Vascular endothelial growth factor receptor 2 (VEGFR-2) plays a critical role in tumor angiogenesis. None therapeutic antibodies targeting VEGFR-2 are available in clinical use. Herein, we describe the screening of a new single-chain antibody fragment (scFv) targeting extracellular domain 3 of human VEGFR-2 (KDR3) from Griffin phage display scFv library. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions. The scFv exerted particular binding sites to KDR3 on Molecular Docking, and the binding affinity was further convinced by binding analysis both in quantitative ELISA and real-time kinetic determination by biosensors (K(D)= 40 nM).Finally, the scFv was revealed to inhibit VEGF-stimulated proliferation of human umbilical vein endothelial cells (HUVECs)(IC(50)= 5 nM) and to inhibit HUVEC migration significantly at 17 nM. Taken together, our results indicate that we have successfully isolated a scFv which differentially recognizes KDR3 and has potential clinical applications in the treatment of angiogenesis related diseases. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012.

A family of water/oil interfaces is introduced to provide effective platforms for rapid fabrication of large-area self-assembled nanofilms composed of various nanosized building blocks, including nanoparticles (NPs), nanocubes (NC), nanowires (NWs), and nanosheets, at room temperature. As a general interfacial assembly method, NWs and NPs are co-assembled at the liquid/liquid interface. The as-prepared co-assembled Ag NW and Ag NC films show high surface-enhanced Raman spectroscopy (SERS) intensity, the SERS performance being strongly dependent on the number ratio of the two kinds of nanosized building blocks. The results demonstrate that this interfacial system provides a general method for the assembly of various nanosized building blocks with different shapes and dimensionalities, and thus paves an alternative pathway for further applications of macroscopic assemblies with different functionalities.

Summary:  Operation of the immune system critically depends on intercellular communication among multiple cell types, frequently in the form of physical cell-cell interactions. Germinal centers (GCs) are highly organized tissue microdomains in which high affinity, class-switched, antibody-producing cells and humoral immune memory are generated. Critical underlying cell-cell interaction events include at the minimum binary interactions between CD4(+) T-helper cells and antigen-presenting dendritic cells (DCs), which ensure proper T-cell activation and acquisition of effecter potentials, and those between T-helper cells and antigen-activated B cells whereby the latter cells receive helper signals (e.g. CD40L) important for their proliferation, survival, and differentiation. How these critical cellular interaction events are molecularly regulated and dynamically orchestrated to support GC formation and function is still a study in progress. Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) has recently been defined as a pivotal molecule that controls cognate T-B interactions and GC formation. Detailed analysis of interaction and migration dynamics of SAP-deficient T cells has raised the interesting possibility that T cell:antigen-presenting cell interactions underlying GC development and function are regulated in a cell type- and spatiotemporal stage-specific manner. This has important implications for our understanding of synapse formation, helper signal delivery to B cells, follicular helper T-cell differentiation, and quality control of the GC reaction in general. A model of selective T-B interactions involving bi-directional feedback and feed-forward logic is proposed to underlie GC development and function.

Influenza A viruses, the pathogens responsible for the recent swine flu outbreak and many historical pandemics, remain a threat to the public health. We report herein the fabrication of self-disinfecting surfaces from photoactive building nanocrystals, which can inactivate influenza viruses rapidly, spontaneously and continuously under visible light illumination.

A new methodology is developed to conjugate hyaluronic acid (HA) hydrogel with novel nano-fibrous architectures via non-covalent assembly that specifically allows for targeted adipose-derived stem cells (ASCs) differentiation and soft tissue engineering. The assembly of non-covalently associated hydrogel network produced via the interaction of a low molecular weight heparin (LMWH) modified HA derivative and heparin interacting protein (HIP). The multifunctional star poly(ethylene glycol)(PEG) and HIP copolymer has the capability to mediate the non-covalent assembly of nano-fibrous HA hydrogel networks via affinity interactions with LMWH. The effect of the HIP mediation on in vitro gelation, rheological characteristics, degradation, equilibrium swelling, adipose-derived stem cells (ASCs) proliferation and differentiation of nano-fibrous hydrogel is examined. The results suggest the potential utility of this unique design of the bioactive nano-fibrous HA hydrogel in directing the differentiation of ASCs and adipogenesis in ECM-mimetic scaffolds in vitro. These studies demonstrate that this nano-fibrous HA hydrogel can render the formulation of a therapeutically effective platform for in vitro adipogenesis applications.

In this study, we evaluated the ability of 8.8 mT static magnetic fields (SMF) to enhance the in vitro action of a chemotherapeutic agent, paclitaxel, against K562 human leukemia cells. We analyzed the cell proliferation, cell cycle distribution, DNA damage and alteration of cell surface and cell organelle ultrastructure after K562 cells were exposed to paclitaxel in the presence or absence of 8.8 mT SMF. The results showed that in the presence of SMF, the efficient concentration of paclitaxel on K562 cells was decreased from 50 to 10 ng/ml. Cell cycle analysis indicated that K562 cells treated with SMF plus paclitaxel were arrested at the G2 phase, which was mainly induced by paclitaxel. Through comet assay, we found that the cell cycle arrest effect of paclitaxel with or without SMF on K562 cells was correlated with DNA damage. The results of atomic force microscopy and transmission electron microscopy observation showed that the cell ultrastructure was altered in the group treated with the combination of SMF and paclitaxel, holes and protuberances were observed, and vacuoles in cytoplasm were augmented. Our data indicated that the potency of the combination of SMF and paclitaxel was greater than that of SMF or paclitaxel alone on K562 cells, and these effects were correlated with DNA damage induced by SMF and paclitaxel. Therefore, the alteration of cell membrane permeability may be one important mechanism underlying the effects of SMF and paclitaxel on K562 cells.

We investigated the molecular mechanisms underlying phloxine B (PhB)-induced photocytotoxicity in human T lymphocytic leukemia Jurkat cells. In addition to apoptosis-related biochemical events, photo-irradiated PhB generated intracellular reactive oxygen species (ROS), induced phosphorylation of c-Jun-N-terminal kinase (JNK) in an oxidative stress-dependent manner and up-regulated the gene expression of interferon (IFN)-γ, an inducer of diverse apoptosis-related molecules in activated T cells. PhB-induced apoptosis was significantly inhibited by N-acetyl-l-cysteine, but not by catalase, indicating that ROS generation occurred intracellularly, and by SP600125 and AG490, specific inhibitors of JNK and IFN-γ signaling, respectively, confirming their roles in the apoptotic pathway. IFN-γ up-regulation was also inhibited by SP600125, indicating that it was downstream of JNK activation. These results suggest that PhB-induced apoptosis in Jurkat cells partially involves the intracellular oxidative stress-sensitive and T cell-specific IFN-γ pathway. These data present a novel insight into the mechanisms of photocytotoxicity induced by artificial food colorants in human T lymphocytic leukemia cells.

The M(3) subtype of muscarinic acetylcholine receptors (M(3)-mAChR) plays a protective role in myocardial ischemia and microRNAs (miRNAs) participate in many cardiac pathophysiological processes, including ischemia-induced cardiac injury. However, the role of miRNAs in M(3)-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M(3)-mAChR against myocardial ischemia and elucidate the underlying mechanisms. We established rat model of myocardial ischemia and performed miRNA microarray analysis to identify miRNAs involved in the cardioprotection of M(3)-mAChR. In H9c2 cells, the viability, intracellular free Ca(2+) concentration ([Ca(2+)]i), intracellular reactive oxygen species (ROS), miR-376b-5p expression level, brain derived neurophic factor (BDNF) and nuclear factor kappa-B (NF-κB) levels were measured. Our results demonstrated that M(3)-mAChR protected myocardial ischemia injury. Microarray analysis and qRT-PCR revealed that miR-376b-5p was significantly up-regulated in ischemic heart tissue and the M(3)-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H(2)O(2)-induced H9c2 cell injuries measured by cells viability,[Ca(2+)]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M(3)-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M(3)-mAChR/NF-κB/miR-376b-5p/BDNF axis plays an important role in modulating cardioprotection. MiR-376b-5p promotes myocardial ischemia injury possibly by inhibiting BDNF expression and M(3)-mAChR provides cardioprotection at least partially mediated by the downregulation of miR-376b-5p through NF-κB. These findings provide new insight into the potential mechanism by which M(3)-mAChR provides cardioprotection against myocardial ischemia injury.

Recent work has identified a new subset of CD4+ T cells named as Tfh cells that are localized in germinal centers and critical in germinal-center formation. Tfh cell differentiation is regulated by IL-6 and IL-21, possibly via STAT3 factor, and B-cell lymphoma 6 (Bcl6) is specifically expressed in Tfh cells and required for their lineage specification. In current study, we characterized the role of STAT5 in Tfh cell development. We found that a constitutively active form of STAT5 effectively inhibited Tfh differentiation by suppressing the expression of Tfh-associated factors (C-X-C motif) receptor 5 (CXCR5),(musculoaponeurotic fibrosarcoma (c-Maf), Bcl6, basic leucine zipper transcription factor (Batf) and IL-21, and STAT5 deficiency greatly enhanced Tfh gene expression. Importantly, STAT5 regulated the expression of Tfh cell suppressor factor B lymphocyte-induced maturation protein 1 (Blimp-1); STAT5 deficiency impaired Blimp-1 expression and resulted in elevated expression of Tfh specific genes. Similarly, inhibition of IL-2 potentiated Tfh generation, associated with dampened Blimp-1 expression; Blimp-1 overexpression inhibited Tfh gene expression in Stat5-deficient T cells, suggesting that IL-2/STAT5 axis functions to regulate Blimp-1 expression. In vivo, deletion of STAT5 in CD4+ T cells resulted in enhanced development of Tfh cells and germinal center B cells, and led to an impairment of B cell tolerance in a well-defined mouse tolerance model. Taken together, this study demonstrates that STAT5 controls Tfh differentiation.

4-(1H-benzimidazol-2-yl)benzaldehyde (1) has been developed as a new ratiometric fluorescent probe for bisulphite, based on the modulation of intramolecular charge transfer (ICT). Upon mixing with bisulphite in aqueous ethanol, an aldehyde-bisulphite adduct was formed and the ICT of the probe was switched off, which resulted in a ratiometric fluorescence response with an enhancement of the ratios of emission intensities at 368 and 498 nm. The detection range of the probe for bisulphite is in the 2.0-200 µmol/L concentration range and the detection limit is 0.4 µmol/L. Probe 1 produces a ratiometric fluorescent response to bisulphite with a marked emission wavelength shift (130 nm) and displays high selectivity for bisulphite over other anions. Copyright © 2012 John Wiley & Sons, Ltd.