The simulated data are clinically acceptable but there is still a need to further improve the models in the transport code PHITS. More reliable experimental data are therefore needed.

Comprehensive Proteomic Investigation of Ebf1 Heterozygosity in Pro-B Lymphocytes Utilizing Data Independent Acquisition.

Early B cell factor 1 (EBF1) is one of the key transcription factors required for orchestrating B-cell lineage development. Although studies have shown that Ebf1 haploinsufficiency is involved in the development of leukemia, no study has been conducted that characterizes the global effect of Ebf1 heterozygosity on the proteome of pro-B lymphocytes. Here, we employ both data independent acquisition (DIA) and shotgun data dependent acquisition (DDA) workflows for profiling proteins that are differently expressed between Ebf1+/+ and Ebf1+/- cells. Both DDA and DIA were able to reveal the downregulation of the EBF1 transcription factor in Ebf1+/- pro-B lymphocytes. Further examination of differentially expressed proteins by DIA revealed that, similar to EBF1, the expression of other B-cell lineage regulators, such as TCF3 and Pax5, is also downregulated in Ebf1 heterozygous cells. Functional DIA analysis of differentially expressed proteins showed that EBF1 heterozygosity resulted in the deregulation of at least eight transcription factors involved in lymphopoiesis and the deregulation of key proteins playing crucial roles in survival, development, and differentiation of pro-B lymphocytes.

Comparative study of the effects of different radiation qualities on normal human breast cells.

BACKGROUND: As there is a growing number of long-term cancer survivors, the incidence of carcinogenesis as a late effect of radiotherapy is getting more and more into the focus. The risk for the development of secondary malignant neoplasms might be significantly increased due to exposure of healthy tissue outside of the target field to secondary neutrons, in particular in proton therapy. Thus far, the radiobiological effects of these neutrons and a comparison with photons on normal breast cells have not been sufficiently characterised. METHODS: MCF10A cells were irradiated with doses of up to 2 Gy with neutrons of different energy spectra and X-rays for comparison. The biological effects of neutrons with a broad energy distribution ( = 5.8 MeV), monoenergetic neutrons (1.2 MeV, 0.56 MeV) and of the mixed field of gamma's and secondary neutrons ( = 70.5 MeV) produced by 190 MeV protons impinging on a water phantom, were analysed. The clonogenic survival and the DNA repair capacity were determined and values of relative biological effectiveness were compared. Furthermore, the influence of radiation on the sphere formation was observed to examine the radiation response of the potential fraction of stem like cells within the MCF10A cell population. RESULTS: X-rays and neutrons caused dose-dependent decreases of survival fractions after irradiations with up to 2 Gy. Monoenergetic neutrons with an energy of 0.56 MeV had a higher effectiveness on the survival fraction with respect to neutrons with higher energies and to the mixed gamma - secondary neutron field induced by proton interactions in water. Similar effects were observed for the DNA repair capacity after exposure to ionising radiation (IR). Both experimental endpoints provided comparable values of the relative biological effectiveness. Significant changes in the sphere formation were notable following the various radiation qualities. CONCLUSION: The present study compared the radiation response of MCF10A cells after IR with neutrons and photons. For the first time it was shown that monoenergetic neutrons with energies around 1 MeV have stronger radiobiological effects on normal human breast cells with respect to X rays, to neutrons with a broad energy distribution ( = 5.8 MeV), and to the mixed gamma - secondary neutron field given by interactions of 190 MeV protons in water. The results of the present study are highly relevant for further investigations of radiation-induced carcinogenesis and are very important in perspective for a better risk assessment after secondary neutron exposure in the field of conventional and proton radiotherapy.

Relative biological effectiveness in a proton spread-out Bragg peak formed by pencil beam scanning mode.

In recent years, there is an increased interest in using scanning modes in proton therapy, due to the more conformal dose distributions, thanks to the spot-weighted dose delivery. The dose rate in each spot is however much higher than the dose rate when using passive irradiation modes, which could affect the cell response. The purpose of this work was to investigate how the relative biological effectiveness changes along the spread-out Bragg peak created by protons delivered by the pencil beam scanning mode. Cell survival and micronuclei formation were investigated in four positions along the spread-out Bragg peak for various doses. Monte Carlo simulations were used to estimate the dose-averaged linear energy transfer values in the irradiation positions. The cell survival was found to decrease the deeper the sample was placed in the spread-out Bragg peak, which corresponds to the higher linear energy transfer values found using Monte Carlo simulations. The micronuclei frequencies indicate more complex cell injuries at that distal position compared to the proximal part of the spread-out Bragg peak. The relative biological effectiveness determined in this study varies significantly and systematically from 1.1, which is recommended value by the International Commission on Radiation Units, in all the studied positions. In the distal position of spread-out Bragg peak the relative biological effectiveness values were found to be 2.05 ± 0.44, 1.85 ± 0.42, 1.53 ± 0.38 for survival levels 90, 50 and 10%, respectively.

PHITS simulations of absorbed dose out-of-field and neutron energy spectra for ELEKTA SL25 medical linear accelerator.

Monte Carlo (MC) based calculation methods for modeling photon and particle transport, have several potential applications in radiotherapy. An essential requirement for successful radiation therapy is that the discrepancies between dose distributions calculated at the treatment planning stage and those delivered to the patient are minimized. It is also essential to minimize the dose to radiosensitive and critical organs. With MC technique, the dose distributions from both the primary and scattered photons can be calculated. The out-of-field radiation doses are of particular concern when high energy photons are used, since then neutrons are produced both in the accelerator head and inside the patients. Using MC technique, the created photons and particles can be followed and the transport and energy deposition in all the tissues of the patient can be estimated. This is of great importance during pediatric treatments when minimizing the risk for normal healthy tissue, e.g. secondary cancer. The purpose of this work was to evaluate 3D general purpose PHITS MC code efficiency as an alternative approach for photon beam specification. In this study, we developed a model of an ELEKTA SL25 accelerator and used the transport code PHITS for calculating the total absorbed dose and the neutron energy spectra infield and outside the treatment field. This model was validated against measurements performed with bubble detector spectrometers and Boner sphere for 18 MV linacs, including both photons and neutrons. The average absolute difference between the calculated and measured absorbed dose for the out-of-field region was around 11%. Taking into account a simplification for simulated geometry, which does not include any potential scattering materials around, the obtained result is very satisfactorily. A good agreement between the simulated and measured neutron energy spectra was observed while comparing to data found in the literature.

NUNDO: a numerical model of a human torso phantom and its application to effective dose equivalent calculations for astronauts at the ISS.

The health effects of cosmic radiation on astronauts need to be precisely quantified and controlled. This task is important not only in perspective of the increasing human presence at the International Space Station (ISS), but also for the preparation of safe human missions beyond low earth orbit. From a radiation protection point of view, the baseline quantity for radiation risk assessment in space is the effective dose equivalent. The present work reports the first successful attempt of the experimental determination of the effective dose equivalent in space, both for extra-vehicular activity (EVA) and intra-vehicular activity (IVA). This was achieved using the anthropomorphic torso phantom RANDO(®) equipped with more than 6,000 passive thermoluminescent detectors and plastic nuclear track detectors, which have been exposed to cosmic radiation inside the European Space Agency MATROSHKA facility both outside and inside the ISS. In order to calculate the effective dose equivalent, a numerical model of the RANDO(®) phantom, based on computer tomography scans of the actual phantom, was developed. It was found that the effective dose equivalent rate during an EVA approaches 700 µSv/d, while during an IVA about 20 % lower values were observed. It is shown that the individual dose based on a personal dosimeter reading for an astronaut during IVA results in an overestimate of the effective dose equivalent of about 15 %, whereas under an EVA conditions the overestimate is more than 200 %. A personal dosemeter can therefore deliver quite good exposure records during IVA, but may overestimate the effective dose equivalent received during an EVA considerably.

Voltammetric detection of S100B protein using His-tagged receptor domains for advanced glycation end products (RAGE) immobilized onto a gold electrode surface.

In this work we report on an electrochemical biosensor for the determination of the S100B protein. The His-tagged VC1 domains of Receptors for Advanced Glycation End (RAGE) products used as analytically active molecules were covalently immobilized on a monolayer of a thiol derivative of pentetic acid (DPTA) complex with Cu(II) deposited on a gold electrode surface. The recognition processes between the RAGE VC1 domain and the S100B protein results in changes in the redox activity of the DPTA-Cu(II) centres which were measured by Osteryoung square-wave voltammetry (OSWV). In order to verify whether the observed analytical signal originates from the recognition process between the His6-RAGE VC1 domains and the S100B protein, the electrode modified with the His6-RAGE C2 and His6-RAGE VC1 deleted domains which have no ability to bind S100B peptides were applied. The proposed biosensor was quite sensitive, with a detection limit of 0.52 pM recorded in the buffer solution. The presence of diluted human plasma and 10 nM Aß(1-40) have no influence on the biosensor performance.

The MATROSHKA experiment: results and comparison from extravehicular activity (MTR-1) and intravehicular activity (MTR-2A/2B) exposure.

Astronauts working and living in space are exposed to considerably higher doses and different qualities of ionizing radiation than people on Earth. The multilateral MATROSHKA (MTR) experiment, coordinated by the German Aerospace Center, represents the most comprehensive effort to date in radiation protection dosimetry in space using an anthropomorphic upper-torso phantom used for radiotherapy treatment planning. The anthropomorphic upper-torso phantom maps the radiation distribution as a simulated human body installed outside (MTR-1) and inside different compartments (MTR-2A: Pirs; MTR-2B: Zvezda) of the Russian Segment of the International Space Station. Thermoluminescence dosimeters arranged in a 2.54 cm orthogonal grid, at the site of vital organs and on the surface of the phantom allow for visualization of the absorbed dose distribution with superior spatial resolution. These results should help improve the estimation of radiation risks for long-term human space exploration and support benchmarking of radiation transport codes.

Astronaut's organ doses inferred from measurements in a human phantom outside the international space station.

Space radiation hazards are recognized as a key concern for human space flight. For long-term interplanetary missions, they constitute a potentially limiting factor since current protection limits for low-Earth orbit missions may be approached or even exceeded. In such a situation, an accurate risk assessment requires knowledge of equivalent doses in critical radiosensitive organs rather than only skin doses or ambient doses from area monitoring. To achieve this, the MATROSHKA experiment uses a human phantom torso equipped with dedicated detector systems. We measured for the first time the doses from the diverse components of ionizing space radiation at the surface and at different locations inside the phantom positioned outside the International Space Station, thereby simulating an extravehicular activity of an astronaut. The relationships between the skin and organ absorbed doses obtained in such an exposure show a steep gradient between the doses in the uppermost layer of the skin and the deep organs with a ratio close to 20. This decrease due to the body self-shielding and a concomitant increase of the radiation quality factor by 1.7 highlight the complexities of an adequate dosimetry of space radiation. The depth-dose distributions established by MATROSHKA serve as benchmarks for space radiation models and radiation transport calculations that are needed for mission planning.