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The JAK/STAT pathway plays an important role in the development and immune responses of animals. In vertebrates, families of cytokines or growth factors act as activators of the JAK/STAT pathway; however, the activators for the JAK/STAT signaling pathway in arthropods are largely unknown. Herein we report a new ligand, peroxiredoxin 4 (Prx4), for the Domeless in the JAK/STAT pathway of shrimpMarsupenaeus japonicus. Prx4 was induced to secrete into the extracellular surroundings uponVibriochallenge, which then facilitated the anti-Vibrioactivity of shrimp by activating the phosphorylation and nuclear translocation of STAT and the expression of STAT-responsive antimicrobial peptides. Blocking the expression of Prx4in vivoabrogated the activation of the JAK/STAT pathway byVibrioinfection, while injection of Prx4 protein activated the pathway. The interaction between Prx4 and Domeless was proved by immuno-precipitation and protein pull-down assays. Moreover, two cysteine residues in Prx4 that are critical for the interaction and Prx4’s anti-Vibriorole were identified, and the binding site in Domeless for Prx4 was proved to be the cytokine-binding homology module fragment. Taken together, our study revealed a new function for Prx4 enzyme and established a new enzyme-type ligand for the activation of the JAK/STAT pathway in an aquatic arthropod.

The Toll signaling pathway plays an important role in animal innate immunity. However, its activation and signal transmission greatly differ across species and need to be investigated. Shrimp farming is a worldwide economic activity affected by bacterial disease from the 1990s, which promoted research on shrimp immunity. In this study, we first proved that, among the three identified Toll receptors in Marsupenaeus japonicus kuruma shrimp, Toll 3 plays a pivotal role in initiating the antibacterial response in vivo, especially upon anti-Staphylococcus aureus infection. Further research showed that this result was due to the activation of the Dorsal transcription factor, which induced the expression of two anti-lipopolysaccharide factors (Alfs). Moreover, the evolutionarily conserved signaling intermediate in Toll pathways, ECSIT, was proved to be needed for signal transmission from Toll 3 to Dorsal and the expression of anti-lipopolysaccharide factors. Finally, the mortality assay showed that a Toll3-ECSIT-Dorsal-Alf axis was functional in the anti-S.aureus immunity of M. japonicus shrimp. The results provide new insights into the function and signal transduction of the Toll pathway in aquatic species and offer basic knowledge for shrimp disease control and genetic breeding.

Asthma is an inflammatory syndrome characterized by airway hyperresponsiveness, bronchial inflammation, and airway remodeling. Abnormal proliferation of airway smooth muscle cells (ASMCs) is the main pathological feature of asthma. This study investigated the function and mechanism of serine arginine-rich splicing factor 1 (SRSF1) in ASMC proliferation in asthma.SRSF1 expressions in the bronchi of ovalbumin-induced asthmatic mice and IgE-treated mouse ASMCs (mASMCs) were evaluated using quantitative real-time PCR and Western blot. The localization and expression of SRSF1 in the bronchi of asthmatic mice were assessed by immunohistochemistry. Functionally, gain- and loss-of-function assays, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were conducted. Mechanistically, RNA degradation assay, RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter gene assays were carried out.SRSF1 was highly expressed in the bronchi of ovalbumin-induced asthma mice and IgE-treated mASMCs and was mainly located in the nucleus. Experiments on the function of SRSF1 showed that the silencing of SRSF1 induced the cell cycle of mASMC arrest and restrained mASMC proliferation. Investigations into the mechanism of SRSF1 revealed that SRSF1 and miR-135a are competitively bound to the 3'UTR region of Cyclin D2 (CCND2). SRSF1 overexpression repressed the degradation of CCND2 mRNA, and miR-135a negatively regulated CCND2 expression. Furthermore, SRSF1 knockdown inhibited ASMC proliferation in asthma mouse models by regulating the levels of miR-135a and CCND2.SRSF1 knockdown repressed ASMC proliferation in asthma by regulating miR-135a/CCND2 levels.

LGR4-6 (leucine-rich repeat-containing G-protein-coupled receptors 4, 5, and 6) are three related receptors with an upregulated expression in gastrointestinal cancers to various extents, and LGR5 is enriched in cancer stem cells. Antibody-drug conjugates (ADCs) targeting LGR5 showed a robust antitumor effect in vivo but could not eradicate tumors due to plasticity of LGR5-positive cancer cells. As LGR5-negative cancer cells often express LGR4 or LGR6 or both, we reasoned that simultaneous targeting of all three LGRs may provide a more effective approach. R-spondins (RSPOs) bind to LGR4-6 with high affinity and potentiate Wnt signaling. We identified an RSPO4 furin domain mutant (Q65R) that retains potent LGR binding but no longer potentiates Wnt signaling. Drug conjugates of a peptibody comprising the RSPO4 mutant and IgG1-Fc showed potent cytotoxic effects on cancer cell lines expressing any LGR in vitro and suppressed tumor growth in vivo without inducing intestinal enlargement or other adverse effects.

Activated neutrophil-derived exosomes reportedly contribute to the proliferation of airway smooth muscle cells (ASMCs), thereby aggravating the airway wall remodeling during asthma; however, the specific mechanism remains unclear. Lipopolysaccharide (LPS)-EXO and si-CRNDE-EXO were extracted from the media of human neutrophils treated with LPS and LPS + si-CRNDE (a siRNA targets long non-coding RNA CRNDE), respectively. Human ASMCs were co-cultured with LPS-EXO or si-CRNDE-EXO, and cell viability, proliferation and migration were measured. The interplay of colorectal neoplasia differentially expressed (CRNDE), inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ) and nuclear receptor subfamily 2 group C member 2 (TAK1) was explored using RNA immunoprecipitation (RIP) and Co-IP assays. A mouse model of asthma was induced using ovalbumin. CRNDE was upregulated in LPS-EXO and successfully transferred from LPS-treated neutrophils to ASMCs through exosome. Mechanically, CRNDE loaded in LPS-EXO reinforced TAK1-mediated IKKβ phosphorylation, thereby activating the nuclear factor kappa B (NF-κB) pathway. Functionally, silencing CRNDE in LPS-EXO, an IKKβ inhibitor, and an NF-κB inhibitor all removed the upregulation of cell viability, proliferation and migration induced by LPS-EXO in ASMCs. In the end, the in vivo experiment demonstrated that CRNDE knockdown in neutrophils effectively reduced the thickness of bronchial smooth muscle in a mouse model for asthma. Activated neutrophils-derived CRNDE was transferred to ASMCs through exosomes and activated the NF-κB pathway by enhancing IKKβ phosphorylation. The latter promoted the proliferation and migration of ASMCs and then contributed to airway remodeling in asthma.

As an important disulfide reductase of the intracellular antioxidant system, Thioredoxin (Trx) plays an important role in maintaining oxidative stress balance and protecting cells from oxidative damage. In recent years, there is increasing evidence that Trx is a key molecule in the pathogenesis of various diseases and a potential therapeutic target for major diseases including lung, colon, cervical, gastric and pancreatic cancer. However, few knowledge is known about the function of Trx in virus infection. In this study, we reported the cloning and functional investigation of a Trx homologue gene, named MjTrx, in shrimp Marsupenaeus japonicus suffered white spot syndrome virus (WSSV) infection. MjTrx is a 105-amino acid polypeptide with a conservative Cys-Gly-Pro-Cys motif in the catalytic center. Phylogenetic trees analysis showed that MjTrx has a higher relationship with Trx from other invertebrate and clustered with Trx1 from arthropod. MjTrx transcripts is abundant in the gill and intestine tissues and can be detected in the hemocytes, heart, stomach, and hepatopancreas tissues. The transcription levels of MjTrx in hemocytes, gills and intestine tissues of shrimp were significantly up-regulated after white spot syndrome virus infection. MjTrx was recombinant expressed in vitro and exhibited obvious disulfide reductase activity. In addition, overexpression MjTrx in shrimp resulted in the increase of hydrogen peroxide (H2O2) concentration in vivo. All these results strongly suggested that MjTrx functioned in redox homeostasis regulating and played an important role in shrimp antiviral immunity.

Babesia gibsoni (B. gibsoni), an intracellular apicomplexan protozoan, poses great threat to canine health. Currently, little information is available about the B. gibsoni (WH58) endemic to Wuhan, China. Here, the mitochondrial (mt) genome of B. gibsoni (WH58) was amplified by five pairs of primers and sequenced and annotated by alignment with the reported mt genome sequences of Babesia canis (B. canis, KC207822), Babesia orientalis (KF218819), Babesia bovis (AB499088), and Theileria equi (AB499091). The evolutionary relationships were analyzed with the amino acid sequences of cytochrome c oxidase I (cox1) and cytochrome b (cob) genes in apicomplexan parasite species. Additionally, the mt genomes of Babesia, Theileria, and Plasmodium spp. were compared in size, host infection, form, distribution, and direction of the protein-coding genes. The full size of the mt genome of B. gibsoni (WH58) was 5865 bp with a linear form, containing terminal-inverted repeats on both ends, six large subunit ribosomal RNA fragments, and three protein-coding genes: cox1, cob, and cytochrome c oxidase III (cox3). Babesia, Theileria, and Plasmodium spp. had a similar mt genome size of about 6000 bp. The mt genomes of parasites that cause canine babesiosis showed a slightly smaller size than the other species. Moreover, Babesia microti (R1 strain) was about 11,100 bp in size, which was twice larger than that of the other species. The mt form was linear for Babesia and Theileria spp. but circular for Plasmodium falciparum and Plasmodium knowlesi. Additionally, all the species contained the three protein-coding genes of cox1, cox3, and cob except Toxoplasma gondii (RH strain) which only contained the cox1 and cob genes. The phylogenetic analysis indicated that B. gibsoni (WH58) was more identical to B. gibsoni (AB499087), B. canis (KC207822), and Babesia rossi (KC207823) and most divergent from Babesia conradae in Babesia spp. Despite the highest similarity to B. gibsoni (AB499087) reported in Japan, B. gibsoni (WH58) showed notable differences in the sequence of nucleotides and amino acids and the property in virulence to host and in vitro cultivation. This study compared the mt genomes of the two B. gibsoni isolates and other parasites in the phylum Apicomplexa and provided new insights into their differences and evolutionary relationships.

Babesia gibsoni is a protozoan parasite responsible for the majority of reported cases of canine babesiosis in China. Currently, microscopic examination of the Giemsa-stained thin blood smears is the main diagnosis method in clinic. Here, we report the recombinase polymerase amplification-lateral flow (LF-RPA) dipstick detection method for targeting B. gibsoni cytochrome c oxidase subunit I (cox I) gene. The reaction takes only 20-30 min under isothermal temperatures between 30 and 45 °C. Specificity was evaluated using DNA from related apicomplexan parasites and their host, while the sensitivity was calculated based on the DNA from the experimental B. gibsoni-infected dogs. Results indicated that the LF-RPA method is 20 times more sensitive than the conventional PCR based on 18S rRNA and has no cross reaction with any other test DNAs. The applicability of the LF-RPA method was further evaluated using 15 samples collected from clinic. Thirteen of the 15 samples (86.67%) were detected as positive by LF-RPA, while 10 of them (66.67%) were found positive by conventional PCR. Overall, the novel LF-RPA assay is effective for the detection of B. gobsini and has considerable advantages over the conventional PCR in sensitivity, specificity, simplicity in operation, less time consumption, and visual detection. The LF-RPA method may facilitate the surveillance and early detection of B. gibsoni infection in dogs.

Background: Previous studies have revealed the important role of alveolar macrophages (AMs) in the pathogenesis of acute respiratory distress syndrome (ARDS) and potential anti-inflammatory propert...

Babesia microti is one of the most important pathogens causing humans and rodents babesiosis—an emerging tick-borne disease that occurs worldwide. At present, the gold standard for the detection of Babesia is the microscopic examination of blood smears, but this diagnostic test has several limitations. The recombinase polymerase amplification with lateral flow (LF-RPA) assay targeting the mitochondrial cytochrome oxidase subunit I (cox I) gene of B. microti was developed in this study. The LF-RPA can be performed within 10–30 min, at a wide range of temperatures between 25 and 45 °C, which is much faster and easier to perform than conventional PCR. The results showed that the LF-RAP can detect 0.25 parasites/μl blood, which is 40 times more sensitive than the conventional PCR based on the V4 variable region of 18S rRNA. Specificity assay showed no cross-reactions with DNAs of related apicomplexan parasites and their host. The applicability of the LF-RPA method was further evaluated using two clinical human samples and six experimental mice samples, with seven samples were positively detected, while only three of them were defined as positive by conventional PCR. These results present the developed LF-RPA as a new simple, specific, sensitive, rapid and convenient method for diagnosing infection with B. microti. This novel assay was the potential to be used in field applications and large-scale sample screening.

Babesiosis caused by Babesia orientalis, an intraerythrocytic apicomplexan protozoan, is one of the most important diseases for water buffalo in central and southern China, leading to huge economic losses, and its main diagnostic method is microscopic examination. In this study, a recombinase polymerase amplification - lateral flow dipstick (RPA-LF) assay, targeting the mitochondrial COXI gene of B. orientalis, was developed to detect B. orientalis in water buffalo. The RPA-LF assay was carried out as an isothermal reaction at 37 °C within 15 min. The specificity assay showed no cross-reactivity with other protozoa, and the sensitivity assay revealed the minimum detection limit was 0.25 parasite/μL, which was 40-fold more sensitive than that of conventional PCR (0.25 versus10 parasites/μL blood). Moreover, the RPA-LF method was successfully applied to test clinical samples, with no significant difference being observed between RPA-LF and conventional PCR results. Compared with conventional PCR, the novel RPA-LF method had the advantages of simple operation, short time, high sensitivity, and high specificity for B. orientalis detection, indicating the potential use of RPA-LF for rapid field detection of B. orientalis.

Opioid neuropeptides are developed early in the course of a long evolutionary process. As the endogenous messengers of immune system, opioid neuropeptides participate in regulating immune response. In this study, the mechanism that Met-enkephalin (M-ENK) inhibits ROS production through Wnt/β-catenin signaling was investigated in the ZF4 cells of zebrafish. ZF4 cells were exposed to 0, 10, 20, 40, 80, and 160 μM Met-enkephalin (M-ENK) for 24 h, and the cell viability was detected with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The cell viability was significantly increased by 10, 20, 40, 80, and 160 μM M-ENK. After ZF4 cells were exposed to 0, 20, 40, and 80 μM M-ENK for 24 h, the mRNA expression of Wnt10b, β-catenin, and CCAAT/enhancer binding protein α (C/EBPα) was significantly increased by 40 and 80 μM M-ENK. However, the mRNA and protein expression of GSK-3β was significantly decreased by 40 and 80 μM M-ENK. The protein expression of β-catenin was significantly induced by 40 and 80 μM M-ENK, while the protein expression of p-β-catenin was significantly decreased by 20, 40, and 80 μM M-ENK. In addition, the mRNA expression of CAT, SOD, and GSH-PX was significantly increased by 40 and 80 μM M-ENK. The levels of H2O2, ·OH, and O2·- were significantly decreased, but the activity of CAT, SOD, and GSH-PX was significantly increased by 40 and 80 μM M-ENK. The fluorescence intensity of reactive oxygen species (ROS) was decreased, and that of mitochondrial membrane potential (MMP) was increased with the increase of M-ENK concentration in ZF4 cells. The results showed that M-ENK could induce Wnt/β-catenin signaling, which further inhibited ROS production through the induction of C/EBPα, MMP, and the activities of antioxidant enzymes.

β-Catenin is a multifunctional protein that is involved in many physiological processes, including development, cell proliferation, cell migration, and apoptosis. However, the function of β-Catenin in crustacean is unknown. In this study, the first shrimp homologous gene of β-catenin in Marsupenaeus japonicus (i.e., Mjβ-catenin) was identified and characterized. The full-length of the complementary DNA of Mjβ-catenin is 3130 bp, including a 2463 bp open reading frame that encodes 821 amino acid. Multiple alignment of β-Catenin proteins suggested that the Armadillo/β-Catenin-like repeat domains were conserved. Phylogenetic analysis showed that β-Catenin from shrimp was clustered into one group with invertebrate β-Catenin. The transcription of β-catenin in various development stages of shrimp was detected and persistently increased as the shrimp matured. In adult shrimp, β-catenin was widely distributed in detected tissues and has the relatively high expression level in gills, hemocytes, testes, and ovaries. The transcripts of β-catenin in tissues of adult shrimp were significantly up-regulated at various time points after infecting with Staphylococcus aureus, Vibrio anguillarum, and white-spot syndrome virus. Furthermore, knockdown of β-catenin resulted in impaired bacterial clearance ability and increased virus copy in shrimp in vivo. Therefore, β-Catenin in shrimp participates in the development and immune response of shrimps.

Aims To explore the role of long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) in the cell proliferation of airway smooth muscle cells (ASMCs) in asthma. Materials and methods An asthma rat model was established by ovalbumin sensitization and challenge. The expression of GAS5, miR-10a and BDNF mRNA and protein was determined with qRT-PCR and western blot, separately. The targeting relationship between GAS5 and miR-10a was examined with RNA immunoprecipitation and RNA pull-down assay; the interaction between miR-10a and BDNF was evaluated by luciferase reporter assay. Cell Proliferation Assay (MTS) was used for ASMC proliferation detection. Knock-down of GAS5 was performed in asthmatic rats to determine the effects of GAS5 in vivo. Key findings Compared with control group, the inspiratory resistance and expiratory resistance were increased in asthma group; and the expression of GAS5, miR-10a and BDNF was higher, lower and higher, respectively. The expression of GAS5 and miR-10a was elevated and repressed, respectively, by platelet-derived growth factor-BB (PDGF-BB). GAS5 functioned as a bait of miR-10a. GAS5 regulates BDNF expression through miR-10a. PDGF-BB promotes the cell proliferation of ASMCs through miR-10a/BDNF. Knock-down of GAS5 significantly decreased airway hyperresponsiveness in asthmatic rats. Significance The lncRNA GAS5/miR-10a/BDNF regulatory axis played an important role in promoting ASMCs proliferation, thus contributing to asthma.

Syntenin is a multifunctional cytosolic adaptor protein that contributes to cell migration, proliferation, attachment, and apoptosis, as well as immune response to virus, in vertebrates. However, the functions of syntenin in the antibacterial response of invertebrates remain unclear. In this study, we identified a syntenin-like gene (MjSyn) from the kuruma shrimp (Marsupenaeus japonicus) and detected its function in the antibacterial immunity of shrimp. The full-length MjSyn was 1223 bp with a 963 bp open reading frame that encodes 320 amino acids. The deduced MjSyn proteins contained two atypical PDZ domains (sequence repeat that was first reported in the postsynaptic density protein or PSD-95, DlgA, and ZO-1 protein), an N-terminal domain, and a C-terminal domain. Reverse transcription (RT)-PCR results showed that MjSyn was expressed in all tested tissues. Quantitative real-time PCR analysis revealed that MjSyn transcripts in the hemocyte, gill, and intestine were significantly induced at various time points after infection with Staphylococcus aureus and Vibrio anguillarum. The knockdown of the expression of MjSyn by RNA interference resulted in a significant decrease in the phagocytic ability and increased bacteria number in vivo of shrimp. Moreover, the expression of MjCnx, a cytoplasma and membrane location lectin chaperone protein, was inhibited in the MjSyn-knocked down shrimp, which indicated a possible calnexin-related way. Thus, the MjSyn participates in the bacterial clearance response of kuruma shrimp, thereby providing new insight into the function of this kind of important adaptor protein., Highlights • A syntenin (MjSyn) gene was identified from Marsupenaeus japonicus. • MjSyn was expressed ubiquitously with the highest level in gill. • MjSyn expression was significantly upregulated by both Gram-positive and Gram-negative bacterial infections. • MjSyn participates in the bacterial clearance response of shrimp.

ObjectivesWe investigated the analgesic effects of lidocaine (LDC) and lidocane derivative, QX-572, co-application on the evoked pain behaviour (complete Freund's Adjuvant (CFA)-induced) and spontaneous pain behaviour (formalin-induced) in mice.MethodsThe experiments were performed using adult male Kunming mice. Formalin-induced acute pain model and CFA-induced chronic pain model was established by injecting formalin and CFA, respectively. Separate injections of LDC and QX-572, or co-injection of LDC and QX-572, were performed to observe the differences in neurobehavioural responses, paw withdrawal latency (PWL) and mechanical withdrawal threshold (MWT).Key findingsQX-572 injection alone did not influence PWL and MWT, but injection of LDC alone led to a substantial, but short-lived, elevation in PWL and MWT (45 min). Co-injection of LDC and QX-572, however, resulted in a significant increase in PWL and MWT (120 min) compared with the LDC group. Injection of LDC and QX-572 combination in the adjacent sciatic nerve also produced a long-lasting sensory-specific nerve block. Additionally, intraplantar co-injection of LDC and QX-572 combination inhibited spontaneous pain in formalin-treated mice, but did not detectably attenuated hyperalgesia and allodynia in CFA-treated mice.ConclusionsOur results provide evidence that QX-572 induced sensory-selective blockade and co-injection of QX-572 and LDC enhance pain blockade, as evident from formalin-treated mice.

The aim of this study was to investigate the synergistic antioxidant potential and protective effect of grape seed procyanidins (GSP) in combination with Auricularia auricular-judae polysaccharides (AAP IV) on radiation injury in splenocytes. Rat splenocyte irradiation resulted in significantly higher apoptosis rate, malondialdehyde (MDA) (p &lt, 0.005), reactive oxygen species (ROS) (p &lt, 0.01), cell viability, total superoxide dismutase (T-SOD) (p &lt, 0.01), catalase (CAT) (p &lt, 0.01), glutathione peroxidase (GSH-PX) (p &lt, 0.05), activity and glutathione (GSH) (p &lt, 0.01) levels were significantly reduced, compared with the control group. “GSP + AAP IV” treatment of rat splenocytes at doses of “GSP (0.3 μg/mL) + AAP IV (50 μg/mL)” displayed higher radioprotective and antioxidative effects than the administration of either GSP or AAP IV, as evident by lower levels of MDA (p &lt, 0.001) concentration, as well as higher cell viability and T-SOD (p &lt, 0.05), CAT (p &lt, 0.005), GSH-PX (p &lt, 0.01) and GSH content compared to the radiation group. In addition, in vivo studies have shown that “GSP + AAP IV” significantly ameliorated the decrease of spleen index (p &lt, 0.005) and spleen GSH (p &lt, 0.005) levels and significantly inhibited the increase of MDA (p &lt, 0.005) levels of spleen with radiation-induced damage, compared with the non-treated group. The in vivo and in vitro results suggested that GSP and AAP IV have a synergistic protective effect against radiation-induced injury by improving the antioxidant and immunomodulation activities.

This study aimed to validate whether transient receptor potential channel1 (TRPC1) and TRPC3 participate in the regulation the proliferation of airway smooth muscle cells (ASMCs) through modulating calcium ion (Ca2+ ) influx in vitro. Chronic model of murine asthma was induced and ASMCs isolated from asthmatic mice were used in this whole study. TRPC1 and TRPC3 were upregulated in asthmatic mouse ASMCs and selected for further investigation. Ca2+ concentration and the cell viability of asthmatic mouse ASMCs were significantly higher than that from non- asthma mice, however, TRPC channels blocker SKF96365 alleviated these effects. Furthermore, TRPC1 or TRPC3 overexpression markedly increased Ca2+ concentration and significantly induced the viability of ASMCs; whereas TRPC1 or TRPC3 knockdown exerted the completely conversed effects. Moreover, knockdown of TRPC1 and TRPC3 also exerted different effects on the protein expression of growth-related proteins p-p38, p-JNK, cleaved caspase-3 and Bcl-2, as well as on cell cycle. Finally, we found Ca2+ chelator EGTA or BAPTA-AM significantly diminished the effects of si-TRPC1 and si-TRPC3 on the cell viability, cell cycle, and the protein expression of p-p38, p-JNK, cleaved caspase-3, and Bcl-2 in asthmatic mouse ASMCs. Our findings demonstrated that the effects of TRPC1 and TRPC3 on the cell viability and cell cycle of ASMCs were, at least partially, through regulating Ca2+ influx.

Objective The mechanism of Schisandrin B on the proliferation and migration of airway smooth muscle cells (ASMCs) in asthmatic rats was explored. Methods SD rats were divided into three groups: control (group 1), model (group 2) and model + Schisandrin B (group 3). miR-150 and lncRNA BCYRN1 levels were measured by qRT-PCR. The combination of BCYRN1 and miR-150 was detected by RNA pull down. ASMCs’ viability/proliferation/migration were examined by WST-1 assay and 24-well Transwell system. Results Schisandrin B up-regulated miR-150 expression and down-regulated BCYRN1 expression in sensitized rats. Schisandrin B reversed the expression of miR-150 and BCYRN1 in MV-treated ASMCs. In addition, Schisandrin B inhibited the viability, proliferation and migration of MV-induced ASMCs. We also found miR-150 inhibited BCYRN1 expression which was proved by experiments using ASMCs transfected with miR-150 inhibitor. Conclusion Schisandrin B increased miR-150 expression and decreased BCYRN1, and BCYRN1 expression was inhibited by miR-150, which indicated that Schisandrin B could regulate BCYRN1 through miR-150.

The purpose of this study is to investigate the effect of coenzyme Q10 (CoQ10) on focal cerebral ischemia/reperfusion (I/R) injury in hyperglycemic rats and the possible involved mechanisms. In this study, we established the transient middle cerebral artery occlusion (MCAO) for 30min in the rats with diabetic hyperglycemia. The neurological deficit score, 2,3,5-triphenyltetrazolium chloride (TTC) staining and pathohistology are applied to detect the extent of the damage. The expression of Fis1, Mfn2 and Lc3 in the brain is investigated by immunohistochemical and Western blotting techniques. The results showed that the streptozotocin-induced diabetic hyperglycemia and MCAO-induced focal cerebral ischemia were successfully prepared in rats. In the hyperglycemic group, the neurological deficit scores, infarct volumes, and number of pyknotic cells were higher than that in the normalglycemic group at 24h and/or 72h reperfusion. Pretreated with CoQ10 (10mg/kg) for four weeks could significantly reduce the neurological scores, infarct volume, and pyknotic cells at 24h and/or 72h reperfusion of the hyperglycemic rats compared with non-CoQ10 pretreated hyperglycemic animals. Immunohistochemistry and Western blotting showed that pretreatment with CoQ10 or insulin could significantly reduce the expression of Fis1 protein in the brain at 24h and 72h reperfusion. Inversely, a significantly increased expression of Mfn2 was observed in the rats CoQ10 or insulin pretreated at 24h and/or 72h reperfusion when compared with matched hyperglycemic rats. These results demonstrated that hyperglycemia could aggravate ischemic brain injury. Pretreatment with CoQ10 might ameliorate the diabetic hyperglycemia aggravated I/R brain damage in the MCAO rats by maintain the balance between mitochondrial fission and fusion.

Death-associated protein 1 (DAP1) is a small proline-rich cytoplasmic protein that functions both in the apoptosis and autophage process of mammalian and in the clinical cancer of human. However, little knowledge is known about the homologue gene of DAP1 and its roles in the physiological process of invertebrates. In this paper, we report a novel function of DAP1 in the antivirus immunity of shrimp. A homologue gene of DAP1 was cloned from Marsupenaeus japonicus and named as Mjdap-1. The full-length of Mjdap-1 was 1761 bp with a 309 bp open reading frame that encoded 102 amino acids. Reverse transcription-PCR results showed that Mjdap-1 was expressed in all tested tissues, including hemocytes, gills, intestines, stomach, heart, hepatopancreas, testes, and ovaries. In shrimp, Mjdap-1 transcripts were up-regulated by white spot syndrome virus (WSSV) infection; Mjdap-1 knockdown decreased the virus copy in vivo and the mortality of M. japonicus to WSSV challenge. Conversely, injecting the purified recombinant MjDAP1 protein promoted the amplification of virus in shrimp. Flow cytometric assay showed, the virus infection-induced apoptosis of hemocytes was enhanced by MjDAP1 protein injection and inhibited in MjDAP1 knockdown shrimp. Furthermore, the expression of apoptosis-inducing factor (AIF) was regulated by Mjdap-1, but the caspase transcripts were not affected. Our results suggested that MjDAP1 facilitated the amplification of virus in shrimp, which may be attributed to the promotion of hemocyte apoptosis in an AIF-dependent manner. These results provided a new insight into the function of this protein that may be used for virus disease control.

Accumulating evidence suggests that peroxiredoxins (Prx) are key molecules in the pathogenesis of various infectious diseases and are potential therapeutic targets for major diseases such as cancers. In this study, we report a peroxiredoxin IV (Prx IV) in Marsupenaeus japonicus, designated as MjPrx IV, which exhibited peroxidase activity and participated in the anti-white spot syndrome virus (WSSV) immune response. MjPrx IV is a 245-amino acid polypeptide with a predicted 19-amino acid signal peptide, an Ahpc-TSA domain, and a 1-Cys PrxC domain. Phylogenetic analysis revealed that the protein belongs to the Prx IV subfamily. MjPrx IV transcripts were detected in the gills, hepatopancreas, heart, stomach, ovaries, spermary, and intestine tissues, and are upregulated in the gonads, gills and hemocytes of shrimp after WSSV challenge. The mature MjPrx IV peptide was recombinantly expressed in an Escherichia coli system. The protein exhibited peroxidase activity. Furthermore, dsRNA suppression of MjPrx IV increased WSSV replication in shrimp, whereas rMjPrx IV injection into shrimp decreased WSSV replication. These data suggest that MjPrx IV has an important role in shrimp antiviral immunity. To our knowledge, this study is the first to report a shrimp Prx IV that has anti-WSSV activity.

Airway smooth muscle cell (ASMC) was known to involve in the pathophysiology of asthma. Schisandrin B was reported to have anti-asthmatic effects in a murine asthma model. However, the molecular mechanism involving in the effect of Schisandrin B on ASMCs remains poorly understood. Sprague-Dawley rats were divided into three groups: rats as the control (Group 1), sensitized rats (Group 2), sensitized rats and intragastric-administrated Schisandrin B (Group 3). The expression of miR-135a and TRPC1 was detected in the rats from three groups. Platelet-derived growth factor (PDGF)-BB was used to induce the proliferation of isolated ASMCs, and the expression of miR-135a and TRPC1 was detected in PDGF-BB-treated ASMCs. Cell viability was examined in ASMCs transfected with miR-135a inhibitor or si-TRPC1. The expression of TRPC1 was examined in A10 cells pretreated with miR-135a inhibitor or miR-135a mimic. In this study, we found that Schisandrin B attenuated the inspiratory and expiratory resistances in sensitized rats. Schisandrin B upregulated the mRNA level of miR-135a and decreased the expression of TRPC1 in sensitized rats. In addition, Schisandrin B reversed the expression of miR-135a and TRPC1 in PDGF-BB-induced ASMCs. Si-TRPC1 abrogated the increasing proliferation of ASMCs induced by miR-135a inhibitor. We also found that miR-135a regulated the expression of TRPC1 in the A10 cells. These results demonstrate that Schisandrin B inhibits the proliferation of ASMCs via miR-135a suppressing the expression of TRPC1.

Soluble dietary fiber (SDF) was prepared by Trichoderma viride fermentation by using apricot pulp as the raw material. A four-factor and three-level response surface methodology was applied to optimize the fermentation conditions affecting the extraction rate of SDF. The optimum fermentation conditions were listed: crude enzyme volume, 9.59 mL g(-1); fermentation temperature, 43 °C; initial pH, 5.36; fermentation time, 6.47 h. Under these conditions, 15.69% yield was obtained and its relative error with the predicted theoretical value (15.87%) was 1.14%. The dietary fiber content of SDF was 84.0% whereas it was found to be 43.1% in apricot pulp flour. The anti-diabetic effect of apricot pulp SDF on rat models of diabetes was investigated. Both the blood glucose level and body weight were significantly changed in apricot pulp SDF-treated groups compared with the diabetic group (p < 0.01) after intragastric administration for 28 days. In addition, SDF elicited inhibitory effects on the α-glucosidase activity with an IC50 of 17.458 mg mL(-1). These results implied that apricot pulp SDF relieved the symptoms of diabetic rats.

A new member of antimicrobial peptide genes of the penaeidin family, Ch-penaeidin, has been cloned from the haemocytes of Chinese shrimp, Fenneropenaeus chinensis, by reverse transcription PCR (RT-PCR), 3'-rapid amplification of cDNA end (3'-RACE) and smart cDNA methods. The Ch-penaeidin cDNA was 655 bp and the open reading frame of the cDNA encoded a 71 amino acid peptide. Ch-penaeidin contained a putative NH2-terminal signal Sequence (1-19) followed by a mature peptide (20-71). The sequence identify with other penaeidins from Litopenaeus vannamei and Litopenaeus setiferus is between 48% and 71%. The signal sequence of Ch-penaeidin is almost completely identical to that of other penaeidins, while differing relatively in the N-terminal domain of the mature peptide. Ch-penaeidin was designated as a novel member of class penaeidin 3 according to phylogenetic analysis. The Mature peptide. with a predicted molecular weight of 5589.32 Da, and a pI of 9.77, has eight positively charged amino acids and no negatively charged amino acids. The expression and distribution of Ch-penaeidin in Unchallenged shrimps were studied by RT-PCR, Northern blot and in situ hybridisation. The results showed that the Ch-penaeidin transcripts were detected in haemocytes (granular haemocytes), heart, gill, intestine, and subcuticular epithelia of the shrimp. and that Ch-penaeidin was constitutively expressed mainly in haemocytes. (C) 2003 Elsevier Ltd. All rights reserved.

C-type lectins (CTLs) are a superfamily of calcium-dependent carbohydrate binding proteins containing at least one carbohydrate-recognition domain (CRD) and they are present in almost all metazoans. Insect CTLs may function as pattern-recognition receptors and play important roles in innate immunity. In this study, we selected five AsCTLs from the mosquito Armigeres subalbatus, a natural vector of filarial nematodes, and performed both in vitro and in vivo studies to elucidate their functions in innate immunity. AsCTLMA15, AsCTLGA5 and AsCTL15 were mainly expressed in hemocytes, AsCTL16 was expressed in fat body, while AsCTLMA11 was expressed in both hemocytes and fat body, and only AsCTLMA11 and AsCTL16 were expressed at high levels in adult females. In vitro binding assays showed that all five recombinant AsCTLs could bind to different microbial cell wall components, including lipopolysaccharide (LPS), lipid A, peptidoglycan (PG), lipoteichoic acid (LTA), zymosan and laminarin (beta-1,3-glucan). Recombinant AsCTLs also bound to several Gram-negative and Gram-positive bacteria, and could agglutinate bacterial cells. Injection of double-stranded RNAs (dsRNAs) could significantly reduce expression of the five AsCTL mRNAs, and the survival of mosquitoes treated with dsRNA to AsCTLGA5 was significantly decreased after Escherichia coli infection, but did not change significantly after Micrococcus luteus infection compared to the control groups, suggesting that Ar. subalbatus AsCTLGA5 may participate in innate immunity against E. coli.

Objectives To determine the role of serine protease in the disruption of the blood–brain barrier (BBB) during Cryptococcus neoformans meningitis. Methods Reverse transcription–polymerase chain reaction and immunohistochemistry were used to determine the production of serine protease by different strains of C. neoformans. BBB permeability in immunosuppressed rats inoculated with C. neoformans or C. neoformans plus aprotinin was examined via Evans blue staining. In vitro BBB permeability (transwell passage of horseradish peroxidase) was determined in human brain microvascular endothelial cells (BMECs) cultured with serine protease or serine protease plus aprotinin. Electron microscopy of rat brain tissue was used to visualise C. neoformans infection. Results Serine protease mRNA and protein were detected in all C. neoformans serotypes. C. neoformans infection increased BBB permeability in vivo, but this effect was ameliorated by aprotinin. Treatment of BMECs with serine protease increased permeability in vitro. This effect was reversed by aprotinin. Conclusion Serine protease secreted by C. neoformans leads to BBB disruption during Cryptococcus meningitis. Serine protease may be a novel treatment target for Cryptococcus meningitis .

The Toll/Toll-like receptor (TLR) signaling pathway has an important role in the innate immunity of animals. Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) is a protein that functions as an adaptor protein for the Toll/TLR and bone morphogenetic protein signaling pathways. ECSIT is also a key component in the macrophage bactericidal activity of mammals. However, the function of ECSIT in crustaceans remains unclear. In this study, we cloned and identified a functional ECSIT homologue, MjECSIT 1, from kuruma shrimp Marsupenaeus japonicus. The complementary DNA of MjEcsit 1 is 1442 base pairs long, with an open reading frame of 1221 base pairs that encodes a 407-residue polypeptide. Transcripts of MjEcsit 1 are detected in hemocytes, gills, hepatopancreas, stomach, heart, intestines, testes, and ovaries. Such transcripts are upregulated by Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Vibrio anguillarum) injections. The knockdown of MjEcsit 1 by double-stranded RNA injection increases the sensitivity of M. japonicus to S. aureus challenge and weakens the bacterial clearance ability of M. japonicus in vivo. In addition, suppressing MjEcsit 1 restrains the upregulation of two anti-lipopolysaccharide factors by S. aureus injection. The results indicate that MjECSIT 1 is important in the antibacterial immunity of M. japonicus.

To establish a method of acutely isolating dorsal root ganglion (DRG) neurons for patch clamp study of single-channel.DRG neurons of rats were acutely isolated by enzymatic digestion and mechanical blowing.The acutely isolated DRG cells were easy to form the higher sealing resistance (5G Omega), which lowered noise level, so that pA-level single channel currents could be recorded.The acutely isolated DRG neurons in this study are an ideal for patch-clamp study of single-channel.

Autophagy represents an alternative tumor-suppressing mechanism that overcomes the dramatic resistance of malignant gliomas to radiotherapy and proapoptotic-related chemotherapy. This study reports that valproic acid (VPA), a widely used anti-epilepsy drug, induces autophagy in glioma cells. Autophagy, crucial for VPA-induced cell death, is independent of apoptosis, even though apoptotic machinery is proficient. Oxidative stress induced by VPA occurs upstream of autophagy. Oxidative stress also activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, whereas blocking this pathway inhibits autophagy and induces apoptosis. VPA-induced autophagy cannot be alleviated by inositol, suggesting a mechanism different from that for lithium. Moreover, VPA potentiates autophagic cell death, but not apoptosis, when combined with other autophagy inducers such as rapamycin, Ly294002, and temozolomide in glioma cells both in vitro and in vivo, which may warrant further investigation toward possible clinical application in patients with malignant gliomas.

Penaeidins are members of a special family of antimicrobial peptides existing in penaeid shrimp and play an important role in the immunological defence of shrimp. Here, we report one penaeidin with a putative isotype newly cloned from fleshy prawn Fenneropenaeus chinensis. The penaeidin open reading frame encodes a 79 amino acid peptide while two exons and an intron were identified within the 1126 bp genomic sequence of Fenchi-penaeidin 5. Phylogenetic analysis and sequence comparison with other known penaeidins suggest the new gene belongs to a novel subfamily of penaeidins and the two isoforms were named Fenchi-penaeidin 5-1 and 5-2, respectively. Fenchi-penaeidin 5 mRNA was examined in normal and microbial challenged shrimp and was found to be constitutively expressed in heamocytes, heart, gill, intestine and ovary. Bacterial challenge resulted in mRNA up-regulation, inducing expression in hepatopancreas and stomach. Fenchi-penaeidin 5-1 was also expressed in Pichia pastoris, and recombinant Fenchi-penaeidin 5-1 exhibited activities against Gram-positive and -negative bacteria and fungi.

Thrombospondins (TSPs) are extracellular, multidomain, calcium-binding glycoproteins that modulate cell behavior in homeostasis and during development, wound-healing, immune response and tumor growth of adult tissues in vertebrates. In invertebrates these proteins are a major component of cortical rods in mature oocytes. A fragment of a thrombospondin-like gene was generated by screening a subtractive cDNA library constructed from the hemocytes of Chinese shrimp, Fennerpenaeus chinensis. The full length F. chinensis cDNA of thrombospondin was cloned by 3'- and 5'-rapid amplification of cDNA ends (3'- and 5'-RACE). The complete cDNA sequence, named Fc-TSP, is 2886 bp and the open reading frame of the cDNA encodes a 938-residue protein that contains three ChtBD2 domains, an EGF domain, a TSP-3 domain and a common TSP-C (CTD) domain. The protein shares a high sequence identity with the mj-TSPa (46.3%), mj-TSPb (46.9%) and mj-TSPc (51.9%) of Marsupenaeus japonicus. The expression and distribution of Fc-TSP in both challenged and unchallenged shrimps were studied by Northern blot, RT-PCR and in situ hybridization. Northern blot analysis showed that the Fc-TSP transcripts were detected in the hemocytes, heart, intestine, stomach and ovary of both challenged and unchallenged shrimps, but the signal was much stronger in the challenged tissues. A strong hybridization signal was detected only in challenged hepatopancreas, with no signal in the unchallenged tissue. The RT-PCR showed that the Fc-TSP was detected in both challenged and unchallenged tissues including the hemocytes, heart, hepatopancreas, stomach, gills, intestine, spermary and ovary. Except for the ovary and spermary, the signal of challenged tissues was relatively stronger than that of unchallenged ones, especially in hepatopancreas. These results suggest that the thrombospondin was upregulated in the hemocytes, heart, intestine and stomach of challenged shrimp, and induced in the hepatopancreas of challenged shrimps. Therefore, Fc-TSP may be involved in the defense responses of the shrimp.

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a similar to6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use. (C) 2004 Elsevier Inc. All rights reserved.