A DNA aptamer reveals an allosteric site for inhibition in metallo-ß-lactamases.

The hydrolysis of ß-lactam antibiotics by ß-lactamase enzymes is the most prominent antibiotic resistance mechanism for many pathogenic bacteria. Out of this broad class of enzymes, metallo-ß-lactamases are of special clinical interest because of their broad substrate specificities. Several in vitro inhibitors for various metallo-ß-lactamases have been reported with no clinical efficacy. Previously, we described a 10-nucleotide single stranded DNA aptamer (10-mer) that inhibits Bacillus cereus 5/B/6 metallo-ß-lactamase very effectively. Here, we find that the aptamer shows uncompetitive inhibition of Bacillus cereus 5/B/6 metallo-ß-lactamase during cefuroxime hydrolysis. To understand the mechanism of inhibition, we report a 2.5 Å resolution X-ray crystal structure and solution-state NMR analysis of the free enzyme. Chemical shift perturbations were observed in the HSQC spectra for several residues upon titrating with increasing concentrations of the 10-mer. In the X-ray crystal structure, these residues are distal to the active site, suggesting an allosteric mechanism for the aptamer inhibition of the enzyme. HADDOCK molecular docking simulations suggest that the 10-mer docks 26 Å from the active site. We then mutated the three lysine residues in the basic binding patch to glutamine and measured the catalytic activity and inhibition by the 10-mer. No significant inhibition of these mutants was observed by the 10-mer as compared to wild type. Interestingly, mutation of Lys50 (Lys78; according to standard MBL numbering system) resulted in reduced enzymatic activity relative to wild type in the absence of inhibitor, further highlighting an allosteric mechanism for inhibition.

Inhibition of Bacillus anthracis metallo-ß-lactamase by compounds with hydroxamic acid functionality.

Metallo-ß-lactamases (MBLs) that catalyze hydrolysis of ß-lactam antibiotics are an emerging threat due to their rapid spread. A strain of the bacterium Bacillus anthracis has its ability to produce and secrete a MBL, referred to Bla2. To address this challenge, novel hydroxamic acid-containing compounds such as 3-(heptyloxy)-N-hydroxybenzamide (compound 4) and N-hydroxy-3-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide (compound 7) were synthesized. Kinetic analysis of microbial inhibition indicated that the both sides of hydroxamic acids containing compound 7 revealed a reversible, competitive inhibition with a Ki value of 0.18 ± 0.06 µM. The result has reflected that the both sides of dihydroxamic acids in a molecule play a crucial role in the binding affinity rather than monohydroxamic containing compound 4 which was unable to inhibit Bla2. In addition, in silico analysis suggested that compound 7 was coordinated with a zinc ion in the active site of enzyme. These observations suggest that the dihydroxamic acid-containing compound may be a promising drug candidate, and a further implication for designing new inhibitors of Bla2.

Resolving transitions in the mesoscale domain configuration in VO2 using laser speckle pattern analysis.

The configuration and evolution of coexisting mesoscopic domains with contrasting material properties are critical in creating novel functionality through emergent physical properties. However, current approaches that map the domain structure involve either spatially resolved but protracted scanning probe experiments without real time information on the domain evolution, or time resolved spectroscopic experiments lacking domain-scale spatial resolution. We demonstrate an elegant experimental technique that bridges these local and global methods, giving access to mesoscale information on domain formation and evolution at time scales orders of magnitude faster than current spatially resolved approaches. Our straightforward analysis of laser speckle patterns across the first order phase transition of VO2 can be generalized to other systems with large scale phase separation and has potential as a powerful method with both spatial and temporal resolution to study phase separation in complex materials.

Influence of solute charge and pyrrolidinium ionic liquid alkyl chain length on probe rotational reorientation dynamics.

In recent years, the effect of molecular charge on the rotational dynamics of probe solutes in room-temperature ionic liquids (RTILs) has been a subject of growing interest. For the purpose of extending our understanding of charged solute behavior within RTILs, we have studied the rotational dynamics of three illustrative xanthene fluorescent probes within a series of N-alkylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([Cnmpyr][Tf2N]) RTILs with different n-alkyl chain lengths (n = 3, 4, 6, 8, or 10) using time-resolved fluorescence anisotropy decay. The rotational dynamics of the neutral probe rhodamine B (RhB) dye lies between the stick and slip boundary conditions due to the influence of specific hydrogen bonding interactions. The rotation of the negatively charged sulforhodamine 640 (SR640) is slower than that of its positively charged counterpart rhodamine 6G (R6G). An analysis based upon Stokes-Einstein-Debye hydrodynamics indicates that SR640 adheres to stick boundary conditions due to specific interactions, whereas the faster rotation of R6G is attributed to weaker electrostatic interactions. No significant dependence of the rotational dynamics on the solvent alkyl chain length was observed for any of the three dyes, suggesting that the specific interactions between dyes and RTILs are relatively independent of this solvent parameter.

The influence of circulating anti-Müllerian hormone on ovarian responsiveness to ovulation induction with gonadotrophins in women with polycystic ovarian syndrome: a pilot study.

BACKGROUND: Women with polycystic ovarian syndrome (PCOS) are known to have elevated circulating Anti-Müllerian hormone (AMH), which has been found to desensitize ovarian follicles to follicle stimulating hormone (FSH). The purpose of this study was to investigate the impact of high circulating AMH on ovarian responsiveness to ovulation induction with gonadotrophins in PCOS women. METHODS: This prospective observational pilot study was conducted in two collaborating Fertility Centres in the UK and Egypt. The study included 20 consecutive anovulatory women with PCOS who underwent 34 cycles of human menopausal gonadotrophin (hMG) ovarian stimulation using chronic low-dose step up protocol. Blood samples were collected for the measurement of serum AMH concentrations in the early follicular (day 2-3) phase in all cycles of hMG treatment. The serum levels of AMH were compared between cycles with good vs. poor response. The good response rates and the total dose and duration of hMG treatment were compared between cycles with high vs. low serum AMH concentrations. RESULTS: Cycles with poor response (no or delayed ovulation requiring >20 days of hMG treatment) had significantly (p = .007) higher median{range} serum AMH concentration (6.5{3.2-13.4}ng/ml) compared to that (4.0{2.2-10.2}ng/ml) of cycles with good response (ovulation within 20 days of hMG treatment). ROC curve showed AMH to be a useful predictor of poor response to hMG stimulation (AUC, 0.772; P = 0.007). Using a cut-off level of 4.7 ng/ml, AMH had a sensitivity of 100% and specificity of 58% in predicting poor response. The good response rate was significantly (p < .001) greater in cycles with lower AMH (<4.7 ng/ml) compared to that in those with AMH > = 4.7 ng/ml (100% vs. 35%, respectively). All cycles with markedly raised serum AMH levels (> 10.2 ng/ml) were associated with poor response. Cycles with high AMH (> = 4.7 ng/ml) required significantly (p < .001) greater amounts (median {range}, 1087{450-1650}IU) and longer duration (20 {12-30}days) of hMG stimulation than cycles with lower AMH (525 {225-900}IU and 8{6-14}days). CONCLUSIONS: PCOS women with markedly raised circulating AMH seem to be resistant to hMG ovulation induction and may require a higher starting dose.

The predictive value of circulating anti-Müllerian hormone in women with polycystic ovarian syndrome receiving clomiphene citrate: a prospective observational study.

CONTEXT: Elevated serum anti-Müllerian hormone (AMH) concentration in women with polycystic ovarian syndrome (PCOS) is known to lower sensitivity of ovarian follicles to circulating FSH. This effect may compromise the outcome of clomiphene citrate (CC) ovulation induction. OBJECTIVE: The objective of the study was to investigate the impact of high circulating AMH on the outcome of CC ovulation induction in women with PCOS. DESIGN: This was a prospective cohort observational study. SETTING: The study was conducted at the Fertility Unit, Derby, United Kingdom. PATIENTS: Sixty anovulatory women with PCOS participated in the study. INTERVENTIONS: Serum AMH concentrations were measured on cycle day 2 during 187 CC cycles. These concentrations were compared between responders and nonresponders. The receiver-operating characteristic curve was used to evaluate the prognostic value of circulating AMH. The success rates of CC were compared between patients with high vs low AMH levels. The dose of CC required to achieve ovulation was correlated with serum AMH concentrations. MAIN OUTCOME MEASURES: Ovulation and pregnancy rates were measured. RESULTS: Serum AMH concentrations were significantly (P < .001) lower in responders (achieving ovulation) vs nonresponders (mean ± SEM, 2.5 ± 0.1 vs 5.8 ± 0.7 ng/mL, respectively). Similarly, serum AMH concentrations were significantly (P = .046) lower in pregnant (3.0 ± 0.4 ng/mL) vs nonpregnant patients (4.4 ± 0.5 ng/mL). There was a significant (P = .02) gradient increase of serum AMH levels with the increasing dose of CC required to achieve ovulation. The receiver-operating characteristic curve showed AMH to be a useful predictor of no ovulation (area under the curve, 0.809; P < .001) with a useful cutoff level of 3.4 ng/mL. Ovulation and pregnancy rates were significantly higher (97%, P < .001, and 46%, P = .034) in patients with low AMH (<3.4 ng/mL) vs women with AMH 3.4 ng/mL or greater (48% and 19%). CONCLUSION: PCOS women with high circulating AMH (≥ 3.4 ng/mL) seem to be resistant to CC and may require a higher starting dose.

A role for two-pore potassium (K2P) channels in endometrial epithelial function.

The human endometrial epithelium is pivotal to menstrual cycle progression, implantation and early pregnancy. Endometrial function is directly regulated by local factors that include pH, oxygen tension and ion concentrations to generate an environment conducive to fertilization. A superfamily of potassium channels characterized by two-pore domains (K2P) and encoded by KCNK genes is implicated in the control of the cell resting membrane potential through the generation of leak currents and modulation by various physicochemical stimuli. The aims of the study were to determine the expression and function of K2P channel subtypes in proliferative and secretory phase endometrium obtained from normo-ovulatory women and in an endometrial cancer cell line. Using immunochemical methods, real-time qRT-PCR proliferation assays and electrophysiology. Our results demonstrate mRNA for several K2P channel subtypes in human endometrium with molecular expression of TREK-1 shown to be higher in proliferative than secretory phase endometrium (P < 0.001). The K2P channel blockers methanandamide, lidocaine, zinc and curcumin had antiproliferative effects (P < 0.01) in an endometrial epithelial cancer cell line indicating a role for TASK and TREK-1 channels in proliferation. Tetraethylammonium- and 4-aminopyridine-insensitive outwards currents were inhibited at all voltages by reducing extracellular pH from 7.4 to 6.6. Higher expression of TREK-1 expression in proliferative phase endometrium may, in part, underlie linked to increased cell division. The effects of pH and a lack of effect of non-specific channel blockers of voltage-gated potassium channels imply a role for K2P channels in the regulation of human endometrial function.

Excited-state dynamics of water-soluble polythiophene derivatives: temperature and side-chain length effects.

We report synthesis and detailed spectroscopic study of three water-soluble polythiophene derivatives with distinct homologous oligo(ethylene oxide) side-chain lengths and lower critical solution temperatures (LCSTs). The linear absorption spectra exhibit reversible shifts and broadening with the variation of their aqueous solution temperature, whereas the corresponding steady-state fluorescence emission spectra were found to show negligible shifts and only minor changes in their line shape. Measurements of picosecond time-resolved fluorescence at chosen emission wavelengths reveal a strong dependence of the isotropic decays on both side-chain length and aqueous solution temperature. With lengthening of the side chain, the isotropic decays become not only remarkably slow but also increasingly complex. Except for the polymer with the shortest side chain, significant acceleration of the isotropic decays was found when the solution temperature was raised to the corresponding LCSTs and beyond, which further causes formation of large aggregates as evident by the physical appearance change from clear solutions to turbid suspensions. Direct evidence for a temperature-induced change of polymer chain conformation was obtained through measurements of time-resolved fluorescence anisotropies, which are characterized by a substantial increase of the initial values from ~0.2 to 0.4 and the appearance of a pronounced fast decay component with an estimated lifetime of 36 ps. The high initial anisotropy of ~0.4 observed for the two polymers with longer side-chains above their LCSTs suggests that the polymer chains are highly ordered in the aggregates. The observed effects of side-chain length and solution temperature are discussed by considering the conformational relaxation of the polymer backbones and occurrence of interchain energy transfer.

Rotational and translational dynamics of rhodamine 6G in a pyrrolidinium ionic liquid: a combined time-resolved fluorescence anisotropy decay and NMR study.

NMR spectroscopy and time-resolved fluorescence anisotropy decay (TRFAD) are two of the most commonly used methods to study solute-solvent interactions. However, only a few studies have been reported to date using a combined NMR and TRFAD approach to systematically investigate the overall picture of diffusional and rotational dynamics of both the solute and solvent. In this paper, we combined NMR and TRFAD to probe fluorescent rhodamine dye in a pyrrolidinium-based room temperature ionic liquid (RTIL), an emergent environmentally friendly solvent type used in several energy-related applications. A specific interaction of the R6G cation and [Tf2N] anion was identified, resulting in near-stick boundary condition rotation of R6G in this RTIL. The diffusional rates of the R6G solute and [C4mpyr][Tf2N] solvent derived from (1)H NMR suggest the rates are proportional to their corresponding hydrodynamic radii. The (1)H and (13)C NMR studies of self-rotational dynamics of [C4mpyr][Tf2N] showed that the self-rotational correlation time of [C4mpyr](+) is 47 ± 2 ps at 300 K. At the same temperature, we find that the correlation time for N-CH3 rotation in [C4mpyr](+) is 77 ± 2 ps, comparable to overall molecular reorientation. This slow motion is attributed to properties of the cation structure.

Bead-based microfluidic immunoassay for diagnosis of Johne's disease.

Microfluidics technology offers a platform for development of point-of-care diagnostic devices for various infectious diseases. In this study, we examined whether serodiagnosis of Johne's disease (JD) can be conducted in a bead-based microfluidic assay system. Magnetic micro-beads were coated with antigens of the causative agent of JD, Mycobacterium avium subsp. paratuberculosis. The antigen-coated beads were incubated with serum samples of JD-positive or negative serum samples and then with a fluorescently-labeled secondary antibody (SAB). To confirm binding of serum antibodies to the antigen, the beads were subjected to flow cytometric analysis. Different conditions (dilutions of serum and SAB, types of SAB, and types of magnetic beads) were optimized for a large degree of differentiation between the JD-negative and JD-positive samples. Using the optimized conditions, we tested a well-classified set of 155 serum samples from JD-negative and JD-positive cattle by using the bead-based flow cytometric assay. Of 105 JD-positive samples, 63 samples (60%) showed higher antibody binding levels than a cut-off value determined by using antibody binding levels of JD-negative samples. In contrast, only 43-49 JD-positive samples showed higher antibody binding levels than the cut-off value when the samples were tested using commercially-available immunoassays. Microfluidic assays were performed by magnetically immobilizing a number of beads within a microchannel of a glass microchip and detecting antibody on the collected beads using laser-induced fluorescence. Antigen-coated magnetic beads treated with the bovine serum sample and fluorescently-labeled SAB were loaded into a microchannel to measure the fluorescence (reflecting level of antibody binding) on the beads in the microfluidic system. When the results of five bovine serum samples with the microfluidic system were compared to those analyzed with the flow cytometer, a high level of correlation (linear regression, r(2)=0.994) was observed. In a further experiment, we magnetically immobilized antigen-coated beads in a microchannel, reacted the beads with serum and SAB in the channel, and detected antibody binding to the beads in the microfluidic system. A strong antibody binding in JD-positive serum was detected, whereas there was only negligible binding in negative control experiments. Our data suggest that the bead-based microfluidic system may form a basis for development of an on-site serodiagnosis of JD.

Fluorescence correlation spectroscopy evidence for structural heterogeneity in ionic liquids.

In this work, we provide new experimental evidence for chain length-dependent self-aggregation in room temperature ionic liquids (RTILs) using fluorescence correlation spectroscopy (FCS). In studying a homologous series of N-alkyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl) imide, [C(n)MPy][Tf(2)N] RTILs of varying alkyl chain length (n = 3, 4, 6, 8, and 10), biphasic rhodamine 6G solute diffusion dynamics were observed; both the fast and slow diffusion coefficients decreased with increasing alkyl chain length, with the relative contribution from slower diffusion increasing for longer-chain [C(n)MPy][Tf(2)N]. We propose that the biphasic diffusion dynamics originate from self-aggregation of the nonpolar alkyl chains in the cationic [C(n)MPy](+).

Surface coating effects on the assembly of gold nanospheres.

Optical spectra and atomic force microscopy (AFM) images of individually selected spheres and mechanically assembled silica-coated gold nanosphere pairs were recorded. The shell served as a means of rigid control of the minimum spacing between the metal cores. The spectra of the assembled spheres were simulated using classical electrodynamics. The observed spectra resulted in superior characterization of the particle assembly geometry, relative to the AFM data. Experimental investigations regarding less-rigid polyvinylpyrrolidone (PVP) sphere coatings were also performed and some comparisons were made.

Combined apertureless near-field optical second-harmonic generation/atomic force microscopy imaging and nanoscale limit of detection.

A dual function atomic force/near-field scanning optical microscope (AFM/NSOM) with an ultrafast laser excitation source was used to investigate apertureless, tip enhanced second-harmonic generation (SHG) of ZnO nanowires with spatial resolution below the optical diffraction limit. Single-wire SHG spectra show little to no contribution from bandgap or other emission. Polarization data established values for chi(33)/chi(31) close to previous estimates and confirm the SHG process. Experimental results indicate that the SHG signal was reduced for nanowires after exposure to an atmosphere of carbon dioxide and water vapor. An equation was derived for estimating the minimum chi(2) detectable using apertureless SHG NSOM.

Antibiotic resistance in bacteria: novel metalloenzyme inhibitors.

Beta-lactam antibiotics are among the most important drugs used to fight bacterial infection. Overuse and misuse of beta-lactam antibiotics has caused the evolution of resistance mechanisms, allowing pathogenic bacteria to survive antibiotic treatment. The major source of resistance to beta-lactam antibiotics occurs through production of enzymes called beta-lactamases capable of catalyzing hydrolysis of the beta-lactam rings in these drug compounds. The metallo-beta-lactamases have become a major threat due to their broad substrate specificities; there are no clinically useful inhibitors for these metalloenzymes. We have obtained single-stranded DNA's that are potent inhibitors of the Bacillus cereus 5/B/6 metallo-beta-lactamase. These are rapid, reversible, non-competitive inhibitors of the metalloenzyme, with K(i) and K(i)' values in the nanomolar range. The inhibition patterns and metal ion dependence of their inhibition suggest that the oligonucleotides alter the coordination of the active site metal ion(s); inhibition is efficient and highly specific. Microbiological growth experiments, using combinations of ssDNA with the beta-lactam antibiotic cephalexin, reveal that the inhibitor is capable of causing cell death in liquid cultures of both Gram-positive and Gram-negative metallo-beta-lactamase producing bacteria in the micromolar concentration range.

Computational and experimental evaluation of nanoparticle coupling.

We present theoretical and experimental studies on the optical properties of dimers composed of octahedron-shaped, gold nanoparticles. The experimental measurements show that the photoluminescence varies quite dramatically as two octahedra are brought into close proximity. AFM images and optical emission have been recorded for dimers in uncoupled and strongly coupled configurations. The former displays a single emission peak, while the latter shows two peaks with the new feature at longer wavelengths. Calculations indicate that the red-shifted peak originates from a strongly coupled plasmon state that oscillates along the extended axis of the dimer. Theoretically, we investigate the distances over which the dimers couple and find this to be particularly plasmon mode dependent. The anisotropic morphology and sharp apexes contribute significantly to the orientational dependence of the interparticle couplings and field properties.

Optical absorption measurements with parametric down-converted photons.

It has been known for some time that correlated detection of pairs of photons generated by parametric down-conversion can eliminate several sources of error that occur in single-beam measurements. In the correlated photon measurements, the down-converted photons are separated into two beams with one photon of a pair in each beam. The absolute detection efficiency of a detector in one beam can be determined from the count rate of a detector in the other beam and the coincidence rate for the two detectors. These ideas can be used to measure the optical absorbance of a sample placed in front of one of the detectors. Errors due to stray light and dark counts are substantially reduced and fluctuations in pump intensity largely eliminated.

Interleukin-1 beta secretion from cord blood mononuclear cells in vitro involves P2X 7 receptor activation.

The purinergic receptor P2X(7) is activated by extracellular adenosine 5'-triphosphate (ATP) and promotes the efficient release of interleukin-1 beta. The authors examine protein and molecular expression of the P2X(7) receptor and its ability to stimulate interleukin-1 beta release in cord blood mononuclear cells (CBMCs) from placentae of term nonlaboring and laboring women. They show both mRNA and protein (78 kDa) expression for the P2X( 7) receptor in CBMCs of parturient and nonparturient women. Costimulation of CBMCs in vitro with bacterial endotoxin and ATP resulted in significantly (P < .05) enhanced interleukin-1 beta release in laboring (54.17 +/- 24.78 pg/mL(-1); n = 8) compared with nonlaboring (13.60 +/- 3.20 pg/mL(-1); n = 7) deliveries. Release of interleukin-1 beta in both groups was blocked by preincubation with oxidized ATP, a P2X(7) receptor antagonist. The authors provide evidence for a novel inflammatory pathway in the release of interleukin-1 beta, which may be linked to the onset of labor.

Randomised comparative trial of thermal balloon ablation and levonorgestrel intrauterine system in patients with idiopathic menorrhagia.

AIMS: To compare the effectiveness of thermal balloon ablation (TBA) and levonorgestrel intrauterine system (LNG-IUS) in the management of idiopathic menorrhagia and changes in pictorial blood loss assessment chart (PBAC) scores in patients who had failed on oral medical treatment. METHODS: Phase III, single-centre, open randomised controlled trial. Following full screening and evaluation of 104 women, 33 were randomised to TBA and 33 to LNG-IUS. Primary outcomes were changes in PBAC scores from baseline to 12 months. Secondary outcomes were changes in haemoglobin and serum ferritin, at six months, continuation with treatment and hysterectomy rates at two years and changes in PBAC scores at three, six and nine months. RESULTS: All patients randomised had a PBAC score of > or = 120. At all assessment times, median PBAC scores were less than baseline, the greatest reductions being seen at 12 months for both treatments. When the median PBAC for the LNG-IUS (26 (0-68)) was significantly different to the median PBAC for the TBA cohort (62 (0-142)) P < 0.001. Irregular bleeding problems were the most common reason for discontinuation of the LNG-IUS and resulted in more women (39.8%) seeking other treatment by two years than the TBA (23.1%) (P < 0.05) and more undergoing a hysterectomy (20.7% vs 13.3%, respectively) (p > 0.05). Patient acceptability of the LNG-IUS and TBA was similar at 12 and 24 months in terms of their perceived satisfaction of effect on menorrhagia. CONCLUSIONS: Both TBA and LNG-IUS achieved significant decreases in PBAC scores, with those for the LNG-IUS being significantly greater at 12 months. However, prolonged days of bleeding resulted in fewer women continuing with the LNG-IUS at two years.

Hydrogen peroxide and superoxide anion modulate pregnant human myometrial contractility.

Reactive oxygen species (ROS) have the propensity to cause macromolecular damage with consequent modification of cellular function. We investigated the effects of two particular oxidants, superoxide (O2(-)) anions and hydrogen peroxide (H2O2), on oxytocin-induced myometrial contractility using biopsies from women undergoing Caesarean section at term gestation. Isometric tension recordings were performed and concentration-response curves derived after addition of test agents. A maximal reduction in myometrial contractility to 27.2 +/- 4.5% of control was observed followed application of H2O2. The enzyme scavenger catalase (CAT) reduced the inhibitory effect of H2O2 but had little effect at 10-fold lower concentrations. Addition of dialysed xanthine oxidase +/- hypoxanthine significantly inhibited contractility to 23.8.0 +/- 4.2% compared with control. Pre-incubation with superoxide dismutase and CAT diminished this effect. The non-specific potassium channel blocker, tetraethylammonium chloride (1 mM), had no effect on myometrial contractility. We conclude that human myometrium is susceptible to the effects of ROS, which may be produced by reperfusion-ischaemic episodes during labour. Our findings could, in part, explain the weak or prolonged depression of contractions characteristic of myometrial dysfunction culminating in difficult labours.