| 研究課題/領域番号 |
20K08832
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| 研究機関 | 国立研究開発法人理化学研究所 |
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研究代表者 |
アブケセーサ イマド 国立研究開発法人理化学研究所, 生命医科学研究センター, 上級研究員 (10749906)
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| 研究期間 (年度) |
2020-04-01 – 2023-03-31
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| キーワード | Mycology / Galleria mellonella / Mycetoma / Eumycetoma / Antifungal / Madurella mycetomatis / RNA-Seq |
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| 研究実績の概要 |
1-Madurella mycetomatis genome strain was used to infect Galleria mellonella larvae. The larvae were infected with a final concentration of 4mg wet weight fungus per larvae.2-Larvae were treated at 4, 24 and 48 hours after infection, with itraconazole 400 mg/kg and ravuconazole 300 mg/kg. These concentrations were chosen as they resemble the concentrations used in human for itraconazole.3-We determined the toxicity of these drugs for the larvae. For that we treated uninfected larvae and monitor their survival for 5 days. At this stage no toxicity was noted in thedrug concentrations tested.
4-Larvae were infected with M. mycetomatis via the last left proleg and treated with [400 mg/kg] itraconazole or [300 mg/kg] ravuconazole for three days.5-Survival of the larvae was monitored for a duration of 10 days after infection.6-Histology slides have been prepared, stained with HE& Grocott, for grain counts analysis.7-RNA isolation was performed according the previously described methodology. In short, Larval content was harvested and frozen using liquid nitrogen, followed by mechanical crushing using a pestle and mortar. The crushed powder was collected and used for RNA extracted, using the TRIZOL Chloramphenicol method.8-RNA QC was performed using the Nanodrop and measured all RNA standard QC values. Samples were stored at -80oC.9-In total we have prepared 72 samples for RNA-Seq library preparation.For each sample type we have four timepoints and in each timepoint we have triplicate therefore. All samples passed RNA QC check.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
We recovered from the delay for some tasks in FY2020 (due to the lock-dwon and pandemic)
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| 今後の研究の推進方策 |
1- Histology slides have been prepared, stained with HE& Grocott, for grain counts analysis. Grain counts are ongoing according previous established methodology [PMID: 29698504].
2- Determination of the pharmacokinetics(PK) will be performed according previously methodology at time points 5 min, 30 min, 1h, 2h, 4h, 6h and 24h [PMID: 28992315]. Currently Hemolymph of larvae, treated with the respective concentrations, is stored for further processing. This will be continued in the near future.
3- RNA-Seq library preparation
4- Sequencing of RNA library
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| 次年度使用額が生じた理由 |
Due to the impact of the COVID-19 lockdown, we asked to Incurring FY2021 budget to FY2022. The Incurring will be used for RNA-Seq library preparation, Library QC and RNA sequencing as planned.
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