Artificial Vision Adaption Mimicked by an Optoelectrical In2O3 Transistor Array.

Simulation of biological visual perception has gained considerable attention. In this paper, an optoelectrical In2O3 transistor array with a negative photoconductivity behavior is designed using a side-gate structure and a screen-printed ion-gel as the gate insulator. This paper is the first to observe a negative photoconductivity in electrolyte-gated oxide devices. Furthermore, an artificial visual perception system capable of self-adapting to environmental lightness is mimicked using the proposed device array. The transistor device array shows a self-adaptive behavior of light under different levels of light intensity, successfully demonstrating the visual adaption with an adjustable threshold range to the external environment. This study provides a new way to create an environmentally adaptive artificial visual perception system and has far-reaching significance for the future of neuromorphic electronics.

A robust underwater superoleophobic aminated polyacrylonitrile membrane embedded with CNTs-COOH for durable oil/water and dyes/oil emulsions separation.

Considering the emulsified oil and water-soluble dyes in wastewater, the exploitation of easy-manufacturing, energy-saving and high-efficiency separation materials is urgently required. In this work, integrating the positively charged polyethyleneimine (PEI) with negatively charged CNTs-COOH constructed the superhydrophilic Cassie-Baxter structure onto the electrospun polyacrylonitrile (PAN) membrane surface by ultrasonic, electrostatic interaction and thermal treatment. Based on it, the PEN@CNTs membrane achieved efficient separation for surfactant-free, tween 80-stabilized, SDS-stabilized, and CTAB-stabilized emulsions (the fluxes reached 508-3158 L m-2 h-1, the separation efficiency reached 99.42%) by the splendid water-penetration and oil-repellency, electrostatic interaction, and "aperture sieve". Moreover, because of the porosity and strong charged surface of PEN@CNTs membrane, the anionic dyes can be quickly removed by one-step filtrate method (∼403 L m-2 h-1). Meanwhile, the PEN@CNTs membrane also achieved synchronous and efficient remediation for oil/dye mixture emulsions after many cycles. More importantly, facing the complex physical and chemical environments, the combination of the stabilized PEN membrane, inactive CNTs-COOH layer, and the bond of embedding method between CNTs-COOH and PEN nanofibers made the PEN@CNTs membrane demonstrated robust stability and durable separation capability.

Designing nanofibrous membrane with biomimetic caterpillar-like structured for highly-efficient and simultaneous removal of insoluble emulsified oils and soluble dyes towards sewage remediation.

Purification of insoluble emulsified oils and soluble organic pollutants from sewage has attracted tremendous attention in today's society. Herein, a stable and environmentally friendly nanofibrous membrane with hierarchical caterpillar-like structure was fabricated via in-situ hydrothermal growing the nickel-cobalt layered double hydroxides (NiCo-LDHs) on tche polyacrylonitrile (PAN) electrospun nanofibers. The wrapped hydrophilic NiCo-LDHs constructed the hierarchical structure and endowed the membrane attractive superhydrophilicity (≈ 0°)/underwater superoleophobicity (≈ 161°) and enhanced oil-repellency performance. Meanwhile, the NiCo-LDH@PANI/oPAN NFMs can display the ultra-fast flux of SSEs (xylene/water emulsion, 4175 L m-2 h-1) and satisfactory separation efficiency (99.07%). Moreover, the introduction of positively charged NiCo-LDHs increased plentiful adsorption active sites for membranes, which is beneficial to demulsify ionic SSEs and adsorb organic pollutants. Finally, for simultaneous purification of complex sewage by the dead-end and cross-flow filtration experiment, the composite membrane both displayed splendid removal rate of oil (> 99.0%) and dyes (> 99.0%), robust regeneration recycle-ability and no secondary pollution. Hence, it is expected that such strategy of combining electrospun and chelating-assisted in-situ hydrothermal can provide a low energy consumption and high decontamination technology for severe environmental crisis.

An early event in the transport mechanism of LacY protein: interaction between helices V and I.

Helix V in LacY, which abuts and crosses helix I in the N-terminal helix bundle of LacY, contains Arg(144) and Trp(151), two residues that play direct roles in sugar recognition and binding, as well as Cys(154), which is important for conformational flexibility. In this study, paired Cys replacement mutants in helices V and I were strategically constructed with tandem factor Xa protease cleavage sites in the loop between the two helices to test cross-linking. None of the mutants form disulfides spontaneously; however, three mutants (Pro(28) → Cys/Cys(154), Pro(28) → Cys/Val(158) → Cys, and Phe(29) → Cys/Val(158) → Cys) exhibit cross-linking after treatment with copper/1,10-phenanthroline (Cu/Ph) or 1,1-methanediyl bismethanethiosulfonate ((MTS)(2)-1), 3-4 Å), and cross-linking is quantitative in the presence of ligand. Remarkably, with one mutant, complete cross-linking with (MTS)(2)-1 has no effect on lactose transport, whereas quantitative disulfide cross-linking catalyzed by Cu/Ph markedly inhibits transport activity. The findings are consistant with a number of previous conclusions suggesting that sugar binding to LacY causes a localized scissors-like movement between helices V and I near the point where the two helices cross in the middle of the membrane. This ligand-induced movement may act to initiate the global conformational change resulting from sugar binding.

Site-directed alkylation studies with LacY provide evidence for the alternating access model of transport.

In total, 59 single Cys-replacement mutants in helix VII and helix X of the lactose permease of Escherichia coli were subjected to site-directed fluorescence labeling in right-side-out membrane vesicles to complete the testing of Cys accessibility or reactivity. For both helices, accessibility/reactivity is relatively low at the level of the sugar-binding site where the helices are tightly packed. However, labeling of Cys substitutions in helix VII with tetramethylrhodamine-5-maleimide decreases from the middle toward the cytoplasmic end and increases toward the periplasmic end. Helix X is labeled mainly on the side facing the central hydrophilic cavity with relatively small or no changes in the presence of ligand. In contrast, sugar binding causes a significant increase in accessibility/reactivity at the periplasmic end of helix VII. When considered with similar findings from N-ethylmaleimide alkylation studies, the results confirm and extend support for the alternating access model.

The alternating access transport mechanism in LacY.

Lactose permease of Escherichia coli (LacY) is highly dynamic, and sugar binding causes closing of a large inward-facing cavity with opening of a wide outward-facing hydrophilic cavity. Therefore, lactose/H(+) symport via LacY very likely involves a global conformational change that allows alternating access of single sugar- and H(+)-binding sites to either side of the membrane. Here, in honor of Stephan H. White's seventieth birthday, we review in camera the various biochemical/biophysical approaches that provide experimental evidence for the alternating access mechanism.

Sugar binding induces the same global conformational change in purified LacY as in the native bacterial membrane.

Many independent lines of evidence indicate that the lactose permease of Escherichia coli (LacY) is highly dynamic and that sugar binding causes closing of a large inward-facing cavity with opening of a wide outward-facing hydrophilic cavity. Therefore, lactose/H(+) symport catalyzed by LacY very likely involves a global conformational change that allows alternating access of single sugar- and H(+)-binding sites to either side of the membrane (the alternating access model). The x-ray crystal structures of LacY, as well as the majority of spectroscopic studies, use purified protein in detergent micelles. By using site-directed alkylation, we now demonstrate that sugar binding induces virtually the same global conformational change in LacY whether the protein is in the native bacterial membrane or is solubilized and purified in detergent. The results also indicate that the x-ray crystal structure reflects the structure of wild-type LacY in the native membrane in the absence of sugar.

Fluorescence Detection of Heavy Atom Labeling (FD-HAL): a rapid method for identifying covalently modified cysteine residues by phasing atoms.

Membrane protein crystallography frequently stalls at the phase determination stage due to poor crystal diffraction and the inability to identify heavy atom derivatization prior to data collection. Thus, a majority of time, effort and resources are invested preparing potential derivatized crystals for synchrotron data collection and analysis without knowledge of heavy atom labeling. To remove this uncertainty, we introduce Fluorescence Detection of Heavy Atom Labeling (FD-HAL) using tetramethylrhodamine-5-maleimide (a fluorescent maleimide compound) to monitor in-gel cysteine residue accessibility and ascertain covalent modification by mercury, platinum and gold compounds. We have tested this technique on three integral membrane proteins (LacY, vSGLT and mVDAC1) and can quickly assess the optimal concentrations, time and heavy atom compound to derivatize free cysteine residues in order to facilitate crystal phasing. This, in conjunction with cysteine scanning for incorporating heavy atoms at strategic positions, is a useful tool that will considerably assist in phasing membrane protein structures.

Residues gating the periplasmic pathway of LacY.

X-ray crystal structures of LacY (lactose permease of Escherichia coli) exhibit a large cytoplasmic cavity containing the residues involved in sugar binding and H(+) translocation at the apex and a tightly packed side facing the periplasm. However, biochemical and biophysical evidence provide a strong indication that a hydrophilic pathway opens on the external surface of LacY with closing of the cytoplasmic side upon sugar binding. Thus, an alternating-access mechanism in which sugar- and H(+)-binding sites at the approximate middle of the molecule are alternatively exposed to either side of the membrane is likely to underlie LacY-catalyzed sugar/H(+) symport. To further investigate periplasmic opening, we replaced paired residues on the tightly packed periplasmic side of LacY with Cys, and the effect of cross-linking was studied by testing the accessibility/reactivity of Cys148 with the elongated ( approximately 29 A), impermeant hydrophilic reagent maleimide-PEG2-biotin. When the paired-Cys mutant Ile40-->Cys/Asn245-->Cys containing native Cys148 is oxidized to form a disulfide bond, the reactivity of Cys148 is markedly inhibited. Moreover, the reactivity of Cys148 in this mutant increases with the length of the cross-linking agent. In contrast, maleimide-PEG2-biotin reactivity of Cys148 is unaffected by oxidation of two other paired-Cys mutants at the mouth of the periplasmic cavity. The data indicate that residues Ile40 and Asn245 play a primary role in gating the periplasmic cavity and provide further support for the alternating-access model.

Clogging the periplasmic pathway in LacY.

The lactose permease of Escherichia coli (LacY) is a highly dynamic membrane transport protein. Crystal structures of wild-type and mutant LacY all exhibit an inward-facing conformation with an open cytoplasmic pathway and a tightly packed periplasmic side, which makes the binding site inaccessible from the outside. However, biochemical and biophysical findings provide strong evidence that occupation of the sugar-binding site leads to an increased probability of opening of a hydrophilic pathway on the periplasmic side and closing of the cytoplasmic cavity. By this means, the sugar-binding site becomes accessible to either side of the membrane in alternating fashion. To extend studies on the relationship between the periplasmic pathway and transport activity, engineered single-Cys replacements in the periplasmic pathway were reacted to completion with thiol reagents, and the effects on transport and sugar binding were tested. Inactivation correlates for the most part with the size of the modifying reagent, although the position of the Cys replacement is also important. However, sugar binding is unaffected. The results suggest that placement of a relatively large moiety in the putative periplasmic cleft of LacY likely prevents closure, an essential step in the transport cycle, without significantly altering access of sugar to the binding site.

The Cys154-->Gly mutation in LacY causes constitutive opening of the hydrophilic periplasmic pathway.

The lactose permease of Escherichia coli (LacY) is a highly dynamic membrane transport protein, while the Cys154-->Gly mutant is crippled conformationally. The mutant binds sugar with high affinity, but catalyzes very little translocation across the membrane. In order to further investigate the defect in the mutant, fluorescent maleimides were used to examine the accessibility/reactivity of single-Cys LacY in right-side-out membrane vesicles. As shown previously, sugar binding induces an increase in reactivity of single-Cys replacements in the tightly packed periplasmic domain of wild-type LacY, while decreased reactivity is observed on the cytoplasmic side. Thus, the predominant population of wild-type LacY in the membrane is in an inward-facing conformation in the absence of sugar, sugar binding induces opening of a hydrophilic pathway on the periplasmic side, and the sugar-binding site is alternatively accessible to either side of the membrane. In striking contrast, the accessibility/reactivity of periplasmic Cys replacements in the Cys154-->Gly background is very high in the absence of sugar, and sugar binding has little or no effect. The observations indicate that an open hydrophilic pathway is present on the periplasmic side of the Cys154-->Gly mutant and that this pathway is unaffected by ligand binding, a conclusion consistent with findings obtained from single-molecule fluorescence and double electron-electron resonance.

Site-directed alkylation of LacY: effect of the proton electrochemical gradient.

Previous N-ethylmaleimide-labeling studies show that ligand binding increases the reactivity of single-Cys mutants located predominantly on the periplasmic side of LacY and decreases reactivity of mutants located for the most part of the cytoplasmic side. Thus, sugar binding appears to induce opening of a periplasmic pathway with closing of the cytoplasmic cavity resulting in alternative access of the sugar-binding site to either side of the membrane. Here we describe the use of a fluorescent alkylating reagent that reproduces the previous observations with respect to sugar binding. We then show that generation of an H(+) electrochemical gradient (Delta(mu (H)+), interior negative) increases the reactivity of single-Cys mutants on the periplasmic side of the sugar-binding site and in the putative hydrophilic pathway. The results suggest that Delta(mu (H)+), like sugar, acts to increase the probability of opening on the periplasmic side of LacY.

Energetics of ligand-induced conformational flexibility in the lactose permease of Escherichia coli.

Isothermal titration calorimetry has been applied to characterize the thermodynamics of ligand binding to wild-type lactose permease (LacY) and a mutant (C154G) that strongly favors an inward facing conformation. The affinity of wild-type or mutant LacY for ligand and the change in free energy (DeltaG) upon binding are similar. However, with the wild type, the change in free energy upon binding is due primarily to an increase in the entropic free energy component (TDeltaS), whereas in marked contrast, an increase in enthalpy (DeltaH) is responsible for DeltaG in the mutant. Thus, wild-type LacY behaves as if there are multiple ligand-bound conformational states, whereas the mutant is severely restricted. The findings also indicate that the structure of the mutant represents a conformational intermediate in the overall transport cycle.

Expression and purification of functional ligand-binding domains of T1R3 taste receptors.

Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.

Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli.

Animals utilize hundreds of distinct G protein-coupled receptor (GPCR)-type chemosensory receptors to detect a diverse array of chemical signals in their environment, including odors, pheromones, and tastants. However, the molecular mechanisms by which these receptors selectively interact with their cognate ligands remain poorly understood. There is growing evidence that many chemosensory receptors exist in multimeric complexes, though little is known about the relative contributions of individual subunits to receptor functions. Here, we report that each of the two subunits in the heteromeric T1R2:T1R3 sweet taste receptor binds sweet stimuli though with distinct affinities and conformational changes. Furthermore, ligand affinities for T1R3 are drastically reduced by the introduction of a single amino acid change associated with decreased sweet taste sensitivity in behaving mice. Thus, individual T1R subunits increase the receptive range of the sweet taste receptor, offering a functional mechanism for phenotypic variations in sweet taste.

Identification of Novel Regulatory Elements of Human Stem Cell Factor Gene in MCF Cell.

Human stem cell factor(hSCF)is a pluripotent growth factor that regulates proliferation, differentiation and migration of certain mammalian stem cells, such as primordial germ cells etc. It is shown that hSCF and its receptor are commonly co-expressed in human breast cancer cells. Up to now, the definite regulatory mechanism of hSCF gene in breast cancer cells is unclear, except that its 5'flanking sequence contains essential elements for regulating transcription. To localize the regulatory elements responsible for the regulation of the hSCF gene, we performed transient transfection study in MCF cells, with a series of luciferase reporter gene constructs, containing different 5x end deletions of hSCF gene. This study indicates that the region of -1190 -853 significantly enhanced the luc gene expression, while the region of -339 -162 inhibited the expression. Eletrophoretic mobility shift assay confirmed that MCF nuclear extract proteins bound to both -1190 -853 and -339 -273 regions, forming specific DNA-protein complexes, indicating that there were nuclear protein binding sites in these regions. The results suggest that both -1190 -853 and -339 -273 DNA fragments of the hSCF 5'flanking sequence may be novel regulatory elements, and may play a role in the regulation of hSCF gene expression in MCF cells.