绿脓杆菌Pseudomonas aeruginosa BTE-1直接电催化特征

本文研究厌氧条件下产电绿脓杆菌P. aeruginosa BTE-1的电化学催化特征。研究结果表明,P. aeruginosa BTE-1菌株在厌氧条件下,不能分泌可充当电子介体的绿脓菌素,但可通过在电极表面形成生物膜呈现了直接电催化性能。P. aeruginosa BTE-1在电极表面形成生物膜与其在特定电极电位下向电极传递电子的过程直接相关,适宜的电位为+0.2 V (vs. SCE),电位过高可能会损害P. aeruginosa BTE-1细胞。室温范围内升高温度可增强P. aeruginosa BTE-1生物膜电催化活性,但过高的温度(>60℃)会抑制生物膜电催化活性。循环伏安曲线显示,在厌氧条件下形成的P. aeruginosa BTE-1生物膜,具有与典型产电菌株G. sulfurreducens相近的氧化还原电位(-0.4 V~ -0.2 V vs. SCE)。P. aeruginosa BTE-1生物膜可电催化酵母抽取物和葡萄糖,但不能电催化醋酸盐。     

绿脓杆菌阳极的微生物燃料电池含溶解氧性能研究

本文研究了绿脓杆菌分泌的电子中介体绿脓菌素与电极之间的反应,并探讨了溶解氧的影响. 通过循环伏安曲线、测试电极开路电位等方法,确定绿脓菌素阳极反应是受扩散控制的可逆反应. 菌液的溶解氧浓度在一定范围内（0 ~ 1.6 mg.L^-1）对绿脓菌素和电极之间的反应影响不大. 微生物燃料电池的极化曲线表明,当溶解氧为1.6 mg.L^-1时,微生物燃料电池输出电流下降了7%,对绿脓杆菌阳极的微生物燃料电池影响不大.     

高效离子交换液相色谱法分离绿脓杆菌外膜蛋白

利用高效ＤＥＡＥ离子交换液相色谱法分离纯化了绿脓杆菌标准菌株ＰＡＯ１外膜蛋白。流动相为ｐＨ８．０Ｔｒｉｓ－ＨＣｌ缓冲液,色谱柱为ＴＳＫｇｅｌ－ＤＥＡＥ－５ＰＷ（０．７５ｃｍ×７．５ｃｍｉ．ｄ．）。通过纯化外膜蛋白,可以为研究绿脓杆菌外膜通透性与抗生素耐药性之间的关系及缩短研究周期提供有效的方法。     

Inhibition of Swarming motility of Pseudomonas aeruginosa by Methanol extracts of Alpinia officinarum Hance. and Cinnamomum tamala T. Nees and Eberm

Bacterial drug resistance is a challenge in clinical settings, especially in countries like India. Hence, discovery of novel alternative therapeutics has become a necessity in the fight against drug resistance. Compounds that inhibit bacterial virulence properties form new therapeutic alternatives. Pseudomonas aeruginosa is an opportunistic, nosocomial pathogen that infects immune-compromised patients. Swarming motility is an important virulence property of Pseudomonas which aids it in reaching host cells under nutrient limiting conditions. Here, we report the screening of five plant extracts against swarming motility of P. aeruginosa and show that methanol extracts of Alpinia officinarum and Cinnamomum tamala inhibit swarming motility at 5 μg mL^?1 without inhibiting its growth. These extracts did not inhibit swimming and twitching motilities indicating a mode of action specific to swarming pathway. Preliminary experiments indicated that rhamnolipid production was not affected. This study reveals the potential of the two plants in anti-virulence drug discovery.     

Direct Electrochemical Sensing of Phenazine‐1‐carboxylic Acid Secreted by Pseudomonas chlororaphis subsp. aureofaciens BCRC 11057T Using Disposable Screen‐printed Carbon Electrode

A novel electrochemical approach for direct recognition of antibiotic phenazine‐1‐carboxylic acid (PCA) was developed. PCA was electropolymerized on preanodized screen‐printed carbon electrode (SPCE*‐PCA) through repetitive cyclic voltammetry and characterized by XPS and FESEM. Electron transfer involved intermediate phenomenon of diffusion‐controlled redox process and surface bound redox reaction. At pH 8 (optimum), SPCE*‐PCA had a detection limit of 0.51±0.04 μM, a quantification limit of 1.7±0.13 μM, linearity of up to 50 µM, a repeatability of 15.5 % and a reproducibility of 1.7 %. PCA secreted by Pseudomonas chlororaphis subsp. aureofaciens BCRC 11057^T was investigated successfully using present single run approach.     

Versatile online SPE–HPLC method for the analysis of Irinotecan and its clinically relevant metabolites in biomaterials

Monitoring levels of Irinotecan and its metabolites during cancer therapy could help link broad interpatient variations in antitumor activity and toxicity to the patient's metabolic status. We have developed and validated a versatile and highly sensitive method for the simultaneous determination of Irinotecan and its clinically relevant metabolites 7‐ethyl‐10‐hydroxy‐camptothecin (SN‐38) and SN‐38 glucuronide. Sample clean‐up involves precipitation by acetone/methanol/0.5 M trichloroacetic acid at 4:4:2 v/v followed by extraction of the metabolites on an SPE column by 20% methanol in 25 mM KH₂PO₄ pH 2.9. Online transfer to an analytical μBondapak C18 column, elution with 24% acetonitrile (ACN) in 0.1 M KH₂PO₄ pH 2.9 and fluorescence detection with excitation at 375 nm and emission at 430 nm for SN‐38 glucuronide and Irinotecan or 540 nm for SN‐38 results in high sensitivity (1–2 pg) and short (～10 min) run times. The method was used to determine the degree of SN‐38 glucuronidation in mice after Irinotecan administration and in cultured cancer cells exposed to SN‐38. The method may be used to better understand Irinotecan metabolism, personalize therapy, and develop Irinotecan‐based tumor targeting therapies.     

Combining Topology and Sequence Design for the Discovery of Potent Antimicrobial Peptide Dendrimers against Multidrug‐Resistant Pseudomonas aeruginosa

Multidrug‐resistant opportunistic bacteria, such as Pseudomonas aeruginosa, represent a major public health threat. Antimicrobial peptides (AMPs) and related peptidomimetic systems offer an attractive opportunity to control these pathogens. AMP dendrimers (AMPDs) with high activity against multidrug‐resistant clinical isolates of P. aeruginosa and Acinetobacter baumannii were now identified by a systematic survey of the peptide sequences within the branches of a distinct type of third‐generation peptide dendrimers. Combined topology and peptide sequence design as illustrated here represents a new and general strategy to discover new antimicrobial agents to fight multidrug‐resistant bacterial pathogens.     

The Synthesis of Chitosan Decorated Reduced Graphene Oxide‐Ferrocene Nanocomposite and its Application in Electrochemical Detection Rhodamine B

Chitosan (CS) decorated reduced graphene oxide‐Ferrocene nanocomposite (RGO?Fc?CS) was synthesized by a one‐pot reaction. In the synthesis process of RGO?Fc?CS, GO?Fc was successfully self‐assembled via the π–π interaction, which had the features of large surface area and the high load. Then GO‐Fc and CS linked via one‐pot under alkaline conditions. The FT?IR, TGA, SEM were employed to characterize the successful synthesis of RGO?Fc?CS composites. A miniature electrochemical system was fabricated by RGO?Fc?CS modified glassy carbon electrode (expressed as RGO?Fc?CS/GCE) for the sensitive detection of RhB enantiomers via DPV. Electrochemical results revealed that the RGO?Fc?CS/GCE exhibited high molecular recognition toward RhB. Due to the synergy between the RGO?Fc and CS, the RGO?Fc?CS/GCE showed a linear range of 0.001–70 μM with a LOD of 0.5 nM, indicating that RGO?Fc?CS/GCE has broad application prospects to simplify monitor RhB real‐time.     

Biological activity of chitosan–sugar hybrids: specific interaction with lectin

The specific interactions between lectins and chitosan–sugar hybrids, the synthesized chitosan derivatives linking carbohydrate residue to the amino group of chitosan, were investigated. The specific bindings of chitosan‐L ‐fucose (Fuc) hybrid with Ulex europaeus agglutinin I (UEA I, a lectin specific to L ‐Fuc), and chitosan‐N‐acetyl‐D ‐glucosamine (D ‐GlcNAc) hybrid with Concanavalin A (Con A, a lectin specific to D ‐glucose, D ‐mannose and D ‐GlcNAc), were confirmed by a surface plasmon resonance technique. The microscopic observation of Pseudomonas aeruginosa, which was preincubated with the fluorescein isothiocyanate‐labeled chitosan‐L ‐Fuc hybrid, showed bacteria aggregation. The aggregation was thought to be resulted from the specific interaction of the L ‐Fuc residue of the hybrid with PA‐II lectin on the surface of P. aeruginosa. The chitosan‐L ‐Fuc hybrid inhibited P. aeruginosa growth more effectively in comparison with the other hybrids or unmodified chitosan. The enhancement of antimicrobial activity of chitosan‐L ‐Fuc hybrid could be attributed to the specific binding between PA‐II lectin of P. aeruginosa and L ‐Fuc residue of the L ‐Fuc hybrid. Copyright © 2000 John Wiley & Sons, Ltd.     

Synthesis of Multivalent Carbohydrate‐Centered Glycoclusters as Nanomolar Ligands of the Bacterial Lectin LecA from Pseudomonas aeruginosa

A family of fifteen glycoclusters based on a cyclic oligo‐(1→6)‐β‐D ‐glucosamine core has been designed as potential inhibitors of the bacterial lectin LecA with various valencies (from 2 to 4) and linkers. Evaluation of their binding properties towards LecA has been performed by a combination of hemagglutination inhibition assays (HIA), enzyme‐linked lectin assays (ELLA), and isothermal titration microcalorimetry (ITC). Divalent ligands displayed dissociation constants in the sub‐micromolar range and tetravalent ligands displayed low nanomolar affinities for this lectin. The influence of the linker could also be demonstrated; aromatic moieties are the best scaffolds for binding to the lectin. The affinities observed in vitro were then correlated with molecular models to rationalize the possible binding modes of these glycoclusters with the bacterial lectin.     

Microbial transformation of tributyltin chloride by Pseudomonas aeruginosa strain USS25 NCIM‐5224

A bacterial isolate capable of utilizing tributyltin chloride (TBTC) as sole source of carbon was isolated from marine water samples. The isolate, producing a soluble green pigment, was identified as Pseudomonas aeruginosa strain USS25 NCIM‐5224. The isolate showed maximum growth with 2 mM of TBTC in mineral salt medium. Time course results showed complete elimination of TBTC after 75 days of incubation. During the growth on TBTC, a product (280 mg) was found to accumulate, which was extracted with chloroform and detected on thin‐layer chromatography. Based on the IR, NMR spectra and GC‐MS analysis, the isolated product was identified as monobutyltin dichloro hydride. Copyright © 2005 John Wiley & Sons, Ltd.     

Toward the Rational Design of Galactosylated Glycoclusters That Target Pseudomonas aeruginosa Lectin A (LecA): Influence of Linker Arms That Lead to Low‐Nanomolar Multivalent Ligands

Anti‐infectious strategies against pathogen infections can be achieved through antiadhesive strategies by using multivalent ligands of bacterial virulence factors. LecA and LecB are lectins of Pseudomonas aeruginosa implicated in biofilm formation. A series of 27 LecA‐targeting glycoclusters have been synthesized. Nine aromatic galactose aglycons were investigated with three different linker arms that connect the central mannopyranoside core. A low‐nanomolar (K_d=19 nm , microarray) ligand with a tyrosine‐based linker arm could be identified in a structure–activity relationship study. Molecular modeling of the glycoclusters bound to the lectin tetramer was also used to rationalize the binding properties observed.     

Voltammetric Behavior of Antileukemia Drug Glivec. Part I – Electrochemical Study of Glivec

The electrochemical behavior of the antileukemia drug glivec was investigated at a glassy carbon electrode (GCE). The oxidation is a complex, pH‐dependent, irreversible electrode process involving the transfer of 2 electrons and 2 protons and the formation of an electroactive product, P_glivec, which strongly adsorbs on the GCE surface and undergoes reversible oxidation. The adsorption of P_glivec at the GCE surface yields a compact monolayer that inhibits further oxidation of glivec. The electrochemical reduction is a simple pH dependent irreversible process involving the transfer of 2 electrons and 2 protons and occurs with the formation of a nonelectroactive product. The diffusion coefficient of glivec was calculated to be D_O=7.35×10^?6 cm² s^?1 in pH 4.5 0.1 M acetate buffer.     

Electrochemical Behavior and Amperometric Detection of 4‐Chlorophenol on Nano‐Au Thin Films Modified Glassy Carbon Electrode

An amperometric sensor based on nano‐Au thin films was fabricated, by means of which a fast response to 4‐chlorophenol (4‐CP) can be achieved in the range of mM concentrations. The nanostructured Au thin film was prepared on glassy carbon electrodes by a template‐free, double‐potential step electrodeposition technique. Its structural feature can be controlled well by adjusting the deposition time. The amperometric detection of 4‐CP was performed at +0.85 V with a linear detection range from 0.2 to 4.8 mM and a detection limit of 0.11 mM (S/N=3). Besides, the effect of concentrations on the electrochemical behavior of 4‐CP on the Au thin film was investigated by linear sweep voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy.     

Electrochemical Studies for the Interaction of DNA with an Irreversible Redox Compound – Hoechst 33258

An electrochemical equation suitable for examining the interaction of irreversible redox compounds with DNA is established. According to the equation, diffusion coefficients of both free and binding compounds (D_f , D_b), binding constant (K) and binding site size (s) of compounds with DNA could be obtained simultaneously by nonlinear fit analysis of electrochemical data. Bis‐benzimidazole derivative (Hoechst 33258), as an irreversible redox compound, was investigated for its electrochemical behavior and the interaction with natural fish sperm DNA (fsDNA) using cyclic voltammetry, chronocoulometry, bulk electrolysis and scanning electrochemical microscope technique. A nonlinear fit analysis of the experimental data yielded: D_f=8.3×10^?5 cm² s^?1, D_b=6.0×10^?6 cm² s^?1, K=2.1×10⁸ cm³ mol^?1, s=3.9. The overall results suggest that Hoechst 33258 binds tightly to the minor groove of fsDNA and covers four base pairs.     

Voltammetric Behavior of Antileukemia Drug Glivec. Part II – Redox Processes of Glivec Electrochemical Metabolite

Glivec is a newly developed drug that belongs to the class of 2‐phenylaminopyrimidine. It is a potent inhibitor of ABL‐kinase, the main clinical manifestation of chronic myelogenous leukemia (CML). Based on its activity on CML, glivec is undergoing extensive evaluation for its activity against other tumor types. Detection and quantitation of glivec in biological fluids or cells is thus very important. The antileukemia drug glivec undergoes oxidation at glassy carbon electrodes and involves the formation of an oxidation product, P_glivec. The adsorption of P_glivec at the GCE surface yields a compact monolayer allowing an electrochemical study of this compound adsorbed at the GCE surface. The reversible redox reaction of the adsorbed P_glivec is pH dependent and occurs with the transfer of 2 electrons and 2 protons. The surface standard potential and the rate constant of the heterogeneous electrochemical reaction were calculated using cyclic voltammetry to be E^θ′=+180 mV and k=15.5 s^?1, respectively. The total surface concentration of adsorbed P_glivec is 2.5×10^?12 mol cm^?2. The analytical determination of glivec was carried out by differential pulse voltammetric measurement of the anodic peak current corresponding to either the oxidation peak of glivec or the oxidation peak of P_glivec adsorbed on the GCE surface. The limits of detection of glivec and adsorbed P_glivec based on three times the noise level are 3.3×10^?8 M and 2.9×10^?10 M, respectively.     

Measurement of ceftazidime concentration in human plasma by ultra‐performance liquid chromatography–tandem mass spectrometry. Application to critically ill patients and patients with osteoarticular infections

Ceftazidime is an antibiotic belonging to the third generation of the cephalosporin family. It is indicated in the treatment of serious, simple or mixed bacterial infections, and its administration in continuous or intermittent infusion allows optimization of the concentration of antibiotic to keep it above the minimum inhibitory concentration. We developed and validated a chromatographic method by ultra‐performance liquid chromatography–tandem mass spectrometry to measure ceftazidime concentration in human plasma. Following extraction with acetonitrile and 1,2‐dichloroethane, the chromatographic separation was achieved using an Acquity ® UPLC ® BEH^TM (2.1 × 100 mm i.d., 1.7 µm) reverse‐phase C₁₈ column, with a water–acetonitrile linear gradient containing 0.1% formic acid at a 0.4 mL/min flow rate. Ceftazidime and its internal standard (cefotaxime) were detected by electrospray ionization mass spectrometry in positive ion multiple reaction monitoring mode using mass‐to‐charge transitions of 547.0 → 467.9/396.1 and 456.0 → 395.8/324.1, respectively. The limit of quantification was 0.58 mg/L and linearity was observed in the range 0.58–160 mg/L. Coefficients of variation and absolute relative biases were <9.8 and 8.4%. The mean recovery for ceftazidime was 74.4 ± 8.1%. Evaluation of the matrix effect showed ion enhancement, and no carry‐over was observed. The validated method could be applied to daily clinical laboratory practice to measure the concentration of ceftazidime in plasma. Copyright © 2015 John Wiley & Sons, Ltd.     

Self‐Assembling of Redox‐Active Atrazine Poly(ethylenimine) Conjugates – Interfacial Electrochemical and Spectroscopic Characterization

Thin films of polymers tethered with both atrazine haptens and ferrocenyl (Fc) probes were formed on polycrystalline gold electrodes by taking advantage of the facile formation of self‐assembled monolayers (SAMs) of thiolates. These films were characterized by polarization modulation Fourier transform infrared reflection–absorption spectrometry (PM‐IRRAS), X‐ray photoelectron spectroscopy (XPS) and cyclic voltammetry. The combination of these techniques gave a full insight into the structure and the binding mode of the polymers and provided useful quantitative information about both Fc entity and atrazine hapten surface density. This may open the way to a new type of immunosensor for atrazine monitoring.