Molecular cloning and functional characterization of peroxiredoxin 4 (prx 4) in freshwater crayfish, Procambarus clarkii.

Peroxiredoxin (Prx), which is a newly discovered member of the antioxidant protein family, performs important biological functions in intracellular signal transduction. In the present study, a peroxiredoxin 4 gene was cloned from crayfish for the first time and named Pc-prx 4. According to the amino acid sequence signature, Pc-Prx 4 was identified as the typical 2-Cys Prx molecule, which possessed two conserved cysteines (Cys98 and Cys219). Time-course expression patterns post V. harveyi infection revealed that Pc-prx 4 was likely related to crayfish innate immune defense responses. In particular, the highest fold upregulation of the Pc-prx 4 mRNA transcript reached approximately 170 post V. harveyi infection in the crayfish hepatopancreas. The results of the mixed functional oxidase assay showed that rPc-Prx 4△ could resist the damaging effect of reactive oxygen species generated from the thiol/Fe3+/O2- reaction system to some extent. In addition, the results of the RNAi assay revealed that the crayfish survival rate was obviously increased post injection of V. harveyi when Pc-prx 4 was knocked down. Further study revealed that both hemolymph melanization and PO activity were strengthened to different degrees in the RNAi assay. Therefore, we speculated that the increase in the crayfish survival rate was likely due to the increase in hemolymph melanization. The obviously reinforced hemolymph melanization was directly caused by the upregulation of hemolymph PO activity, which was induced by the knockdown of Pc-prx 4. However, further studies are still indispensable for illuminating the molecular mechanism of Pc-prx 4 in the crayfish innate immune defense system.

Influence of nano-Fe3O4 biochar on the methanation pathway during anaerobic digestion of chicken manure.

Volatile fatty acids and ammonia nitrogen (AN) accumulate during anaerobic digestion (AD) of high N substrates, such as chicken manure (CM), causing decreases in methane yield. Previous research found that the addition of nano-Fe3O4 biochar can alleviate the inhibition caused by acids and ammonia and increase methane production. The mechanism of enhanced methane production in nano-Fe3O4 biochar-mediated AD of CM was explored in depth in this study. The results showed the lowest AN concentration in the control and nano-Fe3O4 biochar addition groups were 8,229.0 mg/L and 7,701.5 mg/L, respectively. Methane yield of volatile solids increased from 92.0 mL/g to 219.9 mL/g in the nano-Fe3O4 biochar treatment, which was attributed to the enrichment of unclassified Clostridiales and Methanosarcina. The mechanism of nano-Fe3O4 biochar in AD of CM under high AN level was to improve methane production by promoting syntrophic acetate oxidation and facilitating direct electron transfer between microorganisms.

Oxygen dynamics, organic matter degradation and main gas emissions during pig manure composting: Effect of intermittent aeration.

To investigate the effect of intermittent aeration on oxygen dynamics, organic matter degradation and main gas emissions, a lab-scale pig manure composting experiment was conducted with intermittent aeration (I_A, 30-min on and 30-min off) and continuous aeration (C_A). Although aeration volume and oxygen supply of I_A was only half of C_A, I_A could obviously enhance the oxygen utilization efficiency by 96.67 % and reduce energy dissipation for aeration by 50.87 %. Based on the comprehensive analysis of total organic matter, total carbon, total nitrogen, cellulose, hemicellulose and lignin contents, there was no significant difference in organic matter degradation between I_A and C_A (p > 0.05). Moreover, a reduction of 21.71 %, 38.93 %, 44.40 % and 62.19 % of CH4, N2O and the total GHG emission equivalent as well as NH3 emissions was realized, respectively, in I_A compared with C_A. Therefore, adopting intermittent aeration was a useful strategy and choice for high-efficiency, high-quality and environment-friendly composting.

A comprehensive assessment on bioavailability, leaching characteristics and potential risk of polycyclic aromatic hydrocarbons in biochars produced by a continuous pyrolysis system.

Biochar application as a soil amendment has attracted worldwide attention. Nevertheless, polycyclic aromatic hydrocarbons (PAHs) formed during biochar production might enter into ecosystems and threaten human health after application to soil. Continuous pyrolysis systems tend to cause an accumulation of PAHs in biochar owing to short residence time and rapid cooling. This study conducted a comprehensive assessment regarding potential risk of PAHs in biochars produced by a continuous pyrolysis system based on bioavailability, leaching behavior, toxic equivalent quantity, health risk and phytotoxicity of PAHs. Results showed that the concentrations of total PAHs in biochars were in the range of 93.40-172.40 mg/kg, exceeding the European Biochar Certificate standard. 3-rings PAHs were the predominant groups. The percentages of total freely dissolved and leachable PAHs were lower than 1%. RH contained the least bioavailable and leachable PAHs concentration and phytotoxicity compared with CS and PS, which might attribute to the characteristic of three biochars. CS and PS were acidic and exhibited high levels of DOC and VFAs, while RH was strongly alkaline and presented greater aromaticity and higher surface area, which might have resulted in high adsorptive capacity and decreased bioavailability of PAHs. When the biochar application rate was higher than 0.6 t/ha, the incremental lifetime cancer risk value for human exposure to biochar-borne PAHs through the biochar-amended soil was over 10-6, suggesting carcinogenic risks. Germination index values of biochars ranged from 25.66 to 88.95%. Phytotoxicity mainly was caused by bioavailable PAHs and dissolved organic compounds. Overall, these findings highlighted that although the percentage of bioavailable PAHs was low, the potential health risk and phytotoxicity of PAHs in biochars produced by a continuous pyrolysis system was of a great concern. High biochar application rates should be avoided without processing both for soil safety and human health.

Anaerobic co-digestion of corn stover and wastewater from hydrothermal carbonation.

This study aimed to investigate the interactions between wastewater of hydrothermal carbonation (W-HTC) and corn stover (CS) during anaerobic co-digestion. The results showed the maximum cumulative methane production of co-digestion was 280.7 ± 3.2 mL/g VS, and it increased by 5.84% and 10.69% compared with mono-digestion of CS and W-HTC, respectively. Increasing the HTC temperature and excess addition of W-HTC inhibits early and middle stage of co-digestion due to toxic organic inhibitors, and the negative effect of phenols is substantially more than furans. The microbial analysis illustrated the addition of W-HTC can promote the growth of Clostridia and Bacteroidia. The growth of Methanomassiliicoccus and Methanosarcina was more vigorous in most of co-digestions, which was positively correlated with methane production. The study concluded methanogenesis can be enhanced by the co-digestion of W-HTC and CS, which provide optimization of process conditions and some reaction mechanism for application of W-HTC in anaerobic digestion.

Molecular characterization and expression analysis of tumor necrosis factor receptor-associated factor 6 (traf6) like gene involved in antibacterial innate immune of fresh water crayfish, Procambarus clarkii.

In invertebrates, innate immunity was the crucial defending pattern against pathogenic microorganisms. For the past few years, Toll or Toll like receptors (TLRs) signaling pathway was studied extensively in crustaceans. Among the components of Toll or Toll like receptors (TLRs) signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6) acted as an important cytoplasmic adaptor, which was conserved from Drosophila to human. In this study, a new traf6 like gene was cloned from hepatopancreas of P. clarkii. After challenged respectively by S. aureus or E. ictaluri, the expression profiles were studied. And the results showed that the mRNA transcript of Pc-traf6 like gene was up-regulated significantly in the hemocytes, hepatopancreas, gills, and intestine of crayfish. After Pc-traf6 like gene was knocked down, the expression levels of transcription factor (Dorsal) and some crucial immunity effectors (ALF 3, Lysozyme 1, Lectin 1, and Crustin 2) in TLRs signaling pathway were dramatically suppressed. Simultaneously, the survival rate of crayfish challenged respectively by S. aureus or E. ictaluri was significantly decreased in RNAi assay. All these results indicated that Pc-traf6 like gene played an important role in regulating the expression of downstream effectors in the TLRs signaling pathway of crayfish.

Molecular cloning, expression and biological activity of rhesus macaque interleukin-17A and interleukin-17F.

Interleukin-17A (IL-17A) and interleukin-17F (IL-17F) as two potent proinflammatory cytokines and the signature cytokines of Th17 cells play important roles in human autoimmune diseases, inflammation and host defenses. In this study, rhesus macaque IL-17A (rhIL-17A) and IL-17F (rhIL-17F) were cloned and expressed, and their biological activities and in vivo distribution were examined. The resulting data showed that both the rhIL-17A and rhIL-17F genes were consisted of three exons and two introns. RhIL-17A and rhIL-17F shared 96.8% and 93.9% amino acid sequence identity with human IL-17A (huIL-17A) and IL-17F (huIL-17F) respectively and the sequences also shared one N-glycosylation site and six conserved cysteine residues with huIL-17A and huIL-17F. IL-17A and IL-17F transcripts were highly expressed in lymphoid tissues and the intestinal tract of rhesus macaques. Functionally, recombinant rhIL-17A and rhIL-17F showed similar effect on Act1 levels and NF-κB phosphorylation compared with that of commercial human IL-17A and IL-17F. Moreover, the antibacterial proteins (such as ß-defensin 2, S100A8, S100A9, RegIIIα and Muc1) and the tight junction associated genes (including CLDN1, CLDN4, OCLN, and ZO1) expressed by Caco-2 cells were largely enhanced after treatment with rhIL-17A and rhIL-17F. Meanwhile, purified rhIL-17A and rhIL-17F could also induce the expression of IL-6 and TNF-α by THP-1 cells. These data indicated that rhesus macaque IL-17A and IL-17F are highly similar to that of humans in both structure and function. Studies on rhIL-17A/rhIL-17F are promising approach to contribute to the understanding of human IL-17A and IL-17F-related intestinal diseases.

IL-17A and IL-17F repair HIV-1 gp140 damaged Caco-2 cell barriers by upregulating tight junction genes.

It is widely accepted that impairment of the intestinal epithelial barrier from HIV/AIDS contributes significantly to microbial translocation and systemic immune activation. Such factors present potential targets for novel treatments aimed toward a functional cure. However, the extracellular mechanisms of intestinal barrier repair are poorly understood. In the current study, we investigated the abilities of IL-17A and IL-17F to repair the damaged barrier caused by HIV-1 gp140 using Caco-2 monolayers. It was found that HIV-1 gp140 downregulated the expression of tight junction-associated genes and disrupted the barrier integrity of Caco-2 monolayers. However, IL-17A and IL-17F treatment reversed the HIV-1 gp140-induced barrier dysfunction by upregulating the expression of tight junction-associated genes, the combination of which resulted in a stronger induction of barrier repair. Furthermore, the effects of IL-17A and IL-17F were reduced by downregulation of Act1 with siRNA and inhibition of NF-κB and MAPK pathways with BAY11-7082 and U0126, respectively. These data indicated that the NF-κB and MAPK pathways are involved in the repair of barrier integrity mediated by IL-17A and IL-17F, and IL-17 pathways are potential targets for gut barrier restoration therapies during HIV/AIDS.

A new crustin homologue (SpCrus6) involved in the antimicrobial and antiviral innate immunity in mud crab, Scylla paramamosain.

Crustins play important roles in defending against bacteria in the innate immunity system of crustaceans. In present study, we identified a crustin gene in Scylla paramamosain, which was named as SpCrus6. The ORF of SpCrus6 possessed a signal peptide sequence (SPS) at the N-terminus and a WAP domain at the C-terminus. And there were 5 Proline residues, 5 Glycine and 4 Cysteine residues between SPS and WAP domain in SpCrus6. These features indicated that SpCrus6 was a new member of crustin family. The SpCrus6 mRNA transcripts were up-regulated obviously after bacteria or virus challenge. These changes showed that SpCrus6 was involved in the antimicrobial and antiviral responses of Scylla paramamosain. Recombinant SpCrus6 (rSpCrus6) showed strong inhibitory abilities against Gram-positive bacteria (Bacillus megaterium, Staphylococcus aureus, and Bacillus subtilis). But the inhibitory abilities against four Gram-negative bacteria (Vibrio parahemolyticus, Vibrio alginolyticus, Vibrio harveyi and Escherichia coli) and two fungi (Pichia pastoris and Candida albicans) were not strong enough. Besides, rSpCrus6 could strongly bind to two Gram-positive bacteria (B. subtilis and B. megaterium) and three Gram-negative bacteria (V. alginolyticus, V. parahemolyticus, and V. harveyi). And the binding levels to S. aureus and two fungi (P. pastoris and C. albicans) were weak. The polysaccharides binding assays' results showed rSpCrus6 had superior binding activities to LPS, LTA, PGN and ß-glucan. Through agglutinating assays, we found rSpCrus6 could agglutinate well three Gram-positive bacteria (S. aureus, B. subtilis and B. megaterium). And the agglutinating activities to Gram-negative bacteria and fungi were not found. In the aspect of antiviral functions, rSpCrus6 could bind specifically to the recombinant envelop protein 26 (rVP26) of white spot syndrome virus (WSSV) but not to recombinant envelop protein 28 (rVP28), whereas GST protein could not bind to rVP26 or rVP28. Besides, rSpCrus6 could suppress WSSV reproduction to some extent. Taken together, SpCrus6 was a multifunctional immunity effector in the innate immunity defending response of S. paramamosain.

Spatial and temporal distribution of pore gas concentrations during mainstream large-scale trough composting in China.

With the advantages of high treatment capacity and low operational cost, large-scale trough composting has become one of the mainstream composting patterns in composting plants in China. This study measured concentrations of O2, CO2, CH4 and NH3 on-site to investigate the spatial and temporal distribution of pore gas concentrations during mainstream large-scale trough composting in China. The results showed that the temperature in the center of the pile was obviously higher than that in the side of the pile. Pore O2 concentration rapidly decreased and maintained <5% (in volume) for 38 days or more in both the center and side of the pile and effective O2 diffusion occurred at most in every two contiguous layers. Pore CO2 and CH4 concentrations at each measurement point were positively correlated (0.436 ≤ r ≤ 0.570, P < 0.01) and the concentrations in the side of the pile were obviously lower than those in the center. The top layer exhibited highest pore O2 concentration and lowest CO2 and CH4 concentrations, and the bottom layer was on the contrary. No significant differences in pore NH3 concentrations between different layers or between different measurement points in the same layer were found. Therefore, mixing the center and the side of the pile when mechanical turning and adjusting the height of the pile according to the physical properties of bulking agents are suggested to optimize the oxygen distribution and promote the composting process during large-scale trough composting when the pile was naturally aerated, which will contribute to improving the current undesirable atmosphere environment in China.

Identification and characterization of intestine microRNAs and targets in red swamp crayfish, Procambarus clarkii infected with white spot syndrome virus.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that play important roles in the innate immunity system of invertebrates, especially in the aspect of antivirus. In the present study, high-throughput small RNA Illumina sequencing systems were used to identify differentially expressed miRNAs (DEMs) from the intestines of Procambarus clarkii that were infected with white spot syndrome virus (WSSV). As a result, 39 known and 12 novel miRNAs were identified in both NG and WG small RNA libraries. Seven DEMs were determined to be involved in the antiviral innate immunity in the intestines of P. clarkii. The results of the target gene predictions of the DEMs showed that the putative target genes of these 7 DEMs are related to tight junctions, vascular smooth muscle contraction regulation of the actin cytoskeleton, focal adhesion, RNA transport, mRNA surveillance, viral carcinogenesis, and Salmonella infection. These results provide theoretical insights for future studies on the antiviral immunity of crustaceans.