Search

PURPOSE: Track structure Monte Carlo (MC) codes have achieved successful outcomes in the quantitative investigation of radiation-induced initial DNA damage. The aim of the present study is to extend a Geant4-DNA radiobiological application by incorporating a feature allowing for the prediction of DNA rejoining kinetics and corresponding cell surviving fraction along time after irradiation, for a Chinese hamster V79 cell line, which is one of the most popular and widely investigated cell lines in radiobiology. METHODS: We implemented the Two-Lesion Kinetics (TLK) model, originally proposed by Stewart, which allows for simulations to calculate residual DNA damage and surviving fraction along time via the number of initial DNA damage and its complexity as inputs. RESULTS: By optimizing the model parameters of the TLK model in accordance to the experimental data on V79, we were able to predict both DNA rejoining kinetics at low linear energy transfers (LET) and cell surviving fraction. CONCLUSION: This is the first study to demonstrate the implementation of both the cell surviving fraction and the DNA rejoining kinetics with the estimated initial DNA damage, in a realistic cell geometrical model simulated by full track structure MC simulations at DNA level and for various LET. These simulation and model make the link between mechanistic physical/chemical damage processes and these two specific biological endpoints.

Heavy charged particles induce severe damage in DNA, which is a radiobiological advantage when treating radioresistant tumors. However, these particles can also induce cancer in humans exposed to them, such as astronauts in space missions. This damage can be directly induced by the radiation or indirectly by the attack of free radicals mainly produced by water radiolysis. We previously studied the impact of a proton on a DNA base pair, using the Time Dependent-Density Functional Theory (TD-DFT). In this work, we go a step further and study the attack of the OH· radical on the Guanine nucleotide to unveil how this molecule subsequently dissociates. The OH· attack on the H1′, H2′, H3′, and H5′ atoms in the guanine was investigated using the Ehrenfest dynamics within the TD-DFT framework. In all cases, the hydrogen abstraction succeeded, and the subsequent base pair dissociation was observed. The DNA dissociates in three major fragments: the phosphate group, the deoxyribose sugar, and the nitrogenous base, with slight differences, no matter which hydrogen atom was attacked. Hydrogen abstraction occurs at about 6 fs, and the nucleotide dissociation at about 100 fs, which agrees with our previous result for the direct proton impact on the DNA. These calculations may be a reference for adjusting reactive force fields so that more complex DNA structures can be studied using classical molecular dynamics, including both direct and indirect DNA damage. [ABSTRACT FROM AUTHOR]

The use of Monte Carlo (MC) simulations remains a powerful tool to study the biological effects induced by ionizing radiation on living beings. Several MC codes are commonly used in research fields such as nanodosimetry, radiotherapy, radiation protection, and space radiation. This work presents an enhancement of an existing model [1] for radiobiological purposes, to account for the indirect DNA damage induced by ionizing particles. The Geant4-DNA simulation toolkit was used to simulate the physical, pre-chemical, and chemical stages of early DNA damage induced by protons and α -particles. Liquid water was used as the medium for simulations. Two phase-space files were generated, one containing the energy deposition events and another with the position of chemical species produced by water radiolysis from 0.1 ps up to 1 ns. These files were used as input in the radiobiological code that contains the genetic material model with atomic resolution, consisting of several copies of 30 nm chromatin fibers. The B-DNA configuration was used. This work focused on the indirect damage produced by the hydroxyl radical ( OH ) attack on the sugar-phosphate group. The approach followed to account for the indirect DNA damage was the same as those used by other radiobiological codes [2,3] . The critical parameter considered here was the reaction radius, which was calculated from the Smoluchowski’s diffusion equation. Single, double, and total strand break yields produced by direct, indirect, and mixed mechanisms are reported. The obtained results are consistent with experimental and calculation data sets published in the literature. [ABSTRACT FROM AUTHOR]

Targeted α -therapy (TAT) could be delivered early to patients who are at a high-risk for developing brain metastases, targeting the areas of the vasculature where tumor cells are penetrating into the brain. We have utilized a Monte Carlo model representing brain vasculature to calculate physical dose and DNA damage from the α -emitters 225 Ac and 212 Pb. The micron-scale dose distributions from all radioactive decay products were modeled in Geant4, including the eV-scale interactions using the Geant4-DNA models. These interactions were then superimposed on an atomic-scale DNA model to estimate strand break yields. In addition to 225 Ac having a higher dose per decay than 212 Pb, it also has a double strand break yield per decay that is 4.7  ±  0.5 times that of 212 Pb. However, the efficacy of both nuclides depends on retaining the daughter nuclei at the target location in the brain vasculature. The relative biological effectiveness (RBE) of 225 Ac and 212 Pb are similar when the entire decay chains are included, with maxima of 2.7  ±  0.6 and 2.5  ±  0.5 (respectively), and RBE values of about 2 to a depth of 80 μm. If the initial daughter is lost, the RBE of 212 Pb is completely reduced to 1 or lower and the RBE of 225 Ac is approximately 2 only for the first 40 μm. [ABSTRACT FROM AUTHOR]

The interaction of heavy charged particles with DNA is of interest for hadrontherapy and the aerospace industry. Here, a time-dependent density functional theory study on the interaction of a 4 keV proton with an isolated DNA base pair (bp) was carried out. Ehrenfest dynamics was used to study the evolution of the system up to about 193 fs. It was observed that the dissociation of the target occurs between 80 and 100 fs. The effect of bp linking to the DNA double helix was emulated by fixing the four O3' atoms responsible for the attachment. The bp tends to dissociate into its main components, namely, the phosphate groups, sugars, and nitrogenous bases. A central impact with an energy transfer of 17.9 eV only produces a base damage while keeping the backbone intact. An impact on a phosphate group with an energy transfer of about 60 eV leads to a backbone break at that site together with a base damage, and the opposite backbone site integrity is kept. As the whole system is perturbed during this collision, no atom remains passive. These results suggest that base damage accompanies all backbone breaks as the hydrogen bonds that keep bases together are much weaker that those between the other components of the DNA. [ABSTRACT FROM AUTHOR]

High dose rate brachytherapy (HDR) using 192Ir sources is well accepted as an important treatment option and thus requires an accurate dosimetry standard. However, a dosimetry standard for the direct measurement of the absolute dose to water for this particular source type is currently not available. An improved standard for the absorbed dose to water based on Fricke dosimetry of HDR 192Ir brachytherapy sources is presented in this study. The main goal of this paper is to demonstrate the potential usefulness of the Fricke dosimetry technique for the standardization of the quantity absorbed dose to water for 192Ir sources. A molded, double-walled, spherical vessel for water containing the Fricke solution was constructed based on the Fricke system. The authors measured the absorbed dose to water and compared it with the doses calculated using the AAPM TG-43 report. The overall combined uncertainty associated with the measurements using Fricke dosimetry was 1.4% for k = 1, which is better than the uncertainties reported in previous studies. These results are promising; hence, the use of Fricke dosimetry to measure the absorbed dose to water as a standard for HDR 192Ir may be possible in the future. [ABSTRACT FROM AUTHOR]

Purpose: Monte Carlo track structures (MCTS) simulations have been recognized as useful tools for radiobiological modeling. However, the authors noticed several issues regarding the consistency of reported data. Therefore, in this work, they analyze the impact of various user defined parameters on simulated direct DNA damage yields. In addition, they draw attention to discrepancies in published literature in DNA strand break (SB) yields and selected methodologies. Methods: The MCTS code Geant4-DNA was used to compare radial dose profiles in a nanometer-scale region of interest (ROI) for photon sources of varying sizes and energies. Then, electron tracks of 0.28 keV-220 keV were superimposed on a geometric DNA model composed of 2.7 x 106 nucleosomes, and SBs were simulated according to four definitions based on energy deposits or energy transfers in DNA strand targets compared to a threshold energy ETH. The SB frequencies and complexities in nucleosomes as a function of incident electron energies were obtained. SBs were classified into higher order clusters such as single and double strand breaks (SSBs and DSBs) based on inter-SB distances and on the number of affected strands. Results: Comparisons of different nonuniform dose distributions lacking charged particle equilibrium may lead to erroneous conclusions regarding the effect of energy on relative biological effectiveness. The energy transfer-based SB definitions give similar SB yields as the one based on energy deposit when ETH≈10.79 eV, but deviate significantly for higher ETH values. Between 30 and 40 nucle-osomes/Gy show at least one SB in the ROI. The number of nucleosomes that present a complex damage pattern of more than 2 SBs and the degree of complexity of the damage in these nucleosomes diminish as the incident electron energy increases. DNA damage classification into SSB and DSB is highly dependent on the definitions of these higher order structures and their implementations. The authors' show that, for the four studied models, different yields are expected by up to 54% for SSBs and by up to 32% for DSBs, as a function of the incident electrons energy and of the models being compared. Conclusions: MCTS simulations allow to compare direct DNA damage types and complexities induced by ionizing radiation. However, simulation results depend to a large degree on user-defined parameters, definitions, and algorithms such as: DNA model, dose distribution, SB definition, and the DNA damage clustering algorithm. These interdependencies should be well controlled during the simulations and explicitly reported when comparing results to experiments or calculations [ABSTRACT FROM AUTHOR]

Simple Summary: High-Z gold nanoparticles show potential as radiosensitizers in the radiotherapy of cancer. In this paper, we introduce the benefits and procedures for the application of gold nanoparticles in targeted cancer radiotherapy. Based on microscopic images of the distribution of antibody-conjugated nanoparticles, we established pharmacokinetic models simulating the biodistribution of nanoparticle conjugates in the tumor and tumor environment in preclinical models. This information has been implemented in radiation transport Monte Carlo simulation codes for further investigating physical and chemical enhancement and radiobiological effects, such as DNA strand breaks and cell survival. Future perspectives and challenges of translating this promising gold nanoparticle-aided radiotherapy into clinical practice are also discussed. High-Z gold nanoparticles (AuNPs) conjugated to a targeting antibody can help to improve tumor control in radiotherapy while simultaneously minimizing radiotoxicity to adjacent healthy tissue. This paper summarizes the main findings of a joint research program which applied AuNP-conjugates in preclinical modeling of radiotherapy at the Klinikum rechts der Isar, Technical University of Munich and Helmholtz Zentrum München. A pharmacokinetic model of superparamagnetic iron oxide nanoparticles was developed in preparation for a model simulating the uptake and distribution of AuNPs in mice. Multi-scale Monte Carlo simulations were performed on a single AuNP and multiple AuNPs in tumor cells at cellular and molecular levels to determine enhancements in the radiation dose and generation of chemical radicals in close proximity to AuNPs. A biologically based mathematical model was developed to predict the biological response of AuNPs in radiation enhancement. Although simulations of a single AuNP demonstrated a clear dose enhancement, simulations relating to the generation of chemical radicals and the induction of DNA strand breaks induced by multiple AuNPs showed only a minor dose enhancement. The differences in the simulated enhancements at molecular and cellular levels indicate that further investigations are necessary to better understand the impact of the physical, chemical, and biological parameters in preclinical experimental settings prior to a translation of these AuNPs models into targeted cancer radiotherapy. [ABSTRACT FROM AUTHOR]

Simple Summary: DNA damage caused by ionizing radiation in a human fibroblast cell evaluated by the Geant4-DNA Monte Carlo toolkit is presented. A validation study using a computational geometric human DNA model was then carried out, and the calculated DNA damage as a function of particle type and energy is presented. The results of this work showed a significant improvement on past work and were consistent with recent radiobiological experimental data, such as damage yields. This work and the developed methodology could impact a broad number of research fields in which the understanding of radiation effects is crucial, such as cancer radiotherapy, space science, and medical physics. Accurately modeling the radiobiological mechanisms responsible for the induction of DNA damage remains a major scientific challenge, particularly for understanding the effects of low doses of ionizing radiation on living beings, such as the induction of carcinogenesis. A computational approach based on the Monte Carlo technique to simulate track structures in a biological medium is currently the most reliable method for calculating the early effects induced by ionizing radiation on DNA, the primary cellular target of such effects. The Geant4-DNA Monte Carlo toolkit can simulate not only the physical, but also the physico-chemical and chemical stages of water radiolysis. These stages can be combined with simplified geometric models of biological targets, such as DNA, to assess direct and indirect early DNA damage. In this study, DNA damage induced in a human fibroblast cell was evaluated using Geant4-DNA as a function of incident particle type (gammas, protons, and alphas) and energy. The resulting double-strand break yields as a function of linear energy transfer closely reproduced recent experimental data. Other quantities, such as fragment length distribution, scavengeable damage fraction, and time evolution of damage within an analytical repair model also supported the plausibility of predicting DNA damage using Geant4-DNA.The complete simulation chain application "molecularDNA", an example for users of Geant4-DNA, will soon be distributed through Geant4. [ABSTRACT FROM AUTHOR]

This work focuses on the calculation of S-values and radial energy profiles for radionuclides emitting high (Y-90, Sr-89), medium (Re-186, Sm-153) and low-energy (Er-169, Lu-177) β-particles, Auger electrons (In-111, Ga-67, I-123) and α-particles (At-211, Ac-225). Simulations were performed using the EGSnrc and GEANT4-DNA Monte Carlo (MC) codes for a spherical cell geometry. S-values were computed using decay spectra available in literature for Tc-99m and In-111. To investigate the effect on S-value when the same emission spectrum is used in two different MC codes. Internal modules of the MC codes were used to simulate the decay of other radionuclides mentioned above. Radial energy profiles for uniformly distributed radioactive sources in the cell nucleus and cytoplasm were calculated and results were compared with the literature. For S-values calculated using the same emission spectrum, the results showed good agreement with each other and with the literature. Whereas, the S-values calculated using the internal decay data of the MC codes, for instance, for Ga-67 and Y-90, showed discrepancies up to 40%. Radial energy profiles were also different from those reported in the literature. Our results show that well validated radiation emission spectra must be used for such calculations and internal decay spectra of MC codes should be used with caution. The normalized probability density functions must be used to sample points uniformly into spherical volumes and the methodology proposed here can be used to correctly determine radial energy profiles. • S-value and Radial energy calculation for high-, medium-, low-energy β-particles, Auger electrons and α-particle emitting radionuclides. • Comparison between condensed history based code and track structure code. • Check of reliability of internal decay spectrum of Monte Carlo codes. • New methodology to calculate radial dose profiles. [ABSTRACT FROM AUTHOR]

The objective of this work was to study the differences in terms of early biological effects that might exist between different X-rays energies by using a mechanistic approach. To this end, radiobiological experiments exposing cell monolayers to three X-ray energies were performed in order to assess the yields of early DNA damage, in particular of double-strand breaks (DSBs). The simulation of these irradiations was set in order to understand the differences in the obtained experimental results. Hence, simulated results in terms of microdosimetric spectra and early DSB induction were analyzed and compared to the experimental data. Human umbilical vein endothelial cells (HUVECs) were irradiated with 40, 220 kVp, and 4 MV X-rays. The Geant4 Monte Carlo simulation toolkit and its extension Geant4-DNA were used for the simulations. Microdosimetric calculations aiming to determine possible differences in the variability of the energy absorbed by the irradiated cell population for those photon spectra were performed on 10,000 endothelial cell nuclei representing a cell monolayer. Nanodosimetric simulations were also carried out using a computation chain that allowed the simulation of physical, physico-chemical, and chemical stages on a single realistic endothelial cell nucleus model including both heterochromatin and euchromatin. DNA damage was scored in terms of yields of prompt DSBs per Gray (Gy) and per giga (109) base pair (Gbp) and DSB complexity was derived in order to be compared to experimental data expressed as numbers of histone variant H2AX (γ-H2AX) foci per cell. The calculated microdosimetric spread in the irradiated cell population was similar when comparing between 40 and 220 kVp X-rays and higher when comparing with 4 MV X-rays. Simulated yields of induced DSB/Gy/Gbp were found to be equivalent to those for 40 and 220 kVp but larger than those for 4 MV, resulting in a relative biological effectiveness (RBE) of 1.3. Additionally, DSB complexity was similar between the considered photon spectra. Simulated results were in good agreement with experimental data obtained by IRSN (Institut de radioprotection et de sûreté nucléaire) radiobiologists. Despite differences in photon energy, few differences were observed when comparing between 40 and 220 kVp X-rays in microdosimetric and nanodosimetric calculations. Nevertheless, variations were observed when comparing between 40/220 kVp and 4 MV X-rays. Thanks to the simulation results, these variations were able to be explained by the differences in the production of secondary electrons with energies below 10 keV. [ABSTRACT FROM AUTHOR]

Many patients with breast cancer develop brain metastases, which are typically treated through whole-brain irradiation. As a potential alternative treatment, targeted radionuclide therapy (TRT) could be delivered early, targeting the areas of the vasculature where tumor cells are penetrating into the brain. We have developed a Monte Carlo model representing brain vasculature to evaluate and understand a variety of potential therapeutic nuclides: (alpha emitters) Pb-212, At-211, Ac-225, Bi-213, and Tb-149; (beta/Auger electron emitters) Lu-177, Tb-161, I-124, In-111, Y-90, Zr-89, and Ga-67. The micron-scale dose distributions from all radioactive decay products were modeled in Geant4, as well as eV-scale interactions through the G4DNA models [1] . These interactions were then superimposed on an atomic-scale DNA model [2] to estimate strand break yields. Some of the alpha emitters have decay chains with multiple daughter nuclei; we investigate the change in dose profiles and biological effectiveness as a function of time. Alpha emitters have higher doses per decay, and the depth-dose profiles fall off less quickly. The general qualities of the depth-dose profiles are maintained through biologically-relevant variations in vasculature geometry. [ABSTRACT FROM AUTHOR]

This work presents a model previously developed for estimating relative biological effectiveness (RBE) associated with high-LET particles. It is based on the combination of Monte Carlo simulations of particle interactions when traversing an atomic resolution DNA geometrical model. In addition, the model emulates the induction of lethal damage from the interaction of two sublethal lesions, taken as double-strand breaks. The Geant4-DNA package was used for simulations with liquid water as the transport medium. The RBE of neutron beams with energies ranging from 0.1 MeV up to 14 MeV was studied. The model succeeded in reproducing the general behavior of RBE as a function of neutron energy, including the RBE peak reported by experiments at approximately 0.4 MeV. Furthermore, the results of the model agree rather well with some experimental works. However, our results underestimate RBE for neutron energies above approximately 5 MeV due to the current limitations of Geant4-DNA for the tracking of heavy ions below 0.5 MeV/u. • A biophysical model based on the Dual Radiation Action Theory of Kellerer and Rossi is used for estimating RBE for fast neutrons. • A whole cell DNA model with atomic resolution is irradiated with neutron beams. • Linear and quadratic components of the dose-effect formula are reproduced. • RBE consistent with experiments were obtained. • The general behavior of RBE versus neutron energy was successfully reproduced. [ABSTRACT FROM AUTHOR]

This study proposes an innovative approach to estimate relative biological effectiveness (RBE) of fast neutrons using the Geant4 toolkit. The Geant4-DNA version cannot track heavy ions below 0.5 MeV/nucleon. In order to explore the impact of this issue, secondary particles are simulated instead of the primary low-energy neutrons. The Evaluated Nuclear Data File library is used to determine the cross sections for the elastic and inelastic interactions of neutrons with water and to find the contribution of each secondary particle spectrum. Two strategies are investigated in order to find the best possible approach and results. The first one takes into account only light particles, protons produced from elastic scattering, and α particles from inelastic scattering. Geantino particles are shot instead of heavy ions; hence all heavy ions are considered in the simulations, though their physical effects on DNA not. The second strategy takes into account all the heavy and light ions, although heavy ions cannot be tracked down to very low energies (E<0.5 MeV/nucleon). Our model is based on the combination of an atomic resolution DNA geometrical model and a Monte Carlo simulation toolkit for tracking particles. The atomic coordinates of the DNA double helix are extracted from the Protein Data Bank. Since secondary particle spectra are used instead of simulating the interaction of neutrons explicitly, this method reduces the computation times dramatically. Double-strand break induction is used as the end point for the estimation of the RBE of fast neutrons. 60Co γ rays are used as the reference radiation quality. Both strategies succeed in reproducing the behavior of the RBEmax as a function of the incident neutron energy ranging from 0.1 to 14 MeV, including the position of its peak. A comparison of the behavior of the two strategies shows that for neutrons with energies less than 0.7 MeV, the effect of heavy ions would not be very significant, but above 0.7 MeV, heavy ions have an important role in neutron RBE. [ABSTRACT FROM AUTHOR]

Purpose: Track structure Monte Carlo (MC) codes have achieved successful outcomes in the quantitative investigation of radiation-induced initial DNA damage. The aim of the present study is to extend a Geant4-DNA radiobiological application by incorporating a feature allowing for the prediction of DNA rejoining kinetics and corresponding cell surviving fraction along time after irradiation, for a Chinese hamster V79 cell line, which is one of the most popular and widely investigated cell lines in radiobiology., Methods: We implemented the Two-Lesion Kinetics (TLK) model, originally proposed by Stewart, which allows for simulations to calculate residual DNA damage and surviving fraction along time via the number of initial DNA damage and its complexity as inputs., Results: By optimizing the model parameters of the TLK model in accordance to the experimental data on V79, we were able to predict both DNA rejoining kinetics at low linear energy transfers (LET) and cell surviving fraction., Conclusion: This is the first study to demonstrate the implementation of both the cell surviving fraction and the DNA rejoining kinetics with the estimated initial DNA damage, in a realistic cell geometrical model simulated by full track structure MC simulations at DNA level and for various LET. These simulation and model make the link between mechanistic physical/chemical damage processes and these two specific biological endpoints., (Copyright © 2022 Associazione Italiana di Fisica Medica e Sanitaria. Published by Elsevier Ltd. All rights reserved.)

Targeted α-therapy (TAT) could be delivered early to patients who are at a high-risk for developing brain metastases, targeting the areas of the vasculature where tumor cells are penetrating into the brain. We have utilized a Monte Carlo model representing brain vasculature to calculate physical dose and DNA damage from the α-emitters 225 Ac and 212 Pb. The micron-scale dose distributions from all radioactive decay products were modeled in Geant4, including the eV-scale interactions using the Geant4-DNA models. These interactions were then superimposed on an atomic-scale DNA model to estimate strand break yields. In addition to 225 Ac having a higher dose per decay than 212 Pb, it also has a double strand break yield per decay that is 4.7 ± 0.5 times that of 212 Pb. However, the efficacy of both nuclides depends on retaining the daughter nuclei at the target location in the brain vasculature. The relative biological effectiveness (RBE) of 225 Ac and 212 Pb are similar when the entire decay chains are included, with maxima of 2.7 ± 0.6 and 2.5 ± 0.5 (respectively), and RBE values of about 2 to a depth of 80 μm. If the initial daughter is lost, the RBE of 212 Pb is completely reduced to 1 or lower and the RBE of 225 Ac is approximately 2 only for the first 40 μm., (Copyright © 2018 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.)

Brain metastases develop frequently in patients with breast cancer, and present a pressing therapeutic challenge. Expression of vascular cell adhesion molecule 1 (VCAM-1) is upregulated on brain endothelial cells during the early stages of metastasis and provides a target for the detection and treatment of early brain metastases. The aim of this study was to use a model of early brain metastasis to evaluate the efficacy of α-emitting radionuclides, 149 Tb, 211 At, 212 Pb, 213 Bi and 225 Ac; β-emitting radionuclides, 90 Y, 161 Tb and 177 Lu; and Auger electron (AE)-emitters 67 Ga, 89 Zr, 111 In and 124 I, for targeted radionuclide therapy (TRT)., Methods: Histologic sections and two photon microscopy of mouse brain parenchyma were used to inform a cylindrical vessel geometry using the Geant4 general purpose Monte Carlo (MC) toolkit with the Geant4-DNA low energy physics models. Energy deposition was evaluated as a radial function and the resulting phase spaces were superimposed on a DNA model to estimate double-strand break (DSB) yields for representative β- and α-emitters, 177 Lu and 212 Pb. Relative biological effectiveness (RBE) values were determined by only evaluating DNA damage due to physical interactions., Results: 177 Lu produced 2.69 ± 0.08 DSB per GbpGy, without significant variation from the lumen of the vessel to a radius of 100 µm. The DSB yield of 212 Pb included two local maxima produced by the 6.1 MeV and 8.8 MeV α-emissions from decay products, 212 Bi and 212 Po, with yields of 7.64 ± 0.12 and 9.15 ± 0.24 per GbpGy, respectively. Given its higher DSB yield 212 Pb may be more effective for short range targeting of early micrometastatic lesions than 177 Lu., Conclusion: MC simulation of a model of early brain metastases provides invaluable insight into the potential efficacy of α-, β- and AE-emitting radionuclides for TRT. 212 Pb, which has the attributes of a theranostic radionuclide since it can be used for SPECT imaging, showed a favorable dose profile and RBE., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.