Binding Activity Difference of Anti-CD20 scFv-Fc Fusion Protein Derived from Variable Domain Exchange

Two novel engineered antibody fragments binding to antigen CD20 were generated by fusing a murine IgM-type anti-CD20 singie-chain Fv fragment （scFv） to the human IgG1 CH2 （i.e., Cγ2） and CH3 （i.e., Cγ3） domains with the human IgG1 hinge （i.e. Hγ）. Given the relationship between structure and function of protein, the 3-D structures of the two engineered antibody fragments were modeled using computer-aided homology modeling method. Furthermore, the relationship between 3-D conformation and their binding activity was evaluated theoretically. Due to the change of active pocket formed by CDRs, the HL23 （VH-Linker-VL-Hγ-Cγ2-Cγ3） remained its activity because of its preserved conformation, while the binding activity of the LH23 （VL-Linker-VH-Hγ-Cγ2-Cγ3） was impaired severely. Experimental studies by flow cytometry and fluorescence microscopy showed that HL23 possessed significantly superior binding activity to CD20-expressing target cells than LH23. That is to say, the order of variable regions could influence the binding activity of the fusion protein to CD20^＋ cell lines, which was in accordance with the theoretical results. The study highlights the potential relationship between the antibody binding activity and their 3-D conformation, which appears to be worthwhile in providing direction for future antibody design of recombinant antibody.

Design,synthesis and evaluation of PPAR gamma binding activity of 2-thioxo-4-thiazolidinone derivatives

We designed and synthesized a series of 2-thioxo-4-thiazolidinone derivatives and evaluated them on peroxisome proliferator activated receptor γ（PPARγ） binding activities.Through the biological assays,compounds 18 and 38 were highlighted with K_i values of 12.15 nmol/Land 14.46 nmol/L,respectively.Then structure-activity relationship（SAR） was analyzed to screen privileged structural modifications.Moreover,molecular fitting of these compounds onto the approved drug Rosightazone in the PPARγligand binding domain was performed to elucidate the SAR and explore potential receptor-ligand interactions.These results demonstrate that the 2-thioxo-4-thiazolidinones can be considered as new promising molecular probes with excellent binding activities to PPARγ.

Endoplasmic reticulum stress transducer old astrocyte specifically induced substance contributes to astrogliosis after spinal cord injury

Old astrocyte specifically induced substance （OASIS） is an endoplasmic reticulum （ER） stress transducer specifically expressed in astrocytes and osteoblasts. OASIS regulates the differentiation of neural precursor cells into astrocytes in the central nervous system. This study aimed to elucidate the involvement of ER stress responses stimulated via OASIS in astrogliosis following spinal cord injury. In a mouse model of spinal cord contusion injury, OASIS mRNA and protein expression were evaluated at days 7 and 14. A significant increase in OASIS mRNA on day 7 and an increase in protein on days 7 and 14 was observed in injured spinal cords. Immunostaining on day 7 revealed co-localization of OASIS and astrocytes in the periphery of the injury site. Furthermore, anti-OASIS small interfering RNA （siRNA） was injected at the injury sites on day 5 to elucidate the function of OASIS. Treatment with anti-OASIS siRNA caused a significant decrease in OASIS mRNA on day 7 and protein on days 7 and 14, and was associated with the inhibition of astrogliosis and hindlimb motor function recovery. Results of our study show that OASIS expression synchronizes with astrogliosis and is functionally associated with astrogliosis after spinal cord injury.

Anticancer activity and DNA binding property of the trimers of triphenylethylene-coumarin hybrids

Novel trimers of triphenylethylene-coumarin hybrid containing two amino side chains （5a-d and 6a-d） were designed and synthesized by the condensation of 1,3,5-benzenetricarboxylic acid with the varied amino monomeric hybrids catalyzed by HATU and DIPEA at room temperature. The extended trimeric compound 6a （R = piperidinyl） exhibited significant anti-proliferative activity against three cancer cells at IC5o of near 10 μmol/L. UV-vis, fluorescence （lifetime） and thermal denaturation exhibited that 6a had significant interaction with Ct-DNA by the intercalative mode of binding. The order of their anti- proliferative activities was 6（a, d） 〉 5（a, d） and （5-6）a 〉 （5-6）d, respectively, in accordance with that of their DNA binding properties, which suggested that the prolonged linker （six carbons） and piperidinyl ~roun on the side chains are beneficial to DNA binding and the anti-tumor activity.

Edaravone protects against oxygen-glucose-serum deprivation/restoration-induced apoptosis in spinal cord astrocytes by inhibiting integrated stress response

We previously found that oxygen-glucose-serum deprivation/restoration（OGSD/R） induces apoptosis of spinal cord astrocytes, possibly via caspase-12 and the integrated stress response, which involves protein kinase R-like endoplasmic reticulum kinase（PERK）, eukaryotic initiation factor 2-alpha（eIF2α） and activating transcription factor 4（ATF4）. We hypothesized that edaravone, a low molecular weight, lipophilic free radical scavenger, would reduce OGSD/R-induced apoptosis of spinal cord astrocytes. To test this, we established primary cultures of rat astrocytes, and exposed them to 8 hours/6 hours of OGSD/R with or without edaravone（0.1, 1, 10, 100 μM） treatment. We found that 100 μM of edaravone significantly suppressed astrocyte apoptosis and inhibited the release of reactive oxygen species. It also inhibited the activation of caspase-12 and caspase-3, and reduced the expression of homologous CCAAT/enhancer binding protein, phosphorylated（p）-PERK, p-eIF2α, and ATF4. These results point to a new use of an established drug in the prevention of OGSD/R-mediated spinal cord astrocyte apoptosis via the integrated stress response.

Human IgG1 C_γ1 Domain Is Crucial for the Bioactivity of the Engineered Anti-CD20 Antibodies

In this study, we discussed the necessity of human IgG1 Cγ1 domain for recombinant antibody using computeraided homology modeling method and experimental studies. The heavy （VH） and light （VL） chain variable regions of 1-28, a murine IgM-type anti-CD20 mAb, were ligated by linker peptide （Gly4Ser）3 to form the single-chain Fv fragment （scFv）. Then, the engineered antibody （LH1-3） was generated by fusing scFv with the entire IgG1 heavy constant regions. The 3-D structure of LH1-3 was modeled using computer-aided homology modeling method and the binding activity of LH1-3 was evaluated theoretically. Compared to the 3-D structure of the Fv fragment of the parent antibody, the conformation of the active pocket of LH1-3 was remained because of the rigid support of Cγ1. Further experimental results of flow cytometry showed that the engineered anti-CD20 antibody possessed specifically binding activity to CD20-expressing target cells. The anti-CD20 antibody fragments could also mediate complement-dependent cytotoxicity （CDC） of human B-lymphoid cell lines. Our study highlights some interests and advantages of a methodology based on the homology modeling and analysis of molecular structural properties.

Expression and Purification of Soluble Human Programmed Death-1 in Escherichia coli

Programmed death-1 （PD-1）, a member of CD28 family, is able to negatively regulate the TCR complex-initiated signaling by interacting with its cognate ligands （PD-L1 and/or PD-L2）. PD-1/PD-L1 pathway plays an important role in down-regulating the effective phase of adaptive immune responses and the blockade of this pathway has been proved to enhance antiviral and antitumoral immunity, suggesting that it might be a potential target for the development of therapies to improve T cell responses in patients with virus infections or malignancies. In present study, the extracellular domain of human PD-1 with a carboxyl terminal His-tag （designated as sPD-1） was expressed as inclusion bodies in Escherichia coll. The product was on-column refolded, purified by immobilized metal affinity chromatography, and characterized by Western blotting. Furthermore, the soluble PD-1 with high purity possessed specific binding activity with its cognate ligand PD-L1, and the dissociation constant was 0.43 nmol/L as determined by Scatchard plot analysis. These results suggest that refolded sPD-1 from prokaryotic cells may be of therapeutic interest in enhancing antivirus and antitumoral immune responses.

Adsorption and desorption of SO_2, NO and chlorobenzene on activated carbon

Activated carbon（AC） is very effective for multi-pollutant removal; however, the complicated components in flue gas can influence each other＇s adsorption. A series of adsorption experiments for multicomponents, including SO_2, NO, chlorobenzene and H2 O,on AC were performed in a fixed-bed reactor. For single-component adsorption, the adsorption amount for chlorobenzene was larger than for SO_2 and NO on the AC. In the multi-component atmosphere, the adsorption amount decreased by 27.6% for chlorobenzene and decreased by 95.6% for NO, whereas it increased by a factor of two for SO_2,demonstrating that a complex atmosphere is unfavorable for chlorobenzene adsorption and inhibits NO adsorption. In contrast, it is very beneficial for SO_2 adsorption. The temperature-programmed desorption（TPD） results indicated that the binding strength between the gas adsorbates and the AC follows the order of SO_2〉 chlorobenzene 〉 NO. The adsorption amount is independent of the binding strength. The presence of H2 O enhanced the component effects, while it weakened the binding force between the gas adsorbates and the AC. AC oxygen functional groups were analyzed using TPD and X-ray photoelectron spectroscopy（XPS） measurements. The results reveal the reason why the chlorobenzene adsorption is less affected by the presence of other components. Lactone groups partly transform into carbonyl and quinone groups after chlorobenzene desorption. The chlorobenzene adsorption increases the number of C = O groups, which explains the positive effect of chlorobenzene on SO_2 adsorption and the strong NO adsorption.

Discovery of novel inhibitors of anti-apoptotic Bcl-2 proteins derived from Bim BH3 domain

The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and/or modification are described. The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Of particular interest were the analogue SM-5（KD= 9.48 nmol/L for Bcl-2） and SM-6（KD= 0.08 nmol/L for Bcl-xL）, which exhibited improved binding affinity compared with the lead Bim（KD= 16.90 nmol/L for Bcl-2 and 22.2 nmol/L for Bcl-xL, respectively）. These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core.

Construction and Application of Efficient Ac-Ds Transposon Tagging Vectors in Rice

Transposons are effective mutagens alternative to T-DNA for the generation of insertional mutants in many plant species including those whose transformation is inefficient. The current strategies of transposon tagging are usually slow and labor-intensive and yield low frequency of tagged lines. We have constructed a series of transposon tagging vectors based on three approaches： （i） AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain （GVG） chemical-inducible expression system; （ii） deletion of AcTPase via Cre-lox site-specific recombination that was initially triggered by Ds excision; and （iii） suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element （HPT-Ds）, We tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful resources for functional genomics of rice and other crop plants. The vectors are freely available for the community,