scholarly journals Proteomic Analysis and Cell Viability of Nine Amnion, Chorion, Umbilical Cord, and Amniotic Fluid–Derived Products

Cartilage ◽  
2020 ◽  
pp. 194760352097676
Author(s):  
Liliya Becktell ◽  
Andrea M. Matuska ◽  
Stephanie Hon ◽  
Michelle L. Delco ◽  
Brian J. Cole ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Western Blot ◽  
Cell Viability ◽  
Blot Analysis ◽  
Proteomic Analysis ◽  
Western Blot Analysis ◽  
Antibody Binding ◽  
Matrix Metalloproteinase 2 ◽  
Live Cells ◽  
Enzyme Linked Immunosorbent Assay

Objective Amnion products are used in various musculoskeletal surgeries and as injections for joint pain with conflicting reports of cell viability and protein contents. The objective of this study was to determine the full proteome and examine cell viability in 9 commercial amnion products using an unbiased bottom-up shotgun proteomics approach and confocal microscopy. Design Products were subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis and searched against a UniProt Homo sapiens database. Relative protein abundance was determined for each sample. Based on proteomics results, lumican was measured by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis was performed for interleukin-1 receptor antagonist (IL-1Ra) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). Cell viability was determined by calcein AM (live) and ethidium homodimer (dead) staining and confocal microscopy. Results Proteomic analysis revealed 919 proteins in the nine products. Proteins were primarily collagens, keratin, and albumin. Lumican, a small leucine-rich proteoglycan (SLRP) was found in all samples. Western blot analysis for IL-1Ra and TIMP-2 indicated presence of both proteins, with nonspecific antibody binding also present in all samples. No live cells were identified in any product. Conclusions Several novel proteins were identified through proteomics that might impart the beneficial effects of amnion products, including SLRPs, collagens, and regulators of fibroblast activity. IL-1Ra and TIMP-2 were identified, but concentrations measured by ELISA may be falsely increased due to nonspecific antibody binding. The concept that the amnion tissues provide live cells to aid in tissue regeneration cannot be supported by the findings of this study.

scholarly journals MicroRNA-145 attenuates IL-6-induced enhancements of sensitivity to UVB irradiation by suppressing MyD88 in HaCaT cells

2018 ◽  
Vol 32 ◽  
pp. 205873841879594 ◽  
Author(s):  
Hui Dong ◽  
Wei Jiang ◽  
Hongquan Chen ◽  
Shui Jiang ◽  
Yunshu Zang ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Lupus Erythematosus ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Western Blot Analysis ◽  
Ultraviolet B ◽  
Hacat Cells ◽  
Related Factors ◽  
Uvb Irradiation

MicroRNAs (miRNAs/miRs) play vital roles in various immune diseases including systemic lupus erythematosus (SLE). The current study aimed to assess the role of miR-145 in interleukin-6 (IL-6)-treated HaCaT cells under ultraviolet B (UVB) irradiation and further explore the potential regulatory mechanism. HaCaT cells were pretreated with IL-6 and then exposed to UVB to assess the effect of IL-6 on sensitivity of HaCaT cells to UVB irradiation. The levels of miR-145 and MyD88 were altered by transfection and the transfected efficiency was verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR)/western blot analysis. Cell viability, percentage of apoptotic cells and expression levels of apoptosis-related factors were measured by trypan blue assay, flow cytometry assay, and western blot analysis, respectively. In addition, the levels of c-Jun N-terminal kinases (JNK) and nuclear factor-κB (NF-κB) signaling pathway-related factors were assessed by western blot analysis. IL-6 treatments significantly aggravated the reduction of cell viability and promotion of cell apoptosis caused by UVB irradiation in HaCaT cells. Interestingly, miR-145 level was augmented by UVB exposure and miR-145 mimic alleviated IL-6-induced increase of sensitivity to UVB irradiation in HaCaT cells, as dramatically increased cell viability and reduced cell apoptosis. Opposite effects were observed in miR-145 inhibitor-transfected cells. Meanwhile, MyD88 was negatively regulated by miR-145 and MyD88 mediated the regulatory effect of miR-145 on IL-6- and UVB-treated cells. In addition, miR-145 mimic inhibited the JNK and NF-κB pathways by down-regulating MyD88. In conclusion, the present study demonstrated that miR-145 alleviated IL-6-induced increase of sensitivity to UVB irradiation by down-regulating MyD88 in HaCaT cells.

scholarly journals Investigations on ornithobacterium rhinotracheale in broiler flocks in elazig province located in the east of turkey

2012 ◽  
Vol 49 (No. 8) ◽  
pp. 305-311 ◽  
Author(s):  
G. Ozbey ◽  
H. Ongor ◽  
D. T Balik ◽  
V. Celik ◽  
A. Kilic ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rrna Gene ◽  
Major Band ◽  
Cell Extracts ◽  
Sds Page ◽  
Ornithobacterium Rhinotracheale

In the present study, lung, trachea and serum samples from broiler flocks slaughtered at an abattoir in Elazig province located in the East of Turkey were examined for the presence of Ornithobacterium rhinotracheale using culture and enzyme-linked immunosorbent assay (ELISA). The identity was latter proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis, and polymerase chain reaction (PCR) assays. A total of 324 serum and 250 lung and trachea samples were collected from 10 commercially reared chicken flocks showing respiratory manifestations. The samples were obtained from different flocks. The causative agent (ORT) was isolated from trachea (1.5%) of five chickens and from both lung and trachea (0.4%) of only one chicken in the bacteriological examination of tissues. The presence of antibodies against ORT was detected in 33 (10.2%) of the 324 sera by ELISA. A 784 bp fragment of the 16S rRNA gene was amplified using specific primers in the PCR. All ORT isolates that were positive by culture were also detected to be positive by the PCR. SDS-PAGE protein profiles of whole cell extracts showed a high similarity for all the isolates with a major band of the molecular weight of 33 kDa (kiloDalton). Results of Western blot analysis indicate four antigenic fractions predominantly with molecular weights of 33, 42, 52 and 66 kDa.

scholarly journals Proteomic analysis of brush-border membrane vesicles isolated from purified proximal convoluted tubules

2010 ◽  
Vol 298 (6) ◽  
pp. F1323-F1331 ◽  
Author(s):  
Scott J. Walmsley ◽  
Corey Broeckling ◽  
Ann Hess ◽  
Jessica Prenni ◽  
Norman P. Curthoys
Keyword(s):  
Western Blot ◽  
Brush Border ◽  
Blot Analysis ◽  
Proteomic Analysis ◽  
Western Blot Analysis ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Proximal Convoluted Tubule ◽  
Brush Border Membrane Vesicles ◽  
Convoluted Tubules

The renal proximal convoluted tubule is the primary site of water, electrolyte and nutrient reabsorption and of active secretion of selected molecules. Proteins in the apical brush-border membrane facilitate these functions and initiate some of the cellular responses to altered renal physiology. The current study uses two-dimensional liquid chromatography/mass spectrometry to compare brush border membrane vesicles isolated from rat renal cortex (BBMVCTX) and from purified proximal convoluted tubules (BBMVPCT). Both proteomic data and Western blot analysis indicate that the BBMVCTX contain apical membrane proteins from cortical cells other than the proximal tubule. This heterogeneity was greatly reduced in the BBMVPCT. Proteomic analysis identified 193 proteins common to both samples, 21 proteins unique to BBMVCTX, and 57 proteins unique to BBMVPCT. Spectral counts were used to quantify relative differences in protein abundance. This analysis identified 42 and 50 proteins that are significantly enriched (p values ≤0.001) in the BBMVCTX and BBMVPCT, respectively. These data were validated by measurement of γ-glutamyltranspeptidase activity and by Western blot analysis. The combined results establish that BBMVPCT are primarily derived from the proximal convoluted tubule (S1 and S2 segments), whereas BBMVCTX include proteins from the proximal straight tubule (S3 segment). Analysis of functional annotations indicated that BBMVPCT are enriched in mitochondrial proteins and enzymes involved in glucose and organic acid metabolism. Thus the current study reports a detailed proteomic analysis of the brush-border membrane of the rat renal proximal convoluted tubule and provides a database for future hypothesis-driven research.

Effect of nicotine on chemoresistant phenotype as mediated through Src-dependent Id1 expression in pancreatic adenocarcinoma cells.

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 216-216
Author(s):  
J. G. Trevino ◽  
S. R. Pillai ◽  
S. P. Chellappan
Keyword(s):  
Pancreatic Cancer ◽  
Western Blot ◽  
Cell Viability ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Pancreatic Tumor ◽  
Pancreatic Cancer Cells ◽  
Dependent Pathway

216 Background: The signaling pathways contributing to DNA-binding protein inhibitor Id1 expression and chemoresistance in pancreatic cancer remain unknown. Id1 plays a role in pancreatic tumor progression with tumor-promoting effects of nicotine regulating protein tyrosine kinase Src activation and Id1 expression, both associated with chemoresistance in other systems. We hypothesize Id1 expression regulates chemoresistance in pancreatic cancer through a nicotine-promoting Src-dependent pathway. Methods: We probed pancreatic cancer cell lines (L3.6pl, PANC-1, Mia-PaCa-2) for innate gemcitabine chemoresistance with cell viability MTT assay and western blot analysis of PARP cleavage programmed cell death. Gemcitabine-sensitive cells were exposed to rising gemcitabine concentrations to establish a resistant subtype, L3.6plGemRes. Protein analysis and mRNA expression were determined by western blot analysis and RT-PCR respectively. Induction of Src phosphorylation or Id1 expression was performed with nicotine (1 μM). Results: Inhibition of c-Src expression was performed with short-interfering RNA (siRNA). Nicotine-induced Src phosphorylation and Id1 expression. Inhibition of Src by siRNA resulted in decreased nicotine-induced Id1 expression. Inhibition of Src and Id1 expression by siRNA in innate or established gemcitabine resistant pancreatic cancer cells resulted in gemcitabine sensitization. To determine if nicotine contributes to gemcitabine chemoresistance, we exposed gemcitabine-sensitive cells to nicotine with subsequent exposure to gemcitabine IC50, 250 ng/ml, and cell viability assays confirmed a 2-fold increase in cell prolilferation and a 4.5-fold reduction in apoptosis. Further, nicotine induced phosphorylation of key signaling enzymes involved in proliferation and apoptosis, Erk1/2 and Akt respectively. Conclusions: In summary, we demonstrate that Id1, through a nicotine-promoting Src-dependent pathway, is necessary for establishment of a chemoresistant phenotype in pancreatic cancer cells. Understanding the signaling pathways involved in pancreatic tumor chemoresistance will lead to therapies resulting in improved tumor responses. No significant financial relationships to disclose.

scholarly journals The diurnal pattern of salivary IL-1β in healthy young adults

2017 ◽  
Vol 31 (5) ◽  
Author(s):  
Nur Basirah Ghazali ◽  
Michael Steele ◽  
David Koh ◽  
Adi Idris
Keyword(s):  
Young Adults ◽  
Circadian Rhythm ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Cytokine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diurnal Pattern ◽  
Healthy Individuals

Abstract Disruption in circadian rhythm affects the production of inflammatory cytokines. Understanding how it behaves in diseased conditions is essential. Despite the role of the interleukin-1β (IL-1β), a potent inflammatory cytokine, in human diseases, little is known about the steady-state circadian rhythm of IL-1β in healthy individuals. This short study investigates the diurnal pattern of salivary IL-1β throughout the day in healthy young adults. Twelve participants provided saliva samples at various times throughout the day. Salivary IL-1β were assessed using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Salivary IL-1β levels were highest at 0430 h and lowest at 0000 h and shared a similar diurnal pattern to that of salivary IL-6. Western blot analysis showed that these levels correspond to the mature form of IL-1β. Our findings are important as it established the diurnal pattern of salivary IL-1β is fluctuating normally throughout the day. The findings also open an incredible opportunity for developing research conducted in the field with saliva as the diagnostic tool.

Em18 and Em16, new serologic marker epitopes for alveolar echinococcosis in Western blot analysis, are the only two epitopes recognized by commercially available weak positive (cut off) sera for Em2plus-ELISA

1995 ◽  
Vol 69 (4) ◽  
pp. 369-371 ◽  
Author(s):  
A. Ito ◽  
Y. Osawa ◽  
M. Nakao ◽  
T. Horii ◽  
M. Okamoto ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Alveolar Echinococcosis ◽  
Specific Antigen ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay ◽  
Molecular Weights ◽  
Serologic Marker

AbstractThe assay system for antibody responses against Em2, the most specific antigen for serodiagnosis of alveolar echinococcosis (AE), has been established by enzyme-linked immunosorbent assay (ELISA) but not by Western blot assay, since Em2 antigen is not protein but carbohydrate in nature. Recently we reported that previously undescribed protein epitopes, designated Em18 and Em16 due to their molecular weights, were good serologic markers for AE by Western blot analysis. It has been shown that Em18 and Em16 are the only two epitopes recognized by commercially available weak positive (cut off) sera for the Em2plus-ELISA.

scholarly journals Quercetin Protects RIMVECs From Inflammatory Damage, Pyroptosis and Cell Migration by Inhibiting the TLR4/NF-κB/NLRP3 Pathway

2021 ◽  
Author(s):  
Huixin Zhang ◽  
Yeye Li ◽  
Zhongjie Liu
Keyword(s):  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Inflammatory Factors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Caspase 1 ◽  
Inflammatory Damage

Abstract Background: Intestinal mucosal microvascular endothelial cells (MEC) have multiple functions and play an important role in intestinal bowel diseases (IBD). Quercetin is a flavonoid found in many plants and fruits. It was reported that quercetin can treat several gastrointestinal cancers, but its effect on bacterial enteritis and pyroptosis-related diseases has been rarely studied. This article aims to explore the effect and mechanism of quercetin on inflammatory injury and pyroptosis of RIMVECs.Methods: The inflammatory damage and pyroptosis in RIMVECs were induced by LPS and ATP. Real-time quantitative polymerase chain reaction (RT-qPCR), western blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence methods were used to detect TLR4/NF-κB/NLRP3 pathways, inflammatory factors (IL-1β and IL-18) and pyroptosis-related proteins (Caspase-1 and GSDMD). The expression and distribution of ZO-1 were detected by western blot analysis and immunofluorescence method. The late apoptosis and necrosis of cells were measured by cell flow cytometry. Results: The results showed that different concentrations (5, 10, 20μM) of quercetin not only significantly reduced the protein and mRNA levels of TLR4, NLRP3, Caspase-1 and GSDMD, but also down-regulated the protein expression, mRNA and secretion of IL-1β and IL-18. Quercetin also inhibited the phosphorylation of NF-κB p65 and the degradation of IκB. At the same time, quercetin increased the cell migration rate and the expression level of ZO-1, and reduced the number of late apoptotic cells (P<0.05). Conclusions: Our data indicated that Quercetin reduced the inflammatory response and pyroptosis induced by LPS/ATP through the TLR4/NF-κB/NLRP3 pathway, and protected the migration and tight junctions of RIMVECs.

scholarly journals Kinetics and Diagnostic and Prognostic Potential of Quantitative Western Blot Analysis and Antigen-Specific Enzyme-Linked Immunosorbent Assay in Experimental Canine Leishmaniasis

2006 ◽  
Vol 13 (2) ◽  
pp. 271-276 ◽  
Author(s):  
D. Talmi-Frank ◽  
D. Strauss-Ayali ◽  
C. L. Jaffe ◽  
G. Baneth
Keyword(s):  
Western Blot ◽  
Immunosorbent Assay ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Relative Increase ◽  
Early Infection ◽  
Canine Leishmaniasis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Time Points ◽  
Experimental Canine

ABSTRACT Quantitative computerized Western blot analysis of antibody responses during experimental canine Leishmania infantum infection distinguished between immunodominant and nonimmunodominant protein bands. Six infected beagles, positive by both PCR and parasite culture, were monitored over 75 weeks postinfection and during a 12-week allopurinol treatment course. All dogs were symptomatic at the time of treatment. Of 12 antigenic bands examined, the immunodominant bands (12, 14, 24, 29, 48, and 68 kDa) showed significantly increased intensities (P < 0.01) and higher frequencies of recognition than the nonimmunodominant bands at all time points. Detection of the former bands at 6 weeks postinfection preceded seroconversion by enzyme-linked immunosorbent assay (ELISA) both on crude Leishmania antigen or the recombinant proteins rK39 and HSP70. Reactivity with the 14-, 48-, and 68-kDa bands signified early infection, whereas increased reactivity with the 14-, 24-, and 29-kDa bands was associated with posttreatment parasite persistence and potential unfavorable prognosis. Total lane intensity (TLI) emerged as a sensitive marker for early infection and increased as early as 4 weeks postinfection. TLI had a significantly higher (P < 0.01) relative increase rate than crude Leishmania antigen or HSP70 or rK39 ELISA at all time points. These immunodominant antigens and TLI, as determined by quantitative Western blotting, will be valuable for early detection and treatment evaluation of canine leishmaniasis.

scholarly journals Liraglutide Inhibits Hepatitis C Virus Replication Through an AMP Activated Protein Kinase Dependent Mechanism

2019 ◽  
Vol 20 (18) ◽  
pp. 4569 ◽  
Author(s):  
Mei-Yueh Lee ◽  
Wei-Chun Chen ◽  
Wei-Hao Hsu ◽  
Szu-Chia Chen ◽  
Jin-Ching Lee
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Western Blot ◽  
Cell Viability ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Adenosine Monophosphate ◽  
Hcv Rna ◽  
Dependent Manner

Insulin resistance and diabetes are both associated with chronic hepatitis C virus (HCV) infection, and the glucagon-like peptide-1(GLP-1) receptor agonist, liraglutide, is a common therapy for diabetes. Our aim was to investigate whether liraglutide treatment can inhibit HCV replication. A cell culture-produced HCV infectious system was generated by transfection of in vitro-transcribed genomic JFH-1 ribonucleic acid (RNA) into Huh-7.5 cells. Total RNA samples were extracted to determine the efficiency of HCV replication. The Ava5 cells were treated with liraglutide and cell viability was calculated. A Western blot analysis of the protein expression was performed. The immunoreactive blot signals were also detected. Liraglutide activated GLP-1 receptors in the HCV infectious system, and inhibited subgenomic HCV RNA replication in the HuH-7.5 cells. The Western blot analysis revealed both HCV protein and replicon RNA were reduced after treatment with liraglutide in a dose-dependent manner. Liraglutide decreased the cell viability of HCV RNA at an optimum concentration of 120 μg/mL, activated the 5′ adenosine monophosphate-activated protein kinase (AMPK) and the phosphorylated- transducer of regulated cyclic adenosine monophosphate (CAMP) response element-binding protein 2 (TORC2), thereby decreasing the cell viability of phosphoenolpyruvate carboxykinase (PEPCK) and G6pase RNA Therefore, we conclude that liraglutide can inhibit HCV replication via an AMPK/TORC2-dependent pathway.

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