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H.-F. Chang, S.-L. Wang*, D.-C. Lee, S. S.-Y. Hsiao, Y. Hashimoto, and K.-C. Yeh* (2020) Assessment of indium toxicity to the model plant Arabidopsis. Journal of Hazardous Materials doi.org/10.1016/j.jhazmat.2019.121983
kcyeh The use of indium in semiconductor products has increased markedly in recent years. The release of indium into the ecosystem is inevitable. Under such circumstances, effective and accurate assessment of indium risk is important. An indispensable aspect of indium risk assessment is to understand the interactions of indium with plants, which are fundamental components of all ecosystems. Physiological responses of Arabidopsis thaliana exposed to indium were investigated by monitoring toxic effects, accumulation and speciation in plants. It was found that indium jeopardized phosphate uptake and translocation by inhibiting the accumulation of phosphate transporters PHOSPHATE TRANSPORTER1 (PHT1;1/4), responsible for phosphate uptake, and PHOSPHATE1 (PHO1), responsible for phosphate xylem loading.

Biljana Cvetanova, Ya-Ching Shen*, and Lie-Fen Shyur* (2019) Cumingianoside A, a phyto-triterpenoid saponin inhibits acquired BRAF inhibitor resistant melanoma growth via programmed cell death. Frontiers in Pharmacology. doi:10.3389/fphar.2019.00030
LFS Mutated proto-oncogene BRAF is a bona fide therapeutic target for melanomas. Regrettably, melanoma acquires resistance to BRAF inhibitors, e.g., vemurafenib (PLX4032) casting doubt on this promising melanoma targeted therapy. In this study, we explored the bioactivity of triterpenoid saponin cumingianoside A (CUMA), isolated from leaves and twigs of Dysoxylum cumingianum against PLX4032-resistant BRAFV600E mutant melanoma A375-R in vitro and in vivo. Our data show that CUMA treatment inhibited A375-R melanoma cell proliferation in a time- and dose-dependent manner. CUMA also suppressed the activity of CDK1/cyclin B1 complex and led to G2/M-phase arrest of A375-R cells. Furthermore, CUMA treatment resulted in induction of apoptosis as shown by the increased activation of caspase 3 and caspase 7, and the proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). We also observed that CUMA induced autophagy-like activity in A375-R cells, as shown by the increased expression of autophagy-related genes and increased formation of autophagosomes. Moreover, we found that CUMA treatment induced ER stress response and co-treatment with an ER stress inhibitor (4-PBA) could attenuate apoptosis induced by CUMA. Importantly, orally administered CUMA as a single agent or in combination with PLX4032 exhibited strong tumor growth inhibition in a PLX4032-resistant A375-R xenograft mouse model, and with little toxicity. This is the first report to explore the anti-tumor activity of CUMA in vitro and in vivo mechanistically, and our results imply that this triterpenoid saponin may be suitable for development into an anti-melanoma agent.

Chih-Ting Chang, Wen-Ni Soo, Yu-Hsin Chen and Lie-Fen Shyur*
(2019) Essential oil of Mentha aquatic var. Kenting Water Mint suppresses two-stage skin carcinogenesis accelerated by BRAF inhibitor vemurafenib. Molecules, 2019, 24, 2344

lfs Epidemiological evidence reveals that a BRAF inhibitor (BRAFi) drug vemurafenib (PLX4032) for treating late-stage melanoma could cause cutaneous squamous cell carcinoma in cancer patients with the most prevalent HRASQ61L mutation. In a two-stage skin carcinogenesis mouse model, the skin papillomas induced by 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) resemble the lesions in BRAF inhibitor-treated patients. We investigated the bioactivity of Mentha aquatica var. Kenting Water Mint essential oil (KWM) against PDV cells, mouse keratinocytes bearing HRASQ61L mutation, and its effect on inhibiting papilloma formation in a two stage skin carcinogenesis mouse model, with or without PLX4032 co-treatment. The results revealed that KWM-EO effectively attenuated cell viability, colony formation, and the invasive and migratory abilities of PDV cells. KWM-EO treatment significantly decreased the formation of cutaneous papilloma in DMBA/TPA mice or in BRAFi PLX4032 further irritated DMBA/TPA mice (DMBA/TPA/BRAFi). Immunohistochemistry analyses showed that infiltration of inflammatory cell infiltration (neutrophils/elastase, macrophages/F4/80), and overexpression of keratin14 and COX-2 in DMBA/TPA and DMBA/TPA/BRAFi mice skins were profoundly suppressed by KWM-EO treatment. This study demonstrates that KWM-EO has chemopreventive effects against PLX4032-induced cutaneous side-effects in a DMBA/TPA-induced two-stage carcinogenesis model and that is worth further exploration for possible application in melanoma patients.

Wen-Wan Chao, Ya-Wen Cheng, Yet-Ran Chen, Shu-Hua Lee, Ching-Yi Chiou, and Lie-Fen Shyur* (2019) Phyto-sesquiterpene lactone deoxyelephantopin and cisplatin synergistically suppress lung metastasis of B16 melanoma in mice with reduced nephrotoxicity Phytomedicine, 2019, 56: 194–206; US patent US9173868.
LFS Cisplatin (CP) is a chemotherapeutic drug for treating melanoma that also causes adverse side effects in cancer patients. This study investigated the bioefficacy of a phytoagent deoxyelephantopin (DET) in inhibiting B16 melanoma cell activity, its synergism with CP against metastatic melanoma, and its capability to attenuate CP side effects in animals. In vivo bioluminescence imaging was used to detect lung metastasis of B16 cells carrying COX-2 reporter gene in syngeneic mice. Nephrotoxicity caused by CP treatment in mice was evaluated by UPLC/ESI-QTOF MS based metabolomics and haematometry. DET, alone or in combination with cisplatin, inhibited B16 cell proliferation, migration, and invasion, and induced cell-cycle arrested at the G2/M phase and de-regulated cell-cycle mediators in cancer cells. In a murine B16COX-Luc metastatic allograft model, CP2 (2 mg/kg) treatment inhibited B16 lung metastasis accompanied by severe body weight loss, renal damage and inflammation, and haematological toxicity. DET10 and CP cotreatment (DET10+CP1) or sequential treatment (CP2_DET10) significantly inhibited formation of pulmonary melanoma foci and reduced renal damage. DET pretreatment (Pre-DET10) or CP2_DET10 treatment had the longest survival (52 vs. 37 days for tumor control mice). CP treatment caused abnormally accumulated urea cycle metabolites and serotonin metabolite hippuric acid in renal tissues that were not seen with DET alone or in combination with CP. The CP and DET combination may be an effective intervention for melanoma with reduced side effects.

Hsu CT, Cheng YJ, Yuan YH, Hung WF, Cheng QW, Wu FH, Lee LY, Gelvin SB, Lin CS. (2019) Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco. Plant Mol Biol. 2019 Nov;101(4-5):355-371.
cslin Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.

Liao, Ya-Yun, Li, Jia-Ling, Pan, Rong-Long* and Chiou, Tzyy-Jen* (2019) Structure-Function Analysis Reveals Amino Acid Residues of Arabidopsis Phosphate Transporter AtPHT1;1 Crucial for Its Activity. Frontiers in Plant Science 10:1158
tjc Phosphorus (P), an essential plant macronutrient, is acquired in the form of inorganic phosphate (Pi) by transporters located at the plasma membrane of root cells. To decipher the Pi transport mechanism, Arabidopsis thaliana Pi transporter 1;1 (AtPHT1;1), the most predominantly H+-coupled Pi co-transporter in the root, was selected for structure-function analysis. We first predicted its secondary and tertiary structures on the basis of the Piriformospora indica Pi transporter (PiPT) and identified 28 amino acid residues potentially engaged in the activity of AtPHT1;1. We then mutagenized these residues into alanine and expressed them in the yeast pam2 mutant defective in high-affinity Pi transporters and Arabidopsis pht1;1 mutant, respectively, for functional complementation validation. We further incorporated the functional characterization and structure analyses to propose a mechanistic model for the function of AtPHT1;1. We showed that D35, D38, R134 and D144, implicated in H+ transfer across the membrane, and Y312 and N421, involved in initial interaction and translocation of Pi, are all essential for its transport activity. When Pi enters the binding pocket, the two aromatic moieties of Y145 and F169 and the hydrogen bonds generated from Q172, W304, Y312, D308, and K449 can build a scaffold to stabilize the structure. Subsequent interaction between Pi and the positive residue of K449 facilitates its release. Furthermore, D38, D93, R134, D144, D212, R216, R233, D367, K373, and E504 may form internal electrostatic interactions for structure ensemble and adaptability. This study offers a comprehensive model for elucidating the transport mechanism of a plant Pi transporter.

Hsing‐Yi Cho, Elena Loreti, Ming‐Che Shih, Pierdomenico Perata (2019) Energy and Sugar Signaling during Hypoxia. New Phytologist https://doi.org/10.1111/nph.16326
MCS The major consequence of hypoxia is a dramatic reduction in energy production. At the onset of hypoxia, both oxygen and ATP availability decrease. Oxygen and energy sensing therefore converge to induce an adaptive response at both the transcriptional and translational levels. Oxygen sensing results in stabilization of the transcription factors that activate hypoxia-response genes, including enzymes required for efficient sugar metabolism, allowing plants to produce enough energy to ensure survival. The translation of the resulting mRNAs is mediated by SnRK1, acting as an energy sensor. However, as soon as the sugar availability decreases, a homeostatic mechanism, detecting sugar starvation, dampens the hypoxia-dependent transcription to reduce energy consumption and preserves carbon reserves for regrowth when oxygen availability is restored.

Ying-Lan Chen, Wei-Hung Chang, Chi-Ying Lee and Yet-Ran Chen* (2019) An Improved Scoring Method for the Identification of Endogenous Peptides based on Mascot MS/MS Ion Search. Analyst. 2019 Apr 23;144(9):3045-3055. doi: 10.1039/c8an02141d.
Yet-Ran Chen To identify the endogenous peptides using MS/MS analysis and searching against a polypeptide sequence database, a non-enzyme specific (NES) search considering all of the possible proteolytic cleavages is required. However, the use of a NES search generates more false positive hits than an enzyme specific search, and therefore has lower identification performance. In this study, the use of the sub-ranked matches for improving the identification performance of Mascot NES search was investigated and a new scoring method was developed that considered the contribution of all sub-ranked random match probabilities, named contribution score (CS). The CS showed the highest identification sensitivity using Mascot NES search with a full protein database when compared to the use of the Mascot first ranked score and delta score (DS). The confident peptides identified by DS and CS were showed to be complementary. When applied to plant endogenous peptide identification, the identification numbers of tomato endogenous peptides using DS and CS were 176.3% and 184.2%, respectively, higher than the use of the first ranked score of Mascot. The combination of DS and CS identified 200.0% and 8.6% more tomato endogenous peptides than the use of Mascot and DS, respectively. This method by combining the CS and DS can significantly improve the identification performance of endogenous peptides without complex computational steps and is also able to improve the identification performance of enzyme specific search. In addition to the application on the plant peptidomics analysis, this method may be applied to the improvement of peptidomics studies in different species. A web interface for calculating the DS and CS based on Mascot search results was developed herein.

Ying‐Lan Chen, Kai‐Ting Fan, Sheng‐Chi Hung and Yet-Ran Chen* (2019) The role of peptides cleaved from protein precursors in eliciting plant stress reactions. New Phytologist, 2019 Oct, doi: 10.1111/nph.16241
Yet-Ran Chen As sessile organisms, plants are exposed to diverse abiotic and biotic stresses, and thus have developed complex signaling mechanisms that orchestrate multiple stress responses. Plant peptides have recently emerged as key signaling molecules of stress responses, not only to mechanical wounding and pathogen infection but also to nutrient imbalance, drought and high salinity. The currently identified stress-related signaling peptides in plants are derived from proteolytic processing of protein precursors. Here, we review these protein-derived peptides and the evidence for their functions in stress signaling. We recommend potential research directions that could clarify their roles in stress biology, and propose possible crosstalk with regard to the physiological outcome. The stress-centric perspective allows us to highlight the crucial roles of peptides in regulating the dynamics of stress physiology. Inspired by historic and recent findings, we review how peptides initiate complex molecular interactions to coordinate biotic and abiotic stress responses in plants.

Apaya MK, Shiau JY, Liao GS, Liang YJ, Chen CW, Yang HC, Chu CH, Yu JC, Shyur LF* (2019). Integrated omics-based pathway analyses uncover CYP epoxygenase-associated networks as theranostic targets for metastatic triple negative breast cancer. J Exp Clin Cancer Res. 38(1):187. doi: 10.1186/s13046-019-1187-y.
LFS Current prognostic tools and targeted therapeutic approaches have limited value for metastatic triple negative breast cancer (TNBC). There is an ultimate need to provide novel and effective avenues for molecular stratification and personalized therapy for TNBC patients. We hypothesized that uncovering the role of oxylipins, a class of bioactive lipids derived by the action of CYP450 cyclooxygenases and lipoxygenases may offer new insight towards this direction. In this study, we utilized integrative genomics, proteomics and oxylipin metabolomics technology platform to decipher the potential theranostic roles of oxylipins in TNBC. Patient-derived tissue specimens, public cohorts, i.e., The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), and a panel of mammary-derived cell lines were utilized in this study. Comparison of oxylipin metabolome profile, tumor tissue immunoreactivity, and gene expression analyses showed that arachidonic acid epoxygenation products, epoxyeicosatrienoic acid metabolites (EETs), by CYP epoxygenases are strongly associated with TNBC metastasis. We further identified EETs as metastasis drivers in mesenchymal-like TNBC cells. Pathway networking analysis revealed that, in hormone-positive breast cancer subtype, CYP epoxygenase overexpression is more related to immune cell-associated signaling, while EET-mediated Myc, Ras, MAPK, EGFR, HIF-1α, and NOD1/2 signaling are the molecular vulnerabilities of metastatic CYP epoxygenase-overexpressing TNBC tumors. This study identifies a special stratification of triple negative breast tumors according to their EET metabolite ratio classifiers and CYP epoxygenase profiles that may provide a useful prognostic and therapeutic target for the development of personalized intervention strategy for TNBC.
* 相關連結

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*2020/10/19 11:00 AM
李文立博士 (行政院農業委員會農業試驗所 鳳山熱帶園藝試驗分所 分所長)
Auditorium A134, Agricultural Technology Building

*2020/11/09 11:00 AM
戴廷恩博士 (行政院農業委員會農業試驗所花卉研究中心 研究員兼主任)
Auditorium A134, Agricultural Technology Building

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