Simultaneous aerobic nitrogen and phosphate removal capability of novel salt-tolerant strain, Pseudomonas mendocina A4: Characterization, mechanism and application potential.

A salt-tolerant strain, Pseudomonas mendocina A4, was isolated from brackish-water ponds showing simultaneous heterotrophic nitrification-aerobic denitrification and phosphorus removal capability. The optimal conditions for nitrogen and phosphate removal of strain A4 were pH 7-8, carbon/nitrogen ratio 10, phosphorus/nitrogen ratio 0.2, temperature 30 °C, and salinity range of 0-5 % using sodium succinate as the carbon source. The nitrogen and phosphate removal efficiencies were 96-100 % and 88-96 % within 24 h, respectively. The nitrogen and phosphate removal processes were matched with the modified Gompertz model, and the underlying mechanisms were confirmed by the activities of key metabolic enzymes. Under 10 % salinity, the immobilization technology was employed to enhance the nitrogen and phosphate removal efficiencies of strain A4, achieving 87 % and 76 %, respectively. These findings highlight the potential application of strain A4 in both freshwater and marine culture wastewater treatment.

Growth Axis Somatostatin, Growth Hormone Receptor, and Insulin-like Growth Factor-1 Genes Express and Are Affected by the Injection of Exogenous Growth Hormone in Chinemys reevesii.

In this study, to explore the effect of growth hormone changes on the related genes and regulatory roles of the turtle, PCR amplification, real-time fluorescence quantitative analysis, and enzyme cutting technology were used to clone and sequence the somatostatin (SS) gene, growth hormone receptor (GHR), and insulin-like growth factor-1 (IGF-I) sequence of Chinemys reevesii. The effects of human growth hormone on the mRNA expression of growth-axis-related genes SS, GHR, and IGF-1 in different sexes were observed. The study of the SS gene in turtles using real-time fluorescence quantitative PCR showed that the SS gene was mainly expressed in the nervous system and the digestive system, with the highest expression found in the brain, while the GHR gene and the IGF-I gene were expressed in all tissues of Chinemys reevesii. The SS gene was expressed in the brain, pituitary, liver, stomach, and intestine, with the highest expression in the brain and the lowest expression in the liver. Within 4 weeks of the injection of exogenous growth hormone, the expression level of the SS gene in the brain of both sexes first increased and then decreased, showing a parabolic trend, and the expression level of the experimental group was lower than that of the control group. After the injection of growth hormone (GH), the expression of the GHR gene in the liver of both sexes showed a significant increase in the first week, decreasing to the control group level in the second week, and then gradually increasing. Finally, a significant level of difference in the expression of the GHR gene was reached at 3 and 4 weeks. In terms of the IGF-I gene, the changing trend of the expression level in the liver was the same as that of the GHR gene. After the injection of exogenous growth hormone, although the expression of the SS gene increased the inhibition of the secretion of the GHR gene by the Reeves' turtle, exogenous growth hormone could replace the synthesis of GH and GHR, accelerating the growth of the turtle. The experiments showed that the injection of recombinant human growth hormone affects the expression of SS, GHR, and IGF-1 genes, and promotes the growth of the Reeves' turtle.

Rapid adaptive and acute stimulatory responses to low salinity stress in Pacific white shrimp (Litopenaeus vannamei): Insights from integrative transcriptomic and proteomic analysis.

The Pacific white shrimp (Litopenaeus vannamei) is a euryhaline crustacean capable of tolerating a wide range of ambient salinity, but the strategies of hepatopancreas to rapid adaptive or acute stimulatory responses to extremely low salinity fluctuations remains unclear. In this study, we integrated transcriptomic and proteomic analyses on the hepatopancreas derived from rapid adaptative (RA) and acute stimulatory (AS) responses to extremely low salinity stress (0.3 ppt) to unveil specific regulatory mechanisms. The RA group displayed normal epithelial cells and tubule structures, while the AS group showed histological changes and lesions. A total of 754 and 649 differentially expressed genes (DEGs) were identified in RA and AS treatments, respectively. For proteome, a total of 206 and 66 differentially expressed proteins (DEPs) were obtained in the RA/CT and AS/CT comparison groups, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted among the DEGs and DEPs, revealing that metabolic related pathways were significantly enriched pathways in both comparison groups. In addition, correlation analysis of transcriptomic and proteomic results showed that 20 and 3 pairs of DEGs/DEPs were identified in RA vs. CT and AS vs. CT comparison groups, respectively. This study is the first report on the rapid adaptive and acute stimulatory transcriptomic and proteomic responses of L. vannamei to extremely low salinity, shedding light on the mechanisms underlying osmoregulation in euryhaline crustaceans.

The antibacterial activity of a novel NK-lysin homolog and its molecular characterization and expression in the striped catfish, Pangasianodon hypophthalmus.

Aeromonas hydrophila was a common bacterial pathogen in aquaculture resulting in considerable losses to the striped catfish aquaculture industry. As an emergent antimicrobial peptide (AMP), NK-lysin (NKL) had activity against various microorganisms. However, the antibacterial activity of NKL from striped catfish (Pangasianodon hypophthalmus) both in vitro and vivo remains unclear. In this study, the cDNA sequence of P. hypophthalmus NK-lysin gene (PhNK-lysin) was cloned and characterized. The amino acid sequence of PhNK-lysin contains a signal peptide sequence of 17 amino acid (aa) residues and a mature peptide composed of 130 aa. The saposin B domain of mature peptide comprised six conserved cysteines forming three putative disulfide bonds. Phylogenetic analysis revealed that the PhNK-lysin was most closely related to that of the channel catfish (Ictalurus punctatus) NK-lysin. The transcriptional levels of the PhNK-lysin were significantly upregulated in response to A. hydrophila infection in various tissues including heart, liver, spleen, head kidney, trunk kidney and gill. The synthetic PhNK-lysin-derived peptide consisting of 38aa showed antibacterial activity against Vibrio harveii, Aeromonas hydrophila and Escherichia coli. The MIC for V. harveii, A. hydrophila and E. coli were 15.625 µM, 250 µM and 31.25 µM respectively. Besides, the synthetic PhNK-lysin decreased the bacterial load of liver and trunk kidney in vivo as well as increased the survival rate of A. hydrophila infected striped catfish. Hence, these data suggest that PhNK-lysin had antimicrobial effect and protects the host from pathogenic infection.

dPRLR causes differences in immune responses between early and late feathering chickens after ALV-J infection.

To understand the differences in immune responses between early feathering (EF) and late feathering (LF) chickens after infection with avian leukosis virus, subgroup J (ALV-J), we monitored the levels of prolactin, growth hormone and the immunoglobulins IgG and IgM in the serum of LF and EF chickens for 8 weeks. Moreover, we analysed the expression of immune-related genes in the spleen and the expression of PRLR, SPEF2 and dPRLR in the immune organs and DF-1 cells by qRT-PCR. The results showed that ALV-J infection affected the expression of prolactin, growth hormone, IgG and IgM in the serum. Regardless of whether LF and EF chickens were infected with ALV-J, the serum levels of the two hormones and two immunoglobulins in EF chickens were higher than those in LF chickens (P < 0.05). However, the expression of immune-related genes in the spleen of positive LF chickens was higher than that in the spleen of positive EF chickens. In the four immune organs, PRLR and SPEF2 expression was also higher in LF chickens than in EF chickens. Furthermore, the dPRLR expression of positive LF chickens was higher than that of negative LF chickens. After infection with ALV-J, the expression of PRLR in DF-1 cells significantly increased. In addition, overexpression of PRLR or dPRLR in DF-1 cells promoted replication of ALV-J. These results suggested that the susceptibility of LF chickens to ALV-J might be induced by dPRLR.

Cytokine inducible SH2-containing protein potentiate J subgroup avian leukosis virus replication and suppress antiviral responses in DF-1 chicken fibroblast cells.

Cytokine-inducible Srchomology2 (SH2)-containing protein (CIS) belongs to the suppressors of cytokine signaling (SOCS) protein family function as a negative feedback loop inhibiting cytokine signal transduction. J subgroup avian leukosis virus (ALV-J), a commonly-seen avian virus with a feature of immunosuppression, poses an unmeasurable threat to the poultry industry across the world. However, commercial medicines or vaccines are still no available for this virus. This study aims to evaluate the potential effect of chicken CIS in antiviral response and its role on ALV-J replication. The results showed that ALV-J strain SCAU-HN06 infection induced CIS expression in DF-1 cells, which was derived from chicken embryo free of endogenous avian sarcoma-leukosis virus (ASLV) like sequences. By overexpressing CIS, the expression of chicken type I interferon (IFN-I) and interferon-stimulated genes (ISGs; PKR, ZAP, CH25H, CCL4, IFIT5, and ISG12) were both suppressed. Meanwhile, data showed that CIS overexpression also increased viral yield. Interestingly, knockdown of CIS enhanced induction of IFN-I and ISGs and inhibited viral replication. Collectively, we proved that modulation of CIS expression not only affected SCAU-HN06 replication in vitro but also altered the expression of IFN-I and ISGs that act as an essential part of antiviral innate immune system. Our data provide a potential target for developing antiviral agents for ALV-J.

Identification of the cDNA Encoding the Growth Hormone Receptor (GHR) and the Regulation of GHR and IGF-I Gene Expression by Nutritional Status in Reeves' Turtle (Chinemys reevesii).

Chinemys reevesii (Reeves' turtle) is a slow-growing reptile that is distributed widely across China. Prior to this study, the cDNA sequence of the growth hormone receptor (GHR) in the Reeve's turtle, or how periods of starvation might influence the gene expression of GHR and insulin-like growth factor I (IGF-I) in this species, were unknown. Here, we identified the full-length sequence of the cDNA encoding GHR in Reeves' turtle by using RT-PCR and RACE. The full-length GHR cDNA was identified to be 3936 base-pairs in length, with a 1848 base-pair open reading frame (ORF) that encodes a 615 amino acid protein. Analysis showed that GHR mRNA was detectable in a wide range of tissues; the highest and lowest levels of expression were detected in the liver and the gonad, respectively. IGF-I was also expressed in a range of tissues, but not in the gonad; the highest levels of IGF-I expression were detected in the liver. After 4 weeks of fasting, the expression levels of GHR and IGF-I in the liver had decreased significantly; however, these gradually returned to normal after refeeding. We report the first cloned cDNA sequence for the GHR gene in the Reeve's turtle. Our findings provide a foundation from which to investigate the specific function of the GHR in Reeve's turtle, and serve as a reference for studying the effects of different nutrient levels on GHR expression in this species.

Cholesterol-25-hydroxylase Is a Chicken ISG That Restricts ALV-J Infection by Producing 25-hydroxycholesterol.

The avian leukosis virus subgroup J (ALV-J) belongs to the chicken retrovirus that causes enormous economic losses in the poultry industry. Interferon-stimulated genes (ISGs) are critical for controlling virus infections. Here, we identified 897 type I ISGs induced by interferon-α (IFN-α) in chicken peripheral blood mononuclear cell (PBMC) by RNA-Seq. In addition, we further identified 152 potential anti-ALV-J chicken type I ISGs. Among these potential anti-ALV-J ISGs, chicken cholesterol 25-hydroxylase (chCH25H) was selected for further antiviral mechanism studies in chicken embryo fibroblast cell lines (DF1). The gene chCH25H is located on chromosome 6 and clustered in a distinct group with mammals CH25H in the phylogenetic tree. The core promoter region of chCH25H was located within -75/-1 sequence. We found that chCH25H was induced by chicken IFN-α and ALV-J in DF1 cells. The overexpression of chCH25H significantly inhibited ALV-J replication in DF1 cells at 48 h post infection (hpi). In addition, ALV-J replication was significantly enhanced in the chCH25H- knockout DF1 cells. Furthermore, we demonstrated that chCH25H restricted ALV-J infection through the production of 25-hydroxycholesterol (25HC), rather than type I and II interferon. Our results identified 152 potential anti-ALV-J chicken type I ISGs and revealed that 25HC, the product of chCH25H, could be used as a natural antiviral agent to control ALV-J infection.

Identification of a novel antisense RNA that regulates growth hormone receptor expression in chickens.

Natural antisense transcripts (NATs) are widely present in mammalian genomes and act as pivotal regulator molecules of gene expression. However, studies on NATs in the chicken are relatively rare. We identified a novel antisense transcript in the chicken, designated GHR-AS-EST, transcribed from the growth hormone receptor (GHR) locus, which encodes a well-known regulatory molecule of muscle development and fat deposition. GHR-AS-EST is predominantly expressed in the chicken liver and muscle tissues. GHR-AS-EST sequence conservation among vertebrates is weak. GHR-AS-EST forms an RNA-RNA duplex with GHBP to increase its stability, and regulates the expression of GHR sense transcripts at both the mRNA and protein levels. Further, GHR-AS-EST promotes cell proliferation by stimulating the expression of signaling factors in the JAK2/STAT pathway, and contributes to fat deposition via downregulating the expression of signaling factors in the JAK2/SOCS pathway in LMH hepatocellular carcinoma cells. We expect that the discovery of a NAT for a regulatory gene associated with cell proliferation and lipolysis will further our understanding of the molecular regulation of both muscle development and fat deposition.

ALV-J infection induces chicken monocyte death accompanied with the production of IL-1ß and IL-18.

Immunosuppression induced by avian leukosis virus subgroup J (ALV-J) causes serious reproduction problems and secondary infections in chickens. Given that monocytes are important precursors of immune cells including macrophages and dendritic cells, we investigated the fate of chicken monocytes after ALV-J infection. Our results indicated that most monocytes infected with ALV-J including field or laboratory strains could not successfully differentiate into macrophages due to cells death. And cells death was dependent upon viral titer and accompanied with increased IL-1ß and IL-18 mRNA levels. In addition, ALV-J infection up-regulated caspase-1 and caspase-3 activity in monocytes. Collectively, we found that ALV-J could cause cell death in chicken monocytes, especially pyroptosis, which may be a significant reason for ALV-J induced immunosuppression.