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High-performance liquid tandem mass spectrometry (HPLC-MS) is widely employed for protein analysis in biological systems. However, conventional proteomic sample pretreatment methods suffer from multiple steps and poor reproducibility. In this study, we introduce EPURISp (Enzymatic Digestion with Ultrafiltration and Rapid In-situ Sample Purification), a novel proteomic pretreatment technique that combines enzymatic digestion, ultrafiltration, and one-step temperature-controlled vacuum drying for efficient desalting. The EPURISp method exhibits excellent protein recovery rates across a wide range of molecular weights and hydrophilicity, surpassing traditional C18 desalting approaches. Practical proteomic analysis (PXD044209) utilizing EPURISp demonstrates the highest protein identification yield with remarkable reproducibility, which is particularly advantageous in membrane protein identification. Notably, EPURISp exhibits superior performance in minimizing oxidation and deamidation modifications compared with conventional FASP methods. This innovative EPURISp method represents a significant advancement in proteomics analysis, providing reliable and efficient results for mass spectrometry.
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This work reports a portable, origami-type paper device with a plasmonic fluor-labeled microneedle sensing module for the multiplexed quantification of anemia biomarkers in whole blood. Sequential steps, including serum separation, target enrichment, and multiplexed readout by a gel imager, are rapidly accomplished with the flexible and highly integrated device. The microneedle array enabled efficient sampling of trace targets from ng mL-1 to pg mL-1 level. Combined with the plasmonic fluor label, the signal is improved by ≈7.6 folds compared with the flat substrate-based assay. The device is applied to simultaneously quantify hemoglobin (Hb), ferritin, folic acid (FA), and vitamin B12 (VB12 ), which are four anemia biomarkers distributed in different environments with different concentration ranges. Featured by the small sample volume (150 µL), short assay time (20 min), low cost (2 $), robust stability, and user-friendliness, the device is promising for the rapid and accurate diagnosis of anemia in real practice.
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Anemia , Humanos , Anemia/diagnóstico , Ácido Fólico , Vitamina B 12 , Hemoglobinas , BiomarcadoresRESUMEN
Cronobacter sakazakii is an opportunistic pathogen capable of causing severe infections, particularly in neonates. Despite the bacterium's strong pathogenicity, the pathogenicity of C. sakazakii is not yet well understood. Using a comparative proteomic profiling approach, we successfully identified pdxY, encoding a pyridoxal kinase involved in the recycling of pyridoxal 5'-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in slower growth and reduced virulence. Our study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii. The identification of pdxY as gene essential for successful pathogenesis provides a potential target for the development of new antibiotic treatments. IMPORTANCE The opportunistic pathogen Cronobacter sakazakii is known to cause severe infections, particularly in neonates, and can result in high mortality rates. In this study, we used a comparative proteomic profiling approach to identify genes essential for the successful pathogenesis of C. sakazakii. We successfully identified pdxY, encoding a pyridoxal kinase involved in the salvage pathway of pyridoxal 5'-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in impaired growth and reduced virulence. This study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii, which can be a potential target for the development of new antibiotic treatments. This study highlights the importance of comparative proteomic profiling in identifying virulence factors that can be targeted for the development of new antibiotics.
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Cronobacter sakazakii , Cronobacter , Recién Nacido , Humanos , Vitamina B 6 , Virulencia , Piridoxal Quinasa/genética , Cronobacter sakazakii/genética , Cronobacter sakazakii/metabolismo , Proteómica , Fosfato de Piridoxal/metabolismo , Piridoxina , Antibacterianos , Fosfatos , VitaminasRESUMEN
Cronobacter sakazakii is a Gram-negative bacterium that causes infections in individuals of all ages, with neonates being the most vulnerable group. The objective of this study was to explore the function of the dnaK gene in C. sakazakii and to elucidate the impact of alterations in the protein composition regulated by dnaK on virulence and stress adaptation. Our research demonstrates the critical role of the dnaK gene in various key virulence factors, including adhesion, invasion, and acid resistance in C. sakazakii. Through the use of proteomic analysis, we discovered that deletion of the dnaK gene in C. sakazakii leads to an upregulation of protein abundance and increased levels of deamidated posttranscriptional modifications, suggesting that DnaK may play a role in maintaining proper protein activity by reducing protein deamidation in bacteria. These findings indicate that DnaK-mediated protein deamidation may be a novel mechanism for virulence and stress adaptation in C. sakazakii. These findings suggest that targeting DnaK could be a promising strategy for developing drugs to treat C. sakazakii infections. IMPORTANCE Cronobacter sakazakii can cause disease in individuals of all ages, with infections in premature infants being particularly deadly and resulting in bacterial meningitis and sepsis with a high mortality rate. Our study demonstrates that dnaK in Cronobacter sakazakii plays a critical role in virulence, adhesion, invasion, and acid resistance. Using proteomic analysis to compare protein changes in response to dnaK knockout, we found that dnaK knockout significantly upregulates the abundance of some proteins but also results in the deamidation of many proteins. Our research has identified a connection between molecular chaperones and protein deamidation, which suggests a potential future drug development strategy of targeting DnaK as a drug target.
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Proteínas Bacterianas , Cronobacter sakazakii , Chaperonas Moleculares , Cronobacter sakazakii/patogenicidad , Cronobacter sakazakii/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mutación , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Técnicas de Inactivación de Genes , Proteínas/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Adaptación FisiológicaRESUMEN
A straightforward, largely instrument-free, smartphone-based analytical strategy for hexavalent chromium and lysine (Lys) on-site detection via exploitation of dual-emission carbon quantum dots (DECQDs) has been demonstrated. DECQDs show dual-emission peaks at 439 and 630 nm with the excitation at 375 nm. As a dual-mode detection probe, the fluorescence and ultraviolet adsorption spectra of DECQDs vary with hexavalent chromium concentrations. Most importantly, Lys can restore the fluorescence of the hexavalent chromium added DECQD nanoprobe and change the color of the probe under natural light. At the same time, based on the participation of smartphones, the prepared DECQD probes favor the establishment of visual smart sensors that can also be used for the in-situ detection of targets. The on-site quantitative analysis exhibited a linear range of 5.3-320 µM with a detection limit of 1.6 µM towards Cr(VI) and the differentiation of Lys variation from 1 to 75 mM with a detection limit of 0.3 mM. The probe has been applied for the first time to enable vision-based colorimetric in complex samples such as water, milk and egg. The recoveries of Cr(VI) and Lys in real samples were between 90 and 104%, and the relative standard deviation (RSD) was as low as 0.4%. This work offers new perspectives for fundamental understanding and new design of functional luminescent materials that are applicable for food-safety and rapid and intelligent inspection. A straightforward, large instrument-free, smartphone-based analytical strategy with dual-emission carbon quantum dots was developed for hexavalent chromium and Lys on-site detection via fluorescent and colorimetric twofold readout measure.
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Puntos Cuánticos , Carbono , Cromo , Lisina , Teléfono InteligenteRESUMEN
Trophoblast cell-surface antigen 2 (Trop-2) is a tumor-associated antigen that is connected with the development of various tumors and has been identified as a promising target for tumor immunotherapy. To date, the immunotherapy against Trop-2 mainly relies on the specific targeting by monoclonal antibody in antibody-drug conjugate (ADC). Alternatively, the single domain antibodies of nanobodies (Nbs) possesses unique properties such as smaller size, better tissue penetration, etc., to make them good candidates for tumor targeting. Thus, it was proposed to develop anti-Trop-2 Nbs for tumor targeting in this study. Generally, three consecutive rounds of bio-panning were performed against immobilized recombinant Trop-2, and yielded three Nbs (Nb60, Nb65, and Nb108). The affinity of selected Nbs was determined in the nanomolar range, especially the good properties of Nb60 were verified as a promising candidate for tumor labeling. The binding to native Trop-2 was confirmed by flow cytometry against tumor cells. The inhibitory effects of the selected Nbs on tumor cell proliferation and migration were confirmed by wound healing and Transwell assay. The clear localization of the selected Nbs on the surface of tumor cells verified the potent labeling efficiency. In conclusion, this study provided several Nbs with the potential to be developed as targeting moiety of drug conjugates.
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Inmunoconjugados , Neoplasias , Anticuerpos de Dominio Único , Anticuerpos Monoclonales , Citometría de Flujo , Humanos , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológicoRESUMEN
Functional nanoprobes which detect specific food hazards quickly and simply are still in high demand in the field of food-safety inspection research. In the present work, a dual-emission metal-organic framework-based ratiometric fluorescence probe was integrated to detect Cu2+ and Pb2+ with rapidness and ease. Specifically, quantum dots (QDs) and carbon quantum dots (CQDs) were successfully embedded into zeolitic imidazolate framework-67 (ZIF-67) to function as a novel ratiometric fluorescent sensing composite. The ratiometric fluorescence signal of CQDs/QDs@ZIF-67 was significantly aligned with the concentration of metal ions to give an extremely low detection limit of 0.3324 nM. The highly sensitive and selective CQDs/QDs@ZIF-67 composite showed potential for the rapid and cost-effective detection of two metal ions.
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Estructuras Metalorgánicas , Puntos Cuánticos , Carbono , Colorantes Fluorescentes , Iones , Límite de DetecciónRESUMEN
Markerless genetic engineering of bacterial genomes is commonly performed by two-step homologous recombination methods using vectors carrying flanking regions of the target gene for site-specific vector integration and counterselection markers to provide positive selection pressure on the second recombination step resulting in vector excision. Here, we provide the proof-of-principle of a novel counterselection method that exploits antagonistic activities between bactericidal and bacteriostatic antibiotics and which can provide selection pressure on the second recombination step by selective killing of bacteria retaining the antibiotic selection marker. This method was optimized for the bacterial pathogens Listeria monocytogenes and Neisseria meningitidis by screening for antagonistic activities between the bactericidal aminoglycosides kanamycin, streptomycin, and gentamicin in combination with the bacteriostatic antibiotics chloramphenicol and erythromycin. The largest difference in selective killing of both L. monocytogenes and N. meningitidis containing an antibiotic selection marker versus wild-type bacteria was observed for the combination of erythromycin, gentamicin, and ermC as antibiotic selection marker. Therefore, this combination was used to generate two markerless deletion mutants for both L. monocytogenes and N. meningitidis. After applying the dual-antimicrobial selection pressure on cultures during the second recombination step, surviving colonies were replica plated on agar with and without erythromycin. On average, 12-13% of the randomly selected bacterial colonies had lost the selection marker due to a second recombination event and approximately half of these colonies were the desired markerless in-frame deletion mutants. Therefore, this method proved to be easy and fast and should be applicable to a wide variety of bacterial species.
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Antibacterianos/farmacología , Secuencia de Bases/genética , Ingeniería Genética/métodos , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/genética , Neisseria meningitidis/efectos de los fármacos , Neisseria meningitidis/genética , Eliminación de Secuencia/efectos de los fármacos , Cloranfenicol/farmacología , Combinación de Medicamentos , Eritromicina/farmacología , Genoma Bacteriano/genética , Gentamicinas/farmacología , Kanamicina/farmacología , Metiltransferasas/genética , Eliminación de Secuencia/genética , Estreptomicina/farmacologíaRESUMEN
Cronobacter sakazakii (C. sakazakii) is a notorious pathogen responsible for infections in infants and newborns, often transmitted through contaminated infant formula. Despite the use of traditional pasteurization methods, which can reduce microbial contamination, there remains a significant risk of pathogenic C. sakazakii surviving due to its exceptional stress tolerance. In our study, we employed a comparative proteomic approach by comparing wild-type strains with gene knockout strains to identify the essential genes crucial for the successful survival of C. sakazakii during desiccation. Our investigation revealed the significance of envZ-ompR, recA, and flhD gene cassettes in contributing to desiccation tolerance in C. sakazakii. Furthermore, through our comparative proteomic profiling, we identified the maltodextrin-binding protein encoded by ESA_03421 as a potential factor influencing dry tolerance. This protein is regulated by EnvZ-OmpR, RecA, and FlhD. Notably, the knockout of ESA_03421 resulted in a 150% greater reduction in Log CFU compared to the wild-type C. sakazakii. Overall, our findings offer valuable insights into the mechanisms underlying C. sakazakii desiccation tolerance and provide potential targets for the development of new antimicrobial strategies aimed at reducing the risk of infections in infants and newborns.
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Cronobacter sakazakii , Desecación , Polisacáridos , Recién Nacido , Lactante , Humanos , Cronobacter sakazakii/metabolismo , Proteínas Portadoras , ProteómicaRESUMEN
The widespread dissemination of carbapenem-resistant Klebsiella pneumoniae (CRKP) and its drug resistance transfer poses a global public health threat. While previous studies outlined CRKP's drug resistance mechanism, there is limited research on strategies inhibiting CRKP drug resistance spread. This study investigates the potential of Bifidobacterium longum (B. longum) FB1-1, a probiotic, in curbing the spread of drug resistance among CRKP by evaluating its cell-free supernatant (CFS) for antibacterial activity. Evaluating the inhibitory effect of FB1-1 CFS on CRKP drug resistance spread involved analyzing its impact on drug resistance and virulence gene expression; drug resistance plasmid transfer FB1-1 CFS exhibited an MIC range of 125 µL/mL against CRKP. After eight hours of co-culture, CFS achieved a 96% and 100% sterilization rate at two and four times the MIC, respectively. At sub-inhibitory concentrations (1/2× MIC), FB1-1 CFS reduced the expression of the bla_KPC gene, which is pivotal for carbapenem resistance, by up to 62.13% across different CRKP strains. Additionally, it markedly suppressed the expression of the uge gene, a key virulence factor, by up to 91%, and the fim_H gene, essential for bacterial adhesion, by up to 53.4%. Our study primarily focuses on determining the inhibitory effect of FB1-1 CFS on CRKP strains harboring the bla_KPC gene, which is a critical resistance determinant in CRKP. Furthermore, FB1-1 CFS demonstrated the ability to inhibit the transfer of drug resistance plasmids among CRKP strains, thus limiting the horizontal spread of resistance genes. This study highlights FB1-1 CFS's inhibitory effect on CRKP drug resistance spread, particularly in strains carrying the bla_KPC gene, thus offering a novel idea and theoretical foundation for developing antibacterial drugs targeting CRKP resistance.
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Cronobacter sakazakii is a notorious foodborne opportunistic pathogen, particularly affecting vulnerable populations such as premature infants, and poses significant public health challenges. This study aimed to elucidate the role of the envZ/ompR genes in environmental tolerance, pathogenicity, and protein regulation of C. sakazakii. An envZ/ompR knockout mutant was constructed and assessed for its impact on bacterial growth, virulence, environmental tolerance, and protein regulation. Results demonstrate that deletion of envZ/ompR genes leads to reduced growth rate and attenuated virulence in animal models. Additionally, the knockout strain exhibited compromised environmental tolerance, particularly in desiccation and oxidative stress conditions, along with impaired adhesion and invasion abilities in epithelial cells. Proteomic analysis revealed significant alterations in protein expression and phosphorylation patterns, highlighting potential compensatory mechanisms triggered by gene deletion. Furthermore, investigation into protein deamidation and glucose metabolism uncovered a link between envZ/ompR deletion and energy metabolism dysregulation. Interestingly, the downregulation of MalK and GrxC proteins was identified as contributing factors to altered desiccation tolerance and disrupted redox homeostasis, respectively, providing mechanistic insights into the phenotypic changes observed. Overall, this study enhances understanding of the multifaceted roles of envZ/ompR in C. sakazakii physiology and pathogenesis, shedding light on potential targets for therapeutic intervention and food safety strategies.
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Proteínas Bacterianas , Cronobacter sakazakii , Regulación Bacteriana de la Expresión Génica , Cronobacter sakazakii/genética , Cronobacter sakazakii/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Virulencia , Humanos , Animales , Infecciones por Enterobacteriaceae/microbiología , Ratones , Estrés OxidativoRESUMEN
Previous studies have shown that advanced glycation end products (AGEs) are implicated in the occurrence and progression of numerous diseases, with dietary AGEs being particularly associated with intestinal disorders. In this study, methylglyoxal-beta-lactoglobulin AGEs (MGO-ß-LG AGEs) were utilized as the exclusive nitrogen source to investigate the interaction between protein-bound AGEs and human gut microbiota. The high-resolution mass spectrometry analysis of alterations in peptides containing AGEs within metabolites before and after fermentation elucidated the capacity of intestinal microorganisms to enzymatically hydrolyze long-chain AGEs into short-chain counterparts. The 16S rRNA sequencing revealed Klebsiella, Lactobacillus, Escherichia-Shigella, and other genera as dominant microbiota at different fermentation times. A total of 187 potential strains of AGE-metabolizing bacteria were isolated from the fermentation broth at various time points. Notably, one strain of Klebsiella exhibited the most robust growth capacity when AGEs served as the sole nitrogen source. Subsequently, proteomics was employed to compare the changes in protein levels of Klebsiella X15 following cultivation in unmodified proteins and proteins modified with AGEs. This analysis unveiled a remodeled amino acid and energy metabolism pathway in Klebsiella in response to AGEs, indicating that Klebsiella may possess a metabolic pathway specifically tailored to AGEs. This study found that fermenting AGEs in healthy human intestinal microbiota altered the bacterial microbiota structure, especially by increasing Klebsiella proliferation, which could be a key factor in AGEs' role in causing diseases, particularly intestinal inflammation.
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Productos Finales de Glicación Avanzada , Piruvaldehído , Humanos , Productos Finales de Glicación Avanzada/metabolismo , ARN Ribosómico 16S , Piruvaldehído/química , Bacterias/metabolismo , NitrógenoRESUMEN
Single nutrient likes polyphenol or dietary fiber have been exhaustively investigated to validate their positive intervention in health or disease. Meanwhile, the common interaction of inner systems with the nutrient complex has not been well elucidated, which raises the scientific issue of the modulatory effect of the nutrient complex on immunity. The representative prebiotics of epigallocatechin-3-gallate (EGCG), ginseng extract, and polydextrose (PDX) were selected on behalf of the classification of polyphenol, flavone or polysaccharides, and dietary fiber to generally cover the daily food intake in this study to explore their intervention in inflammation and macrophage polarization. The intervention of selected nutrients on inflammation and macrophage polarization has been evaluated against macrophages to unveil their comprehensive effects. The synergistic effect of selected nutrients was demonstrated by inhibiting M1 macrophage polarization and the promotion of M2 macrophage polarization. Then, the nutrient formula was set up to verify the intervention effect, and the results revealed the significant inhibition of cell inflammation and the effect on cell proliferation through promoting the cell cycle in the G2 phase. The nutrient complex could inhibit M1 macrophage polarization to inhibit M1-mediated inflammation and promote M2 macrophages for anti-inflammatory effect and enhance cell phagocytosis. Moreover, the varied intervention effects of the nutrient complex with different formulas could be summarized. In general, the formula containing EGCG, ginseng extract, and PDX was demonstrated to possess an enhanced immunomodulatory effect on cell inflammation and macrophage polarization, which could potentially inspire the investigation of complex nutrients in health and diseases.
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This study explores the influence of DnaK on intracellular proteins in Cronobacter sakazakii, a Gram-negative bacterium known for causing infections, particularly in neonates. Proteomic and post-translational modification analyses were performed, revealing a potential link between DnaK and protein deamidation.
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Cronobacter sakazakii (C. sakazakii), a food-borne pathogen, can infect neonates, elderly and immunocompromised populations with a high infection and mortality rate. However, the specific molecular mechanism of its motility, biofilm formation, cell adhesion, and desiccation resistance remains unclear, and flagellum hook associated protein (FlgK), a main component of the flagellar complex, may be an important determinant of its virulence and desiccation resistance. In this study, the flgK mutant strain (ΔflgK) was constructed using the homologous recombination method, and the cpflgK complementary strain was obtained by gene complementation, followed by analysis of the difference between the wild type (WT), mutant, and complementary strains in mobility, biofilm formation, cell adhesion, and desiccation resistance. Results indicated that flgK gene played a positive role in motility and invasion, with no significant effect on biofilm formation. Interestingly, flagellar assembly gene deletion showed increased resistance of C. sakazakii to dehydration. The mechanism underlying the negative correlation of flgK gene with dehydration resistance was further investigated by using the high-throughput sequencing technology to compare the gene expression between WT and ΔflgK strains after drying. The results revealed up-regulation in the expression of 54 genes, including genes involved in osmosis and formate dehydrogenase, while down-regulation in the expression of 50 genes, including genes involved in flagellum hook and nitrate reductase. qRT-PCR analysis of the RNA-seq data further indicated that the flgK gene played an important role in the environmental stress resistance of C. sakazakii by up-regulating the formate dehydrogenase, betaine synthesis, and arginine deiminase pathways, due to dynamic proton imbalance caused by lack of flagella. This study facilitates our understanding of the roles of flgK in motion-related functions and the molecular mechanism of desiccation resistance in C. sakazakii.
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Proteínas Bacterianas , Cronobacter sakazakii , Humanos , Recién Nacido , Anciano , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cronobacter sakazakii/metabolismo , Deshidratación , Desecación , Formiato Deshidrogenasas/metabolismoRESUMEN
Cronobacter sakazakii is an opportunistic Gram-negative pathogen that has been identified as a causative agent of severe foodborne infections with a higher risk of mortality in neonates, premature infants, the elderly, and immunocompromised populations. The specific pathogenesis mechanisms of C. sakazakii, such as adhesion and colonization, remain unclear. Previously, we conducted comparative proteomic studies on the two strains with the stronger and weaker infection ability, respectively, and found an interesting protein, ESA_00986, which was more highly expressed in the strain with the stronger ability. This unknown protein, predicted to be a type of invasitin related to invasion, may be a critical factor contributing to its virulence. This study aimed to elucidate the precise roles of the ESA_00986 gene in C. sakazakii by generating gene knockout mutants and complementary strains. The mutant and complementary strains were assessed for their biofilm formation, mobility, cell adhesion and invasion, and virulence in a rat model. Compared with the wild-type strain, the mutant strain exhibited a decrease in motility, whereas the complementary strain showed comparable motility to the wild-type. The biofilm-forming ability of the mutant was weakened, and the mutant also exhibited attenuated adhesion to/invasion of intestinal epithelial cells (HCT-8, HICE-6) and virulence in a rat model. This indicated that ESA_00986 plays a positive role in adhesion/invasion and virulence. This study proves that the ESA_00986 gene encodes a novel virulence factor and advances our understanding of the pathogenic mechanism of C. sakazakii.
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The biotoxins with high toxicity have the potential to be manufactured into biochemical weapons, seriously threatening international public security. Developing robust and applicable sample pretreatment platforms and reliable quantification methods has been recognized as the most promising and practical approach to solving these problems. Through the integration of the hollow-structured microporous organic networks (HMONs) as the imprinting carriers, we proposed a molecular imprinting platform (HMON@MIP) with enhanced adsorption performance in terms of specificity, imprinting cavity density as well as adsorption capacity. The HMONs core of MIPs provided a hydrophobic surface that enhanced the adsorption of biotoxin template molecules during the imprinting process, resulting in an increased imprinting cavity density. The HMON@MIP adsorption platform could produce a series of MIP adsorbents by changing the biotoxin template, such as aflatoxin and sterigmatocystin, and showed promising generalizability. The limits of detection (LOD) of the HMON@MIP-based preconcentration method for AFT B1 and ST were 4.4 and 6.7 ng L-1, respectively, and the method was applicable to food sample with satisfied recoveries of 81.2-95.1%. And the specific recognition and adsorption sites left on HMON@MIP by the imprinting process can achieve outstanding selectivity for AFT B1 and ST. The developed imprinting platforms hold great potential for application in the identification and determination of various food hazards in complex food sample matrices and contribute to precise food safety inspection.
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Impresión Molecular , Polímeros Impresos Molecularmente , Aflatoxina B1 , Esterigmatocistina , Extracción en Fase Sólida/métodos , Polímeros/química , Impresión Molecular/métodos , AdsorciónRESUMEN
Peanut is widely used for food supplementation with potential allergic reactions in infants and adults, which prompted the development of reliable and accurate detection of peanut allergens with emphasis on Ara h 1. In this study, a nanobody (Nb)-based micro-total electrochemical immunoassay (Nb-µTEI) was proposed to be generated. Generally, an alpaca was immunized with Ara h 1 to yield a Nb reservoir for selection of four specific Nbs. Nb-mediated immunocapturing allowed the identification of the target as Ara h 1. The Nb-based electrochemical immunoassay was developed by constructing a capturing electrode with cycles of signal enhancement. After construction of the capturing electrode, Nb152 with HA-tag was directly applied to connect immobilized anti-HA IgG for the capture of different concentrations of Ara h 1, which was labeled by biotinylated Nb152 to facilitate signal development with alkaline phosphatase conjugated streptavidin (SA-ALP). A linear range from 4.5 to 55 ng/mL was acquired with LOD and LOQ of 0.86 and 2.10 ng/mL, respectively, with an 11-fold increase of the sensitivity compared with the established sandwich ELISA. The dedicated immunoassay was verified by detecting the antigen spiked in food samples and demonstrated the successful conjugation of Nb with advanced detecting techniques.
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Arachis , Hipersensibilidad al Cacahuete , Adulto , Humanos , Antígenos de Plantas , Inmunoensayo/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Alérgenos , Proteínas de PlantasRESUMEN
Screening drugs and compounds to fight against Cronobacter sakazakii (C. sakazakii), one of the most common pathogens that can cause fatal necrotizing enterocolitis, septicema and meningitis, is still needed. We found that p-coumaric acid (pCA) has an inhibitory effect on C. sakazakii in vitro and in vivo. Proteomic changes of C. sakazakii BAA-894 exposed to pCA were studied to reveal the antibacterial mechanisms involved. A total of 1,553 proteins were identified in C. sakazakii BAA-894 by label-free proteomics analysis. Fuzzy cluster analysis showed that 33 were up-regulated, and 110 were down-regulated with pCA treatment. Gene Ontology (GO) analysis concluded that pCA caused the change of metabolic state of bacteria and generally in the state of metabolic inhibition. KEGG Enrichment Analysis (KEGG) analysis showed that pCA inhibited energy metabolism and distorted the balance of amino acid metabolism. Posttranslational modification analysis showed that pCA affected the deamidation of three proteins, including Elongation factor Tu, one of the vital proteins in bacteria. Molecular docking suggested the hydrogen bond between the pCA carboxyl group and Elongation factor Tu Asn-64 might contribute to deamidation. Overall, we found that pCA interfered with cellular energy and amino acid metabolism and promoted elongation factor Tu deamidation, suggesting that pCA can be an effective natural substitute to control C. sakazakii.
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Aflatoxin (AFT) contamination, commonly in foods and grains with extremely low content while high toxicity, has caused serious economic and health problems worldwide. Now researchers are making an effort to develop nanomaterials with remarkable adsorption capacity for the identification, determination and regulation of AFT. Herein, we constructed a novel hollow-structured microporous organic networks (HMONs) material. On the basis of Fe3O4@MOF@MON, hydrofluoric acid (HF) was introduced to remove the transferable metal organic framework (MOF) to give hollow MON structures. Compared to the original Fe3O4@MOF@MON, HMON showed improved surface area and typical hollow cavities, thus increasing the adsorption capacity. More importantly, AFT is a hydrophobic substance, and our constructed HMON had a higher water contact angle, greatly enhancing the adsorption affinity. From that, the solid phase extraction (SPE-HPLC) method developed based on HMONs was applied to analyze four kinds of actual samples, with satisfied recoveries of 85-98%. This work provided a specific and sensitive method for the identification and determination of AFT in the food matrix and demonstrated the great potential of HMONs in the field of the identification and control of mycotoxins.