miR-130a targets CiGadd45bb to modulate the inflammatory response to bacterial infection in Ctenopharyngodon idella kidney (CIK) cells.

Septicemia is a systemic inflammatory response to bacterial infection that results in a hyper-inflammatory state, which could lead to septic shock and death in grass carp (Ctenopharyngodon idella). The aim of this study was to determine the underlying mechanism of microRNA (miR-130a) in bacteria-infected grass carp. Expression levels of miR-130a against Aeromonas hydrophila (A. hydrophila) infection in Ctenopharyngodon idella kidney cells (CIK) were analyzed. Luciferase reporter assay, quantitative reverse transcription-polymerase chain reaction were performed to explore whether Ctenopharyngodon idella growth arrest and DNA damage-inducible 45 (CiGadd45bb) was a target of miR-130a. MiR-130a mimic, inhibitor and miR-control were transfected to CIK respectively. After transfection, the expression levels of proinflammatory genes were determined. Here we show that CiGadd45bb as a target of miR-130a. We also confirmed that miR-130a levels were significantly higher after being stimulated for 4 h and lower after 12 h (P < 0.01). Overexpressing miR-130a strikingly inhibited p38, JNK, ERK and TNF-a genes (P < 0.01) and silencing miR-130a activated p38, JNK, ERK, TNF-a, IFN and IL-8 (P < 0.01). Our results provide a theoretical basis for studying the molecular mechanism underlying the regulation of inflammation by miR-130a in grass carp.

Association of physical activity and the risk of COVID-19 hospitalization: A dose-response meta-analysis.

BACKGROUND: Many people have experienced a high burden due to the spread of the coronavirus disease (COVID-19) and its serious consequences for health and everyday life. Prior studies have reported that physical activity (PA) may lower the risk of COVID-19 hospitalization. The present meta-analysis explored the dose-response relationship between PA and the risk of COVID-19 hospitalization. METHODS: Epidemiological observational studies on the relationship between PA and the risk of COVID-19 hospitalization were included. Categorical dose-response relationships between PA and the risk of COVID-19 hospitalization were assessed using random effect models. Robust error meta-regression models assessed the continuous relationship between PA (metabolic equivalent [Met]-h/wk) and COVID-19 hospitalization risk across studies reporting quantitative PA estimates. RESULTS: Seventeen observational studies (cohort\case-control\cross-section) met the criteria for inclusion in the meta-analysis. Categorical dose-relationship analysis showed a 40% (risk ratio [RR] 0.60, 95% confidence interval [CI]: 0.48-0.71) reduction in the risk of COVID-19 hospitalization compared to the lowest dose of PA. The results of the continuous dose-response relationship showed a non-linear inverse relationship (Pnon-linearity < .05) between PA and the risk of COVID-19 hospitalization. When total PA was < or >10 Met-h/wk, an increase of 4 Met-h/wk was associated with a 14% (RR = 0.83, 95% CI: 0.85-0.87) and 11% (RR = 0.89, 95% CI: 0.87-0.90) reduction in the risk of COVID-19 hospitalization, respectively. CONCLUSIONS: There was an inverse non-linear dose-response relationship between PA level and the risk of COVID-19 hospitalization. Doses of the guideline-recommended minimum PA levels by the World Health Organization may be required for more substantial reductions in the COVID-19 hospitalization risk.

Insufficient or excessive dietary carbohydrates affect gut health through change in gut microbiota and regulation of gene expression of gut epithelial cells in grass carp (Ctenopharyngodon idella).

Dietary carbohydrate levels can affect gut health, but the roles played by gut microbiota and gut epithelial cells, and their interactions remain unclear. In this experiment, we investigated gut health, gut microbiota, and the gene expression profiles of gut epithelial cells in grass carp consuming diets with different carbohydrate levels. Compared to the moderate-carbohydrate diet, low-carbohydrate diet significantly increased the relative abundance of pathogenic bacteria (Ralstonia and Elizabethkingia) and decreased the abundance of metabolism in cofactors and vitamins, implying a dysregulated gut microbiota and compromised metabolic function. Moreover, low-carbohydrate diet inhibited the expression levels of key genes in autophagy-related pathways in gut epithelial cells, which might directly lead to reduced clearance of defective organelles and pathogenic microorganisms. These aforementioned factors may be responsible for the imperfect organization of the intestinal tract. High-carbohydrate diet also significantly increased the abundance of pathogenic bacteria (Flavobacterium), which directly contributed to a decrease in the abundance of immune system of the microbiota. Furthermore, the active pathways of staphylococcus aureus infection and complement and coagulation cascades, as well as the inhibition of the glutathione metabolism pathway were observed. Above results implied that high-carbohydrate diet might ultimately cause severe gut damage by affecting immune function of microbiota, mentioned immune-related pathways, and the antioxidant capacity. Finally, the correlation network diagram revealed strong correlations of the differentially immune-related gene major histocompatibility complex class I antigen (MR1) with Enhydrobacter and Ruminococcus_gnavus_group in low-carbohydrate diet group, and Arenimonas in high-carbohydrate diet group, respectively, suggesting that MR1 might be a central target for immune responses in gut epithelial cells induced by gut microbiota at different levels of dietary carbohydrate. All these results provided insight in the development of antagonistic probiotics and target genes to improve the utilization of carbohydrate.

GH and GHR gene cloning, expression and their associations with growth-related traits of the barbel chub (Squaliobarbus curriculus).

Growth hormone (ScGH) and growth hormone receptor (ScGHR) genes from the barbel chub (Squaliobarbus curriculus), in addition to their cDNAs, were cloned. The associations between their mRNA expression levels and growth-related traits were analysed, and the differences in the levels of expression of growth regulation-related genes between the largest and smallest individuals were compared. The full-length 1182-bp cDNA of ScGH contained a 633-bp open reading frame (ORF), and the length of the gene had 2492 bp. The full-length 2825-bp cDNA of ScGHRa contained a 1818-bp ORF, and the gene had 6970 bp. The full-length 2822-bp cDNA of ScGHRb contained a 1737-bp ORF, and the gene had 8149 bp. Quantitative real-time PCR revealed that ScGH was only expressed in the pituitary. ScGHRa was expressed predominantly in muscle, and the expression level of ScGHRb was the highest in the liver. The ScGHRa mRNA levels in the muscle were significantly negatively correlated with the caudal peduncle length. However, no correlation between growth-related traits and ScGH and ScGHRb expression levels were found. Pituitary ScGH, liver GHRb and liver insulin-like growth factor I (igf-1) expression levels were significantly higher in the largest individuals than those in the smallest S. curriculus individuals. Contrarily, the largest individuals had significantly lower expression levels of muscle igf-1 and liver myog than the smallest individuals. Overall, our results provide novel molecular information for growth-regulation study of S. curriculus.

Characterization and immune function of the interferon-ß promoter stimulator-1 in the barbel chub, Squaliobarbus curriculus.

To elucidate the immunity-protecting role of the interferon-ß promoter stimulator-1 (ScIPS-1) in barbel chub Squaliobarbus curriculus, the full-length cDNA of ScIPS-1 was cloned and expression levels in response to stimulation were investigated. In addition, the function of ScIPS-1 and its domains were analyzed. The full-length cDNA of ScIPS-1 is 2524 bp and encodes 601 aa. The N-terminal caspase activation and recruitment domain, central proline-rich domain, C-terminal transmembrane domain, C2HC-zinc finger, and Cwf21 domains were identified. The mRNA level of ScIPS-1 was the highest in the kidney, whereas the highest protein level was observed in the liver. The ScIPS-1 expressions were significantly up-regulated after lipopolysaccharide and poly I:C treatment. The ScIPS-1 protein level was up-regulated at 12 h in the head kidney and was up-regulated at 12 h and then down-regulated from 12 to 48 h in the liver after grass carp reovirus (GCRV) infection. The CiIFN and CiMx transcription levels were significantly enhanced in pEGFP-C1-IPS-1 and pcDNA3.1-ΔCwf21 overexpressing cells after GCRV infection. The results indicate that ScIPS-1 may function in the immune response against pathogens and provide a basis for achieving resistance to diseases in fish breeding.

Sequence and expression analysis of the cytoplasmic pattern recognition receptor melanoma differentiation-associated gene 5 from the barbel chub Squaliobarbus curriculus.

MDA5 is a cytoplasmic viral double-stranded RNA recognition receptor that plays a pivotal role in the aquatic animal innate immune system. To decipher the role of MDA5 of Squaliobarbus curriculus (ScMDA5) in the immune response, full-length cDNA of ScMDA5 was cloned using the RACE technology, mRNA and protein expression levels of ScMDA5 signalling pathway members in response to stimulation were detected and effects of overexpression of ScMDA5 on the immune response were investigated. ScMDA5 comprises 3597 bp and is composed of an open reading frame (2958 nucleotides long) that translates into a putative peptide of 985 amino acid residues. ScMDA5 possesses two N-terminal caspase-recruiting domains, DEAD-like helicases superfamily, helicase superfamily C-terminal and RIG-I_C-RD domains, and differences in these domains among species were mainly observed with respect to their length and location. ScMDA5 was closely clustered with those of Carassius auratus, Ctenopharyngodon idellus and Mylopharyngodon piceus. ScMDA5 transcripts were most abundant in the spleen and the lowest in the liver. Expression levels of ScMDA5 in healthy tissues were significantly correlated with those of ScIRF3, ScIRF7 and ScIFN. Besides, mRNA expression levels of ScIRF3 were significantly correlated with those of ScIRF7 (0.956, P < 0.01). Expression level changes, including downregulation, upregulation and initial upregulation followed by downregulation, were found in ScMDA5 signalling pathway molecules in tissues after grass carp reovirus infection. Protein levels of ScMDA5 were the highest in the liver and the lowest in the spleen in detected healthy tissues. Overexpression of ScMDA5 led to significantly enhanced CiIRF7 and CiMx transcription in grass carp ovary cells (P < 0.05). The results of this study helped to clarify the role of ScMDA5 in the immune reaction against grass carp reovirus and provided fundamental information for fish breeding to achieve strong resistance to infection.

Molecular cloning and expression analysis of coagulation factor VIII and plasminogen involved in immune response to GCRV, and immunity activity comparison of grass carp Ctenopharyngodon idella with different viral resistance.

The grass carp reovirus (GCRV) has been shown to cause lethal infections in the grass carp Ctenopharyngodon idella (C. idella). In order to investigate the immune response to GCRV infection, the full-length cDNA sequences of coagulation factor VIII (CiFVIII) and plasminogen (CiPLG) from C. idella were cloned and their involvement in the immune response was studied. The immunity factor levels in C. idella with different GCRV resistances were also analyzed. The full-length 2478 bp cDNA of CiFVIII contained an open reading frame of 1965 bp and encoded a putative polypeptide of 654 amino acid residues. The full-length 2907 bp cDNA of CiPLG contained an open reading frame of 2133 bp and encoded a putative polypeptide of 710 amino acid residues. CiFVIII was closely clustered with that of Clupea harengus. CiPLG was first clustered with those of Cyprinus carpio and Danio rerio. CiFVIII transcripts were most abundant in the liver and least in the skin. The highest expression level of CiPLG was observed in liver and the lowest in muscle. Expression levels of CiFVIII in gill, head kidney and spleen, and expression levels of CiPLG in gill, intestine and liver all reached the maximum at 72 h post GCRV infection. In spleen, expression levels of CiFVIII and CiPLG were significantly positively correlated. The activities of T-AOC, LSZ and IgM in R♂ were significantly higher than those in O♂. Likewise, T-AOC and LSZ activities in F1 were significantly higher than f1 individuals (P < 0.01). These results indicated that CiFVIII and CiPLG may play important roles in the immune response to GCRV infection. In addition, antioxidant ability and serum immune factor activity may confer a different viral resistance to C. idella.

Molecular characterization and expression of TLR7 and TLR8 in barbel chub (Squaliobarbus curriculus): Responses to stimulation of grass carp reovirus and lipopolysaccharide.

The barbel chub (Squaliobarbus curriculus) is a kind of small size commercial fish species that is widely spread in Asia and has shown significant resistance to disease. In this study, the full-length cDNA sequences of Toll-like receptor (TLR) 7 and 8 from S. curriculus, designated as ScTLR7 and ScTLR8, were cloned, and their differences in the structure and the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation were investigated. The full-length 3715 base pair (bp) cDNA of ScTLR7 contained a complete open reading frame of 3162 bp and encoded a putative polypeptide of 1053 amino acid residues. The full-length 4624 base pair (bp) cDNA of ScTLR8 contained a complete open reading frame of 3072 bp and encoded a putative polypeptide of 1023 amino acid residues. ScTLR7 and ScTLR8 consisted of N-terminal signal peptide, leucine-rich repeats (LRRs), and Toll/IL-1 Receptors domain. LRR motifs of ScTLR7 and ScTLR8 bend into horseshoe-like solenoid structure, while the number of LRRs between the two genes is different. Phylogenetic analysis showed that both the ScTLR7 and ScTLR8 were closely clustered with Ctenopharyngodon idellus and Megalobrama amblycephala. Quantitative real-time polymerase chain reaction analysis showed that the expression levels of ScTLR7 in S. curriculus were most abundant in the brain followed by the spleen and heart, and the lowest in the intestine. The highest expression level of ScTLR8 was observed in spleen and the lowest in the liver. After LPS stimulation, the relative expression levels of both ScTLR7 and ScTLR8 exhibited an overall trend of up-regulation. The expression levels of type I-IFN showed an overall trend of down-regulation at time points of 12, 24, 72 and 168 h compared to that of 6 h after LPS stimulation. Compared to 6 h post GCRV infection, the transcription level of ScTLR7 was up-regulated from 12 to 168 h, and transcription levels of ScTLR8, MyD88, and type I-IFN were firstly up-regulated from 12 to 72 h, and then down-regulated from 72 to 168 h. Correlation analysis showed that expression level of ScTLR7 in the spleen was significantly positively correlated with that of MyD88 (Pearson correlation coefficient: 0.909, P: 0.033), and a significantly positive correlation was also observed between expression levels of MyD88 and type I IFN (Pearson correlation coefficient: 0.962, P: 0.009), after GCRV stimulation. These results indicate that ScTLR7 and ScTLR8 may play important roles in the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation and trigger different downstream immune signal pathways.