Molecular characterization and antiparasitic activity analysis of a novel piscidin 5-like type 4 from Larimichthys crocea.

Cryptocaryon irritans is an obligate parasitic ciliate protozoan that can infect various commercially important mariculture teleosts and cause high lethality and economic loss, especially Larimichthys crocea. Current methods of controlling or preventing this parasite with chemicals or antibiotics are widely considered to be environmentally harmful. The antiparasitic activity of some antimicrobial peptides (AMPs) attracted extensive attention of scholars. In the study, a novel piscidin 5-like type 4 (termed Lc-P5L4) excavated from comparative transcriptome of C. irritans - immuned L. crocea was identified and characterized. Sequence analysis shows the full-length cDNA of Lc-P5L4 is 539 bp containing an open reading frame (ORF) of 198 bp which encodes a peptide of 65 amino acid residues. The genome consists of three exons and two introns which exist in its ORF, and all the exon-intron boundaries are in accordance with classical GT-AG rule (GT/intron/AG). Multiple alignments indicate the signal peptides share highly conserved identity, while mature peptides are more diverse. Phylogenetic analysis displays Lc-P5L4 clusters together with other members of piscidin 5-like family. Next, quantitative Real-time PCR (qRT-PCR) detection found C. irritans infection could upregulate Lc-P5L4 expression level in all tested tissues significantly, it appeared earliest upregulation in the theronts infection stage in the head kidney; the expression contents reached to maximum level in the intestine, gill and muscle during trophonts falling off stage; while it was just upregulated during secondary bacterial infection stage in the liver and spleen. The data showed Lc-P5L4 upregulation time points were in accordance with different infection stages. With recombinant Lc-P5L4 (rLc-P5L4) obtained through Escherichia coli system, in vitro assay showed rLc-P5L4 could cause cilia deactivation, cell bodiesclumping and sticking to each other, then cell membrane rupture and contents leakage. The data illustrated Lc-P5L4 played critical roles in the immune defense against C. irritans infection, and provided another proof that piscidins exhibit multiple anti- C. irritans features.

Insight into the magnetic lime coagulation-membrane distillation process for desulfurization wastewater treatment: From pollutant removal feature to membrane fouling.

The high suspended solid (SS) and salts were main issues for flue gas desulfurization wastewater (FGDW). A magnetic lime coagulation (MLC)-membrane distillation (MD) integrated process was firstly applied with a self-made poly (vinylidene fluoride-co-chlorotrifluoroethylene) (PVDF-CTFE) membrane and the pollutants remove feature and membrane fouling were discussed. The SS was nearly 100 % removed and magnetic seed significantly accelerate the settleability. The flux was 43.00 kg/m2 h with a salt rejection >99 %. It was higher than 13 kg/m2 h in the first 125 h during the 18d continuous test, and the rejection for all cations, anions, total organic carbon (TOC) and total inorganic carbon (TIC) were higher than 99.95 %, 99.00 %, 98.81 %, and 99.65 %, respectively. Humic substances and tryptophan with 100-5000 Da were main dissolved organic matter (DOM), which were significantly removed. However, membrane fouling and wetting happened after 150 h. Scaling was the main foulants, while the organic fouling and biofouling were also detected. A new "bricklaying model" was induced to depict the formation of foulant layer, the colloids, organic matters (OMs) and microbe communities act as the "concrete", while the inorganic crystals (magnesium and calcium oxysulphides) were the "bricks". This contribution offers a new method for FGDW treatment and the membrane fouling mechanism of MD process.

Ferritin has an important immune function in the ark shell Scapharca broughtonii.

Ferritin, the principle cytosolic iron storage protein in the majority of living organisms, has important roles during immune process in invertebrates. Detailed information about ferritin in the ark shell Scapharca broughtonii, however, has been very limited. In this study, full-length ferritin (termed SbFer) was cloned by the rapid amplication of cDNA ends (RACE) method based upon the sequence from the transcriptome library. The cDNA contained a 182 bp 5'-untranslated region, a 519 bp open reading frame encoding a polypeptide of 172 amino acids, a 229 bp 3'-untranslated region, and three introns (902, 373 and 402 bp) embedded in four exons. There was an iron response element (IRE) in the 5'-untranslated region. The deduced amino acid sequence of SbFer possessed many characteristics of vertebrate H type ferritin, shared 63%-91% identity with mollusks and greater identity with vertebrate H type ferritin compared to the L type. The SbFer gene expression pattern examined by quantitative real-time PCR showed ferritin mRNA was expressed in all ark shell tissues examined. The highest levels of expression were found in hemocytes with decreasing levels of expression in foot, mantle, gill, adductor muscle and hepatopancreas. A challenge with Vibrio anguillarum resulted in time-dependent significant upregulation of SbFer mRNA, indicating SbFer participated actively in the bacterial defense process. Further analysis of the antibacterial activity indicated recombinant SbFer could function as an immune antibacterial agent to both Gram-positive and Gram-negative bacteria. Taken together, these results suggested strongly that ferritin of the ark shell is involved in immune defense against microbial infection and it is a constitutive and inducible acute-phase protein.

A manganese superoxide dismutase (MnSOD) from ark shell, Scapharca broughtonii: Molecular characterization, expression and immune activity analysis.

Manganese superoxide dismutase (MnSOD) is one of the key members of the antioxidant defense enzyme family, however, data regarding to the immune function of MnSOD in mollusks still remain limited now. In this study, a full-length MnSOD cDNA was identified by rapid amplification of cDNA ends (RACE) method from cDNA library of ark shell Scapharca broughtonii (termed SbMnSOD). The cDNA contained an open reading frame (ORF) of 696 bp which encoded a polypeptide of 232 amino acids, a 5'-UTR with length of 32 bp and a 3'-UTR of 275 bp. Four putative amino acid residues (His-57, His-105, Asp-190 and His-194) responsible for manganese coordination were located in the most highly conserved regions of SbMnSOD and the signature sequence (DVWEHAYY) also existed in SbMnSOD. The deduced amino acid sequence of SbMnSOD shared high homology to MnSOD from other species. All those data revealed that the SbMnSOD was a novel member of the MnSOD family. The mRNA expression profiles of SbMnSOD in tissues of foot, gill, mantle, adductor muscle, hemocytes and hepatopancreas analyzed by quantitative real-time PCR (qRT-PCR) suggested the mRNA transcripts of SbMnSOD distributed in all the examined tissues. Importantly, Vibrio anguillarum challenge resulted in the increased expression of SbMnSOD mRNA with a regular change trend in all examined tissues, indicating SbMnSOD actively participated in the immune response process. What's more, further analysis on the antibacterial activity of the recombinant SbMnSOD showed that the fusion protein could remarkably inhibit growth of both Gram-positive and Gram-negative bacteria. The present results clearly suggested that SbMnSOD was an acute phase protein involved in the immune reaction in S. broughtonii.