Two novel nitrogen-rich metal-organic nanotubes: syntheses, structures and selective adsorption toward rare earth ions.

The construction and development of metal-organic nanotubes (MONTs) with nanoscale interior channel diameters for potential applications is of great interest. An angular nitrogen-rich ligand, 3,6-bis(2-ethylimidazole)-2-methylpyrimidine (beim-CH3), was designed to construct MONTs by coupling with the V-shaped carboxylate ligands of benzophenone 4,4'-dicarboxylic acid (H2bpndc) and 4,4'-oxybisbenzoic acid (H2obba). Two new MONTs were synthesized and named NCD-166 ([Zn(bpndc)(beim-CH3)]·H2O) and NCD-167 ([Zn(obba)(beim-CH3)]·H2O), and they were isostructural and have almost identical tube inner diameters of approximately 1.76 nm. Benefiting from the abundantly exposed nitrogen and oxygen atoms in their tube walls and open nanoporous channels, they display superior adsorption capacities for Eu3+ (150.90 mg g-1) and high adsorption selectivity (>96%) in the low-concentration solutions. Additionally, it was revealed that the adsorption effect of ether oxygen on rare earth elements was significantly better than that of carbonyl oxygen. The adsorption isotherm conformed to the Langmuir model and the adsorption kinetics obeyed the pseudo-second-order model. These results clearly indicate that such novel MONTs are favorable sorbents for REEs.

Evidence for a non-fusogenic aquareovirus encoding a transmembrane protein.

Fusogenic aquareoviruses can induce host cell-cell fusion, forming syncytia via a fusion-associated transmembrane protein. However, there have been very few reports on non-fusogenic aquareoviruses encoding a membrane-associated protein. Previously, sequence-based analysis has indicated that grass carp reovirus strain 104 (GCRV-104), a non-fusogenic aquareovirus, encodes the proteins VP8 (nt 36-263) and VP15 (nt 400-822) in its genome segment S11. Here, we employed a liquid chromatography-tandem mass spectrometry assay to experimentally annotate small coding genes in the GCRV-104 genome and confirmed that segment S11 indeed functions as bicistronic mRNA. Notably, some additional polypeptides were identified that are encoded upstream of the VP15 open reading frame (ORF), which suggests that the virus uses a novel ORF with a non-AUG initiator codon, tentatively named VP15L (nt 274-822), which is longer than the previous putative VP15 ORF. Furthermore, a transmembrane domain was identified at the N-terminus of VP15L, but its function is unclear. Thus, the aquareovirus GCRV-104 potentially encodes a transmembrane protein, which opens a new perspective on the properties of viral proteins and the pathogenesis of this non-fusogenic reovirus.

Identification and characterization of a type I interferon induced by cyprinid herpesvirus 2 infection in crucian carp Carassius auratus gibelio.

Crucian carp (Carassius auratus gibelio) is a popular food fish in Asia, and cyprinid herpesvirus 2 (CyHV-2) is the only known viral pathogen for crucian carp. Type I interferon genes are induced up on host cell recognition of viral nucleic acids and well recognized for their crucial roles in providing local or systemic protection against the viruses in various organisms. In a transcriptome analysis to uncover differentially expressed genes in crucian carp in response to CyHV-2 challenge, a partial interferon transcript was identified to be significantly up-regulated in the kidney of infected fish, which was named as crucian carp IFNc (ccIFNc). The complete ORF of ccIFNc was further determined by RACE technique, which spanned over 546 bp and encoded a polypeptide containing 182 amino acids. Phylogenetic analysis revealed that ccIFNc clustered with known type I IFN genes from other aquatic organisms. Quantitative RT-PCR analysis demonstrated that ccIFNc was constitutively expressed in all investigated tissues with a comparably higher expression level in spleen, gill, kidney, and muscle. Following challenge with CyHV-2, the transcriptional levels of ccIFNc were dramatically up-regulated in all of the tested tissues, especially in the spleen and gill with increased folds of 436 and 158, respectively. The intramuscular (i.m.) injection of a eukaryotic expression plasmid encoding ccIFNc (pEGFP-cIFNc) resulted in increased ccIFNc expression and reduced the mortality after the CyHV-2 challenge significantly. In summary, our data suggested that the ccIFNc belongs to the type I interferon family with a potential role in countering CyHV-2 infection in crucian carp.

Integrated analysis of mRNA and viral miRNAs in the kidney of Carassius auratus gibelio response to cyprinid herpesvirus 2.

MicroRNAs (miRNAs) are small, non-coding single stranded RNAs that play crucial roles in numerous biological processes. Vertebrate herpesviruses encode multiple viral miRNAs that modulate host and viral genes. However, the roles of viral miRNAs in lower vertebrates have not been fully determined. Here, we used high-throughput sequencing to analyse the miRNA and mRNA expression profiles of Carassius auratus gibelio in response to infection by cyprinid herpesvirus 2 (CyHV-2). RNA sequencing obtained 26,664 assembled transcripts, including 2,912 differentially expressed genes. Based on small RNA sequencing and secondary structure predictions, we identified 17 CyHV-2 encoded miRNAs, among which 14 were validated by stem-loop quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and eight were validated by northern blotting. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of miRNAs-mRNA pairs revealed diverse affected immune signalling pathways, including the RIG-I-like receptor and JAK-STAT pathways. Finally, we presented four genes involved in RIG-I-like pathways, including host gene IRF3, RBMX, PIN1, viral gene ORF4, which are negatively regulated by CyHV-2 encoded miRNA miR-C4. The present study is the first to provide a comprehensive overview of viral miRNA-mRNA co-regulation, which might have a key role in controlling post-transcriptomic regulation during CyHV-2 infection.

The superoxide dismutase from red claw crayfish, Cherax quadricarinatus: molecular cloning and characterization analysis.

In the present study, an extracellular copper-zinc superoxide dismutase (ecCuZnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene were cloned from hemocytes of red claw crayfish, Cherax quadricarinatus. The open reading frame (ORF) of ecCuZnSOD is 498 bp and encodes a 166 amino acids (aa) protein, whereas the ORF of mtMnSOD is 654 bp and encodes a 218 aa protein. The amino acid sequences of C. quadricarinatus ecCuZnSOD and mtMnSOD showed high similarities with those of ecCuZnSODs and mtMnSODs of other crustaceans, respectively. Both ecCuZnSOD and mtMnSOD of C. quadricarinatus were highly expressed in hepatopancreas, hemocytes, intestine, and gill; low transcript levels were seen in other tissues (heart, muscle, and nerve). The immune responses of ecCuZnSOD and mtMnSOD were studied following inoculation with Spiroplasma eriocheiris and Aeromonas hydrophila. After S. eriocheiris or A. hydrophila challenge, mRNA transcription of ecCuZnSOD and mtMnSOD in hemocytes and gill was upregulated. mRNA transcription of ecCuZnSOD in the hepatopancreas was also upregulated after S. eriocheiris or A. hydrophila inoculation. mtMnSOD in hepatopancreas was upregulated after A. hydrophila inoculation, whereas this was down-regulated after S. eriocheiris challenge. After S. eriocheiris and A. hydrophila challenge, total SOD activity and CuZnSOD activity both increased compared to control group. The results showed that these SODs from C. quadricarinatus likely play an important role in protecting some tissues from reactive oxygen intermediates produced during challenge from S. eriocheiris and A. hydrophila.

iTRAQ-based proteomic study of the effects of Spiroplasma eriocheiris on Chinese mitten crab Eriocheir sinensis hemocytes.

Spiroplasma eriocheiris is as a novel pathogen of Chinese mitten crab Eriocheir sinensis tremor disease. The hemocytes have been shown to be major target cells in S. eriocheiris infection. The aim of this study was to examine the hemocytes' immune response at the protein levels. The differential proteomes of the crab hemocytes were analyzed immediately prior to injection with the pathogen, and at 10 d post-injection by isobaric tags for relative and absolute quantization (iTRAQ) labeling, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1075 proteins were identified by LC-MS/MS and de novo sequencing data. Using a 1.2-fold change in expression as a physiologically significant benchmark, 76 differentially expressed proteins (7.07%) were reliably quantified by iTRAQ analysis. Thirty-five (3.26%) proteins were up-regulated and 41 (3.81%) proteins were down-regulated resulting from a S. eriocheiris infection. Approximately 20 differential proteins in hemocytes were involved in the stress and immune responses. Up-regulated proteins included alpha-2-macroglobulin (α2M), prostaglandin D synthase (GST), ferritin, and heat shock protein 60. Down-regulated proteins included two lectins (mannose-binding protein and hemocytin), three kinds of serine proteinase inhibitors (two serpins and pacifastin), three different kinds of serine proteases, mitogen-activated protein kinase kinase (MAPKK), and two thioredoxins (Trx), crustin, etc. Selected bioactive factors (α2M, GST, ferritin, tubulin, crustin, thioredoxin, clip domain serine protease and serpin) are verified by their immune roles in the S. eriocheiris infection using Real-time PCR. The variation trend of immune gene's mRNA expression is similar with the result of iTRAQ, except the tubulin. The prophenoloxidase-activating system, antimicrobial action and antioxidant system involved in the immune responses of E. sinensis is believed to be a resistance to S. eriocheiris infection. This is the first report of the proteome response of crab hemocytes against S. eriocheiris infection. These findings contribute to our understanding of tremor disease processes in crabs, and provide the first evidence to promote a search for potential biomarkers of the disease.

Histopathological characterization and fluorescence in situ hybridization of Cyprinid herpesvirus 2 in cultured Prussian carp, Carassius auratus gibelio in China.

Cyprinid herpesvirus 2 (CyHV-2) is an emerging pathogen in the commercially exploited fish, Prussian carp (Carassius auratus gibelio), which has caused huge economic loss in China and appears to be spreading worldwide. In this article, CyHV-2 infection of Prussian carp was confirmed for the first time by polymerase chain reaction (PCR), which gave positive results from the tissue samples dissected from moribund fish including kidney, spleen, liver, and gill. Histological examination showed systemic inflammatory reactions in the infected tissues, with infiltration of hemocytes, hypertrophied nuclei, marginal chromatin and karyorrhexis, epithelial cell shedding, vacuolar degeneration and focal necrosis. Tissue alterations were also evaluated semi-quantitatively by the degree of tissue change. The values of degree of tissue change determined for kidney, spleen, liver, and gill were significantly greater than respective controls and kidney was the most severely damaged organ, with highest degree of tissue change value. In addition, a fluorescence in situ hybridization (FISH) based on oligonucleotide probes to detect the pathogen directly in the tissue, allowing pathogen-lesion correlation, was established. With the advantages of better tissue penetration, potentially more specific and stable, three oligonucleotide probes were designed. Positive reactions to the probes with intense green fluorescence were observed within the infected tissues where PCR and H&E analysis had suggested previously the presence of the virus within these lesions. The probes did not hybridize with host tissues of uninfected fish, nor did they cross-react with 3 other virus samples tested. The current research could facilitate the study of CyHV-2 infection mechanism in Prussian carp, and enhance the early diagnosis of the novel virus.