Bacillus mycoides JA20-1 was screened and identified as a biocontrol bacterium with a high capacity for producing volatile organic compounds(VOCs) in the laboratory. This strain had significant inhibitory effects on various postharvest disease pathogens in crops, such as Botrytis cinerea, as well as soil-borne disease pathogens in ginseng, such as Sclerotinia ginseng. In order to accelerate its industrialization process, in this study, single-factor experiments and response surface optimization methods were used. The fermentation medium and fermentation conditions in the shake flask of strain JA20-1 were systematically optimized by using cell production volume as the response variable. Meanwhile, the biocontrol effect of JA20-1 on B. cinerea of ginseng during the storage period was evaluated by using the method of fumigation in a dry dish in vitro. The results indicated that the optimal fermentation medium formulation for strain JA20-1 was as follows: 1% yeast paste, 1% soluble starch, 0.25% K_2HPO_4·3H_2O, and 0.2% NaCl. The optimal fermentation conditions in the shake flask were vaccination size of 3%, culture volume of 50 mL in a 250 mL Erlenmeyer flask, pH of 6.2, fermentation temperature of 34 ℃, shaking speed of 180 r·min~(-1), and incubation time of 18 hours. The bacteria count in the fermentation broth under these conditions reached 2.17 × 10~8 CFU·mL~(-1), which was 6.58 times higher than before. The average control efficacy of the fermentation broth on Botrytis cinerea of ginseng under in vitro fumigation reached 61.70% and 84.04% respectively, when 20 mL and 30 mL per dish were used. The research provided theoretical support and technical foundation for the development and utilization of Bacillus mycoides JA20-1 and the biocontrol of soil-borne diseases in ginseng and postharvest diseases in crops.
Botrytis/drug effects* ; Fermentation ; Panax/microbiology* ; Plant Diseases/prevention & control* ; Volatile Organic Compounds/metabolism* ; Bacillus/physiology* ; Pest Control, Biological/methods* ; Biological Control Agents/metabolism* ; Culture Media/chemistry*

Aerobic methane oxidizing bacteria (methanotrophs) can use methane as carbon source and energy source, eliminating 10%-20% of global methane. Methanotrophs can also effectively synthesize valuable methane-derived products. This article introduced the methane oxidizing mechanism of methanotrophs, and summarized the practical application and research hotspots of methanotrophs in the field of methane emission reduction in the landfill, ventilation air methane mitigation in coal mines, valuable chemicals biosynthesis, as well as oil and gas reservoir exploration. Main factors influencing the pollutant removal and the biosynthesis efficiency in various applications were also discussed. Based on the study of large-scale cultivation of methanotrophs, some measures to benefit the application and promotion of aerobic methane oxidizing biotechnology were proposed. This includes investigating the effect of intermediate metabolites on methanotrophs activity and population structure, and exploiting economical and efficient alternative culture media and culture techniques.
Biotechnology ; Carbon ; Culture Media/chemistry* ; Methane/metabolism* ; Methylococcaceae/metabolism* ; Oxidation-Reduction

Royal jelly (RJ) from honeybee has been widely used as a health promotion supplement. The major royal jelly proteins (MRJPs) have been identified as the functional component of RJ. However, the question of whether MRJPs have anti-senescence activity for human cells remains. Human embryonic lung fibroblast (HFL-I) cells were cultured in media containing no MRJPs (A), MRJPs at 0.1 mg/ml (B), 0.2 mg/ml (C), or 0.3 mg/ml (D), or bovine serum albumin (BSA) at 0.2 mg/ml (E). The mean population doubling levels of cells in media B, C, D, and E were increased by 12.4%, 31.2%, 24.0%, and 10.4%, respectively, compared with that in medium A. The cells in medium C also exhibited the highest relative proliferation activity, the lowest senescence, and the longest telomeres. Moreover, MRJPs up-regulated the expression of superoxide dismutase-1 (SOD1) and down-regulated the expression of mammalian target of rapamycin (MTOR), catenin beta like-1 (CTNNB1), and tumor protein p53 (TP53). Raman spectra analysis showed that there were two unique bands related to DNA synthesis materials, amide carbonyl group vibrations and aromatic hydrogens. These results suggest that MRJPs possess anti-senescence activity for the HFL-I cell line, and provide new knowledge illustrating the molecular mechanism of MRJPs as anti-senescence factors.
Animals ; Bees ; Cattle ; Cell Line ; Cell Proliferation ; Cellular Senescence/drug effects* ; Culture Media ; Dose-Response Relationship, Drug ; Fatty Acids/chemistry* ; Fibroblasts/drug effects* ; Humans ; Insect Proteins/chemistry* ; Lung/drug effects* ; Serum Albumin/metabolism* ; Spectrum Analysis, Raman ; Superoxide Dismutase-1/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Tumor Suppressor Protein p53/metabolism* ; beta Catenin/metabolism*

Nocathiacin I, a glycosylated thiopeptide antibiotic, displays excellent antibacterial activities against multidrug resistant bacterial pathogens. Previously, a novel nocathiacin I formulation for intravenous administration has been successfully developed and its aqueous solubility is greatly enhanced for clinical application. The purpose of the present study was to increase the fermentation titer of nocathiacin I and reduce or eliminate analogous impurities by screening the medium ingredients using response surface methodology. After a sysmatic optimization, a water-soluble medium containing quality-controllable components was developed and validated, resulting in an increase in the production of nocathiacin I from 150 to 405.8 mg·L at 150-L scale. Meanwhile, the analogous impurities existed in reported processes were greatly reduced or eliminated. Using optimized medium for fermentation, nocathiacin I with pharmaceutically acceptable quality was easily obtained with a recovery of 67%. In conclusion, the results from the present study offer a practical and efficient fermentation process for the production of nocathiacin I as a therapeutic agent.
Actinobacteria ; growth & development ; metabolism ; Anti-Bacterial Agents ; biosynthesis ; chemistry ; Bioreactors ; Culture Media ; Fermentation ; Intercellular Signaling Peptides and Proteins ; Peptides ; chemistry ; metabolism ; Quality Improvement

OBJECTIVETo investigate the effects of oxygen concentration and reactive oxygen species (ROS) on the biological characteristics of hematopoietic stem cells (HSC) and to analyzed the relationship among the oxygen concentration, ROS and the biological characteristics of mouse HSC through simulation of oxygen environment experienced by PB HSC during transplantation.

METHODSThe detection of reactive oxygen species (ROS), in vitro amplification, directional differentiation (BFU-E, CFU-GM, CFU-Mix), homing of adhesion molecules (CXCR4, CD44, VLA4, VLA5, P-selectin), migration rate, CFU-S of NOD/SCID mice irradiated with sublethal dose were performed to study the effect of oxgen concentration and reactive oxygen species on the biological characteristics of mouse BM-HSC and the relationship among them.

RESULTSThe oxygen concentrations lower than normal oxygen concentration (especially hypoxic oxygen environment) could reduce ROS level and amplify more Lin(-) c-kit(+) Sca-1(+) BM HSC, which was more helpful to the growth of various colonies (BFU-E, CFU-GM, CFU-Mix) and to maintain the migratory ability of HSC, thus promoting CFU-S growth significantly after the transplantation of HSC in NOD/SCID mice irradiated by a sublethal dose. BM HSC exposed to oxygen environments of normal, inconstant oxygen level and strenuously thanging of oxygen concentration could result in higher level of ROS, at the same time, the above-mentioned features and functional indicators were relatively lower.

CONCLUSIONThe ROS levels of BM HSC in PB HSCT are closely related to the concentrations and stability of oxygen surrounding the cells. High oxygen concentration results in an high level of ROS, which is not helpful to maintain the biological characteristics of BM HSC. Before transplantation and in vitro amplification, the application of antioxidancs and constant oxygen level environments may be beneficial for transplantation of BMMSC.

Animals ; Cell Differentiation ; Culture Media ; chemistry ; Erythroid Precursor Cells ; cytology ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; metabolism ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Oxygen ; chemistry ; Reactive Oxygen Species ; metabolism

OBJECTIVETo compare several schemes of inducing and expanding the antibody-mediated high efficiency CIK (AMHE-CIK) in vitro, so as to find out a method that can acquire a large number of cells capable to kill the tumor cells in a short time.

METHODSPeripheral blood mononuclear cells (PBMNC) from healthy volunteers was isolated and activated with CD3 antibody, then were cultured with the addition of different cytokines (IL-2, IL-4, G-CSF, GM-CSF, IFN-γ, TNF-α) for 14 days in vitro. The morphological changes of cells were observed by light microscopy. Based on the immunophenotypes of cells in each groups analyzed by flow cytometry, the cytokines capable to induce the dendritic cells and killer cells were screened out, respectively. According to different combination of cytokines, the cells were divided 4 groups: control, IL-2, group 1 (componant A included IL-2, IL-4, and GM-CSF. Componant B included IL-2, G-CSF, IFN-γ, and TNF-α), and group 2 (componant A included IL-2, IL-4, and GM-CSF. Componant B included IL-2, IL-4, G-CSF, IFN-γ, and TNF-α). The proliferation and differentiation of CD3(+) CD8(+) and CD3(+) CD56(+) cells were measured by flow cytometry after culture in vitro for 7 days.

RESULTSAfter inducing and expanding in vitro for 7 days, the cell proliferation rate of control group, IL-2 group, group 1 and group 2 were 1.57 ± 0.01, 4.17 ± 0.16, 5 ± 0.47, 7.17 ± 0.24-folds, respectively. The differences between IL-2 group, group 1, group 2 and control group were statistically significant (P < 0.05). The immunophenotype analysis showed that the proportion of CD3(+) CD8(+) induced by each protocol was 13.96 ± 0.23%, 26.33 ± 0.55%, 36.83 ± 0.34% and 35.88 ± 0.16%, respectively. The proportion of CD3(+) CD8(+) in group 1 and 2 was higher than that in IL-2 group (P < 0.05), but the difference between them was not significant (P < 0.05). The proportions of CD3(+) CD56(+) induced by each protocol were 11.03 ± 0.28%, 29.31 ± 0.60%, 39.96 ± 0.38% and 29.33 ± 0.54%, respectively, the proportion of group 1 was higher than that of IL-2 group and group 2 (P < 0.05), but the difference between IL-2 group and group 2 was not significant (P < 0.05).

CONCLUSIONThe group 1 protocol obtained from this study can promote the proliferation of DC-CIK and also increase the proportion of the tumor killing cells (CD3(+) CD8(+) and CD3(+) CD56(+)).

Cell Culture Techniques ; Cells, Cultured ; Culture Media ; chemistry ; Cytokine-Induced Killer Cells ; cytology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Immunophenotyping ; Interferon-gamma ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-4 ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate the effect of MiR-200b on human retinal endothelial cells (hRECs) cultured in high glucose and explore the mechanism.

METHODShRECs cultured in high glucose or in normal media were examined for MiR-200b mRNA expression using real-time PCR. The effect of MiR-200b transfection on hREC proliferation in high-glucose culture was evaluated with MTT assay, and real-time PCR and Western blotting were performed to determine vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGFβ1) expression in the transfected cells.

RESULTSThe cells in high-glucose culture showed significantly decreased MiR-200b expression and active proliferation. Compared with those in normal control cells, VEGF and TGFβ1 mRNA and protein expressions increased markedly in cells cultured in high glucose (P<0.05). MiR-200b transfection of the cells caused significantly increased cellular expression of MiR-200b but decreased expression levels of VEGF and TGFβ1 mRNA and protein, and suppressed hREC proliferation in high glucose culture (P<0.05).

CONCLUSIONMiR-200b can regulate REC growth and proliferation by changing VEGF and TGFβ1 expressions and thus play a role in the pathogenesis and progression of diabetic retinopathy.

Blotting, Western ; Cell Proliferation ; Cells, Cultured ; Culture Media ; chemistry ; Diabetic Retinopathy ; Endothelial Cells ; cytology ; Glucose ; chemistry ; Humans ; MicroRNAs ; metabolism ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Retina ; cytology ; Transfection ; Transforming Growth Factor beta1 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo explore effects of exendin-4 on the metabolism of extracellular matrix (ECM) in human mesangial cells (HMC) cultured in the presence of high glucose and explore the possible mechanism.

METHODSHuman mesangial cells (HMC) were treated with exendin-4 under high glucose conditions. The cell proliferation was observed using CCK8 assay, and the expressions of collagen type I, fibronectin, transforming growth factor-β1 (TGFβ1) expression and extracellular signal- regulated kinase (ERK) signaling pathway activity were assessed using Western blotting.

RESULTSExendin-4 inhibited cell proliferation and the expressions of collagen type I, fibronectin and TGFβ1 and reversed ERK phosphorylation in high glucose-induced HMC.

CONCLUSIONExendin-4 can regulate ECM metabolism in HMC cultured in high glucose by inhibiting TGFβ1/ERK pathway, suggesting the beneficial effects of exendin-4 in preventing and treating diabetic nephropathy.

Cell Proliferation ; Cells, Cultured ; Collagen Type I ; metabolism ; Culture Media ; chemistry ; Diabetic Nephropathies ; Extracellular Matrix ; metabolism ; Fibronectins ; metabolism ; Glucose ; chemistry ; Humans ; MAP Kinase Signaling System ; Mesangial Cells ; drug effects ; Peptides ; pharmacology ; Phosphorylation ; Signal Transduction ; Transforming Growth Factor beta1 ; metabolism ; Venoms ; pharmacology

To evaluate the effect of laser solid forming (LSF) of porous titanium on receptor activator of NF-κB ligand (RANKL)/osteoprorotegerin (OPG) expression and osteoblast cells growth.
 Methods: The DMEM and sterile saline were used for porous titanium extract. The osteoblast cells were cultured in the extract while equal amount of  DMEM and sterile saline were added to the control group. The growth of the cells were observed under an inverted phase contrast microscope. MTT was used to detect the growth inhibitory rates. The adhesion capacity of osteoblasts were measured. The growth in the material surface was examined by the electron microscope, and the expressions of RANKL and OPG were determined by Westen blot.
 Results: At the first day, the osteoblast proliferation rate was significantly different (P<0.05), at the fourth and seventh day, the osteoblast proliferation rate was not significantly affected in the LSF group (P>0.05); at each time point, the osteoblast proliferation rate were significantly different between the two groups (P<0.05). Compared with the control group, RANKL and OPG protein expression were not significantly different (P>0.05). The laser solid forming of porous titanium showed well bone compatibility.
 Conclusion: The porous titanium did not affect osteoblast proliferation due to its well bone compatibility. It did not affect the OPG/RANKL/RANK-axis system of bone metabolism, exibiting a wide applicable prospect for tissue engineering.
Biocompatible Materials ; chemistry ; Cell Adhesion ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; chemistry ; Ligands ; Osteoblasts ; drug effects ; Osteogenesis ; drug effects ; Osteoprotegerin ; metabolism ; Porosity ; Receptor Activator of Nuclear Factor-kappa B ; metabolism ; Tissue Engineering ; instrumentation ; Tissue Scaffolds ; chemistry ; Titanium ; pharmacology

To explore whether hypoxic condition could promote the olfactory mucosa mesenchymal stem cells (OM-MSCs) to differentiate into neurons with the olfactory ensheathing cells (OECs) supernatant and the potential mechanisms.
 Methods: The OM-MSCs and OECs were isolated and cultured, and they were identified by flow cytometry and immunofluorescence. The OM-MSCs were divided into three groups: a 3%O2+ HIF-1α inhibitors (lificiguat: YC-1) + OECs supernatant group (Group A) , a 3%O2 + OECs supernatant group (Group B) and a 21%O2 + OECs supernatant group (Control group). The neurons, which were differentiated from OM-MSCs, were assessed by immunofluorescence test. The mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α), βIII-tubulin and glial fibrillary acidic portein (GFAP) were detected by quantitative polymerase chain reaction (Q-PCR) and Western blot. The potassium channels were analyzed by patch clamp.
 Results: The neurons differentiated from OM-MSCs expressed the most amount of βIII-tubulin, and the result of Q-PCR showed that HIF-1α expression in the Group B was significantly higher than that in the other groups (all P<0.05). Western blot result showed that the βIII-tubulin protein expression was significantly higher and GFAP protein expression was obviously decreased in the Group B (both P<0.05). The patch clamp test confirmed that the potassium channels in the neurons were activated.
 Conclusion: Hypoxic condition can significantly increase the neuronal differentiation of OM-MSCs by the OECs supernatant and decrease the production of neuroglia cells, which is associated with the activation of HIF-1 signal pathway.
Blotting, Western ; Cell Differentiation ; physiology ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; pharmacology ; Flow Cytometry ; Glial Fibrillary Acidic Protein ; metabolism ; Hypoxia ; physiopathology ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Indazoles ; pharmacology ; Mesenchymal Stem Cells ; physiology ; Neurogenesis ; physiology ; Neuroglia ; metabolism ; physiology ; Neurons ; physiology ; Olfactory Mucosa ; Potassium Channels ; Signal Transduction ; Tubulin ; metabolism