Ammonia (NH3) is a major pollutant in intensive and high-density laying hens farming, primarily originating from the physiological metabolism of laying hens and the decomposition of nitrogen-containing substances in manure. Among them, the decomposition of uric acid in feces by microorganisms is the key generation pathway. Excessive NH3 concentration not only significantly reduces laying performance and egg quality, damages the respiratory mucosal barrier and triggers inflammatory responses, suppresses immune function, and induces oxidative stress, but also contributes to the formation of PM2.5, thereby exacerbating both the farming environment and atmospheric pollution. Currently, a multi-dimensional NH3 mitigation system encompassing source control, process regulation, and end-of-pipe treatment has been established in the layer farming industry. Specific measures include low-protein diet regulation, application of functional feed additives, precise environmental control in hen houses, and harmless manure treatment technologies. This article reviews the generation mechanism of NH3 in laying hens farming, its multiple adverse effects on laying hens, and the principles and application effects of various mitigation technologies, aiming to provide a reference for scientifically formulating NH3 reduction strategies, solving farming pollution problems, and promoting the green and healthy development of the laying hens industry.

DM9 domain containing protein (DM9CP) is a family of newly identified recognition receptors exiting in most organisms except plants and mammals. In the current study, to our knowledge, a novel DM9CP-5 (CgDM9CP-5) with two tandem DM9 repeats and high expression level in gill was identified from the Pacific oyster, Crassostrea gigas. The deduced amino acid sequence of CgDM9CP-5 shared 62.1% identity with CgDM9CP-1 from C. gigas, and 47.8% identity with OeFAMeT from Ostrea edulis. The recombinant CgDM9CP-5 (rCgDM9CP-5) was able to bind d-mannose, LPS, peptidoglycan, and polyinosinic-polycytidylic acid, as well as fungi Pichia pastoris, Gram-negative bacteria Escherichia coli and Vibrio splendidus, and Gram-positive bacteria Staphylococcus aureus. The mRNA transcript of CgDM9CP-5 was highly expressed in gill, and its protein was mainly distributed in gill mucus. After the stimulations with V. splendidus and mannose, mRNA expression of CgDM9CP-5 in oyster gill was significantly upregulated and reached the peak level at 6 and 24 h, which was 13.58-fold (p < 0.05) and 14.01-fold (p < 0.05) of that in the control group, respectively. CgDM9CP-5 was able to bind CgIntegrin both in vivo and in vitro. After CgDM9CP-5 or CgIntegrin was knocked down by RNA interference, the phosphorylation levels of JNK and P38 in the MAPK pathway decreased, and the expression levels of CgIL-17s (CgIL-17-3, -4, -5, and -6), Cg-Defh1, Cg-Defh2, and CgMolluscidin were significantly downregulated. These results suggested that there was a pathway of DM9CP-5-Integrin-MAPK mediated by CgDM9CP-5 to regulate the release of proinflammatory factors and defensins in C. gigas.

B lymphocyte-inducible maturation protein 1 (Blimp-1) is a SET domain and zinc fingers containing transcrip-tional repressor, which is necessary for regulating the development of many immune cell lineages and keeping immune homeostasis. In the present study, a Blimp-1 homologue (designated as CgBlimp-1) was identified from oyster Crassostrea gigas, which contained a conserved SET domain and five ZnF_C2H2 domains and shared high homology with Blimp-1 from other species. The mRNA transcripts of CgBlimp-1 were highly expressed in gill and hepatopancreas. CgBlimp-1 protein was detected to be specifically expressed in granulocytes. After V. splendidus stimulation, the mRNA expression level of CgBlimp-1 in haemocytes up-regulated significantly at 24, 48, and 96 h, which was 4.39-fold (p < 0.05), 7.68-fold (p < 0.01) and 2.65-fold (p < 0.05) of that in control group, respectively. When the expression of CgBlimp-1 was knocked-down in vivo by RNAi, the mRNA expressions of downstream transcription factor CgMyc-A (1.63-fold of that in control group, p < 0.05) and cell cycle related gene CgCDK2 (1.70-fold, p < 0.05) increased significantly at 24 h after V. splendidus stimulation. Concomitantly, the ratio of EdU+ haemocytes increased notably (p < 0.01) while the proportion of haemocytes in G0/G1 phase decreased dramatically (p < 0.001), compared to that in control group. More specifically, the proportion of granulocytes in total haemocytes increased apparently (p < 0.05) in CgBlimp-1-RNAi oysters, together with up-regulation (p < 0.05) of the ratio of EdU+ granulocytes and down-regulation (p < 0.001) of the proportion of granulocytes in G0/G1 phase. Furthermore, the mRNA expression levels of CgIL17-1, CgIL17-2 and CgIL17-4 in haemocytes increased significantly in CgBlimp-1-RNAi oysters, which was 1.71-fold (p < 0.05), 144.70-fold (p < 0.01) and 1.93-fold (p < 0.05) of that in control group, respectively. Aforementioned results suggested that CgBlimp-1 could reduce the proliferation of granulocytes by arresting cell cycle in G1/G0 phase and avoid over-expression of interleukin to maintain homeostasis in the immune response of oyster.

Recent findings regarding the immunomodulatory role of Wnt signaling suggest that it is significant in regulating the differentiation and proliferation of immune cells. In the present study, a Wnt-1 homolog (designated as CgWnt-1) with a conserved WNT1 domain was identified from oyster Crassostrea gigas. The transcripts of CgWnt-1 were barely expressed in egg to gastrula stage during early embryogenesis, and up-regulated significantly in the trochophore to juvenile stage. The mRNA transcripts of CgWnt-1 were detected in different tissues of adult oyster, with an extremely high expression level in the mantle, which was 77.38-fold (p < 0.05) of that in labial palp. After Vibrio splendidus stimulation, the mRNA expression levels of CgWnt-1 and Cgβ-catenin in haemocytes up-regulated significantly at 3, 12, 24, and 48 h (p < 0.05). After injection of recombinant protein (rCgWnt-1) into oyster in vivo, the expressions of Cgβ-catenin, cell proliferation related genes CgRunx-1 and CgCDK-2 in haemocytes significantly up-regulated, which were 4.86-fold (p < 0.05), 9.33-fold (p < 0.05), 6.09-fold (p < 0.05) of those in rTrx group, respectively. The percentage of EDU+ cells in haemocytes also significantly increased (2.88-fold of that in control group, p < 0.05) at 12 h after rCgWnt-1 treatment. When the Wnt signal inhibitor C59 was injected simultaneously with rCgWnt-1, the expressions of Cgβ-catenin, CgRunx-1, and CgCDK-2 were significantly reduced, which were 0.32-fold (p < 0.05), 0.16-fold (p < 0.05), and 0.25-fold (p < 0.05) of that in rCgWnt-1 group, respectively, and the percentage of EDU+ cells in haemocytes was also significantly inhibited (0.15-fold compared with that in rCgWnt-1 group, p < 0.05). These results suggested that the conserved CgWnt-1 could modulate haemocytes proliferation via regulating cell cycle related genes and involved in the immune response of oysters.

The stimulator of interferon gene (STING), an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response, especially the induction of type I interferon (IFN) during pathogenic infection. A STING homologue (CgSTING) regulating the expression of IFN-like protein (CgIFNLP) was previously identified in the Pacific oyster Crassostrea gigas, and its involvement in antibacterial immunity was further investigated in the present study. The mRNA transcripts of CgSTING were ubiquitously detected in all the three subpopulations of haemocytes with the highest expression in semi-granulocytes. After the stimulation with Vibrio splendidus, the mRNA expression of CgSTING in haemocytes was significantly up-regulated and peaked at 72 h, which was 12.91-fold of that in control group (p < 0.01). The CgSTING protein was mainly located in the cytoplasm of haemocytes. After the expression of CgSTING was knocked down (0.12-fold of that in control group, p < 0.05) by RNAi, the mRNA expression levels of interleukin17-1 (CgIL17-1), interleukin17-3 (CgIL17-3), interleukin17-4 (CgIL17-4), defensins (Cgdefh1, Cgdefh2), big defensin (CgBigDef1), interferon-like protein (CgIFNLP), tumor necrosis factor (CgTNF) and nuclear factor-kappa B (CgRel) all decreased significantly at 12 h after V. splendidus stimulation, which was 0.12-fold-0.72-fold < 0.05) of that in control group, respectively. The positive signals of CgRel were observed in the haemocyte nucleus after V. splendidus stimulation. The nuclear translocation of CgRel was suppressed in CgSTING-RNAi oysters, and the green signals of CgRel were mainly observed in the haemocyte cytoplasm after V. splendidus stimulation. Furthermore, the number of V. splendidus in the haemolymph of CgSTING-RNAi oysters increased significantly, which was 26.78-fold (p < 0.01) of that in the control group at 12 h after V. splendidus stimulation. These results indicated that CgSTING played important role in the immune defense against bacterial infection by inducing the expressions of cytokines and defensins.

Inflammatory cytokine interleukin-17 (IL-17) binds its receptors (IL-17Rs) to activate the downstream immune signals and plays an important role in host defense. In the present study, an IL-17 receptor (designated as CgIL-17R1) was identified from oyster Crassostrea gigas with an open reading frame of 3141 bp encoding 1047 amino acids. The amino acid sequence of CgIL-17R1 with two conserved FN3 domains shared higher similarity with other known IL-17Rs from mollusc species. The recombinant CgIL-17R1 protein (rCgIL-17R1) displayed high binding affinity to the recombinant CgIL-17 protein (rCgIL-17) in vitro. The mRNA transcripts of CgIL-17R1 were significantly higher expressed in haemocytes, especially in granunolyctes, compared with that in other tissues. After the stimulation with Vibrio splendidus or rCgIL17-1 in vivo, the expressions of CgIL-17R1 and cell proliferation related genes (CgRunx-1, CgCDC-6, CgCDC-45, and CgCDK-2) were significantly up-regulated in haemocytes (p < 0.01). When the CgIL-17R1 expression was interfered by specific CgIL-17R1-dsRNA, the expressions of these cell proliferation related genes reduced significantly, and the proliferation rate of haemocytes declined dramatically at 6 h post V. splendidus stimulation (p < 0.01), compared to that of blank group. These results collectively indicated that CgIL-17R1 expressed in granulocytes mediated the CgIL-17 induced haemocytes proliferation during immune response in oyster C. gigas, which provided novel information about the regulation of haemocyte proliferation in invertebrates.

Toll like receptor (TLR) signaling plays a key role in the innate immune recognition and inflammatory regulation in both vertebrates and invertebrates. The expanded TLR signaling components, including 83 TLRs and 10 MyD88s, have been reported in the genome of the Pacific oyster Crassostrea gigas. In the present study, one endogenous TLR (designated CgTLR2) and two MyD88s (including a full-length CgMyD88-2 containing intact TIR domain and Death-domain, and a truncated CgMyD88s with only TIR domain) were identified from oyster C. gigas. CgTLR2 was highly expressed in haemocytes, especially in granulocytes. The recombinant protein of the extracellular LRR domains of CgTLR2 recognized and bound a variety of PAMPs with the strongest binding capability to LPS. The recombinant protein of intracellular TIR domain of CgTLR2 was able to bind the re-combinant proteins of rCgMyD88-2 (K-D = 1.96 x 10(-9) M) and rCgMyD88s (K-D = 4.84 x 10(-8) M), with higher affinity towards rCgMyD88-2. After Vibrio splendidus stimulation, the mRNA expression levels of CgTLR2 and CgMyD88-2 were rapidly up-regulated at early stage of immune response (from the 3rd hours after V. splendidus stimulation), while that of CgMyD88s did not change until 24 h post stimulation. When CgTLR2 was knocked-down by siRNA interference, the expression levels of CgMyD88-2 and CgMyD88s decreased significantly, concomitant with the down-regulation of expression of CgIL17-1. After the expression of CgMyD88-2 was interfered, the expressions of CgMyD88s and CgIL17-1 were all decreased. In contrast, after the expression of CgMyD88s was interfered, the expressions of CgMyD88-2 and CgIL17-1 all increased. The results showed that CgMyD88s played a negative role in the regulation of CgTLR2 on inflammatory factor CgIL17-1.

FOXOs belong to the forkhead transcription factor superfamily, several of which are suggested to be involved in the control of food intake. Previously, we proved that the neuropeptide FF (NPFF) peptide was involved in feeding regulation in spotted sea bass. In the present study, seven members of the foxo family were identified in the whole genome of spotted sea bass. The distributions of these genes in different tissues were analyzed by qRT-PCR. Variations in the foxo1a and npff expression profiles during short-term starvation showed similar expression patterns. The colocalization of foxo1a and npff in the telencephalon, hypothalamus, stomach and intestine further provided evidence that foxo1a may act directly to promote the transcription of npff. Thirteen predicted FOXO1 binding sites were found in the 5' upstream region of npff. Luciferase assay results showed that FOXO1A was able to activate npff transcriptional responses by directly binding DNA response elements, and the key regulatory areas and sites of FOXO1A on the npff promoter were confirmed by deletion and site-directed mutagenesis analyses. These findings may help to elucidate the role of FOXO1 in the regulation of feeding processes in teleosts.

Ketamine has gained increasing popularity in adolescent drug abusers worldwide. However, relatively little is known about the long-term effects of recreational ketamine on adolescent hippocampus. The present study investigates the effects of different periods (1, 3 and 6 months) of recreational ketamine administration on locomotor activity and neuron damage in the hippocampus of adolescent cynomolgus monkeys. 32 4-year-old male cynomolgus monkeys were divided into control, 1-month, 3-month and 6-month groups. All animals in ketamine groups received daily intravenous injection with 1 mg/kg ketamine in saline for respective 1, 3 or 6 months while control group received normal saline. Automatic behaviors were recorded for 10 min before and after ketamine and saline administration. Meanwhile, the markers of apoptosis in the hippocampus were assessed using terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL), electron microscopy and western blotting. Results showed that ketamine significantly decreased locomotor activity, increased apoptotic neurons and pro-apoptotic proteins, cleaved Caspase-3 and Bax, while decreased the anti-apoptotic protein Bcl-2 in the hippocampus after 6-month ketamine administration. Our study suggested that chronically recreational ketamine might induce hypolocomotion and neurotoxic effect via apoptotic pathway in adolescent hippocampus of monkeys.

The programmed cell death-ligand-1 (PD-L1) and bromodomain protein 4 (BRD4) are frequently overexpressed in cancer and have even been shown to act synergistically. The aim of this study was to determine their potential oncogenic role .in tongue squamous cell carcinoma (TSCC). We detected significantly higher expression levels of both PD-L1 and BRD4 in TSCC tissues compared to normal tissues (P ≤ .05). In addition, the high levels of PD-L1 were significantly associated with increased tumor lymphatic metastasis (P ≤ .05), tumor staging (P ≤ .01), as well as BRD4 expression (P ≤ .05). Genetic and pharmacological inhibition of BRD4 in TSCC cells not only reduced their growth rate but also PD-L1 levels (P ≤ .05), while overexpression of BRD4 upregulated PD-L1. Bioinformatics analysis showed that c-MYC and CDK9 were interactive partners of both BRD4 and PD-L1. While c-MYC clearly modulated the expression of PD-L1, as well as reversed the inhibitory effects of JQ1, no obvious association was observed between CDK9 and PD-L1. We report a novel regulatory axis consisting of BRD4, PD-L1, and c-MYC that likely drives TSCC progression, and is a potential prognostic marker and/or therapeutic target for TSCC.

Ketamine has become increasingly popular in adolescent drug abusers worldwide. Meanwhile, alcohol is usually used by ketamine users. However, little work has been conducted to examine the chronic combined effects of ketamine and ethanol on adolescent brain. Here we probed into the effects of chronic administration of ketamine at recreational doses alone or combined with ethanol on behaviors and neuron damage in an adolescent rat model. 28-day old rats were treated with either 20 or 30 mg/kg ketamine plus or not plus 10% ethanol daily for 21 days. Depressive like behaviors, anxiety like behavior and memory impairment were tested using open field test, forced swimming test, elevated plus maze and Morris water maze. Apoptosis in prefrontal cortex (PFC) and hippocampus (HIP) were determined by the TdT-mediated dUTP Nick-End Labeling (TUNEL) and protein and mRNA levels of caspase-3, Bax and Bcl-2. Results show that co-application of ketamine and ethanol significantly increased immobility time in the forced swimming test, up-regulated TUNEL positive cells and both protein and mRNA expressions of caspase-3 and Bax, compared with the control group and ketamine and ethanol use alone groups in the PFC, but not in the HIP. Our study suggests that chronic co-administration of ketamine and ethanol results in depressive-like behavior and the caspase-dependent apoptosis in the PFC of adolescent rats' brains.

The ethanol-induced toll-like receptor 4 (TLR4) signal activation of microglia and neuroinflammation are observed in both adolescent and adult rat brains, but the regulatory mechanisms of some TLR4 signaling-related factors in this process are still unclear. SUMO-specific protease 6 (SENP6) inhibits neuroinflammation by dampening nuclear factor kappa-B (NF- κ B) activation via the de-SUMOylation of NF kappa-B essential modulator (NEMO). This study investigates the effects of long-term ethanol consumption on neuroinflammation in the hippocampus of adolescent rats and the regulatory roles of TLR4 and SENP6. Twenty-one days of ethanol exposure in adolescent rats were used to develop an animal model. The number of microglia, microglial activation, and the expression of TLR4 in the hippocampus of adolescent rats were examined by immunoreactivity. The levels of TLR4, activation of NF- κ B including IkB- α and p-NF- κ B-p65, and SENP6 were measured by western blotting. Proinflammatory cytokines including TNF- α , IL-1 β , and IL-6 were measured by enzyme-linked immunosorbent assay. The NF- κ B activation and proinflammatory cytokines released in overexpressed SENP6 and siRNA targeting SENP6 microglial cells after treatment with ethanol were estimated in vitro. This study found that alcohol exposure increased the number of activated microglia and the levels of p-NF- κ B-p65 and proinflammatory cytokines, while it decreased the SENP6 level in wild-type rats, but not in TLR4 knockout rats. The ethanol-induced increases of p-NF- κ B-p65, TNF- α , and IL-1 β were dampened by overxpression of SENP6 and enhanced in SENP6-siRNA microglia. Our data suggest that ethanol exposure during adolescence induces the microglia-mediated neuroinflammation via TLR4 activation, and SENP6 plays an essential role in dampening NF- κ B activation and neuroinflammation.

Neuropeptide FF (NPFF) is a family member of RF-amide peptides, which are suggested to be involved in the control of vertebrate feeding behavior. However, little is known about the effect of the NPFF peptide on feeding-related processes in basal vertebrates. In this study, four full-length cDNAs, npff, npffr1, npffr2-1, and npffr2-2, were cloned from spotted sea bass and characterized. The conserved NPFF peptide is biologically active because it functionally interacts with different receptors expressed in cultured eukaryotic cells to enhance CRE promoter activity. Tissue distribution analysis showed that the highest npff mRNA expression occurred in the telencephalon, hypothalamus, medulla, gonad and muscle, but the npffrs mRNAs were mainly distributed within the central nervous system (CNS). In situ hybridization (ISH) detected npff-expressing cells in several specific regions ranging across the telencephalon and midbrain to the hypothalamus. Incubation of the spotted sea bass conserved NPFF peptide significantly increased the expression of orexin (orx) and neuropeptide Y (npy) mRNA and decreased the expression of leptin (lep), somatostatin (ss), and cholecystokinin (cck) mRNA in brain cells. Similarly, the conserved NPFF peptide also heightened the expression of gastrin (gas), ghrelin (ghrl), and motilin (mtl) mRNA and significantly reduced the expression of cck mRNA in the intestine and stomach. Taken together, these data suggest that the NPFF peptide may play a stimulating role in regulating feeding-related processes in spotted sea bass.

Neurokinin B (NKB) is a member of the tachykinin (tac) family that plays important roles in mammalian growth by modulating prolactin (PRL) synthesis and secretion and causing contraction of the stomach and intestine. However, its potential role in regulating growth of teleosts is less clear. We aimed to explore the role that NKB plays in regulating fish growth using the spotted sea bass (Lateolabrax maculatus) as a model. In the present study, two tac3 and two tacr3 genes were identified in the spotted sea bass. Sequence analysis showed that two tac3 transcripts, tac3a and tac3b, encode four NKBs: NKBa-13, NKBa-10, NKBb-13, and NKBb-10. Expression analysis in different tissues showed that both genes are highly expressed in the brain, stomach and intestine of the spotted sea bass. In situ hybridization indicated that the tac3a and tac3b mRNAs are both localized in several brain regions, such as the telencephalon and hypothalamus, and that tacr3a and tacr3b are localized in the intestinal villus and gastric gland. To investigate the potential role of NKBs in regulating growth, in vitro experiments were performed to detect the effect of NKBs on growth-related gene expression in the brain and brain-gut peptide (BGP)-related genes in the stomach and intestine. NKBb-13 was the most critical ligand in regulating the expression of growth-related genes in the brain and brain-gut peptide (BGP)-related genes in the stomach. The expression of cholecystokinin (cck) was enhanced by NKBa-13, NKBa-10, and NKBb-10 but not NKBb-13 in the intestine. In general, our results showed that NKBs participate in regulating the growth of spotted sea bass.

Ketamine, a non-competitive N-methyl-D-aspartic acid receptor antagonist, has emerged as an increasingly popular drug among young drug abusers worldwide. Available evidence suggests that ketamine produces acute impairments of working, episodic and semantic memory along with psychotogenic and dissociative effects when a single dose is given to healthy volunteers. However, understanding of the possible chronic effects of ketamine on behavior, cognitive anomalies and neurochemical homeostasis is still incomplete. Although previous human studies demonstrate that ketamine could impair a range of cognitive skills, investigation using non-human models would permit more precise exploration of the neurochemical mechanisms which may underlie the detrimental effects. The current study examined the abnormalities in behavior (move, walk, jump and climb) and apoptosis of the prefrontal cortex using terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) and apoptotic markers, including Bax, Bcl-2 and caspase-3 in adolescent male cynomolgus monkeys (Macaca fascicularis) after 1 or 6 months of sub-anesthetic ketamine administration (1 mg/kg, i.v.). Results showed that ketamine decreased locomotor activity and increased cell death in the prefrontal cortex of monkeys with 6 months of ketamine treatment when compared with the control monkeys. Such decreases were not found in the 1-month ketamine-treated group. Our study suggested that ketamine administration of recreational dose in monkeys might produce permanent and irreversible deficits in brain functions due to neurotoxic effects, involving the activation of apoptotic pathways in the prefrontal cortex.

Objective To explore effects of long-term low-dose ketamine treatment on locomotor activity and apoptosis of the thalamus in cynomolgus monkeys.Methods 12 male cynomolgus monkeys [(46.25±5.40)months old] were randomly divided into the ketamine group(n=8) and the control group(n=4).Monkeys in the ketamine group were injected with 1 mg/mL of ketamine,1 mL/kg;while those in the control group were injected with an equal dose of normal saline(1 mL/kg).Locomotor activities of all monkeys were recorded at regular intervals.3 months later,monkeys were sacrificed.The apoptosis of thalamic cells was observed by TUNEL staining.Western blot was used to detect Caspase-3 and Bcl-2 expressions.Results There was no statistically significant difference in locomotor activity between the ketamine group and the control group.The ketamine group showed a statistically significant decrease in locomotor activity with prolongation of treatment time,while the control group had no statistically significant change.The ketamine group showed a statistically significant increase in apoptosis and the Caspase-3 level in the thalamus compared with the control group.There was no statistically significant difference in the Bcl-2 expression between the two groups.Conclusion Chronic repeated treatment of ketamine with recreational dosage decreases locomotor activities which may be caused by drug tolerance,and induces apoptosis of the thalamus which may be associated with up-regulation of Caspase-3.