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The cell as a system of many components, governed by the laws of physics and chemistry drives molecular functions having an impact on the spatial organization of these systems and vice versa. Since the relationship between structure and function is an almost universal rule not only in biology, appropriate methods are required to parameterize the relationship between the structure and function of biomolecules and their networks, the mechanisms of the processes in which they are involved, and the mechanisms of regulation of these processes. Single molecule localization microscopy (SMLM), which we focus on here, offers a significant advantage for the quantitative parametrization of molecular organization: it provides matrices of coordinates of fluorescently labeled biomolecules that can be directly subjected to advanced mathematical analytical procedures without the need for laborious and sometimes misleading image processing. Here, we propose mathematical tools for comprehensive quantitative computer data analysis of SMLM point patterns that include Ripley distance frequency analysis, persistent homology analysis, persistent 'imaging', principal component analysis and co-localization analysis. The application of these methods is explained using artificial datasets simulating different, potentially possible and interpretatively important situations. Illustrative analyses of real complex biological SMLM data are presented to emphasize the applicability of the proposed algorithms. This manuscript demonstrated the extraction of features and parameters quantifying the influence of chromatin (re)organization on genome function, offering a novel approach to study chromatin architecture at the nanoscale. However, the ability to adapt the proposed algorithms to analyze essentially any molecular organizations, e.g., membrane receptors or protein trafficking in the cytosol, offers broad flexibility of use.

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The world is still suffering from the SARS-CoV-2 pandemic, and the number of infected people is still growing in many countries in 2022. Although great strides have been made to produce effective vaccines, efforts in this field should be accelerated, particularly due to the emergence of new variants. Using inactivated viruses is a conventional method of vaccine production. High levels of ionizing radiation can effectively inactivate viruses. Recently, studies on SARS-CoV-2 irradiation using low-LET radiations (e.g., gamma rays) have been performed. However, there are insufficient studies on the impact of charged particles on the inactivation of this virus. In this study, a realistic structure of SARS-CoV-2 is simulated by using Geant4 Monte Carlo toolkit, and the effect of electrons, protons, alphas, C-12, and Fe-56 ions on the inactivation of SARS-CoV-2 is investigated. The simulation results indicated that densely ionizing (high-LET) particles have the advantage of minimum number of damaged spike proteins per single RNA break. The RNA breaks induced by hydroxyl radicals produced in the surrounding water medium were significant only for electron beam radiation. Hence, indirect RNA breaks induced by densely ionizing particles is negligible. From a simulation standpoint, alpha particles (with energies up to 30 MeV) as well as C-12 ions (with energies up to 80 MeV/n), and Fe-56 ions (with any energy) can be introduced as particles of choice for effective SARS-CoV-2 inactivation.

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BACKGROUND AND PURPOSE: Patients who receive carbon-ion radiotherapy (C-ion RT) for primary pancreatic cancer may experience locoregional recurrence; however, the treatment options for such patients are limited. We aimed to investigate the feasibility and efficacy of carbon-ion re-irradiation for patients with pancreatic cancer who experienced recurrence after initial C-ion RT. MATERIALS AND METHODS: Twenty-one patients with recurrent pancreatic cancer who underwent repeat C-ion RT between December 2010 and November 2016 at our institute were retrospectively evaluated. The sites of post-initial C-ion RT failure were in-field central in 16 patients (76.2%) and marginal in 5 (23.8%). The median doses of initial and repeat C-ion RT were both 52.8 Gy (relative biological effectiveness [RBE]). Thirteen patients (61.9%) received concurrent chemotherapy with re-irradiation, while 11 (52.4%) received adjuvant chemotherapy. RESULTS: The median follow-up period after re-irradiation was 11 months. The 1-year local control, progression-free survival, and overall survival rates were 53.5%, 24.5%, and 48.7%, respectively. Toxicity data was obtained from the patients' charts. Only 1 patient (4.8%) developed grade 3 acute toxicities and none developed grade ≥3 late toxicities. Univariate analysis indicated that patients who received adjuvant chemotherapy had significantly improved local control rates compared with those who did not; the 1-year local control rates were 80.0% and 0.0%, respectively (P = 0.0469). CONCLUSION: Repeating C-ion RT may be a reasonable option with tolerable toxicity for patients with recurrent pancreatic cancers. Adjuvant chemotherapy appears to improve the local control rate. This is the first study to examine re-irradiation using C-ion for recurrent pancreatic cancer after initial C-ion RT.

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BACKGROUND AND PURPOSE: Patient-level benefits of proton beam therapy (PBT) relative to photon therapy for prostate cancer (PC) continue to be the focus of debate. Although trials comparing the two modalities are underway, most are being conducted using "conventional" PBT (passive scattering/uniform scanning [PS/US]) rather than pencil beam scanning (PBS). The dosimetric benefits of PBS are well-known, but comparative data are limited. This analysis compares PBS toxicity rates with those of PS/US in a prospective multicenter registry. METHODS: We evaluated acute/late gastrointestinal (GI) and genitourinary (GU) toxicity rates for men with low-to-intermediate risk PC enrolled in PCG 001-09. Acute toxicities with the two techniques were compared using χ2 tests, and the cumulative incidence methods for late toxicity. Multivariable analyses (MVAs) for acute toxicity were performed using logistic regression, and cox proportional hazards models for late toxicity. RESULTS: Patients were treated using PS/US (n = 1105) or PBS (n = 238). Acute grade ≥2 GI toxicity in PBS did not significantly differ from that with PS/US (2.9% and 2.1%, respectively; P = 0.47). Acute grade ≥2 GU toxicity was significantly higher with PBS (21.9% and 15.1%; P < 0.01). In MVA, PBS was significantly associated with increased acute grade ≥2 GU toxicity (RR = 1.57, p < 0.001). Late grade ≥2 GI and GU toxicities did not differ significantly between groups. CONCLUSIONS: This is the first multi-institutional comparative effectiveness evaluation of PBT techniques in PC. Differences in acute GU toxicity warrant further evaluation, and highlight the urgent need for prospective data using PBT.

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BACKGROUND: The role of radiotherapy in malignant melanoma is still in discussion due to its relative resistance to radiation. In various literature, heavy ions show a higher relative biological effectiveness than photons. The aim of this work is to evaluate the radiotherapeutical effect from photons as well as heavy ions on malignant melanoma cells and to indicate the possible radiosensitivity based on its proliferation-inhibitory effect. METHODS: Two different cell lines of malignant melanoma, WM115 (primary tumor) and WM266-4 (metastatic site, skin) were used in this in vitro study. The cells were treated with photons or heavy ions (C12 and O16 ions). Cell-proliferation assay using hemocytometer was used for the quantitative and qualitative evaluation of cell growth. Furthermore, flow cytometry was also used to analyze the cell cycle distribution. RESULTS: Heavy ions compared to photons and between the two heavy ion modalities, O16 ions showed an improved suppression of cell growth in both cell lines. Furthermore, a G2/M arrest was detected in both cell lines after all radiotherapy modalities - with the arrest increasing with the dose applied. CONCLUSION: Heavy ions showed a greater inhibitory effect on cell proliferation compared to photons and an increased G2/M arrest. Therefore, C12 and O16 heavy ions might overcome the relative radioresistance of malignant melanoma to photons. Further research is warranted.

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BACKGROUND AND PURPOSES: Carbon ion radiotherapy (CIRT) with raster scanning technology is a promising treatment for lung cancer and thoracic malignancies. Determining normal tissue tolerance of organs at risk is of utmost importance for the success of CIRT. Here we report the relative biological effectiveness (RBE) of CIRT as a function of dose and fractionation for development of pulmonary fibrosis using well established fibrosis index (FI) model. MATERIALS AND METHODS: Dose series of fractionated clinical quality CIRT versus conventional photon irradiation to the whole thorax were compared in C57BL6 mice. Quantitative assessment of pulmonary fibrosis was performed by applying the FI to computed tomography (CT) data acquired 24-weeks post irradiation. RBE was calculated as the ratio of photon to CIRT dose required for the same level of FI. Further RBE predictions were performed using the derived equation from high-linear energy transfer biologically effective dose (high-LET BED) model. RESULTS: The averaged lung fibrosis RBE of 5-fraction CIRT schedule was determined as 2.75 ±â€¯0.55. The RBE estimate at the half maximum effective dose (RBEED50) was estimated at 2.82 for clinically relevant fractional sizes of 1-6 Gy. At the same dose range, an RBE value of 2.81 ±â€¯0.40 was predicted by the high-LET BED model. The converted biologically effective dose (BED) of CIRT for induction of half maximum FI (BEDED50) was identified to be 58.12 Gy3.95. In accordance, an estimated RBE of 2.88 was obtained at the BEDED50 level. The LQ model radiosensitivity parameters for 5-fraction was obtained as αH = 0.3030 ±â€¯0.0037 Gy-1 and ßH = 0.0056 ±â€¯0.0007 Gy-2. CONCLUSION: This is the first report of RBE estimation for CIRT with the endpoint of pulmonary fibrosis in-vivo. We proposed in present study a novel way to mathematically modeling RBE by integrating RBEmax and α/ßL based on conventional high-LET BED conception. This model well predicted RBE in the clinically relevant dose range but is sensitive to the uncertainties of α/ß estimates from the reference photon irradiation (α/ßL). These findings will assist to eliminate current uncertainties in prediction of CIRT induced normal tissue complications and builds a solid foundation for development of more accurate in-vivo data driven RBE estimates.

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Repair of DNA double strand breaks (DSBs) is influenced by the chemical complexity of the lesion. Clustered lesions (complex DSBs) are generally considered more difficult to repair and responsible for early and late cellular effects after exposure to genotoxic agents. Resection is commonly used by the cells as part of the homologous recombination (HR) pathway in S- and G2-phase. In contrast, DNA resection in G1-phase may lead to an error-prone microhomology-mediated end joining. We induced DNA lesions with a wide range of complexity by irradiation of mammalian cells with X-rays or accelerated ions of different velocity and mass. We found replication protein A (RPA) foci indicating DSB resection both in S/G2- and G1-cells, and the fraction of resection-positive cells correlates with the severity of lesion complexity throughout the cell cycle. Besides RPA, Ataxia telangiectasia and Rad3-related (ATR) was recruited to complex DSBs both in S/G2- and G1-cells. Resection of complex DSBs is driven by meiotic recombination 11 homolog A (MRE11), CTBP-interacting protein (CtIP), and exonuclease 1 (EXO1) but seems not controlled by the Ku heterodimer or by phosphorylation of H2AX. Reduced resection capacity by CtIP depletion increased cell killing and the fraction of unrepaired DSBs after exposure to densely ionizing heavy ions, but not to X-rays. We conclude that in mammalian cells resection is essential for repair of complex DSBs in all phases of the cell-cycle and targeting this process sensitizes mammalian cells to cytotoxic agents inducing clustered breaks, such as in heavy-ion cancer therapy.

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Whole body exposure to low linear energy transfer (LET) ionizing radiations (IRs) damages vital intracellular bio-molecules leading to multiple cellular and tissue injuries as well as pathophysiologies such as inflammation, immunosuppression etc. Nearly 70% of damage is caused indirectly by radiolysis of intracellular water leading to formation of reactive oxygen species (ROS) and free radicals and producing a state of oxidative stress. The damage is also caused by direct ionization of biomolecules. The type of radiation injuries is dependent on the absorbed radiation dose. Sub-lethal IR dose produces more of DNA base damages, whereas higher doses produce more DNA single strand break (SSBs), and double strand breaks (DSBs). The Nrf2-ARE pathway is an important oxidative stress regulating pathway. The DNA DSBs repair regulated by MRN complex, immunomodulation and inflammation regulated by HMGB1 and various types of cytokines are some of the key pathways which interact with each other in a complex manner and modify the radiation response. Because the majority of radiation damage is via oxidative stress, it is essential to gain in depth understanding of the mechanisms of Nrf2-ARE pathway and understand its interactions with MRN complex, HMGB1 and cytokines to increase our understanding on the radiation responses. Such information is of tremendous help in development of medical radiation countermeasures, radioprotective drugs and therapeutics. Till date no approved and safe countermeasure is available for human use. This study reviews the Nrf2-ARE pathway and its crosstalk with MRN-complex, HMGB1 and cytokines (TNF-a, IL-6, IFN-? etc.). An attempt is also made to review the modification of some of these pathways in presence of selected antioxidant radioprotective compounds or herbal extracts.

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Past work has shown that exposure to gamma rays and protons elicit a persistent oxidative stress in rodent and human neural stem cells (hNSCs). We have now adapted these studies to more realistic exposure scenarios in space, using lower doses and dose rates of these radiation modalities, to further elucidate the role of radiation-induced oxidative stress in these cells. Rodent neural stem and precursor cells grown as neurospheres and human neural stem cells grown as monolayers were subjected to acute and multi-dosing paradigms at differing dose rates and analyzed for changes in reactive oxygen species (ROS), reactive nitrogen species (RNS), nitric oxide and superoxide for 2 days after irradiation. While acute exposures led to significant changes in both cell types, hNSCs in particular, exhibited marked and significant elevations in radiation-induced oxidative stress. Elevated oxidative stress was more significant in hNSCs as opposed to their rodent counterparts, and hNSCs were significantly more sensitive to low dose exposures in terms of survival. Combinations of protons and γ-rays delivered as lower priming or higher challenge doses elicited radioadaptive changes that were associated with improved survival, but in general, only under conditions where the levels of reactive species were suppressed compared to cells irradiated acutely. Protective radioadaptive effects on survival were eliminated in the presence of the antioxidant N-acetylcysteine, suggesting further that radiation-induced oxidative stress could activate pro-survival signaling pathways that were sensitive to redox state. Data corroborates much of our past work and shows that low dose and dose rate exposures elicit significant changes in oxidative stress that have functional consequences on survival.

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