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 inhibitory effect of high concentration insulin on K562 cell proliferation and its underlying mechanism.

METHODSK562 cells were treated by different concentration of insulin and/or anti-IGF-1R antibody (IGF-1R-Ab), MTT assay and flow cytometry were used to detect the K562 cells proliferation and apoptosis, respectivety; Western blot was used to measure the expression and phosphorylation level of IGE-IR, Akt, Erk1/2 in K562 cells under the different concentration of insulin.

RESULTSMTT assay showed that less than 40 mU/ml insulin could promote K562 cell proliferation, while high concentration (> 40 mU/ml) insulin has been shown to inhibit K562 cell proliferation; Flow cytometry showed that 40 mU/ml insulin suppressed K562 cell apoptosis (P < 0.05), while 200 mU/ml insulin could significantly induce K562 cell apoptosis (P < 0.01); 0.01 to 1.0 µg/ml IGF-1R-Ab has significantly enhanced the inhibitory and inducing effects of high concentration (> 40 mU/ml) of insulin on K562 cell proliferation and apoptosis respectively (r = 0.962, P < 0.001); Western blot showed that after K562 cells were treated with different concentrations of insulin ERK, and the p-ERK expression did not change significantly, after K562 cells were treated with 200 mU/ml insulin, the expression of IGF-1R and AKT also not were changed obviously, while the phosphorylation level of IGF-1R and AKT increased.

CONCLUSIONHigh concentration (>40 mU/ml) of insulin inhibits K562 cell proliferation and induces its apoptosis, and its mechanism may be related with the binding IGF-1R by insulin, competitively inhibiting the binding of IGF-1 and IGF-1R, the blocking the transduction of PI3K/AKT signal pathway.

Antibodies ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Culture Media ; chemistry ; Humans ; Insulin ; pharmacology ; Insulin-Like Growth Factor I ; metabolism ; K562 Cells ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Receptors, Somatomedin ; immunology ; Signal Transduction ; drug effects

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.
Animals ; Antigens, Helminth ; isolation & purification ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; chemistry ; pharmacology ; Gene Expression Regulation ; Hedgehog Proteins ; agonists ; antagonists & inhibitors ; genetics ; immunology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; parasitology ; prevention & control ; Macrophage Activation ; drug effects ; Macrophages ; cytology ; drug effects ; immunology ; Models, Biological ; Monocytes ; cytology ; drug effects ; metabolism ; Pentoxifylline ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; RNA, Messenger ; genetics ; immunology ; Schistosoma japonicum ; chemistry ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology ; Zinc Finger Protein GLI1 ; genetics ; immunology ; Zygote ; chemistry

OBJECTIVETo investigate the optimal starvation conditions of human umbilical vein endothelial cells (HUVECs) and establish a highly efficient and stable method for separating HUVECs.

METHODSHUVECs harvested from human umbilical cords by digestion with 0.1% collagenase II for 15 min were cultured in endothelial culture medium (ECM) containing 5% fetal bovine serum (FBS), 1% endothelial cell growth factor (ECGS) and 1% penicillin/streptomycin solution(P/S) at 37 degrees celsius; in 5% CO2. The cells were observed for cell morphology under an inverted microscope and identified with immunofluorescence assay. The purity of HUVECs was detected using flow cytometry (FCM). The cell cycles of HUVECs cultured in the presence of 0, 0.1%, 0.5%, and 1% FBS for 0, 6, 12, 18, and 24 h were analyzed with flow cytometry.

RESULTSs The purity of HUVECs harvested by digestion with 0.1% collagenase II reached 99.67%. The primary HUVECs showed a cobblestone or volute appearance in vitro. Immunocytochemistry showed that HUVECs highly expressed VIII-related antigen. Cell culture in the presence of different concentrations of FBS for 6 h resulted in 70% G0/G1 phase cells, which increased to 80%-90% at 12 h of cell culture, and further to around 95% at 18 and 24 h.

CONCLUSIONDigestion with 0.1% collagenase II can obtain high-purity primary HUVECs. Culturing HUVECs in serum-free medium for 12 h can result in a high purity (over 80%) of G0/G1 phase cells.

Cell Culture Techniques ; Cell Cycle ; Cells, Cultured ; Culture Media ; chemistry ; Flow Cytometry ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Matrix Metalloproteinase 8 ; chemistry ; Serum

OBJECTIVETo investigate the effect of prostaglandins E2 (PGE2) in enhancing vascular endothelial growth factor (VEGF) expression in a rat macrophage cell line and the effect of the media from PGE2-inuced rat macrophages on angiogenetic ability of human umbilical vein endothelial cells (HUVECs) in vitro.

METHODSWestern blotting and qPCR were employed to investigate the expressions of VEGF protein and mRNAs in rat macrophage cell line NR8383 stimulated by PGE2 in the presence or absence of EP2 receptor inhibitor (AH6809) and EP4 receptor inhibitor (AH23848). Conditioned supernatants were obtained from different NR8383 subsets to stimulate HUVECs, and the tube formation ability and migration of the HUVECs were assessed with Transwell assay.

RESULTSPGE2 stimulation significantly enhanced the expression of VEGF protein and mRNAs in NR8383 cells in a dose-dependent manner. The supernatants from NR8383 cells stimulated by PGE2 significantly enhanced tube formation ability of HUVECs (P<0.05) and promoted the cell migration. Such effects of PGE2 were blocked by the application of AH6809 and AH23848.

CONCLUSIONPGE2 can dose-dependently increase VEGF expression in NR8383 cells, and the supernatants derived from PGE2-stimulated NR8383 cells can induce HUVEC migration and accelerate the growth of tube like structures. PGE2 are essential to corpus luteum formation by stimulating macrophages to induce angiogenesis through EP2/EP4.

Animals ; Cell Line ; Cell Movement ; Cells, Cultured ; Culture Media, Conditioned ; pharmacology ; Dinoprostone ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Macrophages ; chemistry ; Neovascularization, Pathologic ; RNA, Messenger ; Rats ; Receptors, Prostaglandin E, EP2 Subtype ; metabolism ; Receptors, Prostaglandin E, EP4 Subtype ; metabolism ; Vascular Endothelial Growth Factor A ; Xanthones ; pharmacology

Acetic acid, as a main by-product generated in the pretreatment process of lignocellulose hydrolysis, significantly affects cell growth and lipid synthesis of oleaginous microorganisms. Therefore, we studied the tolerance of Rhodotorula glutinis to acetic acid and its lipid synthesis from substrate containing acetic acid. In the mixed sugar medium containing 6 g/L glucose and 44 g/L xylose, and supplemented with acetic acid, the cell growth was not:inhibited when the acetic acid concentration was below 10 g/L. Compared with the control, the biomass, lipid concentration and lipid content of R. glutinis increased 21.5%, 171% and 122% respectively when acetic acid concentration was 10 g/L. Furthermore, R. glutinis could accumulate lipid with acetate as the sole carbon source. Lipid concentration and lipid yield reached 3.20 g/L and 13% respectively with the initial acetic acid concentration of 25 g/L. The lipid composition was analyzed by gas chromatograph. The main composition of lipid produced with acetic acid was palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid, including 40.9% saturated fatty acids and 59.1% unsaturated fatty acids. The lipid composition was similar to that of plant oil, indicating that lipid from oleaginous yeast R. glutinis had potential as the feedstock of biodiesel production. These results demonstrated that a certain concentration of acetic acid need not to be removed in the detoxification process when using lignocelluloses hydrolysate to produce microbial lipid by R. glutinis.
Acetic Acid ; Biofuels ; Biomass ; Culture Media ; Fatty Acids ; Hydrolysis ; Industrial Microbiology ; Lignin ; chemistry ; Linoleic Acid ; Lipids ; biosynthesis ; Oleic Acid ; Rhodotorula ; metabolism