Proteins become asymmetrically distributed in the one-cell Caenorhabditis elegans embryo thanks to reaction-diffusion mechanisms that are often entangled in complex feedback loops. Cortical polarity drives the enrichment of the RNA-binding proteins MEX-5 and MEX-6 in the anterior cytoplasm through concentration gradients. MEX-5 and MEX-6 promote the patterning of other cytoplasmic factors, including that of the anteriorly enriched mitotic polo-like kinase PLK-1, but also contribute to proper cortical polarity. The gradient of MEX-5 forms through a differential-diffusion mechanism. How MEX-6 establishes a gradient and how MEX-5 and MEX-6 regulate cortical polarity is not known. Here, we reveal that the two MEX proteins develop concentration asymmetries via similar mechanisms, but despite their strong sequence homology, they differ in terms of how their concentration gradients are regulated. We find that PLK-1 promotes the enrichment of MEX-5 and MEX-6 at the anterior through different circuits: PLK-1 influences the MEX-5 gradient indirectly by regulating cortical polarity while it modulates the formation of the gradient of MEX-6 through its physical interaction with the protein. We thus propose a model in which PLK-1 mediates protein circuitries between MEX-5, MEX-6, and cortical proteins to faithfully establish and maintain polarity.

Molecules inside cells are subject to physical forces and undergo biochemical interactions, continuously changing their physical properties and dynamics. Despite this, cells achieve highly ordered molecular patterns that are crucial to regulate various cellular functions and to specify cell fate. In the Caenorhabditis elegans one-cell embryo, protein asymmetries are established in the narrow time window of a cell division. What are the mechanisms that allow molecules to establish asymmetries, defying the randomness imposed by Brownian motion? Mathematical and computational models have paved the way to the understanding of protein dynamics up to the 'single-molecule level' when resolution represents an issue for precise experimental measurements. Here we review the models that interpret cortical and cytoplasmic asymmetries in the one-cell C. elegans embryo.

PurposeThe aim of this study was to explore the effects of chronic low-dose-rate gamma-radiation at a multi-scale level. The specific objective was to obtain an overall view of the endothelial cell response, by integrating previously published data on different cellular endpoints and highlighting possible different mechanisms underpinning radiation-induced senescence.Materials and methodsDifferent datasets were collected regarding experiments on human umbilical vein endothelial cells (HUVECs) which were chronically exposed to low dose rates (0, 1.4, 2.1 and 4.1 mGy/h) of gamma-rays until cell replication was arrested. Such exposed cells were analyzed for different complementary endpoints at distinct time points (up to several weeks), investigating cellular functions such as proliferation, senescence and angiogenic properties, as well as using transcriptomics and proteomics profiling. A mathematical model was proposed to describe proliferation and senescence.ResultsSimultaneous ceasing of cell proliferation and senescence onset as a function of time were well reproduced by the logistic growth curve, conveying shared equilibria between the two endpoints. The combination of all the different endpoints investigated highlighted a dose-dependence for prematurely induced senescence. However, the underpinning molecular mechanisms appeared to be dissimilar for the different dose rates, thus suggesting a more complex scenario.ConclusionsThis study was conducted integrating different datasets, focusing on their temporal dynamics, and using a systems biology approach. Results of our analysis highlight that different dose rates have different effects in inducing premature senescence, and that the total cumulative absorbed dose also plays an important role in accelerating endothelial cell senescence.

Significance Intracellular gradients have essential roles in cell and developmental biology, but their formation is not fully understood. We have developed a computational approach facilitating interpretation of protein dynamics and gradient formation. We have combined this computational approach with experiments to understand how Polo-Like Kinase 1 (PLK-1) forms a cytoplasmic gradient in Caenorhabditis elegans embryos. Although the PLK-1 gradient depends on the Muscle EXcess-5/6 (MEX-5/6) proteins, we reveal differences in PLK-1 and MEX-5 gradient formation that can be explained by a model with two components, PLK-1 bound to MEX-5 and unbound PLK-1. Our combined approach suggests that a weak coupling between PLK-1 and MEX-5 reaction–diffusion mechanisms dictates the dynamic exchange of PLK-1 with the cytoplasm, explaining PLK-1 high diffusivity and smooth gradient.

The cellular uptake of nanoparticles (NPs) represents a critical step in nanomedicine and a crucial point for understanding the interaction of nanomaterials with biological systems. No specific mechanism of uptake has been identified so far, as the NPs are generally incorporated by the cells through one of the few well-known endocytotic mechanisms. Here, an alternative internalization route mediated by microvilli adhesion is demonstrated. This microvillus-mediated adhesion (MMA) has been observed using ceria and magnetite NPs with a dimension of <40 nm functionalized with polyacrylic acid but not using NPs with a neutral or positive functionalization. Such an adhesion was not cell specific, as it was demonstrated in three different cell lines. MMA was also reduced by modifications of the microvillus lipid rafts, obtained by depleting cholesterol and altering synthesis of sphingolipids. We found a direct relationship between MAA, cell cycle, and density of microvilli. The evidence suggests that MMA differs from the commonly described uptake mechanisms and might represent an interesting alternative approach for selective NP delivery.

Cell cycle progression can be studied with computational models that allow to describe and predict its perturbation by agents as ionizing radiation or drugs. Such models can then be integrated in tools for pre-clinical/clinical use, e.g. to optimize kinetically-based administration protocols of radiation therapy and chemotherapy. We present a deterministic compartmental model, specifically reproducing how cells that survive radiation exposure are distributed in the cell cycle as a function of dose and time after exposure. Model compartments represent the four cell-cycle phases, as a fuction of DNA content and time. A system of differential equations, whose parameters represent transition rates, division rate and DNA synthesis rate, describes the temporal evolution. Initial model inputs are data from unexposed cells in exponential growth. Perturbation is implemented as an alteration of model parameters that allows to best reproduce cell-cycle profiles post-irradiation. The model is validated with dedicated in vitro measurements on human lung fibroblasts (IMR90). Cells were irradiated with 2 and 5 Gy with a Varian 6 MV Clinac at IRCCS Maugeri. Flow cytometry analysis was performed at the RadBioPhys Laboratory (University of Pavia), obtaining cell percentages in each of the four phases in all studied conditions up to 72 hours post-irradiation. Cells show early G2-phase block (increasing in duration as dose increases) and later G1-phase accumulation. For each condition, we identified the best sets of model parameters that lead to a good agreement between model and experimental data, varying transition rates from G1- to S- and from G2- to M-phase. This work offers a proof-of-concept validation of the new computational tool, opening to its future development and, in perspective, to its integration in a wider framework for clinical use. Author summary We implemented a computational model able to describe how the progression in the cell cycle is perturbed when cells are exposed to ionizing radiation. It is known that radiation causes delays or arrest in cell cycle progression, and also that cells that are in different phases of the cycle at the time of exposure show different sensitivity to radiation. Chemotherapeutic drugs also affect cell cycle, and their action can be phase-specific. These findings can be exploited to find the optimal protocol of a combined radiotherapy/chemotherapy cancer treatment: to this aim, we need to know not only the effectiveness of an agent (dose/drug) in terms of cell killing, but also how surviving cells are distributed in the cell cycle. With the model we present, this information can be reproduced as a function of dose and time after radiation exposure. To test the model performance we measured distributions of cells in different phases of the cycle (using flow-cytometry) for human healthy fibroblast cells exposed to X-rays. The results of this work constitute a first step for further development of our model and its future integration in a tool for pre-clinical/clinical use.

In vitro co-culture models between tumor cells and peripheral blood mononuclear cells (PBMCs) allow studying the interplay between these cell populations, potentially gaining insight into the in vivo response of the immune system to the presence of the tumor, as well as to possible other agents as radiation used for therapeutic purposes. However, great care is needed in the experimental optimization of models and choice of conditions, as some setups might offer a limited possibility to capture subtle immune perturbations. A co-culture model of PBMCs from healthy donors and colorectal adenocarcinoma Caco-2 cells was successfully adopted in a previous work to measure effects on Caco-2 and modulation of signaling when these latter are irradiated. We here tested if the same experimental setting allows to measure perturbations to the main PBMC subsets: we performed immunophenotyping by means of flow cytometry and quantified helper and cytotoxic T cells, NK cells, and B cells, when PBMCs are cultured alone (control), in presence of non-irradiated Caco-2 cells or when these latter are exposed to a 10 Gy X-ray dose from a conventional radiotherapy accelerator. To measure a baseline response in all experimental conditions, PBMCs were not further stimulated, but only followed in their time-evolution up to 72 h post-irradiation of Caco-2 and assembly of the co-culture. In this time interval PBMCs maintain a high viability (measured via the MTT assay). Caco-2 viability (MTT) is slightly affected by the presence of PBMCs and by the high radiation dose, confirming their radioresistance. Immunophenotyping results indicate a large inter-individual variability for different population subsets already at the control level. We analyzed relative population changes and we detected only a small but significant perturbation to cytotoxic T cells. We conclude that this model, as it is, is not adequate for the measurements of subtler immune perturbations (if any, not washed-out by inter-individual differences). For this purpose, the model needs to be modified and further optimized e.g., including a pre-treatment strategy for PBMCs. We also performed a pooled analysis of all experimental observations with principal component analysis, suggesting the potential of this tool to identify subpopulations of similarly-responding donors.

Stress granules (SGs) are membraneless organelles that form in eukaryotic cells after stress exposure [1] (reviewed in [2-4]). Following translation inhibition, polysome disassembly releases 48S preinitiation complexes (PICs). mRNA, PICs, and other proteins coalesce in SG cores [1, 5-7]. SG cores recruit a dynamic shell, whose properties are dominated by weak interactions between proteins and RNAs [8-10]. The structure and assembly of SGs and how different components contribute to their formation are not fully understood. Using super-resolution and expansion microscopy, we find that the SG component UBAP2L [11, 12] and the core protein G3BP1 [5, 11-13] occupy different domains inside SGs. UBAP2L displays typical properties of a core protein, indicating that cores of different compositions coexist inside the same granule. Consistent with a role as a core protein, UBAP2L is required for SG assembly in several stress conditions. Our reverse genetic and cell biology experiments suggest that UBAP2L forms granules independent of G3BP1 and 2 but does not interfere with stress-induced translational inhibition. We propose a model in which UBAP2L is an essential SG nucleator that acts upstream of G3BP1 and 2 and facilitates G3BP1 core formation and SG assembly and growth.

The PERSEO project (PErsonal Radiation Shielding for intErplanetary missiOns), funded by the Italian Space Agency, has led to the development of a first technological demonstrator of a radiation shielding garment, to be used in a pressurized space habitat, that can be filled at need with on-board water and used for personal protection in case of solar particle events. The collaboration, including academic partners and companies active in space research and technology development, designed and manufactured the prototype that has been successfully tested on board the International Space Station by the European Space Agency astronaut Paolo Nespoli in November 2017, during the VITA mission. The effectiveness of the garment in terms of reduction of the radiation dose to sensitive organs (subject to the occurrence of short-term non-cancer effects following acute exposure) has been evaluated with Monte Carlo simulations with an anthropomorphic phantom. The successful outcome of the experimental session on board has demonstrated the practicality of use and wearability of the prototype, and, in perspective, the feasibility of a personal radiation shielding strategy, complementary to habitat shielding and based on the use of available resources, of fundamental importance also in view of future manned interplanetary missions.

The consideration of how a given technique affects results of experimental measurements is a must to achieve correct data interpretation. This might be challenging when it comes to measurements on biological systems, where it is unrealistic to have full control (e.g. through a software replica) of all steps in the measurement chain. In this work we address how the effectiveness of different radiation qualities in inducing biological damage can be assessed measuring DNA damage foci yields, only provided that artefacts related to the scoring technique are adequately considered. To this aim, we developed a unified stochastic modelling approach that, starting from radiation tracks, predicts both the induction, spatial distribution and complexity of DNA damage, and the experimental readout of foci when immunocytochemistry coupled to 2D fluorescence microscopy is used. The approach is used to interpret γ-H2AX data for photon and neutron exposures. When foci are reconstructed in the whole cell nucleus, we obtain information on damage characteristics “behind” experimental observations, as the average damage content of a focus. We reproduce how the detection technique affects experimental findings, e.g. contributing to the saturation of foci yields scored at 30 minutes after exposure with increasing dose and to the lack of dose dependence for yields at 24 hours.

Glioblastoma multiforme (GBM) is characterized by a poor prognosis and a median survival of ~12-18 months. GBM is usually managed by neurosurgery followed by both chemotherapy and radiotherapy. Since GBM develops resistance to conventional therapies, treatment with C-ions is promising to completely eradicate the tumoural mass. During cranial irradiation, exposure of healthy tissues is inevitable. Because of the presence of neural stem cells, a deep investigation on the effects of C-ion irradiation with respect to X-ray induced damage is mandatory to allow a better definition of treatments. In this work, the comparison of X-rays and C-ion irradiation-induced effects on human neural stem cell, focusing on multiple endpoints, such as cell viability, cytokine secretion and spheroid formation is presented. Results show different temporal and dose responses of human neural stem cells to the different radiation qualities, suggesting different underpinning mechanisms of radiation-induced damages.

Purpose There is an increasing interest in calculating linear energy transfer (LET) distributions for proton therapy treatments in order to assess the influence of this quantity in biological terms. Microdosimetric Monte Carlo (MC) simulations are useful tools to calculate dose-averaged LET, as this has been broadly proposed as the most adequate quantity to characterize these biological effects. However, a straightforward uniform sampling of the scoring site turns out to be computationally unaffordable. In contrast, some issues have been pointed out with the more efficient weighted sampling approach, frequently used in literature. Here, we address the issues associated with the latter method and propose adequate corrections to achieve reliable calculations of dose-averaged LET values from microdosimetry. Methods and materials Proton track structures have been simulated with Geant4-DNA considering two different approaches. One version employs a uniform sampling for placing the spherical site and is used as the reference. The other one uses a weighted sampling by considering the spatial distribution of transfer points. Some corrections are proposed for calculating a dose-averaged LET comparable to the reference case. An additional MC approach is proposed to obtain the weighted mean of the energy imparted per electronic collision of the proton within the site, the delta 2 function, related to the straggling distribution, as an intermediate step in the LET calculation. Results Energy imparted per event distributions are different when employing either sampling methods, due to the different geometrical randomness. We have found an agreement below (0.15 +/- 0.05) keV/mu m in the worst case for uniform and weighted methods in dose-averaged LET values when the weighted sampling results are corrected according to our proposal. Our analysis is restricted to spherical sites of 1 and 10 mu m diameter and monoenergetic beams in the range from 2 to 90 MeV. Conclusions This work shows a reliable and computational-efficient method to perform calculations of track segment dose-averaged LET using MC simulations for proton therapy beams, including the necessary considerations for obtaining the straggling distribution characteristics. The validity of this approach remains as long as the stopping power of the proton can be considered as constant along its track within the site.

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

As manned spaceflights beyond low Earth orbit are in the agenda of Space Agencies, the concerns related to space radiation exposure of the crew are still without conclusive solutions. The risk of long-term detrimental health effects needs to be kept below acceptable limits, and emergency countermeasures must be planned to avoid the short-term consequences of exposure to high particle fluxes during hardly predictable solar events. Space habitat shielding cannot be the ultimate solution: the increasing complexity of future missions will require astronauts to protect themselves in low-shielded areas, e.g. during emergency operations. Personal radiation shielding is promising, particularly if using available resources for multi-functional shielding devices. In this work we report on all steps from the conception, design, manufacturing, to the final test on board the International Space Station (ISS) of the first prototype of a water-filled garment for emergency radiation shielding against solar particle events. The garment has a good shielding potential and comfort level. On-board water is used for filling and then recycled without waste. The successful outcome of this experiment represents an important breakthrough in space radiation shielding, opening to the development of similarly conceived devices and their use in interplanetary missions as the one to Mars.

An approach based on track-structure calculations has been developed to take account of artefacts occurring during γ-H2AX foci detection in 2D images of samples analyzed through immunocytochemistry. The need of this works stems from the observed saturation in foci yields measured after X-ray doses higher than few grays, hindering an unambiguous quantification of DNA damage and of radiation effectiveness. The proposed modelling approach allows to simulate the observer's point of view for foci scoring, mimicking the selection of a slice Δz of the cell nucleus due to the microscope depth of field, and applying a clustering algorithm to group together damages within a resolution parameter r. Calculation results were benchmarked with experimental measurements at an early time-point for mouse breast cancer cells, irradiated with X-ray doses in the range 0-5 Gy. The model is able to reproduce the saturation in experimental data.

When early radiation damage to biological systems is studied based on the formation of foci at the location of DNA double-strand breaks, the foci observed in irradiated cells either may be induced by ionizing radiation (IR) interactions or they may be due to other causes that lead to observation of foci also in unirradiated cells. Generally, to take account of the latter, additional samples are taken where the exposure to IR is skipped in the protocol. The data analysis relies on statistical independence of the frequency distributions of background and radiation-induced foci. In microscopy, however, the observed spatial patterns of foci are 2D projections of the spatial distributions of foci in the observed cell nuclei. This may lead to missing foci when scoring their number, particularly if projections of foci overlap or coincide. This paper investigates to what extent the statistical independence of the frequency distribution of the number of foci coming from IR interaction or other causes is compromised by foci overlapping.

We present predictions of neutron relative biological effectiveness (RBE) for cell irradiations with neutron beams at PTB-Braunschweig. A neutron RBE model is adopted to evaluate initial DNA damage induction given the neutron-induced charged particle field. RBE values are predicted for cell exposures to quasi-monoenergetic beams (0.56 MeV, 1.2 MeV) and to a broad energy distribution neutron field with dose-averaged energy of 5.75 MeV. Results are compared to what obtained with our RBE predictions for neutrons at similar energies, when a 30-cm sphere is irradiated in an isotropic neutron field. RBE values for experimental conditions are higher for the lowest neutron energies, because, as expected, target geometry determines the weight of the low-effectiveness photon component of the neutron dose. These results highlight the importance of characterizing neutron fields in terms of physical interactions, to fully understand neutron-induced biological effects, contributing to risk estimation and to the improvement of radiation protection standards.

Personal radiation shielding is likely to play an important role in the strategy for radiation protection of future manned interplanetary missions. There is potential for the successful adoption of wearable shielding devices, readily available in case of accidental exposures or used for emergency operations in low-shielded areas of the habitat, particularly in case of solar particle events (SPEs). Based on optimization of available resources, conceptual models for radiation protection spacesuits have been proposed, with elements made of different materials, and the first prototype of a water-fillable garment was designed and manufactured in the framework of the PERSEO project, funded by the Italian Space Agency, leading to the successful test of such prototype for ease of use and wearability on-board the International Space Station. We present results of Monte Carlo calculations offering a proof-of-principle validation of the shielding efficacy of such prototype in different SPE environments and shielding conditions.