Logarithmic Growth Period Research Articles

The Inhibitory Effects of Anti-GPC3 Antibody on Wnt/β-Catenin Signaling Pathway as a Biological Therapy in Liver Cancer.

To investigate the effects of anti-GPC3 antibody on the Wnt/catenin pathway in liver cancer biology, thus providing a new target for the biological treatment of the disease. A total of 12 BALB/C experimental nude mice were selected as experimental objects. The mice were all male, weighed 15-20g and aged 4-5weeks. First, mouse liver cancer models were constructed. Then, the HepG2 liver cancer cells in logarithmic growth period were inoculated into the caudal veins of mouse models. On the 3rd day after inoculation, the tumor formations of nude mice were observed. Second, the anti-GPC3 antibody was designed and constructed, and the activity of anti-GPC3 antibody was detected. The mouse models were divided into the experimental group (EG) and the control group (CG), with 6 mice in each group. In the EG, mice were injected with 3mg/kg of anti-GPC3 antibody in the caudal veins once a day for three consecutive days, while in the CG mice were injected with the same amount of saline as the control. Mice in both groups received 3 injections in total. After the last administration, all mice were euthanized using the decapitation method following anesthesia with ethyl ether. The liver cancer cells of nude mice were extracted and cultured in DMEM medium. The effects of anti-GPC3 antibody on Wnt/β-catenin in liver cancer nude mice and the effects of anti-GPC3 antibody on epithelial-mesenchymal transition (EMT) in mouse liver cancer cells were observed. The bodyweight, liver weight and index of mice in the EG increased significantly (P < 0.05). The serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) levels of mice in the EG were reduced than those in the CG (P < 0.05). Compared with the CG, the superoxide dismutase (SOD) concentration increased, while the malonaldehyde (MDA) concentration decreased in the liver tissues of mice in the EG (P < 0.05). There was binding activity between GPC3 recombinant protein and the antibody; the affinity constant was KD = 1.4 × 10-6 M, compared with the commercial anti-GPC3 antibody, the affinity constant was lower. The interference effects of siRNA on anti-GPC3 antibody were detected by Western Blot. After the injection of anti-GPC3 antibody, the expression of β-catenin siRNA in liver cancer cells decreased significantly. The optical microscope images of mouse liver cancer cells in groups were compared. Through down-regulating the Wnt/β-catenin by anti-GPC3 antibody, the morphology of epithelial cells was maintained, the cells were arranged orderly, the cubic structure was kept stable, and the occurrence of EMT was reduced. However, in the CG, the structure of mouse liver cancer cells was disordered, the obvious EMT occurred (P < 0.05). Through down-regulating the expression of Wnt/β-catenin by anti-GPC3 antibody, the invasiveness, and metastasis of liver cancer cells could be effectively inhibited. Compared with the CG, the number of cells passing through the chamber was significantly reduced (P < 0.05). The anti-GPC3 antibody had inhibitory effect on Wnt/β-catenin signaling pathway. The occurrence of EMT and the invasiveness of liver cancer cells were inhibited effectively. Therefore, the anti-GPC3 antibody could be used as a reference for clinical molecular targeted therapy of liver cancer.

A new understanding of the regulatory mechanism by which Fe/Mn nanoparticles boost Bisphenol A removal using Comamonas testosteroni

Green synthesized iron/manganese nanoparticles (Fe/Mn NPs), acted as an exogenous promoter to enhance the lignin-degrading bacteria Comamonas testosteroni FJ17 resulting in more efficient removal of bisphenol A (BPA). Batch experiments demonstrated that removal efficiency of BPA via cells at a BPA concentration of 10 mg·L−1 increased by 20.9 % when exposed to 100 mg·L−1 Fe/Mn NPs after 48 h (93.63 %) relative to an unexposed control group (72.70 %). TEM and 3D-EEM analysis confirmed that the cell membrane thickness increased from 47 to 80 nm under Fe/Mn NPs exposure, and the TB-EPS secretion was promoted. Meanwhile, Fe/Mn NPs facilitated greater electron transfer capacity of c-cytochrome (0.55 V reduction peak) and an unknown cytochrome substance (0.7 V oxidation peak) on the surface of cells. Studies of the effect of Fe/Mn NPs on both the growth and activity of laccase cells showed that both biomass and laccase secretion increased significantly during the logarithmic growth period (6–36 h). LC-MS analysis and toxicity assessment indicated that Fe/Mn NPs decreased the degradation time of BPA and efficiently reduced the toxicity of its by-products. Transcriptomic analysis revealed 315 up-regulation of the key genes associated with energy supply, membrane translocation, and metabolic pathways upon exposure to Fe/Mn NPs. Such as MFS transporter (2.27-fold), diguanylate cyclase (1.76-fold) and protocatechuate-3,4-dioxygenase (1.62-fold). Overall, Fe/Mn NPs accelerated proliferation by enhancing metabolic capacity and nutrient transport processes, which serves to improve the efficiency of BPA removal.

Combined Astragalus, vitamin C, and vitamin E alleviate DEHP-induced oxidative stress and the decreased of insulin synthesis and secretion in INS-1 cells

Di-(2-ethylhexyl)-phthalate (DEHP), a common Phthalic acid ester (PAEs), has been reported to be associated with diabetes mellitus, yet the underlying mechanisms remain unknown. Combined nutrient interventions have been shown to alleviate the diabetic toxicity of DEHP. However, the effects and mechanisms of the combined intervention of Astragalus and vitamins (C and E) are currently unknown. In this study, we investigated the potential mechanisms of DEHP-induced diabetes mellitus through transcriptome analysis and vitro experiments using rat insulinoma cells (INS-1 cells). Furthermore, we explored the protection of the combined Astragalus, vitamin C, and vitamin E on DEHP-induced diabetes mellitus through these mechanisms. INS-1 cells in the logarithmic growth period were exposed to 125 umol/L DEHP followed by high-throughput sequencing analysis. The cell proliferation inhibition rate was determined using MTT assay for each group, and the cell apoptosis rate and intracellular ROS level were measured using flow cytometer. Finally, insulin levels and markers of oxidative stress were detected using ELISA kits in different groups. A total of 372 differentially expressed genes were found between the 125 umol/L DEHP and control groups, subsequent functional enrichment analyses indicated that DEHP induced oxidative stress and disturbed insulin levels. In INS-1 cells, the rate of cell proliferation inhibition, apoptosis, and the degree of oxidative stress increased concentration-dependently with increasing DEHP concentrations, while antioxidant intervention could reverse these changes. Insulin synthesis and secretion decreased after 240 μmol/L DEHP exposure stimulated by 25 mM glucose in INS-1 cells, also could antioxidant intervention alleviate these reductions. Based on these results, the underlying mechanism of DEHP impairing the function of INS-1 cells might be through apoptosis pathways induced by oxidative stress and direct reduction of insulin levels (both synthesis and secretion), while the optimal combination of Astragalus and vitamins (C and E) could exert an alleviating effect.

In Vitro Evaluation of Intestinal Transport and High-Density Fermentation of Lactobacillus acidophilus.

Lactobacillus acidophilus strains have limiting factors such as low cell density and complex nutritional requirements in industrial production, which greatly restricts their industrial application. In this study, fermentation conditions for L. acidophilus were optimized and transcriptomic analysis used to understand growth mechanisms under high-density fermentation conditions. We found that L. acidophilus IMAU81186 has strong tolerance to gastrointestinal juice. In addition, its optimal culture conditions were 3% inoculum (v/v); culture temperature 37 °C; initial pH 6.5; and medium composition of 30.18 g/L glucose, 37.35 g/L soybean peptone, 18.68 g/L fish peptone, 2.46 g/L sodium citrate, 6.125 g/L sodium acetate, 2.46 g/L K2HPO4, 0.4 g/L MgSO4·7H2O,0.04 g/L MnSO4·5H2O, 0.01 g/L serine, and 0.3 g/L uracil. After optimization, viable counts of IMAU81186 increased by 7.03 times. Differentially expressed genes in IMAU81186 were analyzed at different growth stages using transcriptomics. We found that a single carbon source had limitations in improving the biomass of the strain, and terP and bfrA were significantly down-regulated in the logarithmic growth period, which may be due to the lack of extracellular sucrose. After optimizing the carbon source, we found that adding 12 g/L sucrose to the culture medium significantly increased cell density.

Characterization of structure and antioxidant activity of exopolysaccharides from endophytic Lysinibacillus sphaericus Ya6 under acid-base stress

In this study, the structure and antioxidant activity of exopolysaccharides (EPS) produced by Lysinibacillus sphaericus Ya6 under different acid-base stresses were investigated. The structure of EPS was analyzed by UV-visible and mid-IR, and the monosaccharide composition and the molecular weight were determined by High-performance liquid chromatography (HPLC) and high-performance gel permeation chromatography respectively. The findings indicated that EPS produced by Lysinibacillus sphaericus Ya6 was a homogenous polysaccharide with a molecular weight of 79.25 kDa that was composed of mannose and glucose. The molecular weight and monosaccharide composition of polysaccharides changed under acid-base stress conditions. With the addition of the acid-base stress level, the logarithmic growth period of Lysinibacillus sphaericus Ya6 was prolonged for a short while, but later growth rates and EPS production under stress conditions were all higher than under the control. In vitro determinations found that acid-base stress boosted the antioxidant activity of EPS. Particularly under conditions of alkaline stress, DPPH radical scavenging capacity and superoxide anion scavenging of EPS were higher than those of the control, and their capacities to scavenge DPPH was increased from 39.30% (the control) to 95.65% at pH 10.0, which was close to ascorbic acid's (95.77%) as a positive control.

An innovative strategy to control Microcystis growth using tea polyphenols sustained-release particles: preparation, characterization, and inhibition mechanism.

Allelochemicals have been shown to inhibit cyanobacterial blooms for several years. In view of the disadvantages of "direct-added" mode, natural and pollution-free tea polyphenolic allelochemicals with good inhibitory effect on cyanobacteria were selected to prepare sustained-release particles by microcapsule technology. Results showed that the encapsulation efficiency of tea polyphenols sustained-release particles (TPSPs) was 50.6% and the particle size ranged from 700 to 970nm, which reached the nanoscale under optimum preparation condition. Physical and chemical properties of TPSPs were characterized to prove that tea polyphenols were well encapsulated and the particles had good thermal stability. The optimal dosage of TPSPs was determined to be 0.3g/L, at which the inhibition rate on Microcystis aeruginosa in logarithmic growth period could be maintained above 95%. Simultaneous decrease in algal density and chlorophyll-a content indicated that the photosynthesis of algal cells was affected leading to cell death. Significant changes of antioxidant enzyme activities suggested that Microcystis aeruginosa's antioxidant systems had been disrupted. Furthermore, TPSPs increased the concentration of O2- which led to lipid peroxidation of cell membrane and a subsequent increase in malondialdehyde (MDA) content. Meanwhile, the protein content, nucleic acid content, and electrical conductivity in culture medium rose significantly indicating the cell membrane was irreversibly damaged. This work can provide a basis for the utilization of environmentally friendly algal suppressants.

Glutamine alleviated heat stress-induced damage of porcine intestinal epithelium associated with the mitochondrial apoptosis pathway mediated by heat shock protein 70.

The present study aimed to investigate the effect of glutamine (Gln) addition on the damage of porcine intestinal epithelial cells (IPEC-J2) induced by heat stress (HS). IPEC-J2 cultured in logarithmic growth period in vitro were firstly exposed to 42 °C for 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h for cell viability and cultured with 1, 2, 4, 6, 8, or 10 mmol Gln per L of culture media for heat shock protein 70 (HSP70) expression to determine the optimal disposal strategy (HS, 42 °C for 12 h and HSP70 expression, 6 mmol/L Gln treatment for 24 h). Then IPEC-J2 cells were divided into three groups: control group (Con, cultured at 37 °C), HS group (HS, cultured at 42 °C for 12 h), and glutamine group (Gln+HS, cultured at 42 °C for 12 h combined with 6 mmol/L Gln treatment for 24 h). The results showed that HS treatment for 12 h significantly decreased the cell viability of IPEC-J2 (P < 0.05) and 6 mmol/L Gln treatment for 12 h increased HSP70 expression (P < 0.05). HS treatment increased the permeability of IPEC-J2, evidenced by the increased fluorescent yellow flux rates (P < 0.05) and the decreased transepithelial electrical resistance (P < 0.05). Moreover, the downregulated protein expression of occludin, claudin-1, and zonula occludens-1 was observed in HS group (P < 0.05), but Gln addition alleviated the negative effects on permeability and the integrity of intestinal mucosal barrier induced by HS (P < 0.05). In addition, HS resulted in the elevations in HSP70 expression, cell apoptosis, cytoplasmic cytochrome c potential expression, and the protein expressions of apoptosis-related factors (apoptotic protease-activating factor-1, cysteinyl aspartate-specific proteinase-3, and cysteinyl aspartate-specific proteinase-9) (P < 0.05); however, the reductions in mitochondrial membrane potential expression and B-cell lymphoma-2 expression were induced by HS (P < 0.05). But Gln treatment attenuated HS-induced adverse effects mentioned above (P < 0.05). Taken together, Gln treatment exhibited protective effects in protecting IPEC-J2 from cell apoptosis and the damaged integrity of epithelial mucosal barrier induced by HS, which may be associated with the mitochondrial apoptosis pathway mediated by HSP70.

Effects of silencing farnesyltransferase on the migration, invasion, and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells.

This study aimed to investigate the effects of farnesyltransferase (FTase) on the migration, invasion, and epithelial-mesenchymal transition (EMT) of SACC-LM and SACC-83 cells in salivary adenoid cystic carcinoma and determine the relative mechanism. Three small interfering RNA (siRNA) sequences were designed and constructed based on the human FTase gene sequence. The SACC-LM and SACC-83 cells in the logarithmic growth period were used, and the expression of FTase was suppressed by liposomal transient transfection. The tested cells were categorized as the FTase-siRNA-1, FTase-siRNA-2, and FTase-siRNA-3 groups. Both negative control group (NC-siRNA) and blank control group (only transfection reagent was added) were set. The mRNA expression of FTase and HRAS was detected by quantitive real-time polymerase chain reaction, and the silencing efficiency was determined. The expression levels of FTase, HRAS, protein kinase B (AKT), phospho-AKT, p65, phospho-p65 (Ser563), E-cadherin, vimentin, matrix metalloproteinase (MMP)-9 protein, and HRAS membrane protein were detected by Western blot. Transwell assay and wound healing assay were used to detect the invasion and migration abilities of cells. The relative expression of FTase mRNA and protein in the FTase-siRNA-1 group decreased compared with those in the control group (P<0.05). HRAS mRNA and total protein expression had no significant difference (P>0.05), and the relative expression of HRAS membrane protein decreased (P<0.05). The relative expression of E-cadherin increased (P<0.05), vimentin decreased (P<0.05), and MMP-9 decreased (P<0.05). There was no significant difference in the relative expression levels of the RAS/PI3K/AKT/nuclear factor-κB signaling pathway-related proteins AKT and p65 (P>0.05), but the relative expression levels of phospho-AKT and phospho-p65 decreased. The invasion and migration ability of the FTase-siRNA-1 group significantly decreased compared with that in the control group (P<0.05). Silencing FTase in vitro could effectively inhibit the invasion and migration of SACC-LM and SACC-83 cells by interfering with the localization of the HRAS membrane protein and regulating the RAS/PI3K/AKT/nuclear factor-κB signaling pathway to mediate EMT.

Relationship between β-Carotene Accumulation and Geranylgeranyl Pyrophosphate Synthase in Different Species of Dunaliella.

To study the relationship between β-carotene synthesis and geranylgeranyl pyrophosphate synthase (GGPS) activity, 15 species of Dunaliella were used to determine the changes in photosynthetic pigment contents, chlorophyll fluorescence parameters, β-carotene content, and GGPS activity. By observing the morphology and size of 15 species of Dunaliella, D8 has the largest individual algal cell and D9 has the smallest individual. Growth was relatively slow during days one through seven. After about eight days, the cells entered the logarithmic growth period and grew rapidly to a high density. After about 45 days, they entered a mature period, and growth slowed down. The contents of chlorophyll, carotenoids, and β-carotene increased during growth. D1 has the highest accumulation of β-carotene, and GGPS enzyme activity has a positive linear relationship with the β-carotene synthesis content. Phylogenetic analysis showed that the GGPS proteins of the 15 species were highly homologous, and the GGPS protein was not part of the membrane.

Effects of miR-155-3p on the degradation rate of EAF1 mRNA and malignant proliferation in HANK1 cells

Objective: To investigate the effects and mechanisms of miR-155-3p on the malignant behavior of human NK/T cell lymphoma cell line HANK1. Methods: Targetscan database was used to predict the target gene of miR-155-3p. HANK1 cells in logarithmic growth period were cultured, and the cells were divided into blank group, over-expressed group, control group and interference group, which were transfected with pENTER-puro vector, pENTER-miR-155-3p vector, GV248 control vector and GV248-miR-155-3p siRNA interference vector, respectively. Meanwhile, actinomycin D (ActD) was used to treat each group of cells, and the expressions of miR-155-3p, EAf1, β-catenin and c-Myc in each group were detected by real-time fluorescence quantitative PCR (n=5). The degradation rate of EAF1 mRNA, the expressions of EAF1, β-catenin and c-Myc protein were detected by Western blot (n=3), and the malignant proliferation abilities of cells were detected by CCK-8 (n=5). Results: Compared with the blank group, the expression levels of miR-155-3p, β-catenin and c-Myc in the over-expressed group were significantly higher, the expression level of EAF1 was lower, the half-life of EAF1 mRNA was shortened, and the malignant proliferation ability of the cells was strengthened (P＜0.05). Compared with the control group, the expression levels of miR-155-3p, β-catenin and c-Myc in the interference group were significantly lower, and the expression level of EAF1 was increased, the half-life of mRNA was prolonged and the ability of cell proliferation was decreased (P＜0.05). Conclusion: miR-155-3p can promote EAF1 mRNA degradation and proliferation in HANK1 cells.

Identification and fermentative properties of an indigenous strain ofWickerhamomyces anomalusisolated fromRosa roxburghiiTratt

PurposeThe purpose of this paper is to identify and analyse the fermentative properties of a strain of indigenousWickerhamomyces anomalus(W. anomalus) fromRosa roxburghiiTratt (R. roxburghii).Design/methodology/approachMorphological and molecular methods were used to determine the species of the selected strainW. anomalusC11. The physiological tolerances to glucose, ethanol, citric acid and sulphur dioxide (SO2) were further assessed by checking the growth of cells, and the oenological performances were proved to measure the related fermentative properties ofR. roxburghiiwines.FindingsTheW. anomalusC11 strain could be grown faster than commercialS. cerevisiaeX16 in its logarithmic growth period and had preferable tolerances to glucose, ethanol, citric acid and SO2. Moreover, this strain of nativeR. roxburghiiyeastW. anomalusC11 produced less sulphuretted hydrogen and had a higherβ-glucosidase activity. Furthermore,W. anomalusC11 could reduce the volatile acids, reduce the sourness and enhance volatile aroma richness and complexity ofR. roxburghiiwines including types of aroma and content thereof. Taken together, theR. roxburghiinative yeastW. anomalusC11 may have potential for use inR. roxburghiiwinemaking.Originality/value(1) The fermentative properties of a strain of indigenousW. anomalus(named as C11) fromR. roxburghiiwas evaluated. (2) The strain ofW. anomalusC11 had preferable tolerances to glucose, ethanol, citric acid and SO2. (3) This strain of nativeR. roxburghiiyeastW. anomalusC11 produced less sulphuretted hydrogen and had a higherß-glucosidase activity. (4)W. anomalusC11 could reduce the volatile acids, reduce the sourness and enhance volatile aroma richness and complexity ofR. roxburghiiwines including types of aroma and content thereof.

Signs of biofilm formation in the genome of Labrenzia sp. PO1.

Labrenzia sp. are important components of marine ecology which play a key role in biochemical cycling. In this study, we isolated the Labrenzia sp. PO1 strain capable of forming biofilm, from the A. sanguinea culture. Growth analysis revealed that strain reached a logarithmic growth period at 24 hours. The whole genome of 6.21813 Mb of Labrezia sp. PO1 was sequenced and assembled into 15 scaffolds and 16 contigs, each with minimum and maximum lengths of 644 and 1,744,114 Mb. A total of 3,566 genes were classified into five pathways and 31 pathway groups. Of them, 521 genes encoded biofilm formation proteins, quorum sensing (QS) proteins, and ABC transporters. Gene Ontology annotation identified 49,272 genes that were involved in biological processes (33,425 genes), cellular components (7,031genes), and molecular function (7,816 genes). We recognised genes involved in bacterial quorum sensing, attachment, motility, and chemotaxis to investigate bacteria's ability to interact with the diatom phycosphere. As revealed by KEGG pathway analysis, several genes encoding ABC transporters exhibited a significant role during the growth and development of Labrenzia sp. PO1, indicating that ABC transporters may be involved in signalling pathways that enhance growth and biofilm formation.

Influence Scope of Potamogeton crispus Population on Lake Waterquality during the Growth Period in Xuanwu Lake, China

Potamogeton crispus has a significant effect on the purification of contaminated water and can improve the stability of aquatic ecosystems, but much research work was carried out under controlled conditions. The water quality indexes were measured to determine whether the purification effect under natural conditions can achieve the same effect as under controlled conditions in this study. The physical and chemical indicators of the water around the P. crispus population were monitored to study the range of impact of P. crispus population during the logarithmic growth period on the water in Xuanwu Lake, Nanjing City. These indicators included pH, secchi disc transparency, turbidity, dissolved oxygen, total nitrogen, ammonia, and total phosphate. The result showed that P. crispus population played an important role in improving lake water quality. P. crispus population increased dissolved oxygen concentration, improved transparency, reduced turbidity, absorbed nutrients from the water column. The transparency, turbidity, and pH value could be affected to 40% of the width of P. crispus population, while dissolved oxygen concentration could be affected to the same width of the population. But the nitrogen and phosphorus concentration could be affected to 10% of the population. In vertical space, the influence of P. crispus population on the surface water was greater than that of the bottom water. According to this study, the gap between P. crispus population can be controlled at 20% of the population width, which can effectively reduce nutrient concentration, improve water transparency and dissolved oxygen content, improve water quality. In the process of ecological restoration with P. crispus population, the width of P. crispus population should be controlled and prevent the spread by regular harvesting, blocking with the net, and so on. The gap between populations should also be determined according to the population width.

Role of 14-3-3σgene in the regulation of endotoxin/lipopolysaccharide-induced inflammatory responses in human pulmonary epithelial cells

Objective: To explore the mechanism of 14-3-3σgene in regulating inflammatory response of human pulmonary epithelial cells induced by endotoxin/lipopolysaccharide (LPS). Methods: (1) Cells of human normal pulmonary epithelial cell line BEAS-2B cultured in logarithmic growth period were collected and divided into control group and PCMV6-14-3-3σgroup using the random number table, with 3 wells in each group. Cells in control group were transfected with empty plasmid, and cells in PCMV6-14-3-3σgroup were transfected with PCMV6-14-3-3σplasmid. The protein expression of 14-3-3σin cell was detected by Western blotting at 48 hours after transfection. (2) Cells of human normal pulmonary epithelial cell line BEAS-2B cultured in logarithmic growth period were collected and divided into control group, PCMV6-14-3-3σgroup, PCMV6-14-3-3σ+ LPS group, and LPS group using the random number table, with 3 wells in each group. Cells in control group were transfected with empty plasmid for 42 hours. Cells in PCMV6-14-3-3σgroup were transfected with PCMV6-14-3-3σplasmid for 42 hours. Cells in PCMV6-14-3-3σ+ LPS group were stimulated with 1 μg/mL LPS (the same final mass concentration below) for 6 hours after being transfected with PCMV6-14-3-3σplasmid for 42 hours. Cells in LPS group were stimulated by LPS for 6 hours. The protein expressions of Bax and B-cell lymphoma-2 (Bcl-2) were detected by Western blotting, and the ratio of Bax to Bcl-2 was calculated. Apoptotic rate was detected by flow cytometry. The mRNA expressions of tumor necrosis factor alpha (TNF-α) and interleukin 1beta (IL-1β) in cells were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction technique. Content of TNF-α and IL-1β in cell culture supernatant was detected by enzyme-linked immunosorbent assay. Data were statistically analyzed with t test, one-way analysis of variance, and least significant difference test. Results: (1) At 48 hours after transfection, the protein expression of 14-3-3σin cells of PCMV6-14-3-3σgroup (1.05±0.03) was significantly higher than that in control group (0.78±0.04, t=5.41, P<0.01). (2) Compared with those in control group, the ratio of Bax to Bcl-2, apoptotic rate, mRNA expressions of TNF-α and IL-1β, and content of TNF-α and IL-1β in cell supernatant in PCMV6-14-3-3σgroup showed no significant difference (P>0.05); the above-mentioned indexes of cells in LPS group were significantly higher or increased (P<0.01). Compared with those in LPS group, the above-mentioned indexes of cells in PCMV6-14-3-3σ+ LPS group were significantly lower or decreased (P<0.01). Conclusions: 14-3-3σis a key factor in regulating apoptosis. It can alleviate the LPS-induced inflammatory responses by regulating the ratio of apoptotic regulators Bax to Bcl-2 and inhibiting apoptosis of human pulmonary epithelial cells.

Protective effect of hydrogen-rich water on oxidative stress cell model and the impact of the phosphatidylinositol 3 kinase/protein kinase B pathway

To explore the protective effect of hydrogen-rich water on the oxidative stress injury of astrocytes in mice and its effect on phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signal pathway. In vitro, mice astrocytes were cultured and the logarithmic growth period cells were taken for experiment. (1) Experiment one: some cells were acted by 1.25, 2.50, 5.00, 10.00 μmol/L hydrogen peroxide (H2O2) for 20 minutes to determine the appropriate concentration required for astrocyte damage induced by H2O2; cultivating 3, 6, 9, and 12 hours with hydrogen-rich water of 25, 50, 100, and 200 μmol/L, respectively, to determine the concentration and time of hydrogen-rich water pretreatment; the 50 μmol/L hydrogen-rich water was cultured together with PI3K/Akt signal pathway inhibitors wortmannin (WM) 200 nmol/L or 400 nmol/L to determine the best inhibition concentration of wortmannin. Astrocyte activity was detected by methyl thiazolyl tetrazolium (MTT) colorimetry. (2) Experiment two: some cells were divided into blank control group, H2O2 injury group, hydrogen-rich water pretreatment group (HW+H2O2 group), and co-culture of hydrogen-rich water and wortmannin pretreatment group (HW+WM+H2O2 group). The mRNA expressions of PI3K and Akt were detected by reverse transcription-polymerase chain reaction (RT-PCR); the protein expressions of PI3K, Akt and phosphorylated Akt (p-Akt) were detected by Western Blot. (1) Experiment one: the survival rate of the blank control group was 100%. Cell activity gradually decreased with the increase of H2O2 concentration, and the survival rate of the H2O2 action 20 minutes cells of 2.50 μmol/L was reduced to about 50%, so a cell injury model was established at this concentration. With the increase of hydrogen-rich water pretreatment concentration, and the duration of action, the cell survival rate increased first and then decreased. The cell survival rate was highest when 50 μmol/L hydrogen-rich water was pretreated with 9 hours, so a hydrogen-rich water pre-protection model was established. After 200 nmol/L or 400 nmol/L wortmannin was cultured together with hydrogen-rich water, cell activity was inhibited, and the cell survival rate of 200 nmol/L wortmannin group was no significantly different compared with that of H2O2 injury group, so the astrocyte suppression model was established. (2) Experiment two: compared with the blank control group, the mRNA expressions of PI3K and Akt and the protein expressions of PI3K, Akt and p-Akt were significantly decreased in the H2O2 injury group. Compared with the H2O2 injury group, the PI3K, Akt mRNA expressions and PI3K, Akt, p-Akt protein expressions were significantly increased in the HW+H2O2 group [PI3K mRNA (2-ΔΔCT): 0.843±0.019 vs. 0.631±0.038, Akt mRNA (2-ΔΔCT): 0.591±0.025 vs. 0.558±0.037, PI3K/β-actin: 1.277±0.008 vs. 0.757±0.004, Akt/β-actin: 1.308±0.015 vs. 0.682±0.006, p-Akt/β-actin: 1.210±0.005 vs. 0.614±0.005, all P < 0.05]. The mRNA expressions of PI3K, Akt in the HW+WM+H2O2 group was 0.784±0.159 and 0.556±0.037, respectively, and the protein expressions of PI3K, Akt, p-Akt was 0.715±0.006, 0.686±0.005, and 0.606±0.004, respectively, both were significantly lower than those in HW+H2O2 group (all P < 0.05), and there was no significant difference with H2O2 injury group (all P > 0.05). Hydrogen-rich water activates the PI3K/Akt pathway, thereby mediates mice astrocytes to exert the biological function of antioxidant.

Effect of high voltage electric field on cell growth, apoptosis of human pancreatic cancer cell line PANC-1

Objective To investigate the effects of high intensity electric field on cell growth, apoptosis and microstructure of human pancreatic cancer cell line PANC-1. Methods The PANC-1 cells in the logarithmic growth period were selected, and cells in the high voltage electrical treatment group were treated with high voltage electric field 250, 500, 750, 1000 V/cm, respectively. The effects of different high voltage electric fields on cell growth and microstructure of PANC-1 cells were determined by cell viability, cell death staining, apoptosis detection, transmission electron microscopy and scanning electron microscopy. Results Compared with control group, the high voltage electric pulse significantly inhibited the growth of PANC-1 cells in the field dependent manner. Moreover, when the field was more than 500 V/cm, the cell viability was significantly decreased (P<0.05). High voltage electric pulse could induce cell apoptosis. When the field was higher than 750 V/cm, serious necrosis was noticed. In the 1000 V/cm group, the integrity of cell membrane and the structure of organelles was seriously damaged. Conclusion High voltage electric pulse could significantly inhibit the growth of PANC-1 cells and would be a promising method in cancer treatment. Key words: High voltage electric field; Pancreatic cancer cell; Growth; Apoptosis; Cell membrane; Organelle

Effect of microplastics exposure on the photosynthesis system of freshwater algae.

Microplastics are widely distributed in freshwater environments. At present, most of the studies on the toxicity of microplastics are concentrated on aquatic feeding animals, but relatively few have addressed freshwater algae. This study investigated the effect of microplastics (polypropylene (PP) and polyvinyl chloride (PVC)) exposure on the photosynthetic system of freshwater algae over the logarithmic growth period. The results showed that both PVC and PP had a negative effect on chlorophyll a concentrations of Chlorella (C.) pyrenoidosa and Microcystis (M.) flos-aquae; among them, when the concentration of PVC exceeded 250 mg/L, compared with the control group, the chlorophyll a content of C. pyrenoidosa was reduced by 55.23%. For photosynthetic activity, higher concentrations of PVC and PP can induce lower values of Fv/Fm, Fv/F0, and Fv'/Fm', suggesting a larger impact in algae. However, algae were able to adjust, with increased values of Fv/Fm, Fv/F0, and Fv'/Fm'. This dose-negative effect phenomenon also exists in the study of the rapid light-response curves. In addition, comparing the two microplastics, we could see that PVC greatly inhibits the photosynthesis system of freshwater algae. Our study confirmed that microplastics can affect algae growth under certain concentrations, which provides evidence for understanding the risks of microplastics.

Isolation, culture and identification of human adipose-derived stem cells.

The aim of the present study was to improve methods for the isolation and identification of adipose-derived stem cells (ASCs). Human subcutaneous adipose tissue was collected during liposuction surgery, without ultrasound-assisted liposuction and other assisted techniques, and digested with 0.075% collagenase I. First (P1) and second (P2) passage ASCs were applied to the subsequent experiments. ASCs were observed under a microscope, the growth curves of the cells were assessed using a cell counting kit-8 assay and the membrane expression of cell surface antigens, including cluster of differentiation (CD)44, CD105 and CD45, were detected by flow cytometry. In addition, ASCs were induced to differentiate into lipocytes and osteocytes. Oil red staining was applied to examine adipogenic induction, whereas alkaline phosphatase (ALP) staining was used to assess osteogenic induction. Primary ASCs adhered to the culture vessel wall after 72 h, were fusiform in appearance at 5 days and exhibited stable growth with active proliferation. In total, 1×105 stem cells were gained per 50 ml of lipo-aspirate. ASCs were plated in a 25 cm2 culture flask at a density of 5×104/ml; the cells underwent the first logarithmic growth period after 72 h and grew to 90% confluence within 3 days. Flow cytometry demonstrated that the cells were highly positive for CD105 and CD44, and weakly positive for CD45; 18.6% of P1 cells and 90.7% of P2 cells were CD44+CD45-CD105+. Oil red and ALP staining were positive. The results of the present study suggested that ASCs may be considered a promising cell type for tissue engineering. Furthermore, the present study established an effective method for the isolation and identification of ASCs, which reduced damage to the stem cells and simplified the identification procedure.

异养硝化–好氧反硝化细菌X&lt;sub&gt;3&lt;/sub&gt;的氮形态响应

异养硝化–好氧反硝化细菌的氮响应机制对研究生物脱氮技术具有重要意义。通过分析异养硝化–好氧反硝化细菌X3 (Halomonas alkaliphila)生化周期与培养液中氮形态的关系，对该菌的脱氮机制进行了研究。结果表明：1) 含混合形态氮培养液中，细菌X3在对数生长期主要进行有机氮的降解；整个周期中，氨氮先升高后降低，48 h达到最高值，亚硝酸氮先升高后降低，第4天达到最高值，硝酸氮表现为先下降，48 h后上升的变化趋势，96 h总氮去除率为17.07%；2) 仅含无机氮的培养液中，细菌X3在对数生长期主要进行氨氮和硝酸氮的降解；整个周期中，氨氮和硝酸氮持续下降，亚硝酸氮先升高后降低，24 h达到最高值，96 h总氮去除率可达51.13%。结论：细菌X3的氮降解优先顺序和最终效能皆受控于环境氮化合物形态。本研究有望为生物脱氮工程设计提供理论指导。 For the development of biological denitrification technique, study on the response mechanism of heterotrophic nitrifying-aerobic denitrifying bacteria to nitrogen forms has great significance. Herein the relationship between the nitrogen form and the denitrification activity of Halomonas alkaliphila X3 was studied by monitoring the change of different nitrogen forms in the culture so-lution, and the results are as follows. 1) In the culture solution containing both organic nitrogen and inorganic nitrogen, Halomonas alkaliphila X3 mainly degrades organic nitrogen during the logarithmic growth period. Over the whole culture cycle, the concentration of ammonia nitrogen rises up in the first 48 h, followed by a decreasing trend; the concentration of nitrite nitrogen rises up in the first 4 d, followed by a decreasing trend; while the concentration of nitrate nitrogen de-creases in the first 48 h, followed by a decreasing trend. In total 17.07% nitrogen is removed from the culture solution after a treatment for 96 h. 2) In the culture solution containing only inorganic nitrogen, the bacteria mainly degrades ammonia nitrogen during the logarithmic growth period. Over the whole culture cycle, the concentration of ammonia and nitrate nitrogen keeps down; the concentration of nitrite nitrogen rises up in the first 24 h, followed by a decreasing trend. In total 51.13% nitrogen is removed from the culture solution after a treatment for 96 h. In conclusion, the priority and efficiency of the nitrogen degradation by Halomonas alkaliphila X3 both depend on the initial nitrogen forms in the culture solution. These outcomes are promising to help the construction of biological denitrification engineering.

Growth and microcystin production of a Brazilian Microcystis aeruginosa strain (LTPNA 02) under different nutrient conditions

Cyanobacteria are prokaryotic and photosynthetic organisms, which can produce a wide range of bioactive compounds with different properties; including a variety of toxic compounds, also known as cyanotoxins. In this work, we describe the isolation of seven cyanobacterial strains from two reservoirs in São Paulo State, Brazil. Seven different chemical variants of microcystins (MC-RR, MC-LR, MC-YR, MC-LF, MC-LW, and two demethylated variants, dm-MC-RR and dm-MC-LR) were detected in three of the ten isolated strains. One particular Microcystis aeruginosa strain (LTPNA 02) was chosen to evaluate its growth by cell count, and its toxin production under seven different nutritional regimes. We observed different growth behaviors in the logarithmic growth period for only three experiments (p < 0.05). The total growth analysis identified four experiments as different from the control (p < 0.01). Three microcystin variants (MC-RR, MC-LR and MC-YR) were quantified by liquid chromatography-tandem mass spectrometry. At the experimental end, the toxin content was unchanged when comparing cell growth in ASM-1 (N:P = 1), MLA and BG-11 (N:P = 10) medium. In all other experiments, the lowest microcystin production was observed from cells grown in Bold 3N medium during the exponential growth phase. The highest microcystin content was observed in cultures using BG-11(N:P = 100) medium.