Prediction of DNA rejoining kinetics and cell survival after proton irradiation for V79 cells using Geant4-DNA.

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.

Implementation of the EPICS2017 database for photons in Geant4.

This paper describes in detail the implementation of Geant4 Livermore electromagnetic physics models based on the EPICS2017 database for the low energy transport of photons. These models describe four photon processes: gamma conversion, Compton scattering, photoelectric effect and Rayleigh scattering. New parameterizations based on EPICS2017 were performed for scattering functions of Compton effect, subshell cross-sections of the photoelectric effect and form factors of Rayleigh scattering, in order to improve the precision of fitted values compared to tabulated values. Comparisons between new and old parameterizations were also carried out to evaluate the precision of the new parameterizations. The models were tested through a comparative study, in which the mass attenuation coefficient was calculated for both total photon interaction and each process using Geant4 simulations based on EPICS2017 and EPDL97 respectively. The results obtained from the simulations were found in good agreement with the XCOM reference data.

A Geant4 simulation of X-ray emission for three-dimensional proton imaging of microscopic samples.

PURPOSE: Proton computed microtomography is a technique that reveals the inner content of microscopic samples. The density distribution of the material (in g·cm-3) is obtained from proton transmission tomography (STIM: Scanning Transmission Ion Microscopy) and the element content from X-ray emission tomography (PIXE: Particle Induced X-ray Emission). A precise quantification of chemical elements is difficult for thick samples, because of the variations of X-ray production cross-sections and of X-ray absorption. Both phenomena are at the origin of an attenuation of the measured X-ray spectra, which leads to an underestimation of the element content. Our aim is to quantify the accuracy of a specific correction method that we designed for thick samples. METHODS: In this study, we describe how the 3D variations in the mass density were taken into account in the reconstruction code, in order to quantify the correction according to the position of the proton beam and the position and aperture angle of the X-ray detector. Moreover, we assess the accuracy of the reconstructed densities using Geant4 simulations on numerical phantoms, used as references. RESULTS: The correction process was successfully applied and led, for the largest regions of interest (little affected by partial volume effects), to an accuracy ≤ 4% for phosphorus (compared to about 40% discrepancy without correction). CONCLUSION: This study demonstrates the accuracy of the correction method implemented in the tomographic reconstruction code for thick samples. It also points out some advantages offered by Geant4 simulations: i) they produce projection data that are totally independent of the inversion method used for the image reconstruction; ii) one or more physical processes (X-ray absorption, proton energy loss) can be artificially turned off, in order to precisely quantify the effect of the different phenomena involved in the attenuation of X-ray spectra.

Atypical Membrane-Anchored Cytokine MIF in a Marine Dinoflagellate.

Macrophage Migration Inhibitory Factors (MIF) are pivotal cytokines/chemokines for vertebrate immune systems. MIFs are typically soluble single-domain proteins that are conserved across plant, fungal, protist, and metazoan kingdoms, but their functions have not been determined in most phylogenetic groups. Here, we describe an atypical multidomain MIF protein. The marine dinoflagellate Lingulodinium polyedra produces a transmembrane protein with an extra-cytoplasmic MIF domain, which localizes to cell-wall-associated membranes and vesicular bodies. This protein is also present in the membranes of extracellular vesicles accumulating at the secretory pores of the cells. Upon exposure to biotic stress, L. polyedra exhibits reduced expression of the MIF gene and reduced abundance of the surface-associated protein. The presence of LpMIF in the membranes of secreted extracellular vesicles evokes the fascinating possibility that LpMIF may participate in intercellular communication and/or interactions between free-living organisms in multispecies planktonic communities.

Cross-Kingdom Analysis of Diversity, Evolutionary History, and Site Selection within the Eukaryotic Macrophage Migration Inhibitory Factor Superfamily.

Macrophage migration inhibitory factors (MIF) are multifunctional proteins regulating major processes in mammals, including activation of innate immune responses. MIF proteins also play a role in innate immunity of invertebrate organisms or serve as virulence factors in parasitic organisms, raising the question of their evolutionary history. We performed a broad survey of MIF presence or absence and evolutionary relationships across 803 species of plants, fungi, protists, and animals, and explored a potential relation with the taxonomic status, the ecology, and the lifestyle of individual species. We show that MIF evolutionary history in eukaryotes is complex, involving probable ancestral duplications, multiple gene losses and recent clade-specific re-duplications. Intriguingly, MIFs seem to be essential and highly conserved with many sites under purifying selection in some kingdoms (e.g., plants), while in other kingdoms they appear more dispensable (e.g., in fungi) or present in several diverged variants (e.g., insects, nematodes), suggesting potential neofunctionalizations within the protein superfamily.

A Geant4 simulation for three-dimensional proton imaging of microscopic samples.

Proton imaging can be carried out on microscopic samples by focusing the beam to a diameter ranging from a few micrometers down to a few tens of nanometers, depending on the required beam intensity and spatial resolution. Three-dimensional (3D) imaging by tomography is obtained from proton transmission (STIM: Scanning Transmission Ion Microscopy) and/or X-ray emission (PIXE: Particle Induced X-ray Emission). In these experiments, the samples are dehydrated for under vacuum analysis. In situ quantification of nanoparticles has been carried out at CENBG in the frame of nanotoxicology studies, on cells and small organisms used as biological models, especially on Caenorhabditis elegans (C. elegans) nematodes. Tomography experiments reveal the distribution of mass density and chemical content (in g.cm-3) within the analyzed volume. These density values are obtained using an inversion algorithm. To investigate the effect of this data reduction process, we defined different numerical phantoms, including a (dehydrated) C. elegans phantom whose geometry and density were derived from experimental data. A Monte Carlo simulation based on the Geant4 toolkit was developed. Using different simulation and reconstruction conditions, we compared the resulting tomographic images to the initial numerical reference phantom. A study of the relative error between the reconstructed and the reference images lead to the result that 20 protons per shot can be considered as an optimal number for 3D STIM imaging. Preliminary results for PIXE tomography are also presented, showing the interest of such numerical phantoms to produce reference data for future studies on X-ray signal attenuation in thick samples.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis.

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 µg.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

Efficient Encapsulation of Fluorinated Drugs in the Confined Space of Water-Dispersible Fluorous Supraparticles.

Fluorophobic-driven assemblies of gold nanomaterials were stabilized into water-dispersible fluorous supraparticles by the film-forming protein hydrophobin II. The strategy makes use of fluorous nanomaterials of different dimensions to engineer size and inner functionalization of the resulting confined space. The inner fluorous compartments allow efficient encapsulation and transport of high loadings of partially fluorinated drug molecules in water.

In situ quantification of diverse titanium dioxide nanoparticles unveils selective endoplasmic reticulum stress-dependent toxicity.

Although titanium dioxide nanoparticles (TiO2 NPs) have been extensively studied, their possible impact on health due to their specific properties supported by their size and geometry, remains to be fully characterized to support risk assessment. To further document NPs biological effects, we investigated the impact of TiO2 NPs morphology on biological outcomes. To this end, TiO2 NPs were synthesized as nanoneedles (NNs), titanate scrolled nanosheets (TNs), gel-sol-based isotropic nanoparticles (INPs) and tested for perturbation of cellular homeostasis (cellular ion content, cell proliferation, stress pathways) in three cell types and compared to the P25. We showed that TiO2 NPs were internalized at various degrees and their toxicity depended on both titanium content and NPs shape, which impacted on intracellular calcium homeostasis thereby leading to endoplasmic reticulum stress. Finally, we showed that a minimal intracellular content of TiO2 NPs was mandatory to induce toxicity enlightening once more the crucial notion of internalized dose threshold beside the well-recognized dose of exposure.

Single cell in situ detection and quantification of metal oxide nanoparticles using multimodal correlative microscopy.

Assessing in situ nanoparticles (NPs) internalization at the level of a single cell is a difficult but critical task due to their potential use in nanomedicine. One of the main actual challenges is to control the number of internalized NPs per cell. To in situ detect, track, and above all quantify NPs in a single cell, we propose an approach based on a multimodal correlative microscopy (MCM), via the complementarity of three imaging techniques: fluorescence microscopy (FM), scanning electron microscopy (SEM), and ion beam analysis (IBA). This MCM was performed on single targeted individual primary human foreskin keratinocytes (PHFK) cells cultured and maintained on a specifically designed sample holder, to probe either dye-modified or bare NPs. The data obtained by both FM and IBA on dye-modified NPs were strongly correlated in terms of detection, tracking, and colocalization of fluorescence and metal detection. IBA techniques should therefore open a new field concerning specific studies on bare NPs and their toxicological impact on cells. Complementarity of SEM and IBA analyses provides surface (SEM) and in depth (IBA) information on the cell morphology as well as on the exact localization of the NPs. Finally, IBA not only provides in a single cell the in situ quantification of exogenous elements (NPs) but also that all the other endogenous elements and the subsequent variation of their homeostasis. This unique feature opens further insights in dose-dependent response analyses and adds the perspective of a better understanding of NPs behavior in biological specimens for toxicology or nanomedicine purposes.