Multi-step Column Research Articles

Automated multistep column chromatography on ÄKTA pure system using in-line sample dilution

Recent advances in instrumentation of fast protein liquid chromatography and the availability of various pre-packed columns helped to standardize protein purification protocols. However, purification processes are often plagued by inefficient manual procedures between the column purification steps, such as sample transfer, dialysis, concentration and filtration. Working with automation development in lab scale aims at giving immediate increase in research productivity. Here, we describe the automation of a four-column step purification using a modular sample dilution process coupled with ion exchange chromatography on a commonly available ÄKTA™ pure chromatography system. The method was applied to purify detergent-solubilized G protein-coupled receptors. Queuing multiple samples resulted in reduction in experiment time of up to ~58% and a reduction in manual labor time of ~83% while sustaining a comparable protein yield. The resultant combination of in-line dilution and ion-exchange chromatography could function as a generic method for automating other multistep column purifications schemes.

Skeletogenic Capacity of Human Perivascular Stem Cells Obtained Via Magnetic-Activated Cell Sorting.

Human perivascular stem/stromal cells (PSC) are a multipotent mesenchymal progenitor cell population defined by their perivascular residence. PSC are increasingly studied for their application in skeletal regenerative medicine. PSC from subcutaneous white adipose tissue are most commonly isolated via fluorescence-activated cell sorting (FACS), and defined as a bipartite population of CD146+CD34-CD31-CD45- pericytes and CD34+CD146-CD31-CD45- adventitial cells. FACS poses several challenges for clinical translation, including requirements for facilities, equipment, and personnel. The purpose of this study is to identify if magnetic-activated cell sorting (MACS) is a feasible method to derive PSC, and to determine if MACS-derived PSC are comparable to our previous experience with FACS-derived PSC. In brief, CD146+ pericytes and CD34+ adventitial cells were enriched from human lipoaspirate using a multistep column approach. Next, cell identity and purity were analyzed by flow cytometry. In vitro multilineage differentiation studies were performed with MACS-defined PSC subsets. Finally, in vivo application was performed in nonhealing calvarial bone defects in Scid mice. Results showed that human CD146+ pericytes and CD34+ adventitial cells may be enriched by MACS, with defined purity, anticipated cell surface marker expression, and capacity for multilineage differentiation. In vivo, MACS-derived PSC induce ossification of bone defects. These data document the feasibility of a MACS approach for the enrichment and application of PSC in the field of tissue engineering and regenerative medicine. Impact Statement Our findings suggest that perivascular stem/stromal cells, and in particular adventitial cells, may be isolated by magnetic-activated cell sorting and applied as an uncultured autologous stem cell therapy in a same-day setting for bone defect repair.

Multi-step column leaching using low-molecular-weight organic acids for remediating vanadium- and chromium-contaminated soil.

In soil, vanadium (V) contamination is commonly concomitant with chromium (Cr) contamination, which poses potential risks to humans, animals, and plants due to the transfer of toxic metals and the increase in their concentrations via the food chain or through direct exposure. This study applied a multi-step column leaching process using low-molecular-weight organic acids (LMWOAs) to treat V-contaminated soil from a smelter site that contains 2015.1 mg V kg-1 and 1060.3 mg Cr kg-1. After leaching three times with an equivalent solution/soil ratio of 0.3 mL/g using 1.0 M oxalic acid solution, the total removal rates reached 77.2% and 7.2% for V and Cr, respectively, while the removal rates of the extractable fractions reached 118.6% and 99.2% due to the reduction in residual fraction (F4) of toxic metals. Simultaneously, the distribution and redistribution of geochemical fractions of V and Cr were determined with a sequential extraction technique, and the greater proportion of potential mobile fractions of V (65.1%) may increase its leaching from soil relative to Cr (7.1%). In addition, a lower pH of the leaching agent increased the efficiency of the leaching process to an extent. Compared with batch extraction with a typical solution to soil ratio of 10 mL/g, multi-step column leaching used less agent and hence produced less wastewater. This strategy could reduce the mobilization and bioavailability of toxic metals, and potentially enhance in situ soil flushing for the remediation of V- and Cr- contaminated soil.

Spicatoside A derived from Liriope platyphylla root ethanol extract inhibits hepatitis E virus genotype 3 replication in vitro

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans worldwide. Although hepatitis E is self-limiting without chronic infection development, HEV infection often leads to severe liver diseases causing high mortality in pregnant women in addition to chronic hepatitis and cirrhosis in immunosuppressed patients. In this study, we investigated the effect of a Liriope platyphylla ethanol extract (LPE) on HEV replication. Interestingly, LPE suppressed replication of the genotype 3 HEV replicon. Sequential solvent fractionation revealed that the ethyl acetate (EA) fraction of LPE exerts the most potent inhibitory effects. With the aid of activity-guided fractionation and multi-step column chromatography, spicatoside A was subsequently isolated in the EA fraction of LPE and specifically shown to exert inhibitory effects on replication of the genotype 3 HEV replicon. In addition, spicatoside A interfered with replication of the HEV genotype 3 strain 47832c and expression of HEV ORF2 capsid proteins. Our findings clearly support the potential utility of spicatoside A as an effective anti-HEV agent.

Isolation and identification of highly active anticholinesterase ingredients from fermented soybean products

ABSTRACTInhibitory activities of ethanol extracts from seven fermented soybean products, including douchi, sufu, stinky tofu, to acetylcholinesterase were determined, and crude extract from Yongchuan douchi, which showed the strongest inhibitory effect, was further extracted systematically. Highly active anticholinesterase ingredients were mainly found in petroleum ether extracts; after multistep column chromatography, three compounds were purified finally with high-speed countercurrent chromatography. By their spectral data of MS, 1H-NMR, and 13C-NMR, three compounds were identified as genistein, daidzein, and kaempferol, whose further research could lay foundation for research and development of new drugs and foods against Alzheimer’s disease.

Hydrolysis kinetics and radical-scavenging activity of gelatin under simulated gastrointestinal digestion

The hydrolysis kinetics and radical-scavenging activity of gelatin were investigated under simulated gastrointestinal digestion in this study. In the gastric phase, the degree of gelatin hydrolysis increased from 0.17% to 1.20%, while the DPPH radical-scavenging rate increased from 6.27% to 24.56%. Further digestion in the intestinal phase brought the degree of hydrolysis and radical-scavenging rate to 26.08% and 44.76%, respectively. After digestion, the gelatin hydrolysates were separated into two fractions by ultrafiltration. The fraction with an average molecular weight of 312.98Da exhibited the higher yield (78.26%) and radical-scavenging activity (IC50=2.09mg/ml), suggesting the high digestibility and bioactivity of gelatin after oral administration. The fraction was further purified with multi-step column chromatography and identified to be Gly-Pro-Met (303.38Da) by UPLC–ESI-MS. These results may help us to better understand its physiological effects and to use it properly in foods and pharmaceuticals.

Inhibitory Effect on β -Hexosaminidase Release from RBL-2H3 Cells of Extracts and Some Pure Constituents of Benchalokawichian, a Thai Herbal Remedy, Used for Allergic Disorders.

Introduction. Benchalokawichian (BCW), a Thai traditional herbal formulation, has long been used as antipyretic and to treat skin disorders. It comprises roots from five herbs: Ficus racemosa, Capparis micracantha, Clerodendrum petasites, Harrisonia perforata, and Tiliacora triandra. This polyherbal remedy has recently been included in the Thailand National List of Essential Medicines (Herbal Products list). Methodology. A Bioassay-guided fractionation technique was used to evaluate antiallergy activities of crude extracts, and those obtained by the multistep column chromatography isolation of pure compounds. Inhibitory effect on the release of β-hexosaminidase from RBL-2H3 cells was used to determine antiallergic activity. Results. Two pure compounds from BCW formulation showed higher antiallergic activity than crude or semipure extracts. Pectolinarigenin showed the highest antiallergic activity, followed by O-methylalloptaeroxylin, with IC50 values of 6.3 μg/mL and 14.16 μg/mL, respectively. Moreover, the highest activities of pure compounds were significantly higher than chlorpheniramine (16.2 μg/mL). Conclusions. This study provides some support for the use of BCW in reducing itching and treatment of other skin allergic disorders. The two isolated constituents exhibited high antiallergic activity and it is necessary to determine their mechanism of action. Further phytochemical and safety studies of pure compounds are required before development of these as antiallergy commercial remedies.

Analysis of Flexibility and Stability of Crane Telescopic Boom with Elastic Restraint and Second-Order Effect

The cylinder support crane telescopic booms deformation and stability analysis model in the lifting plane is equivalent with the multistep column with elastic restraint. To analyze the lateral flexibility and vertical stability of the telescopic booms with elastic restraint accurately, this paper established the deflection differential equations of multi-sectioned telescopic booms with second-order effect, introduced proper boundary conditions, obtained the precise recurrence lateral deflection differential equations and the buckling characteristic equations of arbitrary sectioned telescopic booms, and some practical applications of the buckling characteristic equations were presented. Took certain five-sectioned telescopic booms as example, by comparing the results with ANSYS method, the accuracy of the equations deduced in this paper was verified.

A new cleanup method of dioxins in sediment using large volume injection gas chromatography online coupled with liquid chromatography

A new cleanup method was developed and validated for the determination of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) in sediment. The sample extract was first treated with sulfuric acid and then cleaned up by a large volume injection gas chromatography online coupled with liquid chromatography (LVI-GC-LC) system. PCDD/Fs in the extract were separated by a GC column (DB-5), selected cut, cool trapped and transferred to a LC column (alumina). The fraction of PCDD/Fs eluted from the alumina column was collected and concentrated for the instrumental analysis. Under the optimized conditions, LVI-GC-LC method achieved the recoveries of 57–102% for 2,3,7,8-substituted PCDD/Fs, which met the requirements of US Environmental Protection Agency (EPA) Method 1613 and were better than those obtained using the conventional multistep column cleanup method. Meanwhile, compared with the conventional method, the limit of detection (LOD) values of 2,3,7,8-substituted PCDD/Fs cleaned up by LVI-GC-LC method were decreased due to the high-efficiency removal of interferents. These results suggested that the LVI-GC-LC cleanup method was a promising alternative to the multistep cleanup procedure for the determination of dioxins in environmental samples.

Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1

The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both enzymes appeared to be homotetramers with a subunit size of 45+/-1 kDa as revealed by SDS-PAGE, which was close to the calculated value (44.8 kDa). The recombinant ADH contained 1.0+/-0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105+/-2 U mg(-1), which was very similar to that of the native enzyme (110+/-3 U mg(-1)). The optimal pH-values for both enzymes for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively. Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there was no activity towards methanol and secondary alcohols. Kinetic parameters of the enzymes showed lower K (m)-values for acetaldehyde and NADPH and higher K (m)-values for ethanol and NADP(+). It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile.

Rapid screening of dioxin-contaminated soil by accelerated solvent extraction/purification followed by immunochemical detection

Since soils at industrial sites might be heavily contaminated with polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), there is a need for large-scale soil pollution surveys and, thus, for cost-efficient, high-throughput dioxin analyses. However, trace analysis of dioxins in complex matrices requires exhaustive extraction, extensive cleanup, and very sensitive detection methods. Traditionally, this has involved the use of Soxhlet extraction and multistep column cleanup, followed by gas chromatography-high-resolution mass spectrometry (GC/HRMS), but bioanalytical techniques may allow much more rapid, cost-effective screening. The study presented here explores the possibility of replacing the conventional method with a novel approach based on simultaneous accelerated solvent extraction (ASE) and purification, followed by an enzyme-linked immunosorbent assay (ELISA). Both the traditional and the novel cleanup and detection approaches were applied to contaminated soil samples, and the results were compared. ELISA and GC/HRMS results for Soxhlet-extracted samples were linearly correlated, although the ELISA method slightly underestimated the dioxin levels. To avoid an unacceptable rate of false-negative results, the use of a safety factor is recommended. It was also noted that the relative abundance of the PCDDs/PCDFs, evaluated by principal component analysis, had an impact on the ELISA performance. To minimize this effect, the results may be corrected for differences between the ELISA cross-reactivities and the corresponding toxic equivalency factor values. Finally, the GC/HRMS and ELISA results obtained following the two sample preparation methods agreed well; and the ELISA and GC/HRMS results for ASE extracts were strongly correlated (correlation coefficient, 0.90). Hence, the ASE procedure combined with ELISA analysis appears to be an efficient approach for high-throughput screening of PCDD-/PCDF-contaminated soil samples.

Cleavage of the Carboxyl-Terminus of LEACS2, a Tomato 1-Aminocyclopropane-1-Carboxylic Acid Synthase Isomer, by a 64-kDa Tomato Metalloprotease Produces a Truncated but Active Enzyme

: 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS (LeACS2) into 52-, 50- and 49-kDa truncated isoforms in ripening tomato (Lycopersicon esculentum Mill. cv. Cooperation 903) fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys438, Glu447, Lys448, Asn456, Ser460, Ser462, Lys463, and Leu474, but does not cleave the N-terminus of LeACS2. Four C-terminus-deleted (26—50 amino acids) LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain (H14) and Ser460 for this metalloprotease. Furthermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo. (Managing editor: Wei WANG)

Rapid and quantitative electrolytic preparation and speciation of neptunium ions of various oxidation states using multi-step column electrodes

Redox behavior of Np(III)–Np(VI) ions in aqueous perchloric, nitric and sulphuric acid solutions was elucidated by using column electrodes connected in series in a flow system (multi-step column electrode system). Using a glassy carbon fiber working electrode as the column electrode was found to be very useful for the investigation of current–potential relations of not only reversible redox processes, such as Np(VI)/Np(V) or Np(IV)/Np(III) couple but also irreversible processes, such as Np(V)/Np(IV) or Np(V)/Np(III) couple, the latter not observed by conventional voltammetry which uses a glassy carbon or platinum electrode. Quantitative (coulometric) electrolysis could be executed even if the redox process was completely irreversible by the use of the column electrodes. By taking advantage of column electrode electrolysis, a novel method was developed for the rapid preparation of a neptunium species of a desired oxidation state, including unstable species. The multi-step column electrode system was demonstrated to be useful for the coulometric determination and speciation of Np(IV)–Np(VI) in aqueous HNO 3 solution as an example.

Inverse Estimation of the Unsaturated Soil Hydraulic Properties from Column Outflow Experiments Using Free-Form Parameterizations

Inverse methods are increasingly used to estimate the hydraulic properties of unsaturated soils. The method generally uses a weighted least-squares approach in which numerically simulated data are fitted to measured data. In this study we used inverse methods to estimate the unsaturated soil hydraulic properties from continuous and multistep column outflow experiments. The method employs piecewise polynomial functions to obtain a free-form parameterization of the hydraulic properties, rather than fixed functional forms typical of the van Genuchten–Mualem and Brooks–Corey–Burdine models. For the polynomial functions we used quadratic B-splines and piecewise cubic Hermite interpolation. The method leads to local parameterizations that can also be hierarchic, depending on the invoked number of degrees of freedom. Since a suitable number of degrees of freedom cannot be defined a priori, we embedded the estimation method into a multilevel procedure, which also included a stability analysis, based on singular value decomposition of the sensitivity matrix. The optimization procedure was made more stable by imposing monotonicity constraints on the hydraulic functions. Tests with synthetic and measured data from column outflow experiments show the validity and robustness of the method.

Inverse Estimation of the Unsaturated Soil Hydraulic Properties from Column Outflow Experiments Using Free‐Form Parameterizations

Inverse methods are increasingly used to estimate the hydraulic properties of unsaturated soils. The method generally uses a weighted least‐squares approach in which numerically simulated data are fitted to measured data. In this study we used inverse methods to estimate the unsaturated soil hydraulic properties from continuous and multistep column outflow experiments. The method employs piecewise polynomial functions to obtain a free‐form parameterization of the hydraulic properties, rather than fixed functional forms typical of the van Genuchten–Mualem and Brooks–Corey–Burdine models. For the polynomial functions we used quadratic B‐splines and piecewise cubic Hermite interpolation. The method leads to local parameterizations that can also be hierarchic, depending on the invoked number of degrees of freedom. Since a suitable number of degrees of freedom cannot be defined a priori, we embedded the estimation method into a multilevel procedure, which also included a stability analysis, based on singular value decomposition of the sensitivity matrix. The optimization procedure was made more stable by imposing monotonicity constraints on the hydraulic functions. Tests with synthetic and measured data from column outflow experiments show the validity and robustness of the method.

Non-conservative stability of multi-step non-uniform columns

The non-conservative stability of non-uniform columns under the combined action of concentrated and variably distributed forces is solved analytically. Two types of follower force system are considered: (i) concentrated follower forces and variably distributed follower forces, (ii) concentrated follower forces and variably distributed conservative forces. The exact solutions for stability of four kinds of one-step non-uniform columns subjected to the two types of follower force system are derived for the first time. Then a new exact approach, which combines the exact solutions of one-step columns and the transfer matrix method, is presented for the non-conservative stability analysis of multi-step non-uniform columns. The advantage of the proposed method is that the resulting eigenvalue equation for a multi-step non-uniform column with any kinds of two end support configurations, an arbitrary number of spring supports and concentrated masses can be conveniently determined from a second order determinant. The decrease in the determinant order, as compared with previously developed procedures, leads to significant savings in the computational effort. A numerical example shows that the results obtained from the proposed method are in good agreement with those determined from the finite element method (FEM), but the proposed method takes less computational time than FEM.

Recombinant fusion proteins for haemagglutination-based rapid detection of antibodies to HIV in whole blood

Recombinant fusion proteins, consisting of a monovalent anti-human RBC monoclonal antibody B6, and conserved immunodominant peptide of HIV-1 envelope glycoprotein gp41 or HIV-2 envelope glycoprotein gp36, have been designed and purified after over-expression in E. coli. These fusion proteins are Fab-based and were obtained by assembling the light chain with Fd (variable domain and the first constant domain of the heavy chain) or Fd fusions containing HIV-derived peptide, and following a protocol of in vitro denaturation of inclusion bodies and subsequent renaturation to assemble functional Fab. Using a multistep column chromatographic procedure, monomeric Fab and Fab fusion proteins containing HIV-derived peptide were purified to high degree, free of aggregates. The yield of various proteins on the laboratory scale (1–2 l of shake flask culture) was in the range of tens of milligram. Purified anti-human RBC Fab fusion proteins containing sequences derived from HIV-1 gp41 and HIV-2 gp36 were highly specific for detection of antibodies to HIV-1 and HIV-2, respectively. The described design, expression and purification protocols will make it possible to produce specific recombinant reagents in large quantities for agglutination-based rapid detection of antibodies to HIV in whole blood.

Buckling of multi-step cracked columns with shear deformation

The governing differential equation for buckling of a one-step cracked column with shear deformation is established and its solution is found first. Then a new approach that combines the exact buckling solution of a one-step column and the transfer matrix method is presented for solving the entire and partial buckling of a multi-step column with various end conditions, with or without cracks and shear deformation, subjected to concentrated axial loads. The main advantage of the proposed method is that the eigenvalue equation for buckling of a multi-step column with an arbitrary number of cracks, any kind of two end supports and various spring supports at intermediate points can be conveniently determined from a system of two linear equations. Due to the decrease in the determinant order as compared with previously developed procedures, the computational time required by the present method for solving the title problem can be reduced significantly. A numerical example is given for explaining the proposed procedure and investigating the effects of shear deformation and the number, depth and location of cracks on critical buckling force of a multi-step column. It is shown through the numerical example that the proposed procedure is a simple, exact and efficient method.

Buckling of elastically restrained non-uniform columns

An exact solution approach is presented to study the buckling of a non-uniform column with spring supports under combined concentrated and distributed loads. In this paper, the expression for describing the distribution of flexural stiffness of a non-uniform column is arbitrary, and the distribution of axial forces of the column is expressed as a functional relation with the distribution of flexural stiffness and vice versa. The governing equation for buckling of a one-step non-uniform column is reduced to Bessel equations and other solvable equations for 13 cases, several of which are important in engineering practices, by means of the functional transformation. The exact buckling solutions of one-step non-uniform columns are thus found. Then the obtained exact solutions are used to derive the eigenvalues equation for buckling of a multi-step non-uniform column with spring supports by using the transfer matrix method. A numerical example shows that the critical buckling force of a non-uniform structure calculated by the proposed method is almost the same as that determined by FEM, but the present method takes much less computational time than the FEM, illustrating the proposed procedure is an exact and efficient method.

FLOW-COULOMETRY OF URANIUM, NEPTUNIUM AND PLUTONIUM IONS WITH MULTI-STEP COLUMN ELECTRODES

The overall feature of the reduction-oxidation behavior of U, Np or Pu ions in acidic aqueous solutions was investigated by flow-coulometry with multi-step column electrodes. Redox potentials, number of electrons involved in the electrode processes, and reversibilities of the processes were determined and the species participating in the reactions were discussed. Methods for the rapid preparation of the ion of a desired oxidation state and for the determination and the speciation of ions were developed.