RESUMEN
A perchlorotriarylmethyl tricarboxylic acid radical 99% enriched in 13C at the central carbon (13C1-PTMTC) was characterized in phosphate buffered saline solution (pH = 7.2) (PBS) at ambient temperature. Samples immobilized in 1:1 PBS:glycerol or in 9:1 trehalose:sucrose were studied as a function of temperature. Isotope enrichment at C1 creates a trityl that can be used to accurately measure microscopic viscosity. Understanding of the impact of the 13C hyperfine interaction on electron spin relaxation is important for application of this trityl in oximetry and distance measurements. The anisotropic 13C1 hyperfine couplings (Ax = Ay = 24 ± 2 MHz, Az = 200 ± 1 MHz) are larger than for the related 13C1-perdeuterated Finland trityl (13C1-dFT) and the g anisotropy (gx = 2.0013, gy = 2.0016, gz = 2.0042) is slightly larger than for 13C1-dFT. The tumbling correlation times (τR) for 13C1-PTMTC are 0.20 ± 0.02 ns in PBS and 0.40 ± 0.05 ns in 3:1 PBS:glycerol, which are shorter than for 13C1-dFT in the same solutions. T1 for 13C1-PTMTC is 3.5 ± 0.5 µs in PBS and 5.3 ± 0.4 µs in 3:1 PBS:glycerol, which are shorter than for 13C1-dFT due to faster tumbling, larger anisotropy of the 13C1 hyperfine, and about 30% larger contribution from the local mode. In immobilized samples T1 for 13C1-PTMTC is similar to that for 13C1-dFT and other trityls without chlorine or 13C1 substituents, indicating that the 13C1 and Cl substituents on the phenyl rings have little impact on T1. The temperature dependence of T1 was modeled with contributions from the direct, Raman, and local mode processes. Broadening of CW linewidths of about 0.6 G in fluid solution and about 2 G in rigid lattice is attributed to unresolved 35,37Cl hyperfine couplings.
RESUMEN
Enzyme-linked immunosorbent assay (ELISA) is the gold standard method for protein biomarkers. However, scaling up ELISA for multiplexed biomarker analysis is not a trivial task due to the lengthy procedures for fluid manipulation and high reagent/sample consumption. Herein, we present a highly scalable multiplexed ELISA that achieves a similar level of performance to commercial single-target ELISA kits as well as shorter assay time, less consumption, and simpler procedures. This ELISA is enabled by a novel microscale fluid manipulation method, composable microfluidic plates (cPlate), which are comprised of miniaturized 96-well plates and their corresponding channel plates. By assembling and disassembling the plates, all of the fluid manipulations for 96 independent ELISA reactions can be achieved simultaneously without any external fluid manipulation equipment. Simultaneous quantification of four protein biomarkers in serum samples is demonstrated with the cPlate system, achieving high sensitivity and specificity (â¼ pg/mL), short assay time (â¼1 h), low consumption (â¼5 µL/well), high scalability, and ease of use. This platform is further applied to probe the levels of three protein biomarkers related to vascular dysfunction under pulmonary nanoparticle exposure in rat's plasma. Because of the low cost, portability, and instrument-free nature of the cPlate system, it will have great potential for multiplexed point-of-care testing in resource-limited regions.
Asunto(s)
Proteína C-Reactiva/análisis , Antígeno Carcinoembrionario/análisis , Ensayo de Inmunoadsorción Enzimática , Interleucina-6/análisis , Técnicas Analíticas Microfluídicas , Antígeno Prostático Específico/análisis , Biomarcadores/análisis , HumanosRESUMEN
BACKGROUND: The environmental gliding bacteria Lysobacter are emerging as a new group of biocontrol agents due to their prolific production of lytic enzymes and potent antibiotic natural products. These bacteria are intrinsically resistant to many antibiotics, but the mechanisms behind the antibiotic resistance have not been investigated. RESULTS: Previously, we have used chloramphenicol acetyltransferase gene (cat) as a selection marker in genetic manipulation of natural product biosynthetic genes in Lysobacter, because chloramphenicol is one of the two common antibiotics that Lysobacter are susceptible to. Here, we found L. enzymogenes, the most studied species of this genus, could still grow in the presence of a low concentration of chloramphenicol. Three chloramphenicol derivatives (1-3) with an unusual acylation pattern were identified in a cat-containing mutant of L. enzymogenes and in the wild type. The compounds included chloramphenicol 3'-isobutyrate (1), a new compound chloramphenicol 1'-isobutyrate (2), and a rare chloramphenicol 3'-isovalerate (3). Furthermore, a mutation of a global regulator gene (clp) or a Gcn5-related N-acetyltransferase (GNAT) gene in L. enzymogenes led to nearly no growth in media containing chloramphenicol, whereas a complementation of clp restored the chloramphenicol acylation as well as antibiotic HSAF production in the clp mutant. CONCLUSIONS: The results indicated that L. enzymogenes contains a pool of unusual acyl donors for enzymatic modification of chloramphenicol that confers the resistance, which may involve the Clp-GNAT regulatory system. Because Lysobacter are ubiquitous inhabitants of soil and water, the finding may have important implications in understanding microbial competitions and bioactive natural product regulation.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/metabolismo , Cloranfenicol/administración & dosificación , Cloranfenicol/farmacocinética , Farmacorresistencia Bacteriana/fisiología , Lysobacter/efectos de los fármacos , Lysobacter/metabolismo , Acilación , Antibacterianos/administración & dosificación , Antibacterianos/farmacocinética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cloranfenicol O-Acetiltransferasa/genética , Lysobacter/clasificación , Especificidad de la EspecieRESUMEN
Lysobacter species are emerging as new sources of antibiotics. The regulation of these antibiotics is not well understood. Here, we identified a small molecule metabolite (LeDSF3) that regulates the biosynthesis of the antifungal antibiotic heat-stable antifungal factor (HSAF), a polycyclic tetramate macrolactam with a structure and mode of action distinct from the existing antifungal drugs. LeDSF3 was isolated from the culture broth of Lysobacter enzymogenes, and its chemical structure was established by NMR and MS. The purified compound induced green fluorescence in a reporter strain of Xanthomonas campestris, which contained a gfp gene under the control of a diffusible signaling factor (DSF)-inducible promoter. Exogenous addition of LeDSF3 in L. enzymogenes cultures significantly increased the HSAF yield, the transcription of HSAF biosynthetic genes, and the antifungal activity of the organism. The LeDSF3-regulated HSAF production is dependent on the two-component regulatory system RpfC/RpfG. Moreover, LeDSF3 upregulated the expression of the global regulator cAMP receptor-like protein (Clp). The disruption of clp led to no HSAF production. Together, the results show that LeDSF3 is a fatty acid-derived, diffusible signaling factor positively regulating HSAF biosynthesis and that the signaling is mediated by the RfpC/RpfG-Clp pathway. These findings may facilitate the antibiotic production through applied genetics and molecular biotechnology in Lysobacter, a group of ubiquitous yet underexplored microorganisms.
Asunto(s)
Antifúngicos/metabolismo , Proteínas Bacterianas/metabolismo , Lysobacter/enzimología , Lysobacter/genética , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Escherichia coli/genética , Genes Reporteros , Espectroscopía de Resonancia Magnética , Transducción de Señal , TemperaturaRESUMEN
Lysobacter enzymogenes is a bacterial biological control agent emerging as a new source of antibiotic metabolites, such as heat-stable antifungal factor (HSAF) and the antibacterial factor WAP-8294A2. The regulatory mechanism(s) for antibiotic metabolite biosynthesis remains largely unknown in L. enzymogenes. Clp, a cyclic adenosine monophosphate (cAMP)-receptor-like protein, is shown to function as a global regulator in modulating biocontrol-associated traits in L. enzymogenes. However, the genetic basis of Clp signaling remains unclear. Here, we utilized transcriptome/microarray analysis to determine the Clp regulon in L. enzymogenes. We showed that Clp is a global regulator in gene expression, as the transcription of 775 genes belonging to 19 functional groups was differentially controlled by Clp signaling. Analysis of the Clp regulon detected previously characterized Clp-modulated functions as well as novel loci. These include novel loci involved in antibiotic metabolite biosynthesis and surface motility in L. enzymogenes. We further showed experimentally that Clp signaling played a positive role in regulating the biosynthesis of HSAF and WAP-8294A2, as well as surface motility which is a type-IV-pilus-dependent trait. The regulation by Clp signaling of antibiotic (HSAF and WAP-8294A2) biosynthesis and surface motility was found to be independent. Importantly, we identified a factor Lysobacter acetyltransferase (Lat), a homologue of histone acetyltransferase Hpa2, which was regulated by Clp and involved in HSAF biosynthesis, but not associated with WAP-8294A2 production and surface motility. Overall, our study provided new insights into the regulatory role and molecular mechanism of Clp signaling in L. enzymogenes.
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Antiinfecciosos/metabolismo , Regulación Bacteriana de la Expresión Génica , Locomoción , Lysobacter/fisiología , Metabolismo Secundario , Transducción de Señal , Factores de Transcripción/metabolismo , Perfilación de la Expresión Génica , Lysobacter/genética , Análisis por MicromatricesRESUMEN
Soluble stable radicals are used as spin probes and spin labels for in vitro and in vivo electron paramagnetic resonance (EPR) spectroscopy and imaging applications. We report the synthesis and characterization of a perchlorinated triarylmethyl radical enriched 99% at the central carbon, 13C1-PTMTC. The anisotropy of the hyperfine splitting with the 13C1 (Ax = 26, Ay = 25, Az = 199.5 MHz) and the g (gx = 2.0015, gy = 2.0015, gz = 2.0040) are responsible for a strong effect of the radical tumbling rate on the EPR spectrum. The rotational correlation time can be determined by spectral simulation or via the line width or the apparent Az after calibration, so the spin probe 13C1-PTMTC can be used to measure media microviscosity with high sensitivity.
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Carbono , Espectroscopía de Resonancia por Spin del Electrón , Marcadores de SpinRESUMEN
Bead-based ELISA in microfluidics is a promising platform for reducing the reagent consumption and improving assay throughput due to its fast binding kinetics and low reagent consumption. Current microfluidic bead-based immunoassay mainly relies on magnetic and centrifugal forces to manipulate microparticles to complete an assay protocol. Here we report an acoustic streaming-based microfluidic method that can perform all the fluid and particle operations of bead-based ELISA. A series of sharp-edge structures are used to generate a long-range coupled acoustic streaming that enables simultaneous particle trapping and active molecular exchange in a dead-end microchannel. The device achieved >99% of molecular exchange in 4â¯min, while maintaining a particle trapping efficiency of 85%. This acoustofluidic-based method demonstrates all three key operations in a bead-based immunoassay: (1) Beads "immobilization"; (2) Active mixing of fluid for bead/target binding; (3) Active molecular exchange for reagent loading and washing. Finally, on-chip quantitative detection of biomarker IL-6 with a limit of detection â¼50â¯pg/mL is achieved using this platform including an enzymatic signal amplification step. The small footprint, simple setup, and continuous flow operation of the acoustic streaming-based method makes it an attractive platform for continuous flow bead-based immunoassay.
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Acústica , Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos/inmunología , Biomarcadores/análisis , Diseño de Equipo , Humanos , Interleucina-6/análisis , Interleucina-6/inmunología , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodosRESUMEN
We report the synthesis of hydroxyethyl tetrathiatriarylmethyl radical OX063 and its deuterated analogue OX071 for biomedical EPR applications.
RESUMEN
The immuno-competence hypothesis proposes that higher levels of testosterone increases the susceptibility to parasitism. Here we examined the testosterone levels in two species of flying squirrels (Glaucomys): one known to regularly host a nematode species (Strongyloides robustus) without ill effects (G. volans) and a closely related species that is considered negatively affected by the parasite. We quantified fecal testosterone levels in northern and southern flying squirrels (G. sabrinus, G. volans) with high-performance liquid chromatography-ultraviolet spectroscopy (HPLC-UV), and compared levels to endoparasites detected in individual squirrels. Qualitatively, we found highest levels of testosterone in male northern flying squirrels infected with Strongyloides robustus. This analytical approach represents an alternative and equally reliable method to using enzyme-linked immunosorbent assay (ELISA), for detecting and quantifying fecal testosterone levels.
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Polyketides (PKs) are a large group of natural products produced by microorganisms and plants. They are biopolymers of acetate and other short carboxylates and are biosynthesized by multifunctional enzymes called polyketide synthases (PKSs). This review discusses the biosynthesis of four toxic PK, aflatoxins, fumonisins, ochratoxins (OTs), and zearalenone. These metabolites are structurally diverse and differ in their mechanisms of toxicity. However, they are all of concern in food safety and agriculture because of their toxic properties and their frequent accumulation in crops used for food and feed. The focus is on the recent advancements in the understanding of the molecular mechanisms for the biosynthesis of these mycotoxins. Several of the mycotoxin PKSs have been genetically and biochemically studied while other PKSs remain to be investigated. Multiple post-PKS modifications are often required for the maturation of the mycotoxins. Many of these modification steps for aflatoxins and fumonisins are well established while the post-PKS modifications for zearalenone and OTs remain to be biochemically characterized. More efforts are needed to completely illustrate the biosynthetic mechanisms for this important group of PKs.
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Macrólidos/metabolismo , Hongos Mitospóricos/metabolismo , Micotoxinas/biosíntesis , Contaminación de Alimentos/prevención & control , Macrólidos/química , Micotoxinas/químicaRESUMEN
A screen for antifungal compounds from Lysobacter enzymogenes strain C3, a bacterial biological control agent of fungal diseases, has previously led to the isolation of heat-stable antifungal factor (HSAF). HSAF exhibits inhibitory activities against a wide range of fungal species and shows a novel mode of antifungal action by disrupting the biosynthesis of a distinct group of sphingolipids. We have now determined the chemical structure of HSAF, which is identical to that of dihydromaltophilin, an antifungal metabolite with a unique macrocyclic lactam system containing a tetramic acid moiety and a 5,5,6-tricyclic skeleton. We have also identified the genetic locus responsible for the biosynthesis of HSAF in strain C3. DNA sequencing of this locus revealed genes for a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), a sterol desaturase, a ferredoxin reductase, and an arginase. The disruption of the PKS-NRPS gene generated C3 mutants that lost the ability to produce HSAF and to inhibit fungal growth, demonstrating a hybrid PKS-NRPS that catalyzed the biosynthesis of the unique macrolactam system that is found in many biologically active natural products isolated from marine organisms. In addition, we have generated mutants with disrupted sterol desaturase, ferredoxin reductase, and arginase and examined the metabolites produced in these mutants. The work represents the first study of the genetic basis for the biosynthesis of the tetramic acid-containing macrolactams. The elucidation of the chemical structure of HSAF and the identification of the genetic locus for its biosynthesis establish the foundation for future exploitation of this group of compounds as new fungicides or antifungal drugs.
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Antifúngicos/metabolismo , Antifúngicos/farmacología , Ascomicetos/efectos de los fármacos , Lysobacter/genética , Lysobacter/metabolismo , Amidas/química , Amidas/metabolismo , Amidas/farmacología , Secuencia de Aminoácidos , Antifúngicos/química , Ascomicetos/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromatografía Líquida de Alta Presión , Lactamas/química , Lactamas/metabolismo , Lactamas/farmacología , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Modelos Genéticos , Datos de Secuencia Molecular , Estructura Molecular , Familia de Multigenes/genética , Mutación , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/metabolismo , Hidrocarburos Policíclicos Aromáticos/farmacología , Sintasas Poliquetidas/genética , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
A phytochemical investigation of the leaves of Hyperbaena valida resulted in the isolation and characterization of two erythrina-type alkaloids, 1 and 2, which were found to be antagonists at nicotinic receptors. Compound 1 was assigned as the new 15-amido-3-demethoxy-2alpha,3alpha-methylenedioxyerythroculine and compound 2 as the known 3-demethoxy-2alpha,3alpha-methylenedioxyerythroculine. Antagonism of a 100 microM nicotine response was observed for alkaloid 1 (IC50 value of 94 +/- 8 microM) and alkaloid 2 (IC50 value of 77 +/- 19 microM).