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In this study, a novel core@shell magnetic nanocomposite Fe3O4/CoFe-layered double hydroxide (Fe3O4@CoFe-LDH) was successfully synthesized by the co-precipitation method, and then employed as an efficient heterogeneous catalyst for activation of peroxymonosulfate (PMS) in removal of azo-dye acid orange 7 (AO7). The as-obtained nanocomposite was characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and vibrating sample magnetometer (VSM). The results from these characterizations showed Fe3O4@CoFe-LDH to possess good ferromagnetism and a perfect crystalline structure with a typical core@shell morphology. The system of Fe3O4@CoFe-LDH11/PMS (cobalt : iron molar ratio of 1 : 1) achieved 95.1% removal rate of AO7 (40 mg L−1) within 15 min under the optimized conditions, which outperformed bare Fe3O4 and raw CoFe-LDH11. Meanwhile, Fe3O4@CoFe-LDH11 displayed good adaptability in a wide pH range from 4 to 9 and relatively low PMS activation energy (39.9 kJ mol−1). The interference tests revealed HCO3− to possess the strongest restriction effect. Only 57.7% AO7 was removed when 10 mM HCO3− was introduced, which was ascribed to HCO3− not only serving as a radical scavenger, but also increasing the pH of the system. The radical quenching tests demonstrated SO4˙− as the dominant reactive species during the catalytic reaction. Based on X-ray photoelectron spectroscopy (XPS) analysis, the core structure of Fe3O4 served as an electron donor for accelerating the cycle of Co(II)/Co(III) at the active site of the LDH outer shell. Also, Fe3O4@CoFe-LDH exhibited outstanding stability and recyclability, and maintained high degradation efficiency of AO7 even after five cycles. In sum, the proposed magnetic Fe3O4@CoFe-LDH nanocomposite has great potential for remediation of wastewater contaminated with synthetic dyes.

Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected ‘single shelled’ intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is ‘collapsed’ compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses., Reoviridae undergo a complex assembly pathway in the host cell. Here the authors use cryo-electron tomography to visualize the assembly stages of mammalian orthoreovirus revealing a single shelled intermediate with gross similarity to an early assembly stage of a family of prokaryotic dsRNA viruses.

The hunger hormone ghrelin activates the ghrelin receptor GHSR to stimulate food intake and growth hormone secretion and regulate reward signaling. Acylation of ghrelin at Ser3 is required for its agonistic action on GHSR. Synthetic agonists of GHSR are under clinical evaluation for disorders related to appetite and growth hormone dysregulation. Here, we report high-resolution cryo-EM structures of the GHSR-Gi signaling complex with ghrelin and the non-peptide agonist ibutamoren as an investigational new drug. Our structures together with mutagenesis data reveal the molecular basis for the binding of ghrelin and ibutamoren. Structural comparison suggests a salt bridge and an aromatic cluster near the agonist-binding pocket as important structural motifs in receptor activation. Notable structural variations of the Gi and GHSR coupling are observed in our cryo-EM analysis. Our results provide a framework for understanding GHSR signaling and developing new GHSR agonist drugs., Ghrelin is a central orexigenic peptide hormone in human energy homeostasis that is also known as ‘hunger hormone’ and signals through its GPCR, GHSR. Here, the authors present the cryo-EM structures of the human GHSR-Gi signaling complex with bound ghrelin and the synthetic non-peptide agonist ibutamoren that are of interest for drug design.

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection., Mendonça et al. determine the structures of both in vitro assemblies of the HIV structural precursor protein Gag and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions, using cryo-electron tomography and subtomogram averaging. This study provides insights into the future development of small molecule inhibitors for HIV-1 infection.

Gag is the major HIV-1 structural polyprotein precursor. The Gag SP1 domain with the last residues of CA have been hypothesized to form a six-helix bundle necessary for particle assembly, but this bundle has not been fully resolved. Here, we determined the structures of complete CA-SP1 six-helix bundle connecting to the NC domain, from both in vitro Gag assemblies and viral-like particles (VLPs) carrying a T8I mutation in SP1, to near-atomic resolutions using cryoET and subtomogram averaging. The structures revealed novel densities, however distinct from IP6, inside the six-helix bundle of Gag assemblies, stabilizing the immature lattice. Interestingly, the T8I mutation impaired proteolytic cleavage of Gag at both SP1 boundaries. Our findings signify the involvement of small molecules in immature Gag assembly and provide the structural basis for development of small molecule inhibitors that stabilize SP1 helix, thus interfere with PR-mediated virus maturation.

Traditionally molecular assembly pathways for viruses have been inferred from high resolution structures of stable intermediates purified in vitro, and from low resolution images of cell sections as well as from genetic approaches including conditionally lethal mutants. Here, we directly visualise a previously unsuspected ‘single shelled’ icosahedral intermediate for a mammalian orthoreovirus, in addition to the expected virions, in cryo-preserved infected cells by cryo-electron tomography of cellular lamellae1,2. Particle classification and averaging yielded structures at resolutions as high as 5.6 Å, sufficient to identify secondary structural elements and place known molecular structures, allowing us to produce an atomic model of the intermediate, comprising 120 copies of protein λ1 and 120 copies of σ2. This λ1 shell is in a ‘collapsed’ form compared to the mature virions, with the molecules pushed inwards at the icosahedral 5-folds by ~100 Å. This grossly indented shell, although produced by a mammalian reovirus, is reminiscent of the first assembly intermediate of prokaryotic dsRNA viruses belonging to a different virus family3, adding weight to the supposition that these diverse viruses share a common ancestor, and suggesting mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds enormous promise for the dissection of the replication cycle of many viruses.

Sorting nexins (SNX) are a family of PX domain-containing proteins with pivotal roles in trafficking and signaling. SNX-BARs, which also have a curvature-generating Bin/Amphiphysin/Rvs (BAR) domain, have membrane-remodeling functions, particularly at the endosome. The minimal PX-BAR module is a dimer mediated by BAR-BAR interactions. Many SNX-BAR proteins, however, additionally have low-complexity N-terminal regions of unknown function. Here, we present the cryo-EM structure of the full-length SNX-BAR Mvp1, which is an autoinhibited tetramer. The tetramer is a dimer of dimers, wherein the membrane-interacting BAR surfaces are sequestered and the PX lipid-binding sites are occluded. The N-terminal low-complexity region of Mvp1 is essential for tetramerization. Mvp1 lacking its N-terminus is dimeric and exhibits enhanced membrane association. Membrane binding and remodeling by Mvp1 therefore requires unmasking of the PX and BAR domain lipid-interacting surfaces. This work reveals a tetrameric configuration of a SNX-BAR protein that provides critical insight into SNX-BAR function and regulation., SNX-BAR proteins are a family of PX and BAR domain-containing proteins with pivotal roles in trafficking processes. Here authors present the cryo-EM structure of the full-length fungal SNX-BAR Mvp1, which is an autoinhibited tetramer and provides critical insight into SNX-BAR function and regulation.

Glycated hemoglobin (HbA1c) has been widely explored as an important marker for monitoring and diagnosing diabetes. Due to the advantages of high selectivity, easy preparation, and convenient preservation of aptamers, research on glycated hemoglobin detection utilizing aptasensors has received much attention in recent years. However, factors such as the pH and the salt concentration of the solution and the structure of the aptamer could influence the interactions between HbA1c and the aptamer. In this study, the factors were evaluated using surface plasmon resonance (SPR). The results show that the pH and the salt concentration can greatly affect the formation of a complex between the aptamer and HbA1c. In the stereostructure of the aptamer, loop L1 may be an important motif for recognizing glycated hemoglobin. In addition, the best condition for detecting HbA1c was at pH 6, with a high sensitivity and a low limit of detection(LOD) (1.06 × 10−3 RU nM /2.55 nM). The results also demonstrated that the use of an SPR aptamer biosensor can be a sensitive technique to improve the accuracy and correctness of HbA1c measurement.

Purpose Hydrogels derived from decellularized tissues provide superior biocompatibility, tenability and tissue-specific extracellular matrix (ECM) components. Based on the preparation of decellularized porcine cornea (DPC), here we developed an injectable and transparent hydrogel for the regeneration of epithelium and stroma in focal corneal defects. Methods The DPC-derived hydrogel was prepared with N-cyclohexyl-N′-(2-morpholinethyl) carbodiimide metho-p-toluenesulfonate/N-hydroxysuccinimide (CMC/NHS) as cross-linkers. The characteristics of the hydrogel were analyzed and its cytocompatibility was assessed by Live/Dead and Cell Counting Kit (CCK)-8 assays. Immunofluorescence staining, quantitative PCR and Western blot analyses were performed to assess the relative protein and gene expression in corneal fibroblasts on hydrogel. The safety and efficiency of the hydrogel for repairing focal corneal defects in rabbit were measured by slit-lamp, anterior segment optical coherence tomography (AS-OCT), confocal microscopy and histological analyses. Results The DPC-derived hydrogel cross-linked with CMC/NHS assumed favorable transparency, exhibited distinct mechanical properties and preserved the ECM components of native porcine cornea (NPC). In vitro experiments showed that the hydrogel maintained the phenotype, supported the proliferation and promoted the ECM synthesis of corneal fibroblasts. When injected onto rabbit corneas, the hydrogel rapidly covered, solidified and formed a smooth surface on the focal defect. Corneal epithelium was fully regenerated within 3 days. The thickness of the corneal epithelium and stroma was restored at 12 weeks after surgery without significant inflammation or scar formation. Notably, the hydrogel showed no harmful effects on the resident stroma and endothelium. Conclusions The DPC-derived hydrogel may represent a promising biomaterial for corneal epithelial and stromal regeneration.

Hydrogels derived from decellularized tissues provide superior biocompatibility, tenability and tissue-specific extracellular matrix (ECM) components. Based on the preparation of decellularized porcine cornea (DPC), here we developed an injectable hydrogel, characterized the biocompatibility and transparency, and evaluated the use to repair corneal stromal defect in rabbit. Upon cross-linking with N-cyclohexyl-N′-(2-morpholinethyl) carbodiimide metho-p-toluenesulfonate (CMC), the DPC-based hydrogel assumed favorable transparency, exhibited distinct mechanical properties and preserved the ECM components of native porcine cornea (NPC). In vitro experiments showed that the hydrogel maintained the phenotype, supported the proliferation and promoted the ECM synthesis of corneal fibroblasts. When injected on rabbit corneas, the hydrogel rapidly covered, solidified and formed a smooth surface upon the focal defect. Corneal epithelium was fully regenerated within 3 days. The thickness of corneal epithelium and stroma was restored at 12 weeks postoperatively without significant inflammation and scar formation. Notably, the hydrogel showed no harmful effects on the resident stroma and endothelium. Considering to the advantages of low cost and ease of production and use, the DPC-based hydrogel may represent a promising biomaterial for corneal epithelial and stromal regeneration.

MiR-155 can inhibit the formation of atherosclerosis by interfering with the transformation of macrophages into foam cells that plays a critical role in the pathogenesis of atherosclerosis, but the precise mechanisms of miR-155 are still unknown. Herein, we observed that mRNA and protein expression levels of CEH were significantly upregulated in a dose- and time-dependent manner by transfected with miR-155 mimics in THP-1 macrophages. Further studies showed that overexpression of miR-155 can significantly inhibit foam cells formation, reduce intracellular CE accumulation and enhance the efflux of FC and cholesterol, result in a decrease of intracellular lipid accumulation; while this effect was significantly reversed by siCEH. Meanwhile, we found that Tim-3 is associated with miR-155-mediated CEH expression in THP-1 macrophage-derived foam cells. Overexpression of Tim-3 can attenuate miR-155-mediated CEH induction. Taken together, our findings demonstrated that miR-155 can inhibit the transformation of macrophages into foam cells by enhancing CEH signaling pathway in macrophages, this effect is likely to be achieved by inhibiting the expression of Tim-3.

Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the underlying mechanism of the RdRP complex is still unclear. Here we report the biochemical characterization of influenza RdRP subcomplex comprising PA, PB1, and N terminus of PB2, which exist as dimer in solution and can assemble into a tetramer state, regulated by vRNA promoter. Using single-particle cryo-electron microscopy, we have reconstructed the RdRP tetramer complex at 4.3 Å, highlighting the assembly and interfaces between monomers within the tetrameric structure. The individual RdRP subcomplex contains all the characterized motifs and appears as a cage-like structure. High-throughput mutagenesis profiling revealed that residues involved in the oligomer state formation are critical for viral life cycle. Our results lay a solid base for understanding the mechanism of replication of influenza and other negative-stranded RNA viruses.

Identifying the structure sensitivity of catalysts in reactions, such as Fischer-Tropsch synthesis from CO and H2 over cobalt catalysts, is an important yet challenging issue in heterogeneous catalysis. Based on a first-principles kinetic study, we find for the first time that CO activation on hexagonal close-packed (HCP) Co not only has much higher intrinsic activity than that of face centered-cubic (FCC) Co but also prefers a different reaction route, i.e., direct dissociation with HCP Co but H-assisted dissociation on the FCC Co. The origin is identified from the formation of various denser yet favorable active sites on HCP Co not available for FCC Co, due to their distinct crystallographic structure and morphology. The great dependence of the activity on the crystallographic structure and morphology of the catalysts revealed here may open a new avenue for better, stable catalysts with maximum mass-specific reactivity.

The structural evolution of the Pt3Ni(111) surface under oxidizing conditions was studied by ab initio atomistic thermodynamics. The thermodynamic phase diagram from Ni-rich to Pt-rich conditions with oxygen coverages up to one monolayer was constructed from their 560 possible surface structures. With an increase in the oxygen chemical potential, there were only two types of thermodynamically stable structures, which were a clean Pt-skin surface and a Ni-skin surface with chemisorbed oxygen, regardless of the underlying Pt-rich or Ni-rich conditions. Bimetallic surfaces with chemisorbed oxygen were only metastable. The detail analysis revealed that the structural evolution is determined by the factors of segregation cost, difference between oxygen-metal (Pt and Ni) bonding strength, and oxygen chemical potential.

Identification of the active sites in heterogeneous catalysis is important for a mechanistic understanding of the structure–reactivity relationship and rationale of the design of new catalysts, but remains challenging. Among others, the boundaries at metal nanoparticles and supported oxides were found to be important and attributed to the active sites in various catalytic reactions. To reveal the nature of the active sites at the boundaries, the catalytic role of the inverse 3d transition-metal oxide nanoislands on Pt(111) for low-temperature CO oxidation was studied by density functional theory calculations. A characteristic Pt–cation ensemble at the oxide/metal boundaries as the active sites is identified. In Pt–cation ensembles, coordinate-unsaturated (CUS) cations exposed at the edges of oxide nanoislands are highly active for O2 adsorption and dissociation, and less-reactive Pt binds modestly with dissociated O responsible for the facile CO oxidation. Inverse VIIIB-oxide/Pt boundaries exhibit high ac...

C2 oxygenate (acetaldehyde, ethanol, etc.) formation from syngas (CO + H2) is an important industrial process for the production of clean liquid energy fuels and valuable chemical feedstocks that a...

Carbon Chain Growth by Formyl Insertion on Rhodium and Cobalt Catalysts in Syngas Conversion

A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3ʹ to 5ʹ direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 A and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25–47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.

Mutation of Pro94 to phenylalanine or alanine significantly alters the redox properties of the type I copper center of amicyanin. Each mutation increases the redox midpoint potential (E(m)) value by at least 140 mV and shifts the pK(a) for the pH dependence of the E(m) value to a more acidic value. Atomic resolution (0.99-1.1 A) structures of both the P94F and P94A amicyanin have been determined in the oxidized and reduced states. In each amicyanin mutant, an electron-withdrawing hydrogen bond to the copper-coordinating thiolate sulfur of Cys92 is introduced by movement of the amide nitrogens of Phe94 and Ala94 much closer to the thiolate sulfur than in wild-type amicyanin. This is the likely explanation for the much more positive E(m) values which result from each of these mutations. The observed decrease in the pK(a) value for the pH dependence of the E(m) value that is seen in the mutants seems to be correlated with steric hindrance to the rotation of the His95 copper ligand which results from the mutations. In wild-type amicyanin the His95 side chain undergoes a redox and pH-dependent conformational change which accounts for the pH dependence of the E(m) value of amicyanin. The reduced P94A amicyanin exhibits two alternate conformations with the positions of the copper 1.4 A apart. In one of these conformations, a water molecule appears to have replaced Met98 as a copper ligand. The relevance of these structures to the electron transfer properties of P94F and P94A amicyanin are also discussed.

Within the methylamine dehydrogenase-amicyanin-cytochrome c-551i complex, electrons are transferred from tryptophan tryptophylquinone (TTQ) to heme via the type I copper center of amicyanin. Mutation of Pro94 of amicyanin to Phe increases the redox potential of the copper center within the protein complex by approximately 195 mV. This introduces a large energy barrier for the second electron transfer (ET) step in this three-protein ET chain. As a consequence of this mutation, the ET rate from TTQ to copper exhibits about a 6-fold increase and the ET rate from copper to heme exhibits about a 100-fold decrease. These changes in ET rate are consistent with the predictions of Marcus theory. Temperature dependence studies of these reactions indicate that the reorganization energies for the ET to and from the copper center are unchanged by the P94F mutation, despite the large change in redox potential that it causes. Steady-state kinetic studies indicate that despite the large energy barrier for the ET from copper to heme, methylamine-dependent reduction of heme by the three-protein complex with P94F amicyanin goes to completion. The turnover number for this steady-state reaction, however, is decreased 50-fold relative to that of the native complex. As a consequence of the P94F mutation, the rate constant for the unfavorable uphill ET reaction from copper to heme has become the rate-limiting step in the overall reaction. The evolutionary implications of the effects of this mutation on the function of this naturally occurring simple ET chain are discussed.

This review covers experimental works which have been carried out on the properties of the tryptophan tryptophylquinone (TTQ) cofactor and the TTQ-containing enzyme, methylamine dehydrogenase (MADH). The kinetic mechanism of MADH catalysed reactions, factors that determine the substrate specificity of MADH and the chemical reaction mechanism of MADH are discussed in detail. Electron transfer theory and kinetic models of interprotein electron transfer are discussed. Studies of electron transfer reactions in the MADH-amicyanin-cytochrome c551i protein complex are reviewed and discussed in terms of electron transfer theory.

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone-dependent enzyme that catalyzes the oxidative deamination of primary amines. Native MADH exhibits a strong preference for methylamine over longer carbon chain amines. Residue alphaPhe(55) controls this substrate specificity. When alphaPhe(55) is replaced with Ala, this preference is reversed with alphaF55A MADH preferring long-chain amines with at least seven carbons (Zhu, Z., Sun, D., and Davidson, V. L. (2000) Biochemistry 39, 11184-11186). To further modulate the substrate specificity of MADH, the side-chain of alphaPhe(55) was repositioned by site-directed mutagenesis of residue betaIle(107). This residue makes close contact with alphaPhe(55) and restricts its movement. betaI107V MADH exhibits a strong preference for propylamine, and betaI107N MADH exhibits a preference for 1-aminopentane. Thus, it has been possible to create forms of MADH that exhibit a preference for amines with carbon chain lengths of one, three, five, or seven carbons. The ability to discriminate between amines of different chain length was essentially abolished by an alphaF55I mutation. Molecular modeling studies with the known crystal structure are described that provide an explanation for these results. These results provide an example of a design-based approach to protein engineering in which site-directed mutagenesis on one residue can be used to reposition another residue to specifically alter enzyme specificity.

Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biological functions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC chemical labeling enrichment method. The sensitive method enables detection of low-abundance 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated a genome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC in non-CpG context exhibit lower abundance, more dynamically, than those in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H = A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite conversion-free method can be applied to biological samples with limited starting material or low-abundance cytosine modifications.

DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5′ half (CAT) and a canonical 3′ half (GTG). DraIII nicks the 3′ canonical half site at a faster rate than the 5′ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ββα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site-directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases. Electronic supplementary material The online version of this article (doi:10.1007/s13238-014-0038-z) contains supplementary material, which is available to authorized users.

Purpose.To find a relatively safe designed stromal bed thickness to avoid endothelial damage for lamellar keratoplasty with an Allegretto Wavelight FS200 femtosecond laser.Methods.Twelve rabbits were randomly divided into 50 μm and 150 μm groups according to the anticipated residue stromal bed thickness preparation with a femtosecond laser. Six rabbits without laser cutting were used as a control group. Central endothelial images were analyzed with in vivo confocal microscopy and scanning electron microscopy. The apoptosis of endothelium was evaluated with Hoechst 33342 staining and a TUNEL assay.Results.The endothelium of the 50 μm group had extensive injuries upon in vivo confocal and scanning electron microscopic observation, and minor injuries were observed in the 150 μm group. Moreover, more apoptotic cells were observed in the 50 μm group.Conclusions.When using a FS200 femtosecond laser assisted anterior lamellar keratoplasty, there was minor endothelium damage with a 150 μm stromal bed, and a more than 150 μm thickness stromal bed design may prevent the damage of corneal endothelium.

Site-directed mutagenesis was used to alter active-site residues of methylamine dehydrogenase (MADH) from Paracoccus denitrificans. Four residues of the beta subunit of MADH which are in close proximity to the tryptophan tryptophylquinone (TTQ) prosthetic group were modified. The crystal structure of MADH reveals that each of these residues participates in hydrogen bonding interactions with other active-site residues, TTQ or water. Relatively conservative mutations which removed the potentially reactive oxygens on the side chains of Thr122, Tyr119, Asp76 and Asp32 each resulted in greatly reduced or undetectable levels of MADH production. The reduction of MADH levels was determined by assays of activity and Western blots of crude extracts with antisera specific for the MADH beta subunit. No activity or cross-reactive protein was detected in extracts of cells expressing D76N, T122A and T122C MADH mutants. Very low levels of active MADH were produced by cells expressing D32N, Y119F, Y119E and Y119K MADH mutants. The Y119F and D32N mutants were purified from cell extracts and found to be significantly less stable than wild-type MADH. Only the T122S MADH mutant was produced at near wild-type levels. Possible roles for these amino acid residues in stabilizing unusual structural features of the MADH beta subunit, protein folding and TTQ biosynthesis are discussed.

The above results indicate that37Cl/35 C1 ratios in saline minerals are higher than those in brine due to chlorine isotopic fractionation during evaporation.37Cl/35 C1 ratios of salt lakes brine reflect the change in hydrochemical condition. The chlorine isotopic composition of saline mineral is associated with environment. Therefore, chlorine isotope may be used to trace evolution of salt lakes and the environment.

SummaryWe describe the use of a unique DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq) to map high-resolution hydroxymethylome of mouse E14 embryonic stem cells. The specificity of AbaSI enables sensitive detection of 5-hydroxymethylcytosine (5hmC) at low-occupancy regions. Bioinformatic analysis suggests 5hmCs in genic regions closely follow the 5mC distribution. 5hmC is generally depleted in CpG islands and only enriched in a small set of repetitive elements. A regularly spaced and oscillating 5hmC pattern was observed at the binding sites of CTCF. 5hmC is enriched at the poised enhancers with the monomethylated histone H3 lysine 4 (H3K4me1) marks, but not at the active enhancers with the acetylated histone H3 lysine 27 (H3K27Ac) marks. Non-CG hydroxymethylation appears to be prevalent in the mitochondrial genome. We propose that some amounts of transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.

The catalytic role of the PtFe cation ensemble presented at the perimeters of the FeO film supported on Pt(111) for low- temperature CO oxidation and the promotion of water on ac- tivity were studied by using DFT calculations. We found that the perimeter sites along the edge of the FeO islands on Pt provided a favorable ensemble that consisted of coordinatively unsaturated ferrous species and nearby Pt atoms for O2 and H2O activation free from CO poison. A dissociative oxygen atom at the PtFe cation ensemble reacts easily with CO ad- sorbed on nearby Pt. The OH group from water dissociation not only facilitates activation of the oxygen molecule, more im- portantly it opens a facile reaction channel for CO oxidation through the formation of the carboxyl intermediate. The pres- ence of the OH group on the FeO film strengthens interfacial interactions between FeO and Pt(111), which would make the FeO film more resistant to further oxidation. The importance of the PtFe cation ensemble and the role of water as a cocata- lyst for low-temperature CO oxidation is highlighted.

The isotopic compositions of boron in Da Qaidam Lake, Quinghai, have been determined by using the technique of thermal ionization mass spectrometry of cesium tetraborate. The brines are enriched in 11B, with an average δ11B of +5.49 ± 2.34%., compared with those of lake sediments and boron minerals produced by the lake. The apparent isotopic fractionation factors between brine and lake sediments as well as boron minerals are 0.988 ± 0.001 and 0.986 ± 0.001, respectively. Source water δ11B values range from −11.72 to + 16.69%., depending on their origin.

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ) dependent enzyme that catalyzes the oxidative deamination of primary amines. Amino acid residues of both the TTQ-bearing beta subunit and the noncatalytic alpha subunit line a substrate channel that leads from the protein surface to the enzyme active site. Phe55 of the alpha subunit is located at the opening of the active site. Conversion of alphaPhe55 to alanine dramatically alters the substrate preference of MADH. The K(m) for methylamine increases from 9 microM to 15 mM. The preferred substrates are now primary amines with chain lengths of at least seven carbons. The K(m) for 1, 10-diaminodecane is 11 microM, compared to 1.2 mM for wild-type MADH. Despite the large variation in K(m) values, k(cat) values are relatively unaffected by the mutation. Molecular modeling of substrates into the crystal structure of the enzyme active site and substrate channel provides an explanation for the dramatic changes in substrate specificity caused by this mutation of a single amino acid residue.

Quinohemoprotein amine dehydrogenase (QHNDH) possesses a cysteine tryptophylquinone (CTQ) prosthetic group that catalyzes the oxidative deamination of primary amines. In addition to CTQ, two heme c cofactors are present in QHNDH that mediate the transfer of the substrate-derived electrons from CTQ to an external electron acceptor. Steady-state kinetic assays yielded relatively small k(cat) values (6 s(-1)), and the rate-limiting step appears to be the interprotein electron transfer from heme in QHNDH to the external electron acceptor. Transient kinetic studies of the CTQ-dependent reduction of heme in QHNDH by amine substrates yielded different rate constants for different substrates (72, 190, and 162 s(-1) for methylamine, butylamine, and benzylamine, respectively). Deuterium kinetic isotope effect (KIE) values of 5.3, 3.9, and 8.5 were observed, respectively, for the reactions of methylamine, butylamine, and benzylamine. These results suggest that the abstraction of a proton from the alpha-methylene group of the substrate, which occurs concomitant with CTQ reduction, is the rate-limiting step in the CTQ-dependent reduction of hemes in QHNDH by these amine substrates. In contrast, the reaction of 2-phenylethylamine with QHNDH does not exhibit a significant KIE ((H)k(3)/(D)k(3) = 1.05) and exhibits a much smaller rate constant of 16 s(-1). This suggests that for 2-phenylethylamine, the rate-limiting step in the single-turnover reaction is either hydrolysis of the imine reaction intermediate from CTQ or product release prior to intraprotein electron transfer. Analysis of the products of the reactions of QHNDH with chiral deuterated 2-phenylethylamines demonstrated that the enzyme abstracts the pro-S proton of the substrate in a highly stereospecific manner. Inspection of the crystal structure of phenylhydrazine-inhibited QHNDH suggests that Asp33(gamma) is the residue that performs the proton abstraction. On the basis of these results, kinetic and chemical reaction mechanisms for QHNDH are proposed and discussed in the context of the crystal structure of the enzyme.

Methylamine dehydrogenase (MADH) possesses an alpha(2)beta(2) structure with each smaller beta subunit possessing a tryptophan tryptophylquinone (TTQ) prosthetic group. Phe55 of the alpha subunit is located where the substrate channel from the enzyme surface opens into the active site. Site-directed mutagenesis of alphaPhe55 has revealed roles for this residue in determining substrate specificity and binding monovalent cations at the active site. It is now shown that the alphaF55A mutation also increases the rate of the true electron transfer (ET) reaction from O-quinol MADH to amicyanin. The reorganization energy associated with the ET reaction is decreased from 2.3 to 1.8 eV. The electronic coupling associated with the ET reaction is decreased from 12 to 3 cm(-1). The crystal structure of alphaF55A MADH in complex with its electron acceptors, amicyanin and cytochrome c-551i, has been determined. Little difference in the overall structure is seen, relative to the native complex; however, there are significant changes in the solvent content of the active site and substrate channel. The crystal structure of alphaF55A MADH has also been determined with phenylhydrazine covalently bound to TTQ in the active site. Phenylhydrazine binding significantly perturbs the orientation of the TTQ rings relative to each other. The ET results are discussed in the context of the new and old crystal structures of the native and mutant enzymes.

Purple Cu A centers are a class of binuclear, mixed-valence copper complexes found in cytochrome c oxidase and nitrous oxide reductase. An engineered Cu A protein was formed by replacing a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Pseudomonas aeruginosa azurin with the corresponding portion of sequence from the Cu A center of cytochrome c oxidase from Paracoccus denitrificans [Proc. Natl. Acad. Sci. USA 93 (1996) 461]. Oxidation–reduction midpoint potential ( E m ) values of the Cu A azurin of +399±10 and +380±2 mV, respectively, were determined by cyclic voltammetry and spectrochemical titration. An n value of one was obtained, indicating that the redox reaction is cycling between the mixed valence and the fully reduced states. Whereas the E m value of native azurin is pH dependent, the E m value of Cu A azurin is not, as expected for the Cu A center. Similarities and differences in the redox properties are discussed in terms of the known crystal structures of Cu A centers in cytochrome c oxidase and Cu A azurin.

Methylamine dehydrogenase (MADH) may be immobilized in a polypyrrole (PPy) film on an electrode surface and used as an amperometric sensor for the determination of histamine. Using site-directed mutagenesis, phenylalanine 55 on the alpha subunit of MADH was converted to alanine. This alphaF55A MADH exhibits a 400-fold lower Km value for histamine than does native MADH when assayed in solution. An alphaF55A MADH-PPy sensor was constructed, and its properties were compared to that of the native MADH-PPy sensor. The alphaF55A MADH immobilized on the electrode exhibited Michaelis-Menten behavior in response to varied concentrations of histamine with an approximately 3-fold lower Km value than that exhibited by the immobilized native MADH. The detection limit for the native MADH-PPy sensor was approximately 20 microM while the alphaF55A MADH-PPy sensor exhibited a detection limit of approximately 5 microM, a 4-fold increase compared to the native MADH-PPy sensor. This work highlights the potential value of using site-directed mutagenesis to engineer enzymes to alter and improve biosensor performance.

Publisher Summary This chapter describes two different procedures that use the combination of lysozyme and osmotic shock for the fractionation of bacterial cells and selective release of periplasmic proteins. Procedure 1 has been used in our laboratory to fractionate E. coli, Paracoccus denitrificans, and Rhodobacter sphaeroides . Procedure 2 was used with Alcaligenesfaecalis , which could not be fractionated by procedure 1. The development of procedure 2 required significant modification of the generally applicable technique that was developed originally for E. coli. It is likely that these procedures will require further modificaiton for optimal effectiveness with other bacteria. As such, a general protocol is also described for optimizing conditions for the fractionation of other bacterial cells that are not efficiently fractionated by either of these two procedures.

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ)-dependent enzyme that catalyzes the oxidative deamination of primary amines. Monovalent cations are known to affect the spectral properties of MADH and to influence the rate of the gated electron transfer (ET) reaction from substrate-reduced MADH to amicyanin. Two putative monovalent cation binding sites in MADH have been identified by X-ray crystallography [Labesse, G., Ferrari, D., Chen, Z.-W., Rossi, G.-L., Kuusk, V., McIntire, W. S., and Mathews, F. S. (1998) J. Biol. Chem. 273, 25703-25712]. One requires cation-pi interactions involving residue alpha Phe55. An alpha F55A mutation differentially affects these two monovalent cation-dependent phenomena. The apparent K(d) associated with spectral perturbations increases 10-fold. The apparent K(d) associated with enhancement of the gated ET reaction becomes too small to measure, indicating that either it has decreased more than 1000-fold or the mutation has caused a conformational change that eliminates the requirement for the cation for the gated ET. These results show that of the two binding sites revealed in the structure, cation binding to the distal site, which is stabilized by the cation-pi interactions, is responsible for the spectral perturbations. Cation binding to the proximal site, which is stabilized by several oxygen ligands, is responsible for the enhancement of the rate of gated ET. Another site-directed mutant, alpha F55E MADH, exhibited cation binding properties that were the same as those of the native enzyme, indicating that interactions with the carboxylate of Glu can effectively replace the cation-pi interactions with Phe in stabilizing monovalent cation binding to the distal site.

Composition-controllable ternary CdSe(x)S(1-x) quantum dots (QDs) with multiple emission colors were obtained via a hot-injection-like method at a relatively low injection temperature (230 ° C) in octadecene. Then highly fluorescent CdSe(x)S(1-x)/ZnS core/shell (CS) QDs were synthesized by a facile single-molecular precursor approach. The fluorescent quantum yield of the resulting green (λ(em) = 523 nm), yellow (λ(em) = 565 nm) and red (λ(em) = 621 nm) emission of CS QDs in toluene reached up to 85%, 55% and 39%, respectively. Moreover, a QDs white light-emitting diode (QDs-WLED) was fabricated by hybridizing green-, yellow- and red-emitting CdSe(x)S(1-x)/ZnS CS QDs/epoxy composites on a blue InGaN chip. The resulting four-band RYGB QDs-WLED showed good performance with CIE-1931 coordinates of (0.4137, 0.3955), an R(a) of 81, and a T(c) of 3360 K at 30 mA, which indicated the combination of multiple-color QDs with high fluorescence QYs in LEDs as a promising approach to obtain warm WLEDs with good color rendering.

[Image: see text] Photoluminescent compounds can undergo various structural changes upon interaction with light. When these changes manifest themselves in the excited state, the resulting emitters can obtain a sensory function. In this work, we designed coordination compounds that can vary their emission color in response to thermal and mechanical stimuli. When embedded in a polymer matrix, Cu–NHC sensors act as mechanophores, and their color-based response can readily describe mechanical stress and phase transition phenomena. A strong practical advantage of new mechanophores over previous generations of organometallic stress sensors stems from their reliance on emission color variations that are easy to detect. In a broad context, our work implies that emission color variations that we often view as thermally governed can also be triggered mechanically and used to generate sensory information.

Cyclopropylamine is a mechanism-based inhibitor of the quinoprotein methylamine dehydrogenase (MADH) from Paracoccus denitrificans. The resulting inactivation is accompanied by the formation of a covalent cross-link between the alpha and beta subunits of MADH. The results of site-directed mutagenesis studies indicate that Phe55 on the alpha subunit is required for this process. No cross-linking is seen with alphaF55A or alphaF55I MADH mutants. In contrast, with alphaF55E MADH cross-linking of subunits is observed. These results suggest a novel mechanistic role for a phenylalanine residue and the possible importance of protein dynamics in this enzyme mechanism.