Nutrient enrichment and probiotics for sea urchin Anthocidaris crassipina larvae in captivity to promote large-scale aquaculture.

Sea urchin contains physiologically active substances, such as amino acids and unsaturated fatty acids, and an important aquatic organism. Purple sea urchin, one of the common edible sea urchins, is an important aquatic product. In order to supply the vast seafood market, large-scale aquaculture of sea urchins is very important. The aim of this study was to optimize the rearing of the Anthocidaris crassipina larvae enhancing the nutrition by mixing feed to improve their growth and survival. The survival rate of Chaetoceros muelleri feeding alone is only 40%. If the survival rate is improved through nutrient enrichment, the large-scale aquaculture of larvae can be promoted. The experiment was divided into two parts. Experiment 1: Two types of commonly used microalgae, Isochrysis galbana tml (I), C. muelleri (C) and two types of probiotics, Rhodopseudomonas palustris (R), and Saccharomyces cerevisiae (S) were used in the. Feeding amounts are 5000, 10,000, and 20,000 cell mL-1, and the control group (N) did not eat. Experiment 2: C. muelleri 20,000 cell mL-1 was mixed with I. galbana tml, R. palustris (R) and S. cerevisiae (S) at 5000 and 10,000 cell mL-1. After the experiment, body length, body width, stomach length, rudiment length, rudiment length, body composition, digestive enzymes and survival rate were measured to evaluate the best feed. The results showed that the mixed feeding of C. muelleri 20,000 cell mL-1 and R. palustris 5000 cell mL-1 can achieve the best development and survival of larval embryos and can promote metamorphosis into juveniles in the shortest time. The research results will be applied to the large-scale aquaculture of A. crassipina larvae to promote the diversity of aquaculture.

Transcriptome analysis reveals modulation of differentially expressed genes in LPS-treated mouse macrophages (RAW264.7 cells) by grouper (Epinephelus coioides) Epinecidin-1.

The marine antimicrobial peptide Epinecidin (Epi)-1 has been shown to exert direct antimicrobial and immunomodulatory actions in teleost, mammalian and avian organisms. For instance, Epi-1 can suppress bacterial endotoxin lipolysachcharide (LPS)-induced proinflammatory cytokines in RAW264.7 murine macrophages. However, it remains unknown how Epi-1 might broadly affect non-activated and LPS-activated macrophages. To address this question, we performed a comparative transcriptomic analysis of non-treated and LPS-treated RAW264.7 cells in the presence and absence of Epi-1. Gene enrichment analysis was conducted on filtered reads, followed by GO and KEGG analyses. The results showed that Epi-1 treatment modulated pathways and genes associated with nucleoside binding, intramolecular oxidoreductase activity, GTPase activity, peptide antigen binding, GTP binding, ribonucleoside/nucleotide binding, phosphatidylinositol binding and phosphatidylinositol-4-phosphate binding. Based on the GO analysis results, we performed real-time PCR at different treatment times to compare expression levels of selected proinflammatory cytokines, anti-inflammatory cytokines, MHC, proliferation and differentiation genes. Epi-1 decreased expression of the proinflammatory cytokines, TNF-α, IL-6 and IL-1ß, and it increased the anti-inflammatory cytokine TGFß and Sytx1. MHC-associated genes, GM7030, Arfip1, Gpb11 and Gem, were induced by Epi-1, which is expected to enhance the immune response against LPS. Immunoglobulin-associated Nuggc was also upregulated by Epi-1. Finally, we found that Epi-1 downregulated the expression of host defense peptides CRAMP, Leap2 and BD3. Taken together, these findings suggest that Epi-1 treatment induces orchestrated changes in the transcriptome of LPS-stimulated RAW264.7 cells.

Lack of Acute Toxicity and Mutagenicity from Recombinant Epinephelus lanceolatus Piscidin Expressed in Pichia pastoris.

The antimicrobial peptide (AMP) piscidin was identified from Epinephelus lanceolatus and demonstrated to possess antimicrobial and immune-related functions. Supplementation of feed with recombinant Epinephelus lanceolatus piscidin (rEP)-expressing yeast pellets may minimize the excessive use of antibiotics and control pathogens in aquaculture or animal husbandry. However, before implementing rEP as a supplement, it is necessary to understand whether it harbors any toxicity. Since toxicological information on the topic is scarce, the present investigation was carried out to test whether rEP exhibits allergenic and/or toxic effects. In an oral acute toxicity test (OECD 425), Sprague Dawley (SD) rats were administered rEP dissolved in reverse osmosis water, yielding an LD50 > 5000 mg/kg (no observed animal death). The compound was therefore classified as non-toxic by oral administration. In an acute respiratory toxicity test (OECD 403), heads and noses of SD rats were exposed to liquid aerosol for 4 h (the highest concentration that could be administered without causing any animal death), and a lethal concentration (LC50) > 0.88 mg/L was obtained. The mass medium aerodynamics diameter (MMAD) of rEP aerosol particles was 8.18 µm and mass medium aerodynamics diameter (GSD) was 3.04, which meant that 25.90% could enter the airway (<4 µm) of a rat, and 58.06% (<10 µm) could be inhaled by humans. An ocular irritation test (OECD 405) with rEP powder was performed on New Zealand White (NZW) rabbits. Signs of irritation included conjunctival swelling and diffuse flushing 1 h after administration. The signs were less apparent after 24 h and disappeared after 72 h. The classification assigned to the powder was mild eye irritation. Skin sensitization was performed for a local lymphoproliferative test (OECD 442B) using BALB/c mice, with the highest soluble concentration of the rEP considered to be 100% test substance; formulations were diluted to 50% and 25%, and bromodeoxyuridine (BrdU) incorporation was used to measure the degree of lymphocyte proliferation. The stimulation indexes (SIs) were 1.06 (100%), 0.44 (50%), and 0.77 (25%), all of which were less than the cutoff value for a positive sensitization result (1.6). Negative response was also seen in the bacterial reverse mutation test (OECD 471), and no chromosomal effects on Chinese hamster ovary (CHO)-K1 cells were observed (OECD 487). Based on these six toxicity tests, rEP showed neither acute toxic effects in experimental animals nor mutagenicity. Thus, rEP can be considered safe for use in subsequent research on its application as a feed additive for poultry, cattle, or aquatic animals.

Epinecidin-1 Protects against Methicillin Resistant Staphylococcus aureus Infection and Sepsis in Pyemia Pigs.

Methicillin resistant Staphylococcus aureus (MRSA) may be found on the skin, nose, and throats of long-term hospitalized patients. While MRSA infections are usually minor, serious infections and death may occur in immunocompromised or diabetic patients, or after exposure of MRSA to blood. This report demonstrates that the antimicrobial peptide (AMP) epinecidin-1 (Epi-1) efficiently protects against MRSA infection in a pyemia pig model. We first found that Epi-1 exhibits bactericidal activity against MRSA. Next, pharmacokinetic analysis revealed that Epi-1 was stable in serum for 4 h after injection, followed by a gradual decrease. This pharmacokinetic profile suggested Epi-1 may bind serum albumin, which was confirmed in vitro. Harmful effects were not observed for doses up to 100 mg/kg body weight in pigs. When Epi-1 was supplied as a curative agent 30 min post-infection, MRSA-induced abnormalities in blood uric acid (UA), blood urea nitrogen (BUN), creatine (CRE), GOT, and GPT levels were restored to normal levels. We further showed that the bactericidal activity of Epi-1 was higher than that of the antibiotic drug vancomycin. Epi-1 significantly decreased MRSA counts in the blood, liver, kidney, heart, and lungs of infected pigs. Elevated levels of serum C reactive protein (CRP), proinflammatory cytokine IL6, IL1ß, and TNFα were also attenuated by Epi-1 treatment. Moreover, the MRSA genes, enterotoxin (et)-A, et-B, intrinsic methicillin resistance A (mecA), and methicillin resistance factor A (femA), were significantly reduced or abolished in MRSA-infected pigs after treatment with Epi-1. Hematoxylin and eosin staining of heart, liver, lung, and kidney sections indicated that Epi-1 attenuated MRSA toxicity in infected pigs. A survival study showed that the pyemia pigs infected with MRSA alone died within a week, whereas the pigs post-treated with 2.5 mg/kg Epi-1 were completely protected against death. The present investigation, thus, demonstrates that Epi-1 effectively protects pyemia pigs against pathogenic MRSA without major toxic side effects.

Transgenic expression of tilapia piscidin 3 (TP3) in zebrafish confers resistance to Streptococcus agalactiae.

To study the biological role of tilapia piscidin 3 (TP3) in Streptococcus agalactiae infection in vivo, TP3/DsRed overexpressing transgenic zebrafish were generated. Under normal growth conditions, TP3/DsRed transgenic zebrafish exhibited an orange-red body color, without any other obvious abnormalities. However, when compared to wild type fish, TP3/DsRed transgenic zebrafish were resistant to S. agalactiae infection. After infection, the TP3 overexpressing fish exhibited higher expression of Toll-like receptor 4a (TLR4a), interleukin (IL)-10, IL-22, and C3b. Furthermore, TP3/DsRed transgenic zebrafish exhibited reduced induction of proinflammatory cytokines, including TNFα, IL-1ß, IL-21, MyD88, and nuclear factor (NF)-κB. Taken together, our data show that TP3 overexpression in zebrafish can effectively suppress proinflammatory responses and enhance production of C3b. Together, these actions are conducive to the resolution of inflammation and bacterial clearance. We further postulate that TP3 may exert its anti-inflammatory effects by enhancing TLR4a-mediated negative regulation of NF-κB.

Transcriptome analysis of the effect of polyunsaturated fatty acids against Vibrio vulnificus infection in Oreochromis niloticus.

Vibrio vulnificus infection causes severe economic losses in Oreochromis niloticus aquaculture by inducing pro-inflammatory cytokines, that lead to inflammation and mortality. Omega-3 fatty acids, such as Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA), have been reported for their anti-inflammatory and antibacterial abilities in murine and zebrafish models. However, the anti-inflammatory and antibacterial functions of DHA and EPA in commercial aquaculture organisms such as Oreochromis niloticus remain unknown. The present study demonstrates antibacterial function and transcriptional modulation of inflammation-associated genes by DHA and EPA in Vibrio vulnificus infection in Oreochromis niloticus fish models. The administration of EPA or DHA improved the Oreochromis niloticus survival rate against Vibrio vulnificus infection. The induction of proinflammatory cytokines, Interleukin (IL)-1ß, IL-6, Tumor necrosis factor (TNF)-α, and Toll-like receptor (TLR)-2 by Vibrio vulnificus was suppressed in fish that were administered DHA. Bacterial membrane disruption and the killing of Vibrio vulnificus by EPA and DHA was observed using SEM, TEM, and cytoplasm leakage studies. In silico analysis of the transcription profile in Ingenuity Pathway Analysis software showed that DHA may enhance anti-Vibrio vulnificus activity in Oreochromis niloticus via the activation of peroxisome proliferator-activated receptor α (PPARα) to inhibit nuclear factor kappa B and suppress hepatocyte nuclear factor 4 α (HNF4α). In summary, the results of the present study demonstrated that DHA and EPA reduce the severity of Vibrio vulnificus infection and increase the survival rate of Oreochromis niloticus.

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.

Streptococcus agalactiae infection is one of the most significant bacterial diseases in tilapia aquaculture. Identification of immune-related genes associated with Streptococcus agalactiae infection may provide a basis for breeding selection or therapeutics to augment disease resistance. Therefore, we utilized transcriptome profiling to study the host response in tilapia following Streptococcus agalactiae infection. Based on GO and KEGG enrichment analyses, we found that differentially expressed genes are widely involved in immune-related pathways, including the induction of antimicrobial peptides. Moreover, the main components of two immune-related pathways (Toll-like receptor signaling and leukocyte transendothelial migration) and four environmental information processing pathways (TNF, PI3K-Akt, Jak-STAT and MAPK) were identified. Finally, a time-course expression profile for several of the identified transcripts including tilapia piscidin 3 (TP3), tilapia piscidin 4 (TP4), TLR2, TLR5, MyD88, TRAF6, p38, and interleukin components was performed by qRT-PCR. Collectively, these results provide a starting point to study molecular mechanisms of tilapia immune response to Streptococcus agalactiae infection and may be applied as a basis for developing disease resistant strains by breeding selection.

Excavatolide B Attenuates Rheumatoid Arthritis through the Inhibition of Osteoclastogenesis.

Osteoclasts are multinucleated giant cells of macrophage/monocyte lineage, and cell differentiation with the upregulation of osteoclast-related proteins is believed to play a major role in the destruction of the joints in the course of rheumatoid arthritis (RA). Pro-inflammatory cytokines, such as interleukin-17A (IL-17A) and macrophage colony-stimulating factor (M-CSF), can be overexpressed in RA and lead to osteoclastogenesis. In a previous study, we found that cultured-type soft coral-derived excavatolide B (Exc-B) exhibited anti-inflammatory properties. In the present study, we thus aimed to evaluate the anti-arthritic activity of Exc-B in in vitro and in vivo models. The results demonstrated that Exc-B inhibits LPS-induced multinucleated cell and actin ring formation, as well as TRAP, MMP-9, and cathepsin K expression. Additionally, Exc-B significantly attenuated the characteristics of RA in adjuvant (AIA) and type II collagen-induced arthritis (CIA) in rats. Moreover, Exc-B improved histopathological features, and reduced the number of TRAP-positive multinucleated cells in the in vivo AIA and CIA models. Immunohistochemical analysis showed that Exc-B attenuated the protein expression of cathepsin K, MMP-2, MMP-9, CD11b, and NFATc1 in ankle tissues of AIA and CIA rats. Level of interleukin-17A and macrophage colony-stimulating factor were also decreased by Exc-B. These findings strongly suggest that Exc-B could be of potential use as a therapeutic agent by inhibiting osteoclast differentiation in arthritis. Moreover, this study also illustrates the use of the anti-inflammatory marine compound, Exc-B, as a potential therapeutic strategy for RA.

Zebrafish fed on recombinant Artemia expressing epinecidin-1 exhibit increased survival and altered expression of immunomodulatory genes upon Vibrio vulnificus infection.

Artemia has been used extensively in aquaculture as fodder for larval fish, shrimp, and shellfish. Epinecidin-1, an antimicrobial peptide, was isolated from grouper (Epinephelus coioides) in 2005. Epinecidin-1 has been previously reported to possess antimicrobial activity against several Gram-positive and Gram-negative bacterial species, including Staphylococcus coagulase, Pseudomonas aeruginosa, Streptococcus pyogenes, and Vibrio vulnificus. In this study, we used electroporation to introduce plasmid DNA encoding a green fluorescent protein (EGFP)-epinecidin-1 fusion protein under the control of the cytomegalovirus (CMV) promoter into decapsulated Artemia cysts. Optimization of various properties (including cyst weight (0.2 g), plasmid concentration (50 µg/100 µl), and pulse voltage (150 V), length (10 ms), and number (2)) resulted in a hatching rate of 41.15%, a transfection efficiency of 49.81%, and a fluorescence intensity (A.U.) of 47.46. The expression of EGFP-epinecidin-1 was first detected by quantitative RT-PCR at 120 h post-electroporation, and protein was identified by Western blot at the same time. Furthermore, the EGFP-epinecidin-1 protein inhibited V. vulnificus (204) growth, as demonstrated by zone of inhibition studies. Zebrafish fed on transgenic Artemia expressing CMV-gfp-epi combined with commercial fodder were more resistant to infection by V. vulnificus (204): survival rate was enhanced by over 70% at 7, 14, and 21 days post-infection, and bacterial numbers in the liver and intestine were reduced. In addition, feeding of transgenic Artemia to zebrafish affected the immunomodulatory response to V. vulnificus (204) infection; expression of immune-responsive genes, including hepcidin and defbl2, was altered, as shown by qPCR. These findings suggest that feeding transgenic Artemia expressing CMV-gfp-epi to larval fish has antimicrobial effects, without the drawbacks of introducing drug residues or inducing bacterial drug resistance.

Piscidin is highly active against carbapenem-resistant Acinetobacter baumannii and NDM-1-producing Klebsiella pneumonia in a systemic Septicaemia infection mouse model.

This study was designed to investigate the antimicrobial activity of two synthetic antimicrobial peptides from an aquatic organism, tilapia piscidin 3 (TP3) and tilapia piscidin 4 (TP4), in vitro and in a murine sepsis model, as compared with ampicillin, tigecycline, and imipenem. Mice were infected with (NDM-1)-producing K. pneumonia and multi-drug resistant Acinetobacter baumannii, and subsequently treated with TP3, TP4, or antibiotics for different periods of time (up to 168 h). Mouse survival and bacterial colony forming units (CFU) in various organs were measured after each treatment. Toxicity was determined based on observation of behavior and measurement of biochemical parameters. TP3 and TP4 exhibited strong activity against K. pneumonia and A. baumannii in vitro. Administration of TP3 (150 µg/mouse) or TP4 (50 µg/mouse) 30 min after infection with K. pneumonia or A. baumannii significantly increased survival in mice. TP4 was more effective than tigecycline at reducing CFU counts in several organs. TP3 and TP4 were shown to be non-toxic, and did not affect mouse behavior. TP3 and TP4 are able at potentiate anti-Acinetobacter baumannii or anti-Klebsiella pneumonia drug activity, reduce bacterial load, and prevent drug resistance, indicating their potential for use in combating multidrug-resistant bacteria.

Enhanced Control of Bladder-Associated Tumors Using Shrimp Anti-Lipopolysaccharide Factor (SALF) Antimicrobial Peptide as a Cancer Vaccine Adjuvant in Mice.

Shrimp anti-lipopolysaccharide factor (SALF) is an antimicrobial peptide with reported anticancer activities, such as suppression of tumor progression. In this study, we prepared a potential cancer vaccine comprised of SALF in conjunction with the cell lysate of inactivated murine bladder carcinoma cells (MBT-2), and evaluated its efficacy in a mouse tumor model. Our study shows that SALF added to cell culture media inhibits growth progression of MBT-2, and that SALF together with inactivated MBT-2 lysate elevates the level of inflammasome activity, and modulates the levels of IL-1ß, MCP-1, IL-6, IL-12, and TNF-α in mouse macrophages. Immunization of 7, 14, and 21 day-old mice with the vaccine prevented growth of MBT-2 cell-mediated tumors. The vaccine was found to enhance expression of T-cell, cytotoxic T cells, and NK cells in the immunized mice groups. Recruitment of macrophages, T-helper cells, and NK cells was enhanced, but levels of VEGF were decreased in immunized mice. This report provides empirical evidence that our SALF as vaccine adjuvant enhances antitumor immunity in mice.

Epinecidin-1 has immunomodulatory effects, facilitating its therapeutic use in a mouse model of Pseudomonas aeruginosa sepsis.

Antimicrobial peptides (AMPs) are garnering attention as possible alternatives to antibiotics. Here, we describe the antimicrobial properties of epinecidin-1 against a multidrug-resistant clinical isolate of P. aeruginosa (P. aeruginosa R) and a P. aeruginosa strain from ATCC (P. aeruginosa ATCC 19660) in vivo. The MICs of epinecidin-1 against P. aeruginosa R and P. aeruginosa ATCC 19660 were determined and compared with those of imipenem. Epinecidin-1 was found to be highly effective at combating peritonitis infection caused by P. aeruginosa R or P. aeruginosa ATCC 19660 in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. Taken together, our results indicate that epinecidin-1 enhances the rate of survival of mice infected with the bacterial pathogen P. aeruginosa through both antimicrobial and immunomodulatory effects.

Use of the antimicrobial peptide pardaxin (GE33) to protect against methicillin-resistant Staphylococcus aureus infection in mice with skin injuries.

Antimicrobial peptides (AMPs) have recently been determined to be potential candidates for treating drug-resistant bacterial infections. Pardaxin (GE33), a marine antimicrobial peptide, has been reported to possess antimicrobial function. In this study, we investigated whether pardaxin promoted healing of contaminated wounds in mice. One square centimeter of outer skin was excised from the ventral region of mice, and a lethal dose of methicillin-resistant Staphylococcus aureus (MRSA) was applied in the presence or absence of methicillin, vancomycin, or pardaxin. While untreated mice and mice treated with methicillin died within 3 days, mice treated with pardaxin survived infection. Pardaxin decreased MRSA bacterial counts in the wounded region and also enhanced wound closure. Reepithelialization and dermal maturation were also faster in mice treated with pardaxin than in mice treated with vancomycin. In addition, pardaxin treatment controlled excess recruitment of monocytes and macrophages and increased the expression of vascular endothelial growth factor (VEGF). In conclusion, these results suggest that pardaxin is capable of enhancing wound healing. Furthermore, this study provides an excellent platform for comparing the antimicrobial activities of peptide and nonpeptide antibiotics.

Expression characterization and promoter activity analysis of the tilapia (Oreochromis niloticus) myosin light chain 3 promoter in skeletal muscle of fish.

A tilapia (Oreochromis niloticus) myosin light chain 3 (Mlc3) promoter region (~4.3 kb) was isolated and characterized. Sequence analysis of the clone revealed high similarity with a tilapia gene encoding the Mlc3 promoter region, exon 1, and intron 1. The clone contained several putative binding sequences for transcription factors, including MEF-2, MYOG, MyoD, PKNOX1, and AREB6. Deletion of a region of the tilapia Mlc3 promoter (801 to -3,881 bp) enhanced promoter activity, as determined by direct injection of a luciferase reporter construct into skeletal muscle of Archocentrus nigrofasciatus. These findings suggest that the region between -801 and -3,881 bp may contain negative regulatory elements. Stable germline transgenic strains of the ornamental fish species A. nigrofasciatus var. carrying the Taiwan coral red fluorescent protein (TcRFP) driven by the Mlc3 promoter were established. F1 adult transgenic A. nigrofasciatus var. exhibited brilliant pink fluorescence in skeletal muscles in the daylight. Therefore, our current study demonstrates the feasibility of using the tilapia Mlc3 promoter to drive fluorescence in new fish species, such as Perciformes.

Padina Minor Extract Confers Resistance against Candida Albicans Infection: Evaluation in a Zebrafish Model.

Padina minor is a seaweed rich in polysaccharides often used in food, feed, fertilizers, and antibacterial drugs. This study is the first to evaluate the effect of feeding zebrafish with Padina minor extract on preventing and treating C. albicans infections. This study evaluated the growth, survival, and disease resistance effects of P. minor extract on zebrafish. The fish were divided into four groups: three groups treated with 1%, 5%, or 10% P. minor extract and one untreated group (c, control). Subsequently, we analyzed how the extract affected the immune function of zebrafish infected with C. albicans. Based on the lethal concentration (LC50) calculated in the first stage, 1% was used as the effective therapeutic concentration. The results showed that the growth rate of the 1% feed group was the best, and no significant difference in survival rates between the four groups was observed. Feeding with 1% P. minor extract downregulated the expression of key inflammatory genes like tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), and IL-10, effectively preventing and treating C. albicans infections in zebrafish. This study is a preliminary evaluation of the therapeutic efficacy of P. minor extracts against C. albicans.

The Antimicrobial Peptide Tilapia Piscidin 4 Induced the Apoptosis of Bladder Cancer Through ERK/SIRT1/PGC-1α Signaling Pathway.

Marine antimicrobial peptides have been demonstrated in numerous studies to possess anti-cancer properties. This research investigation aimed to explore the fundamental molecular mechanisms underlying the antitumor activity of Tilapia piscidin 4 (TP4), an antimicrobial peptide, in human bladder cancer. TP4 exhibited a remarkable inhibitory effect on the proliferation of bladder cancer cells through cell cycle arrest at the G2/M phase. Additionally, TP4 upregulated the expression of cleaved caspase-3, caspase-9, and PARP, leading to the activation of apoptotic pathways in bladder cancer cells. TP4 exhibit a marked rise in mitochondria reactive oxygen species, leading to the subsequent loss of potential for the mitochondrial membrane. Furthermore, the inhibition of mitochondrial oxidative phosphorylation resulted in a decrease in downstream ATP production. Meanwhile, TP4-treated bladder cancer cells showed an increase in Bax and ERK but a decrease in SIRT1, PGC-1α, and Bcl2. ERK activation, SIRT1/PGC-1α-axis, and TP4-induced apoptosis were all significantly reversed by the ERK inhibitor SCH772984. Finally, the inhibitory effect of TP4 on tumor growth has been confirmed in a zebrafish bladder cancer xenotransplantation model. These findings suggest that TP4 may be a potential agents for human bladder cancer through apoptosis induction, ERK activation, and the promotion of SIRT1-mediated signaling pathways.

Oral administration of recombinant epinecidin-1 protected grouper (Epinephelus coioides) and zebrafish (Danio rerio) from Vibrio vulnificus infection and enhanced immune-related gene expressions.

Immunostimulatory effects of the oral administration of the recombinant epinecidin-1 protein from BL21 Escherichia coli (containing the pET28a-epinecidin-1-dsRed plasmid) were studied in grouper (Epinephelus coioides) and zebrafish (Danio rerio). For this purpose, fish were fed diets for 30 days containing the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) at different bacterial numbers (10(4), 10(6), 10(8), and 10(10) colony-forming units (cfu) of BL21 E. coli in 50 ml of LB medium) mixed with 50 g of eel powder as fodder. After 30 days of feeding, immune-related gene expressions for bacterial-infection responses and disease resistance against Vibrio vulnificus (204) were determined. The V. vulnificus (204) injected into the fish abdominal cavity mimicked gram-negative bacterial infections in culture ponds. Experimental results assessed whether the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) has up- (or down-) regulation immune-related genes expression. Results indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement significantly enhanced expressions several immune-related genes such as tumor necrosis factor (TNF)-1 in grouper and Toll-like receptor (TLR)4, interleukin (IL)-1ß, nitric oxide synthase (NOS)2, and nuclear factor (NF)-κB in zebrafish. After being challenged with V. vulnificus (204) for 24, 48, 72, or 96 h, the percentage mortality was significantly reduced in treated fish, which indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement could bring about downregulation of TNF-1 expression and functioned like an antagonist for binding TLR4, which reduced the signal transduction pathway for inhibiting TNF and IL-1ß expressions while reducing binding of the transcription factor, NF-κB, to TNF and the IL-1ß promoter region. The experimental results indicated that dietary intake of the recombinant epinecidin-1 protein from BL21 E. coli modulated immune-related gene expressions and disease resistance of grouper and zebrafish after a V. vulnificus (204) infection.

In vivo screening of zebrafish microRNA responses to bacterial infection and their possible roles in regulating immune response genes after lipopolysaccharide stimulation.

Micro (mi)RNAs are abundant small noncoding RNAs found in plants and animals, the regulatory functions of which are not fully understood in fish. To identify potential miRNAs, we screened an miRNA microarray with total RNA from zebrafish infected with Vibrio harveyi and another from uninfected zebrafish. Six miRNAs were obtained from the microarray screening. We studied miRNA expression patterns of 2 miRNAs (miR-122 and miR-194) after bacterial infection of transgenic zebrafish (containing tilapia hepcidin (TH)2-3) and non-transgenic zebrafish from which the 2 miRNAs were obtained from the microarray experiment. The results indicated that miR-122 and miR-194 were higher in PBS-injected zebrafish compared with TH2-3 zebrafish or wild-type (WT) zebrafish after V. harveyi infection. Overexpression of miRNAs (miR-122, miR-192, and miR-194a) was seen in zebrafish liver (ZFL) cells after lipopolysaccharide (LPS) treatment and in untreated fish. Our results showed that after 24 h of doxycycline treatment without LPS stimulation, interleukin (IL)-22, lysozyme, toll-like receptor (TLR)1, TLR3, TLR4a, and tumor necrosis factor (TNF)-α gene expressions were, respectively, upregulated by ~14-, 22-, 2.2-, 13-, 200-, and 38-fold in miR-122-transfected compared with non-transfected (WT) ZFL cells. In cells transfected with miR-192 and treated with LPS after 8-12 h, IL-22, lysozyme, TLR1, TLR3, TLR4a, and TNF-α expressions significantly differed between WT and miR-192-overexpressing ZFL cells. However, we observed significantly higher IL-22 expression levels after 12 h of LPS treatment in miR-192-transfected ZFL cells compared with non-transfected cells. In contrast, IL-22, lysozyme, and TNF-α were markedly upregulated (>100-fold) after miR-194a transfection and overexpression in ZFL cells and treatment with LPS. Our cloning and expression analyses indicated that miR-122, miR-192, and miR-194a play important roles in zebrafish immunology.

Electrospun Polyacrylonitrile Silver(I,III) Oxide Nanoparticle Nanocomposites as Alternative Antimicrobial Materials.

Infectious microbial diseases can easily be transferred from person to person in the air or via high contact surfaces. As a result, researchers must aspire to create materials that can be implemented in surface contact applications to disrupt pathogen growth and transmission. This study examines the antimicrobial properties of polyacrylonitrile (PAN) nanofibers coated with silver nanoparticles (AgNPs) and silver(I,III) oxide. PAN was homogenized with varied weight concentrations of silver nitrate (AgNO3) in N,N-dimethylformamide solution, a common organic solvent that serves as both an electrospinning solvent and as a reducing agent that forms AgNPs. The subsequent colloids were electrospun into nanofibers, which were then characterized via various analysis techniques, including scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray analysis, dynamic light scattering, and X-ray photoelectron spectroscopy. A total of 10 microbes, including 7 strains of Gram-positive bacteria, 2 strains of Gram-negative bacteria, and Candida albicans, were incubated with cutouts of various PAN-AgNP nanocomposites using disk diffusion methods to test for the nanocomposites' antimicrobial efficiency. We report that our electrospun PAN-AgNP nanocomposites contain 100% AgO, a rare, mixed oxidation state of silver(I,III) oxide that is a better sterilizing agent than conventional nanosilver. PAN-AgNP nanocomposites also retain a certain degree of antimicrobial longevity; samples stored for approximately 90 days demonstrate a similar antimicrobial activity against Escherichia coli (E. coli) and Lactobacillus crispatus (L. crispatus) when compared to their newly electrospun counterparts. Moreover, our results indicate that PAN-AgNP nanocomposites successfully display antimicrobial activity against various bacteria and fungi strains regardless of their resistance to conventional antibiotics. Our study demonstrates that PAN-AgNP nanocomposites, a novel polymer material with long-term universal antimicrobial stability, can potentially be applied as a universal antimicrobial on surfaces at risk of contracting microbial infections and alleviate issues related to antibiotic overuse and microbial mutability.