RESUMEN
Vancomycin resistant enterococci (VRE) constitute a challenging problem in health care institutions worldwide. Novel methods to rapidly identify resistances are highly required to ensure an early start of tailored therapy and to prevent further spread of the bacteria. Here, a spectroscopy-based rapid test is presented that reveals resistances of enterococci towards vancomycin within 3.5â hours. Without any specific knowledge on the strain, VRE can be recognized with high accuracy in two different enterococci species. By means of dielectrophoresis, bacteria are directly captured from dilute suspensions, making sample preparation very easy. Raman spectroscopic analysis of the trapped bacteria over a time span of two hours in absence and presence of antibiotics reveals characteristic differences in the molecular response of sensitive as well as resistant Enterococcus faecalis and Enterococcus faecium. Furthermore, the spectroscopic fingerprints provide an indication on the mechanisms of induced resistance in VRE.
Asunto(s)
Enterococcus faecalis/efectos de los fármacos , Enterococcus faecium/efectos de los fármacos , Vancomicina/farmacología , Enterococcus faecalis/química , Enterococcus faecium/química , Espectrometría Raman , Factores de Tiempo , Resistencia a la VancomicinaRESUMEN
Heme and heme degradation products play critical roles in numerous biological phenomena which until now have only been partially understood. One reason for this is the very low concentrations at which free heme, its complexes and the partly unstable degradation products occur in living cells. Therefore, powerful and specific detection methods are needed. In this contribution, the potential of nondestructive Raman spectroscopy for the detection, quantification and discrimination of heme and heme degradation products is investigated. Resonance Raman spectroscopy using different excitation wavelengths (413, 476, 532, and 752 nm) is employed to estimate the limit of detection for hemin, myoglobin, biliverdin, and bilirubin. Concentrations in the low micromolar range (down to 3 µmol/L) could be reliably detected when utilizing the resonance enhancement effect. Furthermore, a systematic study on the surface-enhanced Raman spectroscopy (SERS) detection of hemin in the presence of other cellular components, such as the highly similar cytochrome c, DNA, and the important antioxidant glutathione, is presented. A microfluidic device was used to reproducibly create a segmented flow of aqueous droplets and oil compartments. Those aqueous droplets acted as model chambers where the analytes have to compete for the colloid. With the help of statistical analysis, it was possible to detect and differentiate the pure substances as well as the binary mixtures and gain insights into their interaction.
Asunto(s)
Bilirrubina/análisis , Biliverdina/análisis , Hemo/análisis , Hemina/análisis , Mioglobina/análisis , Espectrometría Raman/instrumentación , Animales , Diseño de Equipo , Caballos , Límite de Detección , Técnicas Analíticas Microfluídicas/instrumentación , Modelos Moleculares , Espectrofotometría UltravioletaRESUMEN
Body fluids are easily accessible and contain valuable indices for medical diagnosis. Fascinating tools are tumour cells circulating in the peripheral blood of cancer patients. As these cells are extremely rare, they constitute a challenge for clinical diagnostics. In this contribution we present the Raman spectroscopic-based identification of different single cells in suspension that are found in peripheral blood of cancer patients including healthy cells like leukocytes and erythrocytes, and tumour cells like leukaemic cells and cells originating from solid tumours. Leukocytes and erythrocytes were isolated from the peripheral blood of healthy donors while myeloid leukaemia cells (OCI-AML3) and breast carcinoma derived cells (MCF-7, BT-20) were obtained from cell cultures. A laser emitting 785 nm light was used for optical trapping the single cells in the laser focus and to excite the Raman spectrum. Support vector machines were applied to develop a supervised classification model with spectra of 1210 cells originating from three different donors and three independent cultivation batches. Distinguishing tumour cells from healthy cells was achieved with a sensitivity of >99.7% and a specificity of >99.5%. In addition, the correct cell types were predicted with an accuracy of approximately 92%.
Asunto(s)
Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Leucemia Mieloide/sangre , Leucemia Mieloide/patología , Células Neoplásicas Circulantes/patología , Espectrometría Raman/métodos , Células Sanguíneas/citología , Línea Celular Tumoral , Femenino , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
In this work, regulation of organic cation transporter type 2 from rat (rOCT2) stably transfected in HEK293 cells was investigated by microfluorimetry with 4-(4-(dimethylamino)styryl)-N-methylpyridinium as substrate. The transport mediated by rOCT2 was specifically stimulated by PKA, phosphatidylinositol-3-kinase, p56(lck) tyrosine kinase, mitogen-extracellular-signal-regulated-kinase-1/2, calmodulin (CaM), and CaM-kinase-II. The regulatory pattern of rOCT2 differs markedly quantitatively and qualitatively from that of other OCT isoforms. Only CaM-dependent upregulation is conserved throughout the OCT family. For this reason, CaM regulation of rOCT2 was also investigated in isolated S3-segments (known to express only rOCT2) of male and female rat proximal tubules. Inhibition of CaM by calmidazolium significantly decreased rOCT2 activity (-49.0 +/- 13.6%, n = 4) in male but not female (9.0 +/- 13.0%, n = 4) rats. Real-time PCR and Western blot investigations of CaM expression in rat kidneys showed that male animals have significantly higher CaM expression. This is the first study describing post-translational gender-dependent rOCT2 regulation.
Asunto(s)
Calmodulina/genética , Calmodulina/metabolismo , Riñón/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Animales , Transporte Biológico , Calmodulina/antagonistas & inhibidores , Línea Celular , Femenino , Fluorometría , Regulación de la Expresión Génica , Humanos , Riñón/citología , Túbulos Renales Proximales/metabolismo , Masculino , Proteínas de Transporte de Catión Orgánico/agonistas , Transportador 2 de Cátion Orgánico , Compuestos de Piridinio/metabolismo , Ratas , Factores Sexuales , TransfecciónRESUMEN
Tip-enhanced Raman scattering is used to investigate the surface structure of the cell wall of Staphylococcus epidermidis with high lateral resolution and chemical specificity. For most biological samples, the transmission tip-enhanced Raman scattering set-up is ideally suited because it allows the specimen to be kept under specific environmental conditions, whereas it most efficiently collects the signal created at the field-enhancing probe. Special emphasis is given here to the parameters required to reproducibly set up the instrument, such that field-enhancement factors can be estimated properly. Also the importance of control experiments to avoid misinterpretation of signals will be emphasized by an example.
Asunto(s)
Pared Celular/ultraestructura , Espectrometría Raman/instrumentación , Espectrometría Raman/métodos , Staphylococcus epidermidis/ultraestructura , Humanos , Microscopía de Fuerza Atómica/métodosRESUMEN
Increasing resistance of many antibiotics has made the design of new drugs necessary. To assist a target-oriented search for new structures and for the elucidation of the mode of action of existing drugs, powerful analytical techniques are required. In this work, vibrational spectroscopy is used to shed more light on the as-yet elusive interaction of gyrase inhibitors of the fluoroquinolone type with their biological target inside the Gram-positive bacterium Staphylococcus epidermidis by investigating whole-cell changes that occur as a result of the presence of the drug moxifloxacin. IR absorption and Raman spectra with excitation off resonance (lambda exc = 532 nm) and in resonance with the biological targets DNA and the aromatic amino acids of gyrase (lambda exc = 244 nm) were recorded for unperturbed bacteria and bacteria in varying drug concentrations (0.08, 0.16, 0.27, and 0.62 microg moxifloxacin/mL bacterial culture). The spectral changes caused by the action of the drug were analyzed with the help of statistical methods, such as hierarchical cluster analysis (HCA), principal component analysis (PCA), and Fisher's linear discriminant analysis (LDA) combined with variable selection. The wavenumbers mostly affected by the action of the drug could be assigned to protein and DNA moieties, supporting the proposed mechanisms of a tertiary complex of the fluoroquinolone, the enzyme gyrase, and DNA.
Asunto(s)
Antibacterianos/farmacología , Fluoroquinolonas/farmacología , Staphylococcus epidermidis/efectos de los fármacos , Antibacterianos/química , Compuestos Aza/farmacología , Análisis por Conglomerados , ADN/metabolismo , Girasa de ADN/metabolismo , Fluoroquinolonas/química , Modelos Químicos , Modelos Estadísticos , Moxifloxacino , Análisis de Componente Principal , Quinolinas/farmacología , Espectrofotometría Infrarroja , Espectrometría Raman , Factores de Tiempo , Rayos UltravioletaRESUMEN
In this work we monitor the bacterial growth of a Bacillus pumilus batch culture by means of UV resonance Raman spectroscopy. Excitation with a wavelength of 244 nm especially enhances the Raman scattering of the aromatic amino acids and the nucleic acid bases and therefore is a good method to track the metabolic changes that occur during bacterial growth. Furthermore, a drug from the fluoroquinolone group is added to the bacterial suspension at the beginning of the exponential growth phase. With the help of chemometrical methods such as hierarchical cluster analysis (HCA) and principal component analysis (PCA) it is possible to visualize the small changes that occur in the UV resonance Raman spectra due to the interaction of the drug with its biological targets DNA and the enzyme gyrase within the bacterial cell.
Asunto(s)
Antibacterianos/farmacología , Bacillus/efectos de los fármacos , Espectrometría Raman/métodos , Bacillus/crecimiento & desarrollo , Fluoroquinolonas/farmacología , Análisis de Componente Principal , Espectrofotometría Ultravioleta/métodosRESUMEN
Quinolones are important gyrase inhibitors. Even though they are used as active agents in many antibiotics, the detailed mechanism of action on a molecular level is so far not known. It is of greatest interest to shed light on this drug-target interaction to provide useful information in the fight against growing resistances and obtain new insights for the development of new powerful drugs. To reach this goal, on a first step it is essential to understand the structural characteristics of the drugs and the effects that are caused by the environment in detail. In this work we report on Raman spectroscopical investigations of a variety of gyrase inhibitors (nalidixic acid, oxolinic acid, cinoxacin, flumequine, norfloxacin, ciprofloxacin, lomefloxacin, ofloxacin, enoxacin, sarafloxacin and moxifloxacin) by means of micro-Raman spectroscopy excited with various excitation wavelengths, both in the off-resonance region (532, 633, 830 and 1064 nm) and in the resonance region (resonance Raman spectroscopy at 244, 257 and 275 nm). Furthermore DFT calculations were performed to assign the vibrational modes, as well as for an identification of intramolecular hydrogen bonding motifs. The effect of small changes in the drug environment was studied by adding successively small amounts of water until physiological low concentrations of the drugs in aqueous solution were obtained. At these low concentrations resonance Raman spectroscopy proved to be a useful and sensitive technique. Supplementary information was obtained from IR and UV/vis spectroscopy.
Asunto(s)
Fluoroquinolonas/química , Espectrometría Raman/métodos , Compuestos Aza/química , Cinoxacino/química , Ciprofloxacina/análogos & derivados , Ciprofloxacina/química , Girasa de ADN/metabolismo , ADN Bacteriano/metabolismo , Enoxacino/química , Inhibidores Enzimáticos/farmacología , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Modelos Químicos , Modelos Moleculares , Modelos Teóricos , Moxifloxacino , Ácido Nalidíxico/química , Norfloxacino/química , Ofloxacino/química , Ácido Oxolínico/química , Quinolinas/química , Quinolonas/química , Espectrofotometría Infrarroja , Temperatura , Rayos Ultravioleta , Vibración , Agua/químicaRESUMEN
The reactions of CpZr(CH(3))(3), 1, and Cp(2)Zr(CH(3))(2), 2, with partially dehydroxylated silica, silica-alumina, and alumina surfaces have been carried out with careful identification of the resulting surface organometallic complexes in order to probe the relationship between catalyst structure and polymerization activity. The characterization of the supported complexes has been achieved in most cases by in situ infrared spectroscopy, surface microanalysis, qualitative and quantitative analysis of evolved gases during surface reactions with labeled surface, solid state (1)H and (13)C NMR using (13)C-enriched compounds, and EXAFS. 1 and 2 react with silica(500) and silica-alumina(500) by simple protonolysis of one Zr-Me bond by surface silanols with formation of a single well-defined neutral compound. In the case of silica-alumina, a fraction of the supported complexes exhibits some interactions with electronically unsaturated surface aluminum sites. 1 and 2 also react with the hydroxyl groups of gamma-alumina(500), leading to several surface structures. Correlation between EXAFS and (13)C NMR data suggests, in short, two main surface structures having different environments for the methyl group: [Al](3)-OZrCp(CH(3))(2) and [Al](2)-OZrCp(CH(3))(mu-CH(3))-[Al] for the monoCp series and [Al](2)-OZrCp(2)(CH(3)) and [Al]-OZrCp(2)(mu-CH(3))-[Al] for the bisCp series. Ethylene polymerization has been carried out with all the supported complexes under various reaction conditions. Silica-supported catalysts in the absence of any cocatalyst exhibited no activity whatsoever for ethylene polymerization. When the oxide contained Lewis acidic sites, the resulting surface species were active. The activity, although improved by the presence of additional cocatalysts, remained very low by comparison with that of the homogeneous metallocene systems. This trend has been interpreted on the basis of various possible parameters, including the (p-pi)-(d-pi) back-donation of surface oxygen atoms to the zirconium center.
RESUMEN
We investigate the phase diagram of antiferromagnetic spin ladders with additional exchange interactions on diagonal bonds by variational and numerical methods. These generalized spin ladders interpolate smoothly between the [Formula: see text] chain with competing nn and nnn interactions, the [Formula: see text] chain with alternating exchange and the antiferromagnetic (AF) S = 1 chain. The Majumdar - Ghosh ground states are formulated as matrix product states and are shown to exhibit the same type of hidden order as the AF S = 1 chain. Generalized matrix product states are used for a variational calculation of the ground state energy and the spin and string correlation functions. Numerical (Lanczos) calculations of the energies of the ground state and of the low-lying excited states are performed, and compare reasonably with the variational approach. Our results support the hypothesis that the dimer and Majumdar - Ghosh points are in the same phase as the AF S = 1 chain.
RESUMEN
The binding of initiator and elongator tRNAs to 70-S ribosomes and the 30-S subunits was followed by velocity sedimentation in the analytical ultracentrifuge. fMet-tRNAfMet binds to A-U-G-programmed 30-S subunits, but not to free or misprogrammed particles. Both the formylmethione residue and the initiation factors increase the stability of the 30-S x A-U-G x fMet-tRNAfMet complex. fMet-tRNAfMet is bound only to the P site of the 70-S ribosome even in the absence of A-U-G. Two copies of tRNAPhe or Phe-tRNAPhe are bound to the ribosome with similar affinity. In contrast to a recent report [Rheinberger et al. (1981) Proc. Natl Acad. Sci. USA, 78, 5310-5314], it is shown that three copies of tRNA cannot be bound simultaneously to the ribosome with binding constants higher than 2 x 10(4) M-1. Phe-tRNAPhe when present as the ternary complex Phe-tRNAPhe. EF-Tu x guanosine 5'-[beta,gamma-methylene]triphosphate binds exclusively to the A site. The peptidyl-tRNA analogue, acetylphenylalanine-tRNA, can occupy both ribosomal centers, albeit with a more than tenfold higher affinity for the P site. The thermodynamic data obtained under equilibrium conditions confirm the present view of two tRNA binding sites on the ribosome. The association constants determined are discussed in relation to the mechanism of ribosomal protein synthesis.