[Effect of curcumin on radiosensitization of CNE-2 cells and its mechanism].

OBJECTIVE: To investigate the effect of curcumin (Cur) on radiosensitivity of nasopharyngeal carcinoma cell CNE-2 and its mechanism. METHOD: The effect of curcumin on radiosensitivity was determined by the clone formation assay. The cell survival curve was fitted by Graph prism 6. 0. The changes in cell cycle were analyzed by flow cytometry (FCM). The differential expression of long non-coding RNA was detected by gene chip technology. Part of differentially expressed genes was verified by Real-time PCR. RESULT: After 10 micro mol L-1 Cur had worked for 24 h, its sensitization enhancement ratio was 1. 03, indicating that low concentration of curcumin could increase the radiosensitivity of nasopharyngeal carcinoma cells; FCM displayed a significant increase of G2 phase cells and significant decrease of S phase cells in the Cur combined radiation group. In the Cur group, the GUCY2GP, H2BFXP, LINC00623 IncRNA were significantly up-regulated and ZRANB2-AS2 LOC100506835, FLJ36000 IncRNA were significantly down-regulated. CONCLUSION: Cur has radiosensitizing effect on human nasopharyngeal carcinoma CNE-2 cells. Its mechanism may be related to the changes in the cell cycle distribution and the expression of long non-coding IncRNA.

Muscone restores anoikis sensitivity in TMZ-resistant glioblastoma cells by suppressing TOP2A via the EGFR/Integrin ß1/FAK signaling pathway.

BACKGROUND: Temozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of Shexiang (Moschus), can cross the blood-brain barrier (BBB) and is being investigated as an antineoplastic medication. However, muscone treatment for GBM has received little research, and its possible mechanisms are still unclear. PURPOSE: This study aims to evaluate the effect and the potential molecular mechanism of muscone on TMZ-resistant GBM cells. METHODS: The differentially expressed genes (DEGs) between TMZ-resistant GBM cells and TMZ-sensitive GBM cells were screened using GEO2R. By progressively raising the TMZ concentration, a relatively stable TMZ-resistant human GBM cell line was established. The drug-resistance traits of U251-TR cells were assessed via the CCK-8 assay and Western Blot analysis of MGMT and TOP2A expression. Cell viability, cell proliferation, cell migration ability, and drug synergism were detected by the CCK-8 assay, colony formation assay, wound healing assay, and drug interaction relationship test, respectively. Anoikis was quantified by Calcein-AM/EthD-1 staining, MTT assay, and flow cytometry. Measurements of cell cycle arrest, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed using cell cycle staining, Annexin V-FITC/PI labeling, JC-1 assay, and ROS assay, respectively. DNA damage was measured by TUNEL assay, alkaline comet assay, and γ-H2AX foci assay. GEPIA was used to investigate the link between the anoikis marker (FAK)/drug resistance gene and critical proteins in the EGFR/Integrin ß1 signaling pathway. Molecular docking was used to anticipate the probable targets of muscone. The intracellular co-localization and expression of EGFR and FAK were shown using immunofluorescence. The U251-TR cell line stably overexpressing EGFR was constructed using lentiviral transduction to assess the involvement of EGFR-related signaling in anoikis resistance. Western Blot was employed to detect the expression of migration-related proteins, cyclins, anoikis-related proteins, DNA damage/repair-related proteins, and associated pathway proteins. RESULTS: DEGs analysis identified 97 deregulated chemotherapy-resistant genes and 3779 upregulated genes in TMZ-resistant GBM cells. Subsequent experiments verified TMZ resistance and the hyper-expression of DNA repair-related genes (TOP2A and MGMT) in continuously low-dose TMZ-induced U251-TR cells. Muscone exhibited dose-dependent inhibition of U251-TR cell migration and proliferation, and its co-administration with TMZ showed the potential for enhanced therapeutic efficacy. By downregulating FAK, muscone reduced anoikis resistance in anchorage-independent U251-TR cells. It also caused cell cycle arrest in the G2/M phase by upregulating p21 and downregulating CDK1, CDK2, and Cyclin E1. Muscone-induced anoikis was accompanied by mitochondrial membrane potential collapse, ROS production, an increase in the BAX/Bcl-2 ratio, as well as elevated levels of Cytochrome c (Cyt c), cleaved caspase-9, and cleaved caspase-3. These findings indicated that muscone might trigger mitochondrial-dependent anoikis via ROS generation. Moreover, significant DNA damage, DNA double-strand breaks (DSBs), the formation of γ-H2AX foci, and a reduction in TOP2A expression are also associated with muscone-induced anoikis. Overexpression of EGFR in U251-TR cells boosted the expression of Integrin ß1, FAK, ß-Catenin, and TOP2A, whereas muscone suppressed the expression levels of EGFR, Integrin ß1, ß-Catenin, FAK, and TOP2A. Muscone may influence the expression of the key DNA repair enzyme, TOP2A, by suppressing the EGFR/Integrin ß1/FAK pathway. CONCLUSION: We first demonstrated that muscone suppressed TOP2A expression through the EGFR/Integrin ß1/FAK pathway, hence restoring anoikis sensitivity in TMZ-resistant GBM cells. These data suggest that muscone may be a promising co-therapeutic agent for enhancing GBM treatment, particularly in cases of TMZ-resistant GBM with elevated TOP2A expression.

lncRNA expression signatures in response to enterovirus 71 infection.

Outbreaks of hand, foot, and mouth disease caused by enterovirus 71 (EV71) have become considerable threats to the health of infants and young children. To identify the cellular long noncoding RNAs (lncRNAs) involved in the host response to EV71 infection, we performed comprehensive lncRNA and mRNA profiling in EV71-infected rhabdomyosarcoma cells through microarray. We observed the differential expression of more than 4800 lncRNAs during infection. Further analysis showed 160 regulated enhancer-like lncRNA and nearby mRNA pairs, as well as 313 regulated Rinn's lncRNA [M. Guttman I. Amit, M. Garber, C. French, M.F. Lin, D. Feldser, M. Huarte, O. Zuk, B.W. Carey, J.P. Cassady, M.N. Cabili, R. Jaenisch, T.S. Mikkelsen, T. Jacks, N. Hacohen, B.E. Bernstein, M. Kellis, A. Regev, J.L. Rinn, E.S. Lander. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458 (2009) 223-227, A.M. Khalil, M. Guttman, M. Huarte, M. Garber, A. Raj, D. Rivea Morales, K. Thomas, A. Presser, B.E. Bernstein, A. van Oudenaarden, A. Regev, E.S. Lander, J.L. Rinn. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc. Natl. Acad. Sci. USA 106 (2009) 11667-11672] and nearby mRNA pairs. The results provided information for further research on the prevention and treatment of EV71 infection, as well as on distinguishing severe and mild EV71 cases.

Quercetin induces MGMT+ glioblastoma cells apoptosis via dual inhibition of Wnt3a/ß-Catenin and Akt/NF-κB signaling pathways.

BACKGROUND: Surgical resection combined with radiotherapy and chemotherapy remains a common clinical treatment for glioblastoma multiforme (GBM). However, the therapeutic outcomes have not been satisfying due to drug resistance and other factors. Quercetin, a phytoingredient capable of crossing the blood-brain barrier, has shown effectiveness in the treatment of various solid tumors. Nevertheless, the potential of quercetin in GBM treatment has not been adequately explored. PURPOSE: This study aims to investigate the effects and mechanisms of quercetin on MGMT+GBM cells. METHODS: The potential targets and mechanisms of quercetin in glioma treatment were predicted based on network pharmacology and molecular docking. The effects of quercetin on cell inhibition rate, cell migration ability, cell cycle arrest, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), Mitochondrial superoxide formation and apoptosis were measured by the CCK8 assay, wound healing assay, PI/RNase staining, JC-1 assay, DCFH-DA assay, MitoSOX staining and Annexin V-FITC/PI double staining, respectively. The methylation status of the MGMT promoter was assessed through methylation-specific polymerase chain reaction (MS-PCR). DNA damage was quantified by alkaline/neutral comet assay and TUNEL assay. The intracellular localization and expression of NF-κB and MGMT were revealed by immunofluorescence. The expression of migration-related proteins, matrix metalloproteinases, apoptosis-related proteins, cyclins, DNA damage/repair enzymes and related pathway proteins was detected by Western blot. RESULTS: Network pharmacology identified 96 targets and potential molecular mechanisms of quercetin in glioma treatment. Subsequent experiments confirmed the synergistic effect of quercetin in combination with temozolomide (TMZ) on T98G cells. Quercetin significantly suppressed the growth and migration of human GBM T98G cells, induced apoptosis, and arrested cells in the S-phase cell cycle. The collapse of mitochondrial membrane potential, ROS generation, enhanced Bax/Bcl-2 ratio, and strengthened cleaved-Caspase 9 and cleaved-Caspase 3 suggested the involvement of ROS-mediated mitochondria-dependent apoptosis in the process of quercetin-induced apoptosis. In addition, quercetin-induced apoptosis was accompanied by intense DNA double-strand breaks (DSBs), γH2AX foci formation, methylation of MGMT promoter, increased cleaved-PARP, and reduced MGMT expression. Quercetin may influence the expression of the key DNA repair enzyme, MGMT, by dual suppression of the Wnt3a/ß-Catenin and the Akt/NF-κB signaling pathways, thereby promoting apoptosis. Inhibition of Wnt3a and Akt using specific inhibitors hindered MGMT expression. CONCLUSION: Our study provides the first evidence that quercetin may induce apoptosis in MGMT+GBM cells via dual inhibition of the Wnt3a/ß-Catenin pathway and the Akt/NF-κB signaling pathway. These findings suggest that quercetin could be a novel agent for improving GBM treatment, especially in TMZ-resistant GBM with high MGMT expression.

Xihuang Pill-destabilized CD133/EGFR/Akt/mTOR cascade reduces stemness enrichment of glioblastoma via the down-regulation of SOX2.

BACKGROUND: Our previous study found that XHP could induce GBM cells to undergo apoptosis. A lot of evidence suggests that glioma stem-like cells (GSCs) are key factors that contribute to disease progression and poor prognosis of glioblastoma multiforme (GBM). Traditional Chinese medicine has been applied in clinical practice as a complementary and alternative therapy for glioma. PURPOSE: To evaluate the effect and the potential molecular mechanism of Xihuang pill (XHP) on GSCs. METHODS: UPLC-QTOF-MS analysis was used for constituent analysis of XHP. Using network pharmacology and bioinformatics methods, a molecular network targeting GSCs by the active ingredients in XHP was constructed. Cell viability, self-renewal ability, apoptosis, and GSC markers were detected by CCK-8 assay, tumor sphere formation assay and flow cytometry, respectively. The interrelationship between GSC markers (CD133 and SOX2) and key proteins of the EGFR/Akt/mTOR signaling pathway was evaluated using GEPIA and verified by western blot. A GBM cell line stably overexpressing Akt was constructed using lentivirus to evaluate the role of Akt signaling in the regulation of glioma stemness. The effect of XHP on glioma growth was analyzed by a subcutaneously transplanted glioma cell model in nude mice, hematoxylin-eosin staining was used to examine pathological changes, TUNEL staining was used to detect apoptosis in tumor tissues, and the expression of GSC markers in tumor tissues was identified by western blot and immunofluorescence. RESULTS: Bioinformatics analysis showed that 55 matched targets were related to XHP targets and glioma stem cell targets. In addition to causing apoptosis, XHP could diminish the number of GBM 3D spheroids, the proportion of CD133-positive cells and the expression level of GSC markers (CD133 and SOX2) in vitro. Furthermore, XHP could attenuate the expression of CD133, EGFR, p-Akt, p-mTOR and SOX2 in GBM spheres. Overexpression of Akt significantly increased the expression level of SOX2, which was prohibited in the presence of XHP. XHP reduced GSC markers including CD133 and SOX2, and impeded the development of glioma growth in xenograft mouse models in vivo. CONCLUSION: We demonstrate for the first time that XHP down-regulates stemness, restrains self-renewal and induces apoptosis in GSCs and impedes glioma growth by down-regulating SOX2 through destabilizing the CD133/EGFR/Akt/mTOR cascade.

Discovery of novel IDH1-R132C inhibitors through structure-based virtual screening.

Isocitrate dehydrogenase (IDH) belongs to a family of enzymes involved in glycometabolism. It is found in many living organisms and is one of the most mutated metabolic enzymes. In the current study, we identified novel IDH1-R132C inhibitors using docking-based virtual screening and cellular inhibition assays. A total of 100 molecules with high docking scores were obtained from docking-based virtual screening. The cellular inhibition assay demonstrated five compounds at a concentration of 10 µM could inhibit cancer cells harboring the IDH1-R132C mutation proliferation by > 50%. The compound (T001-0657) showed the most potent effect against cancer cells harboring the IDH1-R132C mutation with a half-maximal inhibitory concentration (IC50) value of 1.311 µM. It also showed a cytotoxic effect against cancer cells with wild-type IDH1 and normal cells with IC50 values of 49.041 µM and >50 µM, respectively. Molecular dynamics simulations were performed to investigate the stability of the kinase structure binding of allosteric inhibitor compound A and the identified compound T001-0657 binds to IDH1-R132C. Root-mean-square deviation, root-mean-square fluctuation, and binding free energy calculations showed that both compounds bind tightly to IDH1-R132C. In conclusion, the compound identified in this study had high selectivity for cancer cells harboring IDH1-R132C mutation and could be considered a promising hit compound for further development of IDH1-R132C inhibitors.

Network Pharmacology and Molecular Docking Study of the Chinese Miao Medicine Sidaxue in the Treatment of Rheumatoid Arthritis.

PURPOSE: This study aimed to investigate the molecular mechanisms of Compound Sidaxue (SX), a prescription of Chinese Miao medicine, in treating rheumatoid arthritis (RA) using network pharmacology and in vivo experimental approaches. METHODS: Network pharmacology was adopted to detect the active components of four Traditional Chinese herbal medicine (TCM) of SX, and the key targets and signaling pathways in the treatment of RA were predicted, and the key components and targets were screened for molecular docking. The predicted targets and pathways were validated in bovine type II collagen and incomplete Freund's adjuvant emulsifier-induced rat RA model. RESULTS: In this study, we identified 33 active components from SX, predicted to act on 44 RA-associated targets by network pharmacology. PPI network demonstrated that TNF-α, VEGF-A, IL-2, IL-6, AKT, PI3K, STAT1 may serve as the key targets of SX for the treatment of RA. The main functional pathways involving these key targets include PI3K-AKT signaling pathway, TNF signaling pathway, NF-κB signaling pathway. Molecular docking analysis found that the active components ß-amyrin, cajanin, eleutheroside A have high affinity for TNF-α, VEGFA, IL-2, AKT, and PI3K, etc. SX can improve joint swelling in Collagen-induced arthritis (CIA) rats, reduce inflammatory cell infiltration and angiogenesis in joint synovial tissue, and down-regulate IL-2, IL-6, TNF-α, VEGF, PI3K, AKT, p-AKT, NF-κBp65, the expression of p-NF-κBp65, STAT1, and PTGS2 are used to control the exacerbation of inflammation and alleviate the proliferation of synovial pannus, and at the same time play the role of cartilage protection to achieve the effect of treating RA. CONCLUSION: Through a network pharmacology approach and animal study, we predicted and validated the active compounds of SX and their potential targets for RA treatment. The results suggest that SX can markedly alleviate CIA rat by modulating the VEGF/PI3K/AKT signaling pathway, TNF-α signaling pathway, IL/NF-κB signaling pathway.

Tubeimoside-I sensitizes temozolomide-resistant glioblastoma cells to chemotherapy by reducing MGMT expression and suppressing EGFR induced PI3K/Akt/mTOR/NF-κB-mediated signaling pathway.

BACKGROUND: Glioblastoma multiforme (GBM, World Health Organization [WHO] grade IV) is one of the malignant Central Nerve System (CNS) tumors with high incidence rate and poor prognosis. The use of alkylating agents, such as temozolomide (TMZ), has been the main method of cytotoxic therapy for glioma patients for decades. However, TMZ resistance may be one of the major reasons for treatment failure, so far. In searching for effective agents to reverse TMZ resistance, we found that Tubeimoside-I (TBMS1), a saponin from traditional Chinese medicine, Bolbostemma paniculatum (Maxim.) Franquet, showed activities of reversing TMZ resistance of GBM. However, the ability of TBMS1 enhancing the chemosensitivity of GBM has been rarely studied, and its underlying mechanisms remain unclear. PURPOSE: This study purposes to reveal the synergistic effects and mechanism of TBMS1 and TMZ against TMZ-resistant GBM cells. METHODS: CCK8 assay was used to investigate the anti-proliferative effects on grade IV glioblastoma human T98G and U118 MG cells. Cell proliferation was determined by EdU assay and clonogenic assay after TMZ plus TBMS1 treatment. Apoptosis was analyzed by flow cytometry. DNA damage and DNA Double Strand Break (DSB) were assessed by cleaved Poly (ADP-ribose) polymerase (PARP), γH2AX Foci Assay and Comet Assay, respectively. Expression of proteins associated with apoptosis and DNA repair enzymes were measured by Western blot analysis. The prognostic significance of key proteins of the epidermal growth factor receptor (EGFR) induced PI3K/Akt/mTOR/NF-κB signaling pathway was analyzed using GEPIA (http://gepia.cancer-pku.cn) and validated by Western blotting. RESULTS: Here we demonstrated that TBMS1 sensitized TMZ-resistant T98G and U118 MG glioblastoma cells to chemotherapy and exhibited promotion of apoptosis and inhibition on cell viability, proliferation and clone formation. Coefficient of drug in interaction (CDI) values showed a notable synergistic effect between TBMS1 and TMZ. Moreover, we observed that combination of TBMS1 and TMZ induced apoptosis was accompanied by robust DSB, γH2AX Foci formation and increasing cleaved PARP, as well as the heightened ratio of Bax/Bcl-2, cleavages of caspase-3 and caspase-9. In addition, the synergistic anti-glioma effect between TBMS1 and TMZ was intimately related to the reduction of MGMT expression in TMZ-resistant GBM cells. Moreover, it was also associated with attenuated expression of EGFR, p-PI3K-p85, p-Akt (Ser473), p-mTOR (Ser2481) and p-NF-κB p65(Ser536), which implying deactivation of the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway. CONCLUSION: We first demonstrated that synergistic effects of TBMS1 and TMZ induced apoptosis in GBM cells through reducing MGMT expression and inhibiting the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway. This study provides a rationale for combined application of TMZ and TBMS1 as a potential chemotherapeutic treatment for MGMT+ GBM patients.

Cloning and characterization of a shrimp ML superfamily protein.

ML superfamily proteins, including MD-1, MD-2, Niemann-Pick type C2 (Npc2) protein, GM2 activator protein, phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP) and mite allergen Der p 2, bind to specific lipids and play important roles in lipid-recognition and metabolism. Among these ML (MD-2-related lipid-recognition) proteins, MD-2 is essential for lipopolysaccharide (LPS) signaling and the following secretion of proinflammatory factors. In this report, we identified the cDNA and gene of an ML protein from an important white shrimp Litopenaeus vannamei and named it LvML. The gene consists of four exons and three introns. The putative LvML contains 6 cysteines which may form 3 disulfide bonds that are conserved in ML proteins. Reverse transcription PCR analysis showed that in the examined tissues LvML mRNA is only expressed in the hepatopancreas, while not in hemocytes, eyestalk, gill, heart, stomach, intestine, nerve core, muscle or pyloric caecum. Its expression is positively regulated after injection of LPS. Then enzyme-linked immunosorbent assay showed that the recombinant LvML possessed activity of binding to LPS, and that the binding was inhibited by pre-incubation with LPS. We suggested that the LvML may play roles in the shrimp innate immunity.

Differences in Brain Functional Networks of Executive Function Between Cantonese-Mandarin Bilinguals and Mandarin Monolinguals.

It remains controversial whether long-term logographic-logographic bilingual experience shapes the special brain functional subnetworks underlying different components of executive function (EF). To address this question, this study explored the differences in the functional connections underlying EF between the Cantonese-Mandarin bilinguals and Mandarin monolinguals. 31 Cantonese-Mandarin bilinguals and 31 Mandarin monolinguals were scanned in a 3-T magnetic resonance scanner at rest. 4 kinds of behavioral tasks of EF were tested. Network-based statistics (NBS) was performed to compare the connectomes of fronto-parietal (FP) and cingulo-opercular (CO) network between groups. The results showed that the bilinguals had stronger connectivity than monolinguals in a subnetwork located in the CO network rather than the FP network. The identified differential subnetwork referred to as the CO subnetwork contained 9 nodes and 10 edges, in which the center node was the left mid-insula with a degree centrality of 5. The functional connectivity of the CO subnetwork was significantly negatively correlated with interference effect in bilinguals. The results suggested that long-term Cantonese-Mandarin bilingual experience was associated with stronger functional connectivity underlying inhibitory control in the CO subnetwork.

The Correlation between Chinese Written Vocabulary Size and Cognitive, Emotional and Behavioral Factors in Primary School Students.

Written vocabulary size plays a key role in children's reading development. We aim to study the relationship between Chinese written vocabulary size and cognitive, emotional, and behavioral factors in primary school students. Using stratified cluster sampling, 1162 pupils from Grade 2~5 in Guangzhou were investigated. Chinese written vocabulary size, cognitive, emotional, and behavioral factors were assessed by the Chinese written vocabulary size assessment scale, the dyslexia checklist for Chinese children (DCCC) and the Strengths and Difficulties Questionnaire (SDQ), respectively. The scores of visual word recognition deficit (ß = -3.32, 95% CI: -5.98, -0.66) and meaning comprehension deficit (ß = -6.52, 95% CI: -9.39, -3.64) were negatively associated with Chinese written vocabulary size; the score of visual word recognition deficit (odds ratio (OR) = 1.04, 95% CI: 1.02, 1.07) was the related factor of a delay in written vocabulary size. The score of meaning comprehension deficit was negatively associated with boys' Chinese written vocabulary size, while the score of auditory word recognition deficit was negatively associated with girls' Chinese written vocabulary size. The related factor of a delay in written vocabulary size was spelling deficit in boys and visual word recognition deficit in girls. There is a significant correlation between Chinese written vocabulary size and cognitive factors, but not emotional and behavioral factors in primary school students and these correlations are different when considering gender.

Tumor Associated Macrophages, as the Dominant Immune Cells, Are an Indispensable Target for Immunologically Cold Tumor-Glioma Therapy?

Tumor microenvironment (TME) is the cornerstone of the occurrence, development, invasion and diffusion of the malignant central nerve system (CNS) tumor, glioma. As the largest number of inflammatory cells in glioma TME, tumor associated macrophages (TAMs) and their secreted factors are indispensable to the progression of glioma, which is a well-known immunologically "cold" tumor, including the growth of tumor cells, invasion, migration, angiogenesis, cancer immunosuppression and metabolism. TAMs intimately interface with the treatment failure and poor prognosis of glioma patients, and their density increases with increasing glioma grade. Recently, great progress has been made in TAM-targeting for anti-tumor therapy. According to TAMs' function in tumorigenesis and progression, the major anti-tumor treatment strategies targeting TAMs are to hinder macrophage recruitment in TME, reduce TAMs viability or remodel TAMs phenotype from M2 to M1. Different approaches offer unique and effective anti-tumor effect by regulating the phagocytosis, polarization and pro-tumor behaviors of macrophages in the therapy of glioma. The present review summarizes the significant characteristics and related mechanisms of TAMs and addresses the related research progress on targeting TAMs in glioma.

A novel C-type lectin from the shrimp Litopenaeus vannamei possesses anti-white spot syndrome virus activity.

C-type lectins play key roles in pathogen recognition, innate immunity, and cell-cell interactions. Here, we report a new C-type lectin (C-type lectin 1) from the shrimp Litopenaeus vannamei (LvCTL1), which has activity against the white spot syndrome virus (WSSV). LvCTL1 is a 156-residue polypeptide containing a C-type carbohydrate recognition domain with an EPN (Glu(99)-Pro(100)-Asn(101)) motif that has a predicted ligand binding specificity for mannose. Reverse transcription-PCR analysis revealed that LvCTL1 mRNA was specifically expressed in the hepatopancreas of L. vannamei. Recombinant LvCTL1 (rLvCTL1) had hemagglutinating activity and ligand binding specificity for mannose and glucose. rLvCTL1 also had a strong affinity for WSSV and interacted with several envelope proteins of WSSV. Furthermore, we showed that the binding of rLvCTL1 to WSSV could protect shrimps from viral infection and prolong the survival of shrimps against WSSV infection. Our results suggest that LvCTL1 is a mannose-binding C-type lectin that binds to envelope proteins of WSSV to exert its antiviral activity. To our knowledge, this is the first report of a shrimp C-type lectin that has direct anti-WSSV activity.

Cloning of IRAK1 and its upregulation in symptomatic mandarin fish infected with ISKNV.

Interleukin-1 receptor activated kinases (IRAKs) play crucial roles in the Toll-like receptor (TLR) mediated signal transduction pathways that control host innate immune responses. Here we report the cloning of an IRAK1 cDNA (named ScIRAK1) from the mandarin fish. The predicted ScIRAK1 peptide contains a death domain and a serine/threonine-specific kinase domain. Quantitative RT-PCR showed that ScIRAK1 mRNA was primarily expressed in blood cells and posterior kidney. Seven days following infection with infectious spleen and kidney necrosis virus (ISKNV), the ScIRAK1 mRNA level was significantly higher in the blood cells of clinically symptomatic fish than in the blood cells of asymptomatic fish or control fish injected with phosphate-buffered saline. Additional experiments showed that overexpression of ScIRAK1 in the 293T cells could induce NF-kappaB activation. These results suggest that ScIRAK1 may play a role in the pathology of ISKNV infection in the mandarin fish.

A novel prophenoloxidase 2 exists in shrimp hemocytes.

The prophenoloxidase (proPO)-activating system in crustaceans and other arthropods is regarded as a constituent of the immune system and plays an important role in defense against pathogens. Hitherto in crustaceans, only one proPO gene per species has been identified. Here we report the identification of a novel proPO-2 (LvproPO-2) from the hemocytes of Litopenaeus vannamei, which shows 72% identity to proPO-1 (LvproPO-1) cloned previously. Northern blotting analysis and quantitative real-time PCR reveal that LvproPO-2 is mainly expressed in the hemocytes, and its expression is down-regulated in shrimp challenged with white spot syndrome virus (WSSV). Western blotting analysis shows that most LvproPO-2/LvPO-2 (L. vannamei phenoloxidase-2) exists in the hemocytes, but not in plasma of L. vannamei. LvproPO-2/LvPO-2 could be detected on the hemocyte surface and the nucleus of hemocytes by indirect immunofluorescence assay (IFA). These findings provide insight into the molecular biological basis for further studying on the defense mechanism of shrimp innate immunity, especially on the proPO-activating system and melanization cascade of shrimp.

The alpha inhibitor of NF-kappaB (IkappaBalpha) from the mandarin fish binds with p65 NF-kappaB.

The NF-kappaB/IkappaBalpha pathway plays an important role in the regulation of immune and inflammatory responses. IkappaBalpha is an inhibitory molecule that sequesters transcription activator NF-kappaB dimer in the cytoplasm of unstimulated cells. Here, we isolated the full-length cDNAs of the mandarin fish (Siniperca chuatsi) alpha inhibitor of NF-kappaB (ScIkappaBalpha) and p65 NF-kappaB (Scp65). Multiple sequence alignments showed that the amino acid sequences of both ScIkappaBalpha and Scp65 contain conserved domains similar to those of mammalian counterparts. Protein pull-down and coimmunoprecipitation assays showed that ScIkappaBalpha directly bound with Scp65. Real-time quantitative PCR analysis showed that ScIkappaBalpha mRNA was constitutive in all mandarin fish tissues detected. After challenge with infectious spleen and kidney necrosis virus (ISKNV), the mRNA level of ScIkappaBalpha was decreased nearly 6 fold in the spleen. This result suggests that the NF-kappaB/IkappaBalpha pathway in mandarin fish may play a role in the immune response against ISKNV.

Identification and functional study of a shrimp Relish homologue.

Rel/NF-kappaB transcription factors play central roles in induction and regulation of innate immune responses. Here we describe the identification and functional analysis of a Relish homologue, LvRelish and its shorter isoform sLvRelish, from the Pacific white shrimp, Litopenaeus vannamei. The LvRelish gene has 22 exons in approximately 15 kb genomic sequence. The full-length cDNA of LvRelish is 4071 bp with an open reading frame that encodes 1207 amino acids. LvRelish contains a conserved Rel homology domain (RHD), a nucleus localization signal, an IkappaB-like domain (six ankyrin repeats), and a death domain, suggesting that it belongs to the class I NF-kappaB. sLvRelish cDNA is 1051 bp encoding 317 amino acids. It shares the RHD region with LvRelish. RT-PCR analysis showed that LvRelish and sLvRelish mRNAs were expressed at different levels in tissues. Western blot analysis showed that recombinant intact LvRelish could be cleaved into two fragments in S2 cells, and immunofluorescence assay showed that the plasmid-expressed LvRelish protein was seen both in the cytoplasm and the nucleus. Electrophoretic mobility shift assay showed that recombinant RHD of LvRelish in S2 cells bound specifically with Drosophila melanogaster kappaB motifs in vitro. Both the LvRelish and its RHD domain transactivated the reporter gene controlled by the 5' flanking region of penaeidin 4, an antibacterial peptide of shrimp, suggesting that LvRelish can regulate the transcription of penaeidin 4 gene. Identification of LvRelish will help us better understand shrimp immunity and may help obtain more effective methods to prevent shrimp diseases.

Cloning, characterization and expression analysis of a CXCR1-like gene from mandarin fish Siniperca chuatsi.

In this study we cloned and characterized a CXCR1-like gene (mfCXCR1) from mandarin fish (Siniperca chuatsi). The full-length cDNA of mfCXCR1 is 2,173 bp and contains a 1,056 bp open reading frame (ORF) that encodes a protein of 351 amino acids. The 5' and 3' untranslated regions (UTR) are 57 and 1,080 bp in length, respectively. The coding region of the mfCXCR1 gene consists of a single exon with a 734 bp intron that is two nucleotides upstream of the ATG start codon in the 5' UTR. The mfCXCR1 protein shares a relatively high identity with the CXCR1 and CXCR2 proteins of other fishes (approximately 50-65%). Furthermore, phylogenetic analysis indicates a close relatedness of mfCXCR1 to CXCR1 of other fishes. Many binding sites for stress-inducible transcription factors were present in the promoter region of the mfCXCR1 gene, indicating that it might be activated by certain stressors. The level of mfCXCR1 mRNA, when normalized to that in liver (1-fold), was highest in spleen (approximately 192.9-fold), with intermediate levels in kidney (approximately 163.2-fold), blood (approximately 131.2-fold) and head kidney (approximately 109.4-fold), and relatively low levels in intestine (approximately 34.4-fold) and gill (approximately 16.4-fold) (P < 0.05). Expression of mfCXCR1 during the clinical stage of infectious spleen and kidney necrosis virus (ISKNV) infection showed that its expression was regulated over the course of infection. On day 4 after ISKNV challenge, mfCXCR1 expression was down-regulated in blood (approximately 0.91-fold), spleen (approximately 0.26-fold), head kidney (approximately 0.18-fold) and kidney (approximately 0.82-fold).

Molecular cloning and expression analysis of the ASC gene from mandarin fish and its regulation of NF-kappaB activation.

Apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein that has a bipartite domain structure, an N-terminal PYRIN domain and a C-terminal caspase-recruitment domain (CARD). In this study, we cloned the mandarin fish ASC cDNA (mfASC), which consisted of 899bp with a 115bp 5'-UTR and a 181bp 3'-UTR. The open reading frame encoded 201 amino acids. The mfASC shows 37% identity to an ASC orthologue from zebrafish. The mfASC has two protein-protein interaction domains, an N-terminal PYRIN domain and a C-terminal CARD domain. The mfASC gene structure was determined and had a length of 3954bp with four exons separated by three introns. Northern blot analysis showed that mfASC mRNA is constitutively expressed in the head kidney, gill, hind kidney, spleen and intestine. In vitro studies, mfASC fused with green fluorescent protein appeared as a speck in the transfected 293T cells. When transiently overexpressed in 293T cells, mfASC inhibited NF-kappaB activity with or without tumor necrosis factor (TNFalpha) or lipopolysacharide (LPS) stimulation.

Molecular cloning of two C1q-like cDNAs in mandarin fish Siniperca chuatsi.

C1q, a subunit of the C1 complex, plays a key role in the recognition of immune complexes to initiate the classical complement pathway. In this study, we reported two C1q-like cDNAs from mandarin fish (Siniperca chuatsi), mC1q-like-1 (mC1qL1) and mC1q-like-2 (mC1qL2). The full-length cDNA of mC1qL1was 990bp, containing a 71bp 5'-untranslated region (UTR), an open reading frame (ORF) of 723bp, and a 196bp long 3'-UTR. mC1qL2 cDNA was 1193bp, containing a 100bp 5'-UTR, followed by an ORF of 756bp and a 3'-UTR of 337bp. mC1qL1 and mC1qL2 share 29% identity in amino acid sequence. Both mC1qL1 and mC1qL2 contained three parts: a short amino-terminal region, a collagen-like region and a carboxyl-terminal globular C1q domain. The phylogenetic analysis showed that mC1qL1 clustered with two Danio rerio hypothetical proteins and further grouped with C1q proteins, while mC1qL2 clustered with C1qA proteins from other species. In healthy mandarin fish, mC1qL1 and mC1qL2 were expressed in all tissues tested, including liver, spleen, head kidney, caudal kidney, intestine and gill. mC1qL1 was highly expressed in head kidney, while mC1qL2 was mainly expressed in spleen. The expression level of mC1qL1 and mC1qL2 in liver were not changed obviously and mC1qL2 was significantly changed (p<0.05) in spleen after infectious spleen and kidney necrosis virus (ISKNV) infection. Mandarin fish C1q may play a role in response to ISKNV infection.