Construction of Xylose Dehydrogenase Displayed on the Surface of Bacteria Using Ice Nucleation Protein for Sensitive d-Xylose Detection

A novel method was developed to detect d-xylose (INS 967) sensitively and selectively, which is based on a xylose dehydrogenase (XDH) cell-surface displaying system using a newly identified ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif. With coenzyme NAD⁺, the XDH-displayed bacteria facilitates the catalysis of the oxidization of xylose and the resultant NADH can be detected spectrometrically at 340 nm. The fusion protein was characterized by proteinase accessibility, Western blot, and enzyme activity assays. The established XDH surface displaying system did not inhibit the growth of the recombinant Escherichia coli strain. The XDH was mainly displayed on the surface of host cells, which is of high XDH activity and high d-xylose specificity. The optimal temperature and pH of cell displayed XDH were found at 30 °C and pH 8.0, respectively. The XDH-displayed bacteria can be used directly without further enzyme extraction and purification, and it improved the stability of the enzyme. Moreover, the cell-surface-displayed-protein-based approach showed a wide linear range (5–900 μM) and a low detection limit of 2 μM of d-xylose. More importantly, the recombinant cells could be used for precise detection of d-xylose from the real samples such as foods and degradation products of lignocellulose. The method shown here provides a simple, rapid, and low-cost strategy for the sensitive and selective measurement of d-xylose. In addition, the XDH-displayed bacteria showed an interesting response in developing electrochemical biosensors. Thus, the genetically engineered cells may find broad application in such biosensors and biocatalysts. Similarly, this type of genetic approach may be used for the expression of other intracellular enzymes of interest for certain purposes

Diketopyrrolopyrrole Amphiphile-Based Micelle-Like Fluorescent Nanoparticles for Selective and Sensitive Detection of Mercury(II) Ions in Water

A technique for encapsulating fluorescent organic probes in a micelle system offers an important alternative method to manufacture water-soluble organic nanoparticles (ONPs) for use in sensing Hg²⁺. This article reports on a study of a surfactant-free micelle-like ONPs based on a 3,6-di­(2-thienyl)-2,5-dihydro­pyrrolo­[3,4-<i>c</i>]­pyrrole-1,4-dione (TDPP) amphiphile, (2-(2-(2-methoxy­ethoxy)­ethyl)-3,6-di­(2-thiophyl)-2,5-dihydro­pyrrolo­[3,4-<i>c</i>]­pyrrole-1,4-dione (NDPP) fabricated to monitor Hg²⁺ in water. NDPP was synthesized through a simple one-step modification of a commercially available dye TDPP with a flexible and hydrophilic alkoxy. This study reports, for the first time, that TDPP dyes can respond reversibly, sensitively, and selectively to Hg²⁺ through TDPP–Hg–TDPP complexation, similar to the well-known thymine­(T)–Hg–thymine­(T) model and the accompanying molecular aggregation. Interestingly, transmission electron microscopy (TEM) and dynamic light scattering (DLS) confirmed that, in water, NDPP forms loose micelle-like fluorescent ONPs with a hydrohobic TDPP portion encapsulated inside. These micelle-like nanoparticles offer an ideal location for TDPP–Hg complexation with a modest molecular aggregation, thereby providing both clear visual and spectroscopic signals for Hg²⁺ sensing. An estimated detection limit of 11 nM for Hg²⁺ sensing with this NDPP nanoparticle was obtained. In addition, NDPP ONPs show good water solubility and high selectivity to Hg²⁺ in neutral or alkalescent water. It was superior to most micelle-based nanosensors, which require a complicated process in the selection or synthesis of suitable surfactants. The determinations in real samples (river water) were made and satisfactory results were achieved. This study provides a low-cost strategy for fabricating small molecule-based fluorescent nanomaterials for use in sensing Hg²⁺. Moreover, the NDPP nanoparticles show potential ability in Hg²⁺ ion adsorption and recognization of cysteine using NDPP-Hg composite particle

Enhanced Performance of a Glucose/O₂ Biofuel Cell Assembled with Laccase-Covalently Immobilized Three-Dimensional Macroporous Gold Film-Based Biocathode and Bacterial Surface Displayed Glucose Dehydrogenase-Based Bioanode

The power output and stability of enzyme-based biofuel cells (BFCs) is greatly dependent on the properties of both the biocathode and bioanode, which may be adapted for portable power production. In this paper, a novel highly uniform three-dimensional (3D) macroporous gold (MP-Au) film was prepared by heating the gold “supraspheres”, which were synthesized by a bottom-up protein templating approach, and followed by modification of laccase on the MP-Au film by covalent immobilization. The as-prepared laccase/MP-Au biocathode exihibited an onset potential of 0.62 V versus saturated calomel electrode (SCE, or 0.86 V vs NHE, normal hydrogen electrode) toward O₂ reduction and a high catalytic current of 0.61 mAcm^–2. On the other hand, mutated glucose dehydrogenase (GDH) surface displayed bacteria (GDH-bacteria) were used to improve the stability of the glucose oxidation at the bioanode. The as-assembled membraneless glucose/O₂ fuel cell showed a high power output of 55.8 ± 2.0 μW cm^–2 and open circuit potential of 0.80 V, contributing to the improved electrocatalysis toward O₂ reduction at the laccase/MP-Au biocathode. Moreover, the BFC retained 84% of its maximal power density even after continuous operation for 55 h because of the high stability of the bacterial surface displayed GDH mutant toward glucose oxidation. Our findings may be promising for the development of more efficient glucose BFC for portable battery or self-powered device applications

High Intensity Focused Ultrasound Responsive Metallo-supramolecular Block Copolymer Micelles

The metal–supramolecular diblock copolymer containing mechano-labile bis­(terpyridine)–Cu­(II) complex linkage in the junction point was synthesized. These metal–ligand containing amphiphilic copolymers are able to self-assemble in aqueous solution to form spherical micelles with poly­(propylene glycol) block forming the hydrophobic core. It is found that high intensity focused ultrasound can open the copolymer micelles and trigger the release of the payload in the micelle. The micellar properties and release kinetics of encapsulated guest molecule in response to ultrasound stimuli were investigated. The weak Cu­(II)–terpyridine dynamic bond in the copolymer chain can be cleaved under ultrasound and thus leads to the disruption of the copolymer micelle and the release of loaded cargo. This study will open up a new way for the molecular design of ultrasound modulated drug delivery systems

Effects of loss-of-function mutations.

<p><b>A</b> Effects of the mutations on AP (i-iii) and APD-restitution (iv-vi) properties of human atrial myocytes elicited by updated <i>Colman et al</i>. model, <i>Courtemanche et al</i>. model and <i>Grandi et al</i>. model. <b>B</b> Effects of the mutations on the maximum sustained dominant frequency of excitation waves in the lone AF and chronic AF conditions using (i) <i>Colman et al</i>. model and (ii) <i>Courtemanche et al</i>. model. <b>C</b> Effects of <i>KCNA5</i> mutations on APD heterogeneity and tissue vulnerability window at the CT/PM junction in <i>Colman et al</i>. model. APD distribution among regional cells of whole atria and CT/PM for in (i) isolated single cells and (ii) in coupled tissue; the APD distribution in tissue is shown in boxplots with outlier limits of 1.5×IQR (interquartile range). (iii) Temporal vulnerability window to wave propagation break at the CT/PM junction. In panel B, * indicates cases in which re-entrant waves could not be sustained.</p

Effects of gain-of-function <i>KCNA5</i> mutations.

<p><b>A</b> Effects of the mutations on the AP (i-iii) and APD-restitution (iv-vi) properties of human atrial myocytes elicited by updated <i>Colman et al</i>. model, <i>Courtemanche et al</i>. model and <i>Grandi et al</i>. model. <b>B</b> Effects of the mutations on the maximum sustained dominant frequency of excitation waves under the lone AF and chronic AF conditions using (i) <i>Colman et al</i>. model and (ii) <i>Courtemanche et al</i>. model. <b>C</b> Effects of the mutations on APD heterogeneity and tissue vulnerability window at the CT/PM junction in <i>Colman et al</i>. model. APD distribution among regional cells of whole atria and CT/PM for in (i) isolated single cells and (ii) in coupled tissue; the APD distribution in tissue is shown in boxplots with outlier limits of 1.5×IQR (interquartile range). (iii) Temporal vulnerability window to propagation wave break at the CT/PM junction.</p

Model representation describing <i>I</i>_Kur and simulated voltage clamp.

<p><b>A</b> Steady-state activation and inactivation; <b>B</b> Time constants; <b>C</b> Current trace obtained from a simulated voltage clamp. Inserts: top right–voltage protocol; bottom right–experimental current traces; <b>D</b> I-V relationship. In the Fig the simulation data were shown using lines, and experimental data represented by points. Experimental data were taken from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005587#pcbi.1005587.ref002" target="_blank">2</a>]; specially, for <b>B</b> the time constants were derived from current traces from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005587#pcbi.1005587.ref002" target="_blank">2</a>] by fitting the activation phase of the current trace to a mono-exponential equation.</p

Illustration of the phase distribution method to initiate re-entrant waves in the 3D human atrial model.

<p><b>A</b> The distributed phase map used on the anatomical model, with numbered indications of the stage of the AP which is mapped onto each location; RAA–right atrial appendage, PV–pulmonary vein, LA–left atrium; <b>B</b> Demonstration of the development of a spiral wave in the tissue with mapped initial conditions; the time of screenshot is indicated in the top of each panel.</p

<i>KCNA5</i> loss-of-function mutations induced EADs following the beta-adrenergic stimulation.

<p><b>A</b>(i) In the presence of ISO, EADs were produced by Y155C and P488S, but not in WT or D469E in RA; (ii) EADs were induced by D469E in CT but not in PM. <b>B</b> Under uniform application of ISO in a 1D strand model with D469E, EADs in the CT but not PM induced conduction block at an S2 of 689 ms (i) and success at 690 ms (ii). <b>C</b> 1D conduction patterns under mutations and application of ISO in various configurations. On the left of each panel is a breakdown of the regions in the 1D model and an illustration of the anatomical conduction pathway to which they correspond; on the right is the regions of tissue to which ISO was applied. (i) Regular conduction pattern under WT and uniform ISO application; (ii) Under uniform application of ISO, D469E led to alternating bidirectional conduction block due to EADs in the CT; (iii) D469E and non-uniform ISO can lead to unidirectional conduction block, resulting from EADs in the CT regions with ISO; (iv) Unidirectional block can also be attained through a different pathway to the PM, in which the CT on one side of the SAN is of insufficient extent to develop significant EADs. In the simulations effects of beta-adrenergic stimulation was modelled by simulated application of ISO (1 μM).</p

Changes in parameters of steady-state variables and maximum conductance of <i>I</i>_Kur carried by the <i>KCNA5</i> mutants relative to WT.

<p>Changes in parameters of steady-state variables and maximum conductance of <i>I</i>_Kur carried by the <i>KCNA5</i> mutants relative to WT.</p