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1.
Biochem Biophys Res Commun ; 625: 134-139, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-35961137

RESUMO

Kinds of antibiotics are used to prevent and control bacteria infections, unfortunately, the overuse and misuse of antibiotic have promoted the emergence and spread of antibiotic-resistant bacteria. Therefore, understanding the mechanism of antibiotic resistance is very important. This study explores the combined effection of metal ions and antibiotic to the drug resistance of Escherichia coli. Our results found that the minimum inhibitory concentration (MIC) increased as the ammonium ferric citrate concentration increased, especially for gentamicin antibiotic. When the Fe3+ concentration reached 300 µM, the survival of E. coli was stably restored with the increased gentamicin concentration. Exogenous Fe3+ could decrease intracellular gentamicin concentration. On the other hand, Fe3+ treatment together with gentamicin could reduce reactive oxygen species (ROS) production, characterized by decreased levels of NADH and ATP. Furthermore, ROS-scavenging enzymes of superoxide dismutase (SOD) and catalase (CAT) were up-regulated and H2O2 plus gentamicin-mediated killing was restored by Fe3+. These results may have significant implications in understanding bacterial antibiotic-resistant mechanisms based on the external Fe3+ concentration.


Assuntos
Infecções por Escherichia coli , Gentamicinas , Antibacterianos/farmacologia , Bactérias , Escherichia coli , Gentamicinas/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Ferro/farmacologia , Testes de Sensibilidade Microbiana , Espécies Reativas de Oxigênio
2.
BMC Plant Biol ; 22(1): 274, 2022 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-35659253

RESUMO

BACKGROUND: WRKY transcription factors play key roles in plant development processes and stress response. Kandelia obovata is the most cold-resistant species of mangrove plants, which are the important contributors to coastal marine environment. However, there is little known about the WRKY genes in K. obovata. RESULTS: In this study, a WRKY transcription factor gene, named KoWRKY40, was identified from mangrove plant K. obovata. The full-length cDNA of KoWRKY40 gene was 1420 nucleotide bases, which encoded 318 amino acids. The KoWRKY40 protein contained a typical WRKY domain and a C2H2 zinc-finger motif, which were common signatures to group II of WRKY family. The three-dimensional (3D) model of KoWRKY40 was formed by one α-helix and five ß-strands. Evolutionary analysis revealed that KoWRKY40 has the closest homology with a WRKY protein from another mangrove plant Bruguiera gymnorhiza. The KoWRKY40 protein was verified to be exclusively located in nucleus of tobacco epidermis cells. Gene expression analysis demonstrated that KoWRKY40 was induced highly in the roots and leaves, but lowly in stems in K. obovata under cold stress. Overexpression of KoWRKY40 in Arabidopsis significantly enhanced the fresh weight, root length, and lateral root number of the transgenic lines under cold stress. KoWRKY40 transgenic Arabidopsis exhibited higher proline content, SOD, POD, and CAT activities, and lower MDA content, and H2O2 content than wild-type Arabidopsis under cold stress condition. Cold stress affected the expression of genes related to proline biosynthesis, antioxidant system, and the ICE-CBF-COR signaling pathway, including AtP5CS1, AtPRODH1, AtMnSOD, AtPOD, AtCAT1, AtCBF1, AtCBF2, AtICE1, AtCOR47 in KoWRKY40 transgenic Arabidopsis plants. CONCLUSION: These results demonstrated that KoWRKY40 conferred cold tolerance in transgenic Arabidopsis by regulating plant growth, osmotic balance, the antioxidant system, and ICE-CBF-COR signaling pathway. The study indicates that KoWRKY40 is an important regulator involved in the cold stress response in plants.


Assuntos
Arabidopsis , Rhizophoraceae , Antioxidantes/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Prolina/metabolismo , Rhizophoraceae/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
J Proteome Res ; 20(1): 972-981, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33231461

RESUMO

Antibiotic-resistant bacteria are a serious threat to human and animal health. Metabolite-enabled eradication of drug-resistant pathogens is an attractive strategy, and metabolite adjuvants, such as fumarate, are used for restoring the bactericidal ability of antibiotics. However, we show that metabolites in the TCA cycle increase the viability of Edwardsiella tarda against chloramphenicol (CAP), based on the survival assay of differential metabolites identified by LC-MS/MS. Furthermore, NADPH promotes CAP resistance in the CAP-resistant strain, while oxidants restore the bactericidal ability. Finally, we show that the intracellular redox state determines the sensitivity to CAP, and the total antioxidative capacity is decreased significantly in the antibiotic-resistant strain. Considering that the metabolites promote CAP resistance, metabolite adjuvants should be applied very cautiously. Overall, our research expands on the knowledge that the redox state is related to the bactericidal ability of CAP.


Assuntos
Edwardsiella tarda , Doenças dos Peixes , Animais , Antibacterianos/farmacologia , Cloranfenicol/farmacologia , Cromatografia Líquida , Humanos , Espectrometria de Massas em Tandem
4.
BMC Plant Biol ; 21(1): 10, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407136

RESUMO

BACKGROUND: Low temperature is a major abiotic stress that seriously limits mangrove productivity and distribution. Kandelia obovata is the most cold-resistance specie in mangrove plants, but little is known about the molecular mechanism underlying its resistance to cold. Osmotin is a key protein associated with abiotic and biotic stress response in plants but no information about this gene in K. obovata was reported. RESULTS: In this study, a cDNA sequence encoding osmotin, KoOsmotin (GenBank accession no. KP267758), was cloned from mangrove plant K. obovata. The KoOsmotin protein was composed of 221 amino acids and showed a calculated molecular mass of 24.11 kDa with pI 4.92. The KoOsmotin contained sixteen cysteine residues and an N-terminal signal peptide, which were common signatures to most osmotins and pathogenesis-related 5 proteins. The three-dimensional (3D) model of KoOsmotin, contained one α-helix and eleven ß-strands, was formed by three characteristic domains. Database comparisons of the KoOsmotin showed the closest identity (55.75%) with the osmotin 34 from Theobroma cacao. The phylogenetic tree also revealed that the KoOsmotin was clustered in the branch of osmotin/OLP (osmotin-like protien). The KoOsmotin protein was proved to be localized to both the plasma membrane and cytoplasm by the subcellular localization analysis. Gene expression showed that the KoOsmotin was induced primarily and highly in the leaves of K. obovata, but less abundantly in stems and roots. The overexpressing of KoOsmotin conferred cold tolerance in Escherichia coli cells. CONCLUSION: As we known, this is the first study to explore the osmotin of K. obovata. Our study provided valuable clues for further exploring the function of KoOsmotin response to stress.


Assuntos
Temperatura Baixa/efeitos adversos , Resposta ao Choque Frio/genética , Resposta ao Choque Frio/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Rhizophoraceae/genética , Rhizophoraceae/fisiologia , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/metabolismo , Análise de Sequência de DNA
5.
Proc Natl Acad Sci U S A ; 115(7): E1578-E1587, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29382755

RESUMO

The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in Escherichia coli and Edwardsiella tarda These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism.


Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Edwardsiella tarda/efeitos dos fármacos , Edwardsiella tarda/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Ácido Pirúvico/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Fosfoenolpiruvato/metabolismo
6.
Environ Microbiol ; 22(10): 4295-4313, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32291842

RESUMO

Colistin is a last-line antibiotic against Gram-negative multidrug-resistant bacteria, but the increased resistance poses a huge challenge to this drug. However, the mechanisms underlying such resistance are largely unexplored. The present study first identified the mutations of two genes encoding AceF subunit of pyruvate dehydrogenase (PDH) and TetR family transcriptional regulator in colistin-resistant Vibrio alginolyticus (VA-RCT ) through genome sequencing. Then, gas chromatography-mass spectroscopy-based metabolomics was adopted to investigate metabolic responses since PDH plays a role in central carbon metabolism. Colistin resistance was associated with the reduction of the central carbon metabolism and energy metabolism, featuring the alteration of the pyruvate cycle, a recently characterized energy-producing cycle. Metabolites in the pyruvate cycle reprogramed colistin-resistant metabolome to colistin-sensitive metabolome, resulting in increased gene expression, enzyme activity or protein abundance of the cycle and sodium-translocating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase. This reprogramming promoted the production of the proton motive force that enhances the binding between colistin and lipid A in lipopolysaccharide. Moreover, this metabolic approach was effective against VA-RCT in vitro and in vivo as well as other clinical isolates. These findings reveal a previously unknown mechanism of colistin resistance and develop a metabolome-reprogramming approach to promote colistin efficiency to combat with colistin-resistant bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Colistina/metabolismo , Farmacorresistência Bacteriana Múltipla/fisiologia , Complexo Piruvato Desidrogenase/metabolismo , Quinona Redutases/metabolismo , Vibrio alginolyticus/efeitos dos fármacos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Metabolismo Energético/genética , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Lipídeo A/metabolismo , Potenciais da Membrana/fisiologia , Metaboloma/genética , Metabolômica/métodos , Complexo Piruvato Desidrogenase/genética , Transativadores/genética , Vibrio alginolyticus/genética , Vibrio alginolyticus/isolamento & purificação
7.
J Proteome Res ; 14(3): 1612-20, 2015 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-25675328

RESUMO

Edwardsiella tarda, the causative agent of Edwardsiellosis, imposes medical challenges in both the clinic and aquaculture. The emergence of multidrug resistant strains makes antibiotic treatment impractical. The identification of molecules that facilitate or promote antibiotic efficacy is in high demand. In the present study, we aimed to identify small molecules whose abundance is correlated with kanamycin resistance in E. tarda by gas chromatography-mass spectrometry. We found that the abundance of fructose was greatly suppressed in kanamycin-resistant strains. The incubation of kanamycin-resistant bacteria with exogenous fructose sensitized the bacteria to kanamycin. Moreover, the fructose also functioned in bacteria persisters and biofilm. The synergistic effects of fructose and kanamycin were validated in a mouse model. Furthermore, the mechanism relies on fructose in activating TCA cycle to produce NADH, which generates proton motive force to increase the uptake of the antibiotics. Therefore, we present a novel approach in fighting against multidrug resistant bacteria through exploration of antibiotic-suppressed molecules.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Edwardsiella tarda/efeitos dos fármacos , Frutose/farmacologia , Canamicina/farmacologia , Testes de Sensibilidade Microbiana
8.
Anal Chim Acta ; 1307: 342642, 2024 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-38719399

RESUMO

BACKGROUND: Similar to hypochlorous acid (HClO), hypobromous acid (HBrO) is one of the most notable reactive oxygen species (ROS). Overexpression of HBrO is linked to various diseases causing organ and tissue loss. Due to HBrO's role in the oxidation of micropollutants, real-time monitoring of HBrO in water-based systems is essential. Tetraphenylethylene (TPE)-based organic aggregation-induced emission luminophores (AIEgens) are an emerging category of fluorescent probe materials that have attracted considerable attentions. However, AIE probes are rarely applied to detect HBrO. Developing faster, more precise, and more sensitive AIE probes is thus crucial for detecting biological and environmental HBrO. RESULTS: A small molecule fluorescent probe 4-(1,2,2-triphenylvinyl)benzamidoxime (SWJT-21) was synthesized for the sensitive and selective detection of hypobromous acid (HBrO) based on aggregation-induced emission (AIE). The amidoxime unit of SWJT-21 would undergo an oxidation reaction with HBrO, leading to a structure differentiation between the probe and the product, and therefore the turn-on fluorescence by the AIE effect. The probe could recognize hypobromous acid rapidly (less than 3 s) in high aqueous phase (99 % water) with a turn-on fluorescence response. It was determined that the limit of detection for HBrO was 5.47 nM. Moreover, SWJT-21 demonstrates potential as a test strip for the detection of HBrO. SWJT-21 was also successfully used for the monitoring of HBrO in water samples and for the detection of endogenous/exogenous HBrO in living cells and zebrafish. SIGNIFICANCE: A special AIE fluorescence turn-on probe SWJT-21 based on tetraphenylethylene was designed for detecting HBrO in the environmental and biological systems. This probe has an extremely low detection limit of 5.47 nM and is able to detect HBrO in 99 % aqueous phase in less than 3 s.


Assuntos
Bromatos , Corantes Fluorescentes , Estilbenos , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Bromatos/análise , Bromatos/química , Estilbenos/química , Animais , Humanos , Peixe-Zebra , Espectrometria de Fluorescência , Limite de Detecção , Estrutura Molecular
9.
Bioresour Technol ; 401: 130708, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38636878

RESUMO

In this study, the biochemical response of Phaeodactylum tricornutum to varying concentrations of inorganic selenium (Se) was investigated. It was observed that, when combined with fulvic acid, P. tricornutum exhibited enhanced uptake and biotransformation of inorganic Se, as well as increased microalgal lipid biosynthesis. Notably, when subjected to moderate (5 and 10 mg/L) and high (20 and 40 mg/L) concentrations of selenite under fulvic acid treatment, there was a discernible redirection of carbon flux towards lipogenesis and protein biosynthesis from carbohydrates. In addition, the key parameters of microalgae-based biofuels aligned with the necessary criteria outlined in biofuel regulations. Furthermore, the Se removal capabilities of P. tricornutum, assisted by fulvic acid, were coupled with the accumulation of substantial amounts of organic Se, specifically SeCys. These findings present a viable and successful approach to establish a microalgae-based system for Se uptake and biotransformation.


Assuntos
Benzopiranos , Biocombustíveis , Biotransformação , Diatomáceas , Diatomáceas/metabolismo , Benzopiranos/metabolismo , Ácido Selenioso/metabolismo , Microalgas/metabolismo
10.
Cell Death Dis ; 15(7): 483, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38969650

RESUMO

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, and the expression and function of an uncharacterized protein RNF214 in HCC are still unknown. Phase separation has recently been observed to participate in the progression of HCC. In this study, we investigated the expression, function, and phase separation of RNF214 in HCC. We found that RNF214 was highly expressed in HCC and associated with poor prognosis. RNF214 functioned as an oncogene to promote the proliferation, migration, and metastasis of HCC. Mechanically, RNF214 underwent phase separation, and the coiled-coil (CC) domain of RNF214 mediated its phase separation. Furthermore, the CC domain was necessary for the oncogenic function of RNF214 in HCC. Taken together, our data favored that phase separation of RNF214 promoted the progression of HCC. RNF214 may be a potential biomarker and therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular , Proliferação de Células , Progressão da Doença , Neoplasias Hepáticas , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Humanos , Linhagem Celular Tumoral , Animais , Movimento Celular/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Masculino , Camundongos Nus , Camundongos , Regulação Neoplásica da Expressão Gênica , Feminino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Separação de Fases
11.
Front Pharmacol ; 15: 1393693, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38855753

RESUMO

Colorectal cancer is a common malignant tumor with high mortality, for which chemotherapy resistance is one of the main reasons. The high expression of ABCG2 in the cancer cells and expulsion of anticancer drugs directly cause multidrug resistance (MDR). Therefore, the development of new ABCG2 inhibitors that block the active causes of MDR may provide a strategy for the treatment of colorectal cancer. In this study, we find that dorsomorphin (also known as compound C or BML-275) potently inhibits the transporter activity of ABCG2, thereby preserving the chemotherapeutic agents mitoxantrone and doxorubicin to antagonize MDR in ABCG2-overexpressing colorectal cancer cells. Additionally, dorsomorphin does not alter ABCG2 protein expression. The results of molecular docking studies show that dorsomorphin is bound stably to the ABCG2-binding pocket, suggesting that dorsomorphin is a potent ABCG2 inhibitor that attenuates ABCG2-mediated MDR in colorectal cancer.

12.
Sci Adv ; 9(10): eade8582, 2023 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-36888710

RESUMO

The mechanism(s) of how bacteria acquire tolerance and then resistance to antibiotics remains poorly understood. Here, we show that glucose abundance decreases progressively as ampicillin-sensitive strains acquire resistance to ampicillin. The mechanism involves that ampicillin initiates this event via targeting pts promoter and pyruvate dehydrogenase (PDH) to promote glucose transport and inhibit glycolysis, respectively. Thus, glucose fluxes into pentose phosphate pathway to generate reactive oxygen species (ROS) causing genetic mutations. Meanwhile, PDH activity is gradually restored due to the competitive binding of accumulated pyruvate and ampicillin, which lowers glucose level, and activates cyclic adenosine monophosphate (cAMP)/cAMP receptor protein (CRP) complex. cAMP/CRP negatively regulates glucose transport and ROS but enhances DNA repair, leading to ampicillin resistance. Glucose and Mn2+ delay the acquisition, providing an effective approach to control the resistance. The same effect is also determined in the intracellular pathogen Edwardsiella tarda. Thus, glucose metabolism represents a promising target to stop/delay the transition of tolerance to resistance.


Assuntos
Ampicilina , Bactérias , Espécies Reativas de Oxigênio/metabolismo , Ampicilina/farmacologia , Bactérias/metabolismo , Glucose/metabolismo , Piruvatos
13.
Front Microbiol ; 13: 845173, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35547113

RESUMO

Antibiotic-resistant Pseudomonas aeruginosa is insensitive to antibiotics and difficult to deal with. An understanding of the resistance mechanisms is required for the control of the pathogen. In this study, gas chromatography-mass spectrometer (GC-MS)-based metabolomics was performed to identify differential metabolomes in ciprofloxacin (CIP)-resistant P. aeruginosa strains that originated from P. aeruginosa ATCC 27853 and had minimum inhibitory concentrations (MICs) that were 16-, 64-, and 128-fold (PA-R16CIP, PA-R64CIP, and PA-R128CIP, respectively) higher than the original value, compared to CIP-sensitive P. aeruginosa (PA-S). Upregulation of fatty acid biosynthesis forms a characteristic feature of the CIP-resistant metabolomes and fatty acid metabolome, which was supported by elevated gene expression and enzymatic activity in the metabolic pathway. The fatty acid synthase inhibitor triclosan potentiates CIP to kill PA-R128CIP and clinically multidrug-resistant P. aeruginosa strains. The potentiated killing was companied with reduced gene expression and enzymatic activity and the returned abundance of fatty acids in the metabolic pathway. Consistently, membrane permeability was reduced in the PA-R and clinically multidrug-resistant P. aeruginosa strains, which were reverted by triclosan. Triclosan also stimulated the uptake of CIP. These findings highlight the importance of the elevated biosynthesis of fatty acids in the CIP resistance of P. aeruginosa and provide a target pathway for combating CIP-resistant P. aeruginosa.

14.
Front Oncol ; 12: 938234, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36176418

RESUMO

Metabolomics has been reported as an efficient tool to screen biomarkers that are related to esophageal cancer. However, the metabolic biomarkers identifying malignant degrees and therapeutic efficacy are still largely unknown in the disease. Here, GC-MS-based metabolomics was used to understand metabolic alteration in 137 serum specimens from patients with esophageal cancer, which is approximately two- to fivefold as many plasma specimens as the previous reports. The elevated amino acid metabolism is in sharp contrast to the reduced carbohydrate as a characteristic feature of esophageal cancer. Comparative metabolomics showed that most metabolic differences were determined between the early stage (0-II) and the late stage (III and IV) among the 0-IV stages of esophageal cancer and between patients who received treatment and those who did not receive treatment. Glycine, serine, and threonine metabolism and glycine were identified as the potentially overlapped metabolic pathway and metabolite, respectively, in both disease progress and treatment effect. Glycine, fructose, ornithine, and threonine can be a potential array for the evaluation of disease prognosis and therapy in esophageal cancer. These results highlight the means of identifying previously unknown biomarkers related to esophageal cancer by a metabolomics approach.

15.
Plant Methods ; 17(1): 100, 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34587982

RESUMO

BACKGROUND: Mangroves plants, an important wetland system in the intertidal shores, play a vital role in estuarine ecosystems. However, there is a lack of a very effective method for extracting protein from mangrove plants for proteomic analysis. Here, we evaluated the efficiency of three different protein extraction methods for proteomic analysis of total proteins obtained from mangrove plant Kandelia obovata leaves. RESULTS: The protein yield of the phenol-based (Phe-B) method (4.47 mg/g) was significantly higher than the yields of the traditional phenol (Phe) method (2.38 mg/g) and trichloroacetic acid-acetone (TCA-A) method (1.15 mg/g). The Phe-B method produced better two-dimensional electrophoresis (2-DE) protein patterns with high reproducibility regarding the number, abundance and coverage of protein spots. The 2-DE gels showed that 847, 650 and 213 unique protein spots were separated from the total K. obovata leaf proteins extracted by the Phe-B, Phe and TCA-A methods, respectively. Fourteen pairs of protein spots were randomly selected from 2-DE gels of Phe- and Phe-B- extracted proteins for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) technique, and the results of three pairs were consistent. Further, oxygen evolving enhancer protein and elongation factor Tu could be observed in the 2-DE gels of Phe and Phe-B methods, but could only be detected in the results of the Phe-B methods, showing that Phe-B method might be the optimized choice for proteomic analysis. CONCLUSION: Our data provides an improved Phe-B method for protein extraction of K. obovata and other mangrove plant tissues which is rich in polysaccharides and polyphenols. This study might be expected to be used for proteomic analysis in other recalcitrant plants.

16.
Talanta ; 233: 122567, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215063

RESUMO

Photodynamic therapy (PDT) received great attention in cancer therapy due to the advantages of negligible drug resistance, low side effects, and minimal invasiveness. Development of theranostic nanoprobes with specific imaging-guided PDT is of great significance in the field. Herein we report the fabrication of a novel theranostic nanoprobe porphyrin/G-quadruplex conjugated gold/persistent luminescence nanocomposites for imaging-guided PDT. The developed nanoprobe contains NIR-emitting persistent luminescent nanoparticles (PLNP) as the core for autofluorescence-free bioimaging and Au coating on PLNP for facile subsequent DNA conjugation. The DNA sequence is designed to contain G-rich AS1411 aptamer for recognizing the over-expressed cellular nucleolin of cancer cell and forming a G-quadruplex structure to combine with tetrakis (4-carboxyphenyl) porphyrin (TCPP) to realize PDT. The AS1411 aptamer-contained DNA conjugated Au-coated PLNP is rapidly prepared via a freezing method with high content of DNA and good aqueous stability. Meanwhile, TCPP is easily loaded into the G-quadruplex structure formed from G-rich AS1411 aptamer on the surface of Au/PLNP in presence of K+. The theranostic nanoprobe gives integrated merits of PLNP for autofluorescence-free bioimging, TCPP for PDT and AS1411 aptamer-contained DNA for specific binding to cancer cells. This work provides a new specially designed imaging-guided PDT nanoplatform for theranostics.


Assuntos
Fotoquimioterapia , Porfirinas , Linhagem Celular Tumoral , Ouro , Luminescência , Medicina de Precisão
17.
Front Immunol ; 12: 739591, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34950133

RESUMO

Vaccines are safe and efficient in controlling bacterial diseases in the aquaculture industry and are in line with green farming. The present study develops a previously unreported approach to prepare a live-attenuated V. alginolyticus vaccine by culturing bacteria in a high concentration of magnesium to attenuate bacterial virulence. Furthermore, metabolomes of zebrafish immunized with the live-attenuated vaccines were compared with those of survival and dying zebrafish infected by V. alginolyticus. The enhanced TCA cycle and increased fumarate were identified as the most key metabolic pathways and the crucial biomarker of vaccine-mediated and survival fish, respectively. Exogenous fumarate promoted expression of il1ß, il8, il21, nf-κb, and lysozyme in a dose-dependent manner. Among the five innate immune genes, the elevated il1ß, il8, and lysozyme are overlapped in the vaccine-immunized zebrafish and the survival from the infection. These findings highlight a way in development of vaccines and exploration of the underlying mechanisms.


Assuntos
Vacinas Bacterianas/imunologia , Ciclo do Ácido Cítrico/imunologia , Magnésio/imunologia , Vibrio alginolyticus/imunologia , Animais , Vacinas Atenuadas/imunologia , Peixe-Zebra/imunologia
18.
mSystems ; 6(4): e0069421, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34427511

RESUMO

Misuse and overuse of antibiotics drive the selection and spread of antibiotic-resistant bacteria. Although genetic mutations have been well defined for different types of antibiotic resistance, ways to revert antibiotic resistance are largely unexplored. Here, we adopted a proteomics approach to investigate the mechanism underlying ciprofloxacin resistance in Edwardsiella tarda, a representative pathogen that infects both economic animal species and human beings. By comparing the protein expression profiles of ciprofloxacin-sensitive and -resistant E. tarda, a total of 233 proteins of differential abundance were identified, where 53 proteins belong to the functional categories of metabolism, featuring a disrupted pyruvate cycle and decreased energy metabolism but increased fatty acid biosynthesis. The altered pyruvate cycle and energy metabolism were confirmed by gene expression and biochemical assays. Furthermore, the role of fatty acid biosynthesis and quinolone resistance were explored. The expression level and enzymatic activity of acetyl coenzyme A (acetyl-CoA) carboxylase, the first step of fatty acid biosynthesis, were increased in ciprofloxacin-resistant E. tarda. Treatment of ciprofloxacin-resistant E. tarda with acetyl-CoA carboxylase and 3-oxoacyl-[acyl carrier protein] synthase II inhibitors, 2-aminooxazole and triclosan, respectively, reduced the expression of fatty acid biosynthesis and promoted quinolone-mediated killing efficacy to antibiotic-resistant bacteria. Similar results were obtained in clinically isolated E. tarda strains. Our study suggests that energy metabolism has been reprogramed in ciprofloxacin-resistant bacteria that favor the biosynthesis of fatty acid, presenting a novel target to tackle antibiotic-resistant bacteria. IMPORTANCE Edwardsiella tarda is the causative agent of edwardsiellosis, which imposes huge challenges on clinics and aquaculture. Due to the overuse of antibiotics, the emergence and spread of antibiotic-resistant E. tarda threaten human health and animal farming. However, the mechanism of ciprofloxacin resistance in E. tarda is still lacking. Here, iTRAQ (isobaric tags for relative and absolute quantification)-based proteomics was performed to identify a differential proteome between ciprofloxacin-sensitive and -resistant E. tarda. The fluctuated pyruvate cycle and reduced energy metabolism and elevated fatty acid biosynthesis are metabolic signatures of ciprofloxacin resistance. Moreover, inhibition of biosynthesis of fatty acids promotes quinolone-mediated killing efficacy in both lab-evolved and clinically isolated strains. This study reveals that a ciprofloxacin resistance mechanism is mediated by the elevated biosynthesis of fatty acids and the depressed pyruvate metabolism and energy metabolism in E. tarda. These findings provide a novel understanding for the ciprofloxacin resistance mechanism in E. tarda.

19.
Front Microbiol ; 12: 800442, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35310395

RESUMO

Multidrug-resistant Pseudomonas aeruginosa has become one of global threat pathogens for human health due to insensitivity to antibiotics. Recently developed reprogramming metabolomics can identify biomarkers, and then, the biomarkers were used to revert the insensitivity and elevate antibiotic-mediated killing. Here, the methodology was used to study cefoperazone/sulbactam (SCF)-resistant P. aeruginosa (PA-RSCF) and identified reduced glycolysis and pyruvate cycle, a recent clarified cycle providing respiratory energy in bacteria, as the most key enriched pathways and the depressed glucose as one of the most crucial biomarkers. Further experiments showed that the depression of glucose was attributed to reduction of glucose transport. However, exogenous glucose reverted the reduction to elevate intracellular glucose via activating glucose transport. The elevated glucose fluxed to the glycolysis, pyruvate cycle, and electron transport chain to promote downstream proton motive force (PMF). Consistently, exogenous glucose did not promote SCF-mediated elimination but potentiated aminoglycosides-mediated killing since aminoglycosides uptake is PMF-dependent, where amikacin was the best one. The glucose-potentiated amikacin-mediated killing was effective to both lab-evolved PA-RSCF and clinical multidrug-resistant P. aeruginosa. These results reveal the depressed glucose uptake causes the reduced intracellular glucose and expand the application of metabolome-reprogramming on selecting conventional antibiotics to achieve the best killing efficacy.

20.
J Proteomics ; 212: 103562, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-31733415

RESUMO

Antibiotic-resistant bacteria are an increasingly serious threat to human health and aquaculture. To further explore bacterial antibiotic resistance mechanism, iTRAQ is used to identify a differential proteome in ampicillin-resistant LTB4 (LTB4-RAMP), a strain of Edwardsiella piscicida. A total of 102 differentially proteins with 50 upregulation and 52 downregulation are identified. Since many of these changes are related to metabolism, interactive pathways explorer(iPath) is used to understand a global differentially metabolic response in LTB4-RAMP. This analysis identifies a global depressed metabolic modulation as the most characteristic feature of LTB4-RAMP. Lower membrane potential and ATP in LTB4-RAMP than control support that the central carbon metabolism and energy metabolism are reduced. Since the pyruvate cycle (the P cycle) plays a key role in the central carbon metabolism and energy metabolism, further investigation focuses on the P cycle and shows that expression of genes and activity of enzymes in the P cycle are decreased in LTB4-RAMP. These results support the conclusion that the depressed P cycle contributes to the acquisition of ampicillin resistance in E.piscicida. These findings indicate that the combination of proteomics and iPath analysis can provide a global metabolic profile, which helps us better understand the correlation between ampicillin resistance and cellular metabolism. SIGNIFICANCE: The present study uses iTRAQ to explore ampicillin resistance mechanism in Edwardsiella piscicida and finds many of these differential abundances of proteins are related to metabolism. IPath further identifies a global depressed metabolic modulation and characterizes the reduced pyruvate cycle as the most characteristic feature of the ampicillin-resistant E. piscicida, which is supported by reduced expression of genes and activity of enzymes in the pyruvate cycle. Consisitently, lower membrane potential and ATP are detetced. These results reveal the metabolic mechanism of ampicillin resistance and provide a solid proof to revert the resistance by reprogramming metabolomics.


Assuntos
Resistência a Ampicilina , Ampicilina/farmacologia , Ciclo do Ácido Cítrico , Edwardsiella/patogenicidade , Infecções por Enterobacteriaceae/tratamento farmacológico , Proteoma/metabolismo , Ácido Pirúvico/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antibacterianos/farmacologia , Aquicultura , Proteínas de Bactérias/metabolismo , Edwardsiella/efeitos dos fármacos , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Potenciais da Membrana , Proteoma/análise , Proteômica/métodos , Ácido Pirúvico/antagonistas & inibidores
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