Search

As the great majority of gene expression (GE) biodosimetry studies have been performed using blood as the preferred source of tissue, searching for simple and less-invasive sampling methods is important when considering biodosimetry approaches. Knowing that whole saliva contains an ultrafiltrate of blood and white blood cells, it is expected that the findings in blood can also be found in saliva. This human in vivo study aims to examine radiation-induced GE changes in saliva for biodosimetry purposes and to predict radiation-induced disease, which is yet poorly characterized. Furthermore, we examined whether transcriptional biomarkers in blood can also be found equivalently in saliva. Saliva and blood samples were collected in parallel from radiotherapy (RT) treated patients who suffered from head and neck cancer (n = 8) undergoing fractioned partial-body irradiations (1.8 Gy/fraction and 50–70 Gy total dose). Samples were taken 12–24 h before first irradiation and ideally 24 and 48 h, as well as 5 weeks after radiotherapy onset. Due to the low quality and quantity of isolated RNA samples from one patient, they had to be excluded from further analysis, leaving a total of 24 saliva and 24 blood samples from 7 patients eligible for analysis. Using qRT-PCR, 18S rRNA and 16S rRNA (the ratio being a surrogate for the relative human RNA/bacterial burden), four housekeeping genes and nine mRNAs previously identified as radiation responsive in blood-based studies were detected. Significant GE associations with absorbed dose were found for five genes and after the 2nd radiotherapy fraction, shown by, e.g., the increase of CDKN1A (2.0 fold, P = 0.017) and FDXR (1.9 fold increased, P = 0.002). After the 25th radiotherapy fraction, however, all four genes (FDXR, DDB2, POU2AF1, WNT3) predicting ARS (acute radiation syndrome) severity, as well as further genes (including CCNG1 [median-fold change (FC) = 0.3, P = 0.013], and GADD45A (median-FC = 0.3, P = 0.031)) appeared significantly downregulated (FC = 0.3, P = 0.01–0.03). A significant association of CCNG1, POU2AF1, HPRT1, and WNT3 (P = 0.006-0.04) with acute or late radiotoxicity could be shown before the onset of these clinical outcomes. In an established set of four genes predicting acute health effects in blood, the response in saliva samples was similar to the expected up- (FDXR, DDB2) or downregulation (POU2AF1, WNT3) in blood for up to 71% of the measurements. Comparing GE responses (PHPT1, CCNG1, CDKN1A, GADD45A, SESN1) in saliva and blood samples, there was a significant linear association between saliva and blood response of CDKN1A (R2 = 0.60, P = 0.0004). However, the GE pattern of other genes differed between saliva and blood. In summary, the current human in vivo study, (I) reveals significant radiation-induced GE associations of five transcriptional biomarkers in salivary samples, (II) suggests genes predicting diverse clinical outcomes such as acute and late radiotoxicity as well as ARS severity, and (III) supports the view that blood-based GE response can be reflected in saliva samples, indicating that saliva is a "mirror of the body" for certain but not all genes and, thus, studies for each gene of interest in blood are required for saliva. [ABSTRACT FROM AUTHOR]

2.0 MeV proton beam accelerated at Tandetron is extracted in air through a thin film and allowed to scatter to irradiate the cell culture attached to the polymeric base of a biological flask. The irradiated cells were human medulloblastoma cell line Daoy treated with and without 5 nm sized spherical gold nanoparticles. Proton doses from 0.5 to 1.5 Gy have been employed to irradiate the cultures and to investigate the role of the radiotherapy performed with and without the use of the gold nanoparticles. Results indicated that cell survival is significantly reduced to about 50% when the nanoparticles at a concentration of about 6 × 1013 particles/ml are employed. [ABSTRACT FROM AUTHOR]

Abstract: The wild type lactose repressor of Escherichia coli is a tetrameric protein formed by two identical dimers. They are associated via a C-terminal 4-helix bundle (called tetramerization domain) whose stability is ensured by the interaction of leucine zipper motifs. Upon in vitro γ-irradiation the repressor losses its ability to bind the operator DNA sequence due to damage of its DNA-binding domains. Using an engineered dimeric repressor for comparison, we show here that irradiation induces also the change of repressor oligomerisation state from tetramer to dimer. The splitting of the tetramer into dimers can result from the oxidation of the leucine residues of the tetramerization domain. [Copyright &y& Elsevier]

Hyperthermia along with hydrocortisone (HC) are proven teratogens that can negatively influence embryo development during early pregnancy. Proliferation of cells is one of the main developmental processes during the early embryogenesis. This study was focused on testing the effect of elevated temperature and HC addition on proliferation of cells in in vitro cultures. The V79-4 cell line was treated with HC and cultured in vitro at 37 °C or 39 °C, respectively. To reveal the effect of both factors, the proliferation of cells cultured under different conditions was evaluated using various approaches (colony formation assay, generation of growth curves, computation of doubling times, and mitotic index estimation). Our results indicate that a short-term exposure to elevated temperature slightly stimulates and a long-term exposure suppresses cell proliferation. However, HC (0.1 mg/ml) acts as a stimulator of cell proliferation. Interestingly, the interaction of HC and long-term elevated temperature (39 °C) exposure results in at least partial compensation of the negative impact of elevated temperature by HC addition and in higher proliferation if compared with cells cultured at 39 °C without addition of HC., (© 2024. The Author(s).)

Tick-borne encephalitis virus (TBEV) is a neurotropic orthoflavivirus responsible for severe infections of the central nervous system. Although neurons are predominantly targeted, specific involvement of microglia in pathogenesis of TBE is not yet fully understood. In this study, the susceptibility of human microglia to TBEV is investigated, focusing on productive infection and different immune responses of different viral strains. We investigated primary human microglia and two immortalized microglial cell lines exposed to three TBEV strains (Hypr, Neudörfl and 280), each differing in virulence. Our results show that all microglia cultures tested support long-term productive infections, regardless of the viral strain. In particular, immune response varied significantly with the viral strain, as shown by the differential secretion of cytokines and chemokines such as IP-10, MCP-1, IL-8 and IL-6, quantified using a Luminex 48-plex assay. The most virulent strain triggered the highest cytokine induction. Electron tomography revealed substantial ultrastructural changes in the infected microglia, despite the absence of cytopathic effects. These findings underscore the susceptibility of human microglia to TBEV and reveal strain-dependent variations in viral replication and immune responses, highlighting the complex role of microglia in TBEV-induced neuropathology and contribute to a deeper understanding of TBE pathogenesis and neuroinflammation., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Saliva, as a non-invasive and easily accessible biofluid, has been shown to contain RNA biomarkers for prediction and diagnosis of several diseases. However, systematic analysis done by our group identified two problematic issues not coherently described before: (1) most of the isolated RNA originates from the oral microbiome and (2) the amount of isolated human RNA is comparatively low. The degree of bacterial contamination showed ratios up to 1:900,000, so that only about one out of 900,000 RNA copies was of human origin, but the RNA quality (average RIN 6.7 + /- 0.8) allowed for qRT-PCR. Using 12 saliva samples from healthy donors, we modified the methodology to (1) select only human RNA during cDNA synthesis by aiming at the poly(A)+-tail and (2) introduced a pre-amplification of human RNA before qRT-PCR. Further, the manufacturer's criteria for successful pre-amplification (Ct values ≤ 35 for unamplified cDNA) had to be replaced by (3) proofing linear pre-amplification for each gene, thus, increasing the number of evaluable samples up to 70.6%. When considering theses three modifications unbiased gene expression analysis on human salivary RNA can be performed.

The Tandetron Laboratory of the Nuclear Physics Institute of the Czech Academy of Sciences is equipped with five beam lines associated with a 3 MV tandem electrostatic accelerator model 4130 MC from High Voltage Engineering Europa B.V. This accelerator is coupled with two duoplasmatron sources and a single sputter ion source and provides ions from hydrogen to gold. One of these lines is a nuclear microbeam facility, utilizing ion beams of micro- and sub-micro sizes for materials research by use of particle induced x-ray emission spectroscopy, particle induced gamma emission, Rutherford back-scattering spectroscopy, and scanning transmission ion microscopy methods as well as for ion beam writing. The major advantage of the presented microprobe is a possibility of 3D structure creation not only in polymer materials using light ions but also in other materials such as glass, ceramics, etc. by use of heavy ions. The focusing system allows focusing of charged particles with a maximum rigidity of 11 MeV amu/q 2 . The usual resolution in high and low current modes is 2 × 3 µm 2 for a 100 pA and 0.3 × 0.5 µm 2 for the 2000 ions/s of 2 MeV protons, respectively. A detailed facility description is given in the paper. The applications of focused beams of heavy ions as well as examples of light ions utilizing are also presented in the article.

The increasing risk of acute large-scale radiological/nuclear exposures of population underlines the necessity of developing new, rapid and high throughput biodosimetric tools for estimation of received dose and initial triage. We aimed to compare the induction and persistence of different radiation exposure biomarkers in human peripheral blood in vivo. Blood samples of patients with indicated radiotherapy (RT) undergoing partial body irradiation (PBI) were obtained soon before the first treatment and then after 24 h, 48 h, and 5 weeks; i.e. after 1, 2, and 25 fractionated RT procedures. We collected circulating peripheral blood from ten patients with tumor of endometrium (1.8 Gy per fraction) and eight patients with tumor of head and neck (2.0-2.121 Gy per fraction). Incidence of dicentrics and micronuclei was monitored as well as determination of apoptosis and the transcription level of selected radiation-responsive genes. Since mitochondrial DNA (mtDNA) has been reported to be a potential indicator of radiation damage in vitro, we also assessed mtDNA content and deletions by novel multiplex quantitative PCR. Cytogenetic data confirmed linear dose-dependent increase in dicentrics (p < 0.01) and micronuclei (p < 0.001) in peripheral blood mononuclear cells after PBI. Significant up-regulations of five previously identified transcriptional biomarkers of radiation exposure (PHPT1, CCNG1, CDKN1A, GADD45, and SESN1) were also found (p < 0.01). No statistical change in mtDNA deletion levels was detected; however, our data indicate that the total mtDNA content decreased with increasing number of RT fractions. Interestingly, the number of micronuclei appears to correlate with late radiation toxicity (r2 = 0.9025) in endometrial patients suggesting the possibility of predicting the severity of RT-related toxicity by monitoring this parameter. Overall, these data represent, to our best knowledge, the first study providing a multiparametric comparison of radiation biomarkers in human blood in vivo, which have potential for improving biological dosimetry.

Biological effects of high-LET (linear energy transfer) radiation have received increasing attention, particularly in the context of more efficient radiotherapy and space exploration. Efficient cell killing by high-LET radiation depends on the physical ability of accelerated particles to generate complex DNA damage, which is largely mediated by LET. However, the characteristics of DNA damage and repair upon exposure to different particles with similar LET parameters remain unexplored. We employed high-resolution confocal microscopy to examine phosphorylated histone H2AX (γH2AX)/p53-binding protein 1 (53BP1) focus streaks at the microscale level, focusing on the complexity, spatiotemporal behaviour and repair of DNA double-strand breaks generated by boron and neon ions accelerated at similar LET values (∼135 keV μm -1 ) and low energies (8 and 47 MeV per n, respectively). Cells were irradiated using sharp-angle geometry and were spatially (3D) fixed to maximize the resolution of these analyses. Both high-LET radiation types generated highly complex γH2AX/53BP1 focus clusters with a larger size, increased irregularity and slower elimination than low-LET γ-rays. Surprisingly, neon ions produced even more complex γH2AX/53BP1 focus clusters than boron ions, consistent with DSB repair kinetics. Although the exposure of cells to γ-rays and boron ions eliminated a vast majority of foci (94% and 74%, respectively) within 24 h, 45% of the foci persisted in cells irradiated with neon. Our calculations suggest that the complexity of DSB damage critically depends on (increases with) the particle track core diameter. Thus, different particles with similar LET and energy may generate different types of DNA damage, which should be considered in future research.

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH* radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH. radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.