Visualization of acetylcholine distribution in central nervous system tissue sections by tandem imaging mass spectrometry

Metabolite distribution imaging via imaging mass spectrometry (IMS) is an increasingly utilized tool in the field of neurochemistry. As most previous IMS studies analyzed the relative abundances of larger metabolite species, it is important to expand its application to smaller molecules, such as neurotransmitters. This study aimed to develop an IMS application to visualize neurotransmitter distribution in central nervous system tissue sections. Here, we raise two technical problems that must be resolved to achieve neurotransmitter imaging: (1) the lower concentrations of bioactive molecules, compared with those of membrane lipids, require higher sensitivity and/or signal-to-noise (S/N) ratios in signal detection, and (2) the molecular turnover of the neurotransmitters is rapid; thus, tissue preparation procedures should be performed carefully to minimize postmortem changes. We first evaluated intrinsic sensitivity and matrix interference using Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry (MS) to detect six neurotransmitters and chose acetylcholine (ACh) as a model for study. Next, we examined both single MS imaging and MS/MS imaging for ACh and found that via an ion transition from m/z 146 to m/z 87 in MS/MS imaging, ACh could be visualized with a high S/N ratio. Furthermore, we found that in situ freezing method of brain samples improved IMS data quality in terms of the number of effective pixels and the image contrast (i.e., the sensitivity and dynamic range). Therefore, by addressing the aforementioned problems, we demonstrated the tissue distribution of ACh, the most suitable molecular specimen for positive ion detection by IMS, to reveal its localization in central nervous system tissues.     

A Novel endo-1,4-β-Mannanase from <Emphasis Type="Italic">Bispora antennata</Emphasis> with Good Adaptation and Stability over a Broad pH Range

An endo-β-1,4-mannanase encoding gene, man5, was cloned from Bispora antennata CBS 126.38, which was isolated from a beech stump. The cDNA of man5 consists of 1,299 base pairs and encodes a 432-amino-acid protein with a theoretical molecular mass of 46.6 kDa. Deduced MAN5 exhibited the highest amino acid sequence identity of 58% to a β-mannanase of glycoside hydrolase family 5 from Aspergillus aculeatus. Recombinant MAN5 was expressed in Pichia pastoris and purified to electrophoretic homogeneity. The specific activity of the final preparation towards locust bean gum was 289 U mg⁻¹. MAN5 showed optimal activity at pH 6.0 and 70 °C and had good adaptation and stability over a broad range of pH values. The enzyme showed more than 60% of peak activity at pH 3.0–8.0 and retained more than 80% of activity after incubation at 37 °C for 1 h in both acid and alkaline conditions (pH 4.0–11.0). The K _m and V _max values were 1.33 mg ml⁻¹ and 444 μmol min⁻¹ mg⁻¹ and 1.17 mg ml⁻¹ and 196 μmol min⁻¹ mg⁻¹ for locust bean gum and konjac flour, respectively. Of all tested metal ions and chemical reagents, Co²⁺, Ni²⁺, and β-mercaptoethanol enhanced the enzyme activity at 1 mM, whereas other chemicals had no effect on or partially inhibited the enzyme activity. MAN5 was highly resistant to acidic and neutral proteases (trypsin, α-chymotrypsin, collagenase, subtilisin A, and proteinase K). By virtue of the favorable properties of MAN5, it is possible to apply this enzyme in the paper and food industries.     

Application of affinity selection-mass spectrometry assays to purification and affinity-based screening of the chemokine receptor CXCR4

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.     

Formation Enthalpy Calculation of Oxygen Vacancy Defect in Doped Lithium Niobate Crystals

The relationship between temperature and oxygen vacancy concentration is deduced in this paper. Based on the data of thermal weight-loss experiment, the formation enthalpies of congruent and several doped LN crystals have been calculated. It was found that the formation enthalpy of oxygen vacancies can be decreased evidently by doping valence-changeable ions. The experimental results were discussed and a new reduction process of the photorefractive LN crystal at a relatively low temperature was proposed, and the reduced crystals showed a good effect in practical use.     

Lewis acid catalyzed cyclization of glycals/2-deoxy-D-ribose with arylamines: additional findings on product structure and reaction diastereoselectivity

The cyclization reactions of arylamines with 2-deoxy-D-ribose or glycals were reinvestigated in the current report. In the montmorillonite KSF- or InCl(3)-initiated reactions of 2-deoxy-D-ribose with arylamines, a pair of diastereomeric tetrahydro-2H-pyran-fused tetrahydroquinolines was obtained in a nearly 1:1 ratio where the structure of one diastereomer was incorrectly assigned in the literature. Meanwhile, the diastereoselectivity in InBr(3)-catalyzed cyclization of glycals with arylamines was also incorrectly reported previously. It was found that high diastereomeric selectivity was achieved only when a C5-substituted glycal was used; otherwise, a pair of diastereomers was obtained in moderate yield with 1:1 diastereomeric ratio. Furthermore, tetrahydrofuran-fused tetrahydroquinolines 5b and 5b' were also prepared successfully by using TBDPS-protected ribose as the glycal precursor and montmorillonite KSF as the activator.     

Microbial transformation of deoxyandrographolide by Alternaria alternata AS 3.4578

Biotransformation of deoxyandrographolide (1) by Alternaria alternata AS 3.4578 gave five derivatives identified by spectral methods including 2D NMR as the known dehydroandrographolide (2) and 9beta-hydroxy-dehydroandrographolide (3) and the new compounds 9beta-hydroxy-deoxyandrographolide (4), 3alpha,17,19-trihydroxy-8,13-ent-labdadien-15,16-olide (5) and 3-oxo-9beta-hydroxy-deoxyandrographolide (6).     

Ru-catalyzed asymmetric hydrogenation of γ-heteroatom substituted β-keto esters

A series of enantiomerically pure γ-heteroatom substituted β-hydroxy esters were synthesized with high enantioselectivities (up to 99.1% ee) by hydrogenation of γ-heteroatom substituted β-keto esters in the presence of Ru-(S)-SunPhos catalyst. These asymmetric hydrogenations provide key building blocks for a variety of naturally occurring and biologically active compounds.     

One-pot microwave synthesis of water-dispersible, ultraphoto- and pH-stable, and highly fluorescent silicon quantum dots

Fluorescent silicon quantum dots (SiQDs) are facilely prepared via one-pot microwave-assisted synthesis. The as-prepared SiQDs feature excellent aqueous dispersibility, robust photo- and pH-stability, strong fluorescence, and favorable biocompatibility. Experiments show the SiQDs are superbly suitable for long-term immunofluorescent cellular imaging. Our results provide a new and invaluable methodology for large-scale synthesis of high-quality SiQDs, which are promising for various optoelectronic and biological applications.     

Microbial desulfurization of waste latex rubber with Alicyclobacillus sp.

A microbe with desulfurizing capability, Alicyclobacillus sp., was selected to recycle waste latex rubber (WLR). The growth characteristics of the microorganism and the technical conditions in the co-culture desulfurization process were studied. The desulfurization effect of Alicyclobacillus sp. on the WLR was characterized, and the mechanism for the microbial desulfurization of WLR was tentatively explored. The results showed that adding 5% (w/v) WLR into medium had little effect on the growth of Alicyclobacillus sp. The surfactant polysorbate 80 (Tween 80) had a toxic effect on Alicyclobacillus sp., but the growth of the microbe was vigorous if the proper technique was used: the mixing of WLR with Tween 80, followed by the addition of the mixture into the culture media. With the increase of desulfurization time, the swelling value of desulfurizated waste latex rubber (DWLR) increased, but the crosslink density decreased. After co-culture desulfurization for 8–10 days, a DWLR with good desulfurization effect was obtained. The mechanical properties of natural rubber (NR)/DWLR composite improved significantly over those of NR/WLR composite. XPS and FTIR results revealed that Alicyclobacillus sp. could break the crosslinked sulfur bonds and oxidize them to sulfones groups. The increase of O element content on the surface of DWLR was confirmed by water contact angle measurements. The relationship between the crosslink density and sol fraction of DWLR with different desulfurization times agreed with the Horikx equation, an indication that the microorganisms could break the crosslinked sulfur bonds on the surface of WLR, but leaving the main chains intact.