High-Dose Irradiation Inhibits Motility and Induces Autophagy in Caenorhabditis elegans.

Radiation damages many cellular components and disrupts cellular functions, and was previously reported to impair locomotion in the model organism Caenorhabditis elegans. However, the response to even higher doses is not clear. First, to investigate the effects of high-dose radiation on the locomotion of C. elegans, we investigated the dose range that reduces whole-body locomotion or leads to death. Irradiation was performed in the range of 0-6 kGy. In the crawling analysis, motility decreased after irradiation in a dose-dependent manner. Exposure to 6 kGy of radiation affected crawling on agar immediately and caused the complete loss of motility. Both γ-rays and carbon-ion beams significantly reduced crawling motility at 3 kGy. Next, swimming in buffer was measured as a motility index to assess the response over time after irradiation and motility similarly decreased. However, swimming partially recovered 6 h after irradiation with 3 kGy of γ-rays. To examine the possibility of a recovery mechanism, in situ GFP reporter assay of the autophagy-related gene lgg-1 was performed. The fluorescence intensity was stronger in the anterior half of the body 7 h after irradiation with 3 kGy of γ-rays. GFP::LGG-1 induction was observed in the pharynx, neurons along the body, and the intestine. Furthermore, worms were exposed to region-specific radiation with carbon-ion microbeams and the trajectory of crawling was measured by image processing. Motility was lower after anterior-half body irradiation than after posterior-half body irradiation. This further supported that the anterior half of the body is important in the locomotory response to radiation.

Histone Deacetylase Inhibitors Sensitize Murine B16F10 Melanoma Cells to Carbon Ion Irradiation by Inducing G1 Phase Arrest.

Epigenetic processes, in addition to genetic abnormalities, play a critical role in refractory malignant diseases and cause the unresponsiveness to various chemotherapeutic regimens and radiotherapy. Herein we demonstrate that histone deacetylase inhibitors (HDACis) can be used to sensitize malignant melanoma B16F10 cells to carbon ion irradiation. The cells were first treated with HDACis (romidepsin [FK228, depsipeptide], trichostatin A [TSA], valproic acid [VPA], and suberanilohydroxamic acid [SAHA, vorinostat]) and were then exposed to two types of radiation (carbon ions and gamma-rays). We found that HDACis enhanced the radiation-induced apoptosis and suppression of clonogenicity that was induced by irradiation, having a greater effect with carbon ion irradiation than with gamma-rays. Carbon ion irradiation and the HDACi treatment induced G2/M and G0/G1 cell cycle arrest, respectively. Thus, it is considered that HDACi treatment enhanced the killing effects of carbon ion irradiation against melanoma cells by inducing the arrest of G1 phase cells, which are sensitive to radiation due to a lack of DNA homologous recombination repair. Based on these findings, we propose that pretreatment with HDACis as radiosensitizers to induce G1 arrest combined with carbon ion irradiation may have clinical efficacy against refractory cancer.

Early detection of vulnerable atherosclerotic plaque for risk reduction of acute aortic rupture and thromboemboli and atheroemboli using non-obstructive angioscopy.

The mortality rate due to rupture of aortic dissection and aortic aneurysm is approximately 90%. Acute aortic rupture can be fatal prior to hospitalization and has proven difficult to diagnose correctly or predict. The in-hospital mortality rate of ruptured aortic aneurysm ranges from 53 to 66%. Emergency surgical and endovascular treatments are the only options for ruptured aortic dissection and aortic aneurysm. No method of systematic early detection or inspection of vessel injury is available at the prevention stage. Regardless of the improvement in many imaging modalities, aortic diameter has remained a major criterion for recommending surgery in diagnosed patients. Previous reports have suggested a relationship between vulnerable plaque and atherosclerotic aortic aneurysm. Non-obstructive angioscopy is a new method for evaluating intimal injury over the whole aorta. It has been used to identify many advanced atherosclerotic plaques that were missed on traditional imaging modalities before aneurysm formation. Non-obstructive angioscopy has shown that atherosclerosis of the aorta begins before that of the coronary artery, which had been noted on autopsy "in vivo". Strong or repetitive aortic injuries might cause sudden aortic disruption. Aortic atheroma is also a risk factor of stroke and perivascular embolism. Detecting aortic vulnerable atherosclerotic plaque on non-obstructive angioscopy may not only clarify the pathogenesis of acute aortic rupture and "aortogenic" thromboemboli and atheroemboli but also play a role in the pre-emptive medicine.

Quadricuspid aortic valve complicated with infective endocarditis: report of a case.

Congenital quadricuspid aortic valve is a rare cardiac malformation with an unknown risk of infective endocarditis. We report a case of quadricuspid aortic valve complicated with infective endocarditis. A 53-year-old Japanese woman was hospitalized with leg edema and a fever of unknown origin. Corynebacterium striatum was detected in the blood culture. Echocardiography demonstrated a quadricuspid aortic valve with vegetation and severe functional regurgitation. The condition was diagnosed as a quadricuspid aortic valve with infective endocarditis, for which surgery was performed. The quadricuspid aortic valve had three equal-sized cusps and one smaller cusp (type B according to Hurwitz classification). We dissected the vegetation and infectious focus and implanted a mechanical valve. Following the case report, we review the literature.

Radiation-quality-dependent bystander cellular effects induced by heavy-ion microbeams through different pathways.

We investigated the radiation-quality-dependent bystander cellular effects using heavy-ion microbeams with different ion species. The heavy-ion microbeams were produced in Takasaki Ion Accelerators for Advanced Radiation Application, National Institutes for Quantum Science and Technology. Carbon (12C5+, 220 MeV), neon (20Ne7+, 260 MeV) and argon (40Ar13+, 460 MeV) ions were used as the microbeams, collimating the beam size with a diameter of 20 µm. After 0.5 and 3 h of irradiation, the surviving fractions (SFs) are significantly lower in cells irradiated with carbon ions without a gap-junction inhibitor than those irradiated with the inhibitor. However, the same SFs with no cell killing were found with and without the inhibitor at 24 h. Conversely, no cell-killing effect was observed in argon-ion-irradiated cells at 0.5 and 3 h; however, significantly low SFs were found at 24 h with and without the inhibitor, and the effect was suppressed using vitamin C and not dimethyl sulfoxide. The mutation frequency (MF) in cells irradiated with carbon ions was 8- to 6-fold higher than that in the unirradiated control at 0.5 and 3 h; however, no mutation was observed in cells treated with the gap-junction inhibitor. At 24 h, the MFs induced by each ion source were 3- to 5-fold higher and the same with and without the inhibitor. These findings suggest that the bystander cellular effects depend on the biological endpoints, ion species and time after microbeam irradiations with different pathways.

Theoretical and evolutionary parameter tuning of neural oscillators with a double-chain structure for generating rhythmic signals.

A neural oscillator with a double-chain structure is one of the central pattern generator models used to simulate and understand rhythmic movements in living organisms. However, it is difficult to reproduce desired rhythmic signals by tuning an enormous number of parameters of neural oscillators. In this study, we propose an automatic tuning method consisting of two parts. The first involves tuning rules for both the time constants and the amplitude of the oscillatory outputs based on theoretical analyses of the relationship between parameters and outputs of the neural oscillators. The second involves an evolutionary tuning method with a two-step genetic algorithm (GA), consisting of a global GA and a local GA, for tuning parameters such as neural connection weights that have no exact tuning rule. Using numerical experiments, we confirmed that the proposed tuning method could successfully tune all parameters and generate sinusoidal waves. The tuning performance of the proposed method was less affected by factors such as the number of excitatory oscillators or the desired outputs. Furthermore, the proposed method was applied to the parameter-tuning problem of some types of artificial and biological wave reproduction and yielded optimal parameter values that generated complex rhythmic signals in Caenorhabditis elegans without trial and error.

Ionising irradiation alters the dynamics of human long interspersed nuclear elements 1 (LINE1) retrotransposon.

It is important to identify the mechanism by which ionising irradiation induces various genomic alterations in the progeny of surviving cells. Ionising irradiation activates mobile elements like retrotransposons, although the mechanism of its phenomena consisting of transcriptions and insertions of the products into new sites of the genome remains unclear. In this study, we analysed the effects of sparsely ionising X-rays and densely ionising carbon-ion beams on the activities of a family of active retrotransposons, long interspersed nuclear elements 1 (L1). We used the L1/reporter knock-in human glioma cell line, NP-2/L1RP-enhanced GFP (EGFP), that harbours full-length L1 tagged with EGFP retrotransposition detection cassette (L1RP-EGFP) in the chromosomal DNA. X-rays and carbon-ion beams similarly increased frequencies the transcription from L1RP-EGFP and its retrotransposition. Short-sized de novo L1RP-EGFP insertions with 5'-truncation were induced by X-rays, while full-length or long-sized insertions (>5 kb, containing ORF1 and ORF2) were found only in cell clones irradiated by the carbon-ion beams. These data suggest that X-rays and carbon-ion beams induce different length of de novo L1 insertions, respectively. Our findings thus highlight the necessity to investigate the mechanisms of mutations caused by transposable elements by ionising irradiation.

Primary echocardiographic results of the Carpentier-Edwards Perimount Magna.

PURPOSE: The aim of this study was to investigate the primary echocardiographic results of aortic valve replacement using 21- and 19-mm Carpentier-Edwards Perimount Magna bioprosthesis aortic xenografts in patients with small aortic annulus. METHODS: Twenty patients (mean body surface area 1.63 ± 0.16 m(2)) underwent aortic valve replacement between June 2008 and December 2009. Eight and 12 patients received 21- and 19-mm Magna bioprostheses, respectively. After 12 months, hemodynamic data were obtained by echocardiography to estimate the prosthesis-patient mismatch. RESULTS: At follow-up, significant decreases in peak and mean left ventricular aortic pressure gradients were observed in the 12 patients with aortic stenosis (P < 0.05). Regression of the left ventricular mass was observed in all the patients (P < 0.05). The mean measured effective orifice area (EOA) and EOA index (EOAI) were 1.61 ± 0.28 cm(2) and 0.99 ± 0.16 cm(2)/m(2), respectively. Prosthesis-patient mismatch (EOAI ≤0.85) was documented in three patients. CONCLUSION: The primary echocardiographic findings suggested that the hemodynamic performance of the 19- and 21-mm Carpentier-Edwards Perimount Magna bioprostheses was satisfactory in the patients with a small aortic annulus.

RNA sequencing data for gamma radiation response in the extremotolerant tardigrade Ramazzottius varieornatus.

Tardigrades are microscopic animals of which terrestrial species are capable of tolerating extreme environments by entering a desiccated ametabolic state known as anhydrobiosis. Intriguingly, they survive high dosage gamma rays (>4,000 Gy), possibly through a mechanism known as cross-tolerance. We hypothesized that anhydrobiosis genes are also regulated during cross-tolerance, thus we submitted Ramazzottius varieornatus to 500 Gy 60Co gamma-ray and conducted time-course low-input RNA-Seq. The gene expression was quantified with RSEM and differential expression was determined with DEseq2. Differentially expressed genes were submitted to gene ontology enrichment analysis with GOStat. The transcriptome dynamically shifted nine hours post-exposure.

Enhanced micronucleus formation in the descendants of gamma-ray-irradiated tobacco cells: evidence for radiation-induced genomic instability in plant cells.

Ionizing radiation-induced genomic instability has been documented in various end points such as chromosomal aberrations and mutations, which arises in the descendants of irradiated mammalian or yeast cells many generations after the initial insult. This study aimed at addressing radiation-induced genomic instability in higher plant tobacco cells. We thus investigated micronucleus (MN) formation and cell proliferation in tobacco cells irradiated with gamma-rays and their descendants. In gamma-irradiated cells, cell cycle was arrested at G2/M phase at around 24 h post-irradiation but released afterward. In contrast, MN frequency peaked at 48 h post-irradiation. Almost half of 40 Gy-irradiated cells had MN at 48 h post-irradiation, but proliferated as actively as sham-irradiated cells up to 120 h post-irradiation. Moreover, the descendants that have undergone at least 22 generations after irradiation still showed a two-fold MN frequency compared to sham-irradiated cells. This is the direct evidence for radiation-induced genomic instability in tobacco cells.

Heavy ion irradiation induces autophagy in irradiated C2C12 myoblasts and their bystander cells.

Autophagy is one of the major processes involved in the degradation of intracellular materials. Here, we examined the potential impact of heavy ion irradiation on the induction of autophagy in irradiated C2C12 mouse myoblasts and their non-targeted bystander cells. In irradiated cells, ultrastructural analysis revealed the accumulation of autophagic structures at various stages of autophagy (i.e. phagophores, autophagosomes and autolysosomes) within 20 min after irradiation. Multivesicular bodies (MVBs) and autolysosomes containing MVBs (amphisomes) were also observed. Heavy ion irradiation increased the staining of microtubule-associated protein 1 light chain 3 and LysoTracker Red (LTR). Such enhanced staining was suppressed by an autophagy inhibitor 3-methyladenine. In addition to irradiated cells, bystander cells were also positive with LTR staining. Altogether, these results suggest that heavy ion irradiation induces autophagy not only in irradiated myoblasts but also in their bystander cells.

Insufficient membrane fusion in dysferlin-deficient muscle fibers after heavy-ion irradiation.

Recently, SJL/J mice have been used as an animal model in studies of dysferlinopathy, a spectrum of muscle diseases caused by defects in dysferlin protein. In this study we irradiated muscle fibers isolated from skeletal muscle of SJL/J mice with heavy-ion microbeam, and the ultrastructural changes were observed by electron microscopy. The plasma membrane of heavy-ion beam irradiated areas showed irregular protrusions and invaginations. Disruption of sarcomeric structures and the enhancement of autophagy were also observed. In addition, many vesicles of varying size and shape were seen to be accumulated just beneath the plasma membrane. This finding further supports the recent hypothesis that dysferlin functions as a membrane fusion protein in the wound healing system of plasma membrane, and that the defect in dysferlin causes insufficient membrane fusion resulting in accumulation of vesicles.

Heavy-ion-induced bystander killing of human lung cancer cells: role of gap junctional intercellular communication.

The aim of the present study was to clarify the mechanisms of cell death induced by heavy-ion irradiation focusing on the bystander effect in human lung cancer A549 cells. In microbeam irradiation, each of 1, 5, and 25 cells under confluent cell conditions was irradiated with 1, 5, or 10 particles of carbon ions (220 MeV), and then the surviving fraction of the population was measured by a clonogenic assay in order to investigate the bystander effect of heavy-ions. In this experiment, the limited number of cells (0.0001-0.002%, 5-25 cells) under confluent cell conditions irradiated with 5 or 10 carbon ions resulted in an exaggerated 8-14% increase in cell death by clonogenic assay. However, these overshooting responses were not observed under exponentially growing cell conditions. Furthermore, these responses were inhibited in cells treated with an inhibitor of gap junctional intercellular communication (GJIC), whereas they were markedly enhanced by the addition of a stimulator of GJIC. The present results suggest that bystander cell killing by heavy-ions was induced mainly by direct cell-to-cell communication, such as GJIC, which might play important roles in bystander responses.

Modulatory effect of ionizing radiation on food-NaCl associative learning: the role of gamma subunit of G protein in Caenorhabditis elegans.

Ionizing radiation (IR) is known to impair learning by suppressing adult neurogenesis in the hippocampus. However, in a mature nervous system, IR-induced functional alterations that are independent of neurogenesis remain largely unknown. In the present study, we analyzed the effects of IR on a food-NaCl associative learning paradigm of adult Caenorhabditis elegans that does not undergo neurogenesis. We observed that a decrease in chemotaxis toward NaCl occurs only after combined starvation and exposure to NaCl. Exposure to IR induced an additional decrease in chemotaxis immediately after an acute dose in the transition stage of the food-NaCl associative learning. Strikingly, chronic irradiation induced negative chemotaxis in the exposed animals, i.e., the primary avoidance response. IR-induced additional decreases in chemotaxis after acute and chronic irradiation were significantly suppressed in the gpc-1 mutant, which was defective in GPC-1 (one of the two gamma subunits of the heterotrimeric G-protein). Chemotaxis to cAMP, but not to lysine and benzaldehyde, was influenced by IR during the food-NaCl associative learning. Our novel findings suggest that IR behaves as a modulator in the food-NaCl associative learning via C. elegans GPC-1 and a specific neuronal network and may shed light on the modulatory effect of IR on learning.

The radiobiological effectiveness of carbon-ion beams on growing neurons.

PURPOSE: Recently carbon-ion beams have been reported to be remarkably effective for controlling various cancers with less toxicity and are thought to be a promising modality for cancer treatment. However, the biological effect of carbon-ion beams arising on normal neuron remains unknown. Therefore, this study was undertaken to investigate the effect of carbon-ion beams on neurons by using both morphological and functional assays. MATERIALS AND METHODS: Dorsal root ganglia (DRG) and sympathetic ganglion chains (SYMP) were isolated from day-8 and day-16 chick embryos and cultured for 20 h. Cultured neurons were exposed to carbon-ion beams and X-rays. Morphological changes, apoptosis and cell viability were evaluated with the Growth Cone Collapse (GCC), Terminal deoxynucleotidyl Transferase (TdT)-mediated deoxyUridine TriPhosphate (dUTP) nick End Labeling [TUNEL] assay and 4-[3-(4-iodophenyl)- 2-(4-nitrophenyl)- 2H-5-tetrazolio]- 1,3-benzenedisulfonate [WST-1] assays, respectively. RESULTS: Irradiation caused GCC and neurite destruction on a time- and irradiation dose-dependent manner. Changes in morphological characteristics were similar following either irradiation. Morphological and functional assays showed that day-8 neurons were more radiosensitive than day-16 neurons, whereas, radiosensitivity of DRG was comparable to that of SYMP. The dose-response fitting curve utilising both GCC and TUNEL labeling index showed higher relative biological effectiveness (RBE) values were associated with lower lethal dose (LD) values, while lower RBE was associated with higher LD values. CONCLUSION: Exposure to high-linear energy transfer (LET) irradiation is up to 3.2 more efficient to induce GCC and apoptosis, in early developed neuronal cells, than low-LET irradiation. GCC is a reliable method to assess the radiobiological response of neurons.

Kinetic analysis of double-strand break rejoining reveals the DNA reparability of gamma-irradiated tobacco cultured cells.

The rejoining efficiency of double-strand breaks (DSBs) was quantified by a DNA fragment-size analysis in tobacco protoplasts and CHO-K1 cells following gamma-ray irradiation in order to compare DNA reparability of higher plants with mammals. Results showed that the DSB rejoining efficiency of tobacco protoplasts is dependent on the temperature of post-irradiation cultivation and that it reaches a maximum at 27 degrees C, which represents the most suitable temperature for protoplast cultivation. The DSB rejoining kinetics of tobacco protoplasts were well represented by a biphasic-exponential equation: half of initial-induced DSBs were rejoined for 1 h and the others were almost rejoined within 4 h. We found that the DSB rejoining kinetics of tobacco protoplasts at 27 degrees C are the same as those of CHO-K1 cells at 37 degrees C. These findings indicate that the DSB rejoining efficiency of tobacco protoplasts and CHO-K1 cells are comparable at their respective cell cultivation temperatures, suggesting that DSB rejoining efficiency is little responsible for the higher radiation-tolerance of tobacco protoplasts.

Effects of ionizing radiation on locomotory behavior and mechanosensation in Caenorhabditis elegans.

Locomotory behavior (motility) and mechanosensation are of vital importance in animals. We examined the effects of ionizing radiation (IR) on locomotory behavior and mechanosensation using a model organism, the nematode Caenorhabditis elegans. Bacterial mechanosensation in C. elegans induces the dopamine-mediated slowing of locomotion in the presence of bacteria (food), known as the basal slowing response. We previously reported an IR-induced reduction of locomotory rate in the absence of food. In the present study, we observed a similar IR-induced reduction of locomotory rate in the cat-2 mutant, which is defective in bacterial mechanosensation. The dose response pattern of the locomotory rate in the presence of food was relatively flat in wild-type animals, but not in cat-2 mutants. This suggests that the dopamine system, which is related to bacterial mechanosensation in C. elegans, might have a dominant effect on locomotory rate in the presence of food, which masks the effects of other stimuli. Moreover, we found that the behavioral responses of hydrogen peroxide-exposed wild-type animals are similar to those of IR-exposed animals. Our findings suggest that the IR-induced reduction of locomotory rate in the absence of food is mediated by a different pathway from that for bacterial mechanosensation, at least partially through IR-produced hydrogen peroxide.

Ceramide induces myogenic differentiation and apoptosis in Drosophila Schneider cells.

Cells exposed to genotoxic stress, such as ionizing radiation and DNA damaging reagents, either arrest the cell cycle to repair the genome, or undergo apoptosis, depending on the extent of the DNA damage. DNA damage also has been implicated in various differentiation processes. It has been reported that gamma-ray exposure or treatment with DNA-damaging agents could induce myogenic differentiation in Drosophila Schneider cells. However, the mechanism underlying this process has been poorly understood. In this study, exposure of Schneider cells to X-rays or energetic carbon ion beams caused increase of TUNEL-positive cells and conversion of round-shaped cells to elongated cells. Both upregulation of genes related to myogenesis and increase of myosin indicate that the radiation-induced morphological changes of Schneider cells were accompanied with myogenic differentiation. Because the intracellular ceramide was increased in Schneider cells after exposure to X-ray, we examined whether exogenous ceramide could mimic radiation-induced myogenic differentiation. Addition of membrane-permeable C(2)-ceramide to Schneider cells increased apoptosis and expression of myogenic genes. These results suggest that ceramide plays important roles in both apoptosis and the radiation-induced myogenic differentiation process.

Targeted heavy-ion microbeam irradiation of the embryo but not yolk in the diapause-terminated egg of the silkworm, bombyx mori, induces the somatic mutation.

Using heavy-ion microbeam, we report target irradiation of selected compartments within the diapause-terminated egg and its mutational consequences in the silkworm, Bombyx mori. On one hand, carbon-ion exposure of embryo to 0.5-6 Gy increased the somatic mutation frequency, suggesting targeted radiation effects. On the other, such increases were not observed when yolk was targeted, suggesting a lack of nontargeted bystander effect.

Microbeam irradiation facilities for radiobiology in Japan and China.

In order to study the radiobiological effects of low dose radiation, microbeam irradiation facilities have been developed in the world. This type of facilities now becomes an essential tool for studying bystander effects and relating signaling phenomena in cells or tissues. This review introduces you available microbeam facilities in Japan and in China, to promote radiobiology using microbeam probe and to encourage collaborative research between radiobiologists interested in using microbeam in Japan and in China.