RESUMO
Irritable bowel syndrome (IBS) is a prevalent functional gastrointestinal disease characterized by chronic visceral pain with a complex etiology and challenging treatment. Although accumulating evidence supports the involvement of central nervous system sensitization in the development of visceral pain, the precise molecular mechanisms remain incompletely understood. In this study, we highlight the critical regulatory role of lysine-specific demethylase 6B (KDM6B) in the anterior cingulate cortex (ACC) in chronic visceral pain. To simulate clinical IBS conditions, we utilized the neonatal maternal deprivation (NMD) mouse model. Our results demonstrated that NMD induced chronic visceral pain and anxiety-like behaviors in mice. Notably, the protein expression level of KDM6B significantly increased in the ACC of NMD mice, leading to a reduction in the expression level of H32K7me3. Immunofluorescence staining revealed that KDM6B primarily co-localizes with neurons in the ACC, with minimal presence in microglia and astrocytes. Injecting GSK-J4 (a KDM6B-specific inhibitor) into ACC of NMD mice, resulted in a significant alleviation in chronic visceral pain and anxiety-like behaviors, as well as a remarkable reduction in NR2B expression level. ChIP assay further indicated that KDM6B regulates NR2B expression by influencing the demethylation of H3K27me3. In summary, our findings underscore the critical role of KDM6B in regulating chronic visceral pain and anxiety-like behaviors in NMD mice. These insights provide a basis for further understanding the molecular pathways involved in IBS and may pave the way for targeted therapeutic interventions.
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Type 1 fimbria, the short hair-like appendage assembled on the bacterial surface, plays a pivotal role in adhesion and invasion in Edwardsiella piscicida. The type III secretion system (T3SS), another bacterial surface appendage, facilitates E. piscicida's replication in vivo by delivering effectors into host cells. Our previous research demonstrated that E. piscicida T3SS protein EseJ inhibits adhesion and invasion of E. piscicida by suppressing type 1 fimbria. However, how EseJ suppresses type 1 fimbria remains elusive. In this study, a lacI-like operator (nt -245 to -1 of fimA) upstream of type 1 fimbrial operon in E. piscicida was identified, and EseJ inhibits type 1 fimbria through the lacI-like operator. Moreover, through DNA pull-down and electrophoretic mobility shift assay, an AraC-type T3SS regulator, EsrC, was screened and verified to bind to nt -145 to -126 and nt -50 to -1 of fimA, suppressing type 1 fimbria. EseJ is almost abolished upon the depletion of EsrC. EsrC and EseJ impede type 1 fimbria expression. Intriguingly, nutrition and microbiota-derived indole activate type 1 fimbria through downregulating T3SS, alleviating EsrC or EseJ's inhibitory effect on lacI-like operator of type 1 fimbrial operon. By this study, it is revealed that upon entering the gastrointestinal tract, rich nutrients and indole downregulate T3SS and thereof upregulate type 1 fimbria, stimulating efficient adhesion and invasion; upon being internalized into epithelium, the limit in indole and nutrition switches on T3SS and thereof switches off type 1 fimbria, facilitating effector delivery to guarantee E. piscicida's survival/replication in vivo.IMPORTANCEIn this work, we identified the lacI-like operator of type 1 fimbrial operon in E. piscicida, which was suppressed by the repressors-T3SS protein EseJ and EsrC. We unveiled that E. piscicida upregulates type 1 fimbria upon sensing rich nutrition and the microbiota-derived indole, thereof promoting the adhesion of E. piscicida. The increase of indole and nutrition promotes type 1 fimbria by downregulating T3SS. The decrease in EseJ and EsrC alleviates their suppression on type 1 fimbria, and vice versa.
Assuntos
Aderência Bacteriana , Proteínas de Bactérias , Edwardsiella , Fímbrias Bacterianas , Óperon , Sistemas de Secreção Tipo III , Edwardsiella/genética , Edwardsiella/fisiologia , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Animais , Regulação Bacteriana da Expressão Gênica , Infecções por Enterobacteriaceae/microbiologiaRESUMO
Edwardsiella ictaluri, a Gram-negative intracellular pathogen, is the causative agent of enteric septicemia in channel catfish, and catfish aquaculture in China suffers heavy economic losses due to E. ictaluri infection. Vaccination is an effective control measure for this disease. In this study, an attenuated E. ictaluri strain was acquired through deletion mutation of the T3SS protein eseJei, and the ΔeseJei strain fails to replicate in the epithelioma papillosum of carp cells. The type 1 fimbria plays a pivotal role in the adhesion of E. ictaluri, and it was found in this study that deletion of -245 to -50 nt upstream of fimA increases its adhesion to around five times that of the WT strain. A hyper-adhesive and highly attenuated double mutant (ΔeseJeiΔfimA-245--50 strain) was constructed, and it was used as a vaccine candidate in yellow catfish via bath immersion at a dosage of 1 × 105 CFU/mL. It was found that this vaccine candidate can stimulate protection when challenged with E. ictaluri HSN-1 at 5 × 107 CFU/mL (â¼20 × LD50). The survival rate was 83.61% for the vaccinated group and 33.33% for the sham-vaccinated group. The RPS (relative percent of survival) of the vaccination trial reached 75.41%. In conclusion, the ΔeseJeiΔfimA-245--50 strain developed in this study can be used as a vaccine candidate. It excels in terms of ease of delivery (via bath immersion) and is highly efficient in stimulating protection against E. ictaluri infection.
Assuntos
Vacinas Bacterianas , Peixes-Gato , Infecções por Enterobacteriaceae , Doenças dos Peixes , Animais , Aderência Bacteriana , Peixes-Gato/microbiologia , Edwardsiella ictaluri , Infecções por Enterobacteriaceae/prevenção & controle , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Imersão , Vacinas AtenuadasRESUMO
BACKGROUND: Aminoacyl-tRNA synthetases (ARSs) are enzymes responsible for attaching amino acids to tRNA, which enables protein synthesis. Mutations in isoleucyl-tRNA synthetase (IARS1) have recently been reported to be a genetic cause for growth retardation, intellectual disability, muscular hypotonia, and infantile hepatopathy (GRIDHH). CASE PRESENTATION: In this study, we reported an additional case of compound heterozygous missense variations c.701 T > C (p.L234P) and c.1555C > T (p.R519C) in IARS1, which were identified using medical exome sequencing; c.701 T > C (p.L234P) was a novel variant, and c.1555C > T (p.R519C) was found in GnomAD. Unlike other reported patients, this individual presented prominently with recurrent liver failure, which led to her death at an early age of 19 months. She also had significant growth retardation, muscular hypotonia, chubby and flabby face, recurrent loose stools, and abnormal brain computed tomography (CT), while zinc deficiency and hearing loss were not present. Studies in zebrafish embryo modeling recapitulated some of the key phenotypic traits in embryo development, neurodevelopment, liver development, and myogenesis, demonstrating that these variations caused a loss of gene function in IARS1. CONCLUSIONS: We have found a novel mutation point c.701 T > C (p.L234P) in IARS1. Compound heterozygous mutations of c.701 T > C (p.L234P) and c.1555C > T (p.R519C) in IARS1 are pathogenic, which can cause GRIDHH in child.
Assuntos
Falência Hepática , Hipotonia Muscular , Animais , China , Feminino , Transtornos do Crescimento , Humanos , Falência Hepática/genética , Mutação , Peixe-Zebra/genéticaRESUMO
The type III secretion system effector EseJ plays a regulatory role inside bacteria. It suppresses the adherence of Edwardsiella piscicida (E. piscicida) to host epithelial cells by down regulating type 1 fimbriae. In this study, we observed that more macrophages infected with ΔeseJ strain of E. piscicida detached as compared with those infected with the wild-type (WT) strain. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining and cleaved caspase-3 examination revealed that the detachment is due to increased apoptosis, suggesting that EseJ suppresses macrophage apoptosis. However, apoptosis inhibition by EseJ is not relative to a type III secretion system (T3SS) and is not related to EseJ's translocation. Since EseJ negatively regulates type 1 fimbriae, murine J774A.1 cells were infected with ΔeseJΔfimA or ΔeseJΔfimH strains. It was demonstrated that ΔeseJ stimulates macrophage apoptosis through type 1 fimbriae. Moreover, we found that infecting J774A.1 cells with the ΔeseJ strain increased levels of cleaved caspase-8, caspase-9, and caspase-3, demonstrating that EseJ inhibits apoptosis through either an extrinsic or a combination of extrinsic and intrinsic pathways. Pre-treatment of macrophages with caspase-8 inhibitor prior to infection with the ΔeseJ strain decreased the levels of cleaved caspase-8, caspase-9, and caspase-3, indicating that the ΔeseJ strain stimulates apoptosis, mainly through an extrinsic pathway by up regulating type 1 fimbriae. Zebrafish larvae or blue gourami fish infected with the ΔeseJ strain consistently exhibited higher apoptosis than those infected with the E. piscicida WT strain or ΔeseJΔfimA strain. Taken together, we revealed that the T3SS protein EseJ of E. piscicida inhibits host apoptosis, mainly through an extrinsic pathway by down regulating type 1 fimbriae.
Assuntos
Proteínas de Bactérias/metabolismo , Caspase 8/metabolismo , Edwardsiella/metabolismo , Fímbrias Bacterianas/metabolismo , Animais , Apoptose , Caspase 3 , Caspase 9 , Linhagem Celular , Edwardsiella/patogenicidade , Infecções por Enterobacteriaceae/metabolismo , Epitopos , Doenças dos Peixes/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Larva , Lipopolissacarídeos , Macrófagos , Camundongos , Sistemas de Secreção Tipo III/metabolismo , Peixe-ZebraRESUMO
Aeromonas salmonicida is a gram-negative bacterium that is the causative agent of furunculosis. An A. salmonicida strain was isolated from diseased turbot (Scophthalmus maximus) with the sign of furunculosis from North China. Based on vapA gene, the strain was further classified as A. salmonicida subsp. masoucida RZ6S-1. Culturing RZ6S-1 strain at high temperature (28°C) obtained the virulence attenuated strain RZ6S. Genome sequence comparison between the two strains revealed the loss of the type IV secretion system (T4SS) and type III secretion system (T3SS) from the native plasmid pAsmB-1 and pAsmC-1 of wild-type strain RZ6S-1, respectively. Further study demonstrated that the wild-type strain RZ6S-1, but not its derivative mutant RZ6S, can stimulate apoptosis. Elevated protein level of cleaved caspase-3 was detected from epithelioma papulosum cyprinid (EPC) cells infected with wild-type strain RZ6S-1 as compared with that infected with RZ6S strain. Meanwhile, the invasion of the mutant strain RZ6S was about 17-fold higher than the wild-type strain RZ6S-1, suggesting that some protein(s) from A. salmonicida subsp. masoucida RZ6S-1 suppress its invasion. The RZ6S mutant strain was attenuated, since its LD50 is over 10,000 times higher compared to the wild-type strain as revealed in the turbot infection model.
Assuntos
Aeromonas/patogenicidade , Doenças dos Peixes/microbiologia , Linguados/microbiologia , Furunculose/microbiologia , Aeromonas/classificação , Animais , Sistemas de Secreção Bacterianos/genética , China , Doenças dos Peixes/patologia , Furunculose/patologia , Plasmídeos/genéticaRESUMO
The type III secretion system (T3SS) of Edwardsiella piscicida plays a crucial role in its pathogenesis. Our previous study indicated that the T3SS effector protein EseJ inhibits the bacterium's adhesion to epithelioma papillosum cyprini (EPC) cells, while the mechanism of the inhibition remains elusive. In this study, we revealed that EseJ negatively regulates the fimA gene, as demonstrated by comparative transcription analysis of ΔeseJ and wild-type (WT) strains. As well, the dramatically increased production of FimA was detected in the absence of EseJ compared to that by the WT strain. The adherence of the ΔeseJ strain decreased far below that of the WT strain in the absence of FimA, demonstrating that FimA plays a pivotal role in the hyperadhesion of the ΔeseJ strain. Adherence analysis with a strain with truncated eseJ demonstrated that the C-terminal region of EseJ (Gly1191 to Ile1359) is necessary to inhibit the transcription of the type 1 fimbrial operon. Binding between the EseJ fragment from amino acid residues 1191 to 1359 and the DNA fragment upstream of fimA was not detected, indicating that EseJ might indirectly regulate the type 1 fimbrial operon. Our study reveals that EseJ controls E. piscicida adherence to EPC cells by negatively regulating the type 1 fimbrial operon.
Assuntos
Aderência Bacteriana/fisiologia , Edwardsiella/patogenicidade , Infecções por Enterobacteriaceae/microbiologia , Fímbrias Bacterianas/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Animais , Proteínas de Bactérias/genética , Edwardsiella/genética , Infecções por Enterobacteriaceae/metabolismo , Genes Bacterianos/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Transcrição Gênica/genética , Fatores de Virulência/genéticaRESUMO
The type III secretion system (T3SS) is one of the most important virulence factors of the fish pathogen Edwardsiella piscicida It contains three translocon proteins, EseB, EseC, and EseD, required for translocation of effector proteins into host cells. We have previously shown that EseB forms filamentous appendages on the surface of E. piscicida, and these filamentous structures mediate bacterial cell-cell interactions promoting autoaggregation and biofilm formation. In the present study, we show that EseC, but not EseD, inhibits the autoaggregation and biofilm formation of E. piscicida At 18 h postsubculture, a ΔeseC strain developed strong autoaggregation and mature biofilm formation, accompanied by enhanced formation of EseB filamentous appendages. This is in contrast to the weak autoaggregation and immature biofilm formation seen in the E. piscicida wild-type strain. EseE, a protein that directly binds to EseC and also positively regulates the transcription of the escC-eseE operon, was liberated and showed increased levels in the absence of EseC. This led to augmented transcription of the escC-eseE operon, thereby increasing the steady-state protein levels of intracellular EseB, EseD, and EseE, as well as biofilm formation. Notably, the levels of intracellular EseB and EseD produced by the ΔeseE and ΔeseC ΔeseE strains were similar but remarkably lower than those produced by the wild-type strain at 18 h postsubculture. Taken together, we have shown that the translocon protein EseC inhibits biofilm formation through sequestering EseE, a positive regulator of the escC-eseE operon.IMPORTANCEEdwardsiella piscicida, previously known as Edwardsiella tarda, is a Gram-negative intracellular pathogen that mainly infects fish. The type III secretion system (T3SS) plays a pivotal role in its pathogenesis. The T3SS translocon protein EseB is required for the assembly of filamentous appendages on the surface of E. piscicida The interactions between the appendages facilitate autoaggregation and biofilm formation. In this study, we explored the role of the other two translocon proteins, EseC and EseD, in biofilm formation. We have demonstrated that EseC, but not EseD, inhibits the autoaggregation and biofilm formation of E. piscicida, providing new insights into the regulatory mechanism involved in E. piscicida biofilm formation.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Biofilmes/efeitos dos fármacos , Edwardsiella/efeitos dos fármacos , Sistemas de Secreção Tipo III/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação ao Cálcio , Edwardsiella/genética , Doenças dos Peixes/microbiologia , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Glicoproteínas de Membrana , Óperon/genética , Receptores Citoplasmáticos e Nucleares , Receptores de Peptídeos , Fatores de Virulência/metabolismoRESUMO
One new aspidosperma-type alkaloid, melotenine A (1), together with five known ones (2-6), was isolated from the leaves of Melodinus axillaris. Their structures were elucidated by detailed spectroscopic analysis and quantum chemical ECD calculation. The isolated aspidosperma-type alkaloids were evaluated for their cytotoxicity against four human cancer cell lines and melotenine A showed significant cytotoxicity against all cells with IC50 values ranging from 0.6 to 1.5 µM.
Assuntos
Alcaloides/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Apocynaceae/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Modelos Moleculares , Estrutura MolecularRESUMO
High-speed counter-current chromatography was used to separate and purify galloyl, caffeoyl, and hexahydroxydiphenoyl esters of glucoses from the aerial parts of the parasitic plant Balanophora simaoensis for the first time using n-hexane-ethyl acetate-methanol-water (1:2:1:2, v/v) as the optimum solvent system. Accordingly, 1-O-(E)-caffeoyl-3-O-galloyl-ß-d-glucopyranose (I, 12.5 mg), 1-O-(E)-caffeoyl-3-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-ß-d-glucopyranose (II, 27.2 mg), and 1-O-(E)-caffeoyl-4,6-(S)-hexahydroxydiphenoyl-ß-d-glucopyranose (III, 52.8 mg) with 98.0%, 98.5%, and 98.7% purities, respectively, were purified from 210 mg crude extract of B. simaoensis in a one-step separation. The structures of the glucose esters were identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectra (NMR). Their antioxidant activities were evaluated by measuring their inhibition activity on liver microsomal lipid peroxidation induced by the Fe2+-Cys system in vitro. Compounds Iâ»III showed significant antioxidant activities with IC50 values ranging from 2.51 to 6.68 µm, respectively.
Assuntos
Antioxidantes/química , Antioxidantes/isolamento & purificação , Ésteres/química , Ésteres/isolamento & purificação , Glucose/química , Extratos Vegetais/química , Antioxidantes/farmacologia , Cromatografia Líquida de Alta Pressão , Distribuição Contracorrente , Ésteres/farmacologia , Estrutura Molecular , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Solventes/químicaRESUMO
Tryptanthrin (TRYP) is the main active ingredient in Indigo Naturalis. Studies have shown that TRYP had excellent anti-inflammatory activity, but its specific mechanism has been unclear. In this work, the differentially expressed proteins resulting from TRYP intervention in LPS-stimulated RAW264.7 cells were obtained based on tandem mass tag proteomics technology. The anti-inflammatory mechanism of TRYP was further validated by a combination of experiments using the LPS-induced RAW264.7 cell model in vitro and the DSS-induced UC mouse model (free drinking 2.5% DSS) in vivo. The results demonstrated that TRYP could inhibit levels of NO, IL-6, and TNF-α in LPS-induced RAW264.7 cells. Twelve differential proteins were screened out. And the results indicated that TRYP could inhibit upregulated levels of gp91phox, p22phox, FcεRIγ, IKKα/ß, and p-IκBα and reduce ROS levels in vitro. Besides, after TRYP treatment, the health conditions of colitis mice were all improved. Furthermore, TRYP inhibited the activation of JAK/STAT3, nuclear translocation of NF-κB p65, and promoted the nuclear expression of Nrf2 in vitro and in vivo. This work preliminarily indicated that TRYP might suppress the TLR4/MyD88/ROS/NF-κB and JAK/STAT3 signaling pathways to exert anti-inflammatory effects. Additionally, TRYP could achieve antioxidant effects by regulating the Keap1/Nrf2 signaling pathway.
RESUMO
Type III secretion system (T3SS) facilitates survival and replication of Edwardsiella piscicida in vivo. Identifying novel T3SS effectors and elucidating their functions are critical in understanding the pathogenesis of E. piscicida. E. piscicida T3SS effector EseG and EseJ was highly secreted when T3SS gatekeeper-containing protein complex EsaB-EsaL-EsaM was disrupted by EsaB deficiency. Based on this observation, concentrated secretomes of ΔesaB strain and ΔesaBΔesaN strain were purified by loading them into SDS-PAGE gel for a short electrophoresis to remove impurities prior to the in-the gel digestion and mass spectrometry. Four reported T3SS effectors and two novel T3SS effector candidates EseQ (ETAE_2009) and Trx2 (ETAE_0559) were unraveled by quantitative comparison of the identified peptides. EseQ and Trx2 were revealed to be secreted and translocated in a T3SS-dependent manner through CyaA-based translocation assay and immunofluorescent staining, demonstrating that EseQ and Trx2 are the novel T3SS effectors of E. piscicida. Trx2 was found to suppress macrophage apoptosis as revealed by TUNEL staining and cleaved caspase-3 of infected J774A.1 monolayers. Moreover, Trx2 has been shown to inhibit the p65 phosphorylation and p65 translocation into the nucleus, thus blocking the NF-κB pathway. Furthermore, depletion of Trx2 slightly but significantly attenuates E. piscicida virulence in a fish infection model. Taken together, an efficient method was established in unraveling T3SS effectors in E. piscicida, and Trx2, one of the novel T3SS effectors identified in this study, was demonstrated to suppress apoptosis and block NF- κB pathway during E. piscicida infection. IMPORTANCE Edwardsiella piscicida is an intracellular bacterial pathogen that causes intestinal inflammation and hemorrhagic sepsis in fish and human. Virulence depends on the Edwardsiella type III secretion system (T3SS). Identifying the bacterial effector proteins secreted by T3SS and defining their role is key to understanding Edwardsiella pathogenesis. EsaB depletion disrupts the T3SS gatekeeper-containing protein complex, resulting in increased secretion of T3SS effectors EseG and EseJ. EseQ and Trx2 were shown to be the novel T3SS effectors of E. piscicida by a secretome comparison between ∆esaB strain and ∆esaB∆esaN strain (T3SS mutant), together with CyaA-based translocation assay. In addition, Trx2 has been shown to suppress macrophage apoptosis and block the NF-κB pathway. Together, this work expands the known repertoire of T3SS effectors and sheds light on the pathogenic mechanism of E. piscicida.
Assuntos
Edwardsiella , Sistemas de Secreção Tipo III , Animais , Humanos , Sistemas de Secreção Tipo III/metabolismo , Fatores de Virulência/metabolismo , NF-kappa B , Edwardsiella/metabolismo , PeixesRESUMO
Two new stilbenoids, stemobenoids A (1) and B (2), together with three known compounds were obtained from the roots of Stemona tuberosa. The structures of the new compounds were established by extensive spectroscopic analysis, including HRMS, 1D and 2D NMR data. Compounds 1 and 2 displayed potent quinone reductase inducing activity in Hepa 1c1c7 cells.
Assuntos
Stemonaceae , Estilbenos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Raízes de PlantasRESUMO
Bacterial resistance to drugs is a global problem requiring the urgent development of new antibiotics. Antimicrobial peptides (AMPs) are excellent candidates for the design of novel antibiotics to combat microbial resistance. In this research, we identified four new peptides (U-VVTX-Vp1a, U-VVTX-Vp1b, U-VVTX-Vp2a, and U-VVTX-Vp2b, respectively) from the venom of Vespa velutina, and tested their antimicrobial, antioxidant, and hemolytic effects. All four peptides showed scavenging ability against DPPH, ABTS+, and â¢OH free radicals. Of note, Vp1b strongly inhibited the growth of Staphylococcus aureus and Escherichia coli bacteria at concentrations of 60 and 120 µM. Due to their low hemolytic activity, all four peptides could be utilized in the development of new antioxidants and as candidates for the design of novel antimicrobial agents.
Assuntos
Anti-Infecciosos , Vespas , Animais , Anti-Infecciosos/farmacologia , Hemólise , Testes de Sensibilidade Microbiana , Peptídeos/farmacologia , Venenos de VespasRESUMO
In this study, a hypothetical protein (ORF02740) secreted by Edwardsiella piscicida was identified. We renamed the ORF02740 protein as EvpQ, which is encoded by a mobile genetic element (MGE) in E. piscicida genome. The evpQ gene is spaced by 513 genes from type VI secretion system (T6SS) gene cluster. Low GC content, three tRNA, and three transposase genes nearby evpQ define this MGE that evpQ localizes as a genomic island. Sequence analysis reveals that EvpQ shares a conserved domain of C70 family cysteine protease and shares 23.91% identity with T3SS effector AvrRpt2 of phytopathogenic Erwinia amylovora. Instead, EvpQ of E. piscicida is proved to be secreted at a T6SS-dependent manner, and it can be translocated into host cells. EvpQ is thereof a novel T6SS effector. Significantly decreased competitive index of ΔevpQ strain in blue gourami fish (0.53 ± 0.27 in head kidney and 0.44 ± 0.19 in spleen) indicates that EvpQ contributes to the pathogenesis of E. piscicida. At 8-, 18-, and 24-h post-subculture into DMEM, the transcription of evpQ was found to be negatively regulated by Fur and positively regulated by EsrC, and the steady-state protein levels of EvpQ are negatively controlled by RpoS. Our study lays a foundation for further understanding the pathogenic role of T6SS in edwardsiellosis.
RESUMO
In order to investigate the response of soil respiration, soil microbial biomass carbon and nitrogen, and hydrothermal factors to the addition of biochar and straw, we used an LI-8100 soil carbon flux meter (LI-COR, Lincoln, USA) to study changes in soil respiration and microbial biomass under four treatments:conventional fertilization (CK), conventional fertilization +2.25t·hm-2 biochar-C (T1), conventional fertilizer +2.25t·hm-2 straw-C (T2), and conventional fertilizer +2.25t·hm-2 (biochar-C+straw-C), biochar-C:straw-C=1:1 (T3). The results showed that:â the addition of biochar and straw significantly increased the soil respiration rate and total CO2 emissions, with the largest increase in T3 and the smallest increase in T1. The effect of T1 on soil respiration was promoted in the early stage and later inhibited. â¡ The microbial biomass carbon and nitrogen and the number of functional bacterial colonies increased significantly with biochar and straw amendments. T1 had a significant promotion effect on nitrogen-fixing bacteria, while T2 had no significant effect on the number of fungi, and T3 showed a positive interaction effect. Soil respiration rates were significantly and positively related to soil microbial biomass carbon and nitrogen as well as to the number of bacteria and actinomycetes. ⢠The 5 cm soil temperature of T3 significantly increased by 4.53%. The soil respiration rate and soil temperature showed a significant exponential correlation. To sum up, adding straw and biochar with equal carbon content can significantly increase the soil respiration rate and microbial biomass, and the interaction effect between biochar and straw is positive. Compared with that of the straw treatments, the application of biochar can reduce carbon mineralization to a certain extent, and the effect of carbon sequestration is better.
Assuntos
Carbono , Solo , Agricultura , Biomassa , Carvão Vegetal , Fertilizantes , Nitrogênio/análise , Respiração , Microbiologia do SoloRESUMO
Four new aspidosperma-type alkaloids, melosuavines J-M (1-4) were isolated from the leaves of Melodinus suaveolens. Their structures with absolute configurations were elucidated by extensive HRESIMS and NMR spectroscopic analysis, as well as ECD calculations and Mosher's method. Their cytotoxic activities against four human cancer cell lines were also evaluated.
Assuntos
Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apocynaceae/química , Folhas de Planta/química , Alcaloides/isolamento & purificação , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , China , Humanos , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologiaRESUMO
Two polyketides, stemonones A (1) and B (2) with new skeletons, were isolated from the roots of Stemona tuberosa. Their absolute structures were fully characterized by comprehensive spectroscopic analyses and comparison of experimental electronic circular dichroism (ECD) spectra with calculated ones. The plausible biosynthetic pathways for 1 and 2 were also proposed. Anti-inflammatory assay confirmed that the two compounds showed moderate inhibitory effects on ß-glucuronidase release in rat polymorphonuclear leukocytes (PMNs) induced by platelet-activating factor.
Assuntos
Anti-Inflamatórios/química , Policetídeos/química , Stemonaceae/química , Animais , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Células Cultivadas , China , Glucuronidase/metabolismo , Estrutura Molecular , Neutrófilos/efeitos dos fármacos , Raízes de Plantas/química , Policetídeos/isolamento & purificação , Policetídeos/farmacologia , RatosRESUMO
Type III secretion system (T3SS) is a large macromolecular assembly found on the surface of many pathogenic Gram-negative bacteria. Edwardsiella tarda is an important Gram-negative pathogen that employs T3SS to deliver effectors into host cells to facilitate its survival and replication. EseB, EseC, and EseD, when secreted, form a translocon complex EseBCD on host membranes through which effectors are translocated. The orf19 gene (esaE) of E. tarda is located upstream of esaK, and downstream of esaJ, esaI, esaH and esaG in the T3SS gene cluster. When its domains were searched using Delta-Blast, the EsaE protein was found to belong to the T3SS YscJ/PrgK family. In the present study, it is found that EsaE is not secreted into culture supernatant, and the deletion of esaE abolished the secretion of T3SS translocon proteins EseBCD and T3SS effector EseG. Increased steady-state protein level of EseC and EseD was detected in bacterial pellet of ΔesaE strain although a reduced level was observed for the eseC and eseD transcription. EsaE was found to localize on membrane but not in the cytoplasm of E. tarda by fractionation. In blue gourami fish infection model, 87.88% of blue gourami infected with ΔesaE strain survived whereas only 3.03% survived when infected with wild-type strain. Taken together, our study demonstrated that EsaE is probably an apparatus protein of T3SS, which contributes to the pathogenesis of E. tarda in fish.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Sistemas de Secreção Bacterianos/metabolismo , Edwardsiella tarda , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/fisiopatologia , Virulência/genética , Animais , Proteínas de Bactérias/genética , Edwardsiella tarda/genética , Edwardsiella tarda/patogenicidade , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/fisiopatologia , Doenças dos Peixes/microbiologia , Deleção de GenesRESUMO
BACKGROUND: This study interviewed community pharmacists in Shanghai and Guangzhou for their perception of the popular categories of over-the-counter (OTC) Chinese medicines and the factors affecting customer preferences for OTC Chinese medicines. METHODS: A cross-sectional survey was carried out in six main administrative districts in Guangzhou and eight main administrative districts in Shanghai, China. Descriptive statistical analysis was conducted in this study. RESULTS: OTC Chinese medicines contributed 21-50% among all the pharmaceutical sales by the community pharmacies. The prevalent categories of OTC Chinese medicines were common cold medicines, respiratory system medicines, digestive system agents, gynecological medicines, health tonic medicines, and qing re (heat-clearing) and qu du (detoxifying) medicines. Customers were more concerned about medical factors of OTC Chinese medicines than business factors. Among the medical factors, the most important was drug safety, followed by efficacy, contraindications, indications, and side effects. Among the business factors, the most important were brand and price. CONCLUSIONS: This study identified the top sales categories of OTC Chinese medicines in Shanghai and Guangzhou and the important factors such as drug safety, efficacy, period of validity, contraindications, and indications that are affecting the customer preferences for OTC Chinese medicines.