In vitro observation: the GFP-E. coli adhering to porcine erythrocytes can be removed by porcine alveolar macrophages

Cytological observations of transfer of immune complexes via porcine erythrocytes

10.7287/peerj.preprints.26881 ◽

2018 ◽

Author(s):

Wei Yin ◽

Chun Wang ◽

Kuohai Fan ◽

Na Sun ◽

Yaogui Sun ◽

...

Keyword(s):

Flow Cytometry ◽

Confocal Microscopy ◽

Fluorescence Intensity ◽

Immune Complexes ◽

Observation Time ◽

Control Group ◽

Laser Confocal Microscopy ◽

Functional Protein ◽

Blank Control Group ◽

E Coli

ABSTRACT Although the activation of pathogen phagocytosis via complement system has been studied, erythrocyte-phagocyte interactions in pigs are not clearly understood. Therefore, we sought to investigate the ability of porcine erythrocytes to clear immune complexes by using laser confocal microscopy and flow cytometry to observe the immune adhesion of porcine erythrocytes to fluorescent bacilli and the immune presentation process of transferring fluorescent bacilli to macrophages. Isolated porcine alveolar macrophages (PAMs) had uniform morphology and size, and a survival rate of 97.2%. The phagocytosis rate was 98.8%. After WT E. coli was labeled with FITC, the bacteria showed a bright green fluorescence, and the labeling rate was 92.3%. When laser confocal microscopy was utilized to observe the co-incubation system of porcine erythrocytes, PAM, and fluorescent E. coli, the fluorescence intensity of bacilli decreased with increasing observation time and even disappeared. Flow Cytometry examination showed that the average fluorescence intensity of PAMs co-incubated with porcine erythrocytes adhered to WT-E. coli-FITC, was significantly higher than that of normal PAMs. Furthermore, when porcine erythrocytes adhered to WT E. coli were incubated with PAMs, the surface mean fluorescence intensity of porcine erythrocytes was significantly higher than that of the blank control group. This shows that PAMs can competitively bind to the oposinized E. coli adhered to the surface of porcine erythrocytes, and these oposinized pathogens can enter macrophages by the process of phagocytosis，which promoting the internalization of immune complexes or pathogens. During this process, the physical morphology of porcine erythrocytes was not damaged, but the levels of its main functional protein CR1-like were decreased.

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Cytological observations of transfer of immune complexes via porcine erythrocytes

10.7287/peerj.preprints.26881v1 ◽

2018 ◽

Author(s):

Wei Yin ◽

Chun Wang ◽

Kuohai Fan ◽

Na Sun ◽

Yaogui Sun ◽

...

Keyword(s):

Flow Cytometry ◽

Confocal Microscopy ◽

Fluorescence Intensity ◽

Immune Complexes ◽

Observation Time ◽

Control Group ◽

Laser Confocal Microscopy ◽

Functional Protein ◽

Blank Control Group ◽

E Coli

ABSTRACT Although the activation of pathogen phagocytosis via complement system has been studied, erythrocyte-phagocyte interactions in pigs are not clearly understood. Therefore, we sought to investigate the ability of porcine erythrocytes to clear immune complexes by using laser confocal microscopy and flow cytometry to observe the immune adhesion of porcine erythrocytes to fluorescent bacilli and the immune presentation process of transferring fluorescent bacilli to macrophages. Isolated porcine alveolar macrophages (PAMs) had uniform morphology and size, and a survival rate of 97.2%. The phagocytosis rate was 98.8%. After WT E. coli was labeled with FITC, the bacteria showed a bright green fluorescence, and the labeling rate was 92.3%. When laser confocal microscopy was utilized to observe the co-incubation system of porcine erythrocytes, PAM, and fluorescent E. coli, the fluorescence intensity of bacilli decreased with increasing observation time and even disappeared. Flow Cytometry examination showed that the average fluorescence intensity of PAMs co-incubated with porcine erythrocytes adhered to WT-E. coli-FITC, was significantly higher than that of normal PAMs. Furthermore, when porcine erythrocytes adhered to WT E. coli were incubated with PAMs, the surface mean fluorescence intensity of porcine erythrocytes was significantly higher than that of the blank control group. This shows that PAMs can competitively bind to the oposinized E. coli adhered to the surface of porcine erythrocytes, and these oposinized pathogens can enter macrophages by the process of phagocytosis，which promoting the internalization of immune complexes or pathogens. During this process, the physical morphology of porcine erythrocytes was not damaged, but the levels of its main functional protein CR1-like were decreased.

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Metformin Exerts An Antitumor Effect By Inhibiting Bladder Cancer Cell Migration And Growth, And Promoting Apoptosis Through The PI3K/AKT/mTOR Pathway

10.21203/rs.3.rs-846120/v1 ◽

2021 ◽

Author(s):

Zhiyong Shen ◽

Dong Xue ◽

Kun Wang ◽

Facai Zhang ◽

Jiaqi Shi ◽

...

Keyword(s):

Flow Cytometry ◽

Bladder Cancer ◽

Cell Migration ◽

Caspase 3 ◽

Control Group ◽

Mtor Signaling Pathway ◽

Blank Control Group ◽

Metformin Group ◽

Cell Migration And Proliferation

Abstract Background To observe and explore the effect of metformin on the migration,and proliferation of bladder cancer T24 and 5637 cells in vitro.Methods Bladder cancer T24 and 5637 cell lines were cultured in vitro, and were divided into group A (blank control group) and group B (metformin group: 5, 10, 15, and 20 mmol/L); both groups were plated on 6-well plates at the same time. Culture in 24-well plates was used for wound healing assays and in 96-well plates for Transwell migration and invasion, and Cell Counting Kit-8 proliferation experiments. We observed and detected the cell migration and proliferation ability of each group at 48 hours, and calculated the cell migration area and survival rate. Flow cytometry was used to detect cell apoptosis in the groups. The apoptosis-related protein, cleaved-caspase 3, cleaved-PARP and PI3K/AKT/mTOR signaling pathway member proteins PI3K, phosphorylated (p)-PI3K, AKT, p-AKT, mTOR, and p‑mTOR were detected using western blotting.Results After 48 hours of treatment with different concentrations of metformin, the cell migration and proliferation capabilities were significantly lower than those in the blank control group. The proliferation and migration abilities of T24 and 5637 cells decreased in a metformin concentration-dependent manner (P < 0.05). The apoptosis rate under different concentrations of metformin, as detected by flow cytometry, showed a significantly higher rate in the metformin group than in the control group (P＜0.05). Compared with that in the control group, the level of cleaved‑caspase 3 and cleaved-parp protein in the metformin group was increased in each treatment group, and the levels of p-mTOR, p-AKT, and p-PI3K decreased significantly compared with those in the control group (P < 0.05).Conclusion Metformin inhibit bladder cancer T24 and 5637 cell migration and proliferation, and induced their apoptosis. The mechanism might involve inhibition of the activation of the PI3K/AKT/mTOR signaling pathway.

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Evaluating the Effect of Cinnarizine on Promastigotes and Amastigotes forms of Leishmania major

Infectious Disorders - Drug Targets ◽

10.2174/1871526518666181113114820 ◽

2020 ◽

Vol 20 (4) ◽

pp. 550-555 ◽

Cited By ~ 1

Author(s):

Lima Asgharpour Sarouey ◽

Parvaneh Rahimi-Moghaddam ◽

Fatemeh Tabatabaie ◽

Khadijeh Khanaliha

Keyword(s):

Flow Cytometry ◽

Cutaneous Leishmaniasis ◽

Leishmania Major ◽

Inhibitory Effect ◽

Control Group ◽

Secondary Infections ◽

Ic50 Values ◽

J774 Cells ◽

Mtt Assays

: As an important global disease, cutaneous leishmaniasis is associated with complications such as secondary infections and atrophic scars. The first line treatment with antimonials is expensive and reported to have serious side effects and enhance resistance development. The main objective of this study was to evaluate the effect of Cinnarizine on standard strains of Leishmania major because of paucity of information on this subject. Methods: In this experimental study, four concentrations of the drug (5, 10, 15 and 20 μg/ml) were added to Leishmania major cultures at 24, 48 and 72 hours intervals. MTT assays were performed to determine parasite viability and drug toxicity. Leishmania major promastigotes were augmented to the in vitro cultured macrophages (J774 cells) and then incubated for 72 hours. Half maximal inhibitory concentration (IC50) was ascertained by counting parasites. The inhibitory effect of the drug was compared with that of Glucantime. Flow-cytometry was performed to investigate apoptosis. Each test was repeated thrice. Results: The IC50 values of Cinnarizine after 72 hours were calculated to be 34.76 μg/ml and 23.73 μg/ml for promastigotes and amastigotes, respectively. The results of MTT assays showed 48 % promastigote viability after 72 hour-exposure to Cinnarizine at 20 μg/ml concentration. Programmed cell death in promastigote- and amastigote-infected macrophages was quantified to be 13.66 % and 98.7 %, respectively. Flow- cytometry analysis indicated that Cinnarizine induced early and late apoptosis in parasites. All treatments produced results which differed significantly from control group (P<0.05). Conclusion: Cinnarizine showed low toxicity with anti-leishmanial and apoptosis effects on both promastigote and intracellular amastigote forms. Therefore, we may suggest further assessment on animal models of this drug as candidates for cutaneous leishmaniasis therapy.

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Silica/OCP Affects the Viability of Osteoblasts through ROS-Induced Autophagy

Journal of Nanomaterials ◽

10.1155/2021/3159848 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Jianghao Gong ◽

Shangjun Fu ◽

Zhenghao Zhou

Keyword(s):

Flow Cytometry ◽

Cell Viability ◽

Protein Level ◽

Reactive Oxygen ◽

Octacalcium Phosphate ◽

Control Group ◽

Substrate Protein ◽

Autophagy Inhibitor ◽

Osteoblast Cell Line

Objective. To explore the effects of silicone gel nanoparticles modified with octacalcium phosphate on the surface (silica/OCP) polymer drugs on the proliferation of osteoblasts and autophagy. Method. Silica/OCP was prepared in vitro, and the quality of the sample preparation was tested through characterization experiments. The osteoblast cell line (hFOB1.19) was treated with silica/OCP, autophagy inhibitor (3-methyladenine (3-MA)), and silica/OCP+3-MA, respectively. The proliferation of hFOB1.19 cells was detected through the methylthiazolyldiphenyl-tetrazolium bromide (MTT) kit. Flow cytometry was used to detect the cell apoptosis. The change in protein beclin1 and P62 expression in hFOB1.19 cells was observed in Western blot. An ROS detection kit was used to detect the content of reactive oxygen species in hFOB1.19 cells. Results. Silica/OCP was a sphere with a particle size of 50 nm to 130 nm and had an OCP phase in electron projection microscopy and X-ray diffraction techniques. The results indicated that OCP successfully modified silica and the material was successfully prepared. An MTT kit and flow cytometry test showed that the cell viability of the cells treated with silica/OCP increased significantly ( P < 0.05 ), and the intracellular apoptosis phenomenon was significantly decreased ( P < 0.05 ) compared to the control group. Moreover, the inhibition of cell viability and promotion of apoptosis caused by the autophagy inhibitor 3-MA can be rescued. Western blotting demonstrated that the protein level of beclin1 in osteoblasts reached the highest after six hours of treatment with silica/OCP, and the protein level of p62, the substrate protein of autophagy, reached the lowest. At the same time, treatment of cells with the autophagy inhibitor 3-MA and silica/OCP+3-MA found that the protein levels of beclin1 and p62 in the silica/OCP+3-MA group were adjusted back compared to the 3-MA group. After adding the autophagy inhibitor, the reactive oxygen content in the cell was significantly increased ( P < 0.05 ) in the silica/OCP group. In the presence of intracellular reactive oxygen inhibitors catalase and silica/OCP, the cell viability of osteoblasts was significantly lower than that of the silica/OCP group but significantly higher than that of the silica/OCP+3-MA group. The apoptosis level of the silica/OCP+catalase group was also significantly lower than that of the silica/OCP+3-MA group ( P < 0.05 ) but was significantly higher than that of the silica/OCP group ( P < 0.05 ). Conclusion. Silica/OCP nanoparticles can upregulate the level of autophagy in osteoblasts and promote the proliferation of osteoblasts.

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Effect of Rat C6 Glioma Cells in Varying Concentrations of Cultured in Isorhamnetin

Journal of Clinical and Nursing Research ◽

10.26689/jcnr.v1i1.98 ◽

1970 ◽

Vol 1 (1) ◽

Author(s):

TIAN Rui-rui

Keyword(s):

High Performance ◽

Colorimetric Assay ◽

Glioma Cells ◽

C6 Glioma ◽

C6 Glioma Cells ◽

Control Group ◽

Blank Control Group ◽

Rat Glioma ◽

Solvent Control

Objective:Â To investigate the effects of different concentrationsÂ of isorhamnetin on C6 rat glioma cells in vitro from JanuaryÂ 2015 to June 2015. Methods: The blank control group, blankÂ solvent control group and four concentration groups were usedÂ to observe the cell growth status under a microscope. MTTÂ colorimetric assay was used to detect the effect of isorhamnetin on C6 glioma cells in vitro and the cell inhibition rate AndÂ survival rate were measured. The apoptotic and apoptotic ratesÂ were measured by flow cytometry in the treatment group andÂ the control group. The relationship between the different concentrations of isorhamnetin and C6 glioma cell apoptosis wasÂ analyzed the total protein was extracted and the total AKT protein and Ser473 AKT protein content were detected by WesternÂ blotting. The rat model of glioma was constructed by SD rats.Five days of isorhamnetin was continuously fed and the plasma was detected by high-performance liquid chromatography,liver, brain tissue isorhamnetin content.Â

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Sevoflurane Promotes Bactericidal Properties of Macrophages through Enhanced Inducible Nitric Oxide Synthase Expression in Male Mice

Anesthesiology ◽

10.1097/aln.0000000000002992 ◽

2019 ◽

Vol 131 (6) ◽

pp. 1301-1315 ◽

Cited By ~ 2

Author(s):

Thomas J. Gerber ◽

Valérie C. O. Fehr ◽

Suellen D. S. Oliveira ◽

Guochang Hu ◽

Randal Dull ◽

...

Keyword(s):

Early Stage ◽

No Synthase ◽

Control Group ◽

Male Mice ◽

Proinflammatory Response ◽

E Coli ◽

Inducible No Synthase ◽

Bactericidal Properties

Abstract Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Sevoflurane with its antiinflammatory properties has shown to decrease mortality in animal models of sepsis. However, the underlying mechanism of its beneficial effect in this inflammatory scenario remains poorly understood. Macrophages play an important role in the early stage of sepsis as they are tasked with eliminating invading microbes and also attracting other immune cells by the release of proinflammatory cytokines such as interleukin-1β, interleukin-6, and tumor necrosis factor-α. Thus, the authors hypothesized that sevoflurane mitigates the proinflammatory response of macrophages, while maintaining their bactericidal properties. Methods Murine bone marrow–derived macrophages were stimulated in vitro with lipopolysaccharide in the presence and absence of 2% sevoflurane. Expression of cytokines and inducible NO synthase as well as uptake of fluorescently labeled Escherichia coli (E. coli) were measured. The in vivo endotoxemia model consisted of an intraperitoneal lipopolysaccharide injection after anesthesia with either ketamine and xylazine or 4% sevoflurane. Male mice (n = 6 per group) were observed for a total of 20 h. During the last 30 min fluorescently labeled E. coli were intraperitoneally injected. Peritoneal cells were extracted by peritoneal lavage and inducible NO synthase expression as well as E. coli uptake by peritoneal macrophages was determined using flow cytometry. Results In vitro, sevoflurane enhanced lipopolysaccharide-induced inducible NO synthase expression after 8 h by 466% and increased macrophage uptake of fluorescently labeled E. coli by 70% compared with vehicle-treated controls. Inhibiting inducible NO synthase expression pharmacologically abolished this increase in bacteria uptake. In vivo, inducible NO synthase expression was increased by 669% and phagocytosis of E. coli by 49% compared with the control group. Conclusions Sevoflurane enhances phagocytosis of bacteria by lipopolysaccharide-challenged macrophages in vitro and in vivo via an inducible NO synthase–dependent mechanism. Thus, sevoflurane potentiates bactericidal and antiinflammatory host-defense mechanisms in endotoxemia.

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An in vitro study of the effect of 5-ALA-mediated photodynamic therapy on oral squamous cell carcinoma

BMC Oral Health ◽

10.1186/s12903-020-01239-8 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Ying Ma ◽

Shujuan Qu ◽

Liangpeng Xu ◽

Hongbo Lu ◽

Baoguo Li

Keyword(s):

Squamous Cell Carcinoma ◽

Photodynamic Therapy ◽

Oral Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Laser Irradiation ◽

Squamous Cell ◽

Protoporphyrin Ix ◽

Control Group ◽

Blank Control Group

Abstract Background The primary aim of this study was to observe the effect of 5-ALA-mediated photodynamic therapy on oral squamous cell carcinoma in vitro. Methods SCC25 cells were divided into the observation group and the blank control group. Different concentrations of 5-ALA and SCC25 cells were co-incubated for different times, and the concentration of protoporphyrin IX was detected by flow cytometry. SCC25 cells were divided into the 5-ALA group (100 mg/L), the laser irradiation group alone, the 5-ALA plus laser irradiation group, and the blank control group (0 mg/L 5-ALA), and the methyl thiazolyl tetrazolium (MTT) solution method was used (each group was incubated for 4, 8 and 12 h in turn). The cell survival rate was calculated. Using annexin V-fluorescein isothiocyanate/propidium iodide method, the apoptosis of SCC25 cells was detected by flow cytometry. Results The level of protoporphyrin IX in SCC25 cells increased with increased concentrations of 5-ALA and length of incubation. However, after 12 h, protoporphyrin IX level in SCC25 cells was gradually stabilized, and similar effect was obtained with 100 mg/L or more 5-ALA, indicating that the level of protoporphyrin IX in SCC25 cells was determined by 5-ALA concentration and incubation time. 5-ALA plus laser irradiation exerted an inhibitory effect on the growth of SCC25 cells, which was highly associated with drug dose and incubation time. Compared with the control group, laser irradiation alone or 5-ALA alone had no effect on the apoptosis of SCC25 cells. Different concentrations of 5-ALA combined with laser irradiation showed a remarkable effect of apoptosis, and a higher apoptosis rate was seen with higher drug concentrations. Conclusion 5-ALA-mediated photodynamic therapy affects the growth of SCC25 cells in vitro, which may provide a new idea for the clinical treatment of oral squamous cell carcinoma.

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Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live Escherichia coli

mBio ◽

10.1128/mbio.00300-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 15

Author(s):

Sonisilpa Mohapatra ◽

Heejun Choi ◽

Xueliang Ge ◽

Suparna Sanyal ◽

James C. Weisshaar

Keyword(s):

Escherichia Coli ◽

Spatial Distribution ◽

Polypeptide Chain ◽

Peptide Bond ◽

Functional Protein ◽

E Coli ◽

Ribosome Binding ◽

Elongation Cycle ◽

Trna Translocation

ABSTRACT In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in Escherichia coli by the action of elongation factor EF-P. EF-P and its eukaryotic/archaeal homolog IF5A enhance the peptidyl transfer step of elongation. Here, a superresolution fluorescence localization and tracking study of EF-P–mEos2 in live E. coli provides the first in vivo information about the spatial distribution and on-off binding kinetics of EF-P. Fast imaging at 2 ms/frame helps to distinguish ribosome-bound (slowly diffusing) EF-P from free (rapidly diffusing) EF-P. Wild-type EF-P exhibits a three-peaked axial spatial distribution similar to that of ribosomes, indicating substantial binding. The mutant EF-PK34A exhibits a homogeneous distribution, indicating little or no binding. Some 30% of EF-P copies are bound to ribosomes at a given time. Two-state modeling and copy number estimates indicate that EF-P binds to 70S ribosomes during 25 to 100% of translation cycles. The timescale of the typical diffusive search by free EF-P for a ribosome-binding site is τfree ≈ 16 ms. The typical residence time of an EF-P on the ribosome is very short, τbound ≈ 7 ms. Evidently, EF-P binds to ribosomes during many or most elongation cycles, much more often than the frequency of Pro-Pro motifs. Emptying of the E site during part of the cycle is consistent with recent in vitro experiments indicating dissociation of the deacylated tRNA upon translocation. IMPORTANCE Ribosomes translate the codon sequence within mRNA into the corresponding sequence of amino acids within the nascent polypeptide chain, which in turn ultimately folds into functional protein. At each codon, bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. Here, we use superresolution fluorescence imaging to study the spatial distribution and ribosome-binding dynamics of EF-P in live E. coli cells. We were surprised to learn that EF-P binds to and unbinds from translating ribosomes during at least 25% of all elongation events; it may bind during every elongation cycle. Ribosomes translate the codon sequence within mRNA into the corresponding sequence of amino acids within the nascent polypeptide chain, which in turn ultimately folds into functional protein. At each codon, bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. Here, we use superresolution fluorescence imaging to study the spatial distribution and ribosome-binding dynamics of EF-P in live E. coli cells. We were surprised to learn that EF-P binds to and unbinds from translating ribosomes during at least 25% of all elongation events; it may bind during every elongation cycle.

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Impact of Probiotic on the Number of Lactic Acid Rods Forming Hydrogen Peroxide Isolated From Porkers and on Changes in Drug Resistance of Selected Escherichia Coli Isolates

Bulletin of the Veterinary Institute in Pulawy ◽

10.2478/v10213-012-0004-6 ◽

2012 ◽

Vol 56 (1) ◽

pp. 21-25 ◽

Cited By ~ 1

Author(s):

Marek Selwet ◽

Mariola Galbas ◽

Piotr Dullin

Keyword(s):

Escherichia Coli ◽

Hydrogen Peroxide ◽

Drug Resistance ◽

Lactic Acid ◽

Control Group ◽

Large White ◽

E Coli ◽

Probiotic Preparation ◽

Hydrogen Oxide

Abstract The presented investigations were conducted on a group of 60 porkers of crossbreed Polish Landrace x Large White Polish. The animals were divided into two equal experimental groups. The control group (K) was fed diets without supplementation with probiotics, group (P) - diets with the addition of probiotic (0.2 kg t-1 feed). The aim of the study was to determine the effect of probiotic preparation on total numberof lactic acid rods from the Lactobacillus genus and those forming hydrogen oxide. The second part of experiment concerned the influence of probiotic preparation on the number, haemolytic ability and changes in drug resistance of Escherichia coli isolated from animal faeces. The significantly highest number of Lactobacillus sp. were determined in the saliva of porkers fed diets with the addition of probiotic, while the lowest in the control group. Lactobacillus sp. rods capable of forming hydrogen peroxide were isolated from 17 animals in group K and from three animals in group P. E. coli was determined in each examined sample of faeces. In groups K and P, counts of these bacteria were similar and did not differ statistically. High numbers of haemolytic isolates (haemolysis β) were found in faeces of animals fed diets with the addition of probiotic. Number and proportions of resistant isolates in groups K and P were different. Gentamicin was characterised by exceptionally high in vitro effectiveness. The used probiotic increased drug resistance of E. coli and increased frequency of incidence of haemolysis β.

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