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姓名:陳志毅

職稱:研究員

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專長:Antimicrobial peptides,
Marine biotechnology

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研究人員 / 簡介
  2015-present Research Fellow, Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan
  2011-2015 Associate Research Fellow, Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan
  2007-2011 Assistant Research Fellow, Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan
  2003-2007 Assistant Research Scientist, Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan
  2001-2002 Postdoctoral Research Associate, The Sanger Institute Wellcome Trust, Cambridge U.K.
  2000-2001 Postdoctoral Research Fellow, Institute of Zoology, Academia Sinica, Taiwan
  1997 Ph.D. Institute of Zoology, National Taiwan University, Taiwan

抗微生物胜肽與海洋生物技術實驗室

  • Summary of research accomplishments and future directions

    During the last few decades, the emergence of resistance to multiple antimicrobial agents in pathogenic microbes has become a serious threat. Overreliance on antimicrobial drugs to treat infections has led to resistance in various microorganisms (including bacteria, fungi, enveloped viruses and parasites) and even cancerous cells. This antimicrobial resistance casts a shadow over many of modern medicine’s greatest achievements. Despite the magnitude and seriousness of the problem, antibiotics are inexpensive and widely available in Taiwan. The low price has enabled and encouraged many aquaculture practitioners to use antibiotics for the treatment of diseased fish. However, this widespread practice can lead to the development antibiotic-resistant bacterial strains. Furthermore, consuming fish that have accumulated antibiotics may promote the selection of antibiotic-resistant bacterial strains in humans. Following the food chain, a human face can be put on the problem of antibiotic overuse in aquaculture. Such overuse may eventually lead to antibiotic-resistant bacterial infections in humans that cannot be treated with any currently available drugs.

    In my laboratory, the antimicrobial functions of several marine organism-derived antimicrobial peptides (AMPs) have been characterized against various pathogens. AMPs are part of the innate defense of an organism and protect against bacteria, fungi, viruses, and other harmful microbes. The AMPs that we study have been shown to lyse bacterial membranes, suggesting their potential utility as drugs to combat bacterial infections. With the dual goals of helping the local aquaculture industry and also to identify new drugs for future medical use in humans, we have implemented basic and translational research studies on AMP function. Our recent research results suggest that AMPs can be applied in the aquaculture industry or as alternative drugs to treat antibiotic-resistant infections in animals or humans. In order to accomplish these aims, we have used several different approaches, including transgenic fish, transgenic artemia and recombinant protein expression, among others. Our important contributions are as follows:

     

    1. Transgenic hepcidin-overexpressing fish exhibit enhanced resistance to bacterial infection:

    Fish disease is the greatest problem facing aquaculture and damaging its profitability. Although many fish diseases can be prevented or controlled by vaccinations or antibiotics, there is still no truly effective vaccine or miracle drug that can be used against a variety of infections. Genetic engineering to produce disease-resistant transgenic fish may provide a solution to this challenging problem. In my lab, we have used transgenic technology to produce fluorescent fish, which express TH2-3 (hepcidin isolated form Oreochromis mossambicus). Transgenic TH2-3 zebrafish and convict cichlid exhibited resistance to Vibrio vulnificus infection, as well as immune-related gene expression variations after infection by different bacterial strains. Our results may provide crucial information in the understanding of teleost innate immunity. Furthermore, this transgenic approach may have application in the control of microbial infections in aquaculture species.

    Transgenic tilapia hepcidin (TH2-3) (a) 14-day-old zebrafish containing green fluorescent color expression were retained and raised to the F3 generation under microscopic observation. (b) Bright-field and fluorescence images of the convict cichlid (Archocentrus nigrofasciatus)(F3 generation).

     

    2. Nile tilapia fry fed on epinecidin-1-expressing artemia cysts showed enhanced immunity to acute bacterial infection:

    Artemia is used as a live bait feed for fry in aquaculture. We have previously demonstrated that adult zebrafish fed with artemia that express an Epinephelus coioides-derived AMP, Epinecidin-1 (Epi-1), are protected against bacterial infection, indicating that artemia may be used as a bioreactor for producing bio-functional molecules. Recently, we used a Tol2-transposon system combined with microinjection to generate Epi-1 expressing artemia. Oreochromis niloticus fry were fed on decapsulated transgenic cysts followed by acute challenge with Gram-positive S. iniae or Gram-negative V. vulnificus. Survival analysis revealed that tilapia fry fed with Epi-1 decapsulated transgenic cysts had enhanced resistance to acute bacterial infection. Immune-related gene analysis revealed that distinctive immunomodulatory gene expression profiles were induced by S. iniae and V. vulnificus infection in the tilapia fry. Our findings suggested that feeding of larval fish fry with Epi-1 transgenic artemia cyst enhanced immunity to bacterial challenge. Epi-1 transgenic cyst may therefore be considered as a potential functional feed for larval aquaculture.

     

    3. Recombinant antimicrobial peptide supplementation in fish fodder protected fish from Vibrio vulnificus infection and enhanced immunomodulatory functions:

    In my laboratory, the antimicrobial functions of several AMPs have been characterized against important pathogens. One interesting peptide of 21 amino acids, epinecidin-1, was isolated from marine grouper (Epinephelus coioides); epinecidin-1 was found to exhibit considerable antimicrobial activity against bacteria, fungi, viruses, and other harmful microbes. Since no specific therapeutic methods are available to treat fish disease brought about by V. vulnificus infection, we tested whether epinecidin-1 would have this function. V. vulnificus is the causative agent of vibriosis, a hemorrhagic septicaemia that affects a variety of fish species and other aquatic animals and has brought about large economic losses in the aquaculture industry worldwide. We expressed recombinant epinecidin-1 in E. coli and then used it as a fodder supplement to feed fish. Epinecidin-1 in the diet acted as an immunostimulant and antimicrobial agent against V. vulnificus infection. Our study showed that an E. coli expression system for the large-scale production of the recombinant epinecidin-1/DsRed fusion protein may produce peptide with strong antibacterial activity at microgram concentrations. V. vulnificus numbers were reduced in infected tissues and survival rates were enhanced by the use of recombinant epinecidin-1/DsRed fusion protein mixed with eel powder as fodder for 30 days.

     

    4. Fish AMPs exhibited antitumor function

    We have studied the mechanisms and economic value of naturally-occurring AMPs with activity against various tumor types. In our laboratory, we studied pardaxin (GE33) using molecular approaches that are standard in cancer research. We found pardaxin exerts antitumor function and modulates immune responses in mammals. Our research results demonstrate that pardaxin selectively triggers the death of cancer cells through a molecular mechanism that involves ER targeting and c-FOS induction. Transcriptome analysis of pardaxin-treated HT-1080 fibrosarcoma cells revealed induction of the gene encoding c-FOS, and we found that pardaxin mediates cell death by activating c-FOS but not other AP-1 transcription factors. Overexpression of c-FOS caused a dramatic increase in cell death, while knockdown of c-FOS induced pardaxin resistance; such effects were observed in both an in vitro cell model and an in vivo xenograft tumor model. An antitumor effect was observed when pardaxin (25 mg/kg; 0.5 mg/day) was used to treat mice for 14 days, which caused significant inhibition of murine MN-11 tumor cell growth in mice. To obtain a greater understanding of the antitumor effects of pardaxin, we examined the antitumor activity, toxicity profile, and maximally-tolerated dose in dogs with different refractory tumor types. Local injection of pardaxin resulted in a significant reduction of perianal gland adenoma growth between 28 and 38 days post-treatment. Surgically resected canine histiocytomas appeared as large areas of ulceration, suggesting that pardaxin acts as a lytic peptide. However, pardaxin treatment was not associated with significant variations in blood biochemical parameters or secretion of immune-related proteins. Thus, pardaxin has strong therapeutic potential for treating perianal gland adenomas in dogs. These data not only suggest that pardaxin may be suitable for veterinary application, but also provide valuable information for veterinary medicine and future human clinical trials. We have applied for patents in Taiwan that cover the use of pardaxin in canine cancer therapy, and are now conferring with a biotechnology company for technology licensing and technology transfer.

    In conclusion, my laboratory has focused on elucidating the functions of several AMPs through translational research, thus developing novel drug candidates. Our studies detail application-oriented functions of AMPs from marine organisms. In several systems, we have demonstrated the possibility of using fish AMPs as antimicrobial agents, vaccine adjuvants, inactivated vaccines and antitumor agents. These prospective applications would promote the welfare of humans, mammals and aquaculture.

     

    Future directions:

    Our studies on the molecular mechanisms of AMP action will extend our understanding of how AMPs affect cells and organisms. A more complete understanding of the molecular mechanisms of AMP actions will aid the entry of AMP-based products into the market and acceptance by the public. My studies will focus on the application of AMPs in multiple, highly promising directions. Our research touches on the following themes, which address the growing problem of antibiotic-resistant microbes and the possible application of AMPs in cancer treatment:

     

    1. Study the antitumor function of AMPs from marine organisms

    For several years, we have focused on the mechanisms of the anti-cancer effects of AMPs (Carcinogenesis. 2013, 34:1833-42; Biomaterials. 2013, 34:10151-9; Biomaterials. 2014, 35:3627-40; Oncotarget. 2016, 7:40329-47. Biochim Biophys Acta. 2017, 1863:3028-37). To my mind, the application of AMPs in cancer treatment is interesting topic. Targeted therapies function by recognizing specific molecular targets that distinguish cancer cells from healthy cells. For example, PD-L1 as an ideal molecular target based on its expression on cancer cells and cancer-promoting stromal cells. Antibody-drug conjugates (ADCs) are potential therapeutic drugs for cancer treatment. ADCs are composed of a monoclonal antibody (mAb) that targets an epitope on the cancer cell membrane and a potent cytotoxic agent. Cancer cell membranes tend to be negatively charged, and therefore, anti-cancer agents with positive charges often show enhanced anti-cancer potency. AMPs participate in the innate immune response. Tilapia piscidin 4 (TP4) is an AMP that was originally isolated from the Oreochromis niloticus. We have shown that TP4 exhibits excellent cancer cell killing activity in cell and animal models. The major goal of this research theme will be to develop TP4-PDL1 mAb conjugates. Our preliminary study confirmed that TP4 conjugated to a commercially available monoclonal PD-L1 antibody showed enhanced and dose-dependent cancer cell suppression activity against two PD-L1 positive cancer cell-lines (A549 and HCC827 cells) compared with PD-L1 antibody alone. In addition, bioinformatics analysis allowed us to identify two target sequences on human PD-L1, and these sequences were used for generating polyclonal antibodies. An in vitro cell toxicity assay showed that both of these novel polyclonal antibodies efficiently inhibit cancer cell growth. Therefore, we aim to generate novel monoclonal antibodies to the identified target sequences of PD-L1 and further develop TP4-PDL1 mAb conjugates to be applied in cancer therapy. After characterizing the function of TP4-PDL1 mAbs, we will evaluate the therapeutic potential in pre-clinical models, such as mouse, dog, and eventually human pre-clinical trials.

     

    2. Study the anti-sepsis function of AMPs from marine organisms

    The pathogen Staphylococcus aureus (SA) is commonly found on the skin as well as in the nose and throat of people who are hospitalized for an extended time period. SA infections are usually minor but can occasionally lead to serious infections and mortality in people who are immunocompromised, have diabetes, or have blood exposure to methicillin-resistant SA (MRSA). In future studies, we will evaluate whether epinecidin-1 (Epi-1) can efficiently protect against MRSA infection in a pig pyemia model. First, we will assess whether Epi-1 shows effective in vitro bactericidal activity. Preliminary in vitro and in vivo pharmacokinetic analyses have shown that Epi-1 may be stable for up to 4 h post-injection in serum and become diluted at later time points. We will further evaluate whether Epi-1 may interact with human serum albumin, which could explain the pharmacokinetic characteristics. Moreover, we will use different doses (up to 100 mg/kg of body weight) in order to test whether Epi-1 produces harmful symptoms in pigs. The effects of Epi-1 will be compared to those of the currently available antibiotic, vancomycin, to identify which drug is more effective. In addition, MRSA counts in blood, liver, kidney, heart, and lungs will be estimated in Epi-1-treated pigs. We will then ask if the MRSA-specific genes, enterotoxin A, enterotoxin B and intrinsic methicillin resistance A, are reduced or abolished in MRSA-infected pigs after receiving Epi-1 treatment. A survival study also will be conducted to assess whether post-treatment of pyemic pigs that have been infected with MRSA with 2.5 mg/kg Epi-1 can protect against mortality. The future study is expected to demonstrate that the AMP Epi-1 can efficiently protect pyemic pigs against MRSA infection without toxic effects. After conducting detailed studies on animal models to evaluate AMP pharmacology and the effects of AMPs on sepsis, the next step will be to seek government or venture capital funding to extend our research results. More detailed studies will be guided by drug development experts, such as those in the National Taiwan University Hospital Clinical Trial Center or Center for Drug Evaluation (CDE), Taiwan.

     

    3. Application of AMPs as feed additives for the animal breeding industry

    In the animal breeding industry, worldwide limitations on antibiotics in poultry feed will necessitate the development of alternative measures to maintain poultry health and growth performance. AMPs are part of the nonspecific defense system and are natural antibiotics produced by plants, insects, mammalians, and microorganisms. These molecules may be produced through chemical synthesis as well. In our lab, we have identified several marine AMPs that possess broad antimicrobial activity against various fungi, bacteria and enveloped viruses, suggesting that AMPs may be a viable alternative to conventional antibiotics for use in poultry production and/or aquaculture. In future studies, we will use the methylotrophic yeast, Pichia pastoris, as a powerful and inexpensive heterologous expression system for the production of high levels of functionally active antimicrobial peptides. We will purify the antimicrobial peptide from the culture supernatant, after which, we will examine its antimicrobial activities, structure, mechanism of action, biological activity and toxicity. Tests to optimize the fermentation matrix formulation, feed composition and preparation of small animal feed will also be conducted. The aim of this theme is to provide support for the application of AMPs as feed additives, which may replace antibiotics in poultry nutrition or find other applications in animal breeding industries, such as aquaculture.

     

    Summary

    In 2007, we demonstrated that the fish peptide, epinecidin-1, could protect against bacterial infection in animal models, revealing that antimicrobial peptides had activities distinct from direct antibiotic action. There is now a growing body of evidence (including work from our laboratory) for an impressive variety of activities of marine organism-derived AMPs in addition to direct killing. As such, these peptides have been shown to act directly or indirectly on cells of the immune system. In fact, these immunomodulatory activities occur under the same conditions that inhibit antimicrobial activity. Functional genomics, animal models and biochemical studies conducted in our laboratory have demonstrated that AMPs interact directly with host cells to modulate innate immunity, decreasing potentially harmful inflammation while stimulating protective responses. The most active antimicrobial peptides, epinecidin-1 and TP4, exhibit superior protection in animal models against many different bacterial infections, including antibiotic-resistant strains of MRSA, E. coli and Pseudomonas aeruginosa. These results have provided a new concept in anti-infective therapy and suggest that AMPs may be valuable antimicrobial agents for use in pigs, cattle and mice, as well as in human disease. The peptides isolated from my lab are currently being used in pre-clinical studies to treat animal and human diseases. In the near future, I will seek opportunities to cooperate with biotechnology companies in order to realize the development of many potential applications for these AMPs that were isolated from marine organisms and functionally characterized by my previous research results.

  • 姓名職稱電話Email備註
    陳志毅研究員
    丁振紘博士後研究員
    黃瀚寧博士後研究員
    蘇柏全博士後研究員
    林玟君研究助理
    陳儀君研究助理
    蔡宗祐研究助理
    陳信宏碩士班一年級
  • 1.     Bor-Chyuan Su, Yung-Wei Lai, Jyh-Yih Chen*, Chieh-Yu Pan* (2018). Transgenic expression of tilapia piscidin 3 (TP3) in zebrafish confers resistance to Streptococcus agalactiae. Fish and Shellfish Immunology (in press) *Corresponding author.

    2.    Bor-Chyuan Su, Wen-Chun Lin, Jyh-Yih Chen* (2018). Recombinant Epinephelus lanceolatus serum amyloid A as a feed additive: Effects on immune gene expression and resistance to Vibrio alginolyticus infection in Epinephelus lanceolatus. Fish and Shellfish Immunology (in press) *Corresponding author.

    3.    Chen-Hung Ting, Yi-Chun Chen, Jyh-Yih Chen* (2018) Nile tilapia fry fed on antimicrobial peptide Epinecidin-1-expressing Artemia cyst exhibit enhanced immunity against acute bacterial infection. Fish and Shellfish Immunology (in press) *Corresponding author.

    4.    Han-Ning Huang, Chieh-Yu Pan, Jyh-Yih Chen* (2018). Grouper (Epinephelus coioides) antimicrobial peptide epinecidin-1 exhibits antiviral activity against foot-and-mouth disease virus in vitro. Peptides (in press) *Corresponding author.

     


  • 115台北市南港區研究院路二段128號  Tel: 02-27899515   Fax: 02-27858059  *個人隱私權聲明*
    icob@gate.sinica.edu.tw  Copyright © ICOB 2013. All rights reserved. 最佳瀏覽網頁方式請用最新版IE11或其他瀏覽器 -- 瀏覽人數:1312621
    115台北市南港區研究院路二段128號
    Tel: 02-27899515
    Fax: 02-27858059
    icob@gate.sinica.edu.tw
    Copyright © ICOB 2013. All rights reserved. 最佳瀏覽網頁方式請用最新版IE11或其他瀏覽器 /瀏覽人數:1312621--
     瀏覽人數:1312621