Development of Aptamer Beacons for Rapid Presumptive Detection of <Emphasis Type="Italic">Bacillus</Emphasis> Spores

A library of 92 DNA aptamer sequences was developed against Bacillus anthracis (nonpathogenic Sterne strain) spores and anthrose sugar immobilized on magnetic beads. The selected DNA sequences were studied for similarities and potential binding pockets between the B. anthracis spore and anthrose aptamers. Several recurring loop structures were identified and tested for their potential to act as aptamer beacons when labeled with TYE 665 dye on their 5′ ends and Iowa Black quencher on their 3′ ends. Of these candidate sequences, two beacons designated BAS-6F and BAS-6R emerged which gave strong fluorescence responses at high spore concentrations (greater than 30,000 spores/ml). These aptamer beacons also detect B. cereus and B. thuringiensis spores with greater fluorescence intensity, but do not strongly detect vegetative cells from an array of other bacterial species. BAS-6F and 6R are also not capable of detecting pure anthrose, thereby probably ruling that epitope out as a spore surface target for these particular beacons. While not extremely sensitive, the BAS-6F and 6R aptamer beacons are potentially valuable for rapid presumptive detection of anthrax or Bacillus spores in suspect powders or bioterrorist activity where spore concentrations are anticipated to be high. The sequence similarities of these beacons to other published Bacillus spore aptamers are also discussed.     

A Novel Screening Method for Competitive FRET-Aptamers Applied to <Emphasis Type="Italic">E. coli</Emphasis> Assay Development

A novel high-throughput screening method is described in which a family of DNA aptamers selected against E. coli outer membrane proteins (OMPs) is subjected to PCR in the presence of fluorophore-dUTP conjugates using Deep Vent® exo- polymerase. The fluorophore-doped aptamers and their complementary strands are then heated to render them single-stranded and screened in filter well microtiter plates for fluorescence resonance energy transfer (FRET) assay potential. Using this system, a superior competitive FRET-aptamer designated EcO 4R was identified and the location of its putative binding pocket was determined by individually testing FRET potential in each of the secondary loop structures. By labeling the binding pocket with Alexa Fluor (AF) 647 and binding the aptamer to heavily Black Hole Quencher-3 (BHQ-3)-labeled E. coli bacteria, detection of as few as 30 live unlabeled E. coli per ml was achieved in a competitive displacement FRET assay format. The far red fluorescence emission enables detection in largely blue-green autofluorescent matrices. In addition, the competitive transfer of AF 647-EcO-4R aptamer to unlabeled E. coli cells after a 15 min equilibration period was verified by fluorescence microscopy. The present study also demonstrated that high aptamer affinity is not well correlated with competitive FRET potential.     

Raman spectral studies of Zr4+‐rich BaZrxTi1−xO3(0.5⩽x⩽1.00) phase diagram

This paper reports a systematic study of the composition and the temperature‐dependent‐Raman spectra of Zr⁴⁺‐rich BaZr_xTi_1−xO₃ (BZT) ceramic compositions (0.50⩽x⩽1.00). On the basis of the dielectric behavior of Zr rich BZT ceramics, the observed relaxor behavior has been hypothesized as a result of increasing long‐range interactions of nanosized, Ti⁴⁺‐rich polar regions in a Zr⁴⁺‐rich nonpolar matrix. Beyond an optimum concentration of BaTiO₃ (BT) in the nonpolar matrix of BaZrO₃ (x⩽0.75), a critical size and density of the polar regions is reached when the polar clusters start showing the relaxor like behavior, which finally show classical relaxor behavior for compositions with x = 0.5 and 0.6. This hypothesis is strongly supported from the Raman data on Zr‐rich BZT presented in this paper. Well‐defined BT Raman spectra for 5% BT in BZT composition were recorded, which followed completely up to the 50% Ti addition in the BZT samples. The temperature‐dependent Raman spectra collected on the BZT ceramics far beyond the dielectric transition temperatures supported the existence of the nano‐polar BT regions, like in typical relaxor samples. The full width at half‐maximum (FWHM), integrated intensity of the peaks in the Raman spectra has been analyzed to further support the conclusions. Copyright © 2008 John Wiley & Sons, Ltd.     

Lead‐free piezoelectric (Na0.5Bi0.5)0.94TiO3–Ba0.06TiO3 nanofiber by electrospinning

Lead‐free (Na_0.5Bi_0.5)_0.94TiO₃–Ba_0.06TiO₃ (NBT‐BT6) nanofibers were synthesized by the sol–gel process and electrospinning, and a butterfly‐shaped piezoelectric response was measured by scanning force microscopy. NBT‐BT6 nanofibers with perovskite phase were formed, after being cleaned at 700 °C for 1 hour, and the diameters are in the range of 150 nm to 300 nm. The average value of the effective piezoelectric coefficient d₃₃ is 102 pm/V. The high piezoelectricity may be attributed to the easiness for the electric field to tilt the polar vector of the domain and to the increase of the possible spontaneous polarization direction. There is a potential for the application of NBT‐BT6 nanofibers in nanoscale piezoelectric devices. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A Highly Sensitive and Selective Fluorescent Chemodosimeter for Hg<Superscript>2+</Superscript> in Neutral Aqueous Solution

A fluorescent assay of Hg²⁺ in neutral aqueous solution was developed using N-[p-(dimethylamino)benzamido]-N′-phenylthiourea (1). 1’s fluorogenic chemodosimetric behaviors towards various metal ions were studied and a high sensitivity as well as selectivity was achieved for Hg²⁺. It was because of a strongly fluorescent 1,3,4-oxadiazoles which was produced by the Hg²⁺ promoted desulfurization reaction. The spectra of ESI mass and IR provided evidences for this reaction. According to fluorescence titration, a good linear relationship ranging from 1.0 × 10⁻⁷ to 2.0 × 10⁻⁵ mol l⁻¹ was obtained with the limit of detection as 3.1 × 10⁻⁸ mol l⁻¹. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dielectric, ferroelectric and piezoelectric properties of 0-3 barium titanate–Portland cement composites

In this work, barium titanate (BT) and cement composites of 0-3 connectivity were produced with BT concentrations of 30%, 50% and 70% by volume using the mixing and pressing method. The dielectric constant (ε _r ) and the dielectric loss (tan δ) at room temperature and at various frequencies (0.1–20 kHz) of the ferroelectric BT-Portland cement composites with different BT concentrations were investigated. The results show that the dielectric constant of BT-PC composites was found to increase as BT concentration increases, and that the highest value for ε _r —of 436—was obtained for a BT concentration of 70%. In addition, the dielectric loss tangent decreased with increasing BT concentration. Moreover, several mathematical models were used; the experimental values of the dielectric constants are closest to those calculated from the cube model. The 0-3 cement-based piezoelectric composites show typical ferroelectric hysteresis loops at room temperature. The instantaneous remnant polarization (P _ir ), at an applied external electrical field (E ₀) of 20 kV/cm (90 Hz) of 70% barium titanate composite, was found to have a value ≈3.42 μC/cm². Furthermore, the piezoelectric coefficient (d ₃₃) was also found to increase as BT concentration increases, as expected. The highest value for d ₃₃ was 16 pC/N for 70% BT composite.     

Rapid Fluorescent Detection of Escherichia coli K88 Based on DNA Aptamer Library as Direct and Specific Reporter Combined With Immuno-Magnetic Separation

Nucleic acid aptamers have long demonstrated the capacity to bind cells with high affinity so that they have been utilized to diagnose various important pathogens. In this study, a DNA aptamer library was on initial efforts developed to act as a specific reporter for rapid detection of enter toxigenic Escherichia coli (ETEC) K88 combined with immuno-magnetic separation (IMS). During a Whole-cell Systematic Evolution of Ligands by Exponential Enrichment (CELL-SELEX) procedure, the last selection pool against ETEC K88, which is named “DNA aptamer library” here, was selected and subsequently identified by flow cytometric analysis and confocal imaging. A K88 monoclonal antibody (mAb) with high affinity (K_aff: 1.616?±?0.033?×?10⁸ M^?1) against K88 fimbrial protein was prepared, biotinylated and conjugated to streptavidin-coated magnetic beads (MBs). After the bacteria were effectively captured and enriched from the complex sample by immuno-magnetic beads (IMBs), 5′-FITC modified aptamer library was directly bound to target cells as a specific reporter for its detection. The detection system showed clearly high specificity and sensitivity with the detection limit of 1.1?×?10³ CFU/ml in pure culture and 2.2?×?10³ CFU/g in artificially contaminated fecal sample. The results also indicated that fluorophore-lablled DNA aptamer library as specific reporter could generate more reliable signals than individual aptamer with best affinity against target cells and implied it would have great applied potential in directly reporting bacteria from complex samples combined with IMS technology.     

Magnetic phases in UNi<Subscript>2</Subscript>Si<Subscript>2</Subscript>

The tetragonal compound UNi₂Si₂ exhibits in zero magnetic field three different antiferromagnetic phases belowT _N =124 K. They are formed by ferromagnetic basal planes, which are antiferromagnetically coupled along thec-axis with the propagation vectorq=(0, 0, q _z ). Two additional order-order magnetic phase transitions are observed below T _N , namely atT ₁=108 K and T ₂=40 K in zero magnetic field. All three phases exhibit strong uniaxial anisotropy confining the U moments to a direction parallel to the c-axis. UNi₂Si₂ single crystals were studied in detail by measuring bulk thermodynamic properties, such as thermal expansion, resistivity, susceptibility, and specific heat. A microscopic study using neutron diffraction was performed in magnetic fields up to 14.5 T parallel to the c-axis, and a complex magnetic phase diagram has been determined. Here, we present the analysis of specific-heat data measured in magnetic fields up to 14 T compared with the results of the neutron-diffraction study and with other thermodynamic properties of UNi₂Si₂.     

Magnetic and transport properties of monocrystalline<Emphasis Type="Italic">Fe</Emphasis><Subscript>3</Subscript><Emphasis Type="Italic">O</Emphasis><Subscript>4</Subscript>

A monocrystal ofFe ₃ O ₄ is characterized by resistance, magnetoresistance and magnetic measurements in a temperature range from 4.2 K to 350 K and magnetic field-cycling from −9 T to 9 T. The resistance measurements revealed a metal-insulator Verwey transition (VT) atT _v =123.76 K with activation energy E=92.5 meV at T >T _v and temperature-substitute for the activation energy below the VT,T ₀=E/k _B ≈3800 K within 70 K–110K. The magnetotransport results independently verified the VT at 123.70 K, with discontinuous change in the magnetic moment ΔM≈0.21 ΔM≈0.21μ _B and resistance hysteresis, dependent on the magnetic field in a narrow temperature range of 0.4° around theT _v . The magnetic characterization established self consistentlyT _v as ≈123.67 K, the jump in the magnetization at the VT≈0.25μ _B and confirmed, that the magnetocrystalline anisotropy is the main microscopic mechanism responsible for the magnetization of the monocrystal (88%) with additional natural and imposed defects contributing as 12%.     

Pressure–temperature phase diagrams for four higher members (nonyl–dodecyl) of the homologous series of 4′-alkyl-4-isothiocyanato-biphenyl ( n BT)

Differential thermal analysis (DTA) method was applied to study the pressure–temperature phase diagrams for four members of the 4′-alkyl-4-isothiocyanato-biphenyl (nBT) homologous series (n?=?9–12). These compounds exhibit a smectic E polymorphism enriched by a smectic A phase for two longest members. Measurements were performed in the temperature range 25–110°C and the pressure range up to 165?MPa. For all substances studied the pressure-induced phases were established with one or two triple points in the examined pressure range. In addition, for the 11BT and the 12BT the phases existing under atmospheric pressure disappeared under relatively low pressures. The results are compared with recently studied smectogenic n-alkyl-cyanobiphenyl (nCB) compounds.     

Raman and surface‐enhanced Raman spectroscopic studies of the 15‐mer DNA thrombin‐binding aptamer

Aptamers are single‐stranded oligonucleotides that selectively bind to their target molecules owing to their ability to form secondary structures and shapes. The 15‐mer (5′‐GGTTGGTGTGGTTGG‐3′) DNA thrombin‐binding aptamer (TBA) binds to thrombin following the formation of a quadruplex structure via the Hoogsten‐type G–G interactions. In the present study, Raman and SERS spectra of TBA and thiolated TBA (used to facilitate covalent bonding to metal nanoparticle) in different conditions are investigated. The spectra of the two analogs exhibit vibrations, such as the C8N7 H2 deformation band at ∼1480 cm⁻¹ of the guanine tetrad, that are characteristic of the quadruplex structure in the presence of K⁺ ions or at low temperature. Interestingly, SERS spectra of the two analogs differ markedly from their respective normal Raman spectra, possibly due to changes in the conformation of the aptamer upon binding, as well as to the specific interaction of individual vibrational modes with the metal surface. In addition, the SERS spectra of the thiolated aptamer show significant changes with different concentrations, which may be due to different orientation of the molecule with respect to the metal surface. This study provides useful information for the development of label‐free aptamer‐based SERS sensors and assays. Copyright © 2009 John Wiley & Sons, Ltd.     

Influence of the sample orientation in Sn<Subscript>2</Subscript>P<Subscript>2</Subscript>S<Subscript>6</Subscript> crystals on the hydrostatic piezoelectric coefficients

The influence of the sample orientation on the effective value of the hydrostatic piezoelectric coefficients d _h ⁽ⁱ⁾ of Sn₂P₂S₆ crystals has been studied. The hydrostatic piezoelectric coefficients d _h ⁽¹⁾ and d′ _h ⁽³⁾ , were measured, d _h ⁽¹⁾ =(244±3) pC/N and d′ _h ⁽³⁾ =(92±1) pC/N. The hydrostatic piezoelectric coefficient d _h ⁽³⁾ for orthogonal axis system was calculated to be d _h ⁽³⁾ =(87±2) pC/N. The, optimal orientation of the sample has been found as (Xy l)−20°-cut. Maximal value of the effective hydrostatic piezoelectric coefficient d _h ⁽¹⁾ equals 260 pC/N. Double rotated samples were also studied. The orientation of the samples insensitive to the pressure has been found. The theoretical mean value of hydrostatic piezoelectric coefficient (d _h ) _mean corresponding to randomly oriented Sn₂P₂S₆ grains in a poled composite has been calculated to be (d _h ) _mean =136 pC/N.     

Polarons in axial transport in single-layer high-T<Subscript><Emphasis Type="Italic">c</Emphasis></Subscript> superconductors

The temperatureT dependencies ρ(T) of normal state electric resistivitiesρ _c (axial) andρ _ab (in plane) of single-layer high-T _c superconductors show common trends: AsT is raised, the resistivity first drops steeply before it starts rising αT above an apparent semiconductor-to-metal crossoverT _cross . To analyze ρ(T) we plottT/ρ againstT at various dopingsx for bothρ _c andρ _ab .T/ρ is inversely proportional to the traversal time across a potential barrier as an ionic particle drifts in an electric field. We findT/ρ in good agreement with theT dependence of the quantum rate of migrating particles: AsT is raised, a zero-point rate at the lowestT is extended to a nearly flat plateau before a thermally activated branch sets in. We also find evidence for the admixture of 1- & 2-phonon absorptions below the Arrhenius range. These features shape the semiconductor-like branch below T _cross . AboveT _cross a metallic-like branch sets in, its αT character deriving from the field coupling of the migrating particle. Our analysis suggests that metal physics may not suffice if ionic features play a role in transport. We attribute our conclusions to the drift of strong-coupling polarons along Cu−O bonds. These “bond polarons” originate from carrier scattering by double-well potentials associated with the bonds. A bond polaron dissociates to a free hole as it passes onto a neighboring O-O link.     

SERS‐based assays for sensitive detection of modafinil

In this paper, we proposed two sensitive surface‐enhanced Raman spectroscopy assays for the determination of modafinil in urine matrix. In the first assay, modafinil was extracted by solid phase extraction and determined after sandwiching with silver nanoparticles. In the second assay, modafinil was extracted by non‐specific interaction with magnetic gold nanoparticles and determined by sandwiching with magnetic gold and silver nanoparticles. The non‐specific magnetic extraction does not require any analyte specific agent like antibody, aptamer, or molecularly imprinted polymer, therefore, the cost and complexity of the assay is very low. Both assays are capable for application to urine samples with the detection limits under the minimum required performance limit of modafinil. The assays were validated in terms of accuracy, precision, detection limits, and ranges. Copyright © 2015 John Wiley & Sons, Ltd.     

‘Epixenosomes’: Peculiar epibionts of the protozoon ciliate Euplotidium itoi: Do their cytoplasmic tubules consist of tubulin?

Epixenosomes, epibiontic organisms living on the protozoon ciliate Euplotidium itoi, like prokaryotes, lack a true nucleus surrounded by a nuclear envelope. However, their cytoplasm, at least in the mature state, is somehow compartmentalized by the presence of a complex extrusive apparatus surrounded by a form of basket consisting of bundles of tubules (BT), 20–24 nm in diameter, delineated by a wall made of globular subunits. The nature of these tubules was investigated by means of in vivo experiments, ultrastructural analysis and immunocytochemistry by both fluorescence and electron microscopy. It has been found that BT, besides being well preserved when fixed in the presence of tannic acid, are sensitive to factors known to depolymerize cytoplasmic microtubules in a variety of cells such as nocodazole and cold and show a positive immunoreaction against different antitubulin antibodies. On the basis of all the results obtained, the hypothesis that the epixenosomal BT consist of tubulin acquires a strong credibility. This is particularly interesting if we consider that tubulin in prokaryotes has never been conclusively demonstrated.     

The exclusive reaction of pion quasi-elastic knockout from nucleon <Emphasis Type="Italic">p(e,e′ π)B</Emphasis> to various states of the final baryon-spectator <Emphasis Type="Italic">B</Emphasis> and the quark models for the pion cloud of nucleon

The pion momentum distributions (MDs) in four channels of virtual decay p→B+π, B = N, Δ, N _1/2-(1535), N _1/2+(1440) are calculated in two models, the microscopic model of ³ P ₀ scalar q−q fluctuation with the pion as a composite q−q-system and the chiral semi-microscopic model of πq interaction with the pion as a structureless Goldstone boson. The results of the above models are similar for the baryon states B = N, Δ, N _1/2-(1535) but are rather different for the Roper resonance N _1/2+(1440) which corresponds to excitation of two oscillator quanta in the nucleon. The experimental investigation of pion MDs by means of the reaction of quasi-elastic knockout of pion by an electron of a few GeV energy p(e, e′ π)B may be very suitable for Jefferson Laboratory, Virginia (JLab).     

2<Emphasis Type="Italic">µ</Emphasis>m Laser based on an electro-optical <Emphasis Type="Italic">Q</Emphasis>-switched intracavity KTP optical parametric oscillator

An 2 μm pulse laser based on an intracavity optical parameter oscillator (OPO) pumped by an electrooptical Q-switched 1.064 μm laser is realized. A type-II phase-matched (θ = 51.5° and φ = 0) KTP crystal and a limiting diaphragm with a diameter of 4 mm were used. The parameters of the KTP-OPO tuning curves as a function of the phase matching angles were analyzed for the case of 1.064 μm laser pumping. Experiments based on single and double KTPs for walk-off compensation were performed. An output energy of 26 mJ in the 2 μm wavelength region was obtained with a pump-to-signal conversion efficiency of 26%. The center wavelengths of the signal and idle lights were 2165.4 and 2088.6 nm with a FWHM of 3.8 and 4 nm, respectively.     

Coplanar (<Emphasis Type="Italic">e</Emphasis>, 3<Emphasis Type="Italic">e</Emphasis>) differential cross-section of He atom

We present in this paper the results of our calculation of five-fold differential cross-section (FDCS) for (e,3e) process on He atom in low momentum transfer and high electron impact energy in shake-off mechanism. The formalism has been developed in Born approximation using plane waves, Byron and Joachain as well as Le Sech and correlated BBK-type wave functions respectively for incident and scattered, bound and ejected electrons. The angular distribution of FDCS of our calculation is presented in various modes of coplanar geometry and comparison is made with the available experimental data. We observe that the present calculation is able to reproduce the trend of the experimental data. However, it differs in magnitude from the experiment. The present theory does not predict four-peak structure insummed mutual angle mode for lower excess ejected electron energies. We also discuss the importance of momentum transfer, post-collision interaction (PCI) and ion participation in the (e,3e) process in constant θ₁₂ mode     

Effect of biotite content of hydrogels on enhanced removal of methylene blue from aqueous solution

A series of chitosan-g-poly(acrylic acid)/biotite (CTS-g-PAA/BT) hydrogels with unique clay biotite (BT) were prepared and used to remove cationic dye methylene blue (MB) from aqueous solution by batch adsorption experiments. Variables of the system including BT content, initial pH, contact time, initial concentration, and temperature affecting the adsorption efficiency of MB by CTS-g-PAA/BT hydrogels were investigated. Kinetic studies indicated that the adsorption data well followed pseudo-second-order kinetics. Langmuir and Freundlich isotherm models were applied to experimental equilibrium data of MB adsorption depending on temperature. The adsorption equilibrium data obeyed Langmuir isotherm, and the monolayer adsorption capacity calculated from the Langmuir isotherm was 2,125.70 mg/g for CTS-g-PAA/10% BT at 30 °C. The adsorption capacity was much higher compared with other hydrogels with the same content of other clays. The introduction of BT into the hydrogel could effectively improve its adsorption properties and reduce the cost. Thermodynamic parameters were evaluated for the dye-adsorbent systems and revealed that the adsorption process was spontaneous and exothermic in nature. All the information gave an indication that CTS-g-PAA/10% BT could potentially be applied as an efficient adsorbent for cationic dye removal from aqueous solution.     

Solitonic properties for a forced generalized variable-coefficient Korteweg-de Vries equation for the atmospheric blocking phenomenon

In this paper, investigation is given to a forced generalized variable-coefficient Korteweg-de Vries equation for the atmospheric blocking phenomenon. Based on the Lax pair, under certain variable-coefficient-dependent constraints, we present an infinite sequence of the conservation laws. Through the Riccati equations obtained from the Lax pair, a Wahlquist-Estabrook-type Bäcklund transformation (BT) is derived, based on which the nonlinear superposition formula as well as one- and two-soliton-like solutions are obtained. Via the truncated Painlevé expansion, we give a Painlevé BT, along with the one-soliton-like solutions. With the Painlevé BT, bilinear forms are constructed, and we get a bilinear BT as well as the corresponding one-soliton-like solutions. Bell-type bright and dark soliton-like waves and kink-type soliton-like waves are observed, respectively. Graphic analysis shows that (1) the velocities of the soliton-like waves are related to h(t), d(t), f(t) and R(t), while the soliton-like wave amplitudes just depend on f(t), and (2) with the nonzero f(t) and R(t), soliton-like waves propagate on the varying backgrounds, where h(t), d(t) and f(t) are the dispersive, dissipative and line-damping coefficients, respectively, R(t) is the external-force term, and t is the scaled time coordinate.