Mechanistic insights from high resolution DNA damage analysis to understand mixed radiation exposure.

Cells exposed to densely ionising high and scattered low linear energy transfer (LET) radiation (50 % dose of each) react more strongly than to the same dose of each separately. The relationship between DNA double strand break location inside the nucleus and chromatin structure was evaluated, using high-resolution transmission electron microscopy (TEM) in breast cancer MDA-MB-231 cells at 30 min post 5 Gy. Additionally, response to high and/or low LET radiation was assessed using single (1 ×1.5 Gy) versus fractionated dose delivery (5 ×0.3 Gy). By TEM analysis, the highest total number of γH2AX nanobeads were found in cells irradiated with alpha radiation just prior to gamma radiation (called mixed beam), followed by alpha, then gamma radiation. γH2AX foci induced by mixed beam radiation tended to be surrounded by open chromatin (lighter TEM regions), yet foci containing the highest number of beads, i.e. larger foci representing complex damage, remained in the heterochromatic areas. The γH2AX large focus area was also greater in mixed beam-treated cells when analysed by immunofluorescence. Fractionated mixed beams given daily induced the strongest reduction in cell viability and colony formation in MDA-MB-231 and osteosarcoma U2OS cells compared to the other radiation qualities, as well as versus acute exposure. This may partially be explained by recurring low LET oxidative DNA damage by every fraction together with a delay in recompaction of chromatin after high LET, demonstrated by low levels of heterochromatin marker H3K9me3 at 2 h after the last mixed beam fraction in MDA-MB-231. In conclusion, early differences in response to complex DNA damage may lead to a stronger cell kill induced by fractionated exposure, which suggest a therapeutic potential of combined high and low LET irradiation.

Machine Learning Based Prediction of Gamma Passing Rate for VMAT Radiotherapy Plans.

The use of machine learning algorithms (ML) in radiotherapy is becoming increasingly popular. More and more groups are trying to apply ML in predicting the so-called gamma passing rate (GPR). Our team has developed a customized approach of using ML algorithms to predict global GPR for electronic portal imaging device (EPID) verification for dose different 2% and distance to agreement 2 mm criteria for VMAT dynamic plans. Plans will pass if the GPR is greater than 98%. The algorithm was learned and tested on anonymized clinical data from 13 months which resulted in more than 3000 treatment plans. The obtained results of GPR prediction are very interesting. Average specificity of the algorithm based on an ensemble of 50 decision tree regressors is 91.6% for our criteria. As a result, we can reduce the verification process by 50%. The novel approach described by our team can offer a new insight into the application of ML and neural networks in GPR prediction and dosimetry.

Exosome secretion and cellular response of DU145 and PC3 after exposure to alpha radiation.

Exosomes are spherical membrane nanovesicles secreted from cells, and they play an important role in tumor immune response, metastasis, angiogenesis, and survival. Studies investigating exosomes isolated from cells exposed to photon radiation commonly used in conventional radiotherapy demonstrate the influence of this type of radiation on exosome characteristics and secretion. There is currently no research investigating the effects of densely ionizing particles such as protons and alpha radiation on exosomes. Thus we have evaluated the cellular response of human prostate cancer cells exposed to 0, 2, and 6 Gy of alpha radiation emitted from the Am-241 source. Irradiated PC3 and DU145 cell lines, characterized by differences in radiosensitivity, were studied using apoptosis, LDH, and IL-6 assays. Additionally, the corresponding concentration and size of isolated exosomes were measured using NTA. We found that exposure to ionizing radiation resulted in gross changes in viability and cell damage. There were increased amounts of apoptotic or necrotic cells as a function of radiation dose. We demonstrated that irradiated PC3 cells secrete higher quantities of exosomes compared to DU145 cells. Additionally, we also found no statistical difference in exosome size for control and irradiated cells.

Brownian Motion Influence on AFM Exosomes' Size Measurements.

Extracellular vesicles are evaluated by nanoparticle tracking analysis (NTA), providing information on their hydrodynamic diameters, and by atomic force microscopy (AFM) to calculate their geometric diameters. The aim of this study is to explore the influence of Brownian movements in a sample drop and preparation time on imaging-based measurements and to determine the relationship between the geometric and hydrodynamic sizes of the extracellular vesicles measured by the AFM and the NTA, respectively. Exosomes derived from the human prostate cancer cell line PC3 were evaluated by NTA and AFM, and those results were compared with Monte Carlo simulations. The mean size, evaluated by AFM shortly after application on the mica substrate, is less than its real value. It obtains the correct value faster for a thinner sample drop. Fitting the log-normal distribution to the geometric and hydrodynamic diameters leads to the conclusion that the latter could arise from the former by linear scaling by a factor that could be used to characterize the analyzed extracellular vesicles. The size of the vesicles attached to the mica substrate depends on time. The effect of Brownian motion and stretch of the lipid bilayer should be considered in the context of exosome AFM studies.

Freeware tool for analysing numbers and sizes of cell colonies.

The clonogenic cell survival assay is a basic method to study the cytotoxic effect of radiation and chemical toxins. In large experimental setups, counting of colonies by eye is tiresome and prone to bias. Moreover, it is often interesting to quantify the size of individual colonies. Such analyses are largely facilitated by computerised image analysis systems. Although a number of such systems exist, they all focus on enumerating colonies and not on analysing the colony size. We have developed a new software package for both counting colonies and plotting their size distributions. The software called count and Plot HIstograms of Colony Size (countPHICS) consists of two parts: (1) a macro written for ImageJ which analyses computerised images of cell culture dishes or 6-well plates, counts colonies, estimates their size and saves the results in a text file; (2) a program written with QT Creator which reads the text file, plots histograms of colony size distribution and fits the best function. The full program is freely available at: http://www.fuw.edu.pl/~bbrzozow/FizMed/countPHICS.html . In conclusion, our new publically available software will facilitate colony counting and provide additional information on the colony growth rate, which is relevant especially for radiosensitisation studies.

Transmission of brain activity in cognitive and motor tasks.

The Short-time Directed Transfer Function was used for estimation of dynamical patterns of brain activity propagation. The SDTF is based on the multivariate autoregressive model, where all channels of the process are considered simultaneously. Time-frequency patterns of EEG propagation were found for the task of finger movement and its imagination and for the Continuous Attention Test. The results supported the neurophysiological hypotheses concerning information processing in brain and in particular the theory of active inhibition.

Propagation of EEG activity during finger movement and its imagination.

We investigated the pattern of EEG activity propagation in the beta and gamma band during a finger movement experiment and imagination of that task. The data were analyzed by means of a short-time directed transfer function (SDTF) based on a multivariate autoregressive model. The signals from the right (or left) hemisphere were processed simultaneously (not pairwise), which is crucial for obtaining a correct picture of EEG activity transmissions. The pattern of propagation in the beta band involved for both tasks a decrease of the propagation from the motor areas during the execution of the movement - less pronounced in the case of imagination. The performance of the motion was mainly connected with a short outburst of gamma activity from the hand sensorimotor areas. In case of imagination the gamma outflow lasted longer and concerned larger brain areas.

On the statistical significance of event-related EEG desynchronization and synchronization in the time-frequency plane.

We propose and discuss a complete framework for estimating significant changes in the average time-frequency density of energy of event-related signals. Addressed issues include estimation of time-frequency energy density (matching pursuit and spectrogram), choice of resampling statistics to test the hypothesis of change in one small region (resel), and correction for multiplicity (false discovery rate). We present estimation of the significance of event-related electroencephalograph desynchronization and synchronization (ERD/ERS) in the time-frequency plane.