Institute of Fisheries Science, NTU

Research Interests:
(1) Application of antimicrobial peptides(AMPs)
(2) Marine biotechnology

Summary of research accomplishments

The Marine Research Station (MRS) is located at Jiaushi, Ilan, Taiwan. It is a facility of the Institute of Cellular and Organismic Biology for conducting research and development in the fields of marine molecular biology and biotechnology with specific focus on gene transfer, ecophysiology, immunology and pathology of aquatic organisms. My research performance is listed below.
 

Project 1: Production of transgenic fish with fish or shrimp antimicrobial peptide-fluorescent protein with increased resistance to bacterial pathogens and a bright skin color

Recently, in my laboratory, antimicrobial functions against several pathogens of several antimicrobial peptides (AMPs) were characterized. AMPs are part of organisms’ innate defense against bacteria, fungi, viruses, and other harmful microbes. These AMPs have been shown to lyse bacterial membranes, indicating their potential as drugs against bacterial infections. In Taiwan, many aquaculturists use antibiotics to treat diseased fish, and this practice can lead to antibiotic-resistant bacterial strains. Consuming contaminated fish may induce production of antibiotic-resistant bacterial strains in human. With goal of helping aquaculture industry to find new drugs for the future, we began to study AMP functions with transgenic fish approach. Our results on AMP functions suggest that AMPs can potentially be applied in the aquaculture industry as alternatives to the overuse of antibiotics.

Secondly, ornamental fish are aquaculture species with high economic value. Together with AMP function using transgenic technology, we also produced a large and brightly colour fluorescent fish. We constructed plasmid with mylz promoter driving the green fluorescence protein gene and an antimicrobial peptide gene. The first convict cichlid with the improved muscle-promoter fluorescence protein gene was obtained in my lab on August 15, 2008. These transgenic fluorescent fish were transferred to the Jy Lin Co. In the meantime, the Jy Lin Co. is awaiting approval under laws governing genetically modified organisms (GMOs).

For the above-described project, our important contributions are:

1. One mission of the MRS is to help aquaculture industry improve their products. In my lab, we are the first to produce fluorescent convict cichlid (Cichlasoma nigrofasciatus) in the world and shown below. The transgenic fluorescent convict cichlid can help a Taiwan biotechnology company made earning of NT$100~$200 million over the next few years, as quoted from a fish breeder's speech. The transgenic fluorescent convict cichlid (Cichlasoma nigrofasciatus) helped us won the 2013 Taipei Biotechnology Award shown below.

Project 2: Recombinant antimicrobial peptide protein as fish fodder protected fish from Vibrio vulnificus infection and enhanced immunomodulatory functions 

In my laboratory, the antimicrobial functions of several antimicrobial peptides (AMPs) against certain pathogens were characterized. One interesting peptide of 21 amino acids, named epinecidin-1, was isolated from a marine grouper (Epinephelus coioides); epinecidin-1 was found to exhibit considerable antimicrobial activity against bacteria, fungi, viruses, and other harmful microbes. However, there are no specific therapeutic methods for treating fish disease brought about by Vibrio vulnificus infection. Vibrio vulnificus is the causative agent of vibriosis, a hemorrhagic septicaemia affecting a variety of fish species and other aquatic animals, which brings about large economic losses in the aquaculture industry worldwide. Expression of recombinant epinecidin-1 protein in either an E. coli protein expression system or Artemia, and then using the resulting fodder or Artemia to feed fish, may be of potential benefit for aquaculture, as epinecidin-1 in the diet may act as an immunostimulant and antimicrobial agent against Vibrio vulnificus infection in fish. For the above-described project, our important contributions are as follows:

1) Our study used electroporation technology to improve the efficiency of introduction of plasmid DNA into Artemia, and the bactericidal activity of Artemia-expressed epinecidin-1 in zebrafish against Vibrio vulnificus (204) infection.

2) Zebrafish were fed on Artemia expressing recombinant epinecidin-1 peptide-GFP fusion for 7, 14, or 21 days, and our data suggest that the fusion protein may play a role as an immunostimulant after intake into the intestines by enhancing a specific defense mechanism in this organ, thereby increasing disease resistance.

3) We described an E. coli expression system for the large-scale production of the recombinant epinecidin-1/DsRed fusion protein, which showed strong antibacterial activity at microgram concentrations against several bacterial species.

4) V. vulnificus (204) numbers were reduced and survival rates were enhanced by the use of recombinant epinecidin-1/DsRed fusion protein mixed with eel powder as fodder for 30 days.  

5) One goal of the MRS is to help the aquaculture industry improve their products. My lab was the first in the world to produce recombinant epinecidin-1 protein in an E. coli protein expression system and Artemia. The transgenic Artemia (containing an epinecidin-1 transgene) can help in Taiwan aquaculture to culture fish larvae that are resistant to pathogen infection. Artemia expressing CMV-gfp-epi plasmid conferred efficient antimicrobial activity in larval fish without introducing drug residues or inducing bacterial drug resistance. Older larvae to younger small fish can be fed on fodder mixed with recombinant epinecidin-1/DsRed fusion protein obtained using the E. coli protein expression system. Both technologies may play important roles as a culture chain from larval to fry (or young fish) for use in aquaculture.     
 

Project 3: Demonstration of antitumor function of fish antimicrobial peptides

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 employed in cancer research. We found pardaxin (GE33) exerts antitumor function and modulates immune responses in mammalian immune systems

For the above-described project, our important contributions are as follows:

1) Our results indicate that pardaxin selectively targets human cervical carcinoma HeLa cells in an electrostatic manner, resulting in production of ROS to induce oxidative stress and UPR to trigger signal transduction of JNK/c-Jun and PERK/eIF2α/CHOP; in turn, this leads to caspase activation and AIF-dependent apoptotic events, such as loss of mitochondrial membrane potential, decrease of RhoGDI (which is postulated to induce the initial morphology of apoptosis by regulating actin polymerization), and chromatin condensation. Proteomic analysis revealed that pardaxin triggers apoptotic signaling pathways in human cervical carcinoma HeLa cells (cross talk among the UPR, c-Jun, and ROS).

2) 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 cell line) cells revealed induction of the gene encoding c-FOS, an AP-1 transcription factor. 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.

3) 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 of pardaxin treatment in dogs with different types of refractory tumor. Local injection of pardaxin resulted in a significant reduction of perianal gland adenoma growth between 28 and 38 days post-treatment. Surgical resections of canine histiocytomas appear as large areas of ulceration, suggesting that pardaxin acts like a lytic peptide. However, pardaxin treatment was not associated with significant variations in blood biochemical parameters or secretion of immune-related proteins. Our research results indicate that pardaxin has strong therapeutic potential for treating perianal gland adenomas in dogs. These data suggest that pardaxin may be suitable for veterinary application, and also provide valuable information for veterinary medicine and future human clinical trials. We have applied for Taiwanese patents for the use of pardaxin in dog cancer therapy, and are now conferring with a biotechnological company for technology licensing and technology transfer.

Project 4: Demonstration of antibacterial function and application of fish antimicrobial peptides

Almost all antimicrobial peptides (AMPs) studied in my lab show direct antibacterial or bacteriostatic function against Gram-negative and -positive strains. We studied both the application and basic mechanisms of AMP function.

For the above-described project, our important contributions are as follows:

1) Our research results demonstrated the antimicrobial functions of epinecidin-1, tilapia piscidin 4 (TP4), and tilapia piscidin 3 (TP3) against skin trauma injury-mediated MRSA infection in mice. An excision of one square centimeter was made in the outer skin of mice, and a lethal dose of MRSA was applied in the presence or absence of methicillin, vancomycin, epinecidin-1, TP3, or TP4. Mice that received MRSA or MRSA together with methicillin died in four days, whereas the presence of epinecidin-1, TP3, or TP4 protected the mice against MRSA infection. Epinecidin-1, TP3, or TP4 decreased MRSA bacterial counts in the wounded region, improved wound closure, and increased angiogenesis in the wounded region. In conclusion, these three AMPs may act as an effective antimicrobial agent against clinical antibiotic-resistant MRSA, and may be considered for clinical studies against MRSA infection. These research results were obtained in collaboration with Dr. Chang-Jer Wu (Department of Food Science, National Taiwan Ocean University), as the Marine Research Station is lacking facilities for animal experiments. We have applied for US and Taiwanese patents for these innovations, and are negotiating with biotechnology companies for the technological transfer of epinecidin-1 and TP4 as components of burn treatment medical products.

2) Burns are incredibly painful, and many patients die due to bacterial infections of wounds or burns every year. Pain control and therapeutic treatment for burns and wounds remain challenging due to the complex natural history, unclear aetiology, and poor response towards drugs. Neuropathic pain is a widespread health problem associated with nerve injury, prolonged tissue damage, or injury to the peripheral or central nervous system (CNS), which arise through a number of complex changes occurring at various levels in nociceptive pathways. We cooperated with Dr. Zhi-Hong Wen (Department of Marine Biotechnology and Resources, National Sun Yat-Sen University) and applied his animal system to study the use of the antimicrobial peptide piscidin (PCD)-1 as a novel anti-nociceptive agent. For our in vivo studies, mononeuropathy in rat was induced by chronic constriction injury (CCI), and antinociceptive behaviors were compared between naïve rats and CCI models in the presence or absence of PCD-1 (delivered by intrathecal injection). Similar to gabapentin, PCD-1 exerted antinociceptive activity with an approximately 50% effective dose of 9.5 μg against thermal hyperalgesia in CCI rat models. In CCI rats, PCD-1 had antinociceptive effects against mechanical and cold allodynia, thermal hyperalgesia, and weight-bearing threshold. CCI-mediated activation of microglia, astrocytes, and neuron cells were regulated by PCD-1 in the dorsal horn of rat lumbar spinal cord sections. These research results demonstrated that the marine AMP PCD-1 has anti-nociceptive effects, and thus may have potential for development as an alternative pain-alleviating agent; PCD-1 may be combined with epinecidin-1 or TP4 to exert anti-nociceptive and antimicrobial activity for treatment of wounds or burns. We have applied for US and Taiwanese patents for these inventions.

3)We demonstrated the antimicrobial properties of epinecidin-1 against multi-drug resistant clinical isolates of P. aeruginosa (P. aeruginosa (R)) and P. aeruginosa from ATCC (P. aeruginosa (19660)), both in vitro and in vivo. The minimum inhibitory concentrations (MICs) of the peptide against P. aeruginosa (R) and P. aeruginosa (19660) were studied in comparison with those of imipenem. Our results suggested that P. aeruginosa (R) was more susceptible than P. aeruginosa (19660) to epinecidin-1 in vitro. Epinecidin-1 was highly effective at combating peritonitis infection caused by P. aeruginosa (R) or P. aeruginosa (19660) in mouse models, and did not induce adverse effects on the liver or kidney, or on behavior. These findings indicate that epinecidin-1 enhances the survival rate of mice against the bacterial pathogen P. aeruginosa through both antimicrobial and immunomodulatory roles.  

近年重要著作 (2017- ):

*責任作者

Hsueh-Ming Tai, Ming-Feng You, Chia-Hua Lin, Tsung-Yu Tsai, Chieh-Yu Pan#, Jyh-Yih Chen* (2021). Scale-up production of and dietary  supplementation with the recombinant antimicrobial peptide tilapia piscidin 4 to improve growth performance in Gallus gallus domesticus. Plos one (in press) *Corresponding author.

Pin-Yang Tu, Shin-Jie Huang, Venugopal Rajanbabu, Jen-Leih Wu, Jyh-Yih Chen* (2021). Comparative transcriptome analysis reveals ectopic delta-5 and delta-6 desaturases enhance protective gene expression upon Vibrio vulnificus challenge in Tilapia (Oreochromis niloticusis). BMC genomics (in press) *Corresponding author.

Bor-Chyuan Su, Giun-Yi Hung, Yun-Chieh Tu, Wei-Chen Yeh, Meng-Chieh Lin, Jyh-Yih Chen* (2021). Marine antimicrobial peptide TP4 exerts anticancer effects on human synovial sarcoma cells via calcium overload, reactive oxygen species production and mitochondrial hyperpolarization. Marine drugs (in press) *Corresponding author.

Bor-Chyuan Su, Chao-Chin Li, Chia-Wen Liu, Jyh-Yih Chen* (2020). A pilot safety assessment for recombinant Epinephelus lanceolatus piscidin yeast powder as a drug food additive after subacute and subchronic administration to SD rats. Marine drugs (in press) *Corresponding author.

Bor-Chyuan Su, Yi-Chung Liu, Chen-Hung Ting, Ping-Chiang Ly, Jyh-Yih Chen* (2020). Antimicrobial peptide TP4 targets mitochondrial adenine nucleotide translocator 2. Marine drugs (in press) *Corresponding author.

Bor-Chyuan Su, Chen-Hung Ting, Kang-Yun Lee, Sheng-Ming Wu, Po-Hao Feng, Yao-Fei Chan, Jyh-Yih Chen* (2020). Novel PD-L1 mAb HC16 reveals upregulation of PD-L1 in BAC subtype and early-stage. Histology and Histopathology (in press) *Corresponding author.

Keng-Yu Chiang, Wen-Chun Lin, Tsung-Yu Tsai, Cheng-Wei Lin, Shin-Jie Huang, Ching-Yu Huang, Sheng-Han Wu, Chuian-Fu Ken, Hong-Yi Gong*, Jyh-Yih Chen*, Jen-Leih Wu* (2020). Dual expression of transgenic delta-5 and delta-6 desaturase in tilapia alters gut microbiota and enhances resistance to Vibrio vulnificus infection. PLOS ONE (in press) *Corresponding author.

Prakash Kishore Hazam, Jyh-Yih Chen* (2020). Therapeutic utility of the antimicrobial peptide Tilapia Piscidin 4 (TP4). Aquaculture reports (in press) *Corresponding author.

Bor-Chyuan Su, Chao-Chin Li, Jiun-Lin Horng, Jyh-Yih Chen* (2020). Calcium-dependent calpain activation-mediated mitochondrial dysfunction and oxidative stress are required for cytotoxicity of epinecidin-1 in human synovial sarcoma SW982 cells. International Journal of Molecular Sciences (in press) *Corresponding author.

Hsiao-Ching Chen, Chieh-Yu Pan, Venugopal Rajanbabu, Yen-Yun Lee, Wei-Ren Tsai, Jyh-Yih Chen* (2020). Lack of acute toxicity and mutagenicity from recombinant Epinephelus lanceolatus piscidin expressed in Pichia pastoris. Marine drugs (in press) *Corresponding author.

Hsueh-Ming Tai, Han-Ning Huang, Tsung-Yu Tsai, Ming-Feng You, Hung-Yi Wu, Venugopal Rajanbabu, Hsiao-Yun Chang, Chieh-Yu Pan, Jyh-Yih Chen* (2020). Dietary supplementation of recombinant antimicrobial peptide Epinephelus lanceolatus piscidin improves growth performance and immune response in Gallus gallus domesticus. PLOS ONE (in press). *Corresponding author.

Bor-Chyuan Su, Jyh-Yih Chen* (2020). Pharmacological inhibition of p38 potentiates antimicrobial peptide TP4-induced cell death in glioblastoma cells. Molecular and Cellular Biochemistry 464(1-2):1-9. *Corresponding author.

Bor-Chyuan Su, Tsung-Han Wu, Chun-Hua Hsu, Jyh-Yih Chen* (2020). Distribution of positively charged amino acid residues in antimicrobial peptide epinecidin-1 is crucial for in vitro glioblastoma cytotoxicity and its underlying mechanisms. Chemico-Biological Interactions 315:108904. *Corresponding author.

Bor-Chyuan Su, Jyh-Yih Chen* (2020). Epinecidin-1: an orange-spotted grouper antimicrobial peptide that modulates Staphylococcus aureus lipoteichoic acid-induced inflammation in macrophage cells. Fish & Shellfish Immunology 99:362-367. *Corresponding author.

Han-Ning Huang, Tsung-Yu Tsai, Venugopal Rajanbabu, Chieh-Yu Pan, Jyh-Yih Chen* (2020). Dietary supplementation of recombinant Tilapia Piscidin-4-expressing yeast enhances growth and immune response in Lates calcarifer. Aquaculture Reports (in press) *Corresponding author.

Bor-Chyuan Su, Chieh-Yu Pan, Jyh-Yih Chen* (2019). Antimicrobial peptide TP4 induces ROS-mediated necrosis by triggering mitochondrial dysfunction in wild-type and mutant p53 glioblastoma cells. Cancers (Basel). Cancers (Basel). 2019 Feb 1;11(2). pii: E171. doi: 10.3390/cancers11020171. *Corresponding author.

Han-Ning Huang, Jyh-Yih Chen* (2018). Evaluation of antimicrobial peptides as antiviral agents against foot and mouth disease virus. Peptides (in press) *Corresponding author.

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.

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.

Bor-Chyuan Su, Jyh-Yih Chen* (2017). Antimicrobial peptide epinecidin-1 modulates MyD88 protein expression via the proteasome degradation pathway. Marine drugs (in press) *Corresponding author.

Bor-Chyuan Su, Han-Ning Huang, Tai-Wen Lin, Chwan-Deng Hsiao, Jyh-Yih Chen* (2017). Epinecidin-1 protects mice from LPS-induced endotoxemia and cecal ligation and puncture-induced polymicrobial sepsis. BBA - Molecular Basis of Disease (in press) *Corresponding author.

Chuian-Fu Ken, Chieh-Ning Chen, Chen-Hung Ting, Chieh-Yu Pan*, Jyh-Yih Chen* (2017). Transcriptome analysis of hybrid tilapia (Oreochromis spp.) with Streptococcus agalactiae infection identifies Toll-like receptor pathway-mediated induction of NADPH oxidase complex and piscidins as primary immune-related responses. Fish and Shellfish Immunology (in press) *Corresponding author.

Bor-Chyuan Su, Wen-Chun Lin, Han-Ning Huang, Jyh-Yih Chen* (2017). Recombinant expression of Epinephelus lanceolatus serum amyloid A (ElSAA) and analysis of its macrophage modulatory activities. Fish and Shellfish Immunology 64:276-286. *Corresponding author.

Chieh-Yu Pan, Yu-Hao Liu, Hong-Yi Gong, Jyh-Yih Chen* (2017). Transcriptome analysis of the effect of polyunsaturated fatty acids against Vibrio vulnificus infection in Oreochromis niloticus. Fish and Shellfish Immunology 62:153-163. *Corresponding author.

Han-Ning Huang, Chieh-Yu Pan, Hung-Yi Wu*, Jyh-Yih Chen* (2017). Antimicrobial peptide Epinecidin-1 promotes complete skin regeneration of methicillin-resistant Staphylococcus aureus-infected burn wounds in a swine model. Oncotarget 8(13):21067-21080. *Corresponding author.

Chieh-Yu Pan*, Tsung-Yu Tsai, Bor-Chyuan Su, Cho-Fat Hui, Jyh-Yih Chen* (2017). Study of the antimicrobial activity of tilapia piscidin 3 (TP3) and TP4 and their effects on immune functions in hybrid tilapia (Oreochromis spp.). Plos one 12(1):e0169678. *Corresponding author.