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1.
Wei Sheng Yan Jiu ; 50(1): 116-142, 2021 Jan.
Artículo en Chino | MEDLINE | ID: mdl-33517972

RESUMEN

OBJECTIVE: A method utilizing liquid chromatography-tandem mass spectrometry(LC-MS/MS) coupled with dispersive solid phase extraction for quantitative analysis of domoic acid in four kinds of shellfish was established. METHODS: The sample of 0. 1 g was extracted with 25% methanol aqueous solution, the extract was purified by dispersive solid phase extraction with 50 mg HLB and 5 mg GCB, and then filtered through a PTFE membrane. The analytes were separated on a C_(18) column(100 mm×2. 1 mm, 1. 9 µm), and detected in selected reaction monitoring(SRM) mode via positive electrospray ionization. The matrix matching and external standard method was used for quantitation. RESULTS: Domoic acid showed good linearity in the concentration range between 1. 0 ng/mL and 50. 0 ng/mL with correlation coefficients higher than 0. 9994. The detection limits of domoic acid in shellfish was 5 µg/kg. The inter-and intra-day recoveries were 91. 6%-109. 2% and 90. 9%-109. 3%, respectively. The inter-and intra-day ralitive standard deviations(RSDs) were lower than 8. 2% at spiked concentrations of 20, 50 and 100 µg/kg. CONCLUSION: The method is accurate, fast, easy to operate, which can satisfy the requirements of public health emergency testing or routine testing.


Asunto(s)
Extracción en Fase Sólida , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Ácido Kaínico/análogos & derivados , Mariscos/análisis
2.
Wei Sheng Yan Jiu ; 50(1): 121-128, 2021 Jan.
Artículo en Chino | MEDLINE | ID: mdl-33517973

RESUMEN

OBEJECTIVE: To develop a method for the determination of 13 antibiotics in 8 classes for desinfection products by ulta-high perfomance chromatography-tandem mass spectrometry(UPLC-MS/MS). METHODS: Samples were extracted by methanol or acetonitrile. The target compouds were separated on a Waters HSS T3 column(100 mm×2. 1 mm, 1. 8 µm), and detected by triple quadrupole tandem mass spectrometer. RESULTS: The 13 selected antibiotics showed good linear relationships in the range of 4-100 µg/L and the correlation coefficients(r~2) were all above 0. 991. The limits of detection ranged from 2 to 25 µg/kg. The recovery rates at three spiked levels(low, medium and high) in three dosage forms of disinfection products were in the range of 71. 2%-130. 4%, and the relative standard deviations(RSD) were all less than 11. 3%, which could meet the detection requirements of illegal addition of antibiotics in disinfection products. Ofloxacin at a concentration of 21. 1 mg/kg was found in a cream disinfection product by the developed method, and no related drugs were detected in other samples. CONCLUSION: This method is simple, reliable, reproducible, which covers a wide range of antibiotics, and provides technical support for monitoring the illegal addition of antibiotics in disinfection products.


Asunto(s)
Antibacterianos , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Desinfección
3.
Sci Rep ; 11(1): 3061, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542252

RESUMEN

The outbreak of COVID-19 has raised interest in the kinin-kallikrein system. Viral blockade of the angiotensin-converting enzyme 2 impedes degradation of the active kinin des-Arg(9)-bradykinin, which thus increasingly activates bradykinin receptors known to promote inflammation, cough, and edema-symptoms that are commonly observed in COVID-19. However, lean and reliable investigation of the postulated alterations is currently hindered by non-specific peptide adsorption, lacking sensitivity, and cross-reactivity of applicable assays. Here, an LC-MS/MS method was established to determine the following kinins in respiratory lavage fluids: kallidin, bradykinin, des-Arg(10)-kallidin, des-Arg(9)-bradykinin, bradykinin 1-7, bradykinin 2-9 and bradykinin 1-5. This method was fully validated according to regulatory bioanalytical guidelines of the European Medicine Agency and the US Food and Drug Administration and has a broad calibration curve range (up to a factor of 103), encompassing low quantification limits of 4.4-22.8 pg/mL (depending on the individual kinin). The application of the developed LC-MS/MS method to nasal lavage fluid allowed for the rapid (~ 2 h), comprehensive and low-volume (100 µL) determination of kinins. Hence, this novel assay may support current efforts to investigate the pathophysiology of COVID-19, but can also be extended to other diseases.


Asunto(s)
Bradiquinina/análisis , Sistema Calicreína-Quinina , Líquido del Lavado Nasal/química , Adulto , Cromatografía Liquida , Femenino , Voluntarios Sanos , Humanos , Masculino , Espectrometría de Masas en Tándem , Adulto Joven
4.
Nat Commun ; 12(1): 1020, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589611

RESUMEN

The extracellular matrix (ECM) is unique to each tissue and capable of guiding cell differentiation, migration, morphology, and function. The ECM proteome of different developmental stages has not been systematically studied in the human pancreas. In this study, we apply mass spectrometry-based quantitative proteomics strategies using N,N-dimethyl leucine isobaric tags to delineate proteome-wide and ECM-specific alterations in four age groups: fetal (18-20 weeks gestation), juvenile (5-16 years old), young adults (21-29 years old) and older adults (50-61 years old). We identify 3,523 proteins including 185 ECM proteins and quantify 117 of them. We detect previously unknown proteome and matrisome features during pancreas development and maturation. We also visualize specific ECM proteins of interest using immunofluorescent staining and investigate changes in ECM localization within islet or acinar compartments. This comprehensive proteomics analysis contributes to an improved understanding of the critical roles that ECM plays throughout human pancreas development and maturation.


Asunto(s)
Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Páncreas/metabolismo , Proteoma/genética , Adolescente , Adulto , Niño , Preescolar , Cromatografía Liquida , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/clasificación , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Feto , Técnica del Anticuerpo Fluorescente , Ontología de Genes , Humanos , Masculino , Persona de Mediana Edad , Anotación de Secuencia Molecular , Organogénesis/genética , Páncreas/crecimiento & desarrollo , Proteoma/clasificación , Proteoma/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem
5.
BMC Bioinformatics ; 22(1): 68, 2021 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-33579189

RESUMEN

BACKGROUND: The clustering of data produced by liquid chromatography coupled to mass spectrometry analyses (LC-MS data) has recently gained interest to extract meaningful chemical or biological patterns. However, recent instrumental pipelines deliver data which size, dimensionality and expected number of clusters are too large to be processed by classical machine learning algorithms, so that most of the state-of-the-art relies on single pass linkage-based algorithms. RESULTS: We propose a clustering algorithm that solves the powerful but computationally demanding kernel k-means objective function in a scalable way. As a result, it can process LC-MS data in an acceptable time on a multicore machine. To do so, we combine three essential features: a compressive data representation, Nyström approximation and a hierarchical strategy. In addition, we propose new kernels based on optimal transport, which interprets as intuitive similarity measures between chromatographic elution profiles. CONCLUSIONS: Our method, referred to as CHICKN, is evaluated on proteomics data produced in our lab, as well as on benchmark data coming from the literature. From a computational viewpoint, it is particularly efficient on raw LC-MS data. From a data analysis viewpoint, it provides clusters which differ from those resulting from state-of-the-art methods, while achieving similar performances. This highlights the complementarity of differently principle algorithms to extract the best from complex LC-MS data.


Asunto(s)
Algoritmos , Análisis por Conglomerados , Péptidos , Proteómica , Cromatografía Liquida , Compresión de Datos , Espectrometría de Masas , Péptidos/química , Proteómica/métodos
6.
Environ Monit Assess ; 192(Suppl 1): 812, 2021 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-33443728

RESUMEN

An analytical method was developed by using LC-ESI(-ve)-MS/MS to investigate the residue dynamics of 2,4-D (2,4-dichlorophenoxyacetic acid) in green tea leaves, processed tea, tea liquor, and tea-cropped soil at Singhiajhora Tea Estate and Putinbari Tea Estate at Terai Region, Darjeeling District, West Bengal, India. In this method, an acidified methanol was used for extraction and subsequent clean-up was done by HLB (hydrophilic lipophilic balanced) cartridges. The method was validated as per SANTE guideline (SANTE/11813/2017). The limit of quantification (LOQ) of 2,4-D was 0.05 mgkg-1 and average % recoveries were in the range from 88.05 to 113.28 with relative standard deviation (RSD) 3.46 to 6.43. The dissipation of 2,4-D followed the 1st-order reaction kinetics with a half-life (T1/2) of 1.51-1.61 day at the recommended dose and 2.50-2.72 day for doubled recommended dose in tea for both locations. This method can be applied successfully for the determination of 2,4 D residues in/on tea matrix and subsequent studies on safety evaluation showed that the use of 2,4-D in tea is safe.


Asunto(s)
Camellia sinensis , Residuos de Plaguicidas , Ácido 2,4-Diclorofenoxiacético , Cromatografía Liquida , Monitoreo del Ambiente , India , Residuos de Plaguicidas/análisis , Espectrometría de Masas en Tándem ,
7.
Anticancer Res ; 41(1): 327-334, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33419827

RESUMEN

BACKGROUND/AIM: Pancreaticobiliary maljunction (PBM), a disease with reflux of pancreatic and bile juice in the pancreaticobiliary tract, is a high-risk factor for biliary tract cancer. The aim of this study was to investigate the mechanism of carcinogenesis in PBM using a metabolomics analysis of bile sampled during surgery. PATIENTS AND METHODS: Three patients with PBM without biliary tract cancer, four patients with extrahepatic bile duct cancer (EHBC), and three controls with benign disease were enrolled. Metabolomics analysis of bile samples was performed using capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry to discriminate the amino acid and lipidomic profiles. RESULTS: The principal component analysis in the capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry revealed similar metabolites in patients with PBM and those with EHBC; furthermore, there was a clear difference between patients with PBM or EHBC compared to controls. The amino acid profiles revealed the following 20 potential carcinogenic candidates for PBM: isoleucine, phenylalanine, tyrosine, leucine, tryptophan, arginine, lysine, valine, asparagine, methionine, aspartic acid, serine, threonine, histidine, glutamine, alanine, proline, glutamic acid, and pyruvic acid. The lipidomic profiles revealed the following 11 carcinogenic candidates: lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidyl glycerol, lysophosphatidyl glycerol, triacylglycerol, diacylglycerol, ceramide, sphyngomyeline, fatty acid, hyperforin, and vitamin D. Among these characteristic metabolites, the branched-chain amino acids, methionine and lysophosphatidylcholine are known to be related to carcinogenesis. CONCLUSION: The bile metabolites were extremely similar in patients with PBM and those with EHBC. Furthermore, amino acid and lipid metabolism was markedly different in patients with PBM or EHBC compared to healthy controls.


Asunto(s)
Neoplasias de los Conductos Biliares/etiología , Bilis/metabolismo , Transformación Celular Neoplásica/metabolismo , Susceptibilidad a Enfermedades , Mal funcionamiento Pancreaticobiliar/complicaciones , Mal funcionamiento Pancreaticobiliar/metabolismo , Neoplasias de los Conductos Biliares/diagnóstico , Neoplasias de los Conductos Biliares/terapia , Cromatografía Liquida , Electroforesis Capilar , Femenino , Humanos , Masculino , Espectrometría de Masas , Metabolómica/métodos , Proyectos Piloto , Medición de Riesgo , Factores de Riesgo
8.
BMC Infect Dis ; 21(1): 99, 2021 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-33482745

RESUMEN

BACKGROUND: Treatment monitoring of drug-resistant tuberculosis (DR-TB) in resource-limited settings is challenging. We developed a multi-analyte assay for eleven anti-TB drugs in small hair samples as an objective metric of drug exposure. METHODS: Small hair samples were collected from participants at various timepoints during directly observed RR-TB treatment at an inpatient tertiary referral facility in South Africa (DR-TB cohort). We assessed qualitative determination (i.e., detection above limit of detection) of bedaquiline, linezolid, clofazimine, pretomanid, levofloxacin, moxifloxacin, pyrazinamide, isoniazid, ethambutol, ethionamide, and prothionamide in an LC-MS/MS index panel assay against a reference standard of inpatient treatment records. Because treatment regimens prior to hospitalization were not available, we also analyzed specificity (for all drugs except isoniazid) using an external cohort of HIV-positive patients treated for latent TB infection with daily isoniazid (HIV/LTBI cohort) in Uganda. RESULTS: Among the 57 DR-TB patients (58% with pre-XDR/XDR-TB; 70% HIV-positive) contributing analyzable hair samples, the sensitivity of the investigational assay was 94% or higher for all drugs except ethionamide (58.5, 95% confidence interval [CI], 40.7-99.9). Assay specificity was low across all tested analytes within the DR-TB cohort; conversely, assay specificity was 100% for all drugs in the HIV/LTBI cohort. CONCLUSIONS: Hair drug concentrations reflect long-term exposure, and multiple successive regimens commonly employed in DR-TB treatment may result in apparent false-positive qualitative and falsely elevated quantitative hair drug levels when prior treatment histories within the hair growth window are not known.


Asunto(s)
Antituberculosos/análisis , Monitoreo de Drogas/métodos , Cabello/química , Rifampin/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Adulto , Antituberculosos/uso terapéutico , Cromatografía Liquida , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/efectos de los fármacos , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem , Tuberculosis/tratamiento farmacológico
9.
Sci Rep ; 11(1): 2291, 2021 01 27.
Artículo en Inglés | MEDLINE | ID: mdl-33504824

RESUMEN

Coronavirus disease 2019 (COVID-19) pandemic is affecting millions of patients worldwide. The consequences of initial exposure to SARS-CoV-2 go beyond pulmonary damage, with a particular impact on lipid metabolism. Decreased levels in HDL-C were reported in COVID-19 patients. Since HDL particles display antioxidant, anti-inflammatory and potential anti-infectious properties, we aimed at characterizing HDL proteome and functionality during COVID-19 relative to healthy subjects. HDLs were isolated from plasma of 8 severe COVID-19 patients sampled at admission to intensive care unit (Day 1, D1) at D3 and D7, and from 16 sex- and age-matched healthy subjects. Proteomic analysis was performed by LC-MS/MS. The relative amounts of proteins identified in HDLs were compared between COVID-19 and controls. apolipoprotein A-I and paraoxonase 1 were confirmed by Western-blot analysis to be less abundant in COVID-19 versus controls, whereas serum amyloid A and alpha-1 antitrypsin were higher. HDLs from patients were less protective in endothelial cells stiumalted by TNFα (permeability, VE-cadherin disorganization and apoptosis). In these conditions, HDL inhibition of apoptosis was blunted in COVID-19 relative to controls. In conclusion, we show major changes in HDL proteome and decreased functionality in severe COVID-19 patients.


Asunto(s)
/sangre , Lipoproteínas HDL/sangre , Apolipoproteína A-I/sangre , Arildialquilfosfatasa/análisis , Arildialquilfosfatasa/sangre , /patología , Estudios de Casos y Controles , Cromatografía Liquida/métodos , Células Endoteliales/patología , Femenino , Francia/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Proteoma/metabolismo , Proteómica/métodos , Proteína Amiloide A Sérica/metabolismo , Espectrometría de Masas en Tándem/métodos , Factor de Necrosis Tumoral alfa/sangre , alfa 1-Antitripsina/sangre
10.
J Chromatogr A ; 1637: 461808, 2021 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-33385741

RESUMEN

This study describes the fully automated middle-up characterization of monoclonal antibodies (mAbs) and next-generation variants by online reduction liquid chromatography-mass spectrometry (LC-MS). Proteins were trapped on-column and subjected to online desalting, denaturation and reduction prior to reversed phase elution of the created subunits in the MS. The evaluation of more than 20 different therapeutic proteins including full length mAbs (subclasses IgG1, IgG2 and IgG4), bispecific antibodies, antibody fragments, fusion proteins and antibody-drug conjugates (ADC) revealed that the online reduction method is as powerful as the widely applied offline sample preparation with dithiothreitol (DTT) as reducing agent and guanidine hydrochloride (Gnd.HCl) as denaturant and tackles some major disadvantages associated with the latter method, i.e. corrosion of stainless steel components, adduct formation impacting spectral quality and sample stability. The value of the online reduction LC-MS method is also enforced by its ability to reveal unstable antibody variants such as succinimide intermediates of asparagine deamidation and aspartic acid isomerization which are often lost when using the offline sample preparation method. The performance of the online reduction LC-MS set-up was verified and it was revealed that the method is precise with RSD values below 0.25% and 3.0% for retention time and area, respectively. Carry-over is within acceptable limits (< 0.5%) and the reducing buffer is stable up to 24 hours.


Asunto(s)
Anticuerpos Monoclonales/química , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Inmunoconjugados/química
11.
J Chromatogr A ; 1637: 461779, 2021 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-33385742

RESUMEN

We report on the design and performance of in-house built column cartridges that can be directly screwed into the ports of a commercial rotor-stator valve to minimize extra-column band broadening and pressure-drop losses when pursuing ultra-fast separations such as those needed in 2D and 3D-LC separations. Two basic designs were evaluated and were compared with the results obtained with a commercial screw-in column cartridge. The system produces an extra-column band broadening as low as 0.05 to 0.1 µL2 for the employed UV-detector set-up. Despite these very low values, the obtained separation efficiency of the in-house fabricated cartridge columns was very low, corresponding to a reduced minimal plate height around h=7 at the very best, which, for the 1.7 µm particle and 26.4 mm long columns corresponds to a number of theoretical plates of N=2200 under isocratic conditions. A similar poor performance was obtained with a commercial column cartridge with similar dimensions using the same set-up. One possible explanation of the observed performance could be found in the inner diameter of the column cartridges (i.d. =0.75 mm and 1 mm) which, for the employed sub 2-µm particles, falls into a region of column diameters that, according to literature models, is most likely to suffer from inherent packing problems.


Asunto(s)
Cromatografía Liquida/instrumentación , Diseño de Equipo , Humanos , Espectrofotometría Ultravioleta
12.
Food Chem ; 346: 128898, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33453579

RESUMEN

In 2016, the European Commission recommended the Member States to monitor the content of Δ9-tetrahydrocannabinol and other cannabinoids in food and feed derived from hemp and in food of animal origin for possible transfer from feed. Thus, the Italian Ministry of Health implemented a monitoring plan. To this aim, nine cannabinoids in beverages and food for human consumption and in feed were determined. The method applied, based on rapid clean-up and LC-MS/MS determination, was previously developed and in-house validated, evaluating the analytical performance in the concentration ranges 2-50 µg/L for beverages, 0.020-0.500 mg/kg for food and 0.100-10.0 mg/kg for feed. Then, it was applied to determine the cannabinoids in 78 food, 16 beverage and 6 feed samples, collected from the Italian market since 2017. The results are herein reported, for evaluation of both product characteristics and compliance to national maximum limits. Some study cases are also described.


Asunto(s)
Alimentación Animal/análisis , Bebidas/análisis , Cromatografía Liquida , Dronabinol/análisis , Análisis de los Alimentos/métodos , Espectrometría de Masas en Tándem , Animales , Cannabis/química , Humanos , Italia
13.
Sci Total Environ ; 764: 143963, 2021 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-33385644

RESUMEN

Consumption of licit and/or illicit compounds during sporting events has traditionally been monitored using population surveys, medical records, and law enforcement seizure data. This pilot study evaluated the temporal and geospatial patterns in drug consumption during a university football game from wastewater using liquid chromatography tandem mass spectrometry (LC-MS/MS). Untreated wastewater samples were collected from three locations within or near the same football stadium every 30 min during a university football game. This analysis leveraged two LCMS/ MS instruments (Waters Acquity TQD and a Shimadzu 8040) to analyze samples for 58 licit or illicit compounds and some of their metabolites. Bayesian multilevel models were implemented to estimate mass load and population-level drug consumption, while accounting for multiple instrument runs and concentrations censored at the lower limit of quantitation. Overall, 29 compounds were detected in at least one wastewater sample collected during the game. The 10 most common compounds included opioids, anorectics, stimulants, and decongestants. For compounds detected in more than 50% of samples, temporal trends in median mass load were correlated with the timing of the game; peak loads for cocaine and tramadol occurred during the first quarter of the game and for phentermine during the third quarter. Stadium-wide estimates of the number of doses of drugs consumed were rank ordered as follows: oxycodone (n = 3246) > hydrocodone (n = 2260) > phentermine (n = 513) > cocaine (n = 415) > amphetamine (n = 372) > tramadol (n = 360) > pseudoephedrine (n = 324). This analysis represents the most comprehensive assessment of drug consumption during a university football game and indicates that wastewater-based epidemiology has potential to inform public health interventions focused on reducing recreational drug consumption during large-scale sporting events.


Asunto(s)
Aguas Residuales , Contaminantes Químicos del Agua , Teorema de Bayes , Cromatografía Liquida , Humanos , Proyectos Piloto , Detección de Abuso de Sustancias , Espectrometría de Masas en Tándem , Universidades , Aguas Residuales/análisis , Contaminantes Químicos del Agua/análisis
14.
Sheng Wu Gong Cheng Xue Bao ; 37(1): 276-289, 2021 Jan 25.
Artículo en Chino | MEDLINE | ID: mdl-33501808

RESUMEN

Different microorganisms can cause intraperitoneal infection. This study was to distinguish different microbial infections by urine analysis. Rats were intraperitoneally injected with Escherichia coli, Staphylococcus aureus, and Candida albicans, separately. Urine samples were collected from rats at 0, 12, 36 and 72 h after infection. Urinary proteins were profiled using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Compared with the control (without infection), a total of 69 differential proteins were identified in rats injected with E. coli. A total of 31 differences proteins were identified in rats injected with S. aureus. A total of 38 differential proteins were identified in rats injected with C. albicans. Urine proteome was different when rats were infected by different microorganisms, suggesting that urine may have the potential for differential diagnosis of different intraperitoneal infections.


Asunto(s)
Proteoma , Espectrometría de Masas en Tándem , Animales , Cromatografía Liquida , Escherichia coli , Ratas , Staphylococcus aureus
15.
Water Res ; 190: 116752, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33385875

RESUMEN

The occurrence and removal patterns of 24 antimicrobial agents and antimicrobial resistant determinants namely 6 antibiotic resistance genes (ARGs) and 2 mobile genetic elements (MGEs), and the fecal indicator E. coli were investigated in three full-scale wastewater treatment plants. Their waterlines and biosolids lines (including secondary treatment based on both granular and activated sludge) were sampled monthly throughout one year. Samples were analyzed by means of LC-MS/MS, qPCR and cell enumeration, respectively. The influence of rainfall, temperature, and turbidity on the occurrence and removal of the aforementioned agents was assessed through statistical linear mixed models. Ten of the antimicrobial agents (macrolides, fluoroquinolones, tetracyclines, and sulfonamides) were commonly found in influent in concentrations of 0.1-2 µg L-1, and the predominant ARGs were ermB and sul1 (6.4 and 5.9 log10 mL-1 respectively). Warmer temperatures slightly reduced gene concentrations in influent whilst increasing that of E. coli and produced an uneven effect on the antimicrobial concentrations across plants. Rainfall diluted both E. coli (-0.25 logs, p < 0.001) and antimicrobials but not genes. The wastewater treatment reduced the absolute abundance of both genes (1.86 logs on average) and E. coli (2.31 logs on average). The antimicrobials agents were also partly removed, but 8 of them were still detectable after treatment, and 6 accumulated in the biosolids. ARGs were also found in biosolids with patterns resembling those of influent. No significant differences in the removal of antimicrobials, genes and E. coli were observed when comparing conventional activated sludge with aerobic granular sludge. Irrespective of the type of sludge treatment, the removal of genes was significantly reduced with increasing hydraulic loads caused by rainfall (-0.35 logs per ∆ average daily flow p < 0.01), and slightly decreased with increasing turbidity (-0.02 logs per ∆1 nephelometric turbidy unit p < 0.05) .


Asunto(s)
Genes Bacterianos , Aguas del Alcantarillado , Antibacterianos , Cromatografía Liquida , Escherichia coli/genética , Espectrometría de Masas en Tándem , Eliminación de Residuos Líquidos , Aguas Residuales
16.
Ecotoxicol Environ Saf ; 211: 111908, 2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33440265

RESUMEN

Hexavalent chromium [Cr(VI)] is a common heavy metal pollutant that can cause a number of human disease, including inflammation and cancer. Senescent cells can secrete a variety of molecules known as senescence-associated secretory phenotype (SASP). Our previous studies have confirmed that Cr(VI) can induce premature senescence in L02 hepatocytes, but the composition and the function of the related SASP are still unknown. In order to understand the components of SASP secreted by senescent L02 hepatocytes under the action of Cr(VI), we applied LC-MS/MS-based label-free protein quantification. We found that three SASP components including Coactosin-like protein 1 (COTL1), Alpha-enolase (ENO1), and Peroxiredoxin 2 (PRDX2) were up-regulated, which were confirmed by western blotting and qRT-PCR. Evidence suggested that SASP may promote the development of tumor through chronic inflammatory response, therefore we identified and analyzed the potential biological functions and signaling pathways of these three SASP components using GO and KEGG methods. The interaction between SASP components was analyzed by STRING, and verified by Co-IP. We also found that ENO1 and PRDX2, which have direct interaction, can inhibit the growth and proliferation of wildtype hepatocytes and premature senescent hepatocytes, but can promote the proliferation and behavioral changes of liver tumor cells. The present study provides valuable clues for elucidation of the carcinogenic mechanism of Cr(VI), especially for further prevention and targeted treatment of Cr(VI)-related cancer.


Asunto(s)
Senescencia Celular , Cromo/toxicidad , Línea Celular , Cromatografía Liquida , Hepatocitos/metabolismo , Humanos , Proteínas de Microfilamentos/metabolismo , Fenotipo , Transducción de Señal , Espectrometría de Masas en Tándem , Regulación hacia Arriba
17.
Water Res ; 192: 116835, 2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33486289

RESUMEN

The complexity of production process and chemical compositions of soluble microbial products (SMPs) largely limits the understanding of membrane fouling in membrane bioreactors (MBRs). Herein, we used a model single-strain Escherichia coli to better understand the chemical natures of SMPs and their roles in membrane fouling. The effects of carbon source and growth phase on the chemical compositions of SMPs were identified at both the compound and molecular levels by using advanced techniques including excitation emission matrix and parallel factor analysis (EEM-PARAFAC), size exclusion chromatography coupled with organic carbon detection (LC-OCD), and untargeted ultra-performance liquid chromatography - Q-Exactive - mass spectrometry (UPLC-Q-Exactive-MS). Subsequently, the roles of SMPs in the propensity of membrane fouling during ultrafiltration (UF) were studied. The results showed that the chemical compositions and fouling potentials of SMPs were carbon source- and growth phase-dependent. In the exponential phase, SMPs mainly consisted of utilization-associated products (UAPs) and remaining substrates. As the microorganism progressed into the stationary and senescent phases, UAPs and biomass-associated products (BAPs) were the main components, respectively. The SMP contents generated in glucose medium were higher than those generated in acetate medium, and higher abundances of humic fluorescent components were observed in glucose-fed SMPs. Van Krevelen diagrams of the UPLC-MS results revealed that acetate-fed SMPs contained more carboxylic-rich alicyclic molecules, peptides-like, aromatic, and carbohydrates-like components than glucose-fed SMPs in the stationary and senescent phases. These components played a significant role in irreversible membrane fouling, as evidenced in UF experiments. Standard blocking and cake filtration were the main fouling mechanisms for the filtration of SMPs collected in the exponential and stationary/senescent phases, respectively. Our findings highlight linkages between SMP compositions and membrane fouling at both the compound and molecular levels and suggest that both the carbon source and growth phase strongly determine the production potential, chemical nature, and fouling behavior of SMPs.


Asunto(s)
Membranas Artificiales , Aguas del Alcantarillado , Reactores Biológicos , Cromatografía Liquida , Escherichia coli , Espectrometría de Masas en Tándem
18.
J Chromatogr A ; 1638: 461862, 2021 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-33433374

RESUMEN

This work presents an evaluation of solid-phase microextraction (SPME) SPME in combination with liquid chromatography-high resolution mass spectrometry (LC-HRMS) as an analytical approach for untargeted brain analysis. The study included a characterization of the metabolite coverage provided by C18, mixed-mode (MM, with benzene sulfonic acid and C18 functionalities), and hydrophilic lipophilic balanced (HLB) particles as sorbents in SPME coatings after extraction from cow brain homogenate at static conditions. The effects of desorption solvent, extraction time, and chromatographic modes on the metabolite features detected were investigated. Method precision and absolute matrix effects were also assessed. Among the main findings of this work, it was observed that all three tested coating chemistries were able to provide comparable brain tissue information. HLB provided higher responses for polar metabolites; however, as these fibers were prepared in-house, higher inter-fiber relative standard deviations were also observed. C18 and HLB coatings offered similar responses with respect to lipid-related features, whereas MM and C18 provided the best results in terms of method precision. Our results also showed that the use of methanol is essential for effective desorption of non-polar metabolites. Using a reversed-phase chromatographic method, an average of 800 and 1200 brain metabolite features detected in positive and negative modes, respectively, met inter-fibre RSD values below 30% (n=4) after removal of fibre and solvent artefacts from the associated datasets. For features detected using a lipidomics method, a total of 900 and 1800 features detected using C18 fibers in positive and negative mode, respectively, met the same criteria. In terms of absolute matrix effects, the majority of the model metabolites tested showed values between 80 and 120%, which are within the acceptable range. Overall, the findings of this work lay the foundation for further optimization of parameters for SPME-LC-HRMS methods suitable for in vivo and ex vivo brain (and other tissue) untargeted studies, and support the applicability of this approach for non-destructive tissue metabolomics.


Asunto(s)
Encéfalo/metabolismo , Cromatografía Liquida , Espectrometría de Masas , Microextracción en Fase Sólida , Animales , Bovinos , Interacciones Hidrofóbicas e Hidrofílicas , Metabolómica/métodos , Solventes/química , Manejo de Especímenes
19.
J Chromatogr A ; 1638: 461867, 2021 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-33485029

RESUMEN

Considering that neurotransmitters (NTs) and amino acids (AAs) exert pivotal roles in various neurological diseases, global detection of these endogenous metabolites is of great significance for the treatment of nervous system diseases. Herein, a workflow that could cope with various challenges was proposed to establish an extendable all-in-one injection liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for analyzing these small molecular metabolites with high coverage. To obtain a qualified blank biological matrix for the preparation of standard curves and quality control samples, different absorption solvents, including activated carbon (AC), calcite (Cal) and montmorillonite (Mnt) were systematically evaluated for efficient absorption of endogenous substances with minimum residue. We also firstly proposed a "Collision Energy Defect (CED)" strategy to solve the huge difference of mass signal strength caused by different properties and concentrations of 11 NTs and 17 AAs. The quantitative results were validated by LC-MS/MS. Sensitivity, accuracy, and recovery meeting generally accepted bioanalytic guidelines were observed in a concentration span of at least 100 to 500 times for each analyte. Then the temporal changes of intracerebral and peripheral NTs and AAs in ischemic stroke model and sham operated rats were successfully produced and compared using the described method. All these results suggested that the currently developed assay was powerful enough to simultaneously monitor a large panel of endogenous small molecule metabolites, which was expected to be widely used in the research of various diseases mediated by NTs and AAs.


Asunto(s)
Aminoácidos/química , Cromatografía Liquida , Neurotransmisores/química , Espectrometría de Masas en Tándem , Adsorción , Animales , Masculino , Ratas
20.
Phytochemistry ; 183: 112643, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33421889

RESUMEN

Phytochelatins (PCs) are peptides that play an important role in homeostasis and detoxification of heavy metal in plants. Furthermore, they have been proposed as earlier potential biomarkers of aquatic pollution by heavy metals. Nowadays, several researchers have reported on current methods for quantification of glutathione (GSH) and the PCs (phytochelatin 2, phytochelatin 3, phytochelatin 4) quantification in plants. However, no method has reported the uncertainty of the measurement, which helps to improve the accuracy and quality assurance in the PC quantification. In this work, a new methodology using ultra-high-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) to measure with high precision and accuracy the PCs in aquatic plants, was validated. Selectivity, linearity, limit of detection, limit of quantification, precision, trueness and uncertainty estimation were examined as parts of the method validation. The described method shows excellent linearity in different ranges for all analytes with coefficients of determination higher than 0.99. The relative standard deviation for intra-day precision was <3% and for inter-day <10%. All LOD and LOQ analytes ranged from 0.02 to 0.08 µg ml-1, and from 0.03 to 0.09 µg ml-1, respectively. The recoveries varied from 61% to 89%. In order to obtain an interval of results with the highest confidence levels, the uncertainty associated with the measurements was evaluated. The calibration curve (>50%) and recovery (19-44%) were the most important contributors to the total uncertainty. The proposed method was applied to quantify GSH and PCs in the aquatic plants Lemna gibba L., Myriophyllum heterophyllum Michx., Arenaria paludicola and Hydrocotyle ranunculoides L. fil., showing statistical differences in the mass fraction of the analytes.


Asunto(s)
Fitoquelatinas , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Incertidumbre
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