19514569
Determination of tetracyclines in food samples by molecularly imprinted monolithic column coupling with high performance liquid chromatography.
A novel solid phase extraction (SPE) method for determination of tetracyclines (TCs) in milk and honey samples by molecularly imprinted monolithic column was developed. Using tetracycline (TC) as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, methanol as the solvent, cyclohexanol and dodecanol as the mixed porogenic solvents, a TC imprinted monolithic column was prepared by in situ molecular imprinting technique for the first time, and the optimal synthesis conditions and the selectivity of TC imprinted monolithic column were investigated. The interfering substances in food samples and TCs can be separated successfully on imprinted column. Molecularly imprinted solid phase extraction (MISPE) coupling with C18 column was used to determinate the TCs in milk and honey. The recoveries of this method for six tetracyclines antibiotics such as tetracycline (TC), oxytetracycline (OTC), minocycline (MINO), chlortetracycline (CTC), metacycline (MTC) and doxycycline (DTC) were investigated, and high recoveries of 73.3-90.6% from milk samples and 62.6-82.3% from honey samples were obtained. A method for determination of TCs at low concentration level in milk and honey samples was successfully developed by using the monolithic column as the precolumn for solid phase extraction of six TCs compounds.
8448000
In-column “click” preparation of hydrophobic organic monolithic stationary phases for protein separation
AbstractTwo types of macroporous organic polymer monoliths based on glycidyl methacrylate (GMA), 4-vinylbenzyl chloride (VBC) and divinylbenzene (DVB) were prepared inside stainless-steel tubes. Azide functionalities were firstly introduced on the surfaces of poly(GMA-co-DVB) and poly(VBC-co-DVB) monoliths to provide reactive sites for click chemistry. With the application of copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition, an in-column click-modification approach for covalent attachment of long alkyl chains onto polymer monoliths was developed. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by the scanning electron microscopy, mercury intrusion porosimeter, Fourier transform infrared spectroscopy, and elemental analyses, respectively. The chromatographic performances of the “clicked” stationary phases were demonstrated with the high separation efficiency for a variety of proteins within 4 min.
 FigureWith the application of copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition (CuAAC), an in-column click-modification approach for covalent attachment of long alkyl chains onto polymer monoliths based on poly(GMA-co-DVB)/poly(VBC-co-DVB) was developed. The “clicked” stationary phases were demonstrated with the high separation efficiency for a variety of proteins within 4 min
24997000
Preparation of phenylboronic acid functionalized cation-exchange monolithic columns for protein separation and refolding.
In this study, we described a simple and effective modification procedure to prepare poly (methacrylate-co-ethylene glycol dimethacrylate) monolithic columns functionalized with 3-aminophenylboronic acid. The column morphology, pore size and specific surface area of the fabricated monolith were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, thermogravimetric analysis, and mercury intrusion porosimeter, respectively. The frontal analysis was carried out for dynamic loading capacity of the model protein on the modified column. The chromatographic performance of the cation-exchange monolith was evaluated through separating a mixture of five proteins such as lysozyme, cytochrome c, ribonuclease A, trypsin and bovine serum albumin and one-step purification of lysozyme from egg whites, and the expected results were obtained. In addition, the functionalized column was used to refold ribonuclease A and cytochrome c, and this procedure was monitored by circular dichroism and fluorescence spectroscopy. Compared with the conventional dilution refolding method, the ion-exchange chromatography refolding method developed here is more effective for specific bioactivity recovery.
24110389
[Isolation of a metsulfuron-methyl-resistant bacteria and cloning and expression of the acetohydroxyacid synthase genes ilvlH].
UNLABELLED
Acetohydroxyacid synthase (EC 4.1.3.18) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine in plant, fungi and bacteria, and also is target of the sulfonylurea, imidazolinone, triazolopyrimidine and other acetohydroxyacid synthase inhibitor herbicides.


OBJECTIVE
The purpose of this study is to get the resistant gene, prepare a functional bacteria with acetohydroxyacid synthase, and investigate the relationship between the mutation sites of the acetohydroxyacid synthase and the herbicides-resistant.


METHODS
A metsulfuron-methyl-resistant bacterium Lm10 was isolated from metsulfuron-methyl contaminated soil. Acetohydroxyacid synthase genes ilvIH was amplified from the genome DNA of strains Lm10 by PCR. The ilvI and ilvH were cloned into the bacterial expression vector pET29a(+) respectively.


RESULTS
Strain Lm10 was identified preliminarily as Pseudomonas sp.. It can endure 14000 micromol/L metsulfuron-methyl and showed cross resistance to diffenent acetohydroxyacid synthase inhibitor herbicids, such as chlorsulfuron, imazethapyr, flumetsulam and penoxsulam. The alignment result of the ilvIH amino acid sequence showed that ilvI of strains Lm10 differed from that of strain KT2440 by 6 sites, While the ilvH of the two strains were the same. The gene ilvI and ilvH were functional expressed in the Escherichia coli strain BL21(DE3) respectively. The expressed production pET-I displayed acetohydroxyacid synthase activity and showed resistance to high concentration of metsulfuron-methyl.


CONCLUSION
The ilvI displayed acetohydroxyacid synthase activity. The 6 different sites in ilvI of strains Lml0 probably led to herbicids resistance.
31652498
Performance optimization in electric field gradient focusing.
Electric field gradient focusing (EFGF) is a technique used to simultaneously separate and concentrate biomacromolecules, such as proteins, based on the opposing forces of an electric field gradient and a hydrodynamic flow. Recently, we reported EFGF devices fabricated completely from copolymers functionalized with poly(ethylene glycol), which display excellent resistance to protein adsorption. However, the previous devices did not provide the predicted linear electric field gradient and stable current. To improve performance, Tris-HCl buffer that was previously doped in the hydrogel was replaced with a phosphate buffer containing a salt (i.e., potassium chloride, KCl) with high mobility ions. The new devices exhibited stable current, good reproducibility, and a linear electric field distribution in agreement with the shaped gradient region design due to improved ion transport in the hydrogel. The field gradient was calculated based on theory to be approximately 5.76 V/cm(2) for R-phycoerythrin when the applied voltage was 500 V. The effect of EFGF separation channel dimensions was also investigated; a narrower focused band was achieved in a smaller diameter channel. The relationship between the bandwidth and channel diameter is consistent with theory. Three model proteins were resolved in an EFGF channel of this design. The improved device demonstrated 14,000-fold concentration of a protein sample (from 2 ng/mL to 27 microg/mL).
24839847
Preparation and evaluation of Zr-beta-FeOOH for efficient arsenic removal.
A Zr-beta-FeOOH adsorbent for both As(V) and As(III) removal was prepared by a chemical co-precipitation method. Compared with beta-FeOOH, the addition of Zr enhanced the adsorption capacities for As(V) and As(III), especially As(III). The maximum adsorption capacities for As(III) and As(V) were 120 and 60 mg/g respectively at pH 7.0, much higher than for many reported adsorbents. The adsorption data accorded with Freundlich isotherms. At neutral pH, for As(V), adsorption equilibrium was approached after 3 hr, while for As(III), adsorption equilibrium was approached after 5 hr. Kinetic data fitted well to the pseudo second-order reaction model. As(V) elimination was favored at acidic pH, whereas the adsorption of As(III) by Zr-beta-FeOOH was found to be effective over a wide pH range of 4-10. Competitive anions hindered the adsorption according to the sequence: phosphate > silicate > bicarbonate > sulfate > nitrate, while Ca2+ and Mg2+ increased the removal of As(III) and As(V) slightly. The high adsorption capability and good performance in other aspects make Zr-beta-FeOOH a potentially attractive adsorbent for the removal of both As(III) and As(V) from water.
2135194
A functionally required unfoldome from the plant kingdom: intrinsically disordered N-terminal domains of GRAS proteins are involved in molecular recognition during plant development
The intrinsic disorder is highly abundant in eukaryotic genomes. In the animal kingdom, numerous intrinsically disordered proteins (IDPs) have been characterized, especially in cell signalling and transcription regulation. An intrinsically disordered region often folds in different structures allowing an IDP to recognize and bind different partners at various binding interfaces. In contrast, there have only been a few reports of IDPs from the plant kingdom. Plant-specific GRAS proteins play critical and diverse roles in plant development and signalling and often act as integrators of signals from multiple plant growth regulatory inputs. Using computational and bioinformatics tools, we demonstrate here that the GRAS proteins are intrinsically disordered, thus forming the first functionally required unfoldome in the plant kingdom. Furthermore, the N-terminal domains of GRAS proteins are predicted to contain numerous Molecular Recognition Features (MoRFs), short interaction-prone segments that are located within extended disorder regions and are able to recognize their interacting partners and to undergo disorder-to-order transitions upon binding to these specific partners. Overlapping with the relatively conserved motifs in the N-terminal domains of GRAS proteins, these predicted MoRFs represent the potential protein–protein binding sites and may be involved in molecular recognition during plant development. This study enables us to propose a conceptual framework that guides future experimental approaches to understand structure–function relationships of the entire GRAS family.
8373412
Expression of uncoupling protein 3 is upregulated in skeletal muscle during sepsis.
Uncoupling protein 3 (UCP3) is a member of the mitochondrial transporter superfamily that is expressed primarily in skeletal muscle. UCP3 is upregulated in various conditions characterized by skeletal muscle atrophy, including hyperthyroidism, fasting, denervation, diabetes, cancer, lipopolysaccharide (LPS), and treatment with glucocorticoids (GCs). The influence of sepsis, another condition characterized by muscle cachexia, on UCP3 expression and activity is not known. We examined UCP3 gene and protein expression in skeletal muscles from rats after cecal ligation and puncture and from sham-operated control rats. Sepsis resulted in a two- to threefold increase in both mRNA and protein levels of UCP3 in skeletal muscle. Treatment of rats with the glucocorticoid receptor antagonist RU-38486 prevented the sepsis-induced increase in gene and protein expression of UCP3. The UCP3 mRNA and protein levels were increased 2.4- to 3.6-fold when incubated muscles from normal rats were treated with dexamethasone (DEX) and/or free fatty acids (FFA) ex vivo. In addition, UCP3 mRNA and protein levels were significantly increased in normal rat muscles in vivo with treatment of either DEX or FFA. The results suggest that sepsis upregulates the gene and protein expression of UCP3 in skeletal muscle, which may at least in part be mediated by GCs and FFA.