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1.
Theranostics ; 10(23): 10498-10512, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32929362

RESUMO

Rationale: Current traditional treatment options are frequently ineffective to fight against ovarian cancer due to late diagnosis and high recurrence. Therefore, there is a vital need for the development of novel therapeutic agents. B7H3, an immune checkpoint protein, is highly expressed in various cancers, representing it a promising target for cancer immunotherapy. Although targeting B7H3 by bispecific T cell-engaging antibodies (BiTE) has achieved successes in hematological malignancies during recent years, attempts to use them for the treatment of solid cancers are less favorable, in part due to the heterogeneity of tumors. Sorafenib is an unselective inhibitor of multiple kinases currently being tested in clinical trials for several tumors, including ovarian cancer which showed limited activity and inevitable side effect for ovarian cancer treatment. However, it is able to enhance antitumor immune response, which indicates sorafenib may improve the efficiency of immunotherapy. Methods: We evaluated the expression of B7H3 in ovarian cancer using online database and validated its expression of tumor tissues by immunohistochemistry staining. Then, B7H3 expression and the effects of sorafenib on ovarian cancer cell lines were determined by flow cytometry. In addition, 2D and 3D ovarian cancer models were established to test the combined therapeutic effect in vitro. Finally, the efficiency of B7H3×CD3 BiTE alone and its combination with sorafenib were evaluated both in vitro and in vivo. Results: Our data showed that B7H3 was highly expressed in ovarian cancer compared with normal samples. Treatment with sorafenib inhibited ovarian cancer cell proliferation and induced a noticeable upregulation of B7H3 expression level. Further study suggested that B7H3×CD3 BiTE was effective in mediating T cell killing to cancer cells. Combined treatment of sorafenib and B7H3×CD3 BiTE had synergistic anti-tumor effects in ovarian cancer models. Conclusions: Overall, our study indicates that combination therapy with sorafenib and B7H3×CD3 BiTE may be a new therapeutic option for the further study of preclinical treatment of OC.

2.
Food Funct ; 11(5): 4707-4718, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32409814

RESUMO

Ferulic acid (FA) has been shown to have a neuroprotective effect on Alzheimer's disease induced by amyloid-beta (Aß) neurotoxicity. This work aims to ascertain the structure-activity relationship of FA and its alkyl esters (FAEs) for evaluating the antioxidant activities in PC12 cells and Aß1-42 aggregation inhibitory activities in vitro, as well as the signaling mechanisms against oxidative stress elicited by Aß1-42 in PC12 cells. Our data showed that alterations in the subcellular localization and cytotoxicity of FAEs caused by the lipophilicity of FA were crucial when evaluating their antioxidant capacities. Pre-treating cells with butyl ferulate (FAC4) significantly attenuated Aß1-42-evoked intracellular ROS formation. Besides, FAC4 exhibited the highest Aß1-42 aggregation inhibitory effectiveness. The molecular docking results showed that FAC4 binds to amide NH in Gln15 and Lys16 via a hydrogen bond. Notably, FAC4 could upregulate antioxidant defense systems by modulating the Keap1-Nrf2-ARE signaling pathway. Identification of the functions of FAEs could be useful in developing food supplements or drugs for treating AD.

3.
Food Chem ; 311: 125878, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31771910

RESUMO

ß-Carotene is a natural nutrient that serves as a natural food colorant. However, the weak physical stability restricts its development in food industrial production. Here, the influences of a variety of external environmental conditions on the stability of ß-carotene enriched zein-carboxymethyl chitosan (CMCS)-tea polyphenols (TP) ternary composite nanoparticles were investigated. Compared with zein unitary and zein-CMCS binary complexes, it was interesting to note that ternary complexes had the best stability against color fading and there was little impact on its nanoparticle size during storage with change in temperature. Besides excellent antioxidant properties, ternary complexes were extremely effective in inhibiting ß-carotene color degradation when exposed to ultraviolet light. Based on our results, the novel zein-CMCS-TP nanoparticles are expected to be an effective delivery system to encapsulate hydrophobic bioactive compounds, which is a promising approach to improve their storage stability against external environmental stresses.


Assuntos
Quitosana/análogos & derivados , Nanopartículas/química , Polifenóis/química , Zeína/química , beta Caroteno/química , Antioxidantes/química , Quitosana/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Concentração Osmolar , Tamanho da Partícula , Chá/química , Temperatura , Raios Ultravioleta
4.
Front Physiol ; 10: 1010, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31447699

RESUMO

Background: Small leucine-rich repeat proteins (SLRPs) are highly effective and selective modulators of cell proliferation and differentiation. Podocan is a newly discovered member of the SLRP family. Its potential roles in the differentiation of bovine muscle-derived satellite cells (MDSCs) and its underlying functional mechanism remain unclear. Our study aimed to characterize the function of the podocan gene in the differentiation of bovine MDSCs and to clarify the molecular mechanism by which podocan functions in order to contribute to a better understanding of the molecular mechanism by which extracellular matrix promotes bovine MDSC differentiation and provide a theoretical basis for the improvement of beef quality. Methods: Bovine MDSCs were transfected with vectors to overexpress or inhibit podocan, and podocan protein was added to differentiation culture medium. qRT-PCR, western blotting, and immunofluorescence were performed to investigate the effects of podocan on MDSC differentiation. Confocal microscopy and western blotting were used to assess the nuclear translocation and expression of ß-catenin. An inhibitor and activator of ß-catenin were used to assess the effects of the Wnt/ß-catenin signaling pathway on MDSC differentiation. We inhibited ß-catenin while overexpressing podocan in MDSCs. Then, we performed mass spectrometry to identify which proteins interact with podocan to regulate the Wnt/ß-catenin signaling pathway. Finally, we confirmed the relationship between podocan and Wnt4 by co-immunoprecipitation and western blotting. Results: Podocan protein expression increased significantly during bovine MDSC differentiation. Differentiation of bovine MDSC was promoted and suppressed by podocan overexpression or inhibition, respectively. Podocan was also shown to modulate the Wnt/ß-catenin signaling pathway. Treatment of bovine MDSCs with ß-catenin inhibitor and activator showed that the Wnt/ß-catenin pathway is involved in bovine MDSC differentiation. Furthermore, the effect of podocan on bovine MDSC differentiation was suppressed when this pathway was inhibited. We also found that podocan interacts with Wnt4. When Wnt4 was inhibited, podocan-induced promotion of bovine MDSC differentiation was attenuated through Wnt/ß-catenin signaling. Conclusion: Podocan regulates Wnt/ß-catenin through Wnt4 to promote bovine MDSC differentiation.

5.
Emerg Microbes Infect ; 8(1): 1229-1239, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31448697

RESUMO

Lysine acetylation and succinylation are both prevalent protein post-translational modifications (PTMs) in bacteria species, whereas the effect of the cross-talk between both PTMs on bacterial biological function remains largely unknown. Our previously study found lysine succinylated sites on proteins play important role on metabolic pathways in fish pathogenic Aeromonas hydrophila. A total of 3189 lysine-acetylation sites were further identified on 1013 proteins of this pathogen using LC-MS/MS in this study. Functional examination of these PTMs peptides showed associations with basal biological processes, especially metabolic pathways. Additionally, when comparing the obtained lysine acetylome to a previously obtained lysine succinylome, 1198 sites in a total of 547 proteins were found to be in common and associated with various metabolic pathways. As the autoinducer-2 (AI-2) synthase involved in quorum sensing of bacteria, the site-directed mutagenesis of LuxS at the K165 site was performed and revealed that the cross-talk between lysine acetylation and succinylation exerts an inverse influence on bacterial quorum sensing and on LuxS enzymatic activity. In summary, this study provides an in-depth A. hydrophila lysine acetylome profile and for the first time reveals the role of cross-talk between lysine acetylation and succinylation, and its potential impact on bacterial physiological functions.


Assuntos
Aeromonas hydrophila/metabolismo , Proteínas de Bactérias/metabolismo , Liases de Carbono-Enxofre/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Acetatos/análise , Acetilação , Aeromonas hydrophila/química , Aeromonas hydrophila/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/genética , Cromatografia Líquida , Análise Mutacional de DNA , Lisina/química , Mutagênese Sítio-Dirigida , Percepção de Quorum , Ácido Succínico/análise , Espectrometria de Massas em Tandem
6.
World J Microbiol Biotechnol ; 35(8): 127, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375931

RESUMO

Aeromonas hydrophila is a Gram-negative bacterium that causes serious infections in aquaculture and exhibits significant multidrug resistance. The LysR-type transcriptional regulator (LTTR) family proteins are a well-known group of transcriptional regulators involved in diverse physiological functions. However, the role of LTTRs in the regulation of bacterial resistance to antibiotics is still largely unknown. In this study, to further investigate the role of four putative LTTR family proteins (A0KIU1, A0KJ82, A0KPK0, and A0KQ63) in antibiotic resistance in A. hydrophila, their genes were cloned and overexpressed in engineered Escherichia coli. After the optimization of experimental conditions including incubation time, temperature, and IPTG concentration, these proteins were successfully purified, and their specific antibodies against mice were obtained. Using western blot analysis, we found that these LTTR family proteins were downregulated in A. hydrophila following antibiotic treatment, indicating that they may be involved in the regulation of antibiotic resistance. Additionally, minimum inhibitory concentration (MIC) assays of chloramphenicol (CM), chlortetracycline (CTC), ciprofloxacin (CF), furazolidone (FZ), and balofloxacin (BF) in E. coli showed that overexpression of these LTTRs led to increased sensitivity to several antibiotics. To further validate their functional role in antibiotic resistance, we demonstrated that bacteria with loss of A0KQ63 (ΔAHA_3980) exhibited multi-drug resistance properties. Our results indicate that these LTTR family proteins may play an important role in the antibiotic resistance of A. hydrophila, and the that underlying mechanisms controlling antibiotic resistance should be further investigated.


Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/genética , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Fatores de Transcrição/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Western Blotting , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Expressão Gênica , Perfilação da Expressão Gênica , Genes Bacterianos , Camundongos , Testes de Sensibilidade Microbiana , Fatores de Transcrição/análise , Fatores de Transcrição/genética
7.
Oncol Lett ; 18(1): 792-803, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31289556

RESUMO

The present study assessed whether estrogen receptor (ER)ß1 is associated with the survival of patients with advanced lung adenocarcinoma, with or without mutations of the epidermal growth factor receptor (EGFR) following treatment with EGFR-tyrosine kinase inhibitors (TKIs). Pathologically confirmed stage IV lung adenocarcinomas were assessed for EGFR mutations and ERß1 expression. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method and the log-rank test. A total of 122 out of the 201 (60.7%) patients had EGFR mutations, 64 (31.8%) of which were EGFR Del19 and 58 mutations (28.9%) were EGFR exon 21 L858R mutation. The presence of EGFR mutations was significantly increased in female patients compared with male patients (P<0.001) and in non-smokers compared with smokers (P<0.001). Patients with EGFR mutations had a significantly improved PFS and OS compared with patients without EGFR mutations treated with EGFR-TKIs. Furthermore, ERß1 expression was significantly increased in patients with EGFR mutations compared with patients without EGFR mutations (P=0.001). However, the median PFS (P=0.005) and OS (P=0.002) of patients carrying the EGFR exon 21 L858R mutation was significantly decreased in patients with tumors where ERß1 cytoplasmic expression was high. The multivariate analysis demonstrated that ERß1 expression was the only independent predictor of PFS (P=0.002) and OS (P=0.003) in patients carrying the EGFR exon 21 L858R mutation. The data demonstrated that ERß1 expression may predict outcomes of patients with lung adenocarcinoma treated with EGFR-TKI.

8.
Cell Biol Int ; 43(7): 799-808, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31050064

RESUMO

Muscle satellite cells are usually at rest, and when externally stimulated or regulated, they can be further differentiated by cell fusion to form new myotubes and muscle fibers. WD repeat domain 13 (WDR13) is highly conserved in vertebrates. Studies have shown that mice lacking the Wdr13 gene develop mild obesity, hyperinsulinemia, and increased islet ß cell proliferation. However, the role of WDR13 in bovine cells is unclear. Here, we investigated the effect of WDR13 on bovine skeletal muscle-derived satellite cells (MDSCs). We found that WDR13 was upregulated in bovine MDSCs using western blotting and immunofluorescence experiments. Moreover, activation and inhibition of WDR13 expression increased and decreased cell differentiation, respectively, suggesting that WDR13 promotes bovine MDSC differentiation. To further understand the mechanism of action of WDR13, we examined changes in the PI3K/AKT signaling pathway following WDR13 activation or inhibition. In addition, cells were treated with a phosphoinositide kinase 3 (PI3K) inhibitor, LY294004, to observe cell differentiation. The results showed that activation of WDR13 inhibited the PI3K/AKT signaling pathway and enhanced cell differentiation. These data suggest that WDR13 can promote the differentiation of bovine MDSCs by affecting the PI3K/AKT signaling pathway.


Assuntos
Diferenciação Celular , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Proteínas Nucleares/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Satélites de Músculo Esquelético/citologia , Animais , Bovinos , Proliferação de Células , Células Cultivadas , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Proteínas Nucleares/genética , Células Satélites de Músculo Esquelético/metabolismo , Transdução de Sinais
9.
Molecules ; 24(7)2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970602

RESUMO

Polyamines have been reported to be involved in grain filling and they might contribute to the construction of heat resistance of some cereals. In this study, the hybrid rice 'YLY 689' was used to explore the possible effects of exogenous spermidine (Spd) on seed quality under high temperature during the filling stage. Rice spikes were treated with Spd or its synthesis inhibitor cyclohexylamine (CHA) after pollination, and then the rice plants were transferred to 40 °C for 5-day heat treatment. The results showed that, compared with the control under high temperature, Spd pretreatment significantly improved the germination percentage, germination index, vigor index, seedling shoot height, and dry weight of seeds harvested at 35 days after pollination, while the CHA significantly decreased the seed germination and seedling growth. Meanwhile, Spd significantly increased the peroxidase (POD) activity and decreased the malondialdehyde (MDA) content in seeds. In addition, after spraying with Spd, the endogenous content of spermidine and spermine and the expression of their synthetic genes, spermidine synthase (SPDSYN) and spermine synthase (SPMS1 and SPMS2), significantly increased, whereas the accumulation of amylose and total starch and the expression of their related synthase genes, soluble starch synthase II-3 (SS II-3) and granules bound starch synthase I (GBSSI), also increased to some extent. The data suggests that exogenous Spd pretreatment could alleviate the negative impacts of high temperature stress on rice seed grain filling and improve the rice seed quality to some extent, which might be partly caused by up-regulating endogenous polyamines and starch metabolism.


Assuntos
Germinação/efeitos dos fármacos , Temperatura Alta , Oryza/crescimento & desenvolvimento , Sementes/crescimento & desenvolvimento , Espermidina/farmacologia , Termotolerância/efeitos dos fármacos
10.
J Cell Physiol ; 234(11): 21211-21223, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31025379

RESUMO

The glucose-regulated endoplasmic reticulum chaperone protein 94 (GRP94) is required for many biological processes, such as secretion of immune factors and mesoderm induction. Here, we demonstrated that GRP94 promotes muscle differentiation in vitro and in vivo. Moreover, GRP94 inhibited the PI3K/AKT/mTOR signaling pathway. Using both in vitro and in vivo approaches, in myoblasts, we found that this inhibition resulted in reduced proliferation and increased differentiation. To further investigate the mechanism of GRP94-induced muscle differentiation, we used co-immunoprecipitation and proximity ligation assays and found that GRP94 interacted with PI3K-interacting protein 1 (Pik3ip1). The latter protein promoted muscle differentiation by inhibiting the PI3K/AKT/mTOR pathway. Furthermore, GRP94 was found to regulate Pik3ip1 expression. Finally, when Pik3ip1 expression was inhibited, GRP94-induced promotion of muscle differentiation was diminished. Taken together, our data demonstrated that GRP94 promoted muscle differentiation, mediated by Pik3ip1-dependent inhibition of the PI3K/AKT/mTOR signaling pathway.

11.
Front Immunol ; 10: 256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30833947

RESUMO

In our previous study, several iron-related outer membrane proteins in Aeromonas hydrophila, a serious pathogen of farmed fish, conferred high immunoprotectivity to fish, and were proposed as potential vaccine candidates. However, the protective efficacy of these extracellular proteins against A. hydrophila remains largely unknown. Here, we identified secreted proteins that were differentially expressed in A. hydrophila LP-2 in response to iron starvation using an iTRAQ-based quantitative proteomics method. We identified 341 proteins, of which 9 were upregulated in response to iron starvation and 24 were downregulated. Many of the differently expressed proteins were associated with protease activity. We confirmed our proteomics results with Western blotting and qPCR. We constructed three mutants by knocking out three genes encoding differentially expressed proteins (Δorf01830, Δorf01609, and Δorf03641). The physiological characteristics of these mutants were investigated. In all these mutant strains, protease activity decreased, and Δorf01609, and Δorf01830 were less virulent in zebrafish. This indicated that the proteins encoded by these genes may play important roles in bacterial infection. We next evaluated the immune response provoked by the six iron-related recombinant proteins (ORF01609, ORF01830, ORF01839, ORF02943, ORF03355, and ORF03641) in zebrafish as well as the immunization efficacy of these proteins. Immunization with these proteins significantly increased the zebrafish immune response. In addition, the relative percent survival (RPS) of the immunized zebrafish was 50-80% when challenged with three virulent A. hydrophila strains, respectively. Thus, these extracellular secreted proteins might be effective vaccine candidates against A. hydrophila infection in fish.

12.
Int J Nanomedicine ; 14: 1805-1815, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30880981

RESUMO

Purpose: Resistant strains of Acinetobacter baumannii (AB) that can form biofilms are resistant to polymyxin. Therefore, effective and safe polymyxin preparations against biofilm-producing AB are urgently needed. This study aims to prepare chitosan-modified polymyxin B-loaded liposomes (CLPs) and ultrasound microbubbles (USMBs) and then explore the synergistic antibacterial effects of USMBs combined with CLPs in vitro. Methods: CLPs were prepared using a modified injection method, and microbubbles were prepared using a simple mechanical vibration method. Minimal biofilm inhibitory concentration (MBIC) of CLPs against resistant biofilm-producing AB was determined. Antibacterial activities of CLPs with or without USMBs were analyzed by crystal violet staining and resazurin assays to evaluate biofilm mass and viable counts, respectively. Then, the anti-biofilm effects of CLPs with or without USMBs on biofilm-producing AB were confirmed via scanning electron microscopy (SEM) analysis. Results: We prepared CLPs that were 225.17±17.85 nm in size and carried positive charges of 12.64±1.44 mV. These CLPs, with higher encapsulation efficiency and drug loading, could exhibit a sustained release effect. We prepared microbubbles that were 2.391±0.052 µm in size and carried negative charges of -4.32±0.43 mV. The MBICs of the CLPs on the biofilm-producing AB was 8±2 µg/mL, while that of polymyxin B was 32±2 µg/mL. USMBs in combination with 2 µg/mL of polymyxin B could completely eliminate the biofilm-producing AB and achieve the maximum antimicrobial effects (P>0.05 vs sterile blank control). SEM imaging revealed some scattered bacteria without a biofilm structure in the USMB combined with the CLP group, confirming that this combination has the greatest anti-biofilm effects. Conclusion: In this research, we successfully prepared USMBs and CLPs that have a more significant antibacterial effect on biofilm-forming AB than polymyxin B alone. Experiments in vitro indicate that the synergistic antibacterial effect of combining USMBs with CLPs containing as little as 2 µg/mL of polymyxin B is sufficient to almost eliminate drug-resistant biofilm-producing AB.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/fisiologia , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Quitosana/química , Microbolhas , Polimixina B/farmacologia , Ultrassom , Acinetobacter baumannii/ultraestrutura , Liberação Controlada de Fármacos , Sinergismo Farmacológico , Humanos , Lipossomos , Testes de Sensibilidade Microbiana
13.
Food Funct ; 10(2): 635-645, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30648709

RESUMO

The design of zein-based nanoparticles to encapsulate bioactive molecules has gained great attention in recent years. However, the use of ethanol to dissolve zein presents flammability concerns and the scale-up production of zein-based nanoparticles is also a concern. In our study, propolis loaded zein/caseinate/alginate nanoparticles were fabricated using a facile one-step procedure: a well-blended solution was prepared containing deprotonated propolis, soluble zein, dissociated sodium caseinate micelles (NaCas) and alginate at alkaline pH, and then this alkaline solution was added to 0.1 M citrate buffer (pH 3.8) to fabricate composite nanoparticles without using organic solvents and sophisticated equipment. During acidification, the alginate molecules adsorbed on the zein/NaCas surfaces by electrostatic complexation, which improved the stability towards aggregation of zein/NaCas nanoparticles under gastrointestinal (GI) or acidic pH. The nanoparticles prepared under the optimized method (method 3 sample) were of spherical morphology with a particle size around 208 nm and a negative zeta potential around -27 mV. The encapsulation efficiency (EE) and loading capacity (LC) of propolis reached 86.5% and 59.6 µg mg-1 by zein/NaCas/alginate nanoparticles, respectively. These nanoparticles were shown to be stable towards aggregation over a wide range of pH values (2-8) and salt concentrations (0-300 mM NaCl). Compared to free propolis, the bioaccessibility of propolis encapsulated with nanoparticles was increased to 80%. Our results showed a promising clean and scalability strategy to encapsulate hydrophobic nutraceuticals for applications in foods, supplements, and pharmaceuticals.


Assuntos
Alginatos/química , Caseínas/química , Nanopartículas/química , Própole/química , Zeína/química , Tecnologia de Alimentos , Microscopia Eletrônica de Transmissão
14.
J Proteomics ; 195: 1-10, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30597314

RESUMO

The RNA-binding protein Hfq protein is a well-characterized post-transcriptional regulator and plays an important role in the regulation of various physiological functions. Most bacterial genomes have only one copy of hfq, but a few bacterial species carry another distinct copy of hfq (hfq2) on the chromosome. However, the physiological properties of Hfq2 remain elusive. Here, we successfully constructed an hfq2 knock-out strain of Aeromonas hydrophila ATCC 7966. Phenotype assays showed that hfq2 deletion significantly increased extracellular protease activity, chemotaxis and swarming motility; increased low temperature, acidic pH, and basic pH resistance; and increased sensitivity to H2O2 stress and high temperatures. A SWATH-based quantitative proteomics method was used to compare the differential expression of proteins between the ∆hfq2 mutant and the wild-type strain. Bioinformatic analysis showed that proteins associated with metabolic pathways were mostly upregulated, while those associated with ribosome subunits were mostly downregulated. Moreover, the deletion of hfq2 leads to the increased expression of several DNA- or RNA-binding regulators, including Hfq and the catabolite gene activator (Crp), and the decreased expression of OmpR. To our knowledge, this is the first study to demonstrate the effects of Hfq2 on physiological function at the protein level. BIOLOGICAL SIGNIFICANCE: Most of bacterial genome has only one hfq copy, while only few bacterial species have two distinct copies in chromosome, and there are few documents about the biological functions of Hfq2. The current phenotype assays showed that Hfq2 plays important roles on bacterial physiological functions such as chemotaxis, swarming motility, ECPase activity and response to various environmental stresses. To better understanding the biological behavior of this protein, a SWATH based quantitative proteomics method was used to compare the altered proteins between ∆hfq2 and wide type strain. Bioinformatics analysis showed that ∆hfq2 significantly affects central metabolic pathway and translation related proteins. Moreover, the deletion of hfq2 lead to the increased expression of post-transcriptional regulator Hfq and catabolite gene activator Crp, and the down regulation of two-component regulatory system regulator OmpR. Our results indicate that Hfq2 is not a pseudogene but plays important roles on the essential physiological functions in A. hydrophila. To our knowledge, this is the first report to demonstrate the molecular function of Hfq2 at proteomics level.

15.
Mol Cell Proteomics ; 18(2): 200-215, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30352804

RESUMO

Protein modification by lysine succinylation is a newly identified post-translational modification (PTM) of lysine residues and plays an important role in diverse physiological functions, although their associated biological characteristics are still largely unknown. Here, we investigated the effects of lysine succinylation on the physiological regulation within a well-known fish pathogen, Aeromonas hydrophila A high affinity purification method was used to enrich peptides with lysine succinylation in A. hydrophila ATCC 7966, and a total of 2,174 lysine succinylation sites were identified on 666 proteins using LC-MS/MS. Gene ontology analysis indicated that these succinylated proteins are involved in diverse metabolic pathways and biological processes, including translation, protein export, and central metabolic pathways. The modifications of several selected candidates were further validated by Western blotting. Using site-directed mutagenesis, we observed that the succinylation of lysines on S-ribosylhomocysteine lyase (LuxS) at the K23 and K30 sites positively regulate the production of the quorum sensing autoinducer AI-2, and that these PTMs ultimately alter its competitiveness with another pathogen, Vibrio alginolyticus Moreover, subsequent metabolomic analyses indicated that K30 succinylation on LuxS may suppress the activated methyl cycle (AMC) and that both the K23 and K30 sites are involved in amino acid metabolism. Taken together, the results from this study provide significant insights into the functions of lysine succinylation and its critical roles on LuxS in regulating the cellular physiology of A. hydrophila.


Assuntos
Aeromonas hydrophila/fisiologia , Proteínas de Bactérias/metabolismo , Liases de Carbono-Enxofre/metabolismo , Lisina/química , Metabolômica/métodos , Percepção de Quorum , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/genética , Cromatografia Líquida , Ontologia Genética , Homosserina/análogos & derivados , Homosserina/metabolismo , Lactonas/metabolismo , Lisina/genética , Metaboloma , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem
16.
J Proteomics ; 194: 148-159, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30521976

RESUMO

Previously, a maltose-specific channel porin, LamB was found to be associate with multi-drug resistance in a lamB deleted strain, but the exact mechanisms require further elucidation. Herein, differential protein expression between the Escherichia coli mutant strain ΔlamB and the wild type strain BW25113, with and without ciprofloxacin (CFLX), was identified using iTRAQ based liquid chromatography-tandem mass spectrometry (LC-MS/MS); while differential metabolite expression was examined using gas chromatography-mass spectrometry (GC-MS). Further Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that lamB deletion leads to a decrease in several key metabolic pathways such as tricarboxylic acid (TCA) cycle, pentose phosphate pathway and glycolysis/gluconeogenesis. When examining the ΔlamB strain without CFLX, many aminoacyl-tRNA biosynthesis and pyrimidine metabolism-related proteins were unaltered, but the addition of CFLX resulted in reduced levels. These findings indicate that a lamB deletion may confer antibiotic resistance by relieving the pressure of protein translation and DNA replication. To further examine antibiotic resistance, exogenous metabolites, including maltose, and several amino acids metabolites were evaluated to determine whether the resistance level could be reduced in the presence of CFLX. The obtained results indicate that lamB knockout may increase bacterial antibiotics resistance by decreasing metabolic pathway activity levels. BIOLOGICAL SIGNIFICANCE: An integrated metabonomic-proteomic method was performed to systematically compare the profiles of metabolites and proteins between ΔlamB and its wild type strain, with and without ciprofloxacin (CFLX) treatment. Following bioinformatics analysis showed that lamB deletion in CFLX stress leads to the decreasing of several key metabolic pathways. Many amino acid-tRNA biosynthesis and pyrimidine metabolism related proteins didn't change in ΔlamB strain but largely decreased after treated with CFLX. Further exogenous metabolites addition assay reveals that maltose and several amino acids metabolites contribute to the CFLX resistance mediated by LamB. Our results indicate that the down-regulation of LamB may increase bacterial antibiotics resistance by decreasing the intracellular metabolism pathways.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Ciprofloxacino/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica , Porinas/metabolismo , Proteômica , Receptores Virais/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Redes e Vias Metabólicas/genética , Porinas/genética , Receptores Virais/genética
17.
Fish Shellfish Immunol ; 82: 50-59, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30086377

RESUMO

Iron-related proteins play important roles in iron homeostasis, and they may be potential vaccine candidates against pathogenic Aeromonas hydrophila. In addition, the encapsulation of antigens in single-walled carbon nanotubes (SWCNTs) has recently been shown to effectively stimulate the host immune response. To investigate the immune response of zebrafish to iron-related proteins and SWCNT-encapsulated proteins, we overexpressed and purified four iron-related recombinant proteins (P55870, A0KGK5, A0KPP0, and A0KIY3) from A. hydrophila. We then vaccinated zebrafish with these proteins and their SWCNT-encapsulated counterparts via both intraperitoneal injection and bath immunization. The target proteins evoked an immune response in zebrafish after intraperitoneal injection, and SWCNT-encapsulation significantly increased the immune response after bath immunization. When challenged with virulent A. hydrophila, zebrafish administered 5 µg intraperitoneal injections of SWCNT-P55870, A0KGK5, A0KPP0, or A0KIY3 had remarkably high relative percent survivals (RPSs) (50%, 55.6%, 66.7%, and 94.44% respectively). The RPSs of zebrafish vaccinated via immunization bath with 40 mg/L SWCNT-encapsulated counterparts were also high (52.94%, 55.56%, 61.11%, and 86.11%, respectively). These results indicated that zebrafish vaccinated with P55870, A0KGK5, SWCNT-P55870, and SWCNT-A0KGK5 were partially protected, while A0KPP0 and A0KIY3 were promising vaccine candidates against pathogenic A. hydrophila infection.


Assuntos
Proteínas de Bactérias/farmacologia , Doenças dos Peixes/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Imunidade Inata , Nanotubos de Carbono/análise , Peixe-Zebra , Aeromonas hydrophila/fisiologia , Animais , Infecções por Bactérias Gram-Negativas/imunologia , Injeções Intraperitoneais/veterinária , Ferro , Distribuição Aleatória , Proteínas Recombinantes/farmacologia
18.
Fish Shellfish Immunol ; 68: 1-9, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28676336

RESUMO

The iron-regulated outer membrane protein (OMP) of Aeromonas hydrophila is an effective vaccine candidate, but its intrinsic functional components are largely unknown. In this study, we compared the differentially expressed sarcosine-insoluble fractions of A. hydrophila in iron-limited and normal medium using tandem mass tag labeling-based quantitative proteomics, and identified 91 upregulated proteins including 21 OMPs and 83 downregulated proteins including 10 OMPs. Subsequent bioinformatics analysis showed that iron chelate transport-related proteins were enriched in increasing abundance, whereas oxidoreductase activity and translation-related proteins were significantly enriched in decreasing abundance. The proteomics results were further validated in selected altered proteins by Western blotting. Finally, the vaccine efficacy of five iron-related recombinant OMPs (A0KGW8, A0KFG8, A0KQ46, A0KIU8, and A0KQZ1) that were increased abundance in iron-limited medium, were evaluated when challenged with virulent A. hydrophila against zebrafish, suggesting that these proteins had highly efficient immunoprotectivity. Our results indicate that quantitative proteomics combined with evaluation of vaccine efficacy is an effective strategy for screening novel recombinant antigens for vaccine development.


Assuntos
Aeromonas hydrophila/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/imunologia , Doenças dos Peixes/prevenção & controle , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Peixe-Zebra , Aeromonas hydrophila/genética , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Vacinas Bacterianas/genética , Western Blotting/veterinária , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/prevenção & controle , Infecções por Bactérias Gram-Negativas/veterinária , Proteômica
19.
Neurology ; 88(18): 1727-1735, 2017 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-28381514

RESUMO

OBJECTIVE: To investigate the prevalence of and personal and professional characteristics associated with burnout, psychological morbidity, job stress, and job satisfaction in Chinese neurologists. METHODS: The China Neurologist Association conducted a national cross-sectional study from September 2014 to March 2015. A questionnaire including the Maslach Burnout Inventory, the 12-item General Health Questionnaire, the Consultants' Mental Health Questionnaire, and questions assessing personal and professional characteristics, career satisfaction, and current doctor-patient relationships was administered. RESULTS: A total of 693 directors of neurology departments and 6,111 neurologists in 30 Chinese provinces returned surveys. Overall, 53.2% of responding neurologists experienced burnout, 37.8% had psychological morbidity, 50.7% had high levels of job stress, 25.7% had low levels of job satisfaction, 76.9% had poor doctor-patient relationships, and 58.1% regretted becoming a doctor. Factors independently associated with burnout were lower income, more hours worked per week, more nights on call per month, working in public hospitals, psychological morbidity, high levels of job stress, low levels of job satisfaction, and poor doctor-patient relationships. Factors independently associated with psychological morbidity included lower income, more nights on call per month, working in enterprise-owned hospitals, burnout, high levels of job stress, and low levels of job satisfaction. CONCLUSIONS: Burnout and psychological morbidity are common in Chinese neurologists. Burnout is the single greatest predictor of neurologists' psychological morbidity, high job stress, and low job satisfaction.


Assuntos
Esgotamento Profissional , Satisfação no Emprego , Neurologistas/psicologia , Estresse Psicológico , Adulto , Esgotamento Profissional/epidemiologia , Escolha da Profissão , China/epidemiologia , Estudos Transversais , Feminino , Humanos , Renda , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Relações Médico-Paciente , Prevalência , Estresse Psicológico/epidemiologia , Inquéritos e Questionários , Carga de Trabalho/psicologia , Adulto Jovem
20.
J Sci Food Agric ; 97(4): 1124-1133, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27283044

RESUMO

BACKGROUND: Timely harvest is critical for hybrid rice to achieve maximum seed viability, vigor and yield. However, how to predict the optimum harvest time has been rarely reported so far. RESULTS: The seed vigor of Zhuliangyou 06 (ZLY06) increased and reached the highest level at 20 days after pollination (DAP), when seed moisture content had a lower value, which was maintained until final seed maturation. For Chunyou 84 (CY84), seed vigor, fresh and dry weight had relatively high values at 25 DAP, when seed moisture content reached the lowest value and changed slightly from 25 to 55 DAP. In both hybrid rice varieties, seed glume chlorophyll content declined rapidly from 10 to 30 DAP and remained at a very low level after 35 DAP. Starch content exhibited an increasing trend during seed maturation, while both soluble sugar content and amylase activity decreased significantly at the early stages of seed development. Moreover, correlation analyses showed that seed dry weight, starch content and superoxide dismutase activity were significantly positively correlated with seed vigor. In contrast, chlorophyll content, moisture content, soluble sugar, soluble protein, abscisic acid, gibberellin content, electrical conductivity, catalase and ascorbate peroxidase activities were significantly negatively correlated with seed vigor. Physiological and biochemical parameters were obviously more closely related with seed vigor than with seed germinability during seed development. CONCLUSION: Seed vigor could be better used as a comprehensive factor to predict the optimum seed harvest time. It is suggested that for ZLY06 seeds could be harvested as early as 20 DAP, whereas for CY84 the earliest optimum harvest time was 25 DAP. © 2016 Society of Chemical Industry.


Assuntos
Agricultura/métodos , Aptidão Genética , Germinação , Hibridização Genética , Oryza/crescimento & desenvolvimento , Melhoramento Vegetal , Sementes/crescimento & desenvolvimento , Ácido Abscísico/metabolismo , Biomassa , Metabolismo dos Carboidratos , Catalase/metabolismo , Clorofila/metabolismo , Giberelinas/metabolismo , Humanos , Vigor Híbrido , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Amido/metabolismo , Superóxido Dismutase/metabolismo
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