Stationary phases for the enrichment of glycoproteins and glycopeptides.

The analysis of protein glycosylation is important for biomedical and biopharmaceutical research. Recent advances in LC-MS analysis have enabled the identification of glycosylation sites, the characterisation of glycan structures and the identification and quantification of glycoproteins and glycopeptides. However, this type of analysis remains challenging due to the low abundance of glycopeptides in complex protein digests, the microheterogeneity at glycosylation sites, ion suppression effects and the competition for ionisation by co-eluting peptides. Specific sample preparation is necessary for comprehensive and site-specific glycosylation analyses using MS. Therefore, researchers continue to pursue new columns to broaden their applications. The current manuscript covers recent literature published from 2008 to 2013. The stationary phases containing various chemical bonding methods or ligands immobilisation strategies on solid supports that selectively enrich N-linked or sialylated N-glycopeptides are categorised with either physical or chemical modes of binding. These categories include lectin affinity, hydrophilic interactions, boronate affinity, titanium dioxide affinity, hydrazide chemistry and other separation techniques. This review should aid in better understanding the syntheses and physicochemical properties of each type of stationary phases for enriching glycoproteins and glycopeptides.

Protein enrichment and digestion improvement of napiergrass and pangolagrass with solid-state fermentation.

BACKGROUND AND PURPOSE: Napiergrass (Pennisetum purpureum Schumacher) and pangolagrass (Digitaria decumbens Stent) are two major forage grasses for cow feeding. They possess high yields and high regeneration properties. Inoculation of cellulolytic microbes on herbage could enhance the protein content of herbage and promote digestibility in chickens. METHODS: Cellulolytic microbes were isolated from various sources and cultivated on napiergrass and pangolagrass with solid-state fermentation for protein enrichment and in vitro digestion improvement.The fermented napiergrass and pangolagrass were used as the main protein source in chicken diets to assess the feasibility for non-ruminants feed. RESULTS: After a 42-day fermentation period, napiergrass showed higher protein contents (13.4-13.9%) than those of pangolagrass(11.1-11.7%). The in vitro digestibility of pangolagrass increased from 5.29% to 20.4%, whereas that of napiergrass increased from 5.29% to 19.0%. The average feed conversion efficiencies of chickens were close to the traditional fodder using corn as the main ingredient. CONCLUSION: Inoculation of appropriate cellulolytic microbes to enrich protein content and improve in vitro digestibility of herbage with solid-state fermentation for chicken feed is the prospective technique for agriculture, animal husbandry, and substantial management.

Zirconia nanoparticles-coated column for the capillary electrochromatographic separation of iron-binding- and phosphorylated-proteins.

A ZrO(2) nanoparticles (ZrO(2)NPs)-coated column was prepared through a sol-gel process using zirconium(iv) oxychloride, which reacted with silanol groups of the fused-silica capillary. The condensation reaction was carried out at 350 °C for 8 h. Electroosmotic flow (EOF) measurements and scanning electron microscopy (SEM) images were used to characterize the ZrO(2)NPs fabricated on the inner wall of the capillary. Below the pI value (pH 5-6), cathodic EOF elucidated that the phosphate buffer adsorbs tightly on the zirconia surface, resulting in a negatively charged surface. In this work, iron-binding proteins, phosphorylated proteins and glycoproteins were selected as the model compounds. The effects of pH, concentration, buffer type and the organic modifier were studied to optimize the separation efficiency. Iron-binding proteins exhibited a retention time for myoglobin (Mb) < hemoglobin (Hb), which corresponded to the binding constants for ZrO(2)NPs. The α- and ß-subunit of Hb could be separated in borate buffer (20 mM, pH 9.0) with MeOH (20%, v/v). Greater affinity of α-casein and bovine serum albumin (BSA) for the stationary phase as the pH decreased was found by comparison with that of conalbumin (ConA) and transferrin (Tf). Interestingly, 14 peaks for glycoisoforms of ovalbumin (OVA) were observed using borate buffer (40 mM, pH 9.0). The established method was also applied to the determination of analytes in the egg whites of chicken and duck eggs.

Microbial communities and bacterial diversity of spruce, hemlock and grassland soils of Tatachia Forest, Taiwan.

To evaluate the bacterial diversity of Tatachia Forest soils, 16S rDNA clone libraries of the spruce, hemlock and grassland soils were constructed. Further, the influence of physicochemical and biological properties of soil on microbial ecology, pH, moisture content, microbial population and biomass were also analyzed. The soil pH increased with the increasing of soil depth; whereas the microbial population, biomass, moisture content, total organic carbon and total nitrogen were reverse. Microbial populations were the highest in the summer season which also correlated with the highest moisture content. The phylogenetic analyses revealed that the clones from nine 16S rDNA clone libraries represented Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Gemmatimonadetes, Planctomycetes, Verrucomicrobia, candidate division TG1 and candidate division TM7. Members of Proteobacteria, Acidobacteria and Actinobacteria constituted 42.2, 35.1 and 7.8 % of the clone libraries, respectively; whereas the remaining bacterial divisions each comprised <3 %. The spruce site had the highest bacterial diversity among the tested sites, followed by the hemlock sites and the grassland sites with the least. The bacterial community is the more diverse in the organic layer than that in deeper horizons. Further, bacterial diversity through the gradient horizons was different, indicating that the bacterial diversity in the deeper horizons is not simply the diluted analogs of the surface soils and some microbes dominate only in the deeper horizons.

NaDDBS as a dispersion agent for multiwalled carbon nanotubes in capillary EKC separation of nucleotides.

A novel pseudostationary phase (PSP) of multiwalled carbon nanotubes (MWCNTs) dispersed with sodium dodecylbenzenesulfonate (NaDDBS) was used for the EKC separation of nucleotides. NaDDBS has a long hydrophobic chain and a benzylsulfonate group. It suspends more MWCNTs (about 100-fold) than SDS, and the π-π interaction between the benzene ring of NaDDBS and MWCNTs prolongs the slurry suspension time. Using NaDDBS as a surfactant can reduce the required amount of MWCNTs and decrease the baseline noise. To produce a stable suspension, the optimum ratio (w/w) of MWCNTs to NaDDBS was investigated with turbidimetry. In this context, several parameters affecting EKC separation were studied, including buffer pH, composition, concentration, and the organic modifier. Use of NaDDBS (8 mg/L)/MWCNTs (0.8 mg/L) as the PSP in a phosphate buffer (30 mM, pH 8) yielded complete resolution of seven geometric isomers of a nucleoside monophosphate. In stacking mode, with 10% MeOH in the sample plug, the mixture of nucleoside mono-, di-, and tri-phosphates was satisfactorily separated in phosphate buffer (50 mM, pH 9). The results indicate that nucleotides with bases containing more electron-withdrawing groups interact more strongly with MWCNTs. The system has been used to separate oligonucleotides, and to analyze nucleotides in a complex matrix sample.

Formulation and characterization of the microencapsulated entomopathogenic fungus Metarhizium anisopliae MA126.

Bioinsecticides are expected to be used for controlling major species of aphids. The present study explored a liquid phase coating technique for the formulation of microencapsulated conidia of the entomopathogenic fungus Metarhizium anisopliae MA126. Various parameters for microencapsulation were investigated. The biopolymers sodium alginate, hydroxypropyl methyl cellulose (HPMC) and chitosan were tested as coating materials. Calcium chloride was used as the cross-linking agent for converting soluble sodium alginate into an insoluble form. To improve the efficiency of microencapsulation, the additives of HPMC, dextrin, chitosan or HPMC/chitosan in various ratios (1 : 1, 1 : 3 and 3 : 1) were used as the coating materials. The particle size of a bare microcapsule was less than 30 microm. Larger size microcapsules were produced using vortex method by comparison with that using homogenization method. The latter method, however, was easy to scale up. The effect of coating materials on the morphology and encapsulation efficiency of the microcapsules was also studied. The best encapsulation efficiency (78%) was using HPMC as the additive of the coating material. The next was dextrin (70%). By measuring the germination rate, the results showed that the activity was approximately 80% of the initial after 6 months of storage at 4 degrees C, while that of the bare conidia was less than 50% stored in identical conditions.

Titanium dioxide nanoparticles-coated column for capillary electrochromatographic separation of oligopeptides.

A novel column made through the condensation reaction of TiO2 nanoparticles (TiO2 NPs) with silanol groups of the fused-silica capillary is described. EOF measurements under various buffer constitutions were used to monitor the completion of reactions. The results indicated that the EOF was dependent on the interactions between buffers and the bonded TiO2 NPs. With formate/Tris buffer, EOF reversal at pH below 5 and cathodic EOF at pH above 5 were indicated. The pI of the bonded TiO2 NPs was found at approximately ph 5. Only cathodic EOF was illustrated by substituting the mobile phase with either glutamate or phosphate buffer. It was elucidated that both glutamate and phosphate buffer yield a negative charge layer on the surface of TiO2 NPs attributable to the formation of a titanium complex. The CEC performance of the column was tested with angiotensin-type oligopeptides. Some parameters that would affect the retention behavior were investigated. The interactions between the bonded phases and the analytes were explicated by epitomized acid-base functional groups of the oligopepetides and the speciation of the surface oxide in different pH ranges. The average separation efficiencies of 3.1 x 10(4) plates/m is readily achieved with a column of 70 cm (50 cm) x 50 mum ID under an applied voltage of 15 kV, phosphate buffer (pH 6.0, 40 mM), and UV detection at 214 nm.

Morphology and eicosapentaenoic acid production by Monodus subterraneus UTEX 151.

The optimum culture conditions of Monodus subterraneus UTEX 151 for eicosapentaenoic acid (EPA) production were at 25 degrees C, initial pH 7.0 and continuous illumination at 10 Klux light. The gas chromatographic data indicated that the fatty acid composition 20:5n-3 was predominant, at approximately 31%. In addition, 16:1, was the major monounsaturated fatty acid, while the 22:6n-3 was absent. Cultivation in 20 mM sodium acetate slightly enhanced the content of EPA from 31% up to 34% of the total fatty acids. Light micrographs of M. subterraneus UTEX 151 showed that the cell shape changed from spherical to ellipsoidal, as cell maturity was achieved. When the cells were in stationary phase, these became elongated spindle-shaped ellipses. TEM data demonstrated lipid body formation occurring in the thylakoid space of the chloroplast. Lipid body size varied with growth phase stage and they finally formed round clusters. The ultrastructure of M. subterraneus UTEX 151 vegetative cells of early and stationary growth stage showed the presence of chloroplasts, with many lipid bodies in the cell. Starch granules and lipid bodies occupied an--equal volume in the cell.

Photoassisted synthesis of CdSe and core-shell CdSe/CdS quantum dots.

This paper describes the synthesis of core-shell CdSe/CdS quantum dots (QDs) in aqueous solution by a simple photoassisted method. CdSe was prepared from cadmium nitrate and 1,1-dimethylselenourea precursors under illumination for up to 3 h using a pulsed Nd:YAG laser at 532 nm. The effects that the temperature and the laser irradiation process have on the synthesis of CdSe were monitored by a series of experiments using the precursors at a Cd:Se concentration ratio of 4. Upon increasing the temperature (80-140 degrees C), the size of the CdSe QDs increases and the time required for reaching a maximum photoluminescence (PL) is shortened. Although the as-prepared CdSe QDs possess greater quantum yields (up to 0.072%) compared to those obtained by microwave heating (0.016%), they still fluoresce only weakly. After passivation of CdSe (prepared at 80 degrees C) by CdS using thioacetamide as the S source (Se:S concentration ratio of 1) at 80 degrees C for 24 h, the quantum yield of the core-shell CdSe/CdS QDs at 603 nm is 2.4%. Under UV irradiation of CdSe/CdS for 24 h using a 100-W Hg-Xe lamp, the maximum quantum yield of the stable QDs is 60% at 589 nm. A small bandwidth (W1/2 < 35 nm) indicates the narrow size distribution of the as-prepared core-shell CdSe/CdS QDs. This simple photoassisted method also allows the preparation of differently sized (3.7-6.3-nm diameters) core-shell CdSe/CdS QDs that emit in a wide range (from green to red) when excited at 480 nm.