Characterization of Litopenaeus vannamei secreted protein acidic and rich in cysteine -like in WSSV infection.

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular and non-structural glycoprotein. In shrimp, a significant function of SPARC in WSSV infection remains unclear. In this study, the full-length cDNA sequence of a secreted protein acidic and rich in cysteine -like was cloned from shrimp Litopenaeus vannamei (named as LvSPARC-L). LvSPARC-L contained an open reading frame of 1002 bp, encoding 333 amino acids. Bioinformatics analysis showed that LvSPARC-L contained a SPARC Ca2+-binding region in the C-terminus, a Kazal-type serine protease inhibitor domain and a BUD22 domain. Tissue distribution assay indicated that LvSPARC-L generally expressed in all tissues selected with a higher expression in hemocyte, stomach and pleoplod. In hepatopancreas and intestine, the relative expression of LvSPARC-L was significantly up-regulated following the WSSV challenge. Besides, the relative expression of viral immediately early gene IE1 and a late gene VP28 was significantly increased in the LvSPARCL-silenced shrimp. Furthermore, the relative expression of LvP53 and LvCaspase3 was extremely decreased in the stomach of dsLvSPARC-L treated shrimp, while that of LvP38 was not affected significantly. All data together suggest that LvSPARC-L might play an antiviral role by regulating apoptosis.

LvCSN5 is involved in WSSV infection via interaction with wsv006.

As an extremely virulent pathogen, white spot syndrome virus (WSSV) greatly threatens shrimp aquaculture worldwide. The interaction between virus and host is important for viral infection. In the present study, a yeast two-hybrid (Y2H) library was constructed to clarify the functions of wsv006, and the interaction between wsv006 and shrimp Litopenaeus vannamei (L. vannamei) was analyzed. Furthermore, we explored the role of the wsv006-interacting molecule L. vannamei COP9 constitutive photomorphogenic-like protein subunit 5 (LvCSN5) in WSSV infection. Y2H assay showed that wsv006 interacted with LvCSN5, and co-immunoprecipitation (Co-IP) assay confirmed such interaction. Multiple alignments of amino acid sequences with other species revealed that the LvCSN5 had high identity with Penaeusmonodon CSN5 (PmCSN5). LvCSN5 was mainly expressed in intestine, eye and hepatopancreas. In addition, the relative expression of LvCSN5 was significantly up-regulated both in intestine and hepatopancreas following the WSSV challenge. Besides, the relative expressions of IE1 and VP28, as well as the viral copy numbers were significantly increased in the LvCSN5-silenced shrimp. Our findings suggested that LvCSN5 was involved in WSSV infection by interacting with wsv006.

Cloning and characterization of Litopenaeus vannamei cystainB-like in WSSV infection.

Cystatins B is an endogenous cysteine cathepsin inhibitor. In shrimp, cystatins B-like (CSTB-L) has not been characterized and its role in WSSV infection is largely unknown. In this study, a full-length 699 bp CSTB-L sequence with 291 bp open reading frame encoding a 96 amino acid from L.vannamei (Lv) was first cloned. The tissue distribution assay indicated that LvCSTB-L presented ubiquitous expression in most examined tissues, with the most predominant expression in the hepatopancreas and the weakest expression in the muscles. LvCSTB-L transcripts could be induced in the intestine and hepatopancreas by WSSV challenge. The relative expression level of IE1 and VP28 in the LvCSTB-L knockdown shrimp were increased significantly. In addition, the shrimp cumulative mortality was remarkably (p < 0.01) increased after LvCSTB-L knockdown. Moreover, following the LvCSTB-L silencing, significant decreases in the mRNA levels of p53, p38, caspase3, STAT and ERK were also observed. The results suggested that LvCSTB-L could play positively roles in antiviral immune response by JAK-STAT, MAPK and apoptotic pathway. These findings would further our understanding of shrimp antiviral response, and therefore help for virus control and prevention.

Overexpression of microRNA-301b accelerates hippocampal microglia activation and cognitive impairment in mice with depressive-like behavior through the NF-κB signaling pathway.

Depression is a condition with a complex etiological pattern, whose effective treatments are highly limited. MicroRNAs (miRNAs) have been investigated in intensive studies owing to their involvement in pathophysiology of mood disorders. The current study aimed to elucidate the role of miR-301b in hippocampus in mouse models of depressive-like behavior. Microarray-based prediction identified the differentially expressed gene neuronal pentraxin II (NPTX2) related to mental depression. Next, the putative miR-301b binding sites on the 3'UTR of NPTX2 were verified. Then the effect of miR-301b on cognitive function of mice with depressive-like behavior was analyzed using the Morris water maze test. In addition, the regulation of miR-301b to NPTX2 and activation of NF-κB signaling pathway was assessed. Following that, the microglia activation and inflammation in hippocampus were evaluated, with the expressions of inflammatory factors being examined. At last, microglia were flow cytometrically sorted and the inflammatory reaction was also assessed in vitro. The obtained findings revealed that miR-301b targeted and negatively regulated NPTX2. Moreover, overexpressed miR-301b activated the NF-κB signaling pathway, as reflected by increasing protein expressions of p-NF-κB. Upregulated miR-301b accelerated cognitive impairment in mice with depressive-like behavior. In addition, overexpression of miR-301b activated microglia and stimulated inflammation in hippocampus, accompanied by enhanced release of tumor necrosis factor-α (TNF-α), interleukin-Iß (IL-Iß) and cyclooxygenase-2(COX-2). Taken together, the evidence provided by the current study indicated that overexpression of miR-301b augmented hippocampal microglia activation, thus exacerbating cognitive impairment and inflammation in mice with depressive-like behavior by activating the NF-κB signaling pathway.

[Correlation of the grades of histologic prostatic inflammation with the risk of prostate cancer in biopsy specimens from men with total PSA of 4－10 µg/L].

OBJECTIVE: To investigate the association of the grades of histologic prostatic inflammation (HPI) with prostate cancer in biopsy specimens for male patients with total serum PSA (tPSA) of 4－10 µg/L. METHODS: We performed prostate biopsy for 200 patients with tPSA of 4－10 µg/L from January 2015 to December 2017. We determined the location, extent and intensity of HPI and analyzed the correlation of the grades of HPI with the risk of prostate cancer. RESULTS: Of the 200 biopsy specimens, BPH was detected in 169 (84.5%) and PCa in 31 (15.5%). Statistically significant differences were found in the positive rates of PCa between grades 1, 2 and 3 HPI, which were 19.3%, 25.8% and 54.8% based on the location (P < 0.01), 77.4%, 19.4% and 3.2% based on the extent (P < 0.01), and 51.6%, 29.0% and 19.4% based on the intensity of the lesion (P < 0.01), but not in the positive rates of BPH (P > 0.05). Multivariate logistic regression analysis showed that the risk of PCa was correlated negatively with the location (95% CI: 0.052－0.407, OR = 0.113, P = 0.001, r = －2.078) and extent of HPI (95% CI: 0.068－0.819, OR = 0.231, P = 0.023, r = －1.526) but not correlated with its intensity (95% CI: 0.796－4.193, OR = 1.804, P = 0.215). The positive predictive value, negative predictive value, sensitivity and specificity of the combined application of the location and extent of HPI in differentiating PCa from BPH were 51.2%, 90.3%, 91.5% and 50.8%, respectively. CONCLUSIONS: The location and extent of HPI are negatively while its intensity is not correlated with the risk of PCa. The grading of HPI based on its location and extent could help reduce the repetition of prostate biopsy.

Increased nucleoside diphosphate kinase activity induces white spot syndrome virus infection in Litopenaeus vannamei.

Nucleoside diphosphate kinase (NDK), which has the same sequence as oncoprotein (OP) in humans, can induce nucleoside triphosphates in DNA replication by maintenance of the deoxynucleotide triphosphate (dNTP's) and is known to be regulated by viral infection in the shrimp Litopenaeus vannamei. This paper describes the relationship between NDK and white spot syndrome virus (WSSV) infection. The recombinant NDK was produced by a prokaryotic expression system. WSSV copy numbers and mRNA levels of IE1 and VP28 were significantly increased in shrimp injected with recombinant NDK at 72 h after WSSV infection. After synthesizing dsRNA-NDK and confirming the efficacy of NDK silencing, we recorded the cumulative mortality of WSSV-infected shrimp injected with NDK and dsRNA-NDK. A comparison between the results demonstrated that silencing NDK delayed the death of shrimps. These findings indicate that NDK has an important role influencing the replication of WSSV replication in shrimp. Furthermore, NDK may have potential target as a new therapeutic strategy against WSSV infection in shrimp.

Cloning of Litopenaeus vannamei CD63 and it's role in white spot syndrome virus infection.

White Spot Syndrome Virus (WSSV) is currently the most serious shrimp pathogen, which has brought huge losses to shrimp industry worldwide. CD63 of shrimp belongs to the tetraspanin superfamily, which plays an important role in signal transduction and immune process. In this paper, CD63 cDNA sequence of Litopenaeus vannamei was cloned using RACE method. The amplified sequence is 1472 bp, with its ORF 744 bp, encoding 247 amino acids. Bioinformatics analysis showed that the sequence of LvCD63 has 93% similarity with Penaeus monodon and 92% similarity with Fenneropenaeus chinensis. Real-time PCR analysis showed that the mRNA levels of LvCD63 expressed in the tissues of hemocytes, gill, epithelial tissue, heart, lymphoid, hepatopancreas, stomach, intestines, muscle and nerve. Among these tissues the highest expression level was showed in the tissue of haemolymph, followed by epithelial tissue, hepatopancreas, and nerve. The lowest expression level of LvCD63 was appeared in the muscle tissue. After WSSV challenge, the expression levels of LvCD63 were both up-regulated in the tissues of gill and epithelial. However the expression level of LvCD63 in hepatopancreas was down-regulated. Far-western blot analysis showed that LvCD63 interacts with VP28, and both VP28N and VP28C fragments interact with LvCD63. Flow cytometry analysis showed that LvCD63 was present on the surface of hemocytes and it is required for binding of WSSV virions. Neutral experiments in vivo showed that LvCD63LEL delayed WSSV infection in shrimp.

Effectiveness of varenicline and counselling for smoking cessation in an observational cohort study in China.

OBJECTIVES: To evaluate the effectiveness of varenicline for smoking cessation in Chinese smokers in a real world cessation clinic practice. DESIGN: A prospective observational study. SETTING: Beijing, China. PARTICIPANTS: A total of 924 smokers (883 men and 41 women) who attended a smoking cessation clinic of a large general hospital were assessed with data from structured questionnaires at baseline and follow-up at 1, 3 and 6 months. Trained physician counsellors provided free individual counselling for all subjects and follow-up interviews with brief counselling. 332 subjects additionally prescribed varenicline according to their own choice were compared with those without varenicline. MAIN OUTCOME MEASURES: Primary outcomes were self-reported 7-day point prevalence abstinence rate and 3-month continuous abstinence rate at 6-month follow-up. Secondary outcomes were 7-day point prevalence abstinence rates at 1 and 3-month follow-up, and 1-month continuous abstinence rate at 3-month follow-up. RESULTS: By intention-to-treat, the 7-day point prevalence abstinence rate with varenicline and counselling at 6 months was significantly higher than counselling only (37.0% vs 23.1%; OR, 1.75; 95% CI 1.46 to 2.62; p=0.001). The 3-month continuous abstinence rate at 6 months was higher with varenicline (33.1% vs 18.4%; OR, 2.04; 95% CI 1.61 to 2.99; p<0.001). Varenicline also showed better secondary outcomes. CONCLUSIONS: Varenicline prescription in the smoking cessation clinic appeared to be effective with doubling of quit rates in Chinese smokers in a real world cessation clinic practice. CLINICAL TRIAL REGISTRATION: NCT01935505; Results.

Multiple proteins of White spot syndrome virus involved in recognition of beta-integrin.

The recognition and attachment of virus to its host cell surface is a critical step for viral infection. Recent research revealed that beta-integrin was involved in White spot syndrome virus (WSSV) infection. In this study, the interaction of beta-integrin with structure proteins of WSSV and motifs involved in WSSV infection was examined. The results showed that envelope proteins VP26, VP31, VP37, VP90 and nucleocapsid protein VP136 interacted with LvInt. RGD-, YGL- and LDV-related peptide functioned as motifs of WSSV proteins binding with beta-integrin. The beta-integrin ligand of RGDT had better blocking effect compared with that of YGL- and LDV-related peptides. In vivo assay indicated that RGD-, LDV- and YGL-related peptides could partially block WSSV infection. These data collectively indicate that multiple proteins were involved in recognition of beta-integrin. Identification of proteins in WSSV that are associated with beta-integrin will assist development of new agents for effective control of the white spot syndrome.

Arginine kinase of Litopenaeus vannamei involved in white spot syndrome virus infection.

Virus-host interaction is important for virus infection. White spot syndrome virus VP14 contains transmembrane and signal peptides domain, which is considered to be important for virus infection. Until now, the function of this protein remains undefined. In this study, we explored the interaction of VP14 with host cell. A new shrimp protein (arginine kinase of Litopenaeus vannamei, LvAK) is selected and its localization in shrimp cells is also confirmed. Cellular localization of LvAK protein in shrimp hemocytes showed that LvAK was primarily located at the periphery of hemocytes and was scarcely detectable in the nucleus. Tissue distribution indicated that arginine kinase gene was spread commonly in the tissues and was highly present in shrimp muscle tissue. The expression of LvAK mRNA in muscle was significantly up-regulated after WSSV stimulation. Indirect immunofluorescence assay showed that LvAK interacted with VP14 in WSSV-infected shrimp. Injection of LvAK protein enhanced the mortality of shrimp infected with white spot syndrome virus (WSSV). These results showed that LvAK is involved in WSSV infection. Future research on this topic will help to reveal the molecular mechanism of WSSV infection.

F0ATP synthase b-chain of Litopenaeus vannamei involved in white spot syndrome virus infection.

White Spot Syndrome Virus (WSSV) is one of the most common and distrous diseases for shrimp. In this study, we show that the protein VP292 that is a envelop protein of WSVV interacts with F0ATP synthase b-chain from Litopenaeus vannamei using far-western blot, ELISA, and indirect immunofluorescence analysis. Tissue distribution analysis of F0ATP synthase b-chain showed that it's transcription can be detected in muscle, hepatopancreas, intestine, hemocytes, lymphoid, and gills. Cellular localization of F0ATP synthase b-chain in shrimp hemocytes showed that F0ATP synthase b-chain was primarily located in the cytoplasm of hemocytes. The transcription levels of F0ATP synthase b-chain were significantly upregulated in intestine, hepatopancreas, hemocytes, and gills of WSSV-infected shrimp at 12 h after infection. Far-western, ELISA, and indirect immunefluorescence assay indicated that F0ATP synthase b-chain interacted with VP292. In the in vivo neutralization experiment, F0ATP synthase b-chain attained 18% protection rate of the shrimp challenged by WSSV. To the best of our knowledge, this is the first report to show that F0ATP synthase b-chain is involved in WSSV infection.

Protective effects of hemin in an experimental model of ventilator-induced lung injury.

Mechanical ventilation is an indispensable life-support modality for critically ill patients with acute lung injury or acute respiratory distress syndrome. Unfortunately, mechanical ventilation even the protective ventilation strategies may evoke ventilator-induced lung injury. Heme oxygenase-1 (HO-1) has recently exhibited anti-inflammatory and anti-oxidative properties in vitro and in vivo. The effect of HO-1 in ventilator-induced lung injury has not been fully characterized. In this study, rabbits were subjected to high tidal volume ventilation to induce ventilator-induced lung injury, which was confirmed by histopathological alterations, increased bronchoalveolar lavage fluid protein content and lung wet-to-dry ratio. In contrast to the level of HO-1 expression in high tidal volume group, pretreatment with hemin, an inducer of HO-1, further up-regulated HO-1 expression. At the same time, these lung injury indexes were attenuated markedly. This pulmonary protection was accompanied by a decrease in bronchoalveolar lavage fluid neutrophil count and in lung myeloperoxidase activity. Besides, pretreatment with hemin prohibited the production of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-8, and up-regulated the level of anti-inflammatory cytokine interleukin (IL)-10 in bronchoalveolar lavage fluid. Furthermore, a decreased malondialdehyde activity, a marker of oxidative stress and a robust increase in total antioxidant capacity were observed in hemin-treated animals. Our findings suggest that HO-1 up-regulation by hemin plays a protective role in ventilator-induced lung injury by suppression inflammatory process and oxidative stress.

[Study design and the preliminary results on the modes of smoking cessation in general hospitals].

To study the intervention programs on smoking cessation in a general hospital and to evaluate its effects of the programs. Four methods including: a) the intervention through specialists in the smoking cessation clinic, b) short-time intervention in the out-patient department, c) free medical intervention, d) group intervention, were adopted for different smokers, with health counseling, psychological intervention and drug treatment. Intervention effect was evaluated by standard methods. During the 20-month period of the project, we treated 690 cases and 402 completed 6-month follow-up. Preliminary results in 402 cases showed that the three methods of smoking cessation interventions could reduce the amount of cigarette smoking and increase the quitting rate. Motivation to quit smoking, intervention methods and intensity of intervention seemed the main factors. The quit rate of 6-month follow-up in the 'specialist intervention' in the smoking cessation clinic (31.6%) and in the group intervention (30.9%) was higher than short-time intervention in free medical events (15.1%). The successful rate of smoking cessation depended on the motivation of quitters, and the attitude, methods and intervention skills of the physicians. Therefore, it is necessary to explore and develop smoking cessation service models suitable to national context and individual intervention methods in China.

White spot syndrome virus VP37 interacts with VP28 and VP26.

White spot syndrome virus (WSSV) is one of the most virulent pathogens affecting penaeid shrimp, causing high mortality in infected populations. Interactions between virus structural proteins are likely to be important for virus assembly. Many steps of the WSSV assembly and maturation pathway remain unclear. In the present study, the interaction between VP37 and envelope or nucleocapsid proteins was characterized. VP37 was expressed in Escherichia coli and confirmed by Western blotting. Pure WSSV virions were subjected to Triton X-100 treatment to separate the envelope and nucleocapsid fractions. Overlay assays showed that VP37 interacted with VP28 and VP26. The interaction of VP37 with VP28 and VP26 was confirmed further by His pull-down and matrix-assisted laser desorption ionization (MALDI) mass spectrographic assays. The binding assay of VP37 with VP28 by ELISA confirmed that the 2 proteins had direct interaction in vivo. This discovery will help elucidate the molecular mechanisms of virion morphogenesis.

Characterization of a pattern recognition molecule vitellogenin from carp (Cyprinus carpio).

Pattern recognition proteins function in innate immune responses by binding to molecules on the surface of invading pathogens and initiating host defense reactions. To explore the role of vitellogenin (Vg) in fish innate immunity, we purified Vg from Carp by gel filtration combined with diethylaminoethyl (DEAE) chromatography. The purified Vg was confirmed by MALDI-TOF mass spectrometry. Antibacterial activity analysis showed that Vg inhibited bacterial activity to Escherichia coli and Staphylococcus aureus in a dose-dependent manner. Vg bound to the surface of Gram-negative and Gram-positive bacteria. It also agglutinated E. coli and S. aureus and weakly to Saccharomyces cerevisiae. Vg showed a strong binding activity to lipopolysaccharides from Gram-negative bacteria. Vg-treated macrophage enhanced phagocytosis to E. coli and S. aureus. Vg also bind with macrophage function as opsonins to promote phagocytosis. The results suggest that Vg serves as a pattern recognition molecule and opsonins in antibacterial defense and as an effector in fish innate immunity.

Role of NADPH oxidase in the mechanism of lung neutrophil sequestration and microvessel injury induced by Gram-negative sepsis: studies in p47phox-/- and gp91phox-/- mice.

We addressed the role of O(2) generated by the NADPH oxidase complex in the mechanism of polymorphonuclear leukocyte (PMN) accumulation and transalveolar migration and lung microvascular injury. Studies were made in mice lacking the p47(phox) and gp91(phox) subunits of NADPH oxidase (p47(phox-/-) and gp91(phox-/-)) in which PMN are incapable of the respiratory burst. The mice were challenged i.p. with live Escherichia coli to induce sepsis. We observed time-dependent increases in PMN sequestration and migration from 1 to 6 h after challenge with 2 x 10(8) E. coli. The responses in knockout mice were greater post-E. coli challenge compared with control mice; i.e., transalveolar PMN migration post-E. coli challenge increased by approximately 50% in the null mice above values in wild type. The increased PMN infiltration was associated with decreased lung bacterial clearance. The generation of the chemoattractant macrophage-inflammatory protein-2 in lung tissue was greater in NADPH oxidase-defective mice after E. coli challenge than control mice; moreover, macrophage-inflammatory protein-2 Ab pretreatment prevented the PMN infiltration. We also observed that E. coli failed to increase lung microvascular permeability in p47(phox-/-) and gp91(phox-/-) mice despite the greater lung PMN sequestration. Thus, O(2) production is required for the induction of sepsis-induced lung microvascular injury. We conclude that NADPH oxidase-derived O(2) generation has an important bactericidal role, such that an impairment in bacterial clearance in NADPH oxidase-defective mice results in increased chemokine generation and lung tissue PMN infiltration.