全國中小學科展

生物化學

菇光十色-秀珍菇中醣類與蛋白質含量探討

本研究選擇秀珍菇作為研究的題材,藉由不同波長(白光、藍光、綠光、紅光)與不同瓦數(9W、3W、1W)低溫型 LED 燈照射(每日12小時,共5天)菌菇太空包,發現在高瓦數(9W)的燈照下其生長最快、3W次之、1W最慢;另在藍光下,秀珍菇生長最茂盛-蕈柄最為茂密粗壯、蕈傘顏色最深,次為白光、再者綠光、紅光下的生長則最慢-蕈柄較稀疏且細、蕈傘顏色最淺。 接續將採收的秀珍菇(以低溫烘乾<40℃),取單位乾重(0.5公克)分別檢測其蛋白質及醣類的含量進行比較,其結果發現蕈菇在紅光下生長其含醣量較高、綠光次之、再者白光、末者藍光;而蛋白質含量由高至低依序為藍光、白光、綠光、紅光;相同色光不同瓦數下的含量分析結果顯示,含醣量以低瓦數較佳,蛋白質含量以高瓦數較佳。

香豆素(Coumarin)及其衍生物對人類胰島類澱粉蛋白(IAPP)聚集之影響

目前已有研究指出:胰島類澱粉蛋白(islet amyloid polypeptide;簡稱IAPP)的不正常堆疊與聚集,與罹患第二型糖尿病有很大的關聯。本研究使用一系列有機物香豆素(coumarin)分子,觀測其對胰島類澱粉蛋白聚集之影響;我們成功地自行合成出高純度的IAPP分子,將其與經光學性質篩選過的香豆素有機物,進行硫磺素-T動力學試驗,並輔以電子顯微鏡、圓偏光二色光譜進一步瞭解有機小分子對於IAPP結構之影響。實驗結果得知:具較高極性基團或取代基之香豆素系列分子,可抑制IAPP聚集成纖維;且具特定官能基結構者可有效改變IAPP之纖維型態。期待透過本研究探討有機分子與蛋白質作用之機制,進一步為糖尿病病症提供治療方法與途徑。

針對漸凍症(ALS)致病蛋白TDP-43纖維的專一性抗體篩選與研究

在漸凍症(ALS)以及額顳葉退化症(FTLD)等神經疾病中,病患體內會有不正常的類澱粉蛋白TDP-43錯誤堆疊,在不同的堆積階段有單體(monomer)、多倍體(oligomer)、纖維(fiber)等不同的形態。目前在相關的疾病中,並沒有有效藥物及生物標誌(Biomarker)可以治療及進行檢測。在本研究中我利用由單株抗體技術所製造出對應TDP-43纖維的不同抗體,進行純化及濃縮,並透過酵素結合免疫吸附分析法(ELISA)鑑定這些抗體對於TDP-43不同型態的親和力,並找出專一性最高、結合效率最好的抗體,以作為疾病不同階段的生物標誌(Biomarker),且希望可以以此來預測疾病進展並評估對治療的反應。

利用 CRISPR/Cas 技術重建複雜的DNA 修復機制

本研究利用合成生物學技術開發的創新基因組篩檢平台PRISM(v2.0),採用合成轉錄因子(crispr-TF)辨認DNA但不進行剪切,透過修改CRISPR/Cas的PAM-interacting (PI) domain序列打破其辨認限制,由原本NGG序列變成可以結合NAG的序列,提升crispr-TF在高通量基因組的篩選能力,期待未來可以做出辨認NNN序列,以此技術初步成功建構Chk2/Rad53所參與的基因網絡,讓失去Chk2/Rad53功能的酵母菌能在DNA損傷的情況生存。Chk2/Rad53蛋白磷酸酶在DNA修復的訊息傳遞中調控龐大的基因網絡,其中潛在大量不同調控路徑聯結的組合性,過去的研究都無法有效地分析且重建完整的DNA修復機制。未來將進一步探討Chk2/Rad53調控的下游基因組,並以此新技術和研究應用於複雜腫瘤與老化疾病的預防及治療。

BIOINFORMATIC PREDICTION OF CORONAVIRUS (SARS-COV-2) MUTATIONS THAT INCREASE CONTAGIOUSNESS

Inhibitory effects of the secondary metabolite of actinomycete were examined on cell cycle of the yeasts of S. pombe and S. cerevisiae. The secondary metabolite was obtained from cultivation of the actinomycete isolated from the soil of Owakudani in Hakone, Japan. The fifth fraction of the secondary metabolite by ODS column separation (HK-T5), which was soluble to pure methanol, was used in the present experiments. The HK-T5 brought about the delay of forming colonies of S. pombe for about 11 days compared to that cultivated without the HK-T5. The delay of the colony formation was longer for the S. pombe cultivated with more amount of the HK-T5. The cultivation with HK-T5 also brought about the extension of the lifespan of the S. pombe for more than 10 weeks in a liquidus medium. The cell life recovered the ordinary manner by removal of the HK-T5, meaning that the activities of the HK-T5 is reversible. These facts confirm the suppression of cell cycle, and the delay of cell growth by the HK-T5. These phenomena were similarly observed for S. cerevisiae. Comparison of the action of HK-T5 with hydroxyurea, which is an anticancer drug inhibiting the cell cycle at S phase, clarified that the inhibitory action of HK-T5 worked at the phase earlier than S phase. The combined effects of HK-T5 on the cell cycle were evaluated with triamcinolone acetonide (TA), or aspirin, the former of which is a drug synchronizing cancer cells in S phase, and the latter keeping human cells in G1/G0 phases. The combined use of HK-T5 with TA synchronized the cells at the phase slightly proceeding from G1 to S phase without toxicity. On the other hand, the combined use with aspirin made the inhibitory effect of HK-T5 inactive. Hence, the HK-T5 is attractive as a drug for the extension of cell lifespan, and anticancer therapy.

BIOINFORMATIC PREDICTION OF CORONAVIRUS (SARS-COV-2) MUTATIONS THAT INCREASE CONTAGIOUSNESS

Inhibitory effects of the secondary metabolite of actinomycete were examined on cell cycle of the yeasts of S. pombe and S. cerevisiae. The secondary metabolite was obtained from cultivation of the actinomycete isolated from the soil of Owakudani in Hakone, Japan. The fifth fraction of the secondary metabolite by ODS column separation (HK-T5), which was soluble to pure methanol, was used in the present experiments. The HK-T5 brought about the delay of forming colonies of S. pombe for about 11 days compared to that cultivated without the HK-T5. The delay of the colony formation was longer for the S. pombe cultivated with more amount of the HK-T5. The cultivation with HK-T5 also brought about the extension of the lifespan of the S. pombe for more than 10 weeks in a liquidus medium. The cell life recovered the ordinary manner by removal of the HK-T5, meaning that the activities of the HK-T5 is reversible. These facts confirm the suppression of cell cycle, and the delay of cell growth by the HK-T5. These phenomena were similarly observed for S. cerevisiae. Comparison of the action of HK-T5 with hydroxyurea, which is an anticancer drug inhibiting the cell cycle at S phase, clarified that the inhibitory action of HK-T5 worked at the phase earlier than S phase. The combined effects of HK-T5 on the cell cycle were evaluated with triamcinolone acetonide (TA), or aspirin, the former of which is a drug synchronizing cancer cells in S phase, and the latter keeping human cells in G1/G0 phases. The combined use of HK-T5 with TA synchronized the cells at the phase slightly proceeding from G1 to S phase without toxicity. On the other hand, the combined use with aspirin made the inhibitory effect of HK-T5 inactive. Hence, the HK-T5 is attractive as a drug for the extension of cell lifespan, and anticancer therapy.

AsqJ酵素活性庫之篩選並探討其專一性與反應性之改變

非血基質鐵/α-酮戊二酸依賴型雙氧化酶為巨大酵素家族,其在生物體內進行許多重要反應。我們選用小巢狀麴菌之 AsqJ 蛋白,其與 Fe2+ 及 α-酮戊二酸能催化 cyclopeptin 的去飽和、環氧化,形成 cyclopenin。根據 AsqJ 蛋白結構,我們將與受質結合的 9 個胺基酸位置以不同鹼基對隨機取代,以轉譯不同胺基酸,構築酵素活性庫,送入大腸桿菌篩選並觀察 AsqJ 突變蛋白分解非典型受質之表現。 我們成功篩選出能分解 X-Gal 之 AsqJ 突變株,代表我們能以改變 AsqJ 特定位置胺基酸,進而改變其活性位點,並分解與原先截然不同的受質,故此篩選平台深具生化技術應用潛力。我們並進一步將上述突變株進行純化、晶體培養及結構解析,以釐清反應機制,並與奈米抗體結合以增強活性。目前正嘗試分解環境汙染物結構相似物,期望對環境做出貢獻。

利用虛擬篩選LpxC抑制劑的方式找出對抗多重抗藥性綠膿桿菌的新療法

多重抗藥性(MDR)細菌已經在全世界的範圍內成為了一個重大威脅,而像是多重抗藥性的綠膿桿菌就是其中一種對大多數療法有抵抗力的病原體。在目前的治療方案無效之前,有必要開發出一種新型機制的抗生素能夠作為對抗的手段。我們通過電腦虛擬篩選的方式,並用一個脂多糖脂質A (LipidA)生合成路徑的關鍵蛋白,LpxC,作為篩選的對象。在我們的第一次預測中,ZINC000001587011 (brequinar) 具有較低的結合能和較高的生物利用度。但由於其較高的cLogP值,使我們對其進行了官能團修飾,以期能有所改善。最後,我們在所有衍生物中找到了N11,有最大的潛力能做為抗綠膿桿菌的藥物前驅物。

PMCA 技術在漸凍症(ALS)致病蛋白TDP-43 纖維之高靈敏度偵測開發及應用

TDP-43是漸凍症(ALS)以及額顳葉失智症(FTLD)等神經退化性疾病的致病蛋白,在患者腦內會有不正常的類澱粉蛋白TDP-43錯誤折疊而產生蛋白質堆積,而不同堆積階段依序為單體(monomer)、多倍體(oligomer)、纖維(fiber)。其中纖維為許多神經退行性疾病的病理標誌,且相較於已有的 TDP-43 oligomer 單株抗體(Yu-Sheng Fang et al.(2014)),目前沒有對於 TDP-43 纖維抗體的研究,因此本實驗利用單株抗體技術所製造出對應TDP-43纖維的不同抗體進行純化及濃縮,找出對TDP-43 fiber專一性最高、結合效率及結合率皆最好抗體,以作為能夠檢測漸凍症患者血液中TDP-43 fiber的生物標誌(Biomarker)。 同時為了增加偵測的靈敏度,本實驗成功創新利用PMCA ( protein misfolding cyclic amplification) 技術快速放大纖維的數量以提高檢測準確度,搭配高專一性、高結合效率的編號3-2自製抗體作為Biomarker,只需五個小時便可以將極少量纖維的訊號約提高至原先的17倍,具有極高的靈敏性可以準確辨認出有無TDP-43纖維的樣本差別。

DEVELOPMENT OF PAPER-BASED ORIGAMI BIOSENSOR PLATFORMS FOR COLORIMETRIC DETECTION OF BIOCONTAMINANTS

Infectious diseases caused by bacteria from biological pollutants pose a great burden in terms of diagnosis and treatment, and millions of people worldwide die from bacterial infections. Detection of bacteria plays a critical role in clinical diagnosis and control of contamination, but is not accessible due to the high cost, complex devices and equipment required. In the project, an alternative to existing methods, a paper-based biosensor for the detection of model organism E. coli bacteria, which is visible, low cost, easy to use, can be integrated with a smartphone, is based on rapid color change in the exposed environments, drinking and pool water, wastewater, beverage products. platforms were developed. For the specific detection of E.coli bacteria, two different biosensors have been developed that can perform colorimetric detection in a user-friendly origami design, minimizing microchip and processing steps based on antibody-bound PVDF membrane and filter paper-based immunological method. In the presence and absence of target bacteria E.coli, the lowest detection limit of the biosensors obtained by using paper-based platforms that create a distinctive color on them, depending on the concentration, was 0.9x103 bacteria/ml for origami biosensor, 2.7x103 bacteria/ml for microchip biosensor and the widest dynamic linear operating range was calculated as 103-107 bacteria/ml. With the biosensor platforms we have developed, the use of only one smartphone for both qualitative and quantitative, visible results and analysis within minutes constitutes the originality of our project. With these promising results, the biosensors we have developed can also be used for the detection of different biological pollutants, do not contain complex devices and can be easily produced in large scales. We believe that the biosensors we have developed for the detection of biological pollutants in water and beverages, especially in regions where test laboratory infrastructure is not available, will contribute to the literature, public health, health economy and sustainable development goals such as clean water and sanitation, health and quality life, and life in water.