scholarly journals Human Adenovirus Serotype 3 Infection Modulates the Biogenesis and Composition of Lung Cell-Derived Extracellular Vesicles

2021 ◽  
Vol 2021 ◽  
pp. 1-19
Author(s):  
Ayodeji O. Ipinmoroti ◽  
Brennetta J. Crenshaw ◽  
Rachana Pandit ◽  
Sanjay Kumar ◽  
Brian Sims ◽  
...  
Keyword(s):  
Cell Viability ◽  
Extracellular Vesicles ◽  
Hemorrhagic Cystitis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
A549 Cells ◽  
Control Group ◽  
Epithelial Cell Line ◽  
The Impact

Adenovirus (Ad) is a major causal agent of acute respiratory infections. However, they are a powerful delivery system for gene therapy and vaccines. Some Ad serotypes antagonize the immune system leading to meningitis, conjunctivitis, gastroenteritis, and/or acute hemorrhagic cystitis. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs) may offer a mechanism by which viruses can enter cells via receptor-independent entry and how they influence disease pathogenesis and/or host protection considering their existence in almost all bodily fluids. We proposed that Ad3 could alter EV biogenesis, composition, and trafficking and may stimulate various immune responses in vitro. In the present study, we evaluated the impact of in vitro infection with Ad3 vector on EV biogenesis and composition in the human adenocarcinoma lung epithelial cell line A549. Cells were infected in an exosome-free media at different multiplicity of infections (MOIs) and time points. The cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorometric calcein-AM. EVs were isolated via ultracentrifugation. Isolated EV proteins were quantified and evaluated via nanoparticle tracking, transmission electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting assays. The cell viability significantly decreased with an increase in MOI and incubation time. A significant increase in particle mean sizes, concentrations, and total EV protein content was detected at higher MOIs when compared to uninfected cells (control group). A549 cell-derived EVs revealed the presence of TSG101, tetraspanins CD9 and CD63, and heat shock proteins 70 and 100 with significantly elevated levels of Rab5, 7, and 35 at higher MOIs (300, 750, and 1500) when compared to the controls. Our findings suggested Ad3 could modulate EV biogenesis, composition, and trafficking which could impact infection pathogenesis and disease progression. This study might suggest EVs could be diagnostic and therapeutic advancement to Ad infections and other related viral infections. However, further investigation is warranted to explore the underlying mechanism(s).

scholarly journals Klotho Alleviates Lung Injury Caused by Paraquat via Suppressing ROS/P38 MAPK-Regulated Inflammatory Responses and Apoptosis

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Zhiqiang Zhang ◽  
Qing Nian ◽  
Gang Chen ◽  
Shuqing Cui ◽  
Yuzhen Han ◽  
...  
Keyword(s):  
Lung Injury ◽  
Cell Viability ◽  
P38 Mapk ◽  
Inflammatory Responses ◽  
A549 Cells ◽  
Control Group ◽  
Ros Production ◽  
The Impact

Acute lung injury (ALI) induced by paraquat (PQ) progresses rapidly with high mortality; however, there is no effective treatment, and the specific mechanism is not well understood. The antiaging protein klotho (KL) has multiple functions and exerts significant influences on various pathophysiological processes. This work evaluated the impact of KL on PQ-induced ALI and investigated its underlying mechanisms. As for in vivo research, C57BL/6 mice were treated with PQ (30 mg/kg) intraperitoneal (IP) injection to create a toxicity model of ALI (PQ group). The mice were divided into control group, KL group, PQ group, and PQ+KL group. For in vitro experiment, A549 cells were incubated with or without KL and then treated in the presence or absence of PQ for 24 h. In vivo result indicated that KL reduced the mortality, reduced IL-1β and IL-6 in the bronchoalveolar lavage fluid (BALF), attenuated ALI, and decreased apoptosis in situ. In vitro result revealed that KL significantly improved cell viability, reduced the levels of IL-1β and IL-6 in culture supernatants, suppressed cell apoptosis, inhibited caspase-3 activation, and enhanced mitochondrial membrane potential (ΔΨm) after PQ treatment. Besides, KL effectively abated reactive oxygen species (ROS) production, improved GSH content, and lowered lipid peroxidation in PQ-exposed A549 cells. Further experiments indicated that phosphorylated JNK and P38 MAPK was increased after PQ treatment; however, KL pretreatment could significantly lower the phosphorylation of P38 MAPK. Suppression of P38 MAPK improved cell viability, alleviated inflammatory response, and reduced apoptosis-related signals; however, it had no obvious effect on the production of ROS. Treatment with N-acetylcysteine (NAC), a classic ROS scavenger, could suppress ROS production and P38 MAPK activation. These findings suggested that KL could alleviate PQ-caused ALI via inhibiting ROS/P38 MAPK signaling-regulated inflammatory responses and mitochondria-dependent apoptosis.

VDAC1 Mediated Anticancer Activity of Gallic Acid in Human Lung Adenocarcinoma A549 Cells

2018 ◽  
Vol 18 (2) ◽  
pp. 255-262 ◽  
Author(s):  
Aikebaier Maimaiti ◽  
Amier Aili ◽  
Hureshitanmu Kuerban ◽  
Xuejun Li
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Gallic Acid ◽  
Molecular Mechanisms ◽  
A549 Cells ◽  
Dependent Manner ◽  
Lung Carcinoma Cells ◽  
Induced Apoptosis ◽  
The Impact

Aims: Gallic acid (GA) is generally distributed in a variety of plants and foods, and possesses cell growth-inhibiting activities in cancer cell lines. In the present study, the impact of GA on cell viability, apoptosis induction and possible molecular mechanisms in cultured A549 lung carcinoma cells was investigated. Methods: In vitro experiments showed that treating A549 cells with various concentrations of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA inhibits cell viability, comparative proteomic analysis was applied. The changed proteins were identified by Western blot and siRNA methods. Results: Two-dimensional electrophoresis revealed changes that occurred to the cells when treated with or without GA. Four up-regulated protein spots were clearly identified as malate dehydrogenase (MDH), voltagedependent, anion-selective channel protein 1(VDAC1), calreticulin (CRT) and brain acid soluble protein 1(BASP1). VDAC1 in A549 cells was reconfirmed by western blot. Transfection with VDAC1 siRNA significantly increased cell viability after the treatment of GA. Further investigation showed that GA down regulated PI3K/Akt signaling pathways. These data strongly suggest that up-regulation of VDAC1 by GA may play an important role in GA-induced, inhibitory effects on A549 cell viability.

scholarly journals Influence of Pasteurization and Storage on Dynamic In Vitro Gastric Digestion of Milk Proteins: Quantitative Insights Based on Peptidomics

Foods ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 998 ◽  
Author(s):  
Xing Li ◽  
Yuxiang Gu ◽  
Shudong He ◽  
Olayemi Eyituoyo Dudu ◽  
Qiming Li ◽  
...  
Keyword(s):  
Cold Storage ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Milk Proteins ◽  
Hydrolysis Rate ◽  
Gastric Digestion ◽  
Pasteurized Milk ◽  
The Impact ◽  
And Storage

It is important to evaluate the nutritional quality of milk during the shelf-life, especially during home storage, from a consumer viewpoint. In this study, we investigated the impact of pasteurization (85 °C/15 s) and subsequent storage (at 4 °C for 7 days) on the coagulation behavior of milk and protein digestibility in a dynamic in vitro gastric digestion test. A high level of hydration in curd formed in pasteurized milk upon 7-day cold storage compared to raw and pasteurized milk, indicating fast pepsin diffusion in the interior of curds, increasing the hydrolysis rate. The digesta collected at various time points throughout the gastric digestion were studied using o-phthaldialdehyde (OPA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), liquid chromatography tandem mass spectrometry (LC-MS/MS), and amino acid analysis. These results showed that milk proteins were hydrolyzed quickly upon a long period of cold storage. Additionally, qualitative and quantitative results obtained using LC-MS/MS exhibited significant differences between samples, especially in pasteurized milk upon cold storage. Processing and storage played a decisive role in bioactive peptide generation. Such knowledge could provide insights into and directions for the storage of pasteurized milk for further clinical studies on protein bioavailability and the generation of bioactive peptides for desired health outcomes.

scholarly journals Lipopolysaccharide administration alters extracellular vesicles in human lung-cancer cells and mice

2020 ◽  
Author(s):  
Leandra B. Jones ◽  
Sanjay Kumar ◽  
Courtnee’ R. Bell ◽  
Brennetta J. Crenshaw ◽  
Mamie T. Coats ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Bacterial Infection ◽  
Cell Line ◽  
Extracellular Vesicles ◽  
A549 Cells ◽  
Human Lung Cancer ◽  
Diagnostic Tools ◽  
The Impact

AbstractExtracellular vesicles (EVs) play a fundamental role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. In this study, we explored the in vitro impact of bacterial lipopolysaccharide administration on a cell line that represents a target for bacterial infection in the host. Administration of lipopolysaccharide at varying concentrations to this A549 cell line caused only modest changes in cell death, but EV numbers were significantly changed. After treatment with the highest concentration of lipopolysaccharide, EVs derived from A549 cells packaged significantly less interleukin-6 and lysosomal-associated membrane protein 1. We also examined the impact of lipopolysaccharide administration on exosome biogenesis and cargo composition in BALB/c mice. Serum-isolated EVs from lipopolysaccharide-treated mice showed significantly increased lysosomal-associated membrane protein 1 and toll-like receptor 4 levels compared with EVs from control mice. In summary, this study demonstrated that EV numbers and cargo were altered using these in vitro and in vivo models of bacterial infection.

scholarly journals The Effects of Three Chlorhexidine-Based Mouthwashes on Human Osteoblast-Like SaOS-2 Cells. An In Vitro Study

2021 ◽  
Vol 22 (18) ◽  
pp. 9986
Author(s):  
Giulia Brunello ◽  
Kathrin Becker ◽  
Luisa Scotti ◽  
Dieter Drescher ◽  
Jürgen Becker ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cetylpyridinium Chloride ◽  
Control Cell ◽  
In Vitro Study ◽  
Control Group ◽  
Sterile Saline ◽  
Lack Of Information ◽  
And Control ◽  
The Impact

Several decontamination methods for removing biofilm from implant surfaces during surgical peri-implantitis treatment have been reported, including the intraoperative usage of chlorhexidine (CHX)-based antiseptics. There is a lack of information on possible adverse effects on bone healing. The study aimed to examine the impact of three CHX-based mouthwashes on osteoblast-like cells (SaOS-2) in vitro. Cells were cultured for three days in 96-well binding plates. Each well was randomly treated for either 30, 60 or 120 s with 0.05% CHX combined with 0.05% cetylpyridinium chloride (CPC), 0.1% CHX, 0.2% CHX or sterile saline (NaCl) as control. Cell viability, cytotoxicity and apoptosis were assessed at day 0, 3 and 6. Cell viability resulted in being higher in the control group at all time points. At day 0, the CHX 0.2 group showed significantly higher cytotoxicity values compared to CHX 0.1 (30 s), CHX + CPC (30 s, 60 s and 120 s) and control (60 s and 120 s), while no significant differences were identified between CHX + CPC and both CHX 0.1 and NaCl groups. All test mouthwashes were found to induce apoptosis to a lower extent compared to control. Results indicate that 0.2% CHX presented the highest cytotoxic effect. Therefore, its intraoperative use should be carefully considered.

Hypothyroidism alters diaphragm muscle development

1996 ◽  
Vol 81 (5) ◽  
pp. 1965-1972 ◽  
Author(s):  
Gary C. Sieck ◽  
Louise E. Wilson ◽  
Bruce D. Johnson ◽  
Wen-Zhi Zhan
Keyword(s):  
Muscle Development ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Diaphragm Muscle ◽  
Rat Diaphragm ◽  
Shortening Velocity ◽  
Pregnant Rats ◽  
Isoform Expression ◽  
The Impact

Sieck, Gary C., Louise E. Wilson, Bruce D. Johnson, and Wen-Zhi Zhan. Hypothyroidism alters diaphragm muscle development. J. Appl. Physiol. 81(5): 1965–1972, 1996.—The impact of hypothyroidism (Hyp) on myosin heavy chain (MHC) isoform expression, maximum specific force (Po), fatigability, and maximum unloaded shortening velocity ( V o) was determined in the rat diaphragm muscle (Dia) at 0, 7, 14, 21, and 28 days of age. Hyp was induced by treating pregnant rats with 6- n-propyl-2-thiouracil (0.05% in drinking water) beginning at gestational day 10 and was confirmed by reduced plasma levels of 3,5,3′-triiodothyronine and thyroxine. MHC isoforms were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by densitometry. Isometric Po and fatigue resistance of the Dia were measured in vitro at 26°C, and V o was determined at 15°C with the slack test. Compared with control muscles, expression of MHC-slow was higher and expression of adult fast MHC isoforms was lower in Hyp Dia at all ages. The neonatal isoform of MHC continued to be expressed in the Hyp Dia until day 28. At each age, Po and fatigability were reduced and V o was slower in the Hyp Dia. We conclude that Hyp-induced alterations in MHC isoform expression do not fully predict the changes in Dia contractile properties.

scholarly journals Lipopolysaccharide Administration Alters Extracellular Vesicles in Cell Lines and Mice

2021 ◽  
Author(s):  
Leandra B. Jones ◽  
Sanjay Kumar ◽  
Courtnee’ R. Bell ◽  
Brennetta J. Crenshaw ◽  
Mamie T. Coats ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Bacterial Infection ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
A549 Cells ◽  
Diagnostic Tools ◽  
In Vivo Models ◽  
The Impact

AbstractExtracellular vesicles (EVs) play a fundamental role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. In this study, we explored the in vitro impact of bacterial lipopolysaccharide administration on cell lines that represents a target for bacterial infection in the host. Administration of lipopolysaccharide at varying concentrations to A549 and BV-2 cell lines caused only modest changes in cell death, but EV numbers were significantly changed. After treatment with the highest concentration of lipopolysaccharide, EVs derived from A549 cells packaged significantly less interleukin-6 and lysosomal-associated membrane protein 1. EVs derived from BV-2 cells packaged significantly less tumor necrosis factor after administration of lipopolysaccharide concentrations of 0.1 µg/mL and 1 µg/mL. We also examined the impact of lipopolysaccharide administration on exosome biogenesis and cargo composition in BALB/c mice. Serum-isolated EVs from lipopolysaccharide-treated mice showed significantly increased lysosomal-associated membrane protein 1 and toll-like receptor 4 levels compared with EVs from control mice. In summary, this study demonstrated that EV numbers and cargo were altered using these in vitro and in vivo models of bacterial infection.

scholarly journals Group B Streptococci and Other Gram-Positive Cocci Bind to Cytokeratin 8

2000 ◽  
Vol 68 (4) ◽  
pp. 2129-2134 ◽  
Author(s):  
Glen S. Tamura ◽  
Aphakorn Nittayajarn
Keyword(s):  
Epithelial Cells ◽  
Plasma Membranes ◽  
Capsular Polysaccharide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
A549 Cells ◽  
Immunoblot Analysis ◽  
Epithelial Cell Line ◽  
Group B Streptococci ◽  
Group B

ABSTRACT Group B streptococci (GBS) adhere to surface receptors present on epithelial cells; these receptors include fibronectin and laminin. To identify other possible receptors, plasma membranes from A549 cells, a respiratory tract epithelial cell line, were prepared. These plasma membranes were tested in a protein blot analysis using radiolabeled GBS as a probe. GBS adhered to two species, with molecular masses of 50 kDa (p50) and 57 kDa (p57). We concluded that p50 and p57 correspond to two forms of cytokeratin 8 (CK8) on the basis of the following results: (i) protein blot results demonstrated that p50 and p57 exactly comigrated with two forms of CK8 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE); (ii) p50 and p57 exactly comigrated with CK8 after separation by two-dimensional PAGE; (iii) CK8 in solution bound to GBS, as demonstrated by immunoblot analysis of proteins from A549 lysates that bound to GBS in a liquid-phase assay; and (iv) radiolabeled GBS bound to A549 lysate-derived CK8 that had been captured in anti-CK8-coated microtiter wells. CK8 bound to COH1-13, an acapsular mutant of COH1, demonstrating that adherence is not mediated by capsular polysaccharide. Trypsin-treated GBS did not bind to CK8, indicating that adherence is mediated via a protein on the surface of GBS. Soluble CK8 bound to six of six GBS strains tested. Soluble CK8 also bound toStaphylococcus aureus, Lactococcus lactis,Enterococcus faecalis, and Streptococcus pyogenes. We hypothesize that adherence of GBS to cytokeratin may be important for maintenance of colonization at sites of keratinized epithelium, such as the vagina, or for adherence of these bacteria to damaged epithelial cells at other sites.

scholarly journals In Vitro Cell-Based Biomarkers Study of Vital Organs: Impact of the Biofield Energy Based Test Formulation

2019 ◽  
Vol 1 (2) ◽  
pp. 22-37
Author(s):  
Krista Joanne Callas ◽  
Mahendra Kumar Trivedi ◽  
Alice Branton ◽  
Dahryn Trivedi ◽  
Gopal Nayak ◽  
...  
Keyword(s):  
Cell Viability ◽  
Hepg2 Cells ◽  
Neuroblastoma Cells ◽  
A549 Cells ◽  
Heart Cells ◽  
Serotonin Level ◽  
Human Neuroblastoma ◽  
Test Formulation ◽  
The Impact

The present study was undertaken to evaluate the impact of Biofield Energy Treated test formulation using multiple cell-lines. The test formulation and cell media (Med) was divided into two parts; one part was untreated (UT) and other part received Biofield Energy Treatment remotely by a renowned Biofield Energy Healer, Krista Joanne Callas, USA and labeled as Biofield Energy Treated (BT) test item (TI)/Med. Based on cell viability, test formulation was found safe. Cytoprotective action of test formulation showed significant restoration of cell viability by 89.9% and 106.4% in human cardiac fibroblasts cells (HCF) cells, while improved restoration of cell viability by 77.3% and 69% in HepG2 cells compared to untreated. Cellular restoration in A549 cells was also improved by 141.2% and 157.1% compared to untreated. ALP activity was significantly increased by 118.7% and 140.7% in UT-Med + BT-TI and BT-Med + UT-TI, respectively at 0.1 µg/mL than untreated. Percent cellular protection of HCF (heart) cells (decreased of LDH activity) was significantly increased by 89.9% and 106.4% in UT-Med + BT-TI and BT-Med + BT-TI, respectively than untreated. HepG2 cells protection (decreased ALT activity) was increased by 59.8% in BT-Med + BT-TI than untreated. Superoxide dismutase (SOD) level was increased by 22.8% in BT-Med + BT-TI than untreated. Serotonin level was significantly increased by 361.7% and 197.6% in BT-Med + UT-TI and BT-Med + BT-TI, respectively than untreated in human neuroblastoma cells (SH-SY5Y). However, relative quantification (RQ) of vitamin D receptor (VDR) was significantly increased by 116.5%, 214.7%, and 241.5% in UT-Med + BT-TI, BT-Med + UT-TI, and BT-Med + BT-TI, respectively than untreated in MG-63 cells. Overall, data showed a significant improvement of organ-specific functional enzyme biomarkers. Thus, Biofield Energy Treated Test formulation (the Trivedi Effect®) would be useful for multiple organs health that can be beneficial against coronary artery disease, arrhythmias, congenital heart disease, cardiomyopathy, cirrhosis, liver cancer, hemochromatosis, asthma, chronic bronchitis, cystic fibrosis, osteoporosis, etc.

scholarly journals In Vitro Assessment of the Impact of Nickel on the Viability and Steroidogenesis in the Human Adrenocortical Carcinoma (NCI-H295R) Cell Line

2020 ◽  
pp. 871-883
Author(s):  
Norbert LUKAC ◽  
Z FORGACS ◽  
H DURANOVA ◽  
T JAMBOR ◽  
J ZEMANOVA ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Adrenocortical Carcinoma ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Cytotoxic Action ◽  
Disruptive Effect ◽  
17Β Estradiol ◽  
The Impact

Nickel is a ubiquitous environmental pollutant, which has various effects on reproductive endocrinology. In this study, human adrenocortical carcinoma (NCI-H295R) cell line was used as an in vitro biological model to study the effect of nickel chloride (NiCl2) on the viability and steroidogenesis. The cells were exposed to different concentrations (3.90; 7.80; 15.60; 31.20; 62.50; 125; 250 and 500 μM) of NiCl2 and compared with control group (culture medium without NiCl2). The cell viability was measured by the metabolic activity assay. Production of sexual steroid hormones was quantified by enzyme linked immunosorbent assay. Following 48 h culture of the cells in the presence of NiCl2 a dose-dependent depletion of progesterone release was observed even at the lower concentrations. In fact, lower levels of progesterone were detected in groups with higher doses (≥125 μM) of NiCl2 (P<0.01), which also elicited cytotoxic action. A more prominent decrease in testosterone production (P<0.01) was also noted in comparison to that of progesterone. On the other hand, the release of 17β-estradiol was substantially increased at low concentrations (3.90 to 62.50 μM) of NiCl2. The cell viability remained relatively unaltered up to 125 μM (P>0.05) and slightly decreased from 250 μM of NiCl2 (P<0.05). Our results indicate endocrine disruptive effect of NiCl2 on the release of progesterone and testosterone in the NCI-H295R cell line. Although no detrimental effect of NiCl2 (≤62.50 μM) could be found on 17β-estradiol production, its toxicity may reflect at other points of the steroidogenic pathway.

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