scholarly journals Direct lysis RT-qPCR of SARS-CoV-2 in cell culture supernatant allows for fast and accurate quantification of virus, opening a vast array of applications

2021 ◽  
Author(s):  
Nicky Craig ◽  
Sarah Louise Fletcher ◽  
Alison Daniels ◽  
Caitlin Newman ◽  
Marie O'Shea ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Culture Supernatant ◽  
Viral Rna ◽  
Cell Culture Supernatant ◽  
Rna Purification ◽  
Qpcr Method ◽  
Virus Replication Cycle ◽  
Accurate Quantification ◽  
Cell Supernatant

An enormous global effort is being made to study SARS-CoV-2 and develop safe and effective treatments. Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify host factors and treatments to combat the infection. However, quantification of released virus often requires lengthy procedures, such as endpoint dilution assays or reinfection with engineered reporter viruses. Quantification of viral RNA in cell supernatant is faster and can be performed on clinical isolates. However, viral RNA purification is expensive in time and resources and often unsuitable for high-throughput screening. Here, we show a direct lysis RT-qPCR method allowing sensitive, accurate, fast, and cheap quantification of SARS-CoV-2 in culture supernatant. During lysis, the virus is completely inactivated, allowing further processing in low containment areas. This protocol facilitates a wide array of high- and low-throughput applications from basic quantification to studying the biology of SARS-CoV-2 and to identify novel antiviral treatments in vitro.

P-307 Fatty acid degradation during in vitro decidualization of human endometrial stromal cells

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A C Mestre Citrinovitz ◽  
J Jauckus ◽  
J Hauke ◽  
C D Langhans ◽  
K Schwarz ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Mrna Expression ◽  
Total Protein ◽  
Metabolic Pathways ◽  
Culture Supernatant ◽  
Cell Culture Supernatant ◽  
Endometrial Stromal Cells ◽  
Oxidation Pathway ◽  
Mrna Expression Levels

Abstract Study question Is the activity of the β-oxidation pathway, involved in the degradation of fatty acids, modified during in vitro decidualization of human endometrial stromal cells (HESC)? Summary answer The level of expression of fatty acid´s transporters suggests that the activity of the mitochondrial β-oxidation pathway is increased during in vitro decidualization of HESC. What is known already The differentiation of endometrial stromal cells (ESC), named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption. In the endometrium, glucose is incorporated into ESC by glucose-transporters (GLUT). Fatty acids are another important energy source in living cells. Fatty acids are transported into mitochondria by the carnitine-palmitoyl-transferases 1 and 2 (CPT1 and 2) and are degraded there through the β-oxidation pathway. It has been described that the inhibition of CPT1 affects ESC decidualization. However, it is unknown whether the turn-over of fatty acids degradation is modified during decidualization. Study design, size, duration This study was performed using primary HESC. Endometrial biopsies (mid-late proliferative-phase) were obtained from healthy-regularly-cycling women (33.6±2.2 years-old) after written informed consent was obtained (protocol approved by Ethics committee no. S-239/2005). HESC were decidualized (D) in vitro with a decidualization-cocktail (containing: medroxyprogesterone acetate, estradiol and 8-Bromo-cyclic adenosine monophosphate) for 6 days. Non-decidualized (ND) controls were treated with vehicle solutions. Cell-culture supernatant and cell extracts were collected for the evaluation of protein/gene expression and metabolite content. Participants/materials, setting, methods Decidualization was evaluated by measuring prolactin (PRL) protein levels in cell-culture supernatant (mU/l). Changes in mRNA expression levels of GLUT1, CPT1A and CPT2 were evaluated by real-time polymerase chain reaction (RT-PCR). Analysis was performed by the ΔΔCt method (internal control: RPLP0) (fold change -FC- in D compared to ND cells). Contents of acylcarnitines were evaluated by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) (nmol/mg of total protein). N = 5, mean±SEM. Paired Student’s t-test was used for statistical analysis. Main results and the role of chance PRL protein levels in cell-culture supernatant were significative increased in HESC treated with the decidualization-cocktail compared to ND cells (ND 16.80±0.73 mU/l; D 684.20±219.80 mU/l, *p<0.05). This result confirmed the decidualized state of HESC upon in vitro treatment with the decidualization-cocktail. Additionally, the mRNA expression level of GLUT1 was highly upregulated in D compared to ND cells (FC 10.02±2.90, ***p<0.001), consistent with the increase in glucose consumption characteristic of decidualization. Once confirmed the decidualized state of HESC, the mRNA expression levels of CPTA1 and CPT2 were evaluated. The mRNA expression levels of both fatty acid´s transporters were upregulated in D compared to ND cells (CPTA1: FC 1.84±0.44, **p<0.01; CPT2: FC 2.04±0.49, **p<0.01). Finally, the content levels of different acylcarnitines, intermediate metabolites of the β-oxidation degradation of fatty acids, were evaluated. The concentrations of acetyl- (C2) and butyryl- (C4) acylcarnites were decreased in D compared to ND cells [(C2: ND 1.37±0.10 nmol/mg of total protein; D 1.06±0.20 nmol/mg of total protein, *p<0.05), (C4: ND 0.03±0.01 nmol/mg of total protein; D 0.01±0.00 nmol/mg of total protein, *p<0.05)]. The content levels of other intermediate acylcarnitines measured from cell extracts had no differences between D and ND cells (p > 0,05). Limitations, reasons for caution This study was performed in vitro using primary HESC treated with a decidualization-cocktail. The interconnection of different metabolic pathways within a living cell is very complex. Further studies are necessary to define whether the different intermediate metabolites of the mitochondrial β-oxidation pathway are being used by related-metabolic pathways during decidualization. Wider implications of the findings The regulation of the energy metabolism and its interconnection with other important intra-cellular metabolic pathways is of great importance for cellular function. Our results contribute to highlight the importance of the regulation of fatty acids degradation during decidualization. Further insights into HESC metabolism could facilitate the improvement of womeńs health. Trial registration number not applicable

scholarly journals Overexpression of lncRNA TUG1 Alleviates NLRP3 Inflammasome-Mediated Cardiomyocyte Pyroptosis Through Targeting the miR-186-5p/XIAP Axis in Coronary Microembolization-Induced Myocardial Damage

2021 ◽  
Vol 12 ◽  
Author(s):  
You Zhou ◽  
Tao Li ◽  
Zhiqing Chen ◽  
Junwen Huang ◽  
Zhenbai Qin ◽  
...  
Keyword(s):  
Nlrp3 Inflammasome ◽  
Myocardial Injury ◽  
Culture Supernatant ◽  
Myocardial Damage ◽  
Luciferase Reporter ◽  
Cell Culture Supernatant ◽  
Coronary Microembolization ◽  
Lncrna Tug1

Coronary microembolization (CME) is a complicated problem that commonly arises in the context of coronary angioplasty. The lncRNA taurine-up regulated gene 1 (TUG1), significantly contributes to cardiovascular diseases; however, its contribution to CME-induced myocardial damage remains elusive. Herein, we establish the rat CME model and investigate the role of TUG1 in CME. The cell viability was evaluated via CCK-8 assay. Serum and cell culture supernatant samples were evaluated via ELISA. The dual luciferase reporter (DLR) assay, RIP, and RNA-pull down were conducted to validate the associations between TUG1 and miR-186-5p as well as miR-186-5p and XIAP. The expression of TUG1, miR-186-5p, and XIAP mRNA were determined by RT-qPCR, and proteins were evaluated via immuneblotting. As a result, TUG1 and XIAP were significantly down-regulated, and the miR-186-5p level was found to be remarkably up-regulated in CME myocardial tissues. Overexpression of TUG1 alleviated CME-induced myocardial injury and pyroptosis, whereas TUG1 knockdown showed the opposite effects. The DLR assay, RIP, and RNA-pull down results reveal that TUG1 directly targets miR-186-5p and miR-186-5p directly targets XIAP. In vitro rescue experiments show that TUG1 overexpression alleviates LPS-caused cardiomyocyte injury and pyroptosis via sponging miR-186-5p and regulating XIAP, and depression of miR-186-5p reduces LPS-induced cardiomyocyte injury and pyroptosis by targeting XIAP. Concludingly, the overexpression of TUG1 alleviates NLRP3 inflammasome-mediated cardiomyocyte pyroptosis through targeting the miR-186-5p/XIAP axis in CME-induced myocardial injury.

P–307 Fatty acid degradation during in vitro decidualization of human endometrial stromal cells

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A C Mestr. Citrinovitz ◽  
J Jauckus ◽  
J Hauke ◽  
C D Langhans ◽  
K Schwarz ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Mrna Expression ◽  
Total Protein ◽  
Metabolic Pathways ◽  
Culture Supernatant ◽  
Cell Culture Supernatant ◽  
Endometrial Stromal Cells ◽  
Oxidation Pathway ◽  
Mrna Expression Levels

Abstract Study question Is the activity of the β-oxidation pathway, involved in the degradation of fatty acids, modified during in vitro decidualization of human endometrial stromal cells (HESC)? Summary answer The level of expression of fatty acid´s transporters suggests that the activity of the mitochondrial β-oxidation pathway is increased during in vitro decidualization of HESC. What is known already The differentiation of endometrial stromal cells (ESC), named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption. In the endometrium, glucose is incorporated into ESC by glucose-transporters (GLUT). Fatty acids are another important energy source in living cells. Fatty acids are transported into mitochondria by the carnitine-palmitoyl-transferases 1 and 2 (CPT1 and 2) and are degraded there through the β-oxidation pathway. It has been described that the inhibition of CPT1 affects ESC decidualization. However, it is unknown whether the turn-over of fatty acids degradation is modified during decidualization. Study design, size, duration This study was performed using primary HESC. Endometrial biopsies (mid-late proliferative-phase) were obtained from healthy-regularly-cycling women (33.6±2.2 years-old) after written informed consent was obtained (protocol approved by Ethics committee no. S–239/2005). HESC were decidualized (D) in vitro with a decidualization-cocktail (containing: medroxyprogesterone acetate, estradiol and 8-Bromo-cyclic adenosine monophosphate) for 6 days. Non-decidualized (ND) controls were treated with vehicle solutions. Cell-culture supernatant and cell extracts were collected for the evaluation of protein/gene expression and metabolite content. Participants/materials, setting, methods Decidualization was evaluated by measuring prolactin (PRL) protein levels in cell-culture supernatant (mU/l). Changes in mRNA expression levels of GLUT1, CPT1A and CPT2 were evaluated by real-time polymerase chain reaction (RT-PCR). Analysis was performed by the ΔΔCt method (internal control: RPLP0) (fold change -FC- in D compared to ND cells). Contents of acylcarnitines were evaluated by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) (nmol/mg of total protein). N = 5, mean±SEM. Paired Student’s t-test was used for statistical analysis. Main results and the role of chance PRL protein levels in cell-culture supernatant were significative increased in HESC treated with the decidualization-cocktail compared to ND cells (ND 16.80±0.73 mU/l; D 684.20±219.80 mU/l, *p<0.05). This result confirmed the decidualized state of HESC upon in vitro treatment with the decidualization-cocktail. Additionally, the mRNA expression level of GLUT1 was highly upregulated in D compared to ND cells (FC 10.02±2.90, ***p<0.001), consistent with the increase in glucose consumption characteristic of decidualization. Once confirmed the decidualized state of HESC, the mRNA expression levels of CPTA1 and CPT2 were evaluated. The mRNA expression levels of both fatty acid´s transporters were upregulated in D compared to ND cells (CPTA1: FC 1.84±0.44, **p<0.01; CPT2: FC 2.04±0.49, **p<0.01). Finally, the content levels of different acylcarnitines, intermediate metabolites of the β-oxidation degradation of fatty acids, were evaluated. The concentrations of acetyl- (C2) and butyryl- (C4) acylcarnites were decreased in D compared to ND cells [(C2: ND 1.37±0.10 nmol/mg of total protein; D 1.06±0.20 nmol/mg of total protein, *p<0.05), (C4: ND 0.03±0.01 nmol/mg of total protein; D 0.01±0.00 nmol/mg of total protein, *p<0.05)]. The content levels of other intermediate acylcarnitines measured from cell extracts had no differences between D and ND cells (p > 0,05). Limitations, reasons for caution This study was performed in vitro using primary HESC treated with a decidualization-cocktail. The interconnection of different metabolic pathways within a living cell is very complex. Further studies are necessary to define whether the different intermediate metabolites of the mitochondrial β-oxidation pathway are being used by related-metabolic pathways during decidualization. Wider implications of the findings: The regulation of the energy metabolism and its interconnection with other important intra-cellular metabolic pathways is of great importance for cellular function. Our results contribute to highlight the importance of the regulation of fatty acids degradation during decidualization. Further insights into HESC metabolism could facilitate the improvement of womeńs health. Trial registration number Not applicable

scholarly journals Studies on the Anti-Inflammatory Effect and Its Mechanisms of Sophoridine

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Xiumei Huang ◽  
Bo Li ◽  
Lianzhong Shen
Keyword(s):  
Cell Culture ◽  
Inflammatory Response ◽  
Inflammatory Mediators ◽  
Culture Supernatant ◽  
Cell Culture Supernatant ◽  
In Vitro Cell Culture ◽  
Anti Inflammatory ◽  
Inflammatory Effect

This work is to study the anti-inflammatory effect and its mechanisms of sophoridine in vitro and in vivo. For this aim, the influences of sophoridine on several inflammatory mediators were investigated. Excessive inflammatory response in vitro model was developed by using lipopolysaccharide (LPS) to stimulate the mouse peritoneal macrophages and HL-60 cells to produce IL-6 and IL-8. Carrageenin-induced mouse paw edema model was used as inflammatory response in vivo model. MTT method, ultraviolet spectrophotometric method, and radioimmunoassay were used to measure the changes of TNFα, IL-6, PGE2, and IL-8 in in vitro cell culture supernatant or in the local inflammatory exudates. The results showed that sophoridine inhibited the production of IL-8 in in vitro cell culture supernatant and inhibited the production of TNFα, PGE2, and IL-8 in the local inflammatory exudates but had no significant effects on the production of IL-6 in vitro and in vivo. It is demonstrated that sophoridine’s anti-inflammatory effect was due to its ability to inhibit the production of cytokine and inflammatory mediators.

scholarly journals Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant

Tumor Biology ◽  
2019 ◽  
Vol 41 (8) ◽  
pp. 101042831986636 ◽  
Author(s):  
Abel Jacobus Bronkhorst ◽  
Vida Ungerer ◽  
Stefan Holdenrieder
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Culture Supernatant ◽  
Quantitative Polymerase Chain Reaction ◽  
High Sensitivity ◽  
Cell Types ◽  
Cell Culture Supernatant ◽  
Cell Free Dna ◽  
Free Dna

Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA–based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cell-free DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop Onec. Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.

scholarly journals Human Th17 cells produce a soluble mediator that increases podocyte motility via signaling pathways that mimic PAR-1 activation

2019 ◽  
Vol 317 (4) ◽  
pp. F913-F921 ◽  
Author(s):  
Carl J. May ◽  
Gavin I. Welsh ◽  
Musleeha Chesor ◽  
Phillipa J. Lait ◽  
Lauren P. Schewitz-Bowers ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Th17 Cell ◽  
Th17 Cells ◽  
Culture Supernatant ◽  
Cell Culture Supernatant ◽  
Soluble Mediator ◽  
Stimulation Of

The specific pathogenesis of idiopathic nephrotic syndrome (NS) is poorly understood, and the role of immune mediators remains contentious. However, there is good evidence for the role of a circulating factor, and we recently postulated circulating proteases as candidate factors. Immunosuppressive therapy with glucocorticoids (GCs) and T cell inhibitors are widely used in the clinical treatment of NS. Given that T helper (CD4+) cells expressing IL-17A (so-called Th17 cells) have recently been reported to be resistant to GC treatment, and GC resistance remains a major challenge in the management of NS, we hypothesized that Th17 cells produce a circulating factor that is capable of signaling to the podocyte and inducing deleterious phenotypic changes. To test this, we generated human Th17 cells from healthy volunteers and added the supernatants from these T cell cultures to conditionally immortalized human podocytes in vitro. This demonstrated that podocytes treated with Th17 cell culture supernatant, as well as with patient disease plasma, showed significant stimulation of JNK and p38 MAPK pathways and an increase in motility, which was blocked using a JNK inhibitor. We have previously shown that nephrotic plasma elicits a podocyte response via protease-activated receptor-1 (PAR-1). Stimulation of PAR-1 in podocytes elicited the same signaling response as Th17 cell culture supernatant treatment. Equally, protease inhibitors with Th17 cell culture treatment blocked the signaling response. This was not replicated by the reagents added to Th17 cell cultures or by IL-17A. Hence, we conclude that an undefined soluble mediator produced by Th17 cells mimics the deleterious effect of PAR-1 activation in vitro. Given the association between pathogenic subsets of Th17 cells and GC resistance, these observations have potential therapeutic relevance for patients with NS.

A study of the potential of medicinalplant extracts in suppression of SARS-CoV-2 replication: a pilot study*

2021 ◽  
Vol 23 (9) ◽  
pp. 445-450
Author(s):  
Michela Luisa De Pellegrin ◽  
◽  
Anette Rohrhofer ◽  
Philipp Schuster ◽  
Barbara Schmidt ◽  
...  
Keyword(s):  
Culture Supernatant ◽  
Quantitative Polymerase Chain Reaction ◽  
Vero Cells ◽  
Cell Culture Supernatant ◽  
Medicinal Products ◽  
Viral Etiology ◽  
Chain Reaction ◽  
Coronavirus Infection ◽  
Polymerase Chain

Relevance. Medicinal products of plant origin have long been successfully used in the treatment of various diseases, including those of viral etiology. Nevertheless, their effect on the replication of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has not been studied so far. Objective. To evaluate the potential of drugs based on validated plant extracts – RE (Bronchipret, Bronchipret TP, Tonsilgon N, Sinupret extract, Tonsipret) with respect to their ability to suppress SARS-CoV-2 replication in vitro. Materials and methods. Vero cells (Vero) incubated with RE of different concentrations were infected with SARS-CoV-2 virus. After 48 h the virus replication (the number of viral RNA copies in cell culture supernatant) was determined by quantitative polymerase chain reaction. Results. SARS-CoV-2 RNA copy number under the action of non-cytotoxic concentrations of Bronchipret TP decreased by 1000 times and by 10 times under the action of drugs Tonsilgon N and Tonsipret. Conclusion. Some of the studied REs in vitro demonstrated promising antiviral activity. It is advisable to conduct further, including clinical, studies of their activity against SARS-CoV-2 in the treatment of coronavirus infection (COronaVirus Disease 2019 – COVID-19).

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Inhibitors of Neuroblastoma Cell Colony Growth That Increase Lysosome Accumulation

2020 ◽  
Author(s):  
Daniel Herp ◽  
Johannes Ridinger ◽  
Dina Robaa ◽  
Stephen A. Shinsky ◽  
Karin Schmidtkunz ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
High Throughput Screening ◽  
Neuroblastoma Cell ◽  
Hdac Inhibitors ◽  
Anticancer Therapy ◽  
Colony Growth ◽  
Autophagic Flux ◽  
Enzymatic Conversion ◽  
Lysine Deacetylase

Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, esp. cancer. First HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement e.g. in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of the other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like spermine or spermidine. Hence, it also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin labelled acetyl spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10 mediated spermidine deacetylation in-vitro. Among those are potent inhibitors of neuroblastoma colony growth in culture that show accumulation of lysosomes, implicating disturbance of autophagic flux.

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