[Detection of Gamma-Ray Irradiation History of Dried Plant-Based Foods via the Analysis of 5,6-Dihydrothymidine].

Irradiation is widely used worldwide to sterilize and kill insects in food, and prevent the germination of agricultural products. However, in Japan, food irradiation is prohibited except to prevent potato sprouting. Herein, 5,6-dihydrothymidine (DHdThd) residue-a damaged nucleoside generated from the thymidine (dThd) residue in DNA contained in food upon irradiation-was used as a detection indicator. Eight dried plant-based food samples were gamma ray-irradiated in the range from 3.2 to 8.3 kGy. Subsequently, DNA was extracted from the irradiated sample and digested into nucleosides by the three enzymes, and the test solution was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Evidently, in all samples, the concentration ratio of DHdThd to dThd in the test solution (DHdThd/dThd) was dependent on the irradiation dose; moreover, during storage under frozen conditions for at least 890 d post-irradiation, this concentration ratio was equal to that immediately after irradiation. The irradiation histories of the eight types of dried plant-based food samples were correctly detected.

Trehalose accumulation and radiation resistance due to prior heat stress in Saccharomyces cerevisiae.

In this study, we examined the accumulation of trehalose, a stress-responsive substance, upon gamma-ray irradiation by evaluating the cause of trehalose accumulation and the development of gamma-ray resistance through intracellular trehalose accumulation. Saccharomyces cerevisiae cells cultured to the logarithmic growth phase were irradiated with gamma rays, and the intracellular trehalose content was measured. However, trehalose was not detectable. The yeast cells with trehalose accumulation caused by pre-treatment at 40 °C were irradiated with gamma rays, and the resistance of these cells to gamma radiation was compared with that of cells without heat treatment. Trehalose accumulation resulted in gamma-ray resistance and suppressed the increase in reactive oxygen species, lipid peroxidation, and DNA double-strand break production in yeast cells. The tests were also performed with a trehalose-6-phosphate-synthase (TPS1)-deficient mutant strain (Δtps1) unable to synthesize trehalose, and the results revealed that TPS1 was involved in protection against oxidative stress.

Evaluation of distinct modes of oxidative secondary injury generated in heat-treated cells of Escherichia coli with solid/liquid and complex/semi-synthetic media sets.

AIMS: To characterize and evaluate oxidative secondary injury generated in heat-treated Escherichia coli cells during recovery cultivation either on agar or in a broth of a semi-synthetic enriched M9 (EM9) medium and a complex Luria broth (LB) medium with different types of antioxidants. METHODS AND RESULTS: E. coli cells grown in the EM9 and LB broth were heated at 50°C in a buffer (pH 7.0). Heated cells were recovered on the same kind of agar medium as that used for growth, with or without different antioxidants. Although these antioxidants mostly protected the cells from oxidative secondary injury on the recovery media, sodium thiosulphate and sodium pyruvate were most protective on EM9 and LB agars, respectively. Determination of viability using the most probable number and growth delay analysis methods showed significant reductions in the protective effects of antioxidants in the EM9 and LB media. CONCLUSION: Oxidative secondary injury generated in heated E. coli cells was found to be qualitatively and quantitatively diverse under cellular and environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that different modes of oxidation should be considered in viability determination and injured cell enumeration of heat-treated cells.

Raman optical activity of tetra-alanine in the poly(l-proline) II type peptide conformation.

The poly(l-proline) II (PPII) helix is considered to be a major conformation in disordered polypeptides and unfolded proteins in aqueous solution. The PPII conformation can be identified by using Raman optical activity (ROA), which measures the different intensities of right- and left-circularly polarized Raman scattered light from chiral molecules and provides information on stereochemistry associated with vibrational motions. In the present study, we used tetra-alanine (Ala4) as a model system, since its central amide bond adopts the PPII conformation. The predominance of the PPII conformation was supported by 11 ns molecular dynamics (MD) simulations at 300 K. The MD snapshots were used for subsequent quantum mechanical/molecular mechanical (QM/MM) calculations to compute the Raman and ROA spectra. The present MD + QM/MM analysis leads to a good agreement between the observed and simulated spectra, allowing us to assign most of the spectral features including the ROA band near 1320 cm-1, which has been used as a marker for the PPII conformation. This positive ROA band has three components. The lower frequency component near 1310 cm-1 arises from an internal peptide bond, whereas the higher frequency components around 1320-1335 cm-1 appear due to N- and C-terminal peptide groups. The MD + QM/MM calculations also reproduced the electronic circular dichroism spectra of Ala4. The present results provide a satisfactory framework for future investigations of unfolded/disordered proteins as well as peptides in solutions by chiral spectroscopic methods.

Important role of catalase in the cellular response of the budding yeast Saccharomyces cerevisiae exposed to ionizing radiation.

Ionizing radiation indirectly causes oxidative stress in cells via reactive oxygen species (ROS), such as hydroxyl radicals (OH(-)) generated by the radiolysis of water. We investigated how the catalase function was affected by ionizing radiation and analyzed the phenotype of mutants with a disrupted catalase gene in Saccharomyces cerevisiae exposed to radiation. The wild-type yeast strain and isogenic mutants with disrupted catalase genes were exposed to various doses of (60)Co gamma-rays. There was no difference between the wild-type strain and the cta1 disruption mutant following exposure to gamma-ray irradiation. In contrast, there was a significant decrease in the ctt1 disruption mutant, suggesting that this strain exhibited decreased survival on gamma-ray exposure compared with other strains. In all three strains, stationary phase cells were more tolerant to the exposure of gamma-rays than exponential phase cells, whereas the catalase activity in the wild-type strain and cta1 disruption mutant was higher in the stationary phase than in the exponential phase. These data suggest a correlation between catalase activity and survival following gamma-ray exposure. However, this correlation was not clear in the ctt1 disruption mutant, suggesting that other factors are involved in the tolerance to ROS induced by irradiation.

Modifications of azoxymethane-induced carcinogenesis and 90-day oral toxicities of 2-tetradecylcyclobutanone as a radiolytic product of stearic acid in F344 rats.

A 90-day oral toxicity test in rats was performed to evaluate the toxicity of 2-tetradecylcyclobutanone (2-tDCB), a unique radiolytic product of stearic acid. Six-week-old male and female F344 rats (n=15/group) were given 2-tDCB at concentrations of 0, 12, 60 and 300 ppm in a powder diet for 13 weeks. Slight dose-dependent increases in serum total protein and albumin in male rats were found, but these changes were not considered to be a toxic effect. The fasting, but not non-fasting, blood glucose levels of the male rats in the 300 ppm group and female rats in the 60 and 300 ppm groups were lower than those of the controls. Gas chromatography-mass spectrometry analysis showed dose-dependent accumulation of 2-tDCB in adipose tissue, notably in males. Next, we performed an azoxymethane (AOM)-induced two-stage carcinogenesis study. After injection of 6-week-old male F344 rats (n=30/group) once a week for 3 weeks, the animals received 2-tDCB at concentrations of 0, 10, 50 and 250 ppm in a powder diet for 25 weeks. The incidences of colon tumors for the 2-tDCB dosages were 34%, 45%, 40% and 37%, respectively, and were not statistically significant. These data suggest that 2-tDCB shows no toxic or tumor-modifying effects under the present conditions, and that the no-observed-adverse-effect level for 2-tDCB is 300 ppm in both sexes, equivalent to 15.5 mg/kg b.w./day in males and 16.5 mg/kg b.w./day in females.

Gamma-irradiated Aspergillus conidia show a growth curve with a reproductive death phase.

In this study, we evaluated the effects of gamma irradiation on the germination of Aspergillus conidia and mycelial growth using microscopy and predictive microbiological modeling methods. A dose of 0.4 kGy reduced the germination rate by 20% compared to the untreated control, indicating interphase death due to the high radiation dose. The number of colonies formed (5.5%) was lower than the germination rate (69%), suggesting that most colonies died after germination. Microscopic observations revealed that mycelial elongation ceased completely in the middle of the growth phase, indicating reproductive death. The growth curves of irradiated conidia exhibited a delayed change in the growth pattern, and a decrease in slope during the early stages of germination and growth at low densities. A modified logistic model, which is a general purpose growth model that allows for the evaluation of subpopulations, was used to fit the experimental growth curves. Dose-dependent waveform changes may reflect the dynamics of the subpopulations during germination and growth. These methods revealed the occurrence of two cell death populations resulting from gamma irradiation of fungal conidia and contribute to the understanding of irradiation-induced cell death in fungi.

Excessive ultraviolet C irradiation causes spore protein denaturation and prohibits the initiation of spore germination in Bacillus subtilis.

Ultraviolet (UV) -C is widely used to kill bacteria as it damages chromosomal DNA. We analyzed the denaturation of the protein function of Bacillus subtilis spores after UV-C irradiation. Almost all of the B. subtilis spores germinated in Luria-Bertani (LB) liquid medium, but the colony-forming unit (CFU) of the spores on LB agar plates decreased to approximately 1/103 by 100 mJ/cm2 of UV-C irradiation. Some of the spores germinated in LB liquid medium under phase-contrast microscopy, but almost no colonies formed on the LB agar plates after 1 J/cm2 of UV-C irradiation. The fluorescence of the green fluorescent protein (GFP) -fused spore proteins, YeeK-GFP, YeeK is a coat protein, decreased following UV-C irradiation of over 1 J/cm2, while that of SspA-GFP, SspA is a core protein, decreased following UV-C irradiation of over 2 J/ cm2, respectively. These results revealed that UV-C affected on coat proteins more than core proteins. We conclude that 25 to 100 mJ/cm2 of UV-C irradiation can cause DNA damage, and more than 1 J/cm2 of UV-C irradiation can cause the denaturation of spore proteins involved in germination. Our study would contribute to improve the technology to detect the bacterial spores, especially after UV sterilization.

Different patterns of germination inhibition by carvacrol and thymol in Bacillus subtilis spores.

This study aimed to clarify how the phenolic monoterpene carvacrol and its structural isomer thymol both as essential oil components (EOCs) inhibit the germination of Bacillus subtilis spore. Germination was evaluated by the OD600 reduction rate in a growth medium and phosphate buffer containing either l-alanine (l-Ala) system or l-asparagine, d-glucose, d-fructose plus KCl (AGFK) system. The germination of the wild-type spores in the Trypticase Soy broth (TSB) was found to be greatly inhibited by thymol than by carvacrol. Such a difference in the germination inhibition was confirmed by the dipicolinic acid (DPA) release from germinating spores in the AGFK buffer system, but not in the l-Ala system. Similar to the wild-type spores, no difference in the inhibitory activity between the EOCs was also indicated with the gerB, gerK-deletion mutant spores in the l-Ala buffer system and the above substantial difference was also done with the gerA-deleted mutant spores in the AGFK. Fructose was found to release spores from the EOC inhibition and inversely even stimulated. Increased concentrations of glucose and fructose partially suppressed the germination inhibition by carvacrol. The results obtained should contribute to the elucidation of the control effects of these EOCs on bacterial spores in foods.

Theory and application of growth delay analysis of colony formation for evaluation of injured population of the stressed fungal conidia.

A new concept of injured population assessment is proposed, in which the size of the injured population in stressed mold spores is evaluated by analyzing the colony formation process on a solid agar medium. In this method, a small paper disc containing mold spores is placed on a subculture agar plate, and the linear increase in the radius of the colony formed by development from the spore is measured over time. Then, the principle of the previously reported growth delay analysis (GDA) method originally using a liquid medium is applied to obtain the integrated viable ratio (IV) of the stressed population from the delay time relative to the growth of the unstressed population. On the other hand, the viable ratio (V) to the initial value as the colony count obtained with the stressed culture is obtained; the difference between the logarithms of V and IV is determined as the log number of the injured population. Applying this analysis method to heated spores of Cladosporium sphaerospermum, we determined the size of the injured population that occurred. This method was considered to be effective as a new method for quantifying injured populations using a solid medium.

Lethal and mutagenic effects of different LET radiations on Bacillus subtilis spores.

New, useful microorganism resources have been generated by ionizing radiation breeding technology. However, the mutagenic effects of ionizing radiation on microorganisms have not been systematically clarified. For a deeper understanding and characterization of ionizing radiation-induced mutations in microorganisms, we investigated the lethal effects of seven different linear energy transfer (LET) radiations based on the survival fraction (SF) and whole-genome sequencing analysis of the mutagenic effects of a dose resulting in an SF of around 1% in Bacillus subtilis spores. Consequently, the lower LET radiations (gamma [surface LET: 0.2 keV/µm] and 4He2+ [24 keV/µm]) showed low lethality and high mutation frequency (MF), resulting in the major induction of single-base substitutions. Whereas higher LET radiations (12C5+ [156 keV/µm] and 12C6+ [179 keV/µm]) showed high lethality and low MF, resulting in the preferential induction of deletion mutations. In addition, 12C6+ (111) ion beams likely possess characteristics of both low- and high-LET radiations simultaneously. A decrease in the relative biological effectiveness and an evaluation of the inactivation cross section indicated that 20Ne8+ (468 keV/µm) and 40Ar13+ (2214 keV/µm) ion beams had overkill effects. In conclusion, in the mutation breeding of microorganisms, it should be possible to regulate the proportions, types, and frequencies of induced mutations by selecting an ionizing radiation of an appropriate LET in accordance with the intended purpose.

Preparation of High-quality Oil from Vegetable Oil with High-free Fatty Acid (FFA) Content Using an Ammonia/Methanol Solution.

There are significant concerns regarding the quality of vegetable oils in the food and biofuel industries. In this study, we explored the preparation of high- quality oil from high-free fatty acid (FFA) vegetable oil using an ammonia/MeOH solvent as an alkali base. Among the six tested solvents, MeOH was the most suitable for the separation of the oil and FFAs. Among the three alkali bases, ammonia enhanced the miscibility of FFAs in MeOH by forming ammonium salts. The amounts of FFAs in the upper layer and oil in the lower layer were positively correlated (r = 0.9348 and 0.9617, respectively) with MeOH. With increasing MeOH concentration, the amount of oil in the lower layer increased along with the FFAs in the upper layer. Using the molar ratio of ammonia to FFA 1:1 and the ratio (v/w) of MeOH to oil 4:3, 91.6% FFAs and 97.8% oil in the upper and lower layers, respectively, were produced from 50% FFA oil. Using a relational expression of FFAs and oil in the upper layer, 97.1% FFAs and 99.6% oil in each layer was obtained from 10% FFA oil. The oil in the lower layer was further purified by extraction with MeOH. This method is easy and efficient for the separation and purification of oil, accompanied by the reuse of reagents with almost no loss of raw materials.

Molecular mechanisms of apoptosis induction by 2-dodecylcyclobutanone, a radiolytic product of palmitic acid, in human lymphoma U937 cells.

The irradiation of fat-containing food forms 2-dodecylcyclobutanone (2-DCB) from palmitic acid (PA). In this study, we investigated whether 2-DCB and PA induce apoptosis in human lymphoma U937 cells. We found that cell viability decreased by 2-DCB and apoptosis was induced by 2-DCB and PA. 2-DCB and PA significantly enhanced the formation of intracellular reactive oxygen species (ROS). Apoptosis induced by 2-DCB and PA was strongly prevented by an antioxidant, N-acetyl-L: -cysteine. The treatment with 2-DCB and PA resulted in the loss of mitochondrial membrane potential, and Fas, caspase-8 and caspase-3 activation. Pretreatment with a pan-caspase inhibitor (z-VAD) significantly inhibited apoptosis induced by 2-DCB and PA. Moreover, 2-DCB and PA also induced Bax up-regulation, the reduction in Bcl-2 expression level, Bid cleavage and the release of cytochrome c from the mitochondria to the cytosol. In addition, an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed after the treatment with 2-DCB and PA. Our results indicated that intracellular ROS generation, the modulation of the Fas-mitochondrion-caspase-dependent pathway and the increase in [Ca(2+)](i) involved in apoptosis are induced by 2-DCB and PA in U937 cells.

Theoretical Base for the Application of the DiVSaL Method to Bacterial Spores to Evaluate Injured Populations Occurring After Exposure to Lethal Stress.

To assess injury in bacterial spore populations exposed to lethal stress, we proposed a theoretical basis for applying the DiVSaL method, which has already been reported for general microorganisms as a double subculture method. We constructed a mathematical model in which both injuries to the germination system and the spore body were taken into the theory. In this theory, we reasonably assumed that the viable and germinable spore count is constant before the subsequent vegetative growth and that the delay of germination and outgrowth can be included in the concept of λ injury previously reported as the growth-independent injury. By introducing these assumptions, the double subculture method can be considered to apply to spores as well. As examples of the application of this theory, the growth delays of Bacillus subtilis spores treated with heat and UV irradiation were analyzed and the numbers of injured spores were evaluated. Based on the results obtained, heat is indicated to have a higher injury generation ability than UV irradiation. The applicability of the DiVSaL method as a tool for food preservation and sanitation designs is presented.

Low Temperature Heating-Induced Death and Vacuole Injury in Cladosporium sphaerospermum Conidia.

The mechanism of thermal death of mold conidia has not been understood in detail. The purpose of this study is to analyze the death kinetics of heated conidia of Cladosporium sphaerospermum and to ascertain the expectant cell injury responsible for the death. The death of the dormant (resting) conidia of Cladosporium sphaerospermum was examined at temperatures of between 43 and 54℃ with the conventional colony count method. The death reaction apparently followed the first order kinetics, but the Arrhenius plot of the death rate constant demonstrated seemingly a break. The linearity at temperatures higher than that at the break was lost at lower temperatures, suggesting the involvement of an unusual mechanism in the latter temperatures. In the cell morphology, we observed with quinacrine staining the vacuole rupture at a lower temperature but not at a high temperature. Interestingly, the vacuole rupture by low-temperature heating was found to correlate with the viability loss. Furthermore, active protease originally locating in vacuoles was detected in the cytoplasm of the conidia after heated at a low temperature. The results obtained suggest the involvement of potent autophagic cell death induced by low temperature heating of C. sphaerospermum conidia.

Biological effects of low-dose γ-ray irradiation on chromosomes and DNA of Drosophila melanogaster.

While the damage to chromosomes and genes induced by high-dose radiation (HDR) has been well researched in many organisms, the effects of low-dose radiation (LDR), defined as a radiation dose of ≤100 mSv, are still being debated. Recent research has suggested that the biological effects of LDR differ from those observed in HDR. To detect the effect of LDR on genes, we selected a gene of Drosophila melanogaster, known as the multiple wing hair (mwh) gene. The hatched heterozygous larvae with genotype mwh/+ were irradiated by γ-rays of a 60Co source. After eclosion, the wing hairs of the heterozygous flies were observed. The area of only one or two mwh cells (small spot) and that of more than three mwh cells (large spot) were counted. The ratio of the two kinds of spots were compared between groups irradiated by different doses including a non-irradiated control group. For the small spot in females, the eruption frequency increased in the groups irradiated with 20-75 mGy, indicating hypersensitivity (HRS) to LDR, while in the groups irradiated with 200 and 300 mGy, the frequency decreased, indicating induced radioresistance (IRR), while in males, 50 and 100 mGy conferred HRS and 75 and 200 mGy conferred IRR. For the large spot in females, 75 mGy conferred HRS and 100-800 mGy conferred IRR. In conclusion, HRS and IRR to LDR was found in Drosophila wing cells by delimiting the dose of γ-rays finely, except in the male large spot.

Detection Time Distribution of Microcolonies Formed by Individual Heat-Injured Cells of Escherichia coli.

The microcolony formation at 30℃ on an enriched minimal salts agar plates by individual Escherichia coli cells heated at 50℃ was monitored with a time-lapse shadow image analysis system, MicroBio µ3DTM AutoScanner. While the time course of microcolony count detected every half an hour for the unheated cells seemingly demonstrated a normal distribution, that for the heated cell population demonstrated totally the growth delay probably resulting from cell injury and also interestingly distributed in its rather deformed pattern with a tailing. Those patterns of the cumulative counts of appearing microcolonies during the post-heating cultivation period were expressed in three different mathematical models. This approach may be proposed as a rapid cultivation method predictable for enumeration of viable and repairable injured cells in practical use.

Application of gamma radiation for disinfection of fungi in a large volume of historical archives damaged by flood following Typhoon Hagibis 2019, Japan: A case report.

A large volume of historical archives was extensively damaged by flood water, following Typhoon Hagibis in Fukushima, Japan, in October 2019. They were rescued from the stricken area within a week, however, the prolonged exposure of paper documents to water caused severe biodegradation by fungal growth. To disinfect fungi, the paper documents were exposed to gamma radiations emitted by a source of Cobalt 60 by the industrial irradiation service. The wet paper documents were mainly contaminated with hydrophilic and cellulolytic fungi, including Trichoderma, Stachybotrys, and Fusarium; no fungi grew after irradiation. These results indicated that the average absorbed dosage from 13.1 kGy to 16.1 kGy were sufficient to disinfect paper documents heavily contaminated with fungi. In the present study, we demonstrated the successful practical use of irradiation in fungi-damaged paper documents using a commercial gamma-irradiation facility.

The characterization of tocols in different plants parts of six Japanese rice cultivars relating to their UVB-sensitivity.

There has been significant interest in the photosensitivity, or photo-resistance, of Japanese rice cultivars, which synthesize tocols (Vitamin E), a class of phytochemicals including tocol derivatives tocopherol (T) and tocotrienol (T3). In the present study, the distribution of tocols in the leaves, seeds, stems, and roots of six Japanese rice cultivars was investigated. The relationship between the different tocols in cultivars and their ultraviolet B sensitivity index (USB-SI) was analyzed. The leaves contained the highest average total amount of tocols at 230 µg.fresh-g-1, followed by seeds, stems, and roots. In leaves and stems, the most abundant component was α-T which was more than 85%. On the other hand, the tocols in seeds were 38% δ-T3, 32% α-T, and 20% α-T3. The tocols in roots were 55% α-T, 14% γ-T, and 13% δ-T3. The total tocol content in four plant parts exhibited a negative correlation (P < 0.05) in stem and root, and a negative relationship (r < -0.70) with the UVB-SI of the cultivars, suggesting that the total tocol contents were closely related to the resistance to UVB in Japanese rice plants.

A Modified Double Subculture Method for the Two-Mode Injuries Evaluation in a Stressed Fungal Spore Population.

In order to evaluate injury of a stressed fungal spore population, a modification of formerly presented double subculture method, which consists of both the conventional plate count method and the growth delay analysis method, was proposed. In this method, an apparent logarithmic growth kinetics was assumed and the previous kinetic model was improved to be able to estimate injured subpopulations in two different modes containing early occurring growth-independent and late occurring growth-dependent injuries, called the λ and µ injuries, respectively. Based on the kinetic theory developed here, this novel method was applied to heat-treated conidia of Cladosporium cladosporioides and these two mode injuries were evaluated.