Herein, we report the first characterization of a novel galactokinase from Meiothermus taiwanensis sp. nov. WR-220 (MtGalK), which is overexpressed in E. coli and exhibits a k cat /K m value of 168.47 mM À1 s À1 toward Gal at 75 8C. The thermophilic MtGalK shows specific substrate recognition toward the Gal configuration with limited tolerance for modifications at the C-2 position to form products such as GalN-1-P, GalN 3 -1-P, and GalNAc-1-P. Due to its unique thermostability toward elevated reaction temperatures, MtGalK served as an ideal biocatalyst in the synthesis of useful sugar-1-phosphates and was further combined with glucose-1-phosphate thymidylyltransferase (RmlA) and a-1,4-galactosyltransferase (LgtC) to achieve the efficient preparative-scale synthesis of globotriose analogs (Gb3) using a one-pot three-enzyme system. In combination with a chemical synthetic strategy, a carbohydrate antigen from breast cancer stem cells, stage specific embryonic antigen-3 (SSEA-3) pentasaccharide, was synthesized from Gb3 in ten steps with a 23% overall yield. This flexible chemoenzymatic strategy for the synthesis of SSEA-3 also allowed us to further expand the inventory of valuable natural and non-natural glycans.

Magnetofection has received increasing attention for its great potential on gene therapy. To promote its clinical therapeutic applications, development of safe and effective magnetic nanocarriers is in high demand. Herein, we present a redox-sensitive polymer/metal nanocomplex system (PSPIO) for efficient magnetofection and magnet resonance imaging (MRI) on cancer cells. PSPIO was prepared by modifying SPIO with redox-sensitive polyethylenimine (SSPEI) via a ligand exchange process. PSPIO could efficiently condense plasmid DNA (pDNA) into nanoparticles, which exhibited several favorable properties for gene delivery, including protection of nucleic acids from enzymatic degradation, stable colloids in serum, and redox-responsive pDNA release. As a potential MR imaging agent, PSPIO displayed good magnetization (28.3 emu/g) and dose-dependent T2-weighted imaging contrast (R2 = 291.1 s(-1) mM(-1)) in vitro. The use of redox-sensitive SSPEI polymer contributed to much lower cytotoxicity of PSPIO compared to nondegradable bPEI25k. In vitro transfection efficiency of PSPIO was significantly enhanced under an external magnetic field. In the presence of serum, PSPIO exhibited higher transgene expression than SSPEI or bPEI25k polymer on mouse glioma (ALTS1C1) or human prostate cancer (PC3) cell lines. Taken together, it is demonstrated that PSPIO possess great potential for cancer gene therapy and molecular imaging.

The design of high-affinity lectin ligands is critical for enhancing the inherently weak binding affinities of monomeric carbohydrates to their binding proteins. Glyco-gold nanoparticles (glyco-AuNPs) are promising multivalent glycan displays that can confer significantly improved functional affinity of glyco-AuNPs to proteins. Here, AuNPs are functionalized with several different carbohydrates to profile lectin affinities. We demonstrate that AuNPs functionalized with mixed thiolated ligands comprising glycan (70 mol %) and an amphiphilic linker (30 mol %) provide long-term stability in solutions containing high concentrations of salts and proteins, with no evidence of nonspecific protein adsorption. These highly stable glyco-AuNPs enable the detection of model plant lectins such as Concanavalin A, wheat germ agglutinin, and Ricinus communis Agglutinin 120, at subnanomolar and low picomolar levels through UV/Vis spectrophotometry and dynamic light scattering, respectively. Moreover, we develop in situ glyco-AuNPs-based agglutination on an oriented immobilized antibody microarray, which permits highly sensitive lectin sensing with the naked eye. In addition, this microarray is capable of detecting lectins presented individually, in other environmental settings, or in a mixture of samples. These results indicate that glyconanoparticles represent a versatile and highly sensitive method for detecting and probing the binding of glycan to proteins, with significant implications for the construction of a variety of platforms for the development of glyconanoparticle-based biosensors.

The trifluoroacetic acid adduct of tetranuclear zinc cluster Zn 4 (OCOCF 3 ) 6 O catalysis in per-O-acetylation and de-O-acetylation of carbohydrates at 70 C can be tuned by adjusting the reaction medium. Per-Oacetylation of hexopyranoses with a near stoichiometric amount of acetic anhydride in toluene resulted in the exclusive formation of pyranosyl products as an anomeric mixture, whereas de-O-acetylation of acetates occurred in methanol in high yields. In the latter, methanol acts as both nucleophile and solvent, and the reaction conditions were compatible to acid-and base-sensitive groups and amino acid derivatives.a Yield of crude product with purity higher than 97% according to 1 H NMR spectroscopy. b Yields aer 30 h under the loading of 5 mol% 1. Partial O-acetylated derivative (32%) was also formed.This journal is

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We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells.

The distortion-product otoacoustic emission (DPOAE) is a backward propagating wave generated inside the cochlea during the wave amplification process. The DPOAE signal can be detected rapidly under relatively noisy conditions. In recent years, the earphone industry demonstrated interest in adopting DPOAE as an add-on feature to make their product “intelligent” of inner-ear status. However, a technical challenge remains to be tackled—the loudspeaker in an earphone generates its own cubic distortion at the same frequency as DPOAE. Unfortunately, the intensity of loudspeaker distortion is typically comparable to that of the DPOAE, if not higher. In this research, we propose two strategies, namely compensation and cancellation, to enable DPOAE measurement with a single loudspeaker. The compensation strategy exploits the part of the growth function of the loudspeaker distortion which is almost linear, and thus suppresses the distortion it generates while retaining a larger portion of DPOAE in the residual signal. The cancellation strategy utilizes a one-dimensional Volterra filter to remove the cubic distortion from the loudspeaker. Testing on normal-hearing ears shows that the compensation strategy improved the DPOAE-to-interference ratio by approximately 7 dB, resulting in a cross-correlation of 0.62 between the residual DPOAE level and the true DPOAE level. Meanwhile, the cancellation strategy directly recovered both the magnitude and the phase of DPOAE, reducing the magnitude estimation error from 15.5 dB to 3.9 dB in the mean-square sense. These pilot results suggest that the cancellation strategy may be suitable for further testing with more subjects.