Baosheng Ge

Enhanced photocurrent production by bio-dyes of photosynthetic macromolecules on designed TiO2 film.

The macromolecular pigment-protein complex has the merit of high efficiency for light-energy capture and transfer after long-term photosynthetic evolution. Here bio-dyes of A. platensis photosystem I (PSI) and spinach light-harvesting complex II (LHCII) are spontaneously sensitized on three types of designed TiO2 films, to assess the effects of pigment-protein complex on the performance of bio-dye sensitized solar cells (SSC). Adsorption models of bio-dyes are proposed based on the 3D structures of PSI and LHCII, and the size of particles and inner pores in the TiO2 film. PSI shows its merit of high efficiency for captured energy transfer, charge separation and transfer in the electron transfer chain (ETC), and electron injection from FB to the TiO2 conducting band. After optimization, the best short current (JSC) and photoelectric conversion efficiency (η) of PSI-SSC and LHCII-SSC are 1.31u2005mA cm-2 and 0.47%, and 1.51u2005mA cm-2 and 0.52%, respectively. The potential for further improvement of this PSI based SSC is significant and could lead to better utilization of solar energy.

Preparation of nitrocellulose (NC) immuno‐affinity membrane for purification of rAPC antibody

In this study, recombinant allophycocyanin (rAPC) with a purity of 98% was transferred from a gel to a nitrocellulose (NC) membrane to develop a simple and efficient immuno-affinity membrane. Atomic force microscopy (AFM) was used to investigate the surface topography of the affinity membrane and its characterization indicated that rAPC easily forms trimers or hexamers on the membrane surface on use of the given transfer method. The hydrodynamic radius (R(h)) of the rAPC aggregation was equal to 103 nm or 365 nm according to dynamic light scattering (DLS), which was in agreement with the result obtained by AFM. Based on the specific immunological reaction of antigen and antibody, anti-APC antibodies were purified from rabbit polyclonal serum in a single step. The amount of absorbed antibody was 5.79 mg/g membrane according to analysis by ELISA methods. The purity of antibodies was up to 98% according to SDS-PAGE. The adsorption-desorption cycle of rAPC was repeated six times using the same immuno-affinity membrane, and there was no significant loss in adsorption capacity. The method provides a novel and efficient immunological affinity membrane for the purification of antibodies.

Deep-Red Fluorescent Gold Nanoclusters for Nucleoli Staining: Real-Time Monitoring of the Nucleolar Dynamics in Reverse Transformation of Malignant Cells

Nucleoli are important subnuclear structures inside cells. We report novel fluorescent gold nanoclusters (K-AuNCs) that are able to stain the nucleoli selectively and make it possible to explore the nucleolar morphology with fluorescence imaging technique. This novel probe is prepared through an easy synthesis method by employing a tripeptide (Lys-Cys-Lys) as the surface ligand. The properties, including deep-red fluorescence emission (680 nm), large Stocks shift, broad excitation band, low cytotoxicity, and good photostability, endow this probe with potential for bioanalytical applications. Because of their small size and their positively charged surface, K-AuNCs are able to accumulate efficiently at the nucleolar regions and provide precise morphological information. K-AuNCs are also used to monitor the nucleolar dynamics along the reverse-transformation process of malignant cells, induced by the agonist of protein A, 8-chloro-cyclic adenosine monophosphate. This gives a novel approach for investigating the working mechanism of antitumor drugs.

Effect of surfactants on apparent oxygen consumption of photosystem I isolated from Arthrospira platensis

Surfactants play a significant role in solubilization of photosystem I (PSI) in vitro. Triton X-100 (TX), n-Dodecyl-β-d-maltoside (DDM), and sodium dodecyl sulfate (SDS) were employed to solubilize PSI particles in MES buffer to compare the effect of surfactant and its dosage on the apparent oxygen consumption rate of PSI. Through a combined assessment of sucrose density gradient centrifugation, Native PAGE and 77xa0K fluorescence with the apparent oxygen consumption, the nature of the enhancement of the apparent oxygen consumption activity of PSI by surfactants has been analyzed. Aggregated PSI particles can be dispersed by surfactant molecules into micelles, and the apparent oxygen consumption rate is higher for surfactant-solubilized PSI than for integral PSI particles. For DDM, PSI particles are solubilized mostly as the integral trimeric form. For TX, PSI particles are solubilized as incomplete trimeric and some monomeric forms. For the much harsher surfactant, SDS, PSI particles are completely solubilized as monomeric and its subunit forms. The enhancement of the oxygen consumption rate cannot be explained only by the effects of surfactant on the equilibrium between monomeric and trimeric forms of solubililized PSI. Care must be taken when the electron transfer activity of PSI is evaluated by methods based on oxygen consumption because the apparent oxygen consumption rate is influenced by uncoupled chlorophyll (Chl) from PSI, i.e., the larger the amount of uncoupled Chl, the higher the rate of apparent oxygen consumption. 77xa0K fluorescence spectra can be used to ensure that there is no uncoupled Chl present in the system. In order to eliminate the effect of trace uncoupled Chl, an efficient physical quencher of 1O2, such as 1xa0mM NaN3, may be added into the mixture.

Single-Molecule Imaging Demonstrates Ligand Regulation of the Oligomeric Status of CXCR4 in Living Cells

The role of dimerization and oligomerization of G-protein-coupled receptors in their signal transduction is highly controversial. Delineating this issue can greatly facilitate rational drug design. With single-molecule imaging, we show that chemokine receptor CXCR4 exists mainly as a monomer in normal mammalian living cells and forms dimers and higher-order oligomers at a high expression level, such as in cancer cells. Chemotaxis tests demonstrate that the signal transduction activity of CXCR4 does not depend only on its expression level, indicating a close relation with the oligomeric status of CXCR4. Moreover, binding ligands can effectively upregulate or downregulate the oligomeric level of CXCR4, which suggests that binding ligands may realize their pivotal roles by regulating the oligomeric status of CXCR4 rather than by simply inducing conformational changes.

A novel membrane based process to isolate photosystem-I membrane complex from spinach

The isolation of photosystem-I (PS-I) from spinach has been conducted using ultrafiltration with 300xa0kDa molecular weight cut-off polyethersulfone membranes. The effects of ultrafiltration operating conditions on PS-I activity were optimized using parameter scanning ultrafiltration. These conditions included solution pH, ionic strength, stirring speed, and permeate flux. The effects of detergent (Triton X-100 and n-dodecyl-beta-D-maltoside) concentration on time dependent activity of PS-I were also studied using an O2 electrode. Under optimized conditions, the PS-I purity obtained in the retentate was about 84% and the activity recovery was greater than 94% after ultrafiltration. To our knowledge, this is the first report of the isolation of a membrane protein using ultrafiltration alone.

Single-molecule imaging reveals dimerization/oligomerization of CXCR4 on plasma membrane closely related to its function

Dimerization and oligomerization of G-protein coupled receptors (GPCRs) have emerged as important characters during their trans-membrane signal transduction. However, until now the relationship between GPCR dimerization and their trans-membrane signal transduction function is still uncovered. Here, using pertussis toxin (PTX) to decouple the receptor from G protein complex and with single-molecule imaging, we show that in the presence of agonist, cells treated with PTX showed a decrease in the number of dimers and oligomers on the cell surface compared with untreated ones, which suggests that oligomeric status of CXCR4 could be significantly influenced by the decoupling of G protein complex during its signal transduction process. Moreover, with chlorpromazine (CPZ) to inhibit internalization of CXCR4, it was found that after SDF-1α stimulation, cells treated with CPZ showed more dimers and oligomers on the cell surface than untreated ones, which suggest that dimers and oligomers of CXCR4 tend to internalize more easily than monomers. Taken together, our results demonstrate that dimerization and oligomerization of CXCR4 is closely related with its G protein mediated pathway and β-arrestin mediated internalization process, and would play an important role in regulating its signal transduction functions.

High-Density Super-Resolution Localization Imaging with Blinking Carbon Dots

Molecular fluorescence blinking provides a simple and attractive way to achieve super-resolution localization via conventional fluorescence microscopy. However, success in super-resolution imaging relies heavily on their blinking characteristics. We here report easily prepared and photostable nanoparticles, carbon dots (CDs), with desirable fluorescence blinking for high-density super-resolution imaging. The CDs exhibit a low duty cycle (∼0.003) and high photon output (∼8000) per switching event, as well as show much higher resistance to photobleaching than Alexa 647 or Cy5 typically used in single molecule localization microscopy. The stable blinking of CDs allows to perform high-density localization imaging at a resolution of 25 nm by sequentially recording the particle positions. The CD-based super-resolution imaging is further demonstrated by rendering CD-stained tubular peptide self-assemblies, CD-packed clusters with well-defined patterns, and CD-stained microtubules in a cell. Furthermore, this method has been validated as a valuable tool to detect the clustering and distribution of protein receptors in the plasma membrane that are not discerned with normal fluorescence imaging.

Regulation of the Oligomeric Status of CCR3 with Binding Ligands Revealed by Single-Molecule Fluorescence Imaging

The relationship between the oligomeric status and functions of chemokine receptor CCR3 is still controversial. We use total internal reflection fluorescence microscopy at the single-molecule level to visualize the oligomeric status of CCR3 and its regulation of the membrane of stably transfected T-REx-293 cells. We find that the population of the dimers and oligomers of CCR3 can be modulated by the binding of ligands. Natural agonists can induce an increase in the level of dimers and oligomers at high concentrations, whereas antagonists do not have a significant influence on the oligomeric status. Moreover, monomeric CCR3 exhibits a stronger chemotactic response in the migration assay of stably transfected CCR3 cells. Together, these data support the notion that CCR3 exists as a mixture of monomers and dimers under nearly physiological conditions and the monomeric CCR3 receptor is the minimal functional unit in cellular signaling transduction. To the best of our knowledge, these results constitute the first report of the oligomeric status of CCR3 and its regulation.

Functional expression of CCL8 and its interaction with chemokine receptor CCR3

BackgroundChemokines and their cognate receptors play important role in the control of leukocyte chemotaxis, HIV entry and other inflammatory diseases. Developing an effcient method to investigate the functional expression of chemokines and its interactions with specific receptors will be helpful to asses the structural and functional characteristics as well as the design of new approach to therapeutic intervention.ResultsBy making systematic optimization study of expression conditions, soluble and functional production of chemokine C-C motif ligand 8 (CCL8) in Escherichia coli (E. coli) has been achieved with approx. 1.5xa0mg protein/l culture. Quartz crystal microbalance (QCM) analysis exhibited that the purified CCL8 could bind with C-C chemokine receptor type 3 (CCR3) with dissociation equilibrium constant (KD) as 1.2u2009×u200910−7xa0M in vitro. Obvious internalization of CCR3 in vivo could be detected in 1xa0h when exposed to 100xa0nM of CCL8. Compared with chemokine C-C motif ligand 11 (CCL11) and chemokine C-C motif ligand 24 (CCL24), a weaker chemotactic effect of CCR3 expressing cells was observed when induced by CCL8 with same concentration.ConclusionThis study delivers a simple and applicable way to produce functional chemokines in E. coli. The results clearly confirms that CCL8 can interact with chemokine receptor CCR3, therefore, it is promising area to develop drugs for the treatment of related diseases.