Rapid synthesis of BaMoO4 :Eu3+ red phosphors using microwave irradiation.

This study synthesized BaMoO4:Eu3+ red phosphors using the microwave method. In addition, the phase composition, morphology, and luminescence properties of the red phosphors were characterized using X-ray diffraction, field-scanning electron microscopy, and photoluminescence spectroscopy. The results revealed that doping red phosphors with different concentrations of Eu3+ does not change the crystal structure of the matrix material. The BaMoO4 :Eu3+ phosphors exhibited micron-scale irregular polyhedra, which could be excited by ultraviolet light with a wavelength of 395 nm to induce red-light emission. The optimal dosage of Eu3+ was 0.08, and the chromaticity coordinates of BaMoO4 :0.08Eu3+ phosphors were (0.5869, 0.3099). White light-emitting diode (w-LED) devices manufactured by using a combination of BaMoO4 :0.08Eu3+ phosphor and commercially available phosphors exhibited good white-light emission under the excitation of an ultraviolet chip. The BaMoO4 :0.08Eu3+ red phosphors that rapidly synthesized under the microwave field are expected to be used in w-LED devices.

Development of a concentration method for detection of tobacco mosaic virus in irrigation water.

Tobacco mosaic virus (TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/µL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000 (PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 10(2) viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.

Rapid detection of tobacco viruses by reverse transcription loop-mediated isothermal amplification.

Tobacco viruses may cause a wide range of diseases that heavily reduce tobacco quality and yield worldwide. In order to detect viral diseases in tobacco fields, a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established. Nucleotide amplification could be observed clearly after adding SYBR Green I, within 60 min under isothermal conditions, at 63-65 °C with a set of primers targeting the viral coat protein (CP) genes of tobacco viruses including cucumber mosaic virus (CMV), potato virus Y (PVY), tobacco etch virus (TEV), tobacco mosaic virus (TMV) and tobacco vein banding mosaic virus (TVBMV). This method has high specificity and sensitivity. The sensitivity of the RT-LAMP was 10 to 100 times higher than that of the conventional RT-PCR method. The RT-LAMP assay was proven reliable for virus diagnosis of tobacco samples from the field.

Plasma membrane associated transcription of cytoplasmic DNA.

Cytoplasmic membrane-associated DNA (cmDNA) is a species of DNA that attaches to the plasma membrane and has physical and chemical properties that differ from those of bulk chromosomal and mitochondrial DNAs. Here, we used deep sequencing to analyze cmDNA and showed that satellite DNAs consisting of both of simple (CCATT)(N) repeats from the pericentromere regions of the chromosomes and 171-bp α-satellite repeat sequences from centromeres were highly enriched. Importantly, we found there is a special cytoplasmic membrane-associated transcription system in which DNA-dependent RNA polymerase II, which colocalizes with template cmDNA at the plasma membrane, can transcribe the membrane-associated 171-bp α-satellite repeat sequences into RNA. Analysis of phosphorylation patterns indicated that the RNA polymerase II in the plasma membrane is in a different chemical state from its nuclear counterpart.

The costimulatory immunogen LPS induces the B-Cell clones that infiltrate transplanted human kidneys.

The mechanism of chronic rejection of transplanted human kidneys is unknown. An understanding of this process is important because, chronic rejection ultimately leads to loss of the kidney allograft in most transplants. One feature of chronic rejection is the infiltration of ectopic B-cell clusters that are clonal into the transplanted kidney. We now show that the antibodies produced by these B-cells react strongly with the core carbohydrate region of LPS. Since LPS is a costimulatory immunogen that can react with both the B-cell receptor (BCR) and the Toll-like receptor 4 (TLR4), these results suggest a mechanism for the selective pressure that leads to clonality of these B-cell clusters and opens the possibility that infection and the attendant exposure to LPS plays a role in the chronic rejection of human kidney transplants. If confirmed by clinical studies, these results suggest that treating patients with signs of chronic rejection with antibiotics may improve kidney allograft survival.

A multiplex reverse transcription PCR assay for simultaneous detection of five tobacco viruses in tobacco plants.

Tobacco viruses including Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Tobacco etch virus (TEV), Potato virus Y (PVY) and Tobacco vein banding mosaic virus (TVBMV) are major viruses infecting tobacco and can cause serious crop losses. A multiplex reverse transcription polymerase chain reaction assay was developed to detect simultaneously and differentiate all five viruses. The system used specific primer sets for each virus producing five distinct fragments 237, 273, 347, 456 and 547 bp, representing TMV, CMV subgroup I, TEV, PVY(O) and TVBMV, respectively. These primers were used for detection of the different viruses by single PCR and multiplex PCR and the results were confirmed by DNA sequencing analysis. The protocol was used to detect viruses from different parts of China. The simultaneous and sensitive detection of different viruses using the multiplex PCR is more efficient and economical than other conventional methods for tobacco virus detection. This multiplex PCR provides a rapid and reliable method for the detection and identification of major tobacco viruses, and will be useful for epidemiological studies.

Ectopic B-cell clusters that infiltrate transplanted human kidneys are clonal.

B cells and their immunoglobulin products participate in allograft rejection of transplanted human kidneys in which an interesting feature is the presence of a germinal center like B-cell clusters in the allograft. We report here that the immunoglobulin repertoires of these infiltrating B cells are highly restricted and the B cells within a cluster are clonal. Antibody libraries made from the infiltrating B cells of individual patients unexpectedly revealed that each patient utilizes a particular set of dominant germ line genes as well as dominant complementarity determining region 3. Comparison of kidney and peripheral blood from the same patient showed that the immunoglobulin genes from both compartments had dominant clones, but they differed. The lymphocytes that infiltrate the kidneys express the immunoglobulin gene somatic recombination machinery usually restricted to highly activated lymphocytes in germinal centers and lymphomas. An analogy can be made between the inescapable antigenic drive in chronic infection versus that in an allograft, both of which may lead to emergence of dominant B-cell clones and even lymphoid malignancy.

A new format of single chain tri-specific antibody with diminished molecular size efficiently induces ovarian tumor cell killing.

A combination of bi-specific antibodies (BsAb), anti-tumorxanti-CD3 and anti-tumorxanti-CD28, is effective in vitro and in vivo, whereas production of two kinds of bi-specific antibodies is labor intensive and administration is complicated. Accordingly, we previously developed a new model of single chain tri-specific antibody (scTsAb), sTRI, which linked both the CD3 and CD28 signals for T-cell activation in one molecule, and demonstrated its capacity for triggering T-cells to kill ovary tumor cells. To improve the pharmacokinetics further and decrease the immunogenicity of scTsAb, we have now generated a new format of scTsAb, TR3H, whose molecular size is smaller than sTRI. Here we describe the construction, purification and characterization of TR3H. TR3H scTsAb bound to effector cells and tumor target cells specifically and induced redirected lyses of ovary tumor cells through freshly isolated, unstimulated human peripheral blood lymphocytes (PBLs). This new format of scTsAb possesses properties that support its potential as a new tumor immunotherapeutic agent.

A new model of trispecific antibody resulting the cytotoxicity directed against tumor cells.

Bispecific antibodies (BsAb) with specificity to both tumor cells and CD3 molecule were believed to be promising immunological tools for the therapy with minimal residual diseases by activating cytotoxic T cells. However, without costimulatory molecule CD28, the activated T cells tended to apoptosis. In order to kill tumor cells more efficiently, a recombinant multifunctional single-chain trispecific antibody (scTsAb), which contains anti-ovarian carcinoma (OC) scFv, anti-CD3 scFv and VH domain of anti-CD28 antibody, was constructed and expressed in E. coli BL21 Star strain. The scTsAb showed strong binding avidities to membrane antigen of SK-OV-3 cell, CD3 molecule on Jurkat cell, and recombinant CD28 antigen. It was further demonstrated that this scTsAb could activate peripheral blood T cells to elicit strong cytotoxicity against SK-OV-3 cells. This new type of recombinant scFv antibody set up a new technological platform for T cells based immunotherapy against cancer, especially with the failure on MHC antigen presentation or absence of costimulating signal.

Construction and expression of a reshaped VH domain against human CD28 molecules.

Compared with the amino acid sequence of a mouse anti-human CD28 VH domain antibody, the two most homologous sequences of human antibodies were pulled out from Genbank. One of them was used as the main template for the framework regions of the reshaped VH domain. While the original mouse antibody CDRs were inserted into the human acceptor FRs, some residues in human acceptor FRs, which were different from those of the original mouse FRs in corresponding positions, were then determined or, alternatively, mutagenized to their conservative properties in kappa classification. Based on the amino acid sequences of the designed VH domain, the nucleotide sequence was deduced by using E. coli bias codons. The sequence was split into ten 30 to 60 nucleotide fragments for synthesizing, then annealed and amplified by overlap PCR. Taq DNA polymerase was used in a buffer with high Mg2+ concentration to induce more random mutations, both in FRs and CDRs. A phage display library was constructed by cloning these PCR products. After three rounds of panning, several reshaped VH with high antigen binding activity were obtained. One of them had the same CDR amino acid sequences as that of the original mouse VH domain. Further study showed that it retained a high antigen binding affinity after being expressed in E. coli BL21 (DE3).

A method for constructing reshaping single-domain antibody.

The aim of this research was to demonstrate a novel and practical method for constructing reshaping Single-domain antibodies. Different from other methods, our method does not need to model the configuration of antibodies with specific sequences to determine the sequences of human acceptor FRs and then determine which amino acid residues in human acceptor FRs should be substituted. Most importantly, reshaping and enhancing the antigen binding affinity shared one procedure at the same time. Using this method, the reshaping anti-CD28 single-domain antibodies were constructed. According to the amino acid sequence of a mouse anti-human CD28 monoclonal antibody VH, two most homologous sequences of human antibodies were selected from GenBank and one of them was used as a main framework region for constructing the reshaping antibody. Before the original mouse antibody CDRs were inserted into the human acceptor FRs, some amino acid residues which were different from those of the original mouse antibody in the corresponding positions of the human acceptor FRs were determined or alternatively mutated by their conservative properties in Kabat classification. When the synthesized nucleotide fragments in different length were spliced by overlap PCR into the entire reshaping genes, Taq DNA polymerase and high Mg2+ concentration were used to introduce more mutation in FRs and CDRs randomly. A phage library was constructed using these PCR products and several reshaping Single-domain antibodies with high antigen binding affinity were selected after three rounds of panning. Two of them were expressed in E. coli BL21 (DE3). The antigen-binding affinity of refolded proteins was still in a high level measured by ELISA. These results suggested that this method was feasible and efficient for constructing reshaping Single-domain antibodies.