About 4-day Rhythm of Proliferative Activity of Fibroblast-like Cell Cultures Depends on the Environmental Factor

A study of the 4-day rhythm of the proliferative activity of the embryonic fibroblast-like cells in the logarithmic growth phase was carried out. It was shown that in cell cultures obtained on different days from embryos of different ages, the phase of the 4-day rhythm coincides. In vitro the maxima of the proliferative activity were consistent with the minima of the motor activity of mice. Freezing the culture for 2 or 6 days does not cause a shift in the phase of the 4-day rhythm of cell proliferative activity compare with the unfreezing culture. That indicates the existence of an external synchronizer, which determines the 4-day infradian rhythm of the proliferative activity of embryonic cells. Then we daily thawed samples of single L-929 culture of mice fibroblast-like cells for 22 and 17 days and researched the dynamics of its proliferative activity. We also showed 4-day rhythm of the simultaneous increase in the number of cells for all thawed samples. Taking into account that deep freezing of a culture leads to the cessation of all life processes, the fact we obtained indicates an exogenous mechanism of the formation of about a 4-day rhythm of the proliferative activity of cell culture.

Metformin promotes apoptosis of A549 cells via regulation of p-AMPK protein expression, bax/bcl-2 ratio and ROS levels

Purpose: To investigate the influence of metformin on apoptosis of pulmonary carcinoma cells (A549), and the associated mode of action.Methods: Pulmonary carcinoma cells in logarithmic growth phase were treated with graded concentrations of metformin, and the anti-proliferative and apoptotic effects of the drug were measured using MTT assay and flow cytometry, respectively. The levels of reactive oxygen species (ROS) in A549 cell suspension were determined with 2, 7- dihydrodichlorofluorescein diacetate (DCFH-DA) assay. The expression levels of phosphorylated AMP-activated protein kinase (p-AMPK), mammalian target of rapamycin (mTOR), and bax/bcl-2 ratio were measured using Western blotting and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR).Results: Metformin significantly promoted A549 cell apoptosis, but suppressed its proliferative potential in a dose- and time-based fashion. The levels of ROS, superoxide anion and MDA in A549 cells were significantly and dose-dependently increased by metformin (p < 0.05). Moreover, metformin markedly upregulated the mRNA and protein expressions of p-AMPK as well as bax/bcl-2 ratio, but had no impact on the expression level of mTOR (p < 0.05).Conclusion: Metformin promotes apoptosis in A549 cells via regulation of p-AMPK protein expression, bax/bcl-2 ratio, and ROS levels, and hence may play a role in lung cancer therapy.

Fibonacci Sequence-related Phenomenon During Chondrocyte Proliferation of the Gottingen Pig Knee Tibial Plateau

Background The aim of our study was to observe the quantitative changes in tibial plateau chondrocytes in the proliferation process from normal Gottingen pigs in vitro and compare them with the Fibonacci sequence.Methods Chondrocytes from normal Gottingen pig tibial plateau cartilage were isolated and cultured to the third generation, and the number of chondrocytes was measured to determine whether the chondrocyte growth was at the logarithmic growth phase. Chondrocytes were added to cell culture bottles at very a low density to allow full separation and allow each chondrocyte to grow as independently as possible. Thirty single chondrocytes were selected, and the number of chondrocyte colonies were observed and recorded every day. Results Among the 30 chondrocyte colonies, the changes in the numbers of 16 chondrocyte colonies were 1, 2, 3, 5, 8, 13, 21, 34, 55, 89, 144, 233, 377, 610, and 987, which conformed to the numbers of the Fibonacci sequence.The number of other chondrocyte colonies was lower than corresponding numbers of the Fibonacci sequence at the same time point.Conclusion The numbers of normal Gottingen pig tibial plateau chondrocytes during the proliferation process were in line with the numbers of the Fibonacci sequence.Alignment to the Fibonacci sequence may be the fastest way for changes in the numbers of normal chondrocytes during the proliferation process in vitro.

Thorsmoerkia curvula gen. et spec. nov. (Trebouxiophyceae, Chlorophyta), a semi-terrestrial microalga from Iceland exhibits high levels of unsaturated fatty acids

A terrestrial green alga was isolated at Iceland, and the strain (SAG 2627) was described for its morphology and phylogenetic position and tested for biotechnological capabilities. Cells had a distinctly curved, crescent shape with conical poles and a single parietal chloroplast. Phylogenetic analyses of 18S rDNA and rbcL markers placed the strain into the Trebouxiophyceae (Chlorophyta). The alga turned out to belong to an independent lineage without an obvious sister group within the Trebouxiophyceae. Based on morphological and phylogenetic data, the strain was described as a new genus and species, Thorsmoerkia curvula gen. et sp. nov. Biomass was generated in column reactors and subsequently screened for promising metabolites. Growth was optimized by pH-regulated, episodic CO2 supplement during the logarithmic growth-phase, and half of the biomass was thereafter exposed to nitrogen and phosphate depletion. The biomass yield reached up to 53.5 mg L−1 day−1. Fatty acid (FA) production peaked at 24 mg L−1 day−1 and up to 83% of all FAs were unsaturated. At the end of the log phase, approximately 45% of dry mass were lipids, including eicosapentaenoic acid. Carotenoid production reached up to 2.94 mg L−1 day−1 but it was halted during the stress phase. The N-linked glycans of glycoproteins were assessed to reveal chemotaxonomic patterns. The study demonstrated that new microalgae can be found at Iceland, potentially suitable for applied purposes. The advantage of T. curvula is its robustness and that significant amounts of lipids are already accumulated during log phase, making a subsequent stress exposure dispensable.

The establishment of primary cell culture from canine mammary gland tumor

BACKGROUND: In dogs, an insufficient variety of cell lines commercially available or difficulties in obtaining the existing cell lines developed from various studies results in a limited number of cytotoxicity and related molecular studies integrated with clinical practice. Hence, the doses of many drugs or supportive treatments used in canine tumor cases are adjusted based on studies in humans. OBJECTIVE: A cell line was established from a benign mixed tumor of the canine mammary gland. METHODS: Following surgical removal of the tumor, mechanical dissociation, and PBS washing, a culture process of the tumor cells was performed, including the passaging, freezing, and thawing stages. After several passages, the morphological characteristics of the cells at the logarithmic growth phase were observed under a phase-contrast microscope. RESULTS: The microscopy of the cells cultured on plastic dishes revealed monolayer colonies. The average passage time, which was 5–6 days in the first three passages, decreased to 2–3 days after the third passage. Microscopic examination of tumor cells revealed an adherent, stellated, and spindle-shaped structure. CONCLUSIONS: No difference was observed in the viability and morphology of the cells thawed even after the long period of freezing (∼18 months). The different canine cell lines can provide promising molecular applications that can be adapted into practical clinics in veterinary science.

Plant Polysaccharides Modulate Biofilm Formation and Insecticidal Activities of Bacillus thuringiensis Strains

After the biological pesticide Bacillus thuringiensis (Bt) is applied to the field, it has to remain on the surface of plants to have the insecticidal activities against insect pests. Bt can form biofilms on the surface of vegetable leaves, which were rich in polysaccharides. However, the relationship between polysaccharides of the leaves and the biofilm formation as well as the insecticidal activities of Bt is still unknown. Herein, this study focused on the effects of plant polysaccharides pectin and xylan on biofilm formation and the insecticidal activities of Bt strains. By adding pectin, there were 88 Bt strains with strong biofilm formation, 69 strains with weak biofilm formation, and 13 strains without biofilm formation. When xylan was added, 13 Bt strains formed strong biofilms, 98 strains formed weak biofilms, and 59 strains did not form biofilms. This indicated that two plant polysaccharides, especially pectin, modulate the biofilm formation of Bt strains. The ability of pectin to induce biofilm formation was not related to Bt serotypes. Pectin promoted the biofilms formed by Bt cells in the logarithmic growth phase and lysis phase at the air–liquid interface, while it inhibited the biofilms formed by Bt cells in the sporangial phase at the air–liquid interface. The dosage of pectin was positively correlated with the yield of biofilms formed by Bt cells in the logarithmic growth phase or lysis phase at the solid–liquid interfaces. Pectin did not change the free-living growth and the cell motility of Bt strains. Pectin can improve the biocontrol activities of the spore–insecticidal crystal protein mixture of Bt and BtK commercial insecticides, as well as the biofilms formed by the logarithmic growth phase or lysis phase of Bt cells. Our findings confirmed that plant polysaccharides modulate biofilm formation and insecticidal activities of Bt strains and built a foundation for the construction of biofilm-type Bt biopesticides.

Isolation and Identification of Lactobacillus plantarum C010 and Growth Kinetics of its Batch Fermentation

Chilled pork is pursuit by people due to its delicious and delicate taste, but it is still susceptible to microbial contamination even under refrigerated conditions. Consequently, to explore microbial preservatives for chilled pork, in this study, the specific spoilage organism Pseudomonas koreensis PS1 from spoiled chilled pork as the indicator was used to isolate the bacteriocin-producing lactic acid bacteria from the soils and fresh cow dungs. Among six bacteriocin-producing bacteria from 182 isolates, the strain C010 with higher-yielding, broad-spectrum, subculture stability and protease (pepsin, trypsin and proteinase K) sensitive was selected and identified as Lactobacillus plantarum based on morphological, biochemical and 16S rDNA gene sequence analysis. Simultaneously, the crude bacteriocin of L. plantarum C010 was stable under high temperature and ultraviolet conditions. The kinetics of bacterial growth and bacteriocin production of L. plantarum C010 were analyzed in batch fermentation. Bacteriocin was produced throughout the logarithmic growth phase and the Leudeking-piret model could characterize the synthesis of bacteriocin well. This present study indicates that bacteriocin-producing L. plantarum C010 has promising potentials to control the specific spoilage organism and can be used as the bio-preservative in food.

Visualizing Borrelia burgdorferi infection using a small molecule imaging probe.

In vivo diagnostic imaging of bacterial infections is currently reliant on targeting their metabolic pathways, an ineffective method to identify microbial species with low metabolic activity. Here we establish HS-198 as a small molecule-fluorescent conjugate that selectively targets the highly conserved bacterial protein, HtpG (High temperature protein G), within B. burgdorferi, the bacterium responsible for Lyme Disease. We describe the use of HS-198 to target morphologic forms of B. burgdorferi in both the logarithmic growth phase and the metabolically dormant stationary phase as well as in inactivated spirochetes. Furthermore, in a murine infection model, systemically injected HS-198 identified B. burgdorferi as revealed by imaging in post necropsy tissue sections. These findings demonstrate how small molecule probes directed at conserved bacterial protein targets can function to identify the microbe using non-invasive imaging and potentially as scaffolds to deliver antimicrobial agents to the pathogen.

Antibacterial Activity of Chrysanthemum buds Crude Extract Against Cronobacter sakazakii and Its Application as a Natural Disinfectant

Cronobacter sakazakii is an opportunistic food-borne pathogen that endangers the health of neonates and infants. This study aims to elucidate the antibacterial activity and mechanism of Chrysanthemum buds crude extract (CBCE) against C. sakazakii and its application as a natural disinfectant. The antibacterial activity was evaluated by the determination of the diameter of inhibition zone (DIZ), minimum inhibitory concentration (MIC), and minimum bactericide concentration (MBC). The antibacterial mechanism was explored based on the changes of growth curve assay, intracellular ATP concentration, membrane potential, intracellular pH (pHin), content of soluble protein and nucleic acid, and cell morphology. Finally, the inactivation effects of CBCE against C. sakazakii in biofilm on stainless steel tube, tinplate, glass, and polystyrene were evaluated. The results showed that the DIZ, MIC, and MBC of CBCE against C. sakazakii were 14.55 ± 0.44–14.84 ± 0.38 mm, 10 mg/mL, and 20 mg/mL, respectively. In the process of CBCE acting on C. sakazakii, the logarithmic growth phase of the tested bacteria disappeared, and the concentrations of intracellular ATP, pHin, bacterial protein, and nucleic acid were reduced. Meanwhile, CBCE caused the cell membrane depolarization and leakage of cytoplasm of C. sakazakii. In addition, about 6.5 log CFU/mL of viable C. sakazakii in biofilm on stainless steel tube, tinplate, glass, and polystyrene could be inactivated after treatment with 1 MIC of CBCE for 30 min at 25°C. These findings reveal the antibacterial activity and mechanism of CBCE against C. sakazakii and provide a possibility of using a natural disinfectant to kill C. sakazakii in the production environment, packaging materials, and utensils.