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1.
Mar Drugs ; 17(9)2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31492051

RESUMO

Previously unreported N,N'-ketal quinazolinone enantiomers [(-)-1 and (+)-1] and a new biogenetically related compound (2), along with six known compounds, 2-pyrovoylaminobenzamide (3), N-(2-hydroxypropanoyl)-2 amino benzoic acid amide (4), pseurotin A (5), niacinamide (6), citreohybridonol (7), citreohybridone C (8) were isolated from the ascidian-derived fungus Penicillium sp. 4829 in wheat solid-substrate medium culture. Their structures were elucidated by a combination of spectroscopic analyses (1D and 2D NMR and Electron Circular Dichroism data) and X-ray crystallography. The enantiomeric pair of 1 is the first example of naturally occurring N,N'-ketal quinazolinone possessing a unique tetracyclic system having 4-quinazolinone fused with tetrahydroisoquinoline moiety. The enantiomeric mixtures of 1 displayed an inhibitory effect on NO production in lipopolysaccharide-activated RAW264.7 cells, while the optically pure (-)-1 showed better inhibitory effect than (+)-1.

2.
Front Immunol ; 10: 1785, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31417561

RESUMO

The outbreak of diseases ordinarily results from the disruption of the balance and harmony between hosts and pathogens. Devoid of adaptive immunity, shrimp rely largely on the innate immune system to protect themselves from pathogenic infection. Two nuclear factor-κB (NF-κB) pathways, the Toll and immune deficiency (IMD) pathways, are generally regarded as the major regulators of the immune response in shrimp, which have been extensively studied over the years. Bacterial infection can be recognized by Toll and IMD pathways, which activate two NF-κB transcription factors, Dorsal and Relish, respectively, to eventually lead to boosting the expression of various antimicrobial peptides (AMPs). In response to white-spot-syndrome-virus (WSSV) infection, these two pathways appear to be subverted and hijacked to favor viral survival. In this review, the recent progress in elucidating microbial recognition, signal transduction, and effector regulation within both shrimp Toll and IMD pathways will be discussed. We will also highlight and discuss the similarities and differences between shrimps and their Drosophila or mammalian counterparts. Understanding the interplay between pathogens and shrimp NF-κB pathways may provide new opportunities for disease-prevention strategies in the future.

3.
Fish Shellfish Immunol ; 93: 406-415, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31369857

RESUMO

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity.

5.
Fish Shellfish Immunol ; 93: 361-368, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31326591

RESUMO

C-type lectins (CTLs) are a group of lectins with at least one carbohydrate recognition domain (CRD), the binding of which to carbohydrates requires the presence of calcium ions. CTLs generally function as pattern recognition receptors (PRRs), essentially participating in innate immunity. In the current study, a novel CTL termed LvCTL5 was identified from Pacific white shrimp Litopenaeus vannamei, which shared sequence identities with other crustacean CTLs. LvCTL5 was highly expressed in hepatopancreas and could be activated by infection with bacteria, virus and fungi. The recombinant LvCTL5 protein purified from E. coli showed microbiostatic and agglutination activities against bacteria and fungi in vitro. Silencing of LvCTL5 in vivo could significantly affect expression of a series of immune effector genes and down-regulate the phagocytic activity of hemocytes. Compared with controls, the LvCTL5-silenced shrimp were highly susceptible to Vibrio parahaemolyticus and white spot syndrome virus (WSSV) infections. These suggest that LvCTL5 has microbiostatic and immune regulatory activities and is implicated in antiviral and antibacterial responses.

6.
Fish Shellfish Immunol ; 92: 889-896, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31299465

RESUMO

Ranaviruses belong to the family Iridoviridae, and have become a serious threat to both farmed and natural populations of fish and amphibians. Previous reports showed that ranaviruses could encode viral Bcl-2 family-like proteins (vBcl-2), which play a critical role in the regulation of cell apoptosis. However, the mechanism of ranaviruses vBcl-2 interactions with host protein in mediating apoptosis remains unknown. Tiger frog virus (TFV) belonging to the genus Ranavirus has been isolated from infected tadpoles of Rana tigrina rugulosa, and it causes a high mortality rate among tiger frog tadpoles cultured in southern China. This study elucidated the molecular mechanism underlying the interaction of TFV ORF104R with the VDAC2 protein to regulate cell apoptosis. TFV ORF104R is highly similar to other ranaviruses vBcl-2 and host Mcl-1 proteins, indicating that TFV ORF104R is a postulate vBcl-2 protein. Transcription and protein expression levels showed that TFV orf104r was a late viral gene. Western blot results suggested that TFV ORF104R was a viral structural protein. Subcellular localization analysis indicated that TFV ORF104R was predominantly colocalized with the mitochondria. Overexpressed TFV ORF104R could suppress the release of cytochrome C and the activities of caspase-9 and caspase-3. These results indicated that TFV ORF104R might play an important role in anti-apoptosis. Furthermore, the interaction between TFV ORF104R and VDAC2 was detected by co-immunoprecipitation in vitro. The above observations suggest that the molecular mechanism of TFV-regulated anti-apoptosis is through the interaction of TFV ORF104R with the VDAC2 protein. Our study provided a mechanistic basis for the ranaviruses vBcl-2-mediated inhibition of apoptosis and improved the understanding on how TFV subverts host defense mechanisms in vivo.

7.
J Nat Prod ; 82(7): 1752-1758, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31251621

RESUMO

Three new 12- or 13-membered-ring macrocyclic alkaloids, named ascomylactams A-C (1-3), along with the analogues phomapyrrolidone C (4) and phomapyrrolidone A (5) were isolated from the mangrove endophytic fungus Didymella sp. CYSK-4. Their structures were elucidated by analysis of extensive spectroscopic data and mass spectrometric data. The structures and absolute configurations of 1 and 2 were determined by single-crystal X-ray diffraction experiments, which represents the first crystal structures described for a (6/5/6/5) tetracyclic skeleton fused with a 12- or 13-membered-ring macrocyclic moiety. The configurations of phomapyrrolidone C (4) and phomapyrrolidone A (5) were revised by detailed analysis of the NMR data. In a cytotoxic assay, compounds 1 and 3 showed moderate cytotoxicity against MDA-MB-435, MDA-MB-231, SNB19, HCT116, NCI-H460, and PC-3 human cancer cell lines, with IC50 values in the range of 4.2-7.8 µM.

8.
Fish Shellfish Immunol ; 92: 141-150, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31176007

RESUMO

Mandarin fish (Siniperca chuatsi) is a popular cultured freshwater fish species due to its high market value in China. With increasing density of breeding, mandarin fish is often cultured under low environmental oxygen concentrations (hypoxia). In this study, the relative expression levels of hypoxia response element (HRE)-luciferase reporter and the HIF signaling pathway downstream genes (scldha, scvegf, and scglut-1) were significantly increased by hypoxic stress, thereby indicating that mandarin fish has an HIF signaling pathway. The mandarin fish HIF-1α (scHIF-1α) was also characterized. Multiple sequence alignments showed that scHIF-1α presented similar architectures to other known vertebrates. Subcellular localization analysis showed that scHIF-1α was mainly located in the nucleus of the mandarin fish fry-1 (MFF-1) cells. The role of scHIF-1α in the regulation of the HIF signaling pathway was confirmed. Overexpression of scHIF-1α could induce the HIF signaling pathway, whereas knockdown of scHIF-1α inhibited the activity of the HIF-1 signaling pathway. Tissue distribution analysis showed that schif-1α was significantly highly expressed in the blood, heart, and liver, which indicated that the main function of scHIF-1α was closely related to the circulatory system. Furthermore, scHIF-1α expression was significantly induced by poly I:C, poly dG:dC or PMA, thereby indicating that scHIF-1α was involved in the immune response. HIF-1α plays an important role in pathogen infections in mammals, but its role in fish is rarely investigated. Overexpression of scHIF-1α could inhibit MRV and SCRV infections, whereas knockdown of scHIF-1α could promote such infections. Those results suggested that scHIF-1α played an important role in fish virus infection. Our study will help understand the hypoxia associated with the outbreaks of aquatic viral disease.

9.
Appl Opt ; 58(11): 2773-2781, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-31044876

RESUMO

Concise open-path continuous-wave cavity ring-down spectroscopy (CW-CRDS) with a bow-tie cavity structure is demonstrated in the single- and dual-optical-path experiments for multicomponent gas detection, e.g., greenhouse gas concentration evaluation in ambient air. Owing to its features of optical feedback suppression and small free spectral range (FSR), the bow-tie configuration shows its special advantages in the realization of both a compact arrangement and two counter-propagating non-interference optical paths. The minimum of the Allan deviation reaches 1.6×10-10 cm-1 for an integration time of 100 s, corresponding to the noise equivalent absorption coefficient of 1.6×10-9 cm-1 Hz-1/2. The detection sensitivity of methane is deduced to be 0.9 ppbv with its absorption cross section of 1.48×10-20 cm2/molecule in the 512 decays averaging mode. A wavelength-correction method is proposed to reduce by about 30% the uncertainty in the measurements caused by the deviation in the wavelength resonance between incident laser and ring-down cavity. The concentrations of greenhouse gases in ambient air are measured by the open-path CW-CRDS with the uncertainties of 0.02, 100, and 10 ppmv for CH4, H2O, and CO2, respectively.

10.
Int J Cardiovasc Imaging ; 35(8): 1499-1508, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31037475

RESUMO

Resting two-dimensional speckle tracking echocardiography (2D-STE) identified right ventricular (RV) systolic function were reported to predict exercise capacity in pulmonary hypertension (PH) patients, but little attention had been payed to 2D-STE detected RV diastolic function. Therefore, we aim to elucidate and compare the relations between 2D-STE identified RV diastolic/systolic functions and peak oxygen consumption (PVO2) determined by cardiopulmonary exercise testing (CPET) in pre-capillary PH. 2D-STE was performed in 66 pre-capillary PH patients and 28 healthy controls. Linear correlation and multivariate regression analyses were performed to evaluate and compare the relations between RV 2D-STE parameters and PVO2. Receiver operating characteristic curves were used to compare the predictive value of 2D-STE parameters in predicting the cut-off-PVO2 < 11 ml/min/kg. There were significant differences of all the 2D-STE parameters between PH patients and healthy controls. In patients, RV-peak global longitudinal strain (GLS, rs = - 0.498, P < 0.001), RV- peak systolic strain rate (GSRs, rs = - 0.537, P < 0.001) and RV- peak early diastolic strain rate (GSRe, rs = 0.527, P < 0.001) significantly correlated with PVO2, but no significant correlation was observed between RV- peak late diastolic strain rate (GSRa, rs = 0.208, P = 0.093) and PVO2. The first multivariate regression analysis of clinical data without echocardiographic parameters identified WHO functional class, NT-proBNP and BMI as independent predictors of PVO2 (Model-1, adjusted r2 = 0.421, P < 0.001); Then we added conventional echocardiographic parameters and 2D-STE parameters to the clinical data, identified S,(Model-2,adjusted r2 = 0.502, P < 0.001), RV-GLS (Model-3, adjusted r2 = 0.491, P < 0.001), RV-GSRe (Model-4, adjusted r2 = 0.500, P < 0.001) and RV-GSRs (Model-5, adjusted r2 = 0.519, P < 0.001) as independent predictors of PVO2, respectively. The predictive power was increased, and Model-5 including RV-GSRs showed the highest predictive capability. ROC curves found RV-GSRs expressed the strongest predictive value (AUC = 0.88, P < 0.001), and RV-GSRs > - 0.65/s had a 88.2% sensibility and 82.2% specificity to predict PVO2 < 11 ml/min/kg. 2D-STE assessed RV function improves the prediction of exercise capacity represented by PVO2 in pre-capillary PH.


Assuntos
Ecocardiografia Doppler/métodos , Tolerância ao Exercício , Hipertensão Pulmonar/diagnóstico por imagem , Função Ventricular Direita , Adulto , Estudos de Casos e Controles , Estudos Transversais , Teste de Esforço , Feminino , Humanos , Hipertensão Pulmonar/fisiopatologia , Masculino , Pessoa de Meia-Idade , Consumo de Oxigênio , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Adulto Jovem
11.
J Environ Sci (China) ; 80: 248-256, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30952342

RESUMO

Antibiotic resistance genes (ARGs), human pathogenic bacteria (HPB), and HPB carrying ARGs are public issues that pose a high risk to aquatic environments and public health. Their diversity and abundance in water, intestine, and sediments of shrimp culture pond were investigated using metagenomic approach. A total of 19 classes of ARGs, 52 HPB species, and 7 species of HPB carrying ARGs were found. Additionally, 157, 104, and 86 subtypes of ARGs were detected in shrimp intestine, pond water, and sediment samples, respectively. In all the samples, multidrug resistance genes were the highest abundant class of ARGs. The dominant HPB was Enterococcus faecalis in shrimp intestine, Vibrio parahaemolyticus in sediments, and Mycobacterium yongonense in water, respectively. Moreover, E. faecalis (contig Intestine_364647) and Enterococcus faecium (contig Intestine_80272) carrying efrA, efrB and ANT(6)-Ia were found in shrimp intestine, Desulfosaricina cetonica (contig Sediment_825143) and Escherichia coli (contig Sediment_188430) carrying mexB and APH(3')-IIa were found in sediments, and Laribacter hongkongensis (contig Water_478168 and Water_369477), Shigella sonnei (contig Water_880246), and Acinetobacter baumannii (contig Water_525520) carrying sul1, sul2, ereA, qacH, OXA-21, and mphD were found in pond water. Mobile genetic elements (MGEs) analysis indicated that horizontal gene transfer (HGT) of integrons, insertion sequences, and plasmids existed in shrimp intestine, sediment, and water samples, and the abundance of integrons was higher than that of other two MGEs. The results suggested that HPB carrying ARGs potentially existed in aquatic environments, and that these contributed to the environment and public health risk evaluation.


Assuntos
Resistência Microbiana a Medicamentos/genética , Monitoramento Ambiental/métodos , Metagenoma/fisiologia , Microbiologia da Água , Poluição da Água/análise , Poluição da Água/estatística & dados numéricos
12.
Fish Shellfish Immunol ; 90: 275-287, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30936049

RESUMO

JAK/STAT signaling pathways are associated with the innate immune system and play important roles in mediating immune responses to virus infection. In this study, a Janus kinase gene from Scylla paramamosain (SpJAK) was cloned and characterized. The full length of SpJAK mRNA contains a 5' untranslated region (UTR) of 304 bp, an open reading frame of 3300 bp and a 3' UTR of 302 bp. The SpJAK protein contains seven characteristic JAK homology domains (JH1 to JH7) and showed 60% identity (78% similarity), 20% identity (35% similarity), and 21% identity (37% similarity) to the Litopenaeus vannamei JAK (LvJAK) protein, the Drosophila melanogaster hopscotch protein, and the Homo sapiens JAK2 protein, respectively. The mRNA of SpJAK showed high expression in the brain and nerve but low expression in the hemocyte and muscle. Moreover, the expression of SpJAK was significantly upregulated by stimulation with mud crab reovirus (MCRV), poly(I:C), and Vibrio parahaemolyticus. SpJAK significantly activated the STAT of S. paramamosain (SpSTAT) to translocate to the nucleus of Drosophila Schneider 2 cells. SpJAK significantly enhanced the activity of the promoter of the WSSV wsv069 gene that was activated significantly by SpSTAT by acting on the STAT-binding DNA motif. These results suggest that SpJAK activates the JAK/STAT pathway. Furthermore, silencing SpJAK in vivo resulted in the high mortality rate of MCRV-infected mud crabs and increased the viral load in tissues. Hence, SpJAK could play an important role in defense against MCRV in mud crab.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Janus Quinases/genética , Janus Quinases/imunologia , Reoviridae/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Janus Quinases/química , Filogenia , RNA Mensageiro/análise , RNA Mensageiro/genética , Alinhamento de Sequência , Transdução de Sinais
13.
Fish Shellfish Immunol ; 89: 555-563, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30999041

RESUMO

In shrimp, the JAK-STAT pathway is essentially implicated in both antiviral and antibacterial responses. However, few regulatory target genes of the JAK-STAT pathway in shrimp have been reported so far. In this study, a novel single WAP domain-containing peptide (LvSWD4) was identified from Pacific white shrimp Litopenaeus vannamei. The promoter of LvSWD4 was predicted to harbor multiple STAT-binding DNA motifs. Over-expression of the JAK-STAT pathway components STAT, JAK and Domeless in vitro significantly enhanced the transcriptional activity of the LvSWD4 promoter, and in vivo silencing of STAT and the the JAK-STAT pathway upstream regulator IRF down-regulated the expression of LvSWD4, suggesting that LvSWD4 could be a target gene of the JAK-STAT pathway. The expression of LvSWD4 was significantly increased after infection with Gram-negative and positive bacteria, fungi and virus, and silencing of LvSWD4 increased the susceptibility of shrimp to V. parahaemolyticus and WSSV infections. In vitro experiments also demonstrated that the recombinant LvSWD4 protein had significant inhibitory activities against Gram negative bacteria V. parahaemolyticus and E. coli and Gram positive bacteria S. aureus and B. subtilis. Furthermore, silencing of LvSWD4 in vivo significantly affected expression of various immune functional genes and attenuated the phagocytic activity of hemocytes. These suggested that as a target gene of STAT, LvSWD4 was essentially implicated in shrimp immunity, which could constitute part of the mechanism underlying the immune function of the shrimp JAK-STAT pathway.


Assuntos
Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Sequência de Bases , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência , Transdução de Sinais , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
14.
Int Rev Cell Mol Biol ; 345: 287-360, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30904195

RESUMO

Innate immunity is an ancient and conserved defense mechanism against infectious agents. It is activated after pathogen-associated molecular pattern sensing by germline-encoded pattern-recognition receptors, including specialized nucleic acid sensors. In vertebrates, nucleic acid sensing activates the dominant antiviral pathway that induces interferon (IFN) response and enhances antigen-specific adaptive immunity. Although canonical IFN system is absent in invertebrates, nucleic acid stimulation, and viral infection trigger an inducible non-specific antiviral response that exhibits multiple similarities to vertebrate IFN system. Invertebrates lack the adaptive immunity that provides long-term antigen-specific protection from pathogens. Meanwhile, the RNA interference (RNAi) pathway senses viral nucleic acids and triggers the sequence-specific degradation of viral RNAs, thereby representing a specific antiviral mechanism of invertebrates. RNAi provides the primary antiviral response in some invertebrates but plays minimal or no role in vertebrate antiviral immunity. This review summarizes the nucleic acid sensing-mediated antiviral immunity in invertebrates.

15.
Appl Microbiol Biotechnol ; 103(7): 3111-3122, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30815709

RESUMO

The intestinal microbiota plays crucial roles in host health. The Pacific white shrimp is one of the most profitable aquaculture species in the world. Antibiotic supplement in feed is an optional practice to treat shrimp bacterial diseases. However, little is known about antibiotic effects on intestinal microbiota in pacific white shrimp. Here, shrimps were given feed supplemented with ciprofloxacin (Cip) (40 and 80 mg kg-1) and sulfonamide (Sul) (200 and 400 mg kg-1) to investigate the microbial community by targeting the V4 region of 16S rRNA genes. Within 4 days after feeding with normal feed and with antibiotics, antibiotic concentrations of Cip and Sul groups in the intestine dropped sharply. Significantly, increased abundance of antibiotic resistance genes (ARGs) of ciprofloxacin (qnrB, qnrD, and qnrS) and sulfonamide (sul1, sul2, and sul3) was observed in Cip and Sul groups (P < 0.05). A total of 3191 operational taxonomic units (OTUs) were obtained and 41 phyla were identified from 63 samples in shrimp intestine. The numbers of OTUs and Shannon index decreased rapidly at day 1 (the first day after feeding with antibiotics) and increased at day 3 (the third day after feeding with antibiotics). The relative abundance of dominant phyla and genera in Cip and Sul groups were significantly different from that in the control group (Ctrl). Furthermore, functional potentials that were related to amino acid metabolism, carbohydrate metabolism, and cellular processes and signaling varied significantly in Cip and Sul groups. These results point to an antibiotic-induced shift in shrimp intestinal microbiota, which highlights the importance of considering the microbiota in shrimp health management.


Assuntos
Ração Animal , Antibacterianos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Penaeidae/efeitos dos fármacos , Penaeidae/microbiologia , Animais , Antibacterianos/efeitos adversos , Aquicultura , Bactérias/classificação , Bactérias/efeitos dos fármacos , Ciprofloxacino/efeitos adversos , Ciprofloxacino/farmacologia , Resistência Microbiana a Medicamentos , Monitoramento Ambiental , Genes Bacterianos , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , RNA Ribossômico 16S/genética , Alimentos Marinhos , Sulfonamidas/efeitos adversos , Sulfonamidas/farmacologia
16.
Cell Microbiol ; 21(6): e13014, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30702192

RESUMO

The major virulence determinant of Legionella pneumophila is the type IVB secretion system (T4BSS), which delivers approximately 330 effector proteins into the host cell to modulate various cellular processes. However, the functions of most effector proteins remain unclear. WipA, an effector, was the first phosphotyrosine phosphatase of Legionella with unknown function. In this study, we found that WipA induced relatively strong growth defects in yeast in a phosphatase activity-dependent manner. Phosphoproteomics data showed that WipA was likely involved into endocytosis, FcγR-mediated phagocytosis, tight junction, and regulation of actin cytoskeleton pathways. Western blotting further confirmed WipA dephosphorylates several proteins associated with actin polymerisation, such as p-N-WASP, p-ARP3, p-ACK1, and p-NCK1. Thus, we hypothesised that WipA targets N-WASP/ARP2/3 complex signalling pathway, leading to disturbance of actin polymerisation. Indeed, we demonstrated that WipA inhibits host F-actin polymerisation by reducing the G-actin to F-actin transition during L. penumophila infection. Furthermore, the intracellular proliferation of wipA/legK2 double mutant was significantly impaired at the late stage of infection, although the absence of WipA does not confer any further effect on actin polymerisation to the legK2 mutant. Collectively, this study provides unique insights into the WipA-mediated regulation of host actin polymerisation and assists us to elucidate the pathogenic mechanisms of L. pnuemophila infection.

17.
J Virol ; 93(7)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30651355

RESUMO

Viruses associated with sleeping disease (SD) in crabs cause great economic losses to aquaculture, and no effective measures are available for their prevention. In this study, to help develop novel antiviral strategies, single-particle cryo-electron microscopy was applied to investigate viruses associated with SD. The results not only revealed the structure of mud crab dicistrovirus (MCDV) but also identified a novel mud crab tombus-like virus (MCTV) not previously detected using molecular biology methods. The structure of MCDV at a 3.5-Å resolution reveals three major capsid proteins (VP1 to VP3) organized into a pseudo-T=3 icosahedral capsid, and affirms the existence of VP4. Unusually, MCDV VP3 contains a long C-terminal region and forms a novel protrusion that has not been observed in other dicistrovirus. Our results also reveal that MCDV can release its genome via conformation changes of the protrusions when viral mixtures are heated. The structure of MCTV at a 3.3-Å resolution reveals a T= 3 icosahedral capsid with common features of both tombusviruses and nodaviruses. Furthermore, MCTV has a novel hydrophobic tunnel beneath the 5-fold vertex and 30 dimeric protrusions composed of the P-domains of the capsid protein at the 2-fold axes that are exposed on the virion surface. The structural features of MCTV are consistent with a novel type of virus.IMPORTANCE Pathogen identification is vital for unknown infectious outbreaks, especially for dual or multiple infections. Sleeping disease (SD) in crabs causes great economic losses to aquaculture worldwide. Here we report the discovery and identification of a novel virus in mud crabs with multiple infections that was not previously detected by molecular, immune, or traditional electron microscopy (EM) methods. High-resolution structures of pathogenic viruses are essential for a molecular understanding and developing new disease prevention methods. The three-dimensional (3D) structure of the mud crab tombus-like virus (MCTV) and mud crab dicistrovirus (MCDV) determined in this study could assist the development of antiviral inhibitors. The identification of a novel virus in multiple infections previously missed using other methods demonstrates the usefulness of this strategy for investigating multiple infectious outbreaks, even in humans and other animals.

18.
Circulation ; 139(1): 51-63, 2019 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-30586694

RESUMO

BACKGROUND: Eisenmenger syndrome describes congenital heart disease-associated severe pulmonary hypertension accompanied by right-to-left shunting. The multicenter, double-blind, randomized, placebo-controlled, 16-week, phase III MAESTRO study (Macitentan in Eisenmenger Syndrome to Restore Exercise Capacity) evaluated the efficacy and safety of the endothelin receptor antagonist macitentan in patients with Eisenmenger syndrome. METHODS: Patients with Eisenmenger syndrome aged ≥12 years and in World Health Organization functional class II-III were randomized 1:1 to placebo or macitentan 10 mg once daily for 16 weeks. Patients with complex cardiac defects, Down syndrome and background PAH therapy were eligible. The primary end point was change from baseline to week 16 in 6-minute walk distance. Secondary end points included change from baseline to week 16 in World Health Organization functional class. Exploratory end points included NT-proBNP (N-terminal pro-B-type natriuretic peptide) at end of treatment expressed as a percentage of baseline. In a hemodynamic substudy, exploratory end points included pulmonary vascular resistance index (PVRi) at week 16 as a percentage of baseline. RESULTS: Two hundred twenty six patients (macitentan n=114; placebo n=112) were randomized. At baseline, 60% of patients were in World Health Organization functional class II and 27% were receiving phosphodiesterase type-5 inhibitors. At week 16, the mean change from baseline in 6-minute walk distance was 18.3 m and 19.7 m in the macitentan and placebo groups (least-squares mean difference, -4.7 m; 95% confidence limit (CL), -22.8, 13.5; P=0.612). World Health Organization functional class improved from baseline to week 16 in 8.8% and 14.3% of patients in the macitentan and placebo groups (odds ratio, 0.53; 95% CL, 0.23, 1.24). NT-proBNP levels decreased with macitentan versus placebo (ratio of geometric means, 0.80; 95% CL, 0.68, 0.94). In the hemodynamic substudy (n=39 patients), macitentan decreased PVRi compared with placebo (ratio of geometric means, 0.87; 95% CL, 0.73, 1.03). The most common adverse events with macitentan versus placebo were headache (11.4 versus 4.5%) and upper respiratory tract infection (9.6 versus 6.3%); a hemoglobin decrease from baseline of ≥2 g/dL occurred in 36.0% versus 8.9% of patients. Five patients (3 macitentan; 2 placebo) prematurely discontinued treatment and 1 patient died (macitentan group). CONCLUSIONS: Macitentan did not show superiority over placebo on the primary end point of change from baseline to week 16 in exercise capacity in patients with Eisenmenger syndrome. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT01743001.

19.
Mol Plant Pathol ; 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30499216

RESUMO

Ralstonia solanacearum, one of the most destructive plant bacterial pathogens, delivers an array of effector proteins via its type III secretion system for pathogenesis. However, the biochemical functions of most of these proteins remain unclear. RipN is a type III effector with unknown function(s) from the pathogen R. solanacearum. Here, we demonstrate that RipN is a conserved type III effector found within the R. solanacearum species complex that contains a putative Nudix hydrolase domain and has ADP-ribose/NADH pyrophosphorylase activity in vitro. Further analysis showed that RipN localizes to endoplasmic reticulum (ER) and nucleus in Nicotiana tabacum leave cells and Arabidopsis protoplasts, and truncation of the C-terminal of RipN results in a loss of nuclear and ER targeting. Furthermore, expression of RipN in Arabidopsis suppressed callose deposition and transcription of PAMP-triggered immunity (PTI) marker genes under flg22 treatment, and promoted bacterial growth in plant. In addition, expression of RipN in plant cells alters NADH/NAD+ but not GSH/GSSG ratios, and its activity of Nudix hydrolase was indispensable for such biochemical function. These results suggest that RipN acts as a Nudix hydrolase, alters the NADH/NAD+ ratio of plant, and contributes to R. solanacearum virulence by suppressing the PTI of host. This article is protected by copyright. All rights reserved.

20.
Artigo em Inglês | MEDLINE | ID: mdl-30521966

RESUMO

Nervous necrosis virus (NNV) causes viral nervous necrosis (VNN), a disease that leads to almost 100% mortality among larvae and juvenile fish, severely affecting the aquaculture industry. VNN vaccines based on inactivated viruses or virus-like particles (VLPs) are unsuitable for fish fry with immature adaptive immune systems. Here, we applied an anti-NNV strategy based on affinity peptides (AFPs). Three phage display peptide libraries were screened against RBS, the VLP of orange-spotted grouper nervous necrosis virus (OGNNV). From the positive clones, a dodecapeptide with the highest binding capacity (BC) to RBS was selected. This AFP agglutinated or disrupted virion particles, inhibiting RBS entry into sea bass (SB) cells. To enhance BC and solubility, we amended the AFP sequence as "LHWDFQSWVPLL" and named as 12C. One to three copies of 12C in tandem were prokaryotically expressed with a maltose binding protein (MBP) linked by a flexible peptide. Of the recombinant proteins expressed, MBP-triple-12C (MBP-T12C) exhibited the highest BC, efficiently blocked RBS entry, and strongly inhibited OGNNV infection at viral entry. Moreover, MBP-T12C bound the VLPs of all NNV serotypes, displaying broad-spectrum anti-NNV ability, and recognized only OGNNV and mud crab virus, demonstrating binding specificity. Therefore, these anti-NNV AFPs specifically bound NNV, aggregating or disrupting the viral particles, to reduce the contact probability between the virus and cell surface, subsequently inhibiting viral infection. Our results not only provided a candidate of anti-NNV AFP, but a framework for the development of antiviral AFP.

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