OBJECTIVETo explore the formation of pre-metastatic niche in the mouse lung and to study the underlying molecular mechanisms whereby primary breast carcinoma-derived factors mediate recruitment of bone marrow-derived cells (BMDCs) and affect the formation of pre-metastatic lung environment before the arrival of tumor cells.

METHODSMammary carcinoma 4T1 cells were inoculated into the mammary gland to construct mouse model of breast cancer. Confocal microscopy was used to detect the recruitment of BMDCs in the pre-metastatic lungs. The expression of factors in the mouse sera and 4T1 cell culture media was assayed using RayBio Custom mouse cytokine antibody array kit. The mice were injected daily with recombinant VEGF for 7 consecutive days to observe the effect of VEGF on BMDCs recruitment in the mouse lung.

RESULTSNo BMDCs were observed in the lungs of control and 4T1-tumor-bearing mice on day 0. On day 7 and 14, clusters of BMDCs observed in the lungs of 4T1-tumor-bearing mice were 8.7±2.2/objective field and 48.8±3.2/objective field, respectively, significantly higher than those in the control mice (1.1±0.8/objective field and 3.1±1.7/objective field) (P<0.05 for both). Confocal microscopic observation found that metastatic breast cancer cells preferentially facilitate BMDCs recruitment sites in the pre-metastatic mouse lungs. The levels of VEGF, GM-CSF, and IL-6 in the serum of 4T1-tumor-bearing mice were significantly increased compared with those in the control group (P<0.05 for all). However, VEGF was detected only in the culture media of 4T1 cells. The amount of BMDCs in the mouse lung tissue was (22.8±3.6)/objective field in the VEGF group and (3.1±0.4)/objective field in the control group (P<0.05). There were 36.8±5.4 metastatic foci in the lung tissue of VEGF group and 12.6±2.2 in the control group (P<0.05).

CONCLUSIONSThe results of this study demonstrate that primary breast cancer cells can alter the lung microenvironment during the pre-metastatic phase and induce the formation of pre-metastatic niche. Primary tumor cell-derived VEGF may be a crucial factor responsible for the formation of pre-metastatic niche.

Animals ; Bone Marrow Cells ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; blood ; Humans ; Interleukin-6 ; blood ; Lung ; pathology ; Lung Neoplasms ; secondary ; Mice ; Recombinant Proteins ; administration & dosage ; Time Factors ; Tumor Microenvironment ; Vascular Endothelial Growth Factor A ; administration & dosage ; physiology ; secretion

Constructing ethanologenic strains with cellulose activity is important to achieve consolidated bioprocessing of lignocellulose for ethanol production. In this study, we integrated the pyruvate decarboxylase gene pdc and alcohol dehydrogenase gene adhB from Zymomonas mobilis ZM4 into Escherichia coli JM109 by Red recombination method to generatea recombinant strain E. coli P81 that could produce ethanol from glucose. Abeta-glucosidase gene bglB from Bacillus polymyxa 1.794 was cloned into the recombinant E. coli P81 and beta-glucosidase was expressed to give a new recombinant strain E. coli P81 (pUC19-bglB) with dual functions of cellobiose degradation and ethanol production. The extracellular beta-glucosidaseactivity was 84.78 mU/mL broth and the extracellular cellobiase activity of E. coli P81 (pUC19-bglB) was 32.32 mU/mL broth. E. coli P81 (pUC19-bglB) fermented cellobiose to ethanol with a yield of 55.8% of the theoretical value, and when glucose and cellobiose were co-fermented, the ethanol yield reached 46.5% of thetheoretical value. The construction of consolidated bioprocessing strain opens the possibility to convert cellobiose to ethanol in a single bioprocess.
Bacterial Secretion Systems ; Cellulose ; metabolism ; Escherichia coli ; genetics ; metabolism ; Ethanol ; metabolism ; Fermentation ; Recombinant Proteins ; biosynthesis ; genetics ; beta-Glucosidase ; biosynthesis ; genetics

To develop a method based on immunoreactions for detection of Ectropis obliqua Nucleopolyhedrovirus (EoNPV), the polyhedra of the virus were purified and used to immunize the mouse BALB/c. The spleen cells from the immunized mice were then fused with the myeloma cell line Sp2/0. A hybridoma cell line which can stably secrete the monoclonal antibody against EoNPV was achieved by using indirect ELISA screening and cloning methods, and was named as 7D3. Meanwhile, the polyhedrin gene was cloned from EoNPV and expressed in E. coli. Western blotting analysis showed that the monoclonal antibody prepared from 7D3 could specifically react with the recombinant polyhedrin. An indirect ELISA method based on this monoclonal antibody for detecting EoNPV in infected tea looper was developed.
Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Antibody Specificity ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hybridomas ; secretion ; Lepidoptera ; growth & development ; virology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; immunology

Animals ; Exocytosis ; drug effects ; physiology ; Glucose ; pharmacology ; Insulin ; secretion ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Microscopy, Fluorescence ; methods ; Potassium ; pharmacology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Vesicle-Associated Membrane Protein 2 ; genetics ; metabolism

BACKGROUNDTissue-engineered bioartificial muscle-based gene therapy represents a promising approach for the treatment of heart diseases. Experimental and clinical studies suggest that systemic administration of insulin-like growth factor-1 (IGF-1) protein or overexpression of IGF-1 in the heart exerts a favorable effect on cardiovascular function. This study aimed to investigate a chronic stage after myocardial infarction (MI) and the potential therapeutic effects of delivering a human IGF-1 gene by tissue-engineered bioartificial muscles (BAMs) following coronary artery ligation in Sprague-Dawley rats.

METHODSLigation of the left coronary artery or sham operation was performed. Primary skeletal myoblasts were retrovirally transduced to synthesize and secrete recombinant human insulin-like growth factor-1 (rhIGF-1), and green fluorescent protein (GFP), and tissue-engineered into implantable BAMs. The rats that underwent ligation were randomly assigned to 2 groups: MI-IGF group (n = 6) and MI-GFP group (n = 6). The MI-IGF group received rhIGF-secreting BAM (IGF-BAMs) transplantation, and the MI-GFP group received GFP-secreting BAM (GFP-BAMs) transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups: S-IGF group (n = 6) and S-GFP group (n = 6). The S-IGF group underwent IGF-1-BAM transplantation, and S-GFP group underwent GFP-BAM transplantation. IGF-1-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats after two weeks of operation was performed. Four weeks after the treatment, hemodynamics was performed. IGF-1 was measured by radioimmunoassay, and then the rats were sacrificed and ventricular samples were subjected to immunohistochemistry. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of bax and Bcl-2. TNF-α and caspase 3 expression in myocardium was examined by Western blotting.

RESULTSPrimary rat myoblasts were retrovirally transduced to secrete rhIGF-1 and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted consistent levels of hIGF (0.4 - 1.2 µg×BAM(-1)×d(-1)). When implanted into syngeneic rat, IGF-BAMs secreted and delivered rhIGF. Four weeks after therapy, the hemodynamics was improved significantly in MI rats treated with IGF-BAMs compared with those treated with GFP-BAMs. The levels of serum IGF-1 were increased significantly in both MI and sham rats treated with IGF-BAM. The mRNA expression of bax was lower and Bcl-2 expression was higher in MI-IGF group than MI-GFP group (P < 0.05). Western blotting assay showed TNF-α and caspase 3 expression was lower in MI-IGF group than MI-GFP group after therapy.

CONCLUSIONSrhIGF-1 significantly improves left ventricular function and suppresses cardiomyocyte apoptosis in rats with chronic heart failure. Genetically modified tissue-engineered BAMs provide a method delivering recombinant protein for the treatment of heart failure.

Animals ; Apoptosis ; Caspase 3 ; analysis ; Desmin ; analysis ; Genetic Therapy ; Heart Failure ; pathology ; physiopathology ; therapy ; Insulin-Like Growth Factor I ; genetics ; secretion ; Myoblasts, Skeletal ; metabolism ; Myocytes, Cardiac ; pathology ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; secretion ; Retroviridae ; genetics ; Tissue Engineering ; Tumor Necrosis Factor-alpha ; analysis ; Ventricular Function, Left

Aspergillus niger is an important industrial workhorse with extensive application in the sectors of industrial enzymes, heterogeneous proteins, organic acids and etc. The disclosure of its genomic sequence to the public brought the study of A. niger into the post-genomic era. Diverse omic data are being produced massively and rapidly, which largely upgrades our understanding to the hyperproduction mechanism of A. niger to a systems and molecular level. At meanwhile, its genetic operating system is becoming mature, which enables genome-scale genetic perturbation within A. niger. In conclusion, we are on the right way to redesign and engineer A. niger to an omnipotent cellular factory.
Aspergillus niger ; genetics ; metabolism ; Biotechnology ; methods ; Enzymes ; genetics ; secretion ; Gene Expression Regulation, Fungal ; Genes, Fungal ; Genome, Fungal ; Protein Biosynthesis ; genetics ; Recombinant Proteins ; secretion ; Transcription, Genetic

OBJECTIVETo investigate if granulocyte-macrophage colony stimulating factor (GM-CSF) gene-modified dendritic cells (DC) enhance antitumor immunity in vitro.

METHODSMice were injected with chemokine ligand 3 (CCL3) via the tail vein. Fresh B220(-)CD11c(+) cells were sorted from the peripheral blood mononuclear cells (PBMCs) and cultured into DCs by cytokines.DCs were transfected with AdGM-CSF gene at different ratios of multiplicity of infection (MOI) to determine the optimal gene transfection conditions, and the expression of GM-CSF was detected after transfection. The variation of GM-CSF gene-modifiedDCs were analyzed by morphological examination, phenotype analysis, and mixed lymphocyte reaction (MLR).DCs were loaded with gastric cancer antigen obtained by freezing and thawing method. The killing effect of DCs vaccine-stimulated T lymphocytes on gastric cancer cells was assessed by MTT assay. INF-gamma production was determined with the INF-gamma ELISA kit.

RESULTSB220(-)CD11c(+) cells increased obviously after CCL3 injection. The ELISA results showed that after GM-CSF gene modification, DCs could produce high level of GM-CSF. When DCs were transfected with AdGM-CSF gene at MOI equal to 100, the GM-CSF level in culture supernatants reached saturation [(130.00 +/- 12.61) pg/ml]. After GM-CSF gene-modification, DCs tend to be more maturated as detected by morphological observation and phenotype analysis. At the same time, the capacity of activating the proliferation of allogeneic T lymphocytes was enhanced greatly. T lymphocytes stimulated by DCs transfected with GM-CSF gene showed a specific killing effect on gastric carcinoma cells and produced high level of INF-gamma [(1245.00 +/- 13.75) pg/ml].

CONCLUSIONAfter GM-CSF gene modification, DCs can produce high level of GM-CSF, which tend to be more maturated, and the capacity of activating the proliferation of allogeneic T lymphocytes is enhanced greatly. GM-CSF gene modified DCs can induce specific CTL to target tumor cells in vitro.

Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Histocompatibility Antigens Class II ; metabolism ; Interferon-gamma ; secretion ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; Stomach Neoplasms ; immunology ; metabolism ; pathology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

OBJECTIVETo achieve secretory and extracellular production of recombinant dengue virus serotypes I-IV envelope glycoprotein domain III (DENV-1-4 EDIII) in Pichia pastoris.

METHODSEDIII genes of DENVI-IV were amplified and cloned into vector pPIC9K, respectively. These recombinant plasmids were then linearized and transferred into Pichia pastoris strain GS115. Clones highly produced in 4.0 mg/ml G418 were amplified and induced by methanol to achieve the secreted recombinant proteins. Ni-NTA agarose beads were used for purification, while SDS-PAGE and Western blotting were used for identification.

RESULTSThe recombinant plasmids pPIC9K-DENV-1-4 EDIII were constructed and successfully transferred into Pichia pastoris strain GS115. The recombinant EDIII proteins were expressed in a secretory way with the molecular weight about 12 × 10(3) and specifically identified by anti-His monoclonal antibody and anti-DENVI-IV mice sera.

CONCLUSIONDENVI-IV EDIII proteins are successfully achieved from Pichia pastoris expression system and could be used for development of dengue vaccines, diagnostic reagents and study of biological function of the E protein.

Dengue Virus ; genetics ; Genetic Vectors ; Pichia ; metabolism ; Recombinant Proteins ; genetics ; Viral Envelope Proteins ; secretion

Selection of suitable signal peptides is an important factor for efficient secretion of heterologous proteins. We defined structural fusion degree (SFD) as the compatibility degree of target proteins and signal peptides by a bioinformatics approach. We mathematically analyzed the interaction of fused signal peptides and adjacent residues of proteins, and proposed a mathematical model of extended signal region and the protein. SFD Features was extracted from this model to characterize the secretability of heterologous proteins. Simulation tests showed that SFD features can effectively discriminate high secretory proteins from poor ones in the host Bacillus subtilis. Results from this research will be useful in signal peptide selection and have a better guiding significance for the optimization of heterologous protein secretion.
Amino Acid Sequence ; Bacillus subtilis ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biotechnology ; methods ; Membrane Transport Proteins ; genetics ; metabolism ; Metalloendopeptidases ; genetics ; metabolism ; Molecular Sequence Data ; Protein Sorting Signals ; genetics ; Proteins ; secretion ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells (BMDCs) and T lymphocytes in vitro.

METHODSAfter BMDCs stimulated by E.coli LLO/OVA, their Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) receptor signalling pathway were examined by superarray hybridization; and the priming effect of the vaccine activated BMDCs on CD4(+)T and CD8(+)T was determined by [3H]thymidine uptake and ELISA, the tumor cytotoxic effect of activated CD8(+)T cells was determined by cytotoxic assay.

RESULTSAfter BMDCs were activated by E. coli LLO/OVA via TLR4, NOD1 receptor and NF-κB signalling pathway, the expression of their surface molecules including MHC class I, MHC class II, CD40, CD80 and CD86 significantly up-regulated; the secretion of IL-12 and IFN-γ increased also. The mature BMDCs stimulated the allergic CD4(+)T and CD8(+)T cells proliferation and their IL-2 and IFN-γ secretion, and the activated CD8(+)T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.

CONCLUSIONE.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.

Animals ; Antigens, Neoplasm ; genetics ; pharmacology ; Bacterial Toxins ; genetics ; pharmacology ; Cancer Vaccines ; genetics ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; immunology ; Coculture Techniques ; Cytokines ; immunology ; secretion ; Dendritic Cells ; cytology ; drug effects ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Female ; Flow Cytometry ; Heat-Shock Proteins ; genetics ; pharmacology ; Hemolysin Proteins ; genetics ; pharmacology ; Immunity, Innate ; drug effects ; Mice ; Mice, Inbred C57BL ; Nod1 Signaling Adaptor Protein ; genetics ; physiology ; Ovalbumin ; genetics ; pharmacology ; Recombinant Fusion Proteins ; genetics ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Toll-Like Receptor 4 ; genetics ; physiology