Exposure to benzidine caused apoptosis and malformation of telencephalon region in zebrafish.

Exposure to benzidine has been known to induce human cancers, particularly bladder carcinomas. In this study, the zebrafish model was used to investigate the developmental toxicity of benzidine. Embryos at 6 h postfertilization (hpf) that were exposed to benzidine exhibited embryonic death in a dose- and time-dependent manner. Benzidine induced malformations in zebrafish, such as small brain development, shorter axes, and a slight pericardial edema. High concentrations (50, 100, and 200 µM) of benzidine triggered widespread apoptosis in the brain and dorsal neurons, as evidenced by acridine orange and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays. Real-time polymerase chain reaction analysis also showed that benzidine treatment affected p53, bax, and noxa expression. Decreases in specific brain markers, such as emx1 in the telencephalon, ngn1 in differentiated neurons, and otx2 in the midbrain, were observed in benzidine-treated embryos at 24 hpf. Conversely, no overt changes to pax2.1 expression in the midbrain-hindbrain boundary were found. Moreover, the use of Tg(HuC:GFP) zebrafish showed that benzidine caused a malformation of the telencephalon region. Our findings show that benzidine exposure triggers widespread apoptosis in the zebrafish brain and dorsal neurons, resulting in the development of an abnormal telencephalon.

Progranulin A-mediated MET signaling is essential for liver morphogenesis in zebrafish.

The mechanism that regulates embryonic liver morphogenesis remains elusive. Progranulin (PGRN) is postulated to play a critical role in regulating pathological liver growth. Nevertheless, the exact regulatory mechanism of PGRN in relation to its functional role in embryonic liver development remains to be elucidated. In our study, the knockdown of progranulin A (GrnA), an orthologue of mammalian PGRN, using antisense morpholinos resulted in impaired liver morphogenesis in zebrafish (Danio rerio). The vital role of GrnA in hepatic outgrowth and not in liver bud formation was further confirmed using whole-mount in situ hybridization markers. In addition, a GrnA deficiency was also found to be associated with the deregulation of MET-related genes in the neonatal liver using a microarray analysis. In contrast, the decrease in liver size that was observed in grnA morphants was avoided when ectopic MET expression was produced by co-injecting met mRNA and grnA morpholinos. This phenomenon suggests that GrnA might play a role in liver growth regulation via MET signaling. Furthermore, our study has shown that GrnA positively modulates hepatic MET expression both in vivo and in vitro. Therefore, our data have indicated that GrnA plays a vital role in embryonic liver morphogenesis in zebrafish. As a result, a novel link between PGRN and MET signaling is proposed.

Composition of nanoclay supported silver nanoparticles in furtherance of mitigating cytotoxicity and genotoxicity.

Silver nanoparticle (Ag-NP) is well known for its high antibacterial efficacy. However, its toxicity toward mammalian cells is still a concern in clinical applications. The aim of our study was to evaluate the composition effects of Ag-NP supported by silicate nanoplatelet (NSP) with respect to the cytotoxicity and genotoxicity, and was in reference to the poly (styrene-co-maleic anhydride)-supported Ag-NP (Ag-NP/SMA). The NSP at the geometric dimension of averaged 80 x 80 x 1 nm3 was prepared from the exfoliation of natural clays and used to support different weight ratio of Ag-NP. The supporting limitation of NSP on Ag-NP was below the weight ratio of 15/85 (Ag-NP to NSP), and the detached Ag-NP from the Ag-NP/NSP (30/70) and Ag-NP/SMA hybrids were observed by TEM. Ames test was performed to assess the mutagenic potential of different compositions of Ag-NP/NSP, only Ag-NP/NSP (30/70) and Ag-NP/SMA hybrids exhibited mutagenicity when the concentration was 1.09 ppm or higher. In viewing of cytotoxicity using MTT tests toward HaCaT cells, the IC30 of Ag-NP/NSP (1/99, 7/93 and 15/85) were 1416.7, 243.6, and 148.9 ppm respectively, while Ag-NP/SMA was 64.8 ppm. The IC30 of Ag-NP/NSP (1/99, 7/93 and 15/85) were at least 833, 78 and 7 folds higher than their corresponding minimum inhibitory concentrations (MIC) respectively, and whereas Ag-NP/SMA was 6.4 folds. The Ag-NP/NSP and Ag-NP/SMA hybrids had been further investigated for genotoxicity by chromosomal aberrations and in vivo micronucleus assay within the concentration at IC10 and IC30, only Ag-NP/SMA showed a higher frequency of chromosomal aberrations. Our findings indicated that the viability of utilizing the NSP to maintain Ag-NP for antimicrobial activity, and the high-surface area of NSP served as an excellent support for associating Ag-NP and consequently rendering the mitigation of the inherent toxicity of Ag-NP in clinical uses.

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.

PKC and MEK pathways inhibit caspase-9/-3-mediated cytotoxicity in differentiated cells.

Many studies have indicated that differentiated cells inhibit drug-induced cytotoxicity but undifferentiated cells do not, though the mechanisms are unclear. Currently, HL-60 cells are induced to differentiate into macrophage-like cells with Phorbol-12-myristate-13-acetate (TPA) treatment (TPA-differentiated cells). Our study shows that caspase-9/-3-mediated cytotoxicity can be induced in undifferentiated HL-60 cells but not in TPA-differentiated HL-60 cells. However, caspase-9/-3-mediated cytotoxicity can be induced in TPA-differentiated cells if they are pretreated with a protein kinase C (PKC) or a mitogen activated protein kinase (MEK) inhibitor. Taken together, this study demonstrates that TPA-differentiated HL-60 cells inhibit caspases-9/-3-mediated cytotoxicity through the PKC and MEK signaling pathways.

3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity.

Artificial bone grafting is widely used in current orthopedic surgery for bone defect problems. Unfortunately, surgeons remain unsatisfied with the current commercially available products. One of the major complaints is that these products cannot provide sufficient mechanical strength to support the human skeletal structure. In this study, we aimed to develop a bone scaffold with better mechanical property and good cell affinity by 3D printing (3DP) techniques. A self-developed 3D printer with laser-aided gelling (LAG) process was used to fabricate bioceramic scaffolds with inter-porous structures. To improve the mechanical property of the bioceramic parts after heating, CaCO3 was added to the silica ceramic slurry. CaCO3 was blended into a homogenous SiO2-sol dispersion at weight ratios varying from 0/100 to 5/95 to 9/91 (w/w). Bi-component CaCO3/SiO2-sol was prepared as a biocomposite for the 3DP scaffold. The well-mixed biocomposite was used to fabricate the bioceramic green part using the LAG method. The varied scaffolds were sintered at different temperatures ranging from 900 to 1500°C, and the mechanical property was subsequently analyzed. The scaffolds showed good property with the composite ratio of 5:95 CaCO3:SiO2 at a sintering temperature of 1300°C. The compressive strength was 47 MPa, and the porosity was 34%. The topography of the sintered 3DP bioceramic scaffold was examined by SEM, EDS and XRD. The silica bioceramic presented no cytotoxicity and good MG-63 osteoblast-like cell affinity, demonstrating good biocompatibility. Therefore, the new silica biocomposite is viable for fabricating 3DP bone bioceramics with improved mechanical property and good cell affinity.

Two distinct teleost hepatocyte nuclear factor 1 genes, hnf1alpha/tcf1 and hnf1beta/tcf2, abundantly expressed in liver, pancreas, gut and kidney of zebrafish.

Two distinct forms of zebrafish hepatocyte nuclear factor 1 (hnf1) were identified and referred to as hnf1alpha/tcf1 and hnf1beta/tcf2. Both hnf1 genes were shown to be expressed abundantly in liver, pancreas, gut and kidney. Zebrafish HNF1alpha and HNF1beta proteins contain all HNF1 signature domains including the dimerization domain, POU-like domain and atypical homeodomain. Sequence and phylogenetic analysis reveals that zebrafish hnf1alpha is closer to tetrapodian hnf1alpha than to tetrapodian hnf1beta and zebrafish hnf1beta is highly conserved with tetrapodian hnf1beta. Existences of hnf1alpha and hnf1beta in teleost zebrafish, tilapia and fugu suggest that hnf1 gene duplication might occur before the divergence of teleost and tetrapod ancestors. Zebrafish hnf1alpha and hnf1beta genes were mapped to linkage group LG8 and LG15 in T51 panel by RH mapping and are composed of 10 and 9 exons, respectively. Zebrafish hnf1beta gene with at least 11 genes in LG15 was identified to maintain the conserved synteny with those of human in chromosome 17 and those of mouse in chromosome 11. Our results indicate that distinct hnf1alpha and hnf1beta genes in teleosts had been evolved from the hnf1 ancestor gene of chordate.

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 osmoregulating 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.

Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus).

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.

Evenly distributed thin-film Ag coating on stainless plate by tricomponent Ag/silicate/PU with antimicrobial and biocompatible properties.

A tricomponent nanohybrid dispersion in water comprising silver nanoparticles (AgNP), nanometer-thick silicate platelets (NSP), and water-based polyurethane (PU) was developed for surface coating on orthopedic metal plates. The previously developed AgNP-on-NSP nanohybrid was homogeneously blended into a selected waterborne PU dispersion at varied weight ratios from 1/0.1 to 1/10 (w/w). PU was used to adhere the Ag nanohybrid to the metal surface. The resultant dispersions were analyzed and found to contain AgNP 2-18 nm in diameter and characterized by using UV absorption and TEM micrograph. The subsequent coating of AgNP/NSP-PU dispersion generated a film of 1.5 µm thickness on the metal plate surface, further characterized by an energy dispersive spectroscope (EDS) to show the homogeneous distribution of Ag, Si, and C elements on the metal plates. The surface antimicrobial efficacy was proven for the coating composition of AgNP/NSP to PU ranging from 1/1 to 1/5 by weight ratio but irrelevant to the thickness of the coated materials. The metal plate coated with the high Ag content at 1/1 (w/w) ratio was shown to have very low cytotoxicity toward the contacted mammal fibroblasts. Overall, the optimized tricomponent Ag/silicate/PU in water dispersion from 1/2 to 1/3 (w/w) could generate a stable film on a metal surface exhibiting both antimicrobial and biocompatible properties. The facile coating technique of the AgNP/NSP in waterborne PU is proven to be viable for fabricating infection- and cytotoxicity-free medical devices.

Aquatic birnavirus-induced ER stress-mediated death signaling contribute to downregulation of Bcl-2 family proteins in salmon embryo cells.

Aquatic birnavirus induces mitochondria-mediated cell death, but whether connects to endoplasmic reticulum (ER) stress is still unknown. In this present, we characterized that IPNV infection triggers ER stress-mediated cell death via PKR/eIF2α phosphorylation signaling for regulating the Bcl-2 family protein expression in fish cells. The IPNV infection can induce ER stress as follows: (1) ER stress sensor ATF6 cleavaged; (2) ER stress marker GRP78 upregulation, and (3) PERK/eIF2α phosphorylation. Then, the IPNV-induced ER stress signals can induce the CHOP expression at early (6 h p.i.) and middle replication (12 h p.i.) stages. Moreover, IPNV-induced CHOP upregulation dramatically correlates to apparently downregulate the Bcl-2 family proteins, Bcl-2, Mcl-1 and Bcl-xL at middle replication stage (12 h p.i.) and produces mitochondria membrane potential (MMP) loss and cell death. Furthermore, with GRP78 synthesis inhibitor momitoxin (VT) and PKR inhibitor 2-aminopurine (2-AP) treatment for blocking GRP78 expression and eIF2α phosphorylation, PKR/PERK may involve in eIF2α phosphorylation/CHOP upregulation pathway that enhances the downstream regulators Bcl-2 family proteins expression and increased cell survival. Taken together, our results suggest that IPNV infection activates PKR/PERK/eIF2α ER stress signals for regulating downstream molecules CHOP upregulation and Bcl-2 family downregulation that led to induce mitochondria-mediated cell death in fish cells, which may provide new insight into RNA virus pathogenesis and disease.

RC-RNase-induced cell death in estrogen receptor positive breast tumors through down-regulation of Bcl-2 and estrogen receptor.

RC-RNase exerts anti-cancer effects on many tumors. However, the mechanisms by which RC-RNase induces cytotoxicity in different tumor cells are unclear. Currently, estrogen receptor (ER)-positive and negative breast tumors are treated with RC-RNase. Our data demonstrate that RC-RNase induces cell death on ER-positive but not on ER-negative breast tumors. This study also shows that down-regulation of ER and Bcl-2 is found on RC-RNase-treated ER-positive breast tumors. Additionally, Bcl-2 overxpression can prevent ER-positive breast tumors from cell death treated with RC-RNase. In summary, this study demonstrates that RC-RNase-induced cell death of ER-positive breast tumors is through regulation of ER and Bcl-2.

Immunotherapy of breast cancer by single delivery with rAAV2-mediated interleukin-15 expression.

Recombinant adenovirus-associated vector serotype 2 (rAAV2) is one of the most promising gene transfer vectors due to its advantage of causing non-pathogenic infection, low immunogenicity, and long-term gene expression in human clinical trials. Human interleukin 15 (hIL15) has been implicated in modulation of antitumor activity of lymphokine-activated killer (LAK) cells, including T cells and NK cells. In this study, the rAAV2-hIL15 vector was produced and subjected for treatment with xenograft JC breast cancer model. Results showed that tumor onset was significantly delayed, the tumor growth was suppressed, and the lifespan of tumor-bearing mice were prolonged by rAAV2-hIL15. In addition, rAAV2-hIL15 was able to produce a substantial expression of IL15 protein that ultimately activated the cytotoxic activity of LAK cells. Furthermore, prominent apoptosis was observed in tumor lesions following injection of rAAV2-hIL15. Taken together, our results suggested that rAAV2-hIL15 appears as a new potential therapeutic tool for breast cancer immunotherapy.

Cloning and functional analysis of the proximal promoter region of the three GnRH genes from the silver sea bream (Sparus sarba).

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a major role in releasing pituitary gonadotropin and controlling vertebrate reproduction. In this study, three GnRH cDNAs, GnRH-I (sbGnRH; 348 bp), GnRH-II (cGnRH-II; 557 bp), and GnRH-III (sGnRH; 483 bp), were cloned from the brain of the silver sea bream (Sparus sarba). In order to understand how the expression of the GnRH isoforms was regulated in the brain, the promoter of each gene was cloned and analyzed. We found regulatory motifs in the promoters that were conserved in the GnRH promoters of tilapia and zebrafish, suggesting that these motifs play a critical role in GnRH regulation. We performed functional analyses and examined tissue-specific expression for each GnRH promoter using EGFP reporter fusions in zebrafish. The GnRH-I promoter was active in the forebrain area, including the olfactory bulb-terminal nerve area and peripheral preoptic areas; the GnRH-II promoter was active in the midbrain; and the GnRH-III promoter was active in the olfactory bulb. These results show that the GnRH promoters of the silver sea bream GnRH genes exhibit tissue-specific activity.

XBP-1, a key regulator of unfolded protein response, activates transcription of IGF1 and Akt phosphorylation in zebrafish embryonic cell line.

The unfolded protein response (UPR) is a conserved and adaptive cellular response to increase cell survival during ER stress. XBP-1 spliced form (XBP-1S) generated by IRE1 endoribonuclease is a key transcriptional regulator in UPR to activate genes involved in protein folding and degradation to restore ER function. Although Akt activation was suggested to be a pro-survival pathway activated during ER stress, the signal to trigger Akt is still not clear. In this study, we report IGF1 transcription and Akt phosphorylation are enhanced in XBP-1S stably overexpressed clone of zebrafish embryonic cell line (ZF4). In addition, zebrafish IGF1 intron1 with predicted UPRE (XBP-1S binding sites) and ERSE (ATF6/XBP-1S binding site) linked with basal promoter could be activated by XBP-1S, not by XBP-1 unspliced form (XBP-1U). Furthermore, we demonstrate that expression of endogenous IGF1 is transiently induced as XBP-1 splicing during ER stress in parallel to ER chaperone GRP78/Hspa5 and ER resided E3 ubiquitin ligase Synoviolin in ZF4 cells by quantitative PCR. Our results suggest zebrafish XBP-1S not only activates genes responsible for protein folding, transporting, glycosylation and ER associated degradation but also activates anti-apoptosis signal via IGF1/Akt pathway in unfolded protein response to cope with ER stress.

Co-induction of hepatic IGF-I and progranulin mRNA by growth hormone in tilapia, Oreochromis mossambiccus.

Like IGF-I, progranulin (pgrn) is a growth factor involved in tumorigenesis and wound healing. We report here the identification and characterization of pgrn cDNA in tilapia and the regulation of its expression by growth hormone (GH). The tilapia pgrn cDNA was cloned by RT-PCR amplification, using gene specific oligonucleotides as amplification primers. The cDNA contains an open reading frame encoding a peptide of 206 amino acid residues (aa) that contains a presumptive signal peptide (23 aa) and two repeat units of granulin (grn, 51 and 52 aa, respectively) franked by a GAP of 49 aa and the carboxyl terminus with 31 aa. The two predicted grn peptides are arranged in tandem repeats interrupted by a GAP peptide. RT-PCR analysis revealed that high levels of prgn mRNA were present in several tissues such as spleen, gastric cecum, intestine, fat tissue, gill, kidney, eye and pancreas, and lower levels in liver, muscle, heart, brain, skin and stomach. Administration of a single dose (500 ng/g body weight) of recombinant seabream growth hormone (rbGH) by intraperitoneal (ip) injection into one-month-old tilapia resulted in an obvious increase of IGF-I and pgrn mRNA (2.7-fold and 2.5-fold, respectively) in the liver at three hours post-GH treatment. The peptide levels of pgrn in the liver of GH-treated fish also were substantially induced over controls at 12h post-GH treatment as detected by western immuno-blot analysis. The co-induction of IGF-I and pgrn following GH treatment may suggest the involvement of pgrn in GH regulated growth in tilapia.