Development and Characterization of a Nanobody against Human T-Cell Immunoglobulin and Mucin-3.

Monoclonal antibodies and antibody-derived biologics are essential tools for cancer research and therapy. The development of monoclonal antibody treatments for successful tumor-targeted therapies took several decades. A nanobody constructed by molecular engineering of heavy-chain-only antibody, which is unique in camel or alpaca, is a burgeoning tools of diagnostic and therapeutic in clinic. In this study, we immunized a 4-year-old female alpaca with TIM-3 antigen. Then, a VHH phage was synthesized from the transcriptome of its B cells by nested PCR as an intermediate library; the library selection for Tim-3 antigen is carried out in three rounds of translation. The most reactive colonies were selected by periplasmic extract monoclonal ELISA. The nanobody was immobilized by metal affinity chromatography (IMAC) purification with the use of a Ni-NTA column, SDS-PAGE, and Western blotting. Finally, the affinity of TIM3-specific nanobody was determined by ELISA. As results, specific 15 kD bands representing nanomaterials were observed on the gel and confirmed by Western blotting. The nanobody showed obvious specific immune response to Tim-3 and had high binding affinity. We have successfully prepared a functional anti-human Tim-3 nanobody with high affinity in vitro.

Optimization of chemoenzymatic Baeyer-Villiger oxidation of cyclohexanone to ε-caprolactone using response surface methodology.

ε-Caprolactone (ε-CL) has attracted a great deal of attention and a high product concentration is of great significance for reducing production cost. The optimization of ε-CL synthesis through chemoenzymatic Baeyer-Villiger oxidation mediated by immobilized Trichosporon laibacchii lipase was studied using response surface methodology (RSM). The yield of ε-CL was 98.06% with about 1.2 M ε-CL concentration that has a substantial increase mainly due to both better stability of the cross-linked immobilized lipase used and the optimum reaction conditions in which the concentration of cyclohexanone was 1.22 M, the molar ratio of cyclohexanone:urea hydrogen peroxide (UHP) was 1:1.3, and the reaction temperature was 56.5°C. Based on our experimental results, it can be safely concluded that there are three reactions in this reaction system, not just two reactions, in which the third reaction is that the acetic acid formed reacts with UHP to form peracetic acid in situ catalyzed by the immobilized lipase. A quadratic polynomial model based on RSM experimental results was developed and the R2 value of the equation is 0.9988, indicating that model can predict the experimental results with high precision. The experimental results also show that the molar ratio of cyclohexanone to UHP has very significant impact on the yield of ε-CL (p < .0006).

[Mechanism Study of the Smectite-OR-SH Compound for Reducing Cadmium Uptake by Plants in Contaminated Soils].

Adsorption and desorption experiments, pot experiments and characterization test were performed to investigate the immobilization effect and mechanism of the smectite-OR-SH compound for reducing cadmium uptake by plants in contaminated soils. The results showed that the saturated adsorption capacity for the adsorption of Cd2+ on smectite raised distinctly after functionalized. The adsorption of Cd2+ on smectite-OR-SH compound was very stable and it was difficult for Cd2+ to be desorbed from it. Crop yields promoted differently in original soil, Cd 3 mg x kg(-1) soil and Cd 10 mg x kg(-1) soil after adding the smectite-OR-SH compound. And the cadmium content of the cabbage reduced 61.00%, 62.10% and 83.73% respectively compare with the control. Characterization test analysis showed that Cd was adsorbed by the compound successfully and ligand interaction occurred between Cd and the thiol group. Floc amount on the compound surface increased correspondingly. In addition to electrostatic adsorption, ion exchange and hydroxyl ligand adsorption, the reaction mechanism of smectite-OR-SH compound with Cd was mainly sulfhydryl ligand adsorption.

[Study of adsorption and desorption of behaviors of Pb2+ on thiol-modified bentonite by flame atomic absorption spectrometry].

A comparative analysis of the functional groups and surface structure of the Ca-bentonite (RB) and thiol-modified bentonite (TMB) were characterized by means of FTIR and SEM. The absorptive property of Pb2+ on TMB and RB and its influential factors was studied and the conditions for the adsorption were optimized by using FAAS method. Then the conditions for desorption of Pb2+ from the TMB by using simulated acid rain were studied and the contrast analysis of absorptive stability of Pb2+ on TMB and RB was given. The results showed that the adsorption rate of Pb2+ by TMB could reach more than 98%, when the initial Pb2+ concentration was 100 mg.L-1, the liquid-solid ratio was 5 g.L-1, pH was 6. 0, KNO3 ionic strength was 0. 1 mol.L-1 and adsorption period was 60min at 25 C. The saturated adsorption capacity of TMB was 67.27 mg.g-1; it's much more than that of RB (9.667 mg.g-1). The adsorption of Pb2+ on TMB follows Langmuir and Freundlich isotherm models well. Desorption experiments of Pb2+ from TMB with simulated acid rain (pH 3. 50) were done, and the desorption rate was 0. The results showed that TMB has a strong adsorption and fixation capacity for PbZ+; it is adapted to lead contaminated soil for chemical remediation.

Low volume microwave digestion and direct determination of selenium in biological samples by hydride generation-atomic fluorescence spectrometry.

A low volume microwave digestion (LVMWD) procedure has been developed to have all forms of selenium (Se) compounds in biological samples decomposed to Se (IV) and allow total Se be directly determined by hydride generation-atomic fluorescence spectrometry (HG-AFS), or voltammetrically. Between 0.3 and 0.4 mL of mixed digestion reagents consisting of concentrated (15.4 M) HNO(3)-(18.5 M) H(2)SO(4) (v:v=10:1) and >5 to <40 mg sample were found ideal systems with an optimized MW digestion program. Total Se in five certified reference materials was accurately determined. The results obtained by the conventional and LVMWD techniques agreed well. By avoiding pre-reduction step, this method, suitable for a wide range of biological samples, fully takes the advantages of HG-AFS or voltammetric techniques for their high sensitivity, high tolerance to matrix-related interference and accessibility in instrumentation. LVMWD not only enhances the sample output by 3 times and reduces the operational cost and acid wastes, but also makes the small sample analysis possible for many environmental and medical related research objectives. The digestion pathways of Se containing organic samples are also discussed based on the experimental results.

[Study of DNA damage induced by potassium dichromate and glutathione with atomic force microscope].

OBJECTIVE: To observe calf thymus DNA damage induced by potassium dichromate in combination with glutathione (GSH). METHODS: Atomic force microscope and ultraviolet spectrum (UV) were used to observe the alterations of the DNA ultrastructure and absorption spectrum. RESULTS: Atomic force microscopy revealed no breaks of the DNA strand in response to treatment with potassium dichromate alone, but when coupled with GSH at proper concentrations, potassium dichromate induced alterations in the DNA structure and DNA fragmentation. UV examination also confirmed these findings by showing increased absorption intensity of the maximum UV peak following combined treatment of the DNA with potassium dichromate and GSH. CONCLUSION: These morphological and spectrographic evidences verified the important role of GSH in mediating the generation of various tumor-inducing intermediate products of potassium dichromate.