Fusions of Dendritic Cells and C6 Cells Transfected with TGF-β1 Antisense in Treatment of Intracranial Gliomas

Objective： To investigate the immunotherapy efficacy of fusion cells （dendritic-C6anti-TGF-β1 cells） in the treatment of intraeranial gliomas. Methods： Dendritic cells were isolated from rat bone-marrow precursors stimulated in vitro with granuloeyte-macrophage colony-stimulating factor （GM-CSF） and Interleukin-4 （IL-4）. C6anti-TGF-β1 cells originally from C6 cell line of a rat glioblastoma were transfected with plasmid of TGF-β1 anti-sense gene. Fusions of dendritic cells and C6anti-TGF-β1 cells were prepared by polyethylene glycol （PEG）. The DC/C6anti-TGF-β1 fusion cells were observed and confirmed by fight microscopy and scanning electron microscopy. Experimental rats were divided into three groups at random： C6 cells （Ⅰ）, dendritic-C6anti-TGF-β1 fusion cells and C6 cells （Ⅱ） and IMDM medium only （Ⅲ）. The cells were injected into right parietal lobe region of the rat with stereotaxic technique. Histology, tumor necrosis and survival time were evaluated. Results： Compared with the rats that received C6 cells （survival median time was less than 20 days, tumor region was seen in all fields of observed）, the rats injected with dendritic-C6anti-TGF-β1 fusion cells and C6 cells got a more prolonged life span （more than 59 days）, as well as less tumor region （5.01%-6.2%）. There was no tumor necrosis, but some glias were seen in surroundings. All rats were survived and no necrosis was observed in negative control group. Statistical analysis showed that group Ⅱ had significant difference compared with group Ⅰ. Conclusions： Dendritic-C6anti-TGF-β1 fusion cells could prolong the life span of rats, providing a strategy to achieve an antitumor response against tumors in the central nervous system.

Anti-tumor effects of p53N15-based fusion peptide in the transfected H1299 lung cancer cells

Objective Loss-of-function mutation of p53,a tumor suppressor gene,is an important mechanism for the development of human cancers. In this study we tried to transfect p53N15-based fusion peptide into H1299,a lung cancer cell line,and evaluate the anti-tumor effects of the fusion peptide. Methods Adeno-associated virus (AAV) vectors were used for transfecting p53N15 fusion peptide into p53-null lung adenocarcinoma H1299 cells.

Investigating Müller glia reprogramming in mice: a retrospective of the last decade, and a look to the future

Müller glia,as prominent glial cells within the retina,plays a significant role in maintaining retinal homeostasis in both healthy and diseased states.In lower vertebrates like zebrafish,these cells assume responsibility for spontaneous retinal regeneration,wherein endogenous Müller glia undergo proliferation,transform into Müller glia-derived progenitor cells,and subsequently regenerate the entire retina with restored functionality.Conversely,Müller glia in the mouse and human retina exhibit limited neural reprogramming.Müller glia reprogramming is thus a promising strategy for treating neurodegenerative ocular disorders.Müller glia reprogramming in mice has been accomplished with remarkable success,through various technologies.Advancements in molecular,genetic,epigenetic,morphological,and physiological evaluations have made it easier to document and investigate the Müller glia programming process in mice.Nevertheless,there remain issues that hinder improving reprogramming efficiency and maturity.Thus,understanding the reprogramming mechanism is crucial toward exploring factors that will improve Müller glia reprogramming efficiency,and for developing novel Müller glia reprogramming strategies.This review describes recent progress in relatively successful Müller glia reprogramming strategies.It also provides a basis for developing new Müller glia reprogramming strategies in mice,including epigenetic remodeling,metabolic modulation,immune regulation,chemical small-molecules regulation,extracellular matrix remodeling,and cell-cell fusion,to achieve Müller glia reprogramming in mice.

Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells

Objective; Fusogenic endogenous retroviral syncytin plays an important role in the formation of syncytiotrophoblasts in human placenta.Apart from its expression in placenta,brain and testis,syncytin has also been found in many cancers.Although syncytin has been proposed to serve as a positive prognostic marker in some cancers,the underlying mechanism is unclear.The aim of this study is to evaluate the effects of syncytin expression on the invasive phenotype of melanoma cells.Methods：The eukaryotic expression plasmid for syncytin-EGFP was constructed and transfected into B16F10 melanoma cells.The effect of syncytin on the invasion potential of rumor cells was evaluated in B16F10 subline cells that stably expressed syncytin-EGFP fusion protein or EGFP alone.Results：The B16F10 sublines that stably expressed syncytin-EGFP or EGFP alone were established respectively and confirmed by immunofluorescent and immtmoblotting assay.Syncytin expression in B16F10 cells was associated with decreased cell proliferation,migration and invasion.Multinucleated giant cells that contained as many as five nuclei were induced in syncytin-expressing cells.In addition,syncytin expression did not alter the sensitivity of B16F10 cells to trichosanthin,a toxin that damages syncytiotrophoblasts more efficiently than other tissues.Conclusions：These results suggest that syncytin expression in some cancers may confine their invasion potential and thus serve as a positive prognostic factor.

Roles of cell fusion, hybridization and polyploid cell formation in cancer metastasis

Cell-cell fusion is a normal biological process playing essential roles in organ formation and tissue differentiation,repair and regeneration.Through cell fusion somatic cells undergo rapid nuclear reprogramming and epigenetic modifications to form hybrid cells with new genetic and phenotypic properties at a rate exceeding that achievable by random mutations.Factors that stimulate cell fusion are inflammation and hypoxia.Fusion of cancer cells with non-neoplastic cells facilitates several malignancy-related cell phenotypes,e.g.,reprogramming of somatic cell into induced pluripotent stem cells and epithelial to mesenchymal transition.There is now considerable in vitro,in vivo and clinical evidence that fusion of cancer cells with motile leucocytes such as macrophages plays a major role in cancer metastasis.Of the many changes in cancer cells after hybridizing with leucocytes,it is notable that hybrids acquire resistance to chemo-and radiation therapy.One phenomenon that has been largely overlooked yet plays a role in these processes is polyploidization.Regardless of the mechanism of polyploid cell formation,it happens in response to genotoxic stresses and enhances a cancer cell’s ability to survive.Here we summarize the recent progress in research of cell fusion and with a focus on an important role for polyploid cells in cancer metastasis.In addition,we discuss the clinical evidence and the importance of cell fusion and polyploidization in solid tumors.

Modeling biomembranes and red blood cells by coarse-grained particle methods

In this work, the previously developed coarse-grained （CG） particle models for biomembranes and red blood cells （RBCs） are reviewed, and the advantages of the CG particle methods over the continuum and atomistic simulations for modeling biological phenomena are discussed. CG particle models can largely increase the length scale and time scale of atomistic simulations by eliminating the fast degrees of freedom while preserving the mesoscopic structures and properties of the simulated system. Moreover, CG particle models can be used to capture the microstructural alternations in diseased RBCs and simulate the topological changes of biomembranes and RBCs, which are the major challenges to the typical continuum representations of membranes and RBCs. The power and versatility of CG particle methods are demonstrated：through simulating the dynamical processes mvolving significant topological .changes e.g. lipid self-assembly vesicle fusion and membrane budding.

TRANSFECTION AND CELL FUSION BY FEMTOSECOND LASERS

Biophotonics is an exciting and fast-expanding frontier which involves the fusion of advanced photonics and biology.It has not only created many novel methodologies for biomedical research,but also achieved many significant results as an independentfield.Thanks to femtosecond(fs)laser technologies,important progresses have been made regarding the manipulation,imaging,and engineering of biological samples ranging from single molecules to tissues in the last 20 years.The ultrashort pulses at near-infrared band provide many advantages:high nonlinear efficiency,low absorption by biological samples,high spatial and temporal resolution and confinement,and low phototoxicity.They are noninvasive and easy to control.Although the mechanism of how fs laser pulses interact with cells remains unclear,experimental results have shown that they could open up the cell membrane and hence made optical transfection and optical cell fusion possible.In this review,some of the seminal works on transfection and cell fusion by fs lasers are presented.The ideas behind and the experimental details will be described together with a highlight on their significances.Specifically,the thermal effect is analyzed based on multiphoton excitation and plasma formation in an aqueous environment to explain the nontoxic characteristic of fs laser irradiation.Last,some applications of fs laser induced transfection and cell
cell fusion with potential major impact in biomedical sciences are proposed.

Escherichia coli Protoplast Fusion and Screening of High L-Lysine-Producing Strain

[Objectives]Protoplast fusion of two parental strains with weak L-lysine production ability was carried out to obtain new fusion strains with strong L-lysine production ability.[Methods]The effects of bacterial age,lysozyme concentration,enzymolysis temperature and time on the protoplast formation rate and regeneration rate were investigated by single factor experiments.On this basis,with the protoplast formation rate as an index,the protoplast preparation process was optimized by an orthogonal experiment.[Results]Bacterial age and enzymolysis time had a greater impact on the protoplast formation rate,followed by enzymolysis temperature and lysozyme concentration.The optimal process for the preparation of L-lysine-producing Escherichia coli protoplasts was to prepare parental protoplasts from bacterial cells cultured for 15 h in the late logarithmic growth phase by enzymolysis with 0.8 mg/ml lysozyme at 37℃for 180 min and promote fusion with PEG6000.In order to facilitate the screening of fusion protoplasts,the empty plasmids p ET-28a and p ET-Duet were transformed into L-lysine-producing E.coli,respectively,and strains p ET-28a-lys01 and p ET-Duet-lys01 were obtained.Fusion strains were then obtained through protoplast fusion.Double-resistance KA1-10were screened on plates containing kanamycin and ampicillin,and a high-yielding fusion strain KA8,which produced L-lysine,was screened by fermentation experiments finally.[Conclusions]The results of this study provide a reference method for further improving the yield of L-lysine and other amino acid strains.

Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells

Objectives To construct a recombinant plasmid carrying enhanced green fluore- scent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expre- ssion in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP- C1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluoresce- nce microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48 h after transfection. Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease.

Infection Behaviour of Melampsora larici-populina on the Leaf Surface of Populus purdomii

Behaviours of urediospore germtube in Melampsora larici-populina on the leaf surface of Populus purdomii were studied by light microscope, scanning electron microscope （SEM）, transmission electron microscope （TEM）, and fluorescence microscope. Crab-like fusion cells on leaf surface, intercellular hyphal cells in leaf tissues, as well as nucleus states, were observed and counted up in this study. Under unsaturated humidity, 32% of germinated tubes fused into a distinguishable swollen crab-shaped cell at the merging site, and 10.5% of observed crab-like cells had more than three nuclei. Wedge-shaped mycelia developed and then penetrated the leaf surface directly, or indirectly through stomata. Tips of germtube passed through the intercellular cells of poplar leaves directly were found in TEM. Aniline blue dyeing also showed that the infecting hyphae could invade into the cuticle and epidemic cell wall directly. For the case of infection through stomata, there were two different situations. Short branches and wedge hyphae usually penetrated the leaf surface via opened stomata, whereas, some germtube branches and wedge hyphae penetrated leaves through the guard cell walls or stoma lips. In the latter case, the stomata were always closed. The samples from wild forestlands had the same fused cells and wedge hyphae, but the occurrence rate was much higher than that in the chamber. Even under the saturated air humidity, germtubes could roll back and formed fusion structure, or merged together with their tips. The fusion cells might centralize the plasma of merged germtubes and have a strong survival capacity to protect germtubes from dying under arid circumstances, and provide a chance of genetic variation as well.

Hot-nanoparticle-mediated fusion of selected cells

Complete fusion of two selected ceils allows for the creation of novel hybrid cells with inherited genetic properties from both original cells. Alternatively, via fusion of a selected cell with a selected vesicle, chemicals or genes can be directly delivered into the cell of interest, to control cellular reactions or gene expression. Here, we demonstrate how to perform an optically controlled fusion of two selected cells or of one cell and one vesicle. Fusion is mediated by laser irradiating plasmonic gold nanoparticles optically trapped between two cells （or a vesicle and a cell） of interest. This hot-particle-mediated fusion causes total mixing of the two cytoplasms and the two cell membranes resulting in formation of a new hybrid cell with an intact cell membrane and enzymatic activity following fusion. Similarly, fusion between a vesicle and a cell results in delivery of the vesicle cargo to the cytoplasm, and after fusion, the cell shows signs of viability. The method is an implementation of targeted drug delivery at the single-cell level and has a great potential for cellular control and design.

Primary Study on Experiment of Tetraploid Induction in Haliotis discus hannai with PEG

The experiment was performed on induction tetraploid of Haliotis discus hannai at two-cell stage through cell fusion with PEG treatment. In this paper, the orthogonal experiment of three factors and three levels [L9 （3^4）] was used. Three factors and three levels were molecular weight of PEG： 8 000, 6 000, 4 000 mol ·g^-1; PEG concentration： 45%, 50%, 55%; treatment duration time： 1, 2, 3 min, respectively. The results showed that the optimal pattern of three factors and three levels on inducing tetraploid of Haliotis discus hannai at two-cell stage through using PEG treatment were： molecular weight 4 000, concentration 55%, treatment duration time： 2 min. The highest tetraploid induction rate was 10.8% at embryo period. The three factors treatment sequence was treatment duration time→concentration→molecular weight.

THBS1 regulates trophoblast fusion through a CD36-dependent inhibition of cAMP,and its upregulation participates in preeclampsia

Preeclampsia is a pregnancy complication which threatens the survival of mothers and fetuses.It originates from abnormal placentation,especially insufficient fusion of the cytotrophoblast cells to form the syncytiotrophoblast.In this study,we found that THBS1,a matricellular protein that mediates cell-to-cell and cell-to-matrix interactions,is downregulated during the fusion of primary cytotrophoblast and BeWo cells,but upregulated in the placenta of pregnancies complicated by preeclampsia.Also,THBS1 was observed to interact with CD36,a membrane signal receptor and activator of the cAMP signaling pathway,to regulate the fusion of cytotrophoblast cells.Overexpression of THBS1 inhibited the cAMP signaling pathway and reduced the BeWo cells fusion ratio,while the effects of THBS1 were abolished by a CD36-blocking antibody.Our results suggest that THBS1 signals through a CD36-mediated cAMP pathway to regulate syncytialization of the cytotrophoblast cells,and that its upregulation impairs placental formation to cause preeclampsia.Thus,THBS1 can serve as a therapeutic target regarding the mitigation of abnormal syncytialization and preeclampsia.

Generation ofαGal-enhanced bifunctional tumor vaccine

Hepatocellular carcinoma(HCC)is a common malignant tumor with poor prognosis and high mortality.In this study,we demonstrated a novel vaccine targeting HCC and tumor neovascular endothelial cells by fusing recombinant MHCC97H cells expressing porcine a-1,3-galactose epitopes(a Gal)and endorphin extracellular domains(END)with dendritic cells(DCs)from healthy volunteers.END^(+)/Gal^(+)-MHCC97H/DC fusion cells induced cytotoxic T lymphocytes(CTLs)and secretion of interferon-gamma(IFN-γ).CTLs targeted cells expressing a Gal and END and tumor angiogenesis.The fused cell vaccine can effectively inhibit tumor growth and prolong the survival time of human hepatoma mice,indicating the high clinical potential of this new cell based vaccine.

Monoclonal Antibodies Recognizing HIV-1 gp41 Could Inhibit Env-Mediated Syncytium Formation

Some monoclonal antibodies （mAbs） could inhibit infection by HIV-1. In this study, four mAbs against HIV-1 gp41 were prepared in mice. All four mAbs could bind to the recombinant soluble gp41 and recognize the native envelope glycoprotein gp160 expressed on the HIV-Env^＋ CHO-WT cell in flow cytometry analysis. Interestingly, the results show that all four mAbs purified by affinity chromatography could inhibit HIV-1 Env-mediated membrane fusion （syncytium formation） by 40%-60% at 10 μg/mL, which implies potential inhibitory activities against HIV-1.