Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

金沢大学子どものこころの発達研究センターEfficient screening of unknown DNA variations is one of the substantive matters of molecular biology even today. Historically, SSCP and heteroduplex analysis (HA) are the most commonly used methods for detecting DNA variations everywhere in the world because of their simplicity. However, the sensitivity of these methods is not satisfactory for screening purpose. Recently, several new PCR-based mutation screening methods have been developed, but most of them require special instruments and adjustment of conditions for each DNA sequence to attain the maximum sensitivity; eventually becoming as inconvenient as old methods. Enzyme mismatch cleavage (EMC) is potentially an ideal screening method. With high-performance nucleases and once experimental conditions are optimized, it requires only conventional staff and conditions remain the same for each PCR product. In this study we tested four commercially available endonucleases for EMC and optimized the electrophoresis and developing conditions. We prepared 25 known DNA variations consisting of 18 single base substitutions (8 transitions and 10 transversions, including all possible sets of mismatches) and 7 small deletions or insertions. The combination of CEL nuclease, 12% PAGE and rapid silver staining can detect all types of mutations and achieved 100% sensitivity. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Quantitative environmental DNA metabarcoding shows high potential as a novel approach to quantitatively assess fish community

水に含まれる環境DNAから「どんな魚」が「どれだけいるか」を同時に推定 --定量的な魚類群集モニタリングを容易に実現--. 京都大学プレスリリース. 2023-01-20.The simultaneous conservation of species richness and evenness is important to effectively reduce biodiversity loss and keep ecosystem health. Environmental DNA (eDNA) metabarcoding has been used as a powerful tool for identifying community composition, but it does not necessarily provide quantitative information due to several methodological limitations. Thus, the quantification of eDNA through metabarcoding is an important frontier of eDNA-based biomonitoring. Particularly, the qMiSeq approach has recently been developed as a quantitative metabarcoding method and has attracted much attention due to its usefulness. The aim here was to evaluate the performance of the qMiSeq approach as a quantitative monitoring tool for fish communities by comparing the quantified eDNA concentrations with the results of fish capture surveys. The eDNA water sampling and the capture surveys using the electrical shocker were conducted at a total of 21 sites in four rivers in Japan. As a result, we found significant positive relationships between the eDNA concentrations of each species quantified by qMiSeq and both the abundance and biomass of each captured taxon at each site. Furthermore, for seven out of eleven taxa, a significant positive relationship was observed between quantified DNA concentrations by sample and the abundance and/or biomass. In total, our results demonstrated that eDNA metabarcoding with the qMiSeq approach is a suitable and useful tool for quantitative monitoring of fish communities. Due to the simplicity of the eDNA analysis, the eDNA metabarcoding with qMiSeq approach would promote further growth of quantitative monitoring of biodiversity

Ixia から分離された bean yellow mosaic virus

A strain (Ixia-B) of bean yellow mosaic virus (BYMV) isolated from Ixia hybrida was characterized and compared with other isolates of BYMV and clover yellow vein virus (CYVV). Ixia-B was transmitted by aphids,Myzus presicae in a non-presistent manner and by sap-inoculation to 11 of 46 species in 5 of 10 families tested, and had a similar host range to that of some BYMV isolates, althrough some defferences were detected. Sap from diseased C. quinoa was infective after 10 min heating at 55℃ but not 60℃, after a dilution to 10-3 but not 10-4, and after 2 days but not 4 days at 20℃.The Virus particles were filamentous rods of about 13×820 nm. Ixia-B contaied a single protein species with a molecular weight of 34,000 and a single viral RNA with approximately 9,000 bases. In ultrahtin sections of leaf tissues from infected plants, the virus particles, cylindrical cytoplasmic inclusions and dense bodies were obsserved in the cytoplasm. The antiserum to Ixia-B produced by immunizing a rabbit had a titer of 1/512. A close serological relationship was revealed between Ixia-B and two strains of BYMV from crocus and gladiolus, but no relationship to clover yellow vein virus was found in agar gel diffusion tests. However,Ixia-B could be distinguished from two strains of BYMV by the formation of spurs among them in agar gel and by the differences in the patterns of peptide mapping of coat proteins. From these findings, Ixia-B was identified as a strain of BYMV.1992年に岡山県倉敷市玉島で、葉に斑入りを生じた球根類花卉植物Ixia hybridaからpotyvirus(Ixia-B)が分離され、その諸性質から　bean yellow masaic virus(BYMV)と同定された。本ウイルスを11科47種の植物に接種したとこと、フリージャ、Nicotiana clevelandii、Chenopodium amaranticolar、ソラマメ、クリムソンクローバー、インゲンマメ、ホウレンソウに全身感染し、またC.quinoa、フダンソウ、ツルナ、センニチコウなどに局部感染したが、エンドウ、ササゲ、ダイズなどには感染しなかった。本ウイルスをはモモアカアブラムシにより非永続的伝搬され、C.quinoa の病葉粗汁液中での安定性は耐熱性が55℃～60℃（10分）、耐希釈性10-3～10-4、耐保存性2～4日であった。DN法試料の電顕観察で多くのpotyvirusよりやや長い約820nmのひも状粒子と管状封入体の破片が見られた。感染葉の超薄切片では風車状、層板状の封入体、dence body、細胞質に散在するウイルス粒子が観察された。本ウイルスはfreesia mosaic virusおよびclover yellow vein virusの抗血清と反応せず、また本ウイルス抗血清を用いた寒天ゲル内二重拡散法ではBYMV分離株（Cro-4, BYMV-G）と反応したが、本ウイルスとBYMVのCro-4およびBYMV-G間にspurが形成され、異種抗原の存在が認められた。外被タンパク質の分子量は約34Kで、ssRNAのサイズは約9Kbであった。Papain,chymotrypsin,pepsinを用いた外被タンパク質のぺプタイドマッピングでは、本ウイルス、Cro-4、BYMV-G、Cal-35の部分分解パターンがそれぞれ異なり、外被タンパク質がアミノ酸配列レベルで異なっていることが示唆された

Phellinus Linteus Extract Sensitizes Advanced Prostate Cancer Cells to Apoptosis in Athymic Nude Mice

Phellinus linteus (PL) mushroom possesses anti-tumor property. We previously reported that the treatment with PL caused cultured human prostate cancer cells to undergo apoptosis. To further studying the mechanisms of PL-mediated apoptosis, we performed xenograft assay, together with in vitro assays, to evaluate the effect of PL on the genesis and progression of the tumors formed from the inoculation of prostate cancer PC3 or DU145 cells. After the inoculation, nude mice were injected with PL every two days for 12 days. Although PL treatment did not prevent the formation of the inoculated tumors, the growth rate of the tumors after PL treatment was dramatically attenuated. We then tested the effect of PL on the tumors 12 days after the inoculation. After inoculated tumors reached a certain size, PL was administrated to the mice by subcutaneous injection. The histochemistry or immunochemistry analysis showed that apoptosis occurred with the activation of caspase 3 in the tumors formed by inoculating prostate cancer DU145 or PC3 cells. The data was in a good agreement with that from cultured cells. Thus, our in vivo study suggests that PL not only is able to attenuate tumor growth, but also to cause tumor regression by inducing apoptosis

Ornithine Decarboxylase Antizyme Induces Hypomethylation of Genome DNA and Histone H3 Lysine 9 Dimethylation (H3K9me2) in Human Oral Cancer Cell Line

Background: Methylation of CpG islands of genome DNA and lysine residues of histone H3 and H4 tails regulates gene transcription. Inhibition of polyamine synthesis by ornithine decarboxylase antizyme-1 (OAZ) in human oral cancer cell line resulted in accumulation of decarboxylated S-adenosylmethionine (dcSAM), which acts as a competitive inhibitor of methylation reactions. We anticipated that accumulation of dcSAM impaired methylation reactions and resulted in hypomethylation of genome DNA and histone tails. Methodology/Principal Findings: Global methylation state of genome DNA and lysine residues of histone H3 and H4 tails were assayed by Methylation by Isoschizomers (MIAMI) method and western blotting, respectively, in the presence or absence of OAZ expression. Ectopic expression of OAZ mediated hypomethylation of CpG islands of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2). Protein level of DNA methyltransferase 3B (DNMT3B) and histone H3K9me specific methyltransferase G9a were down-regulated in OAZ transfectant. Conclusions/Significance: OAZ induced hypomethylation of CpG islands of global genome DNA and H3K9me2 by down-regulating DNMT3B and G9a protein level. Hypomethylation of CpG islands of genome DNA and histone H3K9me2 is a potent mechanism of induction of the genes related to tumor suppression and DNA double strand break repair

Power System Development of the AGU Remote Innovative CubeSat Alert System -2 – ARICA-2

We present the power system development of the 2U CubeSat, AGU Remote Innovative CubeSat Alert system -2(ARICA-2). The main goal of the ARICA-2 project is to demonstrate the real-time alert system of transient astronomical sources using commercial satellite network devices. 1U CubeSat ARICA was launched in November 2021. However, we have not been able to send and receive the data at this point. Therefore, we started developing 2U CubeSat ARICA-2, which is an improved version of ARICA, in April 2022. One of the possible causes of the communication problem of ARICA is the power system, such as a negative power budget or a failure in the installation of the inhibit switches. ARICA-2 is upsized from 1U to 2U to ensure a sufficient power generation and is equipped with improved inhibit switches. The calculation of power consumption and simulation of power supply on orbit have been finished. We confirmed the performance of our Electric Power System (EPS) and the health of the installed batteries. We are currently in the EM development phase with the goal of launching in Japanese fiscal year 2024

Development of the ARICA-2 Satellite Using Spresense as an Onboard Computer

Gamma-ray bursts (GRBs) are transient astronomical phenomena that emit enormous amounts of energy in electromagnetic waves, mainly in the gamma-ray range, for several seconds to tens of seconds. GRB observations are challenging because of the difficulty in predicting the location and time of occurrence and its extremely short duration. Therefore, it is necessary to notify about the discovery in space and to conduct follow-up observations by researchers. The AGU Remote Innovative CubeSat Alert system-2 (ARICA-2) has been developed to demonstrate a new alert system using commercial satellite network services. ARICA-2 uses SONY’s Spresense as its onboard computer (OBC). We manufactured the special board to attach two Spresenses as a redundancy of the OBC system. We will present the system development of ARICA-2 using Spresense

Overview and Status of AGU Remote Innovative Cubesat Alert System-2 on 2023

We present the overview of the 2U CubeSat, AGU Remote Innovative Cubesat Alert system - 2 (ARICA-2). ARICA-2 was selected as a feasibility study phase of the JAXA-Small Satellite Rush Program (JAXA-SMASH) and the JAXA Innovative Satellite Technology Demonstration-4 project in 2022. The main goal of ARICA-2 is to demonstrate the real-time alert system of transient astronomical sources, such as gamma-ray bursts, using commercial satellite network services. The first 1U CubeSat ARICA, which had the same mission goal as ARICA-2, was successfully launched in 2021 by the JAXA’s Epsilon rocket No.5. However, communication with ARICA has yet to be established due to severe hardware issues. Therefore, ARICA-2 is the re-challenging mission of ARICA. ARICA-2 has several different features compared to ARICA. First, a transceiver using amateur radio frequency is added to the commercial satellite network devices to communicate directly from the ground. Second, ARICA-2 uses Sony’s low-power board Spresense as an onboard computer. Third, the attitude control system using magnetorquer is installed to establish better communication with the commercial network satellites. Fourth, the size of a gamma-ray detector is 70 mm x 70 mm x 10 mm, which is larger by a factor of 200 in volume compared to ARICA, to enhance the detection rate of gamma-ray bursts. We plan to develop the engineering model (EM) in 2023 and perform thermal vacuum and vibration tests on the EM. We report the current status and a prospect of ARICA-2