Method for identifying neutron beam type

Abstract

The present disclosure provides a method for identifying the neutron beam type, the method including: a step for exposing an algae culture to a neutron beam; a step for measuring the expression level (A) of a marker gene in the algae culture exposed to the neutron beam; and a step for identifying the neutron beam type from the measured value of the expression level (A) on the basis of a predetermined relationship between the expression level change of the marker gene and the neutron beam type to which the algae is exposed.

Description

How to distinguish between neutron radiation types

[0001] The present disclosure relates to a method for determining the type of neutron radiation.

[0002] Neutron radiation refers to the state in which neutrons leave an atomic nucleus and move in one direction. Neutron radiation occurs naturally, and on the Earth's surface it is mainly produced by cosmic rays colliding with the Earth's atmosphere. When neutron radiation hits the Earth's surface, it can have a negative effect on electronic components on the ground. For example, when neutron radiation hits the semiconductor devices of electronic devices, it is known to cause a "soft error," a phenomenon in which stored data is rewritten.

[0003] Two types of neutrons contribute significantly to soft errors: high-energy neutrons and thermal neutrons. High-energy neutrons are known to be generated, for example, from heavy metal targets irradiated with a charged particle beam, while thermal neutrons are obtained by slowing down high-energy neutrons using a neutron moderator or similar material. However, it is not possible to distinguish between the two types of rays from the results of "soft errors" alone, and therefore it is not possible to take appropriate measures to prevent errors.

[0004] A conventional technique for identifying the type of neutron is the time-of-flight method (Non-Patent Document 1), which is a method in which neutrons are made to fly a certain distance, the velocity is calculated from the arrival time of the neutrons, and the calculated neutron velocity is converted into energy. However, measuring the velocity using the time-of-flight method requires a special accelerator, and the neutron source must be a pulsed neutron source.

[0005] Another method for distinguishing neutron types is to use a cadmium shielding material. This method takes advantage of cadmium's ability to absorb thermal neutrons of 0.4 eV or less. By measuring with and without cadmium, it is possible to distinguish between the number of thermal neutrons and neutrons in energy bands above thermal neutrons (Non-Patent Document 2). However, this method has the disadvantage of requiring measurements to be taken twice, once with and once without cadmium.

[0006] RD John Copley and J. Terrence Udovic, "Neutron time-of-flight spectroscopy", J. Res. Natl. Inst. Stand. Technol., vol. 98, no. 1, pp. 71-87, 1993.Measurement and Reporting of Alpha Particle and Terrestrial Cosmic Ray Induced Soft Error in Semiconductor Devices, Standard JEDEC, JESD89A, 2006.

[0007] The present disclosure has been made to solve the above-mentioned problems, and aims to provide a method for identifying the type of neutron radiation.

[0008] According to one embodiment, a method for identifying the type of neutron radiation is provided, comprising the steps of exposing an algal culture to the neutron radiation, measuring the expression level (A) of a marker gene in the algal culture exposed to the neutron radiation, and identifying the type of neutron radiation from the measured value of (A) based on a predetermined relationship between the type of neutron radiation to which the algae is exposed and changes in the expression level of the marker gene.

[0009] According to the present disclosure, a method for determining the type of neutron radiation can be provided.

[0010] Fig. 1 is a flowchart showing an example of a method according to an embodiment, and Fig. 2 is a block diagram showing an apparatus configuration and a flow of samples or data for illustrating the method according to an embodiment.

[0011] In one embodiment, a method for identifying the type of neutron radiation is provided, comprising the steps of exposing an algal culture to the neutron radiation, measuring the expression level (A) of a marker gene in the algal culture exposed to the neutron radiation, and identifying the type of neutron radiation from the measured value of (A) based on a predetermined relationship between the type of neutron radiation to which the algae is exposed and changes in the expression level of the marker gene.

[0012] In the present disclosure, the term "type of neutron beam" refers to a classification of neutron beams according to the energy of the neutron beam. The type of neutron beam in the embodiments may include thermal neutron beams and high-energy neutron beams. As will be understood by those skilled in the art, thermal neutron beams refer to neutron beams whose majority component (i.e., more than half) is neutrons that are in thermal equilibrium with materials in the environment or have low energies close to the energies of neutrons in such thermal equilibrium, and typically contain neutrons with energies of 4 to 100 millielectron volts as a majority component. On the other hand, as will be understood by those skilled in the art, high-energy neutron beams are sometimes called fast neutron beams, and are neutron beams whose majority component is typically neutrons with energies of 0.1 megaelectron volts or more.

[0013] In one embodiment, the method involves exposing algae to neutron radiation and determining the type of neutron radiation to which the algae was exposed based on changes in gene expression levels induced within the cells. This basic feature allows the method to be used, for example, to identify the type of neutron radiation causing an error in electronic equipment, which may lead to the implementation of appropriate countermeasures against the error. Exposing algae to neutron radiation includes placing the algae in a location where neutron radiation is present or irradiating the algae with neutron radiation.

[0014] The type of algae in the present disclosure is not limited. The algae may be non-sessile algae. The algae may be, for example, unicellular algae. Examples of algae that may be used include Aurantiochytrium, Chlamydomonas, Chlorella, Schizon algae, Spirulina, Botryococcus, Euglena, green algae, brown algae, red algae, cyanobacteria, diatoms, xanthophytes, gold algae, dinoflagellates, and seaweed. Schizon algae is a particularly preferred example. The algae in the present disclosure may be a single species of algae or a population containing multiple species of algae. A genetically homogeneous population (pure line) of algae of the same species may be used, or a population of algae with genetic variation may be used.

[0015] In an embodiment, "red algae" refers to at least algae belonging to the genus Cyanidioschyzon. Examples of algae belonging to Cyanidioschyzon include the unicellular red alga Cyanidioschyzon merolae. In an embodiment, "spirulina" refers to at least algae belonging to the genus Arthrospira. Examples of spirulina include Arthrospira ardissonei, Arthrospira erdosensis, Arthrospira fusiformis, Arthrospira indica, Arthrospira innermongoliensis, Arthrospira jenneri, Arthrospira massartii, Arthrospira maxima, and Arthrospira platensis. In an embodiment, "aurantiochytrium" refers to at least algae belonging to the genus Aurantiochytrium. Examples of Aurantiochytrium include Aurantiochytrium limacinum and Aurantiochytrium mangrovei. In an embodiment, "Botryococcus" includes at least algae belonging to the genus Botryococcus. Examples of Botryococcus include Botryococcus australis, Botryococcus balkachicus, Botryococcus braunii, Botryococcus calcareus, Botryococcus canadensis, Botryococcus comperei, Botryococcus fernandoi, Botryococcus neglectus, Botryococcus pila, Botryococcus protuberans, Botryococcus pusillus, Botryococcus terribilis, and Botryococcus terricola. In an embodiment, "Chlamydomonas" includes at least algae belonging to the genus Chlamydomonas.Examples of Chlamydomonas include Chlamydomonas acidophila, Chlamydomonas caudata, Chlamydomonas ehrenbergii, Chlamydomonas elegans, Chlamydomonas moewusii, Chlamydomonas muriella, Chlamydomonas nivalis, Chlamydomonas ovoidae, Chlamydomonas priscuii, Chlamydomonas smithii, Chlamydomonas reinhardtii, etc. In an embodiment, "chlorella" includes at least algae belonging to the genus Chlorella. Examples of Chlorella include Chlorella autotrophica, Chlorella coloniales, Chlorella lewinii, Chlorella minutissima, Chlorella pituita, Chlorella pulchelloides, Chlorella pyrenoidosa, Chlorella rotunda, Chlorella singularis, Chlorella sorokiniana, Chlorella variabilis, Chlorella volutis, and Chlorella vulgaris. In an embodiment, "Euglena" includes at least algae belonging to the genus Euglena. Examples of Euglena include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, and Euglena viridis.

[0016] Next, an example of a method for determining the type of neutron beam according to the embodiment will be described with reference to the flowchart of FIG.

[0017] Step S1 is a step of culturing algae. Algae can be cultured using a medium for algae known to those skilled in the art. The medium for culturing algae in this embodiment can be any aqueous liquid capable of growing natural algae, without particular limitations. Alternatively, an aqueous medium for algae with a defined composition can also be used. The medium can be any medium for culturing algae known to those skilled in the art, such as a medium containing nutrients, a carbon source, rare metals, etc. Specific examples of the aqueous medium include Koren-Hutner (KH) medium, Cramer-Myers (CM) medium, modified Allen's (MA)2 medium (Ohmura et al., Plant and Cell Physiology, Volume 49, Issue 1, January 2008, Pages 117-120), and mixtures thereof. MA2 medium is a medium (pH 2.5) consisting of 40 mM (NH4)2SO4, 4 mM MgSO4, 8 mM KH2PO4, 1 mM CaCl2.2H2O, 0.1 mM FeCl3, 0.075 mM EDTA.2Na, 5.7 g / L H2BO3, 3.6 g / L MnCl2.4H2O, 0.210 g / L ZnCl2, 0.78 g / L Na2MoO4.2H2O, 0.08 g / L CoCl2.6H2O, 0.086 g / L CuCl2, and the remainder water. The algae of the present disclosure may be cultured under natural and / or artificial light, and in a CO2 atmosphere of 0.5% or more, 1% or more, or 2% or more. ２ This may involve ventilation.

[0018] Step S2 is a step of irradiating the algae culture with the neutron beam (or exposing the algae culture to the neutron beam). In an embodiment, the algae can be irradiated with the neutron beam after, for example, culturing the algae until the logarithmic growth phase and transferring it to a container such as a centrifuge tube. The material of the container such as a centrifuge tube can be, for example, glass, polypropylene, polycarbonate, polyethylene, polystyrene, polyethylene terephthalate, etc., or a combination thereof, but is not limited to these. The absorbed dose of the irradiated neutron beam can be, for example, within a range of 0.1 Gy to 100 Gy (0.1 Gy or more and 100 Gy or less), but can be adjusted by changing the irradiation time, the distance between the neutron generation target and the algae culture solution, etc., so as to obtain a dose that does not kill the algae and allows for detection of changes in gene expression levels.

[0019] Step S3 is a step of measuring the expression level (A) of a marker gene in an algal culture irradiated with neutron beams. Embodiments of the present disclosure are based, at least in part, on the discovery that when irradiating algae with neutron beams, it is possible to observe a group of genes whose expression changes differently depending on the type of neutron beam used. The marker genes of the embodiments are not limited as long as their expression levels change upon irradiation with at least one type of neutron beam, and the manner in which the expression levels change differs depending on the type of neutron beam used. Such marker genes can be identified in advance with reference to the disclosure of the present application. Examples of marker genes include the genes CMC010C, CMC125C, CMI306C, CMK006C, CMM184C, CMP092C, CMS431C, CMT597C, CMQ318C, CMT251C, CMF012C, and CML339C in the unicellular red alga Schizon (Cyanidioschyzon merolae Genome Project v3; http: / / czon.jp) and orthologues of these genes. Throughout this specification, except for this section, the gene names CMC010C, CMC125C, CMI306C, CMK006C, CMM184C, CMP092C, CMS431C, CMT597C, CMQ318C, CMT251C, CMF012C, and CML339C are used to collectively refer to these genes in the red alga Schizon macrocarpa as well as their orthologous genes in other algae.

[0020] The method of the embodiment may include determining that the neutron radiation to which the algal culture was exposed is thermal neutron radiation if the marker gene comprises at least one gene selected from the group consisting of CMC010C, CMC125C, CMI306C, CMK006C, CMM184C, CMP092C, CMS431C, and CMT597C, and the measured value of (A) of any of the at least one gene is higher than the corresponding expression level in a control algal culture that is not exposed to neutron radiation (non-irradiated).

[0021] In the genome assembly of the unicellular red algae Schizon Genome Project v3 (Cyanidioschyzon merolae Genome Project v3), the CMC010C gene (NCBI Gene ID: 16992391) is located at positions 26210 to 25689 on chromosome 3 (where transcription occurs from the position corresponding to the first base number to the position corresponding to the second base number; the same applies below), the CMC125C gene (NCBI Gene ID: 16992537) is located at positions 293754 to 295970 on chromosome 3, the CMI306C gene (NCBI Gene ID: 16993624) is located at positions 804566 to 805627 on chromosome 9, and the CMK006C gene (NCBI Gene ID: The CMM184C gene (NCBI Gene ID: 16995011) is located at positions 455177 to 456454 on chromosome 13. The CMP092C gene (NCBI Gene ID: 16996013) is located at positions 225778 to 225224 on chromosome 16. The CMS431C gene (NCBI Gene ID: 16997464) is located at positions 1073608 to 1073375 on chromosome 19. The CMT597C gene (NCBI Gene ID: 16997876) is located at positions 1511244 to 1512593 on chromosome 20. These genes are genes whose expression levels increased fourfold or more specifically in the unicellular red alga Schizon irradiated with thermal neutrons (rather than high-energy neutrons), and the predicted functions of the proteins encoded by these genes are shown in Table 1. When transcripts are used for measurement, sequences corresponding to either the open reading frame or the outside of the open reading frame in the transcripts of these genes can be used to measure the expression level of the genes.

[0022]

[0023] The method of the embodiment may include determining that the neutron radiation to which the algal culture was exposed is high-energy neutron radiation if the marker gene comprises at least one gene selected from the group consisting of CMQ318C and CMT251C, and if the measured value of (A) of any of the at least one gene is higher than the corresponding expression level in a control algal culture that is not exposed to neutron radiation (non-irradiated).

[0024] In the genome assembly of the unicellular red alga Cyanidioschyzon merolae Genome Project v3, the CMQ318C gene (NCBI Gene ID: 16996264) corresponds to an open reading frame located at positions 823276 to 822998 on chromosome 17, and the CMT251C gene (NCBI Gene ID: 16997837) corresponds to an open reading frame located at positions 628379 to 628726 on chromosome 20. These genes were specifically expressed in the unicellular red alga Schizon irradiated with high-energy neutrons (rather than thermal neutrons), with an increase of more than fourfold. The predicted functions of the proteins encoded by these genes are shown in Table 2. When transcripts are used for measurement, sequences corresponding to either the open reading frame or the open reading frame of the transcripts can be used to measure the expression level of the genes.

[0025]

[0026] The method of the embodiment may include determining that the neutron radiation to which the algal culture was exposed is high-energy neutron radiation if the marker gene comprises at least one gene selected from the group consisting of CMF012C and CML339C, and the measured value of (A) of any of the at least one gene is lower than the corresponding expression level in a control algal culture that is not exposed to neutron radiation (non-irradiated).

[0027] In the genome assembly of the unicellular red alga Cyanidioschyzon merolae Genome Project v3, the CMF012C gene (NCBI Gene ID: 16993115) corresponds to an open reading frame located at positions 31642 to 29246 on chromosome 6, and the CML339C gene (NCBI Gene ID: 16994761) corresponds to an open reading frame located at positions 855627 to 853987 on chromosome 12. These genes were specifically expressed in the unicellular red alga Schizon irradiated with high-energy neutrons (rather than thermal neutrons), with expression levels reduced to one-quarter or more. The putative functions of the proteins encoded by these genes are shown in Table 3. When transcripts are used for measurement, sequences corresponding to either the open reading frame or the open reading frame of the transcripts of these genes can be used to measure the expression levels of the genes.

[0028]

[0029] In the embodiment, the expression level of a gene may be the transcription level of the gene or the expression level of a protein encoded by the gene, but it is preferable to use the transcription level of the gene. The expression level of a gene in the embodiment can be measured by methods known to those skilled in the art, such as RNA-sequencing (RNA-seq) using a next-generation sequencer, quantitative reverse transcription PCR, microarray, Northern blotting, mass spectrometry, and Western blotting, but is preferably measured by RNA-seq or quantitative reverse transcription PCR, and from the viewpoint of rapid determination, is preferably measured by quantitative reverse transcription PCR.

[0030] Step S4 is a step of identifying the type of neutron radiation irradiated to (or exposed to) the algal culture from the measured value of (A) based on a predetermined relationship between the type of neutron radiation to which the algae is exposed and changes in the expression level of the marker gene. In this step, the "relationship between the type of neutron radiation to which the algae is exposed and changes in the expression level of the marker gene" can be a relationship in which an increase or decrease in the expression level of the marker gene corresponds to either thermal neutron radiation or high-energy neutron radiation to which the algae is exposed. Such a relationship can differ for each marker gene, and can be one of the following: an increase in expression level corresponds to the neutron radiation being thermal neutron radiation; a decrease in expression level corresponds to the neutron radiation being thermal neutron radiation; an increase in expression level corresponds to the neutron radiation being high-energy neutron radiation; or a decrease in expression level corresponds to the neutron radiation being high-energy neutron radiation.

[0031] Furthermore, in step S4, determining the type of neutron radiation may be determining that the irradiated neutron radiation is thermal neutron radiation or high-energy neutron radiation based on the predetermined relationship as described above, when the measured value of (A) of any one of at least one gene of the plurality of marker genes is lower or higher than the corresponding expression level in a control algal culture not exposed to neutron radiation.

[0032] The corresponding expression level in the control algal culture in step S4 can be the expression level in non-neutron-exposed algae measured in parallel with or prior to the neutron-exposed algae. In some embodiments, the method can include measuring the expression level (B) of the corresponding marker gene in a control algal culture that is not exposed to neutron radiation.

[0033] The method of the embodiment may include identifying the neutron radiation as thermal neutron radiation when the marker gene includes at least one gene selected from the group consisting of CMC010C, CMC125C, CMI306C, CMK006C, CMM184C, CMP092C, CMS431C, and CMT597C, and the measured (A) value of any of the at least one gene is increased by 1.5-fold or more, 2-fold or more, or 4-fold or more compared to the corresponding expression level in a control algal culture not exposed to neutron radiation. Specific methods for expressing an increase or decrease in expression level as a fold change are known to those skilled in the art. Alternatively, the neutron radiation may be identified as thermal neutron radiation when the (A) values ​​of one, two, three, four, five, six, seven, or eight of these genes are statistically significantly increased compared to the corresponding expression level in a control algal culture not exposed to neutron radiation.

[0034] The method of the embodiment may include identifying the neutron radiation as high-energy neutron radiation when the marker gene includes at least one gene selected from the group consisting of CMQ318C and CMT251C, and the measured value of (A) of any of the at least one gene is increased by 1.5-fold or more, 2-fold or more, or 4-fold or more above the corresponding expression level in a control algal culture not exposed to neutron radiation. Alternatively, the neutron radiation may be identified as high-energy neutron radiation when the value of (A) of one or two of these genes is statistically significantly increased above the corresponding expression level in a control algal culture not exposed to neutron radiation.

[0035] The method of the embodiment may include identifying the neutron radiation as high-energy neutron radiation when the marker gene includes at least one gene selected from the group consisting of CMF012C and CML339C, and the measured value of (A) of any of the at least one gene is reduced to at least two-thirds, at least one-half, or at least one-quarter of the corresponding expression level in a control algal culture not exposed to neutron radiation. Alternatively, the neutron radiation may be identified as high-energy neutron radiation when the value of (A) of one or two of these genes is statistically significantly reduced from the corresponding expression level in a control algal culture not exposed to neutron radiation.

[0036] In embodiments, whether there is a significant difference between the expression levels in the neutron-exposed algal cultures and the control algal cultures can be determined by statistical methods known to those skilled in the art, such as testing for significant differences in expression levels using a Student's t-test.

[0037] In step S4 of the method of the embodiment, expression levels in the control algae and the test algae may be expressed as relative values, such as the proportion of the marker gene in the entire transcriptome or the proportion of a standard gene, such as a cytoskeleton gene or a metabolic enzyme gene, whose expression does not change with neutron exposure. Based on the knowledge that the expression levels of marker genes expressed as such relative values ​​increase above the expression levels in the control algae upon neutron irradiation (or decrease, for example, in the case of CMF012C and CML339C), it is possible to determine whether the expression levels are higher or lower than the corresponding expression levels in the control algae culture without having to measure unexposed algae every time by simply measuring the test algae and determining the marker gene expression levels expressed as relative values ​​and determining whether they exceed the known levels in unexposed algae. For this purpose, the marker gene expression levels expressed as relative values ​​in unexposed algae can be calculated in advance based on the expression levels of standard genes and marker genes obtained in a separate experiment.

[0038] Next, referring to FIG. 2 , an apparatus and sample flow used in an example of a method for identifying the type of neutron radiation according to an embodiment will be described. Algae cultured in an algae culture apparatus are transferred to a centrifuge tube. The algae in the centrifuge tube are irradiated (exposed) to neutron radiation in a neutron radiation irradiation apparatus (or a neutron radiation source). The exposed algae are collected using an algae collection apparatus such as a centrifuge, and RNA is extracted from the algae using an RNA collection apparatus. The extracted RNA is used to analyze the expression levels of marker genes using a transcription level analysis apparatus such as a next-generation sequencer. At this time, measurement of the expression levels of standard genes may also be performed simultaneously. The type of neutron radiation is identified in the neutron radiation type identification apparatus based on the transcription level data obtained by the transcription level analysis apparatus and predetermined and stored data on the relationship between the neutron radiation type to which the algae are exposed and changes in the expression levels of marker genes. As will be understood by those skilled in the art, the type identification apparatus may be a general-purpose or dedicated computer. The type determination device may include means for outputting a determination result that the neutron radiation to which the algae culture was exposed is thermal neutron radiation or high-energy neutron radiation.

[0039] Examples of the present disclosure are described below, but the present disclosure is not limited to the examples described below.

[0040] <Materials and Methods> The unicellular red algae, Cyanidioschyzon merolae, was cultured in MA2 medium under CO2 (2% v / v) aeration. 750 (=0.1-0.2) and transferred 45 ml of each to multiple 50 ml centrifuge tubes. These tubes were irradiated with high-energy neutrons (absorbed dose 20 Gy) or thermal neutrons (absorbed dose 26 Gy). The neutron-irradiated cells were collected by centrifugation (3,000 rpm, room temperature, 5 minutes) and stored at -80°C. After thawing, total RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN), and the transcript levels were analyzed by RNA-Seq using a next-generation sequencer.

[0041] <Results and Discussion> Thermal neutron irradiation specifically increased the transcript levels of the genes CMC010C, CMC125C, CMI306C, CMK006C, CMM184C, CMP092C, CMS431C, and CMT597C (http: / / czon.jp / ) by more than fourfold compared to control algae not exposed to neutrons (Table 4). Furthermore, irradiation with high-energy neutrons also increased the transcript levels of other genes, namely CMQ318C and CMT251C, by more than fourfold compared to non-irradiated algae (Table 5). Furthermore, irradiation with high-energy neutrons specifically decreased the transcript levels of the genes CMF012C and CML339C by more than fourfold compared to non-irradiated algae (Table 6).

[0042]

[0043]

[0044]

[0045] The above results illustrate that in algae, it is possible to observe groups of genes whose expression changes differ depending on the type of neutron radiation to which they are exposed, and show that by measuring the amount of transcripts of genes that increase or decrease specifically depending on the type of neutron radiation to which algae are exposed, it is possible to quickly and easily identify the type of neutron radiation in question.

[0046] Although the present disclosure has been described with reference to the above several embodiments, the present disclosure is not limited to the above several embodiments. Various changes can be made to the configuration and details of the present invention within the scope of the present disclosure.

Claims

1. A method for discriminating the type of neutron beam, comprising: exposing an algal culture to the neutron beam; measuring the expression level (A) of a marker gene in the algal culture exposed to the neutron beam; and discriminating the type of the neutron beam from the measured value of (A) based on a previously determined relationship between the type of neutron beam to which the algae is exposed and the change in the expression level of the marker gene.

2. The method according to claim 1, wherein the marker gene comprises at least one gene selected from the group consisting of CMC010C, CMC125C, CMI306C, CMK006C, CMM184C, CMP092C, CMS431C, and CMT597C, and when the measured value of (A) of any of the at least one gene is higher than the corresponding expression level in a control algal culture not exposed to neutron beam, it is discriminated that the neutron beam to which the algal culture is exposed is a thermal neutron beam.

3. The method according to claim 1, wherein the marker gene comprises at least one gene selected from the group consisting of CMQ318C and CMT251C, and when the measured value of (A) of any of the at least one gene is higher than the corresponding expression level in a control algal culture not exposed to neutron beam, it is discriminated that the neutron beam to which the algal culture is exposed is a high-energy neutron beam.

4. The method according to claim 1, wherein the marker gene comprises at least one gene selected from the group consisting of CMF012C and CML339C, and when the measured value of (A) of any of the at least one gene is lower than the corresponding expression level in a control algal culture not exposed to neutron beam, it is discriminated that the neutron beam to which the algal culture is exposed is a high-energy neutron beam.

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