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Ming-Jung Liu*, Koichi Sugimoto, Sahra Uygun, Nicholas Panchy, Michael S. Campbell, Mark Yandell, Gregg A. Howe and Shin-Han Shiu* (2018). Regulatory divergence in wound-responsive gene expression between domesticated and wild tomato. Plant Cell DOI: http://www.plantcell.org/content/early/2018/05/09/tpc.18.00194
Ming-Jung Liu Background: Two related species accumulate differences between each other over time. If one of the species is domesticated due to human selection of desirable traits, the domesticated species experiences a "bottleneck" where many traits present in the related wild species are lost because only a few individuals are targeted for selection. For example, domesticated tomato has a weaker defense response to wounding and insect feeding compared to wild tomato species that separated from the domesticated tomato ~3-7 million years ago. One of the major reasons for this difference lies with genes that are turned on and off differently due to evolution of short DNA sequences that are like molecular switches.

Question: We wanted to know how wounding leads to different genes being turned on and off in domesticated and wild tomatoes, which DNA sequence "switches" are important for this response, and how these sequence switches differ between species.

Findings: We found that thousands of genes were turned on or off differently in response to wounding between domesticated and wild tomato species. We found hundreds of potential DNA switches associated with this set of genes, and nearly half of them were unique to one species or the other. Our findings indicate a large difference in how these two plant species turn genes on/off in response to wounding; for example this difference is much greater than similar comparisons between human and mouse genes, which were separated from a common ancestor roughly 100 million years ago.
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Sakthivel Kailasam, Ying Wang, Jing-Chi Lo, Hsin-Fang Chang and Kuo-Chen Yeh* (2018) S-nitrosoglutathione works downstream of nitric oxide to mediate iron deficiency signaling in Arabidopsis The Plant Journal, DOI: 10.1111/tpj.13850
kcyeh Several studies elucidated the transcriptional responses to iron (Fe) starvation, however, the Fe sensing and signal transduction machineries are poorly resolved thus far in plants. With this focus, by using chemical biology approach, we identified a small molecule R7, whose use inhibited the physiological and molecular responses of Fe starvation in Arabidopsis. R7 blocked the Fe starvation signaling from nitric oxide (NO) to the central transcription factor, FER-LIKE IRON-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). Exogenous applications of NO abduct, S-nitrosoglutathione (GSNO) were able to attenuate the R7 effect. R7 directly or indirectly decreased the endogenous GSNO levels in roots but not the NO levels. Our finding reveals that, under Fe limited situation the starvation signal from NO is passed to FIT through GSNO.
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Choun-Sea Lin*,Chen-Tran Hsu, Ling-Hung Yang, Lan-Ying Lee, Jin-Yuan Fu, Qiao-Wei Cheng, Fu-Hui Wu, Han Chen-Wei Hsiao, Yesheng Zhang, Ru Zhang, Wan-Jung Chang, Chen-Ting Yu, Wen Wang, Li-Jen Liao, Stanton B. Gelvin*,Ming-Che Shih*. (2018) Application of protoplast technology to CRISPR/Cas9 mutagenesis: From single cell mutation detection to mutant plant regeneration. Plant Biotechnol J. 2017 Dec 12. doi: 10.1111/pbi.12870
cslin Plant protoplasts are useful for assessing the efficiency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated Protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.
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Chin-Hsien Tsai, Sheue-Fen Tzeng, Shih-Chuan Hsieh, Chia-Jui Tsai, Yu-Chih Yang, Mong-Hsun Tsai and Pei-Wen Hsiao (2017) A Standardized Wedelia chinensis Extract Overcomes the Feedback Activation of HER2/3 Signaling upon Androgen-Ablation in Prostate Cancer. Frontiers in Pharmacology 2017 Oct; 8:721
pwh Androgen receptor (AR)-mediated signaling play an essential role in prostate cancer for survival, growth, and invasion. Crosstalk between the AR and other signaling pathways in prostate cancer (PCa) severely affects the therapeutic outcome of hormonal therapy. Although anti-androgen therapy prolongs overall survival in PCa patients, resistance rapidly develops and is often associated with increased AR expression and upregulation of the HER2/3-AKT signaling pathway. However, single-agent therapy targeting AR, HER2/3 or AKT usually fails due to the reciprocal feedback loop. Here, we demonstrated that a standardized herbal extract, named WCE, effectively disrupted the AR, HER2/3, and AKT signaling networks and therefore enhanced the therapeutic efficacy of androgen ablation in PCa. Furthermore, WCE remained effective in suppressing AR and HER2/3 signaling in an in vivo adapted castration-resistant PCa cells that were insensitive to androgen withdrawal and a second-line antiandrogen, enzalutamide. This study provides preclinical evidence that the use of a defined, single plant-derived extract can augment the therapeutic efficacy of castration with significantly prolonged progression-free survival. These data also establish a solid basis for using WCE as a candidate agent in clinical studies.
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Chin-Hsien Tsai, Sheue-Fen Tzeng, Shih-Chuan Hsieh, Yu-Chih Yang, Yi-Wen Hsiao, Mong-Hsun Tsai, and Pei-Wen Hsiao (2017) A standardized herbal extract mitigates tumor inflammation and augments chemotherapy effect of docetaxel in prostate cancer. Scientific Reports 2017 Nov; 7: 15624
pwh Docetaxel remains the standard treatment for castration-resistant prostate cancer by improving overall survival but having severe adverse toxicity. Inflammatory cytokines in the tumor microenvironment promote docetaxel resistance through activation of IKKα/β-NFκB pathways. To tackle such chemotherapy-elicited adverse effects, this study demonstrates the standardized Wedelia chinensis herbal extract effectively inhibited the production of tumor-promoting cytokines, therefore inhibiting angiogenesis, tumor growth, and metastasis. Transcriptomic analysis of tumors revealed that WCE modulates inflammatory tumor microenvironment by suppressing the expression of HIF1α, IKKα/β phosphorylation and the downstream cytokines/chemokines, e.g., IL6, CXCL1, and CXCL8, thus reduced tumor-elicited chemotaxis and infiltration of MDSCs, TAMs, and endothelial cells into the tumors. Furthermore, WCE suppresses the expansion and activity of MDSCs by downregulating STAT3 activity and also prevents the differentiation of macrophages toward tumor-promoting M2 phenotype. On the other hand, co-treatment of docetaxel with this botanical preparation caused significant enhancement of the docetaxel-mediated therapeutic efficacy, while prevented the drug resistance and toxicity.
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Jeng-Yuan Shiau, Kyoko Nakagawa-Goto, Kuo-Hsiung Lee, and Lie-Fen Shyur* (2017) Phytoagent deoxyelephantopin derivative inhibits triple negative breast cancer cell activity by inducing oxidative stress-mediated paraptosis-like cell death. Oncotarget, 2017, 8:56942-56958
lfs Triple negative breast cancer (TNBC) is a highly metastatic cancer among the breast cancer subgroups. A thorny issue for clinical therapy of TNBC is lack of an efficient targeted therapeutic strategy. We previously created a novel sesquiterpene lactone analog (named DETD-35) derived from plant deoxyelephantopin (DET) which exhibits potent effects against human TNBC MDA-MB-231 tumor growth in a xenograft mouse model. Here we studied the mechanisms of both DET and DETD-35 against MDA-MB-231 cells. DETD-35 (3-fold decreased in IC50) exhibited better anti-TNBC cell activity than DET as observed through induction of reactive oxygen species production (within 2 h treatment) and damage to the ER structures, resulting in ER-derived cytoplasmic vacuolation and ubiquitinated protein accumulation in the treated cells. Intriguingly, the effects of both compounds were blockaded by pretreatment with ROS scavengers, N-acetylcysteine and reduced glutathione, and protein synthesis inhibitor, cycloheximide. Further, knockdown of MEK upstream regulator RAF1 and autophagosomal marker LC3, and co-treatment with JNK or ERK1/2 inhibitor resulted in the most significant attenuation of DETD-35{induced morphological and molecular or biochemical changes in cancer cells, while the inhibitory effect of DET was not influenced by MAPK inhibitor treatment. Therefore, DETD-35 exerted both ER stress-mediated paraptosis and apoptosis, which may explain its superior activity to DET against TNBC cells. Although the chemotherapeutic drug paclitaxel induced vacuole-like structures in MDA-MB-231 cells, no paraptotic cell death features were detected. This study provides a strategy for combating TNBC through sesquiterpene lactone analogs by induction of oxidative and ER stresses that cause paraptosis-like cell death.
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Cheng-Hsun Ho* and Wolf B. Frommer (2016) Design and Functional Analysis of Fluorescent Nitrate and Peptide Transporter Acativity Sensors in Yeast Cultures Bio-protocol, vol 6, iss 3
何承訓 This protocol describes the methods used to engineer and deploy genetically encoded fluorescence activity reporters for nitrate and peptide transporter activity in yeast cells. Fusion of the dual-affinity nitrate transceptor CHL1/AtNRT1.1/AtNPF6.3 or four different peptide transporters (AtPTR1, 2, 4, and 5) from Arabidopsis to a pair of fluorescent proteins with different spectral properties, enabled us to engineer the NiTracs (nitrate transporter activity tracking sensors) and the PepTracs (peptide transporter activity tracking sensors), ratiometric fluorescence activity sensors that monitor the activity of the plasma membrane nitrate transceptor or the peptide transporters in vivo (Ho et al., 2014). The NiTrac1 sensor responds specifically and reversibly to the addition of nitrate, while the PepTracs respond to addition of dipeptides, either by a reduction in donor and acceptor emission, while acceptor-excited emission remains unaltered, or by a change in ratio of the fluorophore emission. All sensors are suitable for ratiometric imaging. The similarity of the biphasic kinetics of the NiTrac1 sensor response [from µM to mM (Liu and Tsay, 2003)] and the nitrate transport kinetics of the native nitrate transceptor, intimates that NiTrac1 provides information on conformational rearrangements during the transport cycle, thereby reporting transporter activity over a wide range of external nitrate concentrations. Several variants of NiTrac have been engineered, which differ with respect to their affinity for nitrate (NiTrac1: CHL1; NiTracT101A: CHL1T101A). NiTrac also recognizes chlorate. Here we describe a simple method for the design, implementation, and detection of nitrate transceptor activity in yeast cells using a spectrofluorimeter.
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Cindy Ast, Jessica Foret, Luke M. Oltrogge, Roberto De Michele, Thomas J. Kleist, Cheng-Hsun Ho & Wolf B. Frommer (2017) Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins.Nature Communications(2017), doi:10.1038/s41467-017-00400-2
何承訓 Sensitivity, dynamic and detection range as well as exclusion of expression and instrumental artifacts are critical for the quantitation of data obtained with fluorescent protein (FP)-based biosensors in vivo. Current biosensors designs are, in general, unable to simultaneously meet all these criteria. Here, we describe a generalizable platform to create dual-FP biosensors with large dynamic ranges by employing a single FP-cassette, named GO-(Green-Orange) Matryoshka. The cassette nests a stable reference FP (large Stokes shift LSSmOrange) within a reporter FP (circularly permuted green FP). GO- Matryoshka yields green and orange fluorescence upon blue excitation. As proof of concept, we converted existing, single-emission biosensors into a series of ratiometric calcium sensors (MatryoshCaMP6s) and ammonium transport activity sensors (AmTryoshka1;3). We additionally identified the internal acid-base equilibrium as a key determinant of the GCaMP dynamic range. Matryoshka technology promises flexibility in the design of a wide spectrum of ratiometric biosensors and expanded in vivo applications.
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Ji-Rong Yang, Chieh-Yu Cheng, Chih-Yuan Chen, Chao-Hua Lin, Chuan-Yi Kuo, Hsiang-Yi Huang, Fu-Ting Wu, Yu-Chih Yang, Chia-Ying Wu, Ming-Tsan Liu*, and Pei-Wen Hsiao* (2017) A virus-like particle vaccination strategy expands its tolerance to H3N2 antigenic drift by enhancing neutralizing antibodies against hemagglutinin stalk. Antiviral Research, 2017, 140: 62-75
pwh Seasonal influenza viruses impact public health annually due to their continual evolution. However, the current inactivated seasonal vaccines provide poor protection against antigenically drifted viruses and require periodical reformulation through hit-and-miss predictions about which strains will circulate during the next season. To reduce the impact caused by vaccine mismatch, we investigated the drift-tolerance of virus-like particles (VLP) as an improved vaccine candidate. The cross-protective humoral immunity elicited by the H3N2-VLP vaccine constructed for the 2011-2012 season was examined against viruses isolated from 2010 to 2015 in Taiwan evolving chronologically through clades 1, 4, 5, 3B and 3C, as well as viruses that were circulating globally in 2005, 2007 and 2009. Mouse immunization results demonstrated that H3N2-VLP vaccine elicited superior immunological breadth in comparison with the cognate conventional whole-inactivated virus (WIV) vaccine. Titers of neutralizing antibodies against heterologous strains representing each epidemic period in the VLP group were significantly higher than in the WIV group, indicating the antibody repertoire induced by the H3N2-VLPs was insensitive to viral antigenic drift over a span of at least 10 years. Noticeably, H3N2-VLP elicited higher levels of anti-stalk antibodies than H3N2-WIV, which offset the ineffectiveness caused by antigenic drift. This advantageous effect was attributed to the uncleaved precursor of their HA proteins. These results suggest a mechanism through which VLP-induced humoral immunity may better tolerate the evolutionary dynamics of influenza viruses and point to the possible use of a VLP vaccine as a method by which the requirement for annual updates of seasonal influenza vaccines may be diminished.
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Yu-Yi Wu, Bo-Han Hou, Wen-Chi Lee, Shin-Hua Lu, Chen-Jui Yang, Hervé Vaucheret and Ho-Ming Chen (2017) DCL2- and RDR6-dependent transitive silencing of SMXL4 and SMXL5 in Arabidopsis dcl4 mutants causes defective phloem transport and carbohydrate over-accumulation. The Plant Journal, 2017, 90(6):1064-1078

homing Dicer-Like (DCL) enzymes are able to process double-stranded RNA into small RNAs. Arabidopsis DCL4 and DCL2 each allow the post-transcriptional gene silencing (PTGS) of viruses and transgenes, but DCL2 activity is mostly obscured by DCL4. This hierarchy likely prevents DCL2 having any detrimental effects on endogenous genes. Indeed, dcl4 mutants exhibit leaf pigmentation under regular growth conditions. Here we report that the purple phenotype of dcl4 leaves correlates with carbohydrate over-accumulation and defective phloem transport, and depends on the activity of DCL2 and two enzymes, Suppressor of Gene Silencing 3 (SGS3) and RNA-Dependent RNA Polymerase 6 (RDR6), involved in double-stranded RNA synthesis. Further, this phenotype correlates with the down-regulation of two genes expressed in the apex and the vasculature, SMAX1-Like 4 (SMXL4) and SMXL5, and the accumulation of DCL2- and RDR6-dependent small interfering RNAs derived from these two genes. Supporting a causal effect, smxl4 smxl5 double mutants exhibit leaf pigmentation, enhanced starch accumulation and defective phloem transport, similar to dcl4 plants. Overall, this study elucidates the detrimental action of DCL2 when DCL4 is absent, and indicates that DCL4 outcompeting DCL2 in wild-type plants is crucial to prevent the degradation of endogenous transcripts by DCL2- and RDR6-dependent transitive PTGS.
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*2018/07/16 10:30 AM
Dr. Jeng-Yuan Shiau (Dr. Lie-Fen Shyur's Lab)
Molecular insights of phytoagent deoxyelephantopin and its derivative against human triple negative breast cancer cell activities
Auditorium A134, Agricultural Technology Building, Academia Sinica

*2018/08/22 10:30 AM
Dr. Nathaniel Robert Street (Associate Professor, Department of Plant Physiology, Umeå University, Sweden)
Systems genetics and genomics insights into complex traits in aspen and Norway spruce
Auditorium A134, Agricultural Technology Building, Agricultural Biotechnology Research Center

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