Enhanced mycelium biomass and polysaccharide production in genetically modified Pleurotus ostreatus using agricultural wastes.

Edible fungi, healthier for humans and sustainable for the planet, attract unprecedented attention. In the study, the genetically modified Pleurotus ostreatus overexpression phosphoglucomutase (PGM) was constructed. P. ostreatus overexpression PGM (Po::PGM) had 4.96-folds higher expression level of PGM. Po::PGM grew thicker mycelium and more mycelium branches. Additional Ca2+ can inhibit mycelium growth, and cyclic adenosine monophosphate completely inhibited their growth of Po::PGM. Secondly, Overexpression of PGM made P. ostreatus become more sensitive to cell wall disruptors, and caused 12.75 % reduction of ß-1, 3-glucan and 40.53 % increase of chitin in cell wall. In submerged fermentation, the mycelia biomass yield and endopolysaccharide (IPS) production of Po::PGM in basic PDB can reach 11.18 g/l and 2.55 g/l, increasing by 20.86 % and 28.79 %, respectively. Whereas exopolysaccharide (EPS) reduced by 3.28 %. After replacing potato and glucose in PDB by wheat bran, mycelia biomass and EPS production of Po::PGM were all improved. The additional lactose in wheat bran did not only furtherly enhance mycelia biomass yield of Po::PGM to 27.78 g/l by 199.03 %, but IPS production also increased by 277.99 % to 6.07 g/l. The results provided us key ideas and important research directions that at least manipulating the PGM gene could obtain high-efficient use of agricultural wastes producing more fungus-based foods.

Identification, functional characterization and expression pattern of myeloid differentiation factor 88 (MyD88) in Nibea albiflora.

Myeloid differentiation factor 88 (MyD88), composed of an N-terminal death domain and a C-terminal Toll/interleukin (IL)-IR homology domain, is a key connector protein in the TLR signal transduction pathway. In this study a novel isoform of MyD88 in Nibea albiflora (named as NaMyD88) was identified and functionally characterized (GenBank accession no. MN384261.1). Its complete cDNA sequence was 1672 bp and contained an open reading frame of 879 bp encoding 292 amino acid residues, which was similar to its teleost fish counterparts in the length. The theoretical molecular mass was 33.63 kDa and the isoelectric point was 5.24. BLASTp analysis suggested that the deduced amino acids sequence of NaMyD88 shared high identity to the known MyD88, for instance, 94.77% identity with Collichthys lucidus. Sequence analysis showed that NaMyD88 protein was consistent with MyD88 protein of other species at three conserved domains, N-terminal DD, short middle domain and C-terminal TIR, and the TIR domain contained three highly conserved motifs: Box1, Box2, and Box3. NaMyD88 and red fluorescent protein (Dsred) were fused and expressed in the cytoplasm of the epithelioma papulosum cyprini (EPC cells). The NaTLR9-TIR-EGFP fusion protein, which was obtained in our previous studies, showed green fluorescence and mainly distributed in the cytoplasm. After co-transfection, NaMyD88-Dsred and NaTLR9-TIR-EGFP obviously overlapped and displayed orange-yellow color. The results showed that the homologous MyD88-Dsred could interact with NaTLR9-TIR-EGFP. Based on this result pcMV-NaMyD88-TIR-Myc plasmids and the pcDNA3.1-NaTLR9-TIR-flag were constructed and co-transfected into 293T cells for the immunoprecipitation test. According to Western blot, the protein eluted by Flag-beads could be detected by anti-Flag-tag antibody and anti-Myc tag antibody respectively, while the protein without NaTLR9-TIR could not be found, which further proved that TLR and MyD88 could interact each other. The prokaryotic plasmid of MyD88-TIR domain was constructed, expressed in BL21 (DE3) and purified by Ni-NAT super flow resin conforming to the expected molecular weight of 27 kDa with the corresponding active sites for its conferring protein-protein interaction functions. Real-time fluorescence quantitative PCR showed that NaMyD88 could be expressed in intestine, stomach, liver, kidney, gill, heart and spleen, with the highest in the kidney, and it was up-regulated after being infected with Polyinosinic:polycytidylic acid - Poly (I:C) and Pseudomonas plecoglossicida, which showed that NaMyD88 was involved in the immune response of N.albiflora. These data afforded a basis for understanding the role of NaMyD88 in the TLR signaling pathway of N.albiflora.

Optimized pre-treatment of high strength food waste digestate by high content aluminum-nanocluster based magnetic coagulation.

Coagulation-based pre-treatment efficiency of high strength digestate of food waste (HSDFW) anaerobic digestion is negated by organic ligand-catalyzed decomposition of coagulants. In this study, an efficient HSDFW pre-treatment method, magnetic seeds (MS) coagulation, was employed by using highly stable Keggin Al30 nanocluster (PAC30), MS and polyacrylamide (PAM), and its operation was optimized by evaluating the performance of removing turbidity, total suspended solids (TSS), chemical oxygen demand (COD), and total phosphorous (TP) phosphate. Results showed that at the optimum dosage of 4.82 g/L, PAC30 demonstrated excellent removals as high as 98.93% ± 0.1% of turbidity, 98.04% ± 0.1% of TSS, 58.28% ± 0.3% of total COD, 99.98% ± 0.01% of TP and 99.50% ± 0.01% of dissolved phosphate, respectively. Apparent molecular weight (AMW) and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy analyses demonstrated more efficient removal of dissolved organic matter (DOM), particularly non-biodegradable and hydrophobic components by PAC30 than commercial coagulant. The sedimentation was much improved from 40 min by coagulation/flocculation to about 5 min settling by MS coagulation. The PAC30 based magnetic coagulation (MC) presents theoretical guidance on a cost-effective and much less footprint pre-treatment alternative for high strength wastewater.

Characteristics delineation of piscidin 5 like from Larimichthys crocea with evidence for the potent antiparasitic activity.

Several researches reported that piscidin members of teleosts owned strong antiparasitic activity. Cryptocaryon irritans, a type of ectoparasite, could infect most of the marine teleosts. Larimichthys crocea could severely suffer from marine white spot disease caused by C. irritans, and their mortality rate was significantly high. Concentrating on this problem, we have done many related works. Piscidin 5 like (termed Lc-P5L) was another piscidin member isolated from a comparative transcriptome of C. irritans-immuned L. crocea. In the paper, quantitative Real-time PCR (qRT-PCR) showed Lc-P5L was upregulated in examined tissues, including gill, head kidney, muscle, liver, spleen and intestine after challenged by C. irritans, the significant upregulation time was in accordance to key developmental stages of C. irritans, which implied different infection stages could result in host immune response. Furthermore, using microscope techniques, we observed theronts or trophonts became weakly motile, cilia became detached, cells were out of shape, membranes eventually lysed in different cell positions and cytoplasmic contents leaked. Laser confocal scanning microscope (LCSM) observed theronts macronucleus grew swell and depolymerized after treated by recombinant Lc-P5L (rLc-P5L). Data suggested rLc-P5L was significantly lethal to C. irritans, and the death state of the parasite incubated with rLc-P5L was remarkably similar to other piscidin members or other antiparasitic peptides (APPs). Thus, these data provided new insights into L. crocea immunity against C. irritans and potential of rLc-P5L as a therapeutic agent against pathogen invasion.

Identification of a group D anti-lipopolysaccharide factor (ALF) from kuruma prawn (Marsupenaeus japonicus) with antibacterial activity against Vibrio parahaemolyticus.

Anti-lipopolysaccharide factor (ALF), which belongs to the antimicrobial peptide (AMP) family, has become a relatively new weapon to combat severe infections and has been demonstrated to be active against bacteria, fungi and some viruses. In the present study, a new ALF of group D (MjALF-D; GenBank accession No. MN416688) from Marsupenaeus japonicus was detected. MjALF-D encodes a polypeptide with 124 aa, and the peptide contains a 26-residue signal peptide and a lipopolysaccharide-binding domain (LBD). The structure of MjALF-D was found to consist of three α-helices, four ß-sheets and random coils. qRT-PCR analysis revealed that MjALF-D expression was primarily observed in the stomach and was universally upregulated in both the gill and stomach after challenge by lipopolysaccharide (LPS) and Vibrio parahaemolyticus. Moreover, rMjALF-D can inhibit the growth of V. parahaemolyticus. rMjALF-D could destroy the bacterial membrane and lead to cytoplasmic leakage investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), which may be the mechanism by which rMjALF-D inhibits V. parahaemolyticus. Additionally, rMjALF-D showed distinct binding or antibacterial ability after direct incubation with V. parahaemolyticus or bacterial genomic DNA and a certain effect on the protein expression of it. Together, these results indicated that rMjALF-D possessed the antibacterial activity against V. parahaemolyticus and the potential involvement in the innate immune response of M. japonicus.

An FMRFamide Neuropeptide in Cuttlefish Sepia pharaonis: Identification, Characterization, and Potential Function.

Neuropeptides are released by neurons that are involved in a wide range of brain functions, such as food intake, metabolism, reproduction, and learning and memory. A full-length cDNA sequence of an FMRFamide gene isolated from the cuttlefish Sepia pharaonis (designated as SpFMRFamide) was cloned. The predicted precursor protein contains one putative signal peptide and four FMRFamide-related peptides. Multiple amino acid and nucleotide sequence alignments showed that it shares 97% similarity with the precursor FMRFamides of Sepiella japonica and Sepia officinalis and shares 93% and 92% similarity with the SpFMRFamide gene of the two cuttlefish species, respectively. Moreover, the phylogenetic analysis also suggested that SpFMRFamide and FMRFamides from S. japonica and S. officinalis belong to the same sub-branch. Tissue expression analysis confirmed that SpFMRFamide was widely distributed among tissues and predominantly expressed in the brain at the three development stages. The combined effects of SpFMRFamide+SpGnRH and SpFLRFamide+SpGnRH showed a marked decrease in the level of the total proteins released in the CHO-K1 cells. This is the first report of SpFMRFamide in S. pharaonis and the results may contribute to future studies of neuropeptide evolution or may prove useful for the development of aquaculture methods for this cuttlefish species.

White spot syndrome virus entry is dependent on multiple endocytic routes and strongly facilitated by Cq-GABARAP in a CME-dependent manner.

White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, including crayfish. However, the molecular mechanism underlying its cellular entry remains elusive due to the lack of shrimp cell lines for viral propagation. Crayfish hematopoietic tissue (Hpt) cell culture was recently established as a good model for WSSV infection study. Here, we showed that multiple endocytic routes, including clathrin-mediated endocytosis (CME), macropinocytosis and caveolae-mediated endocytosis, were indispensably employed for the viral entry into Hpt cell of the crayfish Cherax quadricarinatus. Intriguingly, cellular autophagic activity was positively correlated with efficient viral entry, in which a key autophagy-related protein, γ-aminobutyric acid receptor-associated protein (Cq-GABARAP), that not only localized but also co-localized with WSSV on the Hpt cell membrane, strongly facilitated WSSV entry by binding to the viral envelope VP28 in a CME-dependent manner that was negatively regulated by Cq-Rac1. Furthermore, cytoskeletal components, including Cq-ß-tubulin and Cq-ß-actin, bound to both recombinant rCq-GABARAP and WSSV envelope proteins, which likely led to viral entry promotion via cooperation with rCq-GABARAP. Even under conditions that promoted viral entry, rCq-GABARAP significantly reduced viral replication at an early stage of infection, which was probably caused by the formation of WSSV aggregates in the cytoplasm.

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.