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1.
Int J Mol Sci ; 17(3): 307, 2016 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-26927094

RESUMEN

To identify potential biomarkers for improving diagnosis of melioidosis, we compared plasma metabolome profiles of melioidosis patients compared to patients with other bacteremia and controls without active infection, using ultra-high-performance liquid chromatography-electrospray ionization-quadruple time-of-flight mass spectrometry. Principal component analysis (PCA) showed that the metabolomic profiles of melioidosis patients are distinguishable from bacteremia patients and controls. Using multivariate and univariate analysis, 12 significant metabolites from four lipid classes, acylcarnitine (n = 6), lysophosphatidylethanolamine (LysoPE) (n = 3), sphingomyelins (SM) (n = 2) and phosphatidylcholine (PC) (n = 1), with significantly higher levels in melioidosis patients than bacteremia patients and controls, were identified. Ten of the 12 metabolites showed area-under-receiver operating characteristic curve (AUC) >0.80 when compared both between melioidosis and bacteremia patients, and between melioidosis patients and controls. SM(d18:2/16:0) possessed the largest AUC when compared, both between melioidosis and bacteremia patients (AUC 0.998, sensitivity 100% and specificity 91.7%), and between melioidosis patients and controls (AUC 1.000, sensitivity 96.7% and specificity 100%). Our results indicate that metabolome profiling might serve as a promising approach for diagnosis of melioidosis using patient plasma, with SM(d18:2/16:0) representing a potential biomarker. Since the 12 metabolites were related to various pathways for energy and lipid metabolism, further studies may reveal their possible role in the pathogenesis and host response in melioidosis.


Asunto(s)
Melioidosis/sangre , Metaboloma , Esfingomielinas/sangre , Bacteriemia/sangre , Biomarcadores/sangre , Carnitina/análogos & derivados , Carnitina/sangre , Estudios de Casos y Controles , Humanos , Fosfatidilcolinas/sangre
2.
J Clin Microbiol ; 53(12): 3750-9, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26378277

RESUMEN

Although tuberculosis (TB) is a reemerging disease that affects people in developing countries and immunocompromised populations in developed countries, the current diagnostic methods are far from optimal. Metabolomics is increasingly being used for studies on infectious diseases. We performed metabolome profiling of plasma samples to identify potential biomarkers for diagnosing TB. We compared the plasma metabolome profiles of TB patients (n = 46) with those of community-acquired pneumonia (CAP) patients (n = 30) and controls without active infection (n = 30) using ultrahigh-performance liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry (UHPLC-ESI-QTOFMS). Using multivariate and univariate analyses, four metabolites, 12R-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid [12(R)-HETE], ceramide (d18:1/16:0), cholesterol sulfate, and 4α-formyl-4ß-methyl-5α-cholesta-8-en-3ß-ol, were identified and found to have significantly higher levels in TB patients than those in CAP patients and controls. In a comparison of TB patients and controls, the four metabolites demonstrated area under the receiver operating characteristic curve (AUC) values of 0.914, 0.912, 0.905, and 0.856, sensitivities of 84.8%, 84.8%, 87.0%, and 89.1%, specificities of 90.0%, 86.7%, 86.7%, and 80.0%, and fold changes of 4.19, 26.15, 6.09, and 1.83, respectively. In a comparison of TB and CAP patients, the four metabolites demonstrated AUC values of 0.793, 0.717, 0.802, and 0.894, sensitivities of 89.1%, 71.7%, 80.4%, and 84.8%, specificities of 63.3%, 66.7%, 70.0%, and 83.3%, and fold changes of 4.69, 3.82, 3.75, and 2.16, respectively. 4α-Formyl-4ß-methyl-5α-cholesta-8-en-3ß-ol combined with 12(R)-HETE or cholesterol sulfate offered ≥70% sensitivity and ≥90% specificity for differentiating TB patients from controls or CAP patients. These novel plasma biomarkers, especially 12(R)-HETE and 4α-formyl-4ß-methyl-5α-cholesta-8-en-3ß-ol, alone or in combination, are potentially useful for rapid and noninvasive diagnosis of TB. The present findings may offer insights into the pathogenesis and host response in TB.


Asunto(s)
Biomarcadores/sangre , Metaboloma , Plasma/química , Tuberculosis/diagnóstico , Tuberculosis/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Cromatografía Liquida , Femenino , Humanos , Masculino , Espectrometría de Masas/métodos , Persona de Mediana Edad , Curva ROC , Sensibilidad y Especificidad , Adulto Joven
3.
Int J Mol Sci ; 16(6): 13850-67, 2015 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-26090713

RESUMEN

Infections related to Aspergillus species have emerged to become an important focus in infectious diseases, as a result of the increasing use of immunosuppressive agents and high fatality associated with invasive aspergillosis. However, laboratory diagnosis of Aspergillus infections remains difficult. In this study, by comparing the metabolomic profiles of the culture supernatants of 30 strains of six pathogenic Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, A. nomius and A. tamarii) and 31 strains of 10 non-Aspergillus fungi, eight compounds present in all strains of the six Aspergillus species but not in any strain of the non-Aspergillus fungi were observed. One of the eight compounds, Leu-Glu-Leu-Glu, is a novel tetrapeptide and represents the first linear tetrapeptide observed in Aspergillus species, which we propose to be named aspergitide. Two other closely related Aspergillus-specific compounds, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid, may possess anti-inflammatory properties, as 2-(sulfooxy)benzoic acid possesses a structure similar to those of aspirin [2-(acetoxy)benzoic acid] and salicylic acid (2-hydroxybenzoic acid). Further studies to examine the potentials of these Aspergillus-specific compounds for laboratory diagnosis of aspergillosis are warranted and further experiments will reveal whether Leu-Glu-Leu-Glu, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid are virulent factors of the pathogenic Aspergillus species.


Asunto(s)
Antiinflamatorios/metabolismo , Aspergilosis/diagnóstico , Aspergillus/patogenicidad , Metaboloma , Metabolómica , Fragmentos de Péptidos/metabolismo , Aspergilosis/metabolismo , Aspergilosis/microbiología , Humanos , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem
4.
Anal Chem ; 86(12): 5688-96, 2014 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-24844867

RESUMEN

The emerging field of sphingolipidomics calls for accurate quantitative analyses of sphingolipidome. Existing analytical methods for sphingolipid (SPL) profiling often suffer from isotopic/isomeric interference, leading to the low-abundance, but biologically important SPLs being undetected. In the current study, we have developed an improved sphingolipidomic approach for reliable and sensitive quantification of up to 10 subclasses of cellular SPLs. By integratively utilizing high efficiency chromatographic separation, quadrupole time-of-flight (Q-TOF) and triple quadrupole (QQQ) mass spectrometry (MS), our approach facilitated unambiguous identification of several groups of potentially important but low-abundance SPLs that are usually masked by isotopic/isomeric species and hence largely overlooked in many published methods. The methodology, which featured a modified sample preparation and optimized MS parameters, permitted the measurement of 86 individual SPLs in PC12 cells in a single run, demonstrating great potential for high throughput analysis. The improved characterization, along with increased sensitivity for low-abundance SPL species, resulted in the highest number of SPLs being quantified in a single run in PC12 cells. The improved method was fully validated and applied to a lipidomic study of PC12 cell samples with or without amyloid ß peptide (Aß) treatment, which presents a most precise and genuine sphingolipidomic profile of the PC12 cell line. The adoption of the metabolomics protocol, as described in this study, could avoid misidentification and bias in the measurement of the analytically challenging low-abundance endogenous SPLs, hence achieving informative and reliable sphingolipidomics data relevant to discovery of potential SPL biomarkers for Aß-induced neurotoxicity and neurodegenerative disease.


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Neuronas/efectos de los fármacos , Esfingolípidos/química , Animales , Límite de Detección , Células PC12 , Ratas , Pruebas de Toxicidad
5.
J Clin Microbiol ; 52(4): 1153-60, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24452174

RESUMEN

Aspergillus nomius and Aspergillus tamarii are Aspergillus species that phenotypically resemble Aspergillus flavus. In the last decade, a number of case reports have identified A. nomius and A. tamarii as causes of human infections. In this study, using an internal transcribed spacer, ß-tubulin, and calmodulin gene sequencing, only 8 of 11 clinical isolates reported as A. flavus in our clinical microbiology laboratory by phenotypic methods were identified as A. flavus. The other three isolates were A. nomius (n = 2) or A. tamarii (n = 1). The results corresponded with those of metabolic fingerprinting, in which the A. flavus, A. nomius, and A. tamarii strains were separated into three clusters based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC MS) analysis. The first two patients with A. nomius infections had invasive aspergillosis and chronic cavitary and fibrosing pulmonary and pleural aspergillosis, respectively, whereas the third patient had A. tamarii colonization of the airway. Identification of the 11 clinical isolates and three reference strains by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed that only six of the nine strains of A. flavus were identified correctly. None of the strains of A. nomius and A. tamarii was correctly identified. ß-Tubulin or the calmodulin gene should be the gene target of choice for identifying A. flavus, A. nomius, and A. tamarii. To improve the usefulness of MALDI-TOF MS, the number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability.


Asunto(s)
Aspergilosis/microbiología , Aspergillus/clasificación , Técnicas Microbiológicas/métodos , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Anciano , Anciano de 80 o más Años , Animales , Aspergillus/química , Aspergillus/genética , Aspergillus/aislamiento & purificación , Calmodulina/genética , Análisis por Conglomerados , ADN de Hongos/química , ADN de Hongos/genética , ADN de Helmintos/química , ADN de Helmintos/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Femenino , Humanos , Masculino , Metaboloma , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Tubulina (Proteína)/genética
6.
J Clin Microbiol ; 51(1): 334-8, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23135942

RESUMEN

We report a pseudo-outbreak of Tsukamurella due to improperly wrapped scissors used for processing of tissue specimens. A polyphasic approach, involving biochemical, genetic, and metabolomic techniques, was used in the laboratory investigation. This report highlights that early recognition of pseudo-outbreaks is important in preventing unnecessary and incorrect treatment of patients.


Asunto(s)
Infecciones por Actinomycetales/epidemiología , Actinomycetales/aislamiento & purificación , Brotes de Enfermedades , Actinomycetales/genética , Actinomycetales/fisiología , Adulto , Técnicas de Tipificación Bacteriana , Electroforesis en Gel de Campo Pulsado , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , Adulto Joven
7.
Anal Chem ; 84(23): 10236-44, 2012 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-23134482

RESUMEN

In this paper, we describe the development of a novel stable isotope N-phosphorylation labeling (SIPL) strategy for peptide de novo sequencing and protein quantification based on organic phosphorus chemistry. The labeling reaction could be performed easily and completed within 40 min in a one-pot reaction without additional cleanup procedures. It was found that N-phosphorylation labeling reagents were activated in situ to form labeling intermediates with high reactivity targeting on N-terminus and ε-amino groups of lysine under mild reaction conditions. The introduction of N-terminal-labeled phosphoryl group not only improved the ionization efficiency of peptides and increased the protein sequence coverage for peptide mass fingerprints but also greatly enhanced the intensities of b ions, suppressed the internal fragments, and reduced the complexity of the tandem mass spectrometry (MS/MS) fragmentation patterns of peptides. By using nano liquid chromatography chip/time-of-flight mass spectrometry (nano LC-chip/TOF MS) for the protein quantification, the obtained results showed excellent correlation of the measured ratios to theoretical ratios with relative errors ranging from 0.5% to 6.7% and relative standard deviation of less than 10.6%, indicating that the developed method was reproducible and precise. The isotope effect was negligible because of the deuterium atoms were placed adjacent to the neutral phosphoryl group with high electrophilicity and moderately small size. Moreover, the SIPL approach used inexpensive reagents and was amenable to samples from various sources, including cell culture, biological fluids, and tissues. The method development based on organic phosphorus chemistry offered a new approach for quantitative proteomics by using novel stable isotope labeling reagents.


Asunto(s)
Química Orgánica , Marcaje Isotópico , Fragmentos de Péptidos/análisis , Fósforo/química , Proteínas/análisis , Animales , Bovinos , Cromatografía Liquida , Humanos , Nanotecnología , Fosforilación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/metabolismo
8.
J Clin Microbiol ; 50(11): 3780-2, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22972831

RESUMEN

Staphylococcus aureus can be distinguished from similar coagulase-positive staphylococci by its absence of ß-galactosidase activity. This is commonly tested using o-nitrophenyl-ß-D-galactopyranoside (ONPG) as the substrate. Unexpectedly, 111 and 58 of 123 isolates displayed apparent ß-galactosidase activity in the ONPG assay and on the Vitek 2 system, respectively. Compositional analysis showed that the yellow coloration of the positive ONPG assay resulted from production of 2-aminophenoxazin-3-one. Alternative ß-galactosidase substrates like X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) should be used for testing staphylococci.


Asunto(s)
Reacciones Falso Positivas , Oxazinas/metabolismo , Staphylococcus aureus/enzimología , Staphylococcus aureus/metabolismo , beta-Galactosidasa/análisis , Animales , Galactósidos/metabolismo , Humanos , Indoles/metabolismo , Nitrofenilgalactósidos/metabolismo , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/aislamiento & purificación
9.
Proteomics ; 10(9): 1875-9, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20209508

RESUMEN

Unusual truncated forms of nucleocapsid protein (NP) were identified in the lysate of MDCK cells infected by Avian influenza virus (H9N2) using MS-based proteomics approach. Moreover, O-sulfonation that was considered as an unusual modification was identified in one of the tryptic peptides from the truncated NP. The findings might have implications on better understanding on the role of nucleoprotein in Avian influenza virus-host interaction.


Asunto(s)
Subtipo H9N2 del Virus de la Influenza A/química , Proteínas de la Nucleocápside/análisis , Sistemas de Lectura Abierta , Secuencia de Aminoácidos , Animales , Línea Celular , Subtipo H9N2 del Virus de la Influenza A/genética , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Proteómica , Transcripción Genética
10.
FASEB J ; 23(10): 3377-82, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19553505

RESUMEN

Influenza A viruses are RNA viruses that contain negative-sense, single-stranded, and segmented RNA genome, which depends on virally encoded RNA-dependent RNA polymerase and cellular DNA-dependent RNA polymerase for replication of viral genome and transcription of viral mRNA, respectively. Hemagglutinin (HA), one of the major surface proteins of the influenza virus, is responsible for virus attachment to the receptor of host cells to initiate an infection. Amino acid (AA) substitutions in HA may cause changes in virus antigenicity and even receptor specificity. To detect the AA substitutions within HA at protein level, nanoelectrospray-MS/MS was used to analyze tryptic digestion of HA antigen directly purified from virus particles of an avian influenza virus, A/WDK/JX/12416/2005 (H1N1), of which the HA gene was sequenced as a reference. The comparison of the sequences obtained from analysis of viral genome and peptide found seven variations between HA gene and protein, namely E103K, R130K, T169I, I338V, N387S, S398I/L, and I399S in HA. Because influenza virus uses different polymerase machineries for replication and transcription, these substitutions could be introduced in the viral genome through replication process but not in viral mRNA in the transcription. The results, for the first time, provided experimental evidence showing differences in AA sequence obtained from direct analysis of viral protein derived from viral genome.


Asunto(s)
Sustitución de Aminoácidos , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Replicación Viral/genética , Secuencia de Aminoácidos , Animales , Aves/virología , Humanos , Gripe Aviar/virología , Gripe Humana/virología , Ratones , Datos de Secuencia Molecular , Mutación , Transcripción Genética
11.
J Am Soc Mass Spectrom ; 20(2): 312-20, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19019697

RESUMEN

Matrix protein 1 (M1), the major structural protein of the avian influenza virus, plays a critical role in regulation of viral RNA transcription via interaction with RNA and transportation of RNP cores. Mutations in M1 have been frequently observed in the highly virulent avian influenza H5N1 virus, which might be crucial to the pathogenic function. Here we report the characterization of mutated peptides in M1 purified from highly pathogenic avian influenza virus H5N1 by nanoelectrospray MS and MS/MS analyses on a quadrupole-time-of-flight mass spectrometer (Q-TOFMS). The specificity of tandem mass spectrometry allowed the identification of six amino acid (AA) substitutions in M1, including R95K, A166V, I168T, N207S, N224S, and R230K. Two commonly observed modifications such as oxidation and deamidation were accurately assigned in the protein. Bioinformatics analysis suggested some relationship between the amino acid substitution and structural property of M1 protein. Discussions on de novo sequencing of MS/MS spectra, especially in dealing with the AA substitutions, were provided.


Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/química , Nanotecnología/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Embrión de Pollo , Datos de Secuencia Molecular
12.
J Chromatogr A ; 1164(1-2): 113-9, 2007 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-17631302

RESUMEN

A new, sensitive and selective HPLC method with fluorescence detector (HPLC-FLD) for the determination of nephrotoxic and carcinogenic aristolochic acid (AA) in herbal medicines by using pre-column derivatization with zinc powder in acetic acid is presented. Variables governing the derivatization reaction, such as the amount of zinc powder and acetic acid, as well as the derivatization time were studied and optimized. An extended linear dynamic range over three orders of magnitude was observed for AA-I and AA-II (R(2)>0.9998). Method accuracy at low, medium and high spiked AA levels determined by the percentage mean deviation was below 4.4% and 7.2% for AA-I and AA-II, respectively. The detection limits of 0.39 ng/mL (AA-I) and 0.52 ng/mL (AA-II) were 2 orders of magnitude lower than those obtained from HPLC-MS or CE-ECD analyses, 3-4 orders of magnitude lower than those from HPLC-UV or CE-UV methods. The developed method has been applied for the determination of AA in herbal medicines. Among the tested samples, Guanmutong had the highest AA concentration (2607.0 microg/g AA-I, 711.2 microg/g AA-II). Comparison studies between HPLC-FLD and HPLC-MS/MS demonstrated that the two methods gave similar quantitative results for the selected herb samples.


Asunto(s)
Ácidos Aristolóquicos/química , Cromatografía Líquida de Alta Presión/métodos , Medicamentos Herbarios Chinos/química , Ácidos Aristolóquicos/análisis , Carcinógenos/análisis , Carcinógenos/química , Medicamentos Herbarios Chinos/análisis , Fluorescencia , Estructura Molecular , Reproducibilidad de los Resultados
13.
Artículo en Inglés | MEDLINE | ID: mdl-16824810

RESUMEN

A liquid chromatography-ion trap mass spectrometry method with three "time segments" has been developed to determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) in edible goldfish muscle. By using the optimized "time segments", MG and LMG as well as the internal standard atrazine-d(5) were analyzed with good sensitivity with positive ESI-MS in a single run. The homogenized fish muscle tissues were extracted with a solution of perchloric acid and acetonitrile, followed by partitioning with dichloromethane. Strata-x polymeric solid-phase extraction column was used for the clean-up process. The determination of MG and LMG was achieved by using a reversed-phase HPLC gradient program coupled with MS/MS in multiple-reaction-monitoring mode. Matrix calibration curves were linear over the ranges of 5-500 ng/ml for MG and 1-100 ng/ml for LMG. Recoveries of the fish tissue extraction at three spiked levels (2, 10 and 30 ng/g for MG as well as 0.4, 2 and 6 ng/g for LMG) were better than 71% and 89%, respectively. Relative standard derivations from six determinations were less than 8%. The method detection limits were 0.13 ng/g for MG and 0.06 ng/g for LMG.


Asunto(s)
Compuestos de Anilina/análisis , Cromatografía Líquida de Alta Presión/métodos , Músculos/química , Colorantes de Rosanilina/análisis , Animales , Carpa Dorada , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem
14.
Diagn Microbiol Infect Dis ; 85(2): 249-54, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-27105773

RESUMEN

Early diagnosis of acute community-acquired pneumonia (CAP) is important in patient triage and treatment decisions. To identify biomarkers that distinguish patients with CAP from non-CAP controls, we conducted an untargeted global metabolome analysis for plasma samples from 142 patients with CAP (CAP cases) and 97 without CAP (non-CAP controls). Thirteen lipid metabolites could discriminate between CAP cases and non-CAP controls with area-under-the-receiver-operating-characteristic curve of >0.8 (P ≤ 10(-9)). The levels of glycosphingolipids, sphingomyelins, lysophosphatidylcholines and L-palmitoylcarnitine were higher, while the levels of lysophosphatidylethanolamines were lower in the CAP cases than those in non-CAP controls. All 13 metabolites could distinguish CAP cases from the non-infection, extrapulmonary infection and non-CAP respiratory tract infection subgroups. The levels of trihexosylceramide (d18:1/16:0) were higher, while the levels of lysophosphatidylethanolamines were lower, in the fatal than those of non-fatal CAP cases. Our findings suggest that lipid metabolites are potential diagnostic and prognostic biomarkers for CAP.


Asunto(s)
Biomarcadores/sangre , Infecciones Comunitarias Adquiridas/diagnóstico , Infecciones Comunitarias Adquiridas/patología , Lípidos/sangre , Neumonía/diagnóstico , Neumonía/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Diagnóstico Precoz , Femenino , Humanos , Masculino , Persona de Mediana Edad , Plasma/química , Pronóstico , Adulto Joven
15.
Diagn Microbiol Infect Dis ; 84(2): 125-34, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26658315

RESUMEN

Thirteen Aspergillus isolates recovered from nails of 13 patients (fingernails, n=2; toenails, n=11) with onychomycosis were characterized. Twelve strains were identified by multilocus sequencing as Aspergillus spp. (Aspergillus sydowii [n=4], Aspergillus welwitschiae [n=3], Aspergillus terreus [n=2], Aspergillus flavus [n=1], Aspergillus tubingensis [n=1], and Aspergillus unguis [n=1]). Isolates of A. terreus, A. flavus, and A. unguis were also identifiable by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The 13th isolate (HKU49(T)) possessed unique morphological characteristics different from other Aspergillus spp. Molecular characterization also unambiguously showed that HKU49(T) was distinct from other Aspergillus spp. We propose the novel species Aspergillus hongkongensis to describe this previously unknown fungus. Antifungal susceptibility testing showed most Aspergillus isolates had low MICs against itraconazole and voriconazole, but all Aspergillus isolates had high MICs against fluconazole. A diverse spectrum of Aspergillus species is associated with onychomycosis. Itraconazole and voriconazole are probably better drug options for Aspergillus onychomycosis.


Asunto(s)
Antifúngicos/farmacología , Aspergillus/clasificación , Aspergillus/aislamiento & purificación , Variación Genética , Onicomicosis/microbiología , Adulto , Anciano , Aspergillus/química , Aspergillus/genética , Femenino , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
16.
Exp Biol Med (Maywood) ; 240(6): 742-51, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25908634

RESUMEN

Melioidosis is an emerging, potentially fatal disease caused by Burkholderia pseudomallei, which requires prolonged antibiotic treatment to prevent disease relapse. However, difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes. Isolation of B. pseudomallei from clinical specimens has been improved with the use of selective media. However, even with positive cultures, identification of B. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. Commercial identification systems may fail to distinguish between B. pseudomallei and closely related species such as Burkholderia thailandensis. Genotypic identification of suspected isolates can be achieved by sequencing of gene targets such as groEL which offer higher discriminative power than 16S rRNA. Specific PCR-based identification of B. pseudomallei has also been developed using B. pseudomallei-specific gene targets such as Type III secretion system and Tat-domain protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolutionary technique for pathogen identification, has been shown to be potentially useful for rapid identification of B. pseudomallei, although existing databases require optimization by adding reference spectra for B. pseudomallei. Despite these advances in bacterial identification, diagnostic problems encountered in culture-negative cases remain largely unresolved. Although various serological tests have been developed, they are generally unstandardized "in house" assays and have low sensitivities and specificities. Although specific PCR assays have been applied to direct clinical and environmental specimens, the sensitivities for diagnosis remain to be evaluated. Metabolomics is an uprising tool for studying infectious diseases and may offer a novel approach for exploring potential diagnostic biomarkers. The metabolomics profiles of B. pseudomallei culture supernatants can be potentially distinguished from those of related bacterial species including B. thailandensis . Further studies using bacterial cultures and direct patient samples are required to evaluate the potential of metabolomics for improving diagnosis of melioidosis.


Asunto(s)
Burkholderia mallei/genética , Burkholderia mallei/metabolismo , Técnicas de Genotipaje , Melioidosis/diagnóstico , Melioidosis/genética , Melioidosis/metabolismo , Metabolómica , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/genética , Burkholderia mallei/patogenicidad , Chaperonina 60/genética , Chaperonina 60/metabolismo , Bases de Datos Factuales , Técnicas de Genotipaje/métodos , Técnicas de Genotipaje/tendencias , Humanos , Melioidosis/microbiología , Metabolómica/métodos , Metabolómica/tendencias , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo
17.
Cell Biosci ; 5: 26, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26097677

RESUMEN

BACKGROUND: Burkholderia pseudomallei is an emerging pathogen that causes melioidosis, a serious and potentially fatal disease which requires prolonged antibiotics to prevent relapse. However, diagnosis of melioidosis can be difficult, especially in culture-negative cases. While metabolomics represents an uprising tool for studying infectious diseases, there were no reports on its applications to B. pseudomallei. To search for potential specific biomarkers, we compared the metabolomics profiles of culture supernatants of B. pseudomallei (15 strains), B. thailandensis (3 strains), B. cepacia complex (14 strains), P. aeruginosa (4 strains) and E. coli (3 strains), using ultra-high performance liquid chromatography-electrospray ionization-quadruple time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS). Multi- and univariate analyses were used to identify specific metabolites in B. pseudomallei. RESULTS: Principal component and partial-least squares discrimination analysis readily distinguished the metabolomes between B. pseudomallei and other bacterial species. Using multi-variate and univariate analysis, eight metabolites with significantly higher levels in B. pseudomallei were identified. Three of the eight metabolites were identified by MS/MS, while five metabolites were unidentified against database matching, suggesting that they may be potentially novel compounds. One metabolite, m/z 144.048, was identified as 4-methyl-5-thiazoleethanol, a degradation product of thiamine (vitamin B1), with molecular formula C6H9NOS by database searches and confirmed by MS/MS using commercially available authentic chemical standard. Two metabolites, m/z 512.282 and m/z 542.2921, were identified as tetrapeptides, Ile-His-Lys-Asp with molecular formula C22H37N7O7 and Pro-Arg-Arg-Asn with molecular formula C21H39N11O6, respectively. To investigate the high levels of 4-methyl-5-thiazoleethanol in B. pseudomallei, we compared the thiamine degradation pathways encoded in genomes of B. pseudomallei and B. thailandensis. While both B. pseudomallei and B. thailandensis possess thiaminase I which catalyzes degradation of thiamine to 4-methyl-5-thiazoleethanol, thiM, which encodes hydroxyethylthiazole kinase responsible for degradation of 4-methyl-5-thiazoleethanol, is present and expressed in B. thailandensis as detected by PCR/RT-PCR, but absent or not expressed in all B. pseudomallei strains. This suggests that the high 4-methyl-5-thiazoleethanol level in B. pseudomallei is likely due to the absence of hydroxyethylthiazole kinase and hence reduced downstream degradation. CONCLUSION: Eight novel biomarkers, including 4-methyl-5-thiazoleethanol and two tetrapeptides, were identified in the culture supernatant of B. pseudomallei.

18.
J Infect ; 70(5): 433-44, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25727996

RESUMEN

OBJECTIVES: Rapid diagnostic tests for bacteremia are important for early treatment to improve clinical outcome. We sought to identify plasma biomarkers that can identify patients with bacteremia using an untargeted global metabolomic analysis. METHODS: Plasma metabolomic profiles were analyzed for 145 adult patients with (cases) and without (controls) bacteremia using ultra-high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). All metabolites were compared between cases and controls using a 2-tier filtering approach, and each metabolite underwent receiver operating characteristic (ROC) curve analysis. Individual metabolites that distinguish between cases and controls were characterized. Subgroup analysis was performed to identify metabolites with prognostic significance. RESULTS: After 2-tier filtering, 128 molecular features were identified to be potential biomarkers that could distinguish cases from controls. Five metabolites had an area under the ROC curve (AUC) of >0.8 in ROC curve analysis, including a sphingolipid, an acylcarnitine, a fatty acid ester, and 2 glycerophosphocholines. These metabolites could distinguish cases from controls in the unsupervised hierarchical clustering analysis. Subgroup analysis of bacteremic patients showed that the level of trans-2,3,4-trimethoxycinnamate was lower in fatal than non-fatal cases. CONCLUSIONS: Plasma lipid mediators of inflammation can distinguish bacteremia cases from non-bacteremia controls. These biomarkers may be used as targets for rapid test in clinical practice.


Asunto(s)
Bacteriemia/diagnóstico , Mediadores de Inflamación/aislamiento & purificación , Lípidos/sangre , Adulto , Anciano , Anciano de 80 o más Años , Bacteriemia/sangre , Biomarcadores/sangre , Carnitina/análogos & derivados , Carnitina/sangre , Cromatografía Líquida de Alta Presión , Ácidos Grasos/sangre , Femenino , Humanos , Masculino , Metabolómica , Persona de Mediana Edad , Pronóstico , Curva ROC , Esfingolípidos/sangre
19.
Sci Rep ; 5: 9114, 2015 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-25766252

RESUMEN

Schisandrin B (SchB) is one of the most abundant bioactive dibenzocyclooctadiene derivatives found in the fruit of Schisandra chinensis. Here, we investigated the potential therapeutic effects of SchB on non-alcoholic fatty-liver disease (NAFLD). In lipidomic study, ingenuity pathway analysis highlighted palmitate biosynthesis metabolic pathway in the liver samples of SchB-treated high-fat-diet-fed mice. Further experiments showed that the SchB treatment reduced expression and activity of fatty acid synthase, expressions of hepatic mature sterol regulatory element binding protein-1 and tumor necrosis factor-α, and hepatic level of palmitic acid which is known to promote progression of steatosis to steatohepatitis. Furthermore, the treatment also reduced hepatic fibrosis, activated nuclear factor-erythroid-2-related factor-2 which is known to attenuate the progression of NASH-related fibrosis. Interestingly, in fasting mice, a single high-dose SchB induced transient lipolysis and increased the expressions of adipose triglyceride lipase and phospho-hormone sensitive lipase. The treatment also increased plasma cholesterol levels and 3-hydroxy-3-methylglutaryl-CoA reductase activity, reduced the hepatic low-density-lipoprotein receptor expression in these mice. Our data not only suggest SchB is a potential therapeutic agent for NAFLD, but also provided important information for a safe consumption of SchB because SchB overdosed under fasting condition will have adverse effects on lipid metabolism.


Asunto(s)
Lignanos/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Metabolómica , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Ácido Palmítico/metabolismo , Compuestos Policíclicos/farmacología , Animales , Ciclooctanos/administración & dosificación , Ciclooctanos/farmacología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Ayuno , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/metabolismo , Lignanos/administración & dosificación , Lípidos/sangre , Lipólisis , Hígado/efectos de los fármacos , Hígado/metabolismo , Cirrosis Hepática/etiología , Masculino , Redes y Vías Metabólicas , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/genética , Compuestos Policíclicos/administración & dosificación , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
20.
Emerg Microbes Infect ; 4(1): e6, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26038762

RESUMEN

Although previous studies have reported the use of metabolomics for Mycobacterium species differentiation, little is known about the potential of extracellular metabolites of Mycobacterium tuberculosis (MTB) as specific biomarkers. Using an optimized ultrahigh performance liquid chromatography-electrospray ionization-quadruple time of flight-mass spectrometry (UHPLC-ESI-Q-TOF-MS) platform, we characterized the extracellular metabolomes of culture supernatant of nine MTB strains and nine non-tuberculous Mycobacterium (NTM) strains (four M. avium complex, one M. bovis Bacillus Calmette-Guérin (BCG), one M. chelonae, one M. fortuitum and two M. kansasii). Principal component analysis readily distinguished the metabolomes between MTB and NTM. Using multivariate and univariate analysis, 24 metabolites with significantly higher levels in MTB were identified. While seven metabolites were identified by tandem mass spectrometry (MS/MS), the other 17 metabolites were unidentified by MS/MS against database matching, suggesting that they may be potentially novel compounds. One metabolite was identified as dexpanthenol, the alcohol analog of pantothenic acid (vitamin B5), which was not known to be produced by bacteria previously. Four metabolites were identified as 1-tuberculosinyladenosine (1-TbAd), a product of the virulence-associated enzyme Rv3378c, and three previously undescribed derivatives of 1-TbAd. Two derivatives differ from 1-TbAd by the ribose group of the nucleoside while the other likely differs by the base. The remaining two metabolites were identified as a tetrapeptide, Val-His-Glu-His, and a monoacylglycerophosphoglycerol, phosphatidylglycerol (PG) (16∶0/0∶0), respectively. Further studies on the chemical structure and biosynthetic pathway of these MTB-specific metabolites would help understand their biological functions. Studies on clinical samples from tuberculosis patients are required to explore for their potential role as diagnostic biomarkers.


Asunto(s)
Biomarcadores/análisis , Metabolómica , Mycobacterium tuberculosis/metabolismo , Cromatografía Líquida de Alta Presión , Humanos , Lípidos/análisis , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/aislamiento & purificación , Micobacterias no Tuberculosas/clasificación , Micobacterias no Tuberculosas/aislamiento & purificación , Micobacterias no Tuberculosas/metabolismo , Ácido Pantoténico/análogos & derivados , Ácido Pantoténico/análisis , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem
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