Early and Late Effects of Low-Dose X-ray Exposure in Human Fibroblasts: DNA Repair Foci, Proliferation, Autophagy, and Senescence.

The effects of low-dose radiation exposure remain a controversial topic in radiation biology. This study compares early (0.5, 4, 24, 48, and 72 h) and late (5, 10, and 15 cell passages) post-irradiation changes in γH2AX, 53BP1, pATM, and p-p53 (Ser-15) foci, proliferation, autophagy, and senescence in primary fibroblasts exposed to 100 and 2000 mGy X-ray radiation. The results show that exposure to 100 mGy significantly increased γH2AX, 53BP1, and pATM foci only at 0.5 and 4 h post irradiation. There were no changes in p-p53 (Ser-15) foci, proliferation, autophagy, or senescence up to 15 passages post irradiation at the low dose.

Radiation-Induced miRNAs Changes and cf mtDNA Level in Trauma Surgeons: Epigenetic and Molecular Biomarkers of X-ray Exposure.

Exposure to ionizing radiation can result in the development of a number of diseases, including cancer, cataracts and neurodegenerative pathologies. Certain occupational groups are exposed to both natural and artificial sources of radiation as a consequence of their professional activities. The development of non-invasive biomarkers to assess the risk of exposure to ionizing radiation for these groups is of great importance. In this context, our objective was to identify epigenetic and molecular biomarkers that could be used to monitor exposure to ionizing radiation. The impact of X-ray exposure on the miRNAs profile and the level of cf mtDNA were evaluated using the RT-PCR method. The levels of pro-inflammatory cytokines in their blood were quantified using the ELISA method. A significant decrease in miR-19a-3p, miR-125b-5p and significant increase in miR-29a-3p was observed in the blood plasma of individuals exposed to X-ray. High levels of pro-inflammatory cytokines and cf mtDNA were also detected. In silico identification of potential targets of these miRNAs was conducted using MIENTURNET. VDAC1 and ALOX5 were identified as possible targets. Our study identified promising biomarkers such as miRNAs and cf mtDNA that showed a dose-dependent effect of X-ray exposure.

Development of an Ex Vivo Functional Assay for Prediction of Irradiation Related Toxicity in Healthy Oral Mucosa Tissue.

Radiotherapy in the head-and-neck area is one of the main curative treatment options. However, this comes at the cost of varying levels of normal tissue toxicity, affecting up to 80% of patients. Mucositis can cause pain, weight loss and treatment delays, leading to worse outcomes and a decreased quality of life. Therefore, there is an urgent need for an approach to predicting normal mucosal responses in patients prior to treatment. We here describe an assay to detect irradiation responses in healthy oral mucosa tissue. Mucosa specimens from the oral cavity were obtained after surgical resection, cut into thin slices, irradiated and cultured for three days. Seven samples were irradiated with X-ray, and three additional samples were irradiated with both X-ray and protons. Healthy oral mucosa tissue slices maintained normal morphology and viability for three days. We measured a dose-dependent response to X-ray irradiation and compared X-ray and proton irradiation in the same mucosa sample using standardized automated image analysis. Furthermore, increased levels of inflammation-inducing factors-major drivers of mucositis development-could be detected after irradiation. This model can be utilized for investigating mechanistic aspects of mucositis development and can be developed into an assay to predict radiation-induced toxicity in normal mucosa.

Chromosomal damage, gene expression and alternative transcription in human lymphocytes exposed to mixed ionizing radiation as encountered in space.

Astronauts travelling in space will be exposed to mixed beams of particle radiation and photons. Exposure limits that correspond to defined cancer risk are calculated by multiplying absorbed doses by a radiation-type specific quality factor that reflects the biological effectiveness of the particle without considering possible interaction with photons. We have shown previously that alpha radiation and X-rays may interact resulting in synergistic DNA damage responses in human peripheral blood lymphocytes but the level of intra-individual variability was high. In order to assess the variability and validate the synergism, blood from two male donors was drawn at 9 time points during 3 seasons of the year and exposed to 0-2 Gy of X-rays, alpha particles or 1:1 mixture of both (half the dose each). DNA damage response was quantified by chromosomal aberrations and by mRNA levels of 3 radiation-responsive genes FDXR, CDKN1A and MDM2 measured 24 h post exposure. The quality of response in terms of differential expression of alternative transcripts was assessed by using two primer pairs per gene. A consistently higher than expected effect of mixed beams was found in both donors for chromosomal aberrations and gene expression with some seasonal variability for the latter. No synergy was detected for alternative transcription.

Evaluation of the protective effect of coenzyme Q10 against x-ray irradiation-induced ovarian injury.

AIM: This study focused on the anti-oxidant and anti-apoptotic effects of CoQ10 in ovaries exposed to pelvic radiation. METHODS: Thirty-two female rats were randomly assigned into four groups. Group I (control group), Group II: Only 2 Gy pelvic x-ray irradiation (IR) was administered as a single fractioned dose. Group III: 30 mg/kg CoQ10 was administered by oral gavage +2 Gy pelvic IR. Group IV: 150 mg/kg CoQ10 was administered by oral gavage +2 Gy pelvic IR. CoQ10 treatment was started 7 days before pelvic IR and completed 7 days later. The rats in Group III and IV were treated with CoQ10 for a total of 14 days. RESULTS: Histopathological analysis showed severe damage to the ovarian tissue in the radiation group, while both doses of CoQ10 showed normal histological structure. Likewise, while there was a high level of staining in the IR group for necrosis and apoptosis, the CoQ10 treated ones were like the control group. Tissue Malondialdehyde (MDA) levels were like the control group in the low-dose CoQ10 group, while the MDA levels of the high dose CoQ10 group were similar to the radiation group. CONCLUSION: Usage of low-dose CoQ10 has a radioprotective effect on radiation-induced ovarian damage. Although the use of high doses is morphologically radioprotective, no antioxidative effect was observed in the biochemical evaluation.

Association of low-dose ionising radiation with site-specific solid cancers: Chinese medical X-ray workers cohort study, 1950-1995.

BACKGROUND: The dose-response relationship between cancers and protracted low-dose rate exposure to ionising radiation is still uncertain. This study aims to estimate quantified relationships between low-dose radiation exposures and site-specific solid cancers among Chinese medical X-ray workers. METHODS: This cohort study included 27 011 individuals who were employed at major hospitals in 24 provinces in China from 1950 to 1980 and had been exposed to X-ray equipment, and a control group of 25 782 physicians who were not exposed to X-ray equipment. Person-years of follow-up were calculated from the year of employment to the date of the first diagnosis of cancer or the end of follow-up, whichever occurred first. All cancers were obtained from medical records during 1950-1995. This study used Poisson regression models to estimate the excess relative risk (ERR) and excess absolute risk (EAR) for incidence of site-specific solid cancers associated with cumulative dose. RESULTS: 1643 solid cancers were developed, the most common being lung, liver and stomach cancer. Among X-ray workers, the average cumulative colon dose was 0.084 Gy. We found a positive relationship between cumulative organ-specific dose and liver (ERR/Gy=1.48; 95% CI 0.40 to 2.83), oesophagus (ERR/Gy=18.1; 95% CI 6.25 to 39.1), thyroid (ERR/Gy=2.96; 95% CI 0.44 to 8.18) and non-melanoma skin cancers (ERR/Gy=7.96; 95% CI 2.13 to 23.12). We found no significant relationship between cumulative organ-specific doses and other cancers. Moreover, the results showed a statistically significant EAR for liver, stomach, breast cancer (female), thyroid and non-melanoma skin cancers. CONCLUSIONS: These findings provided more useful insights into the risks of site-specific cancers from protracted low-dose rate exposure to ionising radiation.

PVP solid dispersions containing Poloxamer 407 or TPGS for the improvement of ursolic acid release

Abstract Solid dispersions (SDs) of ursolic acid (UA) were developed using polyvinylpyrrolidone K30 (PVP K30) in combination with non-ionic surfactants, such as D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) or poloxamer 407 (P407) with the aim of enhancing solubility and in vitro release of the UA. SDs were investigated using a 24 full factorial design, subsequently the selected formulations were characterized for water solubility, X-ray diffractometry (XRD), differential scanning calorimetry (DSC), particle diameter, scanning electron microscopy, drug content, physical-chemical stability and in vitro release profile. SDs showed higher UA water-solubility than physical mixtures (PMs), which was attributed by transition of the drug from crystalline to amorphous or molecular state in the SDs, as indicated by XRD and DSC analyses. SD1 (with P407) and SD2 (with TPGS) were chosen for further investigation because they had higher drug load. SD1 proved to be more stable than SD2, revealing that P407 contributed to ensure the stability of the UA. Furthermore, SD1 and SD2 increased UA release by diffusion and swelling-controlled transport, following the Weibull model. Thus, solid dispersions obtained with PVP k-30 and P407 proved to be advantageous to enhance aqueous solubility and stability of UA.

Radioprotective effects of N-acetylcysteine on rats' brainstem following megavoltage X-irradiations.

PURPOSE: This study aimed to determine the radioprotective effect of N-acetylcysteine (NAC) on the radiation-induced oxidative stress (OS) in the rats' brainstem. MATERIALS AND METHODS: Eighty rats in four identical groups, including vehicle control (VC), irradiation alone (RAD), irradiation with 1 g/kg of NAC treatment (RAN), and NAC treatment without radiation (NAC) were used. Whole-brain irradiation was performed with a single dose of 25 Gy. The rats received the treatments via intraperitoneal (IP) injection 1 h before the irradiation process. Nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (TAC), and glutathione peroxidase (GPx) were measured in the rats' brainstem and compared between the groups. Furthermore, the pathological study was performed to assess tissue damage after 24 h, 72 h, and 5 days of irradiation. RESULTS: The levels of NO and MDA in the brainstem tissue for the RAD group were 60.37 ± 3.35 µmol/L and 45.10 ± 2.48 µM, respectively, which were higher than those of VC group (NO: 30.41 ± 1.83 µmol/L; MDA: 31.02 ± 1.71 µM). The level of SOD, CAT, TAC, and GPx declined in the RAD compared to the VC group. Pre-treatment with NAC decreased the level of NO and MDA and also enhanced the antioxidant activities. The greatest pathological changes in the rats' brainstems were seen in RAD animals compared to the VC group at 24 h, 72 h, and 5 days. Furthermore, the pathological changes were not observed in the NAC group in all the assessed times. CONCLUSION: Based on the results, NAC can decrease the irradiation-induced oxidative stress and pathology damages in the rats' brainstem. It can be concluded that NAC can be an appropriate radioprotection candidate for the human brainstem.

Differential molecular response in mice and human thymocytes exposed to a combined-dose radiation regime.

In the quest for more effective radiation treatment options that can improve both cell killing and healthy tissue recovery, combined radiation therapies are lately in the spotlight. The molecular response to a combined radiation regime where exposure to an initial low dose (priming dose) of ionizing radiation is administered prior to a subsequent higher radiation dose (challenging dose) after a given latency period have not been thoroughly explored. In this study we report on the differential response to either a combined radiation regime or a single challenging dose both in mouse in vivo and in human ex vivo thymocytes. A differential cell cycle response including an increase in the subG1 fraction on cells exposed to the combined regime was found. Together with this, a differential protein expression profiling in several pathways including cell cycle control (ATM, TP53, p21CDKN1A), damage response (γH2AX) and cell death pathways such as apoptosis (Cleaved Caspase-3, PARP1, PKCδ and H3T45ph) and ferroptosis (xCT/GPX4) was demonstrated. This study also shows the epigenetic regulation following a combined regime that alters the expression of chromatin modifiers such as DNMTs (DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3L) and glycosylases (MBD4 and TDG). Furthermore, a study of the underlying cellular status six hours after the priming dose alone showed evidence of retained modifications on the molecular and epigenetic pathways suggesting that the priming dose infers a "radiation awareness phenotype" to the thymocytes, a sensitization key to the differential response seen after the second hit with the challenging dose. These data suggest that combined-dose radiation regimes could be more efficient at making cells respond to radiation and it would be interesting to further investigate how can these schemes be of use to potential new radiation therapies.

Formulation, Optimization, in vitro and in-vivo evaluation of levofloxacin hemihydrate Floating Tablets

Abstract The objective of the present investigation was to design, optimize and characterize the gastro retentive floating levofloxacin tablets and perform in-vivo evaluation using radiographic imaging. The floating tablets were prepared by using polymers i.e hydroxy propyl methyl cellulose (HPMC-K4M) and carbopol-940 individually and in combination by nonaquous granulation method. All the Formulations were evaluated for swelling index (S.I), floating behavior and in-vitro drug release kinetics. The compatibility study of levofloxacin with other polymers was investigated by FTIR, DSC, TGA and XRD. Results from FTIR and DSC revealed no chemical interaction amongst the formulation components. The optimized formulation (F11) showed floating lag time (FLT), total floating time (TFT) swelling index (S.I) of 60 sec, >16h and approximately 75 %, respectively. Moreover, F11 showed zero order levofloxacin release in simulated gastric fluid over the period of 6 h. X-ray studies showed that total buoyancy time was able to delay the gastric emptying of levofloxacin floating tablets in rabbits for more than 4 hours. In conclusion the optimized formulation (F11) can be used for the sustained delivery of levofloxacin for the treatment of peptic ulcer.

Physicochemical characterization and cosmetic applications of Passiflora nitida Kunth leaf extract

Abstract Passiflora nitida Kunth, an Amazonian Passiflora species, is little studied, although the specie's high biological potential. Herein the plant's pharmacognostic characterization, extract production, antioxidant potential evaluation, and application of this extract in cosmetic products is reported. The physical chemical parameters analyzed were particle size by sieve analysis, loss through drying, extractive yield, total ash content, laser granulometry, specific surface area and pore diameter (SBET), differential scanning calorimetry, thermogravimetry (TG), and wave dispersive X-Ray fluorescence (WDXRF). Total phenol/flavonoid content, LC-MS/MS analysis, DPPH and ABTS antioxidant radical assays, cytotoxicity, melanin, and tyrosinase inhibition in melanocytes test provided evidence to determine the content of the major constituent. P. nitida dry extract provided a fine powder with mesopores determined by SBET, with the TG curve showing five stages of mass loss. The antioxidant potential ranged between 23.5-31.5 mg∙mL-1 and tyrosinase inhibition between 400-654 µg∙mL-1. The species presented an antimelanogenic effect and an inhibitory activity of cellular tyrosinase (26.6%) at 25 µg/mL. The LC-MS/MS analysis of the spray-dried extract displayed the main and minor phenolic compounds constituting this sample. The results indicate that P. nitida extract has promising features for the development of cosmetic formulations

Circulating tRNA-Derived Small RNAs as Novel Radiation Biomarkers of Heavy Ion, Proton and X-ray Exposure.

The effective and minimally invasive radiation biomarkers are valuable for exposure scenarios in nuclear accidents or space missions. Recent studies have opened the new sight of circulating small non-coding RNA (sncRNA) as radiation biomarkers. The tRNA-derived small RNA (tsRNA) is a new class of sncRNA. It is more abundant than other kinds of sncRNAs in extracellular vesicles or blood, presenting great potential as promising biomarkers. However, the circulating tsRNAs in response to ionizing radiation have not been reported. In this research, Kunming mice were total-body exposed to 0.05-2 Gy of carbon ions, protons, or X-rays, and the RNA sequencing was performed to profile the expression of sncRNAs in serum. After conditional screening and validation, we firstly identified 5 tsRNAs including 4 tRNA-related fragments (tRFs) and 1 tRNA half (tiRNA) which showed a significant level decrease after exposure to three kinds of radiations. Moreover, the radiation responses of these 5 serum tsRNAs were reproduced in other mouse strains, and the sequences of them could be detected in serum of humans. Furthermore, we developed multi-factor models based on tsRNA biomarkers to indicate the degree of radiation exposure with high sensitivity and specificity. These findings suggest that the circulating tsRNAs can serve as new minimally invasive biomarkers and can make a triage or dose assessment from blood sample collection within 4 h in exposure scenarios.

Effect of X-ray exposure during hysterosalpingography on capabilities of female germ cells.

PURPOSE: To elucidate the effect of X-ray exposure during hysterosalpingography (HSG) on subsequent laboratory outcomes in in vitro fertilization (IVF). METHODS: A total of 1458 oocytes, consisting of 990 oocytes retrieved from 70 women (89 cycles) who underwent HSG prior to IVF and 468 oocytes from 45 women (57 cycles) who underwent IVF without HSG, were evaluated for their retrieval number, maturity, fertilization, and development post fertilization. X-ray exposure during HSG was recorded as reference air kerma (RAK) (mGy). Subjects were stratified according to the amount of RAK (Nil: IVF without HSG, L-RAK: RAK < 16.23, mH-RAK: RAK ≥ 16.23). The number of oocytes retrieved, oocyte maturation, fertilization, and embryo development was compared among 3 groups. Further, multivariate analyses were performed to investigate the effect of X-ray exposure on laboratory outcomes in IVF. RESULTS: There was a statistically significant difference in the fertilization rate among 3 groups (Nil: 71.6%, L-RAK: 80.5%, mH-RAK: 78.3%). The good-quality blastocyst rate in mH-RAK (46.2%) was significantly higher than L-RAK (35.3%) and Nil (32.4%). Multivariate analyses revealed that X-ray exposure was associated with higher fertilization, higher blastocyst development, and higher good-quality blastocyst development rates with adjustment for patient age, BMI, ovarian stimulation types, and fertilization methods. Association between X-ray exposure and the number of oocytes retrieved, and oocyte maturation was not confirmed. CONCLUSIONS: The present study suggests that X-ray exposure of the female reproductive organs during HSG could enhance the potential of oocytes rather than adversely.

Monte Carlo Simulation of Double-Strand Break Induction and Conversion after Ultrasoft X-rays Irradiation.

This paper estimates the yields of DNA double-strand breaks (DSBs) induced by ultrasoft X-rays and uses the DSB yields and the repair outcomes to evaluate the relative biological effectiveness (RBE) of ultrasoft X-rays. We simulated the yields of DSB induction and predicted them in the presence and absence of oxygen, using a Monte Carlo damage simulation (MCDS) software, to calculate the RBE. Monte Carlo excision repair (MCER) simulations were also performed to calculate the repair outcomes (correct repairs, mutations, and DSB conversions). Compared to 60Co γ-rays, the RBE values for ultrasoft X-rays (titanium K-shell, aluminum K-shell, copper L-shell, and carbon K-shell) for DSB induction were respectively 1.3, 1.9, 2.3, and 2.6 under aerobic conditions and 1.3, 2.1, 2.5, and 2.9 under a hypoxic condition (2% O2). The RBE values for enzymatic DSBs were 1.6, 2.1, 2.3, and 2.4, respectively, indicating that the enzymatic DSB yields are comparable to the yields of DSB induction. The synergistic effects of DSB induction and enzymatic DSB formation further facilitate cell killing and the advantage in cancer treatment.

An Enriched Environment Alters DNA Repair and Inflammatory Responses After Radiation Exposure.

After the Fukushima Daiichi Nuclear Power Plant accident, there is growing concern about radiation-induced carcinogenesis. In addition, living in a long-term shelter or temporary housing due to disasters might cause unpleasant stress, which adversely affects physical and mental health. It's been experimentally demonstrated that "eustress", which is rich and comfortable, has beneficial effects for health using mouse models. In a previous study, mice raised in the enriched environment (EE) has shown effects such as suppression of tumor growth and enhancement of drug sensitivity during cancer treatment. However, it's not yet been evaluated whether EE affects radiation-induced carcinogenesis. Therefore, to evaluate whether EE suppresses a radiation-induced carcinogenesis after radiation exposure, in this study, we assessed the serum leptin levels, radiation-induced DNA damage response and inflammatory response using the mouse model. In brief, serum and tissues were collected and analyzed over time in irradiated mice after manipulating the raising environment during the juvenile or adult stage. To assess the radiation-induced DNA damage response, we performed immunostaining for phosphorylated H2AX which is a marker of DNA double-strand break. Focusing on the polarization of macrophages in the inflammatory reaction that has an important role in carcinogenesis, we performed analysis using tissue immunofluorescence staining and RT-qPCR. Our data confirmed that EE breeding before radiation exposure improved the responsiveness to radiation-induced DNA damage and basal immunity, further suppressing the chronic inflammatory response, and that might lead to a reduction of the risk of radiation-induced carcinogenesis.

Assays of genotoxic damage in peripheral blood lymphocytes of individuals occupationally exposed to different x-ray systems in hospital radiology departments.

We obtained peripheral blood lymphocyte samples from individuals occupationally exposed to X-rays in hospital radiology departments that use different radiology systems: analog film (AF), computerized radiology (CR), or digital radiology (DR). The micronucleus test (MNT) and comet assay were performed on the samples. Micronucleus cell counts (means vs. controls, i.e., individuals not occupationally exposed to ionizing radiation) were as follows: AF, 1.96 ± 0.21 vs 1.2 ± 0.25; CR, 1.89 ± 0.15 vs 1.31 ± 0.36; and DR, 1.75 ± 0.11 vs 1.59 ± 0.32. For the comet assay, damage scores were as follows; AF, 0.84 ± 0.22 vs 0.47 ± 0.04; CR, 0.64 ± 0.26 vs 0.43 ± 0.04; and DR, 0.56 ± 0.19 vs 0.49 ± 0035. These findings were consistent with cytogenetic damage due to radiation exposure.

Mast1 mediates radiation-induced gastric injury via the P38 MAPK pathway.

Radiation-induced gastric injury is a serious adverse effect and reduces the efficacy of radiotherapy treatment. However, the mechanisms underlying radiation-induced stomach injury remain unclear. Here, mouse stomach and gastric epithelial cells were irradiated with different doses of X-ray radiation. The results showed that radiation induced gastric injury in vivo and in vitro. Differentially expressed functional mRNAs in irradiation-induced gastric tissues were screened from the Gene Expression Omnibus (GEO) database. We found that the expression of microtubule-associated serine/threonine kinase 1 (Mast1) was downregulated in mouse gastric tissues and gastric epithelial cells after irradiation. Furthermore, functional assays showed that knockdown of Mast1 inhibited growth and promoted apoptosis in gastric epithelial cells, while overexpression of Mast1 protected gastric epithelial cells from radiation damage. Mechanistically, Mast1 negatively regulated radiation-induced injury in gastric epithelial cells by inhibiting the activation of P38. The apoptosis caused by knockdown of Mast1 in gastric epithelial cells could be partially reversed by the P38 inhibitor SB203580. Moreover, data from several gastric cancer cell lines and online databases revealed that Mast1 was not involved in the development of gastric cancer. Collectively, our findings demonstrated that Mast1 is essential for radiation-induced gastric injury, providing a promising prognostic and therapeutic target.

Radiation­induced dysfunction of energy metabolism in the heart results in the fibrosis of cardiac tissues.

Thoracic radiotherapy increases the risk of radiation­induced heart damage (RIHD); however, the molecular mechanisms underlying these changes are not fully understood. The aim of the present study was to investigate the effects of radiation on the mouse heart using high­throughput proteomics. Male C57BL/6J mice were used to establish a model of RIHD by exposing the entire heart to 16 Gy high­energy X­rays, and cardiac injuries were verified using a cardiac echocardiogram, as well as by measuring serum brain natriuretic peptide levels and conducting H&E and Masson staining 5 months after irradiation. Proteomics experiments were performed using the heart apex of 5­month irradiated mice and control mice that underwent sham­irradiation. The most significantly differentially expressed proteins were enriched in 'cardiac fibrosis' and 'energy metabolism'. Next, the cardiac fibrosis and changes to energy metabolism were confirmed using immunohistochemistry staining and western blotting. Extracellular matrix proteins, such as collagen type 1 α 1 chain, collagen type III α 1 chain, vimentin and CCCTC­binding factor, along with metabolism­related proteins, such as fatty acid synthase and solute carrier family 25 member 1, exhibited upregulated expression following exposure to ionizing radiation. Additionally, the myocardial mitochondria inner membranes were injured, along with a decrease in ATP levels and the accumulation of lactic acid in the irradiated heart tissues. These results suggest that the high doses of ionizing radiation used lead to structural remodeling, functional injury and fibrotic alterations in the mouse heart. Radiation­induced mitochondrial damage and metabolic alterations of the cardiac tissue may thus be a pathogenic mechanism of RIHD.

Decreased mitochondrial membrane potential is an indicator of radioresistant cancer cells.

AIMS: To overcome radioresistant cancer cells, clinically relevant radioresistant (CRR) cells were established. To maintain their radioresistance, CRR cells were exposed 2 Gy/day of X-rays daily (maintenance irradiation: MI). To understand whether the radioresistance induced by X-rays was reversible or irreversible, the difference between CRR cells and those without MI for a year (CRR-NoIR cells) was investigated by the mitochondrial function as an index. MAIN METHODS: Radiation sensitivity was determined by modified high density survival assay. Mitochondrial membrane potential (Δψm) was determined by 5,5',6,6'-tetrachloro-1,1', tetraethylbenzimidazolocarbo-cyanine iodide (JC-1) staining. Rapid Glucose-Galactose assay was performed to determine the shift in their energy metabolism from aerobic glycolysis to oxidative phosphorylation in CRR cells. Involvement of prohibitin-1 (PHB1) in Δψm was evaluated by knockdown of PHB1 gene followed by real-time PCR. KEY FINDINGS: CRR cells that exhibited resistant to 2 Gy/day X-ray lost their radioresistance after more than one year of culture without MI for a year. In addition, CRR cells lost their radioresistance when the mitochondria were activated by galactose. Furthermore, Δψm were increased and PHB1 expression was down-regulated, in the process of losing their radioresistance. SIGNIFICANCE: Our finding reveled that tune regulation of mitochondrial function is implicated in radioresistance phenotype of cancer cells. Moreover, as our findings indicate, though further studies are required to clarify the precise mechanisms underlying cancer cell radioresistance, radioresistant cells induced by irradiation and cancer stem cells that are originally radioresistant should be considered separately, the radioresistance of CRR cells is reversible.

Effects of radiation damage and inelastic scattering on single-particle imaging of hydrated proteins with an X-ray Free-Electron Laser.

We present a computational case study of X-ray single-particle imaging of hydrated proteins on an example of 2-Nitrogenase-Iron protein covered with water layers of various thickness, using a start-to-end simulation platform and experimental parameters of the SPB/SFX instrument at the European X-ray Free-Electron Laser facility. The simulations identify an optimal thickness of the water layer at which the effective resolution for imaging the hydrated sample becomes significantly higher than for the non-hydrated sample. This effect is lost when the water layer becomes too thick. Even though the detailed results presented pertain to the specific sample studied, the trends which we identify should also hold in a general case. We expect these findings will guide future single-particle imaging experiments using hydrated proteins.