Female tilapia, Oreochromis sp. mobilised energy differently for growth and reproduction according to living environment.

This study was conducted to investigate the energy mobilisation preference and ionoregulation pattern of female tilapia, Oreochromis sp. living in different environments. Three different treatments of tilapia as physiology compromising model were compared; tilapia cultured in recirculating aquaculture system (RAS as Treatment I-RAS), tilapia cultured in open water cage (Treatment II-Cage) and tilapia transferred from cage and cultured in RAS (Treatment III-Compensation). Results revealed that tilapia from Treatment I and III mobilised lipid to support gonadogenesis, whilst Treatment II tilapia mobilised glycogen as primary energy for daily exercise activity and reserved protein for growth. The gills and kidney Na+/K+ ATPase (NKA) activities remained relatively stable to maintain homeostasis with a stable Na+ and K+ levels. As a remark, this study revealed that tilapia strategized their energy mobilisation preference in accessing glycogen as an easy energy to support exercise metabolism and protein somatogenesis in cage culture condition, while tilapia cultured in RAS mobilised lipid for gonadagenesis purposes.

Hoven's carp Leptobarbus hoevenii strategized metabolism needs to cope with changing environment.

Current water warming and freshwater acidification undoubtedly affect the life of aquatic animals especially ammonotelic teleost by altering their physiological responses. The effect of temperature (28 °C vs 32 °C) and pH (7 vs. 5) on the metabolic compromising strategies of Hoven's carp (Leptobarbus hoevenii) was investigated in this study. Fishes were conditioned to (i) 28 °C + pH 7 (N28°C); (ii) 32 °C + pH 7 (N32°C); (iii) 28 °C + pH 5 (L28°C) and (iv) 32 °C + pH 5 (L32°C) for 20 days followed by osmorespiration assay. Results showed that feeding performance of Hoven's carp was significantly depressed when exposed to low pH conditions (L28°C and L32°C). However, by exposed Hoven's carp to L32°C induced high metabolic oxygen intake and ammonia excretion to about 2x-folds higher compared to the control group. As for energy mobilization, Hoven's carp mobilized liver and muscle protein under L28°C condition. Whereas under high temperature in both pH, Hoven's carp had the tendency to reserve energy in both of liver and muscle. The findings of this study revealed that Hoven's carp is sensitive to lower water pH and high temperature, thereby they remodeled their physiological needs to cope with the environmental changes condition.

Organic cation transporter 2 activation enhances sensitivity to oxaliplatin in human pancreatic ductal adenocarcinoma.

Oxaliplatin, a third-generation platinum derivative, has become one of the main chemotherapeutic treatments for esophagus, gastric and colorectal cancer; however, it is still unclear the potential effectiveness for pancreatic ductal adenocarcinoma (PDAC) with gemcitabine resistance. Here, we observed that PDAC tumors have low level of organic cation transporter 2 (OCT2, also known as SLC22A2) compared with non-tumor tissues and identified that OCT2 expression is positively correlated with oxaliplatin sensitivity in PDAC cells. Treatment of OCT2 inhibitors or knockdown of OCT2 expression significantly decreased the sensitivity to oxaliplatin in PANC-1 cells. In addition, bisulfite sequencing polymerase chain reaction analysis revealed that higher methylation frequency represses OCT2 expression in gemcitabine-resistant PANC-1 (PANC-1/GR) cells. Moreover, we found that treatment of DNA methyltransferase (DNMT) inhibitors, decitabine or 5-azacytidine recover OCT2 expression and oxaliplatin sensitivity in PANC-1/GR cells, and DNMT1 level has inverse correlation with OCT2 expression in PDAC cells and tumors. Our findings jointly suggest that OCT2 expression is a potential and predictive marker for evaluating oxaliplatin sensitivity and developing alternative treatments for PDAC patients with gemcitabine resistance.

Low water pH depressed growth and early development of giant freshwater prawn Macrobrachium rosenbergii larvae.

Macrobrachium rosenbergii is one of the shellfish species with high aquaculture value due to its increasing market demand. However, the comparatively low production volume compared to demand coupled with the rapid decline of the natural environment, consequently, drives the potential depletion of the wild population. The decrease in water pH related to anthropogenic pollution is one of the most critical factors affecting the early life performances of M. rosenbergii. Therefore, this study was designed to examine the effect of low water pH on feeding, growth and development of M. rosenbergii early life stages. Experimental water pH was set as neutral (7.7 ± 0.4); mild-acidic (6.4 ± 0.5) and acidic (5.4 ± 0.2) with triplication at a stocking density of 2 larvae/L for 30 days. As expected, M. rosenbergii larvae were highly sensitive to acidic pH with no larvae survived beyond 48 h of exposure. Feeding, survival and growth of larvae were adversely affected by mild-acidic pH exposure as compared to neutral pH. Larvae exposed to mild-acidic water pH experienced a prolonged larval period and only metamorphosed to the post-larval stage at day-30. Whilst under neutral water pH, larval that metamorphosed to post-larval was first observed on day-23. The negative impact of decreased pH, even in mild-acidic pH exposure, on the feeding, survival, growth and development of M. rosenbergii larvae highlights the urgency of periodic pH monitoring during M. rosenbergii larviculture.

FUSE binding protein FUBP3 is a potent regulator in Japanese encephalitis virus infection.

BACKGROUND: The JEV genome is a positive-sense RNA with a highly structured capped 5'UTR, 3'UTR and a large open reading frame. 3'UTR is the untranslated region of flavivirus and has various important functions during viral replication, such as translation, replication and encapsidation. During viral replication, the 3'UTR interacts with viral proteins and host proteins and is required for viral RNA replication and translocation. METHODS: The expression level of FUBP3 was knocked down by siRNA and Flag-tagged FUBP3 overexpression plasmid was constructed for overexpression. BHK-21 cells were cultured and infected with JEV to investigate the functional role of FUBP3 in the viral infection cycle. Subcellular localization of FUBP3 and viral replication complexes was observed by dual immunofluorescence staining. RESULTS: Four host proteins were specifically associated with the 3'UTR of JEV, and FUBP3 was selected to further investigate its potential functional role in the JEV infection cycle. Knockdown of FUBP3 protein resulted in a significant decrease in JEV viral titer, whereas ectopic overexpression of FUBP3 resulted in increased JE viral infectivity. In cells stably knocked down for FUBP3 and then infected with JEV, we found almost no detectable viral NS5 protein. In contrast, when cells stably knocking-down of FUBP3 overexpressed FUBP3, we found a significant increase in viral RNA production over time compared to controls. We also demonstrated that FUBP3 re-localized in the cytoplasm after infection with JEV and co-localized with viral proteins. Exogenous overexpression of FUBP3 was also shown to be located in the JE replication complex and to assist viral replication after JEV infection. CONCLUSIONS: The overall results suggest that FUBP3 regulates RNA replication of JEV and promotes subsequent viral translation and viral particle production.

Role of gut microbiota in identification of novel TCM-derived active metabolites.

Traditional Chinese Medicine (TCM) has been extensively used to ameliorate diseases in Asia for over thousands of years. However, owing to a lack of formal scientific validation, the absence of information regarding the mechanisms underlying TCMs restricts their application. After oral administration, TCM herbal ingredients frequently are not directly absorbed by the host, but rather enter the intestine to be transformed by gut microbiota. The gut microbiota is a microbial community living in animal intestines, and functions to maintain host homeostasis and health. Increasing evidences indicate that TCM herbs closely affect gut microbiota composition, which is associated with the conversion of herbal components into active metabolites. These may significantly affect the therapeutic activity of TCMs. Microbiota analyses, in conjunction with modern multiomics platforms, can together identify novel functional metabolites and form the basis of future TCM research.

Like Cures Like: Pharmacological Activity of Anti-Inflammatory Lipopolysaccharides From Gut Microbiome.

Gut microbiome maintains local gut integrity and systemic host homeostasis, where optimal control of intestinal lipopolysaccharides (LPS) activity may play an important role. LPS mainly produced from gut microbiota are a group of lipid-polysaccharide chemical complexes existing in the outer membrane of Gram-negative bacteria. Traditionally, LPS mostly produced from Proteobacteria are well known for their ability in inducing strong inflammatory responses (proinflammatory LPS, abbreviated as P-LPS), leading to septic shock or even death in animals and humans. Although the basic structures and chemical properties of P-LPS derived from different bacterial species generally show similarity, subtle and differential immune activation activities are observed. On the other hand, frequently ignored, a group of LPS molecules mainly produced by certain microbiota bacteria such as Bacteroidetes show blunt or even antagonistic activity in initiating pro-inflammatory responses (anti-inflammatory LPS, abbreviated as A-LPS). In this review, besides the immune activation properties of P-LPS, we also focus on the description of anti-inflammatory effects of A-LPS, and their potential antagonistic mechanism. We address the possibility of using native or engineered A-LPS for immune modulation in prevention or even treatment of P-LPS induced chronic inflammation related diseases. Understanding the exquisite interactive relationship between structure-activity correlation of P- and A-LPS not only contributes to molecular understanding of immunomodulation and homeostasis, but also re-animates the development of novel LPS-based pharmacological strategy for prevention and therapy of chronic inflammation related diseases.

Characterization of betanodavirus quasispecies influences on the subcellular localization and expression of tumor necrosis factor (TNF).

The aim of this study was to investigate the influence of variant coat proteins (CPs) from different quasispecies of betanodavirus on diverse aspects of nodavirus-induced pathogenesis. It is known that variant CPs can acquire either nuclear or cytoplasmic localization, depending on the nodavirus CP genotype, and this variation may arise during viral replication and influence the regulation of host and viral gene transcription. To investigate the role of these variant CPs in pathogenesis, six variant CP expression plasmids were constructed, each containing different quasispecies CP variants from nodavirus genotype red spotted grouper nervous necrosis virus (RGNNV). The CP expression plasmids were transiently transfected into grouper GF-1 cells. At different times, the cell cycle and cell proliferation were assayed using flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. The proportion of G2/M-phase GF-1 cells transfected with CP expression plasmids was higher than that of cells transfected with the blank plasmid, especially in regards to quasispecies 2 (QS2). The proliferation ratio of cells transfected with the CP expression plasmids was significantly higher than that of cells transfected with the blank plasmid, with the exception of QS6. We also found that the different quasispecies CPs downregulated the promoter activity of the tumor necrosis factor (TNF) gene to different degrees. In addition, this is the first report showing the betanodavirus CP derived from different quasispecies of RGNNV provide evidence of a chronically nodavirus-infected grouper. Overall, this study represents the first comprehensive analysis of variant CPs from grouper with persistent nodavirus infections and their effects on different aspects of pathogenesis.

Cloning and characterisation of type I interferon receptor 1 in orange-spotted grouper (Epinephelus coioides) for response to nodavirus infection.

Grouper is known as a highly economical teleost species in the Asian aquaculture industry; however, intensive culture activities easily cause disease outbreak, especially viral disease. For the prevention of viral outbreaks, interferon (IFN) is among the major defence systems being studied in different species. Fish type I IFNs are known to possess antiviral properties similar to mammalian type I IFNs. In order to stimulate antiviral function, IFN will bind to its cognate receptor, the type I interferon receptor (IFNAR), composed of heterodimeric receptor subunits known as IFNAR1 and IFNΑR2. The binding of type I interferon to receptors assists in the transduction of signals from the external to internal environments of cells to activate biological responses. In order to study the function of IFN, we first need to understand IFN receptors. In this study, we cloned and identified IFNAR1 in orange-spotted grouper (osgIFNAR1) and noted the up-regulated mRNA expression of the receptor and downstream effectors in the head kidney cells with cytokine treatment. The transcriptional expression of osgIFNAR1, which is characterised using polyinosinic-polycytidylic acid (poly[I:C]) and lipopolysaccharide (LPS) treatments, indicated the involvement of osgIFNAR1 in the immune response of grouper. The subcellular localisation of osgIFNAR1 demonstrated scattering across the grouper cell. Viral infection showed the negative feedback regulation of osgIFNAR1 in grouper larvae. Further loss of function of IFNAR1 showed a decreased expression of the virus. This study reported the identification of osgIFNAR1 and characterisation of receptor sensitivity towards immunostimulants, cytokine response, and viral challenge in the interferon pathway of orange-spotted grouper and possible different role of the receptor in viral production. Together, these results provide a frontline report of the potential function of osgIFNAR1 in the innate immunity of teleost.

Characterization of gonadal glucocorticoid-induced leucine zipper (GILZ) protein expression during sex change in the protogynous orange-spotted grouper, Epinephelus coioides.

Steroid hormones have been proven as a key drive of sex change in sequentially hermaphroditic organisms. However, the upstream mechanism of sex steroid hormones regulation that affect sex change remain unknown. The main glucocorticoid in teleost fish is cortisol, which both regulates steroidogenesis and has antistress action. Thus, cortisol might be one of the prime factors in sex change. In this study, the glucocorticoid-induced leucine zipper (GILZ) gene, was proven to have a dramatic effect in orange-spotted groupers (Epinephelus coioides) during sex change at the early stage of gonadal transition. The specific action of the GILZ protein is at the pouch-shaped proliferative spermatogonia instead of the degenerative oocyte at the onset of sex change. Immunohistochemical (IHC) evidence revealed that GILZ performs intensively at undifferentiated spermatogonia in the early testis stage. These results imply that cortisol provokes a rise of GILZ through regulation caused by steroid hormones leading to sex change.

Staufen1 Protein Participates Positively in the Viral RNA Replication of Enterovirus 71.

The double-stranded RNA-binding protein Staufen1 (Stau1) has multiple functions during RNA virus infection. In this study, we investigated the role of Stau1 in viral translation by using a combination of enterovirus 71 (EV-A71) infection, RNA reporter transfection, and in vitro functional and biochemical assays. We demonstrated that Stau1 specifically binds to the 5'-untranslated region of EV-A71 viral RNA. The RNA-binding domain 2-3 of Stau1 is responsible for this binding ability. Subsequently, we created a Stau1 knockout cell line using the CRISPR/Cas9 approach to further characterize the functional role of Stau1's interaction with viral RNA in the EV-A71-infected cells. Both the viral RNA accumulation and viral protein expression were downregulated in the Stau1 knockout cells compared with the wild-type naïve cells. Moreover, dysregulation of viral RNA translation was observed in the Stau1 knockout cells using ribosome fractionation assay, and a reduced RNA stability of 5'-UTR of the EV-A71 was also identified using an RNA stability assay, which indicated that Stau1 has a role in facilitating viral translation during EV-A71 infection. In conclusion, we determined the functional relevance of Stau1 in the EV-A71 infection cycle and herein describe the mechanism of Stau1 participation in viral RNA translation through its interaction with viral RNA. Our results suggest that Stau1 is an important host factor involved in viral translation and influential early in the EV-A71 replication cycle.

Viral infection upregulates myostatin promoter activity in orange-spotted grouper (Epinephelus coioides).

Myostatin is a negative regulator of myogenesis and has been suggested to be an important factor in the development of muscle wasting during viral infection. The objective of this study was to characterize the main regulatory element of the grouper myostatin promoter and to study changes in promoter activity due to viral stimulation. In vitro and in vivo experiments indicated that the E-box E6 is a positive cis-and trans-regulation motif, and an essential binding site for MyoD. In contrast, the E-box E5 is a dominant negative cis-regulatory. The characteristics of grouper myostatin promoter are similar in regulation of muscle growth to that of other species, but mainly through specific regulatory elements. According to these results, we conducted a study to investigate the effect of viral infection on myostatin promoter activity and its regulation. The nervous necrosis virus (NNV) treatment significantly induced myostatin promoter activity. The present study is the first report describing that specific myostatin motifs regulate promoter activity and response to viral infection.

Molecular cloning of orange-spotted grouper (Epinephelus coioides) heat shock transcription factor 1 isoforms and characterization of their expressions in response to nodavirus.

Heat shock transcription factor 1 (HSF1) regulates heat shock proteins (HSPs), which assist in protein folding and inhibit protein denaturation following stress. HSF1 was firstly cloned from orange-spotted grouper and exists as two isoforms, one with (osgHSF1a) and one without (osgHSF1b) exon 11. Heat exposure increased the expression of osgHSF1b while cold exposure increased that of osgHSF1a. Both isoforms were mainly expressed in the brains, eyes, and fins. Expression of osgHSF1b was higher than osgHSF1a during development. Poly I:C and LPS could also induce osgHSF1 isoforms expression differentially. Exposure to nervous necrosis virus (NNV) increased the level of both osgHSF1 isoforms at 12 h. GF-1 cells with overexpression of osgHSF1 isoforms enhanced viral loads within 24 h, whereas both pharmacological inhibition and RNA interference of HSF1 reduced virus infection. This study shows that osgHSF1 can support the early stage of virus infection and provides a new insight into the molecular regulation of osgHSF1 between the influence of temperatures and immunity.

Molecular cloning and characterization of orange-spotted grouper (Epinephelus coioides) CXC chemokine ligand 12.

Chemokines are a family of soluble peptides that can recruit a wide range of immune cells to sites of infection and disease. The CXCL12 is a chemokine that binds to its cognate receptor CXCR4 and thus involved in multiple physiological and pathophysiological processes. In this study, we cloned and characterized CXCL12 from Epinephelus coioides (osgCXCL12). We found that the open reading frame of osgCXCL12 consists of 98 amino acid residues with the small cytokine C-X-C domain located between residues 29 and 87. Higher expression levels for osgCXCL12 were detected at the kitting stage, compared with the prolarva and larva shape stages. The expression patterns revealed that osgCXCL12 may play a key role in early grouper development. We detected mRNA transcripts for osgCXCL12 in healthy tissues and found the highest osgCXCL12 expression in the head kidney. Furthermore, a time-course analysis revealed significantly increased osgCXCL12 and osgCXCR4 expression levels after the nervous necrosis virus (NNV) challenge. In addition, expression of osgCXCL12 was affected by injection with microbial mimics [LPS and poly(I:C)]. These results suggest that osgCXCL12 is associated with inflammatory and developmental processes in the grouper.

Salmonids have an extraordinary complex type I IFN system: characterization of the IFN locus in rainbow trout oncorhynchus mykiss reveals two novel IFN subgroups.

Fish type I IFNs are classified into two groups with two (group I) or four (group II) cysteines in the mature peptide and can be further divided into four subgroups, termed IFN-a, -b, -c, and -d. Salmonids possess all four subgroups, whereas other teleost species have one or more but not all groups. In this study, we have discovered two further subgroups (IFN-e and -f) in rainbow trout Oncorhynchus mykiss and analyzed the expression of all six subgroups in rainbow trout and brown trout Salmo trutta. In rainbow trout RTG-2 and RTS-11 cells, polyinosinic-polycytidylic acid stimulation resulted in early activation of IFN-d, whereas the IFN-e subgroup containing the highest number of members showed weak induction. In contrast with the cell lines, remarkable induction of IFN-a, -b, and -c was detected in primary head kidney leukocytes after polyinosinic-polycytidylic acid treatment, whereas a moderate increase of IFNs was observed after stimulation with resiquimod. Infection of brown trout with hemorrhagic septicemia virus resulted in early induction of IFN-d, -e, and -f and a marked increase of IFN-b and IFN-c expression in kidney and spleen. IFN transcripts were found to be strongly correlated with the viral burden and with marker genes of the IFN antiviral cascade. The results demonstrate that the IFN system of salmonids is far more complex than previously realized, and in-depth research is required to fully understand its regulation and function.

Development of pedigree classification using microsatellite and mitochondrial markers for Giant grouper broodstock (Epinephelus lanceolatus) management in Taiwan.

Most giant groupers in the market are derived from inbred stock. Inbreeding can cause trait depression, compromising the animals' fitness and disease resistance, obligating farmers to apply increased amounts of drugs. In order to solve this problem, a pedigree classification method is needed. Here, microsatellite and mitochondrial DNA were used as genetic markers to analyze the genetic relationships among giant grouper broodstocks. The 776-bp fragment of high polymorphic mitochondrial D-loop sequence was selected for measuring sibling relatedness. In a sample of 118 giant groupers, 42 haplotypes were categorized, with nucleotide diversity (π) of 0.00773 and haplotype diversity (HD) of 0.983. Furthermore, microsatellites were used for investigation of parentage. Six out of 33 microsatellite loci were selected as markers based on having a high number of alleles and compliance with Hardy-Weinberg equilibrium. Microsatellite profiles based on these loci provide high variability with low combined non-exclusion probability, permitting practical use in aquaculture. The method described here could be used to improve grouper broodstock management and lower the chances of inbreeding. This approach is expected to lead to production of higher quality groupers with higher disease resistance, thereby reducing the need for drug application.

Functional analysis of an orange-spotted grouper (Epinephelus coioides) interferon gene and characterisation of its expression in response to nodavirus infection.

We cloned and sequenced 2C I-IFN, a two-cysteine containing type I interferon (I-IFN) gene, in orange-spotted grouper (Epinephelus coioides). The cDNA has 769 base pairs, the protein has 172 amino acids, and the predicted signal peptide has 18 amino acids with two cysteines. This gene is similar to I-FNs from sea bass and other teleosts. 2C I-IFN has 5 exons and 4 introns, also similar to other teleost I-IFNs. Immunohistochemical (IHC) analysis indicated that expression is predominantly membrane-localized in healthy grouper, but has a zonal distribution in nodavirus-infected grouper. Grouper infected with nodavirus had elevated levels of 2C I-IFN at 72 h and Mx at days 6-7. Recombinant 2C I-IFN activated grouper Mx, leading to upregulated antiviral activity. The grouper Mx promoter was highly induced after treatment with recombinant 2C I-IFN. The present results suggest that expression of grouper 2C I-IFN may participate in the immunologic barrier function against nodavirus.

Immunity to betanodavirus infections of marine fish.

Betanodaviruses cause viral nervous necrosis in numerous fish species, but some species are resistant to infection by these viruses. It is essential to fully characterize the immune responses that underlie this protective response. Complete characterization of the immune responses against nodaviruses may allow the development of methods that stimulate fish immunity and of an effective betanodavirus vaccine. Such strategies could include stimulation of specific immune system responses or blockage of factors that decrease the immune response. The innate immune system clearly provides a front-line defense, and this includes the production of interferons and other cytokines. Interferons that are released inside infected cells and that suppress viral replication may be the most ancient form of innate immunity. This review focuses on the immune responses of fish to betanodavirus infection.

An automated microfluidic chip system for detection of piscine nodavirus and characterization of its potential carrier in grouper farms.

Groupers of the Epinephelus spp. are an important aquaculture species of high economic value in the Asia Pacific region. They are susceptible to piscine nodavirus infection, which results in viral nervous necrosis disease. In this study, a rapid and sensitive automated microfluidic chip system was implemented for the detection of piscine nodavirus; this technology has the advantage of requiring small amounts of sample and has been developed and applied for managing grouper fish farms. Epidemiological investigations revealed an extremely high detection rate of piscine nodavirus (89% of fish samples) from 5 different locations in southern Taiwan. In addition, positive samples from the feces of fish-feeding birds indicated that the birds could be carrying the virus between fish farms. In the present study, we successfully introduced this advanced technology that combines engineering and biological approaches to aquaculture. In the future, we believe that this approach will improve fish farm management and aid in reducing the economic loss experienced by fish farmers due to widespread disease outbreaks.