Jen-Leih Wu

Suppression of myostatin with vector-based RNA interference causes a double-muscle effect in transgenic zebrafish.

Myostatin belongs to the transforming growth factor (TGF)-beta superfamily and is a potent negative regulator of skeletal muscle development and growth. We utilized microinjection of an antisense RNA-expressing vector to establish a hereditarily stable myostatin gene knockdown zebrafish strain with a double-muscle phenotype. Real-time PCR and immunostaining revealed that the myostatin messenger (m)RNA and protein levels in homozygous transgenic zebrafish were 33% and 26% those of the non-transgenic controls, respectively. Also, the mRNA levels of myogenic regulatory factor markers such as MyoD, myogenin, Mrf4, and Myf5 were dramatically elevated in myostatin-suppressed transgenic fish compared to the non-transgenic controls. Although there was no significant difference in body length, homozygous transgenic zebrafish were 45% heavier than non-transgenic controls. Histochemical analysis showed that the cross-sectional area of the muscle fiber of homozygous transgenic fish was twice as large as that of non-transgenic controls. This is the first model zebrafish with a hereditarily stable myostatin-suppressed genotype and a double-muscle phenotype.

The characterization of prepro-insulin-like growth factor-1 Ea-2 expression and insulin-like growth factor-1 genes (devoid 81 bp) in the zebrafish (Danio rerio).

In this study, we cloned zebrafish (Danio rerio) IGF-1 cDNA and gene from zebrafish brain cDNA library and adult zebrafish genomic library, respectively. Based on two cDNAs sequence with different length of 5- and 3-untranslated region (5UTR and 3UTR) and one nucleotide difference at glutamine (A9, CAG) of A domain represented at IGF-1 sequence. One of zebrafish IGF-1 genes named as IGF-1a gene. The zebrafish IGF-1a gene spanned approximately 15 kb and is divided into five exons. The results of IGF-1 cDNA and genomic Southern blotting, all indicated that the zebrafish have more than one IGF-1 gene. The genomic organization of zebrafish IGF-1a gene in an exon is devoid of 81 bp segment which is located at 3 end of exon 3 encoded 27 amino acid of E domain. The segment of 27 amino acid exists in known teleost IGF-1 genes but is absent in zebrafish IGF-1 gene. The E domain of zebrafish IGF-1 Ea-2 is encoded by 3 end of exon 3 (16 amino acid), full of exon 4 (12 amino acid) and exon 5 (19 amino acid). The sequence data revealed the zebrafish IGF-1a gene encoded IGF-1a Ea-2 mRNA. In combination RT-PCR with Southern blotting, zebrafish IGF-1 genes abundantly expressed IGF-1 Ea-2 mRNA in all tested adult tissues and developmental stages of embryo. The IGF-1 Ea-2 mRNA was first detected during embryo development from blastula stage to hatching, during yolk absorption and at feeding. All these findings suggest that the expression of pro-IGF-1 Ea-2 is not controlled by alternative splicing but alternative gene usage in the zebrafish.

A zebrafish model of intrahepatic cholangiocarcinoma by dual expression of hepatitis B virus X and hepatitis C virus core protein in liver

The mechanisms that mediate the initiation and development of intrahepatic cholangiocarcinoma (ICC) associated with hepatitis B and C virus (HBV and HCV, respectively) infection remain largely unclear. In this study we conditionally coexpressed hepatitis B virus X (HBx) and hepatitis C virus core (HCP) proteins in zebrafish livers, which caused fibrosis and consequently contributed to ICC formation at the age of 3 months. Suppressing the transgene expression by doxycycline (Dox) treatment resulted in the loss of ICC formation. The biomarker networks of zebrafish ICC identified by transcriptome sequencing and analysis were also frequently involved in the development of human neoplasms. The profiles of potential biomarker genes of zebrafish ICC were similar to those of human cholangiocarcinoma. Our data also showed that the pSmad3L oncogenic pathway was activated in HBx and HCP‐induced ICC and included phosphorylation of p38 mitogen‐activated proteinbase (MAPK) and p44/42 mitogen‐activated protein kinase (ERK1/2), indicating the association with transforming growth factor beta 1 (TGF‐β1) signaling pathway in ICC. Bile duct proliferation, fibrosis, and ICC were markedly reduced by knockdown of TGF‐β1 by in vivo morpholinos injections. Conclusion: These results reveal that TGF‐β1 plays an important role in HBx‐ and HCP‐induced ICC development. This in vivo model is a potential approach to study the molecular events of fibrosis and ICC occurring in HBV and HCV infection. (HEPATOLOGY 2012;56:2268–2276)

Activation of cytokine expression occurs through the TNFα/NF-κB-mediated pathway in birnavirus-infected cells

The infectious pancreatic necrosis virus (IPNV) belongs to the Birnaviridae family of viruses and causes acute contagious diseases in a number of economically important freshwater and marine fish. In this study, we infected zebrafish embryonic cells (ZF4) with IPNV and analyzed the gene expression patterns of normal and infected cells using quantitative real-time PCR. We identified a number of immune response genes, including ifna, ifng, mx, irf1, irf2, irf4, tnfa, tnfb, il-1b, il-15, il-26, ccl4 and mmp family genes, that are induced after viral infection. Transcriptional regulators, including cebpb, junb, nfkb and stat1, stat4 and stat5, were also upregulated in IPNV-infected cells. In addition, we used Pathway Studio software to identify TNFα as having the greatest downstream influence among these altered genes. Treating virus-infected cells with an siRNA targeting TNFα inhibited NF-κB expression. To further interrupt the TNFα/NF-κB-mediated pathway, the expression levels of cytokines and metalloproteinases were inhibited in IPNV-infected cells. These data suggest that, during IPNV infection, the expression of cytokines and metalloproteinases might be initiated through the TNFα/NF-κB-mediated pathway. The modulation of TNFα/NF-κB-related mechanisms may provide a therapeutic strategy for inhibiting viral infection in teleosts.

Truncated antimicrobial peptides from marine organisms retain anticancer activity and antibacterial activity against multidrug-resistant Staphylococcus aureus

Antimicrobial peptides (AMPs) were recently determined to be potential candidates for treating drug-resistant bacterial infections. The aim of this study was to develop shorter AMP fragments that combine maximal bactericidal effect with minimal synthesis cost. We first synthesized a series of truncated forms of AMPs (anti-lipopolysaccharide factor from shrimp, epinecidin from grouper, and pardaxin from Pardachirus marmoratus). The minimum inhibitory concentrations (MICs) of modified AMPs against ten bacterial species were determined. We also examined the synergy between peptide and non-peptide antibiotics. In addition, we measured the inhibitory rate of cancer cells treated with AMPs by MTS assay. We found that two modified antibacterial peptides (epinecidin-8 and pardaxin-6) had a broad range of action against both gram-positive and gram-negative bacteria. Furthermore, epinecidin and pardaxin were demonstrated to have high antibacterial and anticancer activities, and both AMPs resulted in a significant synergistic improvement in the potencies of streptomycin and kanamycin against methicillin-resistant Staphylococcus aureus. Neither AMP induced significant hemolysis at their MICs. In addition, both AMPs inhibited human epithelial carcinoma (HeLa) and fibrosarcoma (HT-1080) cell growth. The functions of these truncated AMPs were similar to those of their full-length equivalents. In conclusion, we have successfully identified shorter, inexpensive fragments with maximal bactericidal activity. This study also provides an excellent basis for the investigation of potential synergies between peptide and non-peptide antibiotics, for a broad range of antimicrobial and anticancer activities.

Stage-Specific Expression of TNFα Regulates Bad/Bid-Mediated Apoptosis and RIP1/ROS-Mediated Secondary Necrosis in Birnavirus-Infected Fish Cells

Infectious pancreatic necrosis virus (IPNV) can induce Bad-mediated apoptosis followed by secondary necrosis in fish cells, but it is not known how these two types of cell death are regulated by IPNV. We found that IPNV infection can regulate Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic death pathways via the up-regulation of TNFα in zebrafish ZF4 cells. Using a DNA microarray and quantitative RT-PCR analyses, two major subsets of differentially expressed genes were characterized, including the innate immune response gene TNFα and the pro-apoptotic genes Bad and Bid. In the early replication stage (0–6 h post-infection, or p.i.), we observed that the pro-inflammatory cytokine TNFα underwent a rapid six-fold induction. Then, during the early-middle replication stages (6–12 h p.i.), TNFα level was eight-fold induction and the pro-apoptotic Bcl-2 family members Bad and Bid were up-regulated. Furthermore, specific inhibitors of TNFα expression (AG-126 or TNFα-specific siRNA) were used to block apoptotic and necrotic death signaling during the early or early-middle stages of IPNV infection. Inhibition of TNFα expression dramatically reduced the Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic cell death pathways and rescued host cell viability. Moreover, we used Rip1-specific inhibitors (Nec-1 and Rip1-specific siRNA) to block Rip1 expression. The Rip1/ROS-mediated secondary necrotic pathway appeared to be reduced in IPNV-infected fish cells during the middle-late stage of infection (12–18 h p.i.). Taken together, our results indicate that IPNV triggers two death pathways via up-stream induction of the pro-inflammatory cytokine TNFα, and these results may provide new insights into the pathogenesis of RNA viruses.

Transgenic expression of salmon delta-5 and delta-6 desaturase in zebrafish muscle inhibits the growth of Vibrio alginolyticus and affects fish immunomodulatory activity.

Marine fish are an important nutritional source for highly polyunsaturated fatty acids (PUFAs). PUFA biosynthesis requires the following key enzymes: delta-4 (Δ-4) desaturase, delta-5 (Δ-5) desaturase, delta-6 (Δ-6) desaturase, delta-5 (Δ-5) elongase, and delta-6 (Δ-6) elongase. The effect of overexpressing delta-5 desaturase and/or delta-6 desaturase in zebrafish muscle has not previously been reported. Herein, we investigated the effects of these proteins on antibacterial and immunomodulatory activity in transgenic zebrafish infected with Vibrio alginolyticus. Overexpression of delta-5 and delta-6 desaturase enhanced antibacterial activity at 4 and 12 h after injection of bacteria into muscle, as compared to controls. Furthermore, expression of immune-related genes (IL-1β, IL-22, and TNF-α) was observed to be altered in transgenic fish after 4 h of bacterial infection, resulting in a significant decrease in the inflammatory response, as compared to control fish. These results demonstrate that muscle-specific expression of transgenic desaturases in zebrafish not only enhance PUFA production, but also enhance antibacterial and anti-inflammatory activity. Overall, these results identify delta-5 and delta-6 desaturase as novel candidate genes for use in aquaculture, to enhance both disease resistance and fish oil production.

Aquatic birnavirus infection activates the transcription factor NF-κB via tyrosine kinase signalling leading to cell death

Our previous studies found that infectious pancreatic necrosis virus (IPNV) induces host apoptotic cell death, possibly through a newly synthesized protein trigger. Here, we examine whether IPNV infection can induce NF-kappaB activation through tyrosine kinase signalling of CHSE-214 cell death (host cell death). Using the electrophoretic mobility shift assay (EMSA) to detect transcription factor activation, we found that NF-kappaB is apparently activated 6-8 h post-IPNV infection. Using genistein (100 microg mL(-1); a tyrosine kinase inhibitor) to determine whether NF-kappaB activation requires tyrosine kinase activation, we found genistein blocks NF-kappaB activation at 8 h post-infection (p.i), and either enhances cell viability up to 50% at 12 h p.i. or blocks DNA fragmentation at 24 h p.i. Furthermore, the proteasome inhibitors PSI-I and PSI-II (both at 40 microm) also effectively blocked the NF-kappaB activation as well as stimulating a 30% increase in cell viability (30% decrease in apoptosis) at 8 and 12 h p.i. Taken together our data suggest that IPNV may induce NF-kappaB activation through tyrosine kinase signalling, which may be associated with induction of apoptosis.

Bioenergetics of Adaptation to a Salinity Transition in Euryhaline Teleost (Oreochromis mossambicus) Brain

Freshwater (FW) teleosts are capable of acclimating to seawater (SW) following such a transfer from FW. However, their osmo-regulating mechanisms are still unclear, particularly those in the brain. The present study was conducted to examine acute changes that occur in brain Na+-K+-ATPase activity, creatine kinase (CK) activity, creatine, creatinine contents, and ATP levels of tilapia (Oreochromis mossambicus) in response to this transition. After transfer to SW (25 ppt), the Na+-K+-ATPase activity was maintained for 8 hr at higher levels than that in FW. In contrast, in 35 ppt SW, Na+-K+-ATPase was maintained at a even higher level than in FW for the first 2 hr. Brain Na+-K+-ATPase contents in both the 25 and 35 ppt SW groups were significantly elevated within 1 and 0.5 hr after transfer from FW, respectively. Interestingly, brain CK activities and content (homodimer of the B subunit [BB] form) in both the 25 and 35 ppt SW groups were significantly elevated within 1 hr after transfer from FW. The ATP contents in 35 ppt SW increased abruptly within 0.5 hr, and then gradually decreased during the next 2 hr. Unlike the 35 ppt group that declined in ATP contents, the 25 ppt group leveled off within 24 hr. The elevations in CK activity and creatine levels after transfer from FW to SW imply that abrupt salinity changes alter phosphocreatine/CK ratio. Such changes are needed to satisfy the increases in the energetic requirement of the cotransport mechanisms mediating osmoregulation

The antimicrobial peptide, shrimp anti-lipopolysaccharide factor (SALF), inhibits proinflammatory cytokine expressions through the MAPK and NF-κB pathways in Trichomonas vaginalis adherent to HeLa cells.

Trichomonas vaginalis is a parasitic protozoan that causes sexually transmitted infections (STIs) worldwide. The infection is dangerous and easily spreads within a community. Also, some cases of drug resistance were reported. Previously, we reported that the shrimp anti-lipopolysaccharide factor (SALF), an antimicrobial peptide of 24 amino acids, modulates inflammatory responses and inhibits T. vaginalis growth. To date, there is no report on the mechanism of SALFs actions in T. vaginalis adherence to HeLa cells. In this research using an ELISA, we found that the SALF downregulated the release of proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1) secreted by T. vaginalis which was adhering to HeLa cells. We also performed real-time PCR experiments to determine the roles of the SALF in the expressions of several proinflammatory genes. Through a Western blot analysis, we determined that SALF treatment inhibited T. vaginalis-treated HeLa cells through the p38 and NF-κB pathways. Furthermore, we used different inhibitors to confirm the pathway by ELISA and Western blotting. Taken together, it is apparent that the SALF suppresses T. vaginalis-induced secretion of proinflammatory cytokines by HeLa cells by acting through the p38 and NF-κB pathways.