De novo transcriptome assembly and analysis of differential gene expression following lipopolysaccharide challenge in Pelteobagrus fulvidraco.

The yellow catfish, Pelteobagrus fulvidraco, has been recognized as an important freshwater aquaculture species in Eastern and Southeast Asia. To gain a better understanding of the immune response in P. fulvidraco, we analyzed its transcriptome following stimulation with lipopolysaccharide (LPS). Phosphate buffer saline (PBS) was used as control. Following assembly and annotation, 72,152 unigenes with an average length of 1090 bp were identified. A total of 370 differentially expressed genes (DEGs) in the P. fulvidraco were observed at 12 h post LPS treatment, including 197 up-regulated genes and 173 down-regulated genes. Clusters of Orthologous Groups of proteins (KOG/COG) annotation demonstrated that a total of 18,819 unigenes classified into 26 categories. Gene ontology (GO) analysis revealed 20 biological process subcategories, 7 cellular component subcategories and 20 molecular function subcategories. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified immune responses pathways. Quantitative reverse transcription polymerase chain reaction measured the expression of 18 genes involved in the immune response. CXCL2-like chemokine (CXCL2), goose-type lysozyme (LYZ G), and cathepsin K (CTSK) were significantly up-regulated. This study enriches the P. fulvidraco transcriptome database and provides insight into the immune response of P. fulvidraco against infection.

The complete mitochondrial genome of Clostera anachoreta (Lepidoptera: Notodontidae) and phylogenetic implications for Noctuoidea species.

In this study, the complete mitochondrial genome (mitogenome) of Clostera anachoreta (Lepidoptera: Notodontidae) was sequenced. It comprises 15,456 base pairs (bp), including 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and one non-coding control region (CR), as found in other lepidopterans. Gene order is identical to that of typical lepidopterans. There are 15 intergenic spacers ranging from 2 to 49bp, and 9 overlapping regions ranging from 1 to 8bp, occurring throughout the genome. The CR is 347bp long. All PCGs are initiated by ATN codons. We found a typical gene rearrangement in C. anachoreta (tRNAMet-tRNAIle-tRNAGln), which is different from ancestral insects (tRNAIle-tRNAGln-tRNAMet). The gene rearrangement can be explained by a duplication/random loss model. Phylogenetic analyses indicate that C. anachoreta belongs to Notodontidae, and that the monophyly of Lepidopteran families is well supported.

Characterisation of the complete mitochondrial genome of Helice wuana (Grapsoidea: Varunidae) and comparison with other Brachyuran crabs.

The mitochondrial genome (mitogenome) provides important information for phylogenetic analysis and understanding evolutionary origins. Herein, we sequenced, annotated, and characterised the mitogenome of the crab Helice wuana to better understand its molecular evolution and phylogeny. The 16,359bp mitogenome includes 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and one control region. The genome composition is highly A+T biased 68.42%, and exhibits a negative AT-skew (-0.036) and GC-skew (-0.269) among Brachyura crabs. Gene rearrangements were detected, as was tandem duplication followed by random loss, which explains the translocation of mitochondrial genes. Phylogenetic analysis showed that H. wuana and H. tientsinensis clustered on one branch with high nodal support values. These results confirm that the placement of H. wuana within the Varunidae family of Thoracotrematan crabs. This study will provided a better understanding for gene rearrangements and crab evolution in the further.

cDNA cloning and expression analysis of a hepcidin gene from yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae).

Hepcidin is a small, cysteine-rich antimicrobial peptide with a highly conserved ß-sheet structure that plays a vital role in innate host immunity against pathogenic organisms. In this study, a hepcidin gene was identified in Pelteobagrus fulvidraco, an economically important freshwater fish in China. The gene is named PfHep. The complete PfHep cDNA was 723 bp, including a 5'-untranslated region (UTR) of 102 bp, a 3'-UTR of 339 bp and an open reading frame of 282 bp encoding a polypeptide of 93 amino acids, which includes a predicted signal peptide and the Hepcidin domain. The predicted mature, cationic PfHep protein has a typical hepcidin RX (K/R)R motif and eight conserved cysteine residues. The deduced PfHep protein sequence has 70%, 54% and 39% percent identity with hepcidins from Ictalurus punctatus, Danio rerio, and Homo sapiens, respectively. The predicted tertiary structure of PfHep is very similar to that of hepcidin in other animals. Phylogenetic analysis revealed that PfHep is closely related to the hepcidins of I. punctatus and I. furcatus. Real-time quantitative reverse transcription-PCR showed that the PfHep gene was expressed most in liver of healthy P. fulvidraco, and expressed to some extent in all the tissues tested. After challenge with lipopolysaccharide and polyriboinosinic:polyribocytidylic acid (poly I:C), respectively, the expression levels of PfHep were markedly upregulated in liver, spleen, head kidney and blood at different time points. Together these results imply that PfHep may be an important component of the innate immune system and be involved in immune defense against invading pathogens.

Transcriptome analysis of yellow catfish (Pelteobagrus fulvidraco) liver challenged with polyriboinosinic polyribocytidylic acid (poly I:C).

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important economic freshwater species in China. However, infection by bacterial pathogenic diseases has caused high mortality and great economic loss in aquaculture. It is necessary for disease control to know more about the P. fulvidraco immune system and its related genes in response to bacterial or viral infections. In this study, the transcriptomic profiles of liver from P. fulvidraco stimulated by polyriboinosinic polyribocytidylic acid (poly I:C) was analyzed using high-throughput sequencing method. After assembly and annotation, total 67,447 unigenes were acquired, with an average length of 1091 bp. Under the infection of poly I:C, 522 differentially expressed genes (DEGs) were identified, including 307 up-regulated genes and 215 down-regulated genes. To further investigate the immune-related DEGs, Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed. The result of GO enrichment indicated gene response to external stimulus, regulation of response to stimulus, cellular response to stimulus, immune response and immune system progress. Significant KEGG enrichment analysis identified major immune related pathways. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that 13 immune response genes were identified to be up-regulated after 12 h of poly I:C stimulation compared to controls. Taken together, the results of our study are beneficial for better understanding of the immune system and defense mechanisms of yellow catfish in response to poly I:C infection.

A ferritin gene from Procambarus clarkii, molecular characterization and in response to heavy metal stress and lipopolysaccharide challenge.

Ferritin plays important roles in iron storage, detoxification, and immune response. Here, a ferritin gene (PcFer) was identified in Procambarus clarkii, an economically important freshwater crayfish. Full-length PcFer cDNA was 1022-bp, including a 135-bp 5'-untranslated region (UTR) with a typical iron responsive element, a 374-bp 3'-UTR, and a 513-bp open reading frame encoding a polypeptide of 170 amino acids which contained the Ferritin domain. PcFer has ion binding sites, a ferrihydrite nucleation center, and an iron ion channel. PcFer is phylogenetically closely-related to Pacifastacus leniusculus and Eriocheir sinensis ferritins. Real-time quantitative reverse-transcription PCR analysis showed that PcFer was expressed in all tested P. clarkii tissues, and expressed most in hepatopancreas. After challenge with various heavy metals and lipopolysaccharide, respectively, the hepatopancreatic expression levels of PcFer were markedly upregulated. These results suggest that expression of PcFer might be involved in immune defense and protection of P. clarkii against heavy metal stress.

Flat Chern band in a two-dimensional organometallic framework.

By combining exotic band dispersion with nontrivial band topology, an interesting type of band structure, namely, the flat Chern band, has recently been proposed to spawn high-temperature fractional quantum Hall states. Despite the proposal of several theoretical lattice models, however, it remains doubtful whether such a "romance of flatland" could exist in a real material. Here, we present a first-principles design of a two-dimensional indium-phenylene organometallic framework that realizes a nearly flat Chern band right around the Fermi level by combining lattice geometry, spin-orbit coupling, and ferromagnetism. An effective four-band model is constructed to reproduce the first-principles results. Our design, in addition, provides a general strategy to synthesize topologically nontrivial materials by virtue of organic chemistry and nanotechnology.

Comparative mitochondrial genome analysis of Spilarctia subcarnea and other noctuid insects.

This study was performed to better understand the phylogenetic relationships within the lepidopteran superfamily Noctuoidea. The mitochondrial genome (mitogenome) has been extensively used for studying phylogenetic relationships at different taxonomic levels. In this study, the complete mitogenome of Spilarctia subcarnea (Noctuoidea: Erebidae) was sequenced and annotated. The mitogenome is 15,441bp in length, containing 13 typical protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and a noncoding control region (CR). The order and orientation of genes of S. subcarnea mitogenome with the order trnM-trnI-trnQ-nad2 is different from the ancestral insects in which trnM is located between trnQ and nad2 (trnI-trnQ-trnM-nad2). The phylogenetic relationships based on mitochondrial sequences using Bayesian inference and Maximum likelihood methods showed that S. subcarnea was closely related to Lemyra melli, supporting that S. subcarnea belongs to Erebidae. These analyses confirm that Lymantriidae should be included as subfamilies within Erebidae. The Erebidae was sister to (Nolidae+(Euteliidae+Noctuidae)); Notodontidae is sister to the other families of Noctuoidea in our study.

Upregulation of RASAL2 promotes proliferation and metastasis, and is targeted by miR-203 in hepatocellular carcinoma.

RAS protein activator like 2 (RASAL2) has been reported to be dysregulated in various types of cancer. It has previously been demonstrated that RASAL2 is hypomethylated in hepatocellular carcinoma (HCC). However, the expression pattern of RASAL2 and its potential role in HCC remain to be elucidated. The present study demonstrated that the expression of RASAL2 was upregulated in HCC tissues, compared with in normal liver tissues, by using immunohistochemistry. In addition, Cell Counting Kit-8 assay and invasion assay revealed that knockdown of RASAL2 inhibited the growth and invasion of HCC cells. Western blotting results indicated that the inhibition of RASAL2 reduced the levels of phosphorylated-AKT. Notably, RASAL2 was observed to be a direct target of miR-203 in HCC in luciferase activity assays. Furthermore, overexpression of miR-203 exhibited a similar effect to RASAL2 knockdown in HCC cells. These results indicated that RASAL2 serves a tumor oncogenic role in HCC and may be considered a potential target in HCC.

The complete mitochondrial genome of Sesarmops sinensis reveals gene rearrangements and phylogenetic relationships in Brachyura.

Mitochondrial genome (mitogenome) is very important to understand molecular evolution and phylogenetics. Herein, in this study, the complete mitogenome of Sesarmops sinensis was reported. The mitogenome was 15,905 bp in size, and contained 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a control region (CR). The AT skew and the GC skew are both negative in the mitogenomes of S. sinensis. The nucleotide composition of the S. sinensis mitogenome was also biased toward A + T nucleotides (75.7%). All tRNA genes displayed a typical mitochondrial tRNA cloverleaf structure, except for the trnS1 gene, which lacked a dihydroxyuridine arm. S. sinensis exhibits a novel rearrangement compared with the Pancrustacean ground pattern and other Brachyura species. Based on the 13 PCGs, the phylogenetic analysis showed that S. sinensis and Sesarma neglectum were clustered on one branch with high nodal support values, indicating that S. sinensis and S. neglectum have a sister group relationship. The group (S. sinensis + S. neglectum) was sister to (Parasesarmops tripectinis + Metopaulias depressus), suggesting that S. sinensis belongs to Grapsoidea, Sesarmidae. Phylogenetic trees based on amino acid sequences and nucleotide sequences of mitochondrial 13 PCGs using BI and ML respectively indicate that section Eubrachyura consists of four groups clearly. The resulting phylogeny supports the establishment of a separate subsection Potamoida. These four groups correspond to four subsections of Raninoida, Heterotremata, Potamoida, and Thoracotremata.

Mitochondrial genome of Helice tientsinensis (Brachyura: Grapsoidea: Varunidae): Gene rearrangements and higher-level phylogeny of the Brachyura.

The mitochondrial genome (mt genome) provides important information for understanding molecular evolution and phylogeny. The further understand the molecular evolution and phylogeny of Helice tientsinensis, the complete mt genome was determined. It is 16,212bp long and includes 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes and a control region. The genome composition of H. tientsinensis was highly A+T biased 69.0% and showed negative AT skew (-0.017) and GC skew (-0.289). One PCG, all rRNAs and 12 of the tRNAs appeared to be rearranged with respect to the pancrustacean ground pattern gene order. Tandem duplication, followed by random deletion, is widely considered to explain translocation of mitochondrial genes. Consecutive recombinations events could explain inversions of genes. The phylogenetic analyses showed that H. tientsinensis has close relationships with Eriocheir japonica sinensis, E. j. hepuensis and E. j. japonica; this indicated that H. tientsinensis belongs in the Grapsoidea, part of the Varunidae family. This study provides evidence for a better understanding of gene rearrangements, as well as the evolutionary status of H. tientsinensis and related species.

Complete mitochondrial genome of Clistocoeloma sinensis (Brachyura: Grapsoidea): Gene rearrangements and higher-level phylogeny of the Brachyura.

Deciphering the animal mitochondrial genome (mitogenome) is very important to understand their molecular evolution and phylogenetic relationships. In this study, the complete mitogenome of Clistocoeloma sinensis was determined. The mitogenome of C. sinensis was 15,706 bp long, and its A+T content was 75.7%. The A+T skew of the mitogenome of C. sinensis was slightly negative (-0.020). All the transfer RNA genes had the typical cloverleaf structure, except for the trnS1 gene, which lacked a dihydroxyuridine arm. The two ribosomal RNA genes had 80.2% A+T content. The A+T-rich region spanned 684 bp. The gene order within the complete mitogenome of C. sinensis was identical to the pancrustacean ground pattern except for the translocation of trnH. Additionally, the gene order of trnI-trnQ-trnM in the pancrustacean ground pattern becomes trnQ-trnI-trnM in C. sinensis. Our phylogenetic analysis showed that C. sinensis and Sesarmops sinensis cluster together with high nodal support values, indicating that C. sinensis and S. sinensis have a sister group relationship. The results support that C. sinensis belongs to Grapsoidea, Sesarmidae. Our findings also indicate that Varunidae and Sesarmidae species share close relationships. Thus, mitogenomes are likely to be valuable tools for systematics in other groups of Crustacea.

Transcriptome-Wide Identification of Differentially Expressed Genes in Chinese Oak Silkworm Antheraea pernyi in Response to Lead Challenge.

Antheraea pernyi is a commercially cultivated silk moth and a source of insect food with high-quality protein. Insects suffer oxidative stress on exposure to heavy metals, and reactive oxygen species are cleared by antioxidant enzymes. To gain better understanding of the antioxidant defense system of A. pernyi, we analyzed transcriptomes of pupae after stimulation with lead and phosphate-buffered saline (control). In total, 72 367 unigenes were identified. Gene ontology analysis revealed that these DEGs were in 20 biological process subcategories, 19 cellular component subcategories, and 18 molecular function subcategories. Clusters of orthologous groups of protein annotation placed a total of 528 DEGs into 25 categories. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified antioxidant defense pathways, including "Peroxisome" and "Glutathione metabolism", which are reported for the first time in A. pernyi. Our study enriches A. pernyi transcriptome databases and provides insight into the heavy metal responses of antioxidant systems of this insect fat bodies.

All levels versus alternate levels plate fixation in expansive open door cervical laminoplasty.

BACKGROUND: Expansive open door laminoplasty with the use of titanium miniplate is becoming popular. Usually, the plate is applied at each level to prevent re-closure of the opened lamina. However, it is also used at alternating levels (i.e., C3, C5 and C7) in clinical settings in order to reduce the cost. Whether they have any difference in clinical efficacy? There is a lack of comparative data between the two kinds of plate fixation in the literature. MATERIALS AND METHODS: 83 patients who underwent cervical laminoplasty with alternating levels plate fixation (51 patients in Group A) or all levels plate fixation (32 patients in Group B) between January 2008 and October 2012 were evaluated in our institute retrospectively. Clinical and radiologic outcomes were assessed. RESULTS: No statistical difference was found in the mean operation time, blood loss, incidence of significant axial symptoms and C5 palsy, preoperative anteroposterior diameter (APD) and preoperative Japanese Orthopedic Association score between the two groups. However, Group B showed a higher rate of neurologic recovery after surgery. Postoperative increased APD and open angle in Group B were significantly larger than Group A. The mean cost for Group B (12801 ± 460.6 USD) was higher than Group A (8906 ± 566.7 USD). CONCLUSIONS: Despite the higher cost of all level fixation, it is more effective in maintaining the expansion of the spinal canal and can obtain better clinical improvement compared to alternating levels fixation.

Refolding and characterization of recombinant human GST-PD-1 fusion protein expressed in Escherichia coli.

Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed on activated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibits proliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involved in antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain was amplified and cloned into expression plasmid pGEX-5x-3. The fusion protein GST-PD-1 was effectively expressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was performed to obtain bioactive soluble GST-PD-1. Fusion protein of above 95% purity was acquired by a convenient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependent in vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-PD-1 could competently block the interaction between PD-L1 and PD-1 and increase the production of IL-2 and IFN-gamma of phytohemagglutinin-activated T cells.

[Effect of agonist anti-CD40 mAb 5C11 on the induction and biological characteristics of leukemic dendritic cells].

OBJECTIVE: To study the impact of an agonist anti-CD(40) monoclonal antibody 5C11 on the induction and biological characteristics of leukemic dendritic cells. METHODS: Combinations of 5C11 and different cytokines were used to induce differentiation of leukemic blasts into dendritic cells. Morphology was observed by light microscopy. Surface antigens of the induced cells were analyzed by fluorescence-activated cell sorting (FACS), the yields of dendritic cell by cell counting, the levels of IL-6 and IL-12 by ELISA, T cell proliferating activity by allo-mixed lymphocyte reaction (MLR) in vitro. Allogeneic T cells were stimulated with leukemic dendritic cells and T-cell cytotoxicity was measured by MTT assay. RESULTS: When cultured with combinations of 5C11 and different cytokines, the leukemic cells isolated from the patients could differentiate into dendritic cells. The morphology showed typical features of dendritic cells, which expressed high levels of CD(40), CD(80) and CD(86). In comparison with the original leukemia cells, the leukemic dendritic cells secreted less IL-6 but more IL-12 (P < 0.05). The leukemic dendritic cells were potent to stimulate the proliferation of allogeneic T cells, and the latter was able to lyse the original leukemia cells. CONCLUSION: Leukemic blasts could be induced to differentiate into functional dendritic cells. It may be of great value in the adoptive immunologic therapy of leukemia.