Fabrication of rigid and macroporous agarose microspheres by pre-cross-linking and surfactant micelles swelling method.

A novel combined method of pre-cross-linking and surfactant micelles swelling was proposed in this study to fabricate highly cross-linked and macroporous agarose (HMA) microspheres. Agarose was chemically modified by allylglycidyl ether (AGE) as heterobifunctional cross-linker via its active glycidyl moieties before gel formation and pre-cross-linking was achieved. By this means, the effective concentration of cross-linker presented in agarose gel increased significantly, and thus cross-linking with a high-efficiency was achieved. Further to enhance the intraparticle mass transfer of agarose microspheres, the surfactant micelles swelling method was utilized to create interconnected macropores. Under the optimal condition, HMA microspheres with homogeneous reticular structure and pore size of hundreds nanometers were successfully prepared. They exhibited a low backpressure with a flow velocity as high as 1987 cm/h, which was much higher than that of commercial Sepharose 4 F F. HMA microspheres were then derivatized with carboxymethyl (CM) groups and applied in ion-exchange chromatography. As expected, CM-HMA column separated model proteins effectively even at a flow velocity three times higher than that of commercial CM-4 F F. Visualization of dynamic protein adsorption by confocal laser scanning microscope (CLSM) revealed that the intraparticle mass transfer of CM-HMA microspheres was intensified due to its macroporous structure. All of the results indicated the newly developed agarose microspheres were a promising medium for high-speed chromatography.

Mechanisms of rice straw biochar effects on phosphorus sorption characteristics of acid upland red soils.

An important pathway for biochar to alter the availability of soil phosphorus (P) is to change P sorption characteristics of the soil. The aim of this study was to understand the mechanisms of biochar effects on P sorption in acid upland red soils in the presence of different concentrations of exogenous P. Rice straw biochar (RSB) was prepared and applied at rates of 0, 1%, 3%, and 5% (w/w) to three red soils (MZ1, MZ2, and QY1) differing in initial pH (pH = 4.31, 4.82, and 5.68, respectively). The P sorption characteristics of these red soils were described using the Langmuir and Temkin equations and their relationships with soil basic physicochemical properties were analyzed. Furthermore, a representative red soil (MZ2) was selected to analyze the zeta potential of soil colloids and the chemical properties of sorption equilibrium solution, in order to understand their relationships with P sorption characteristics. Results showed that within a certain range of P concentration in the equilibrium solution, the amount of P sorbed by the three red soils decreased and the corresponding amount of P desorbed increased with increasing amendment rate of RSB. RSB showed the greatest effect on P desorption characteristics of MZ2 soil in the presence of higher exogenous P concentration. With increasing RSB amendment rate, the maximum P sorption of MZ1 soil decreased, while those of MZ2 and QY1 soils increased after an initial decrease. Phosphate sorption equilibrium constant and maximum P buffer capacity of each soil first increased and then decreased. However, a single physicochemical property could not interpret complex changes in multi-factors that jointly determine the P sorption characteristics of red soils. In the case of MZ2 soil, RSB amendment shifted the zeta potential of soil colloids to the negative direction; this decreased the positive charge and increased the negative charge on the soil surface, thus reducing P sorption in the MZ2 soil. In the presence of the same concentration of exogenous P, RSB amendment altered the pH, dissolved organic C (DOC), humification index (HIX), and maximum fluorescence intensity (Fmax) in the sorption equilibrium solution. In most cases, the amount of P sorbed by the MZ2 soil was negatively correlated with the pH value, DOC concentration, HIX value, and Fmax value of humic-like dissolved organic matter (DOM), and positively correlated with the Fmax value of protein-like DOM (P < 0.05 or P < 0.01). The relative fractional distribution of the contents for humic-like and protein-like DOM might determine the difference in the P sorption characteristics of MZ2 soil. In conclusion, different amendment rates of RSB affected the release of phosphate from soil surfaces into the solution by altering basic physicochemical and electrochemical properties of red soils and chemical properties of sorption equilibrium solution.

Multiscale evaluation of pore curvature effects on protein structure in nanopores.

Protein structure in nanopores is an important determinant in porous substrate utilization in biotechnology and materials science. To date, accurate residue details of pore curvature induced protein binding and unfolding were still unknown. Here, a multiscale ensemble of chromatography, NMR hydrogen and deuterium (H/D) exchange, confocal scanning and molecular docking simulations was combined to obtain the protein adsorption information induced by pore size and curvature. Lysozyme and polystyrene microspheres within pores in the 14-120 nm range were utilized as models. With pore size increasing, the bound lysozyme presented a tendency of significantly decreased retention, less unfolding and fewer interacted sites. However, such a significant dependence between pore curvature and protein size only existed in a limited micro-pore range comparable to protein sizes. The mechanism behind the above events could be attributed to the diverse protein interaction area determined by pore curvature and size change, by models calculating the binding of lysozyme onto surfaces. Another surface of opposite curvature for nanoparticles was also calculated and compared, the rules were similar but with opposite direction and such a critical size also existed. These studies of proteins on curved interfaces may ultimately help to guide the design of novel porous materials and assist in the discrimination of the target protein from molecular banks.

A novel matrix derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed immobilized-metal affinity chromatography.

Agarose coated gigaporous polystyrene microspheres were evaluated as a novel matrix for immobilized-metal affinity chromatography (IMAC). With four steps, nickel ions were successfully immobilized on the microspheres. The gigaporous structure and chromatographic properties of IMAC medium were characterized. A column packed with the matrix showed low column backpressure and high column efficiency at high flow velocity. Furthermore, this matrix was used for purifying superoxide dismutase (SOD), which was expressed in Escherichia coli (E. coli) in submerged fermentation, on an Äkta purifier 100 system under different flow velocities. The purity of the SOD from this one-step purification was 79% and the recovery yield was about 89.6% under the superficial flow velocity of 3251 cm/h. In conclusion, all the results suggested that the gigaporous matrix has considerable advantages for high-speed immobilized-metal affinity chromatography.

[Comparison of adsorbent with varying arm length and ligand density for the purification of recombinant hepatitis B virus surface antigen].

Novel hydrophobic absorbents were synthesized by immobilizing butyl derivative onto the highly cross-linked agarose beads manufatured in China, which are used as matrix. The effect of the spacer arm length (3C, 8C and 10C) and ligand density (from 13 to 45 micromol/mL) on the hydrophobicity were investigated using purified Hepatitis B surface antigen (HBsAg) expressed by CHO cell lines. Also considering the effects of salt concentration and pH on HBsAg recovery and purification factor, orthogonal experiment design method was used to evaluated the absorbents. The results showed the butyl-S absorbent with the spacer arm length of C8, the ligand density of 22 micromol/mL gel showed the best performance for the separation of HBsAg. Approximately 100% HBsAg recovery and 60 as purification fold were achieved by this media under the operating condition of pH 7.0 and 9% of salt concentrateion.

[Development of a new hydrophobic interaction chromatography absorbent and its application to the purification of recombinant hepatitis B surface antigen].

A new hydrophobic absorbent based on homemade highly cross-linked agarose beads was synthesized by immobilizing butyl derivative onto the matrix linkage. The density of ligand was controlled by adjusting the concentration of butanethiol and the synthesis route was optimized by evaluating the purification efficiency of recombinant Hepatitis B surface antigen (HBsAg) expressed by Chinese hamster ovary (CHO) cell line. A high performance absorbent was finally screened out with up to 80% of HBsAg recovery and purification-fold (PF) about 20. Furthermore, the column pressure was about 0.06 MPa under the flow rate of 500cm/h, and no leaked butyl were detected after exposing the gel in common buffers, chaotropic agents, high concentrations of denaturing agents such as guanidine hydrochloride, urea and polar organic solvents. These results demonstrated that the absorbent have high physico-chemical stability, so it was available for the downstream process. Finally, after scaled up to 2L wet gel/batch, the absorbent was applied to the integration of three-step chromatography and obtained the purified CHO-HBsAg with 95% purity by SDS-PAGE and HPLC, which meet the requirements of SFDA. The purification efficiency and the reproducible ability of the absorbents were also evaluated from batch-to-batch. The results demonstrated that the absorbent met the requirement of scalable, reproducible, economic effect as well. This absorbent is a promising alternative exported HIC gel for wildly being used in Chinese pharmaceutical industries.

Synthesis and activity of an octapeptide inhibitor designed for SARS coronavirus main proteinase.

The outbreak of SARS, a life-threatening disease, has spread over many countries around the world. So far there is no effective drug for the treatment of SARS. Stimulated by the binding mechanism of SARS-CoV Mpro with the octapeptide AVLQSGFR reported recently as well as the "Chou's distorted key" theory, we synthesized the octapeptide AVLQSGFR for conducting various biochemical experiments to investigate the antiviral potential of the octapeptide against SARS coronavirus (BJ-01). The results demonstrate that, compared with other compounds reported so far, AVLQSGFR is the most active in inhibiting replication of the SARS coronavirus, and that no detectable toxicity is observed on Vero cells under the condition of experimental concentration.

[Polyethylene glycol-accompanied ion-exchange chromatography to purify recombinant hepatitis B virus surface antigen].

The dissociation of virus-like particles of Hepatitis B surface antigen (HBsAg) during the adsorption-desorption on the solid-phase of chromatography is a main challenge for its purification. Herein, poly (ethylene glycol) (PEG) was applied as an additive during the purification of HBsAg from recombinant Chinese hamster ovary (CHO) cell culture to improve the HBsAg recovery and protect its structural assembly. The presence of 1% of PEG10000 in the mobile phase of ion-exchange chromatography (IEC) of DEAE-Sepharose FF column could increase the recovery of HBsAg from about 55% to 80%, with a similar purification (-fold) (about 12) compared with the absence of PEG. Importantly, glycosylated protein forms of HBsAg were reserved well by PEG-accompanied chromatography. Furthermore, size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) analysis was performed on line to monitor the aggregates, particle size and molecular weight distribution of HBsAg. The results demonstrated that the HBsAg particle size and assembly are more homogenous after adding PEG in the mobile phase of IEC than no PEG added in the mobile phase.

[Surgical approach in plastic of shortened lower limb].

OBJECTIVE: To introduce a new surgical approach to rectify the shortened lower limbs. METHODS: From March 1985 to October 2000, 288 cases of shortened lower limbs were treated and reviewed. Closed fracture at the metaphysis was made by a self-made "needle saw", and then the "multiple-plane and double-track elongation instrument" was adopted to elongate the fractured bone. There were totally 161 cases of male and 127 cases of female included, with average age 21.3 years old, ranging from 12 to 29 years old, among which there were 268 cases elongated at the proximal metaphysis of the tibia, 16 cases at the distal femur and 4 cases at the distal tibia. All of the cases were followed up for 6 to 8 months before clinical evaluation. RESULTS: The lower limbs in 288 cases were elongated for 3.0 to 11.5 cm in 24 to 96 days, averaging 47 days, which fulfilled pre-operative plan. In the second week after the operation, new calculus and periosteum formed obviously in the gap between the fractured parts, and in 6 to 8 months bone union was observed at the fractured site in all cases. There was no nerve or blood vessel injury, or non-union of the metaphysis fracture. The function of the manipulated knee joints and ankle joints recovered well. CONCLUSION: It is a practical and safe surgical option to rectify the shortened lower limbs by closed fracture at the metaphysis, followed by elongation of the fractured bone, without any complication such as non-union or atrophy of manipulated bone, and with no need of internal fixation or bone grafting.