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1
Marnis, Huria, Bambang Iswanto, Romy Suprapto, Imron Imron, and Raden Roro Sri Pudji Sinarni Dewi. "IDENTIFIKASI ZIGOSITAS IKAN LELE (Clarias gariepinus) TRANSGENIK F-2 YANG MEMBAWA GEN HORMON (PhGH) DENGAN MENGGUNAKAN METODE REALTIME-qPCR." Jurnal Riset Akuakultur 11, no. 1 (November 14, 2016): 39. http://dx.doi.org/10.15578/jra.11.1.2016.39-46.
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Produktivitas ikan budidaya dapat ditingkatkan melalui teknologi transgenesis. Populasi ikan lele transgenik cepat tumbuh telah dihasilkan dan karakter biologisnya telah diketahui. Namun informasi zigositas ikan lele transgenik perlu ditelaah lebih lanjut. Penelitian ini bertujuan untuk mengidentifikasi zigositas ikan lele transgenik F-2. Zigositas ikan lele transgenik diidentifikasi dengan menggunakan metode real-time qPCR (RT-qPCR) dan uji progeni. Identifikasi zigositas melalui uji progeni, dilakukan dengan mendeteksi transgen (PhGH) pada individu-individu F-3 hasil persilangan transgenik F-2 dengan non-transgenik. Hasil penelitian menunjukkan bahwa zigositas pada ikan lele transgenik F-2 dapat diidentifikasi dengan menggunakan metode RT-qPCR. Semua ikan transgenik F-2 adalah heterozigot, dengan nilai 2-Ct yang hampir sama tiap individu F-2, yaitu berkisar 0,80-0,99. Identifikasi zigositas dengan metode RT-qPCR menunjukkan hasil yang sama dengan uji progeni, semua transgenik F-2 tidak menghasilkan 100% anakan F-3 positif transgen. Pada uji progeni, transmisi transgen pada penelitian ini tidak mengikuti hukum segregasi Mendel, dengan kisaran sebesar 5%-40%.Fish farming productivity can be increased by transgenesis technology. On the previous study, transgenic African catfish population fast growing has been produced and its biological characters has been known. However information of transgenic zygosity of catfish should be examined. The aim of this study was to identify the zygosity of F-2 transgenic African catfish. The zygosity of F-2 transgenic was identified by real time-qPCR (RT-qPCR) method and progeny test. Further, identification of zygosity F-2 transgenic African catfish was confirmed by progeny test, while F-2 transgenic African catfish was mated with non-transgenic. Identification of zygosity F-2 transgenic was conducted by detection PhGH gene (transgene) in F-3 transgenic African catfish population. Transgene transmission was evaluated by PCR method. The result showed that the zygosity F-2 transgenic African catfish could be identified by RT-qPCR method. All F-2 transgenic African catfish were heterozygous, where as the 2-Ct value was almost same for all individual, which ranges from 0.80 to 0.99. The result of zygosity identification using RT-qPCR method was as same as that of progeny test. In the progeny test, transgene transmission in this study was non-Mendelian segregation, with ranges of 5%-40%.
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Hikmawati, Isna, Hendro Wahjono, Martini Martini, Edi Darmana, Soeharyo Hadisaputro, Kisdjamiatun Retna Mustika Djati, and Sugeng Juwono Mardihusodo. "Persistence of Dengue Virus (DENV-1, 2, 3,4) Transovarial-Transgenerational with Realtime Polymerase Chain Reaction (qPCR) in Ae. Aegypti and Ae. Albopictus (Diptera: Culicidae)." Women Health Care and Issues 4, no. 8 (October 30, 2021): 01–09. http://dx.doi.org/10.31579/2642-9756/090.
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Ae. aegypti and Ae. albopictus have an important role in DHF transmission because they can simultaneously transmit the dengue virus vertically / transovarially or horizontally. This phenomenon indicates the persistence of the dengue virus by vectors. The aim of this research was to prove the persistence of the transovarial-transgenerational dengue virus (DENV-1,2,3,4) with real time polymerase chain reaction (qPCR) in Ae. aegypti and Ae. albopictus (Diptera: Culicidae). Quasi experimental design with intervention infects DENV 1-2-3-4 serotypes in Ae. aegypti and Ae. albopictus intratoracally. Research population Ae. aegypti and Ae. albopictus laboratory colony females. Dengue virus detection uses real-time polymerase chain reaction (qPCR). Transovarial detection by qPCR indicates detection of dengue virus in Ae. albopictus DENV-1 to progeny 1 (F1), DENV-2 and DENV-3 to F2, DENV-4 to F3. Next to Ae. aegypti DENV-1 to 1st progeny (F1), DENV-2 to F2, DENV-3 to F4 and DENV-4 to F3. there was no difference in MIR value (p value: 0.356) for the four serotypes in Ae. albopictus and Ae. aegypti. DENV-3 is the most persistent serotype in Ae. aegypti with 83.3% MIR and DENV-4 were the most persistent serotypes in Ae.albopictus with 100% MIR. The need to improve vector control models that focus not only on the main vector, but also other co-vectors.
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Rublenko, N. "Identification of Salmonella spp and serovars Typhimurium, Enteritidis by qPCR." Naukovij vìsnik veterinarnoï medicini, no. 1(154) (May 21, 2020): 21–31. http://dx.doi.org/10.33245/2310-4902-2020-154-1-21-31.
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This article presents the results of the identification of the Salmonella genus as well as serovars Enteritidis and Typhimurium by a real-time polymerase chain reaction. We constructed three pairs of primers and fluorescent probes to simultaneously identify the Salmonella genus, serovars Enteritidis and Typhimurium in a qPCR. The specificity of the primers was evaluated on Salmonella strains of different serovars from the National Center for Strains of Microorganisms (UNCMS) strains of the State Scientific Control Institute of Biotechnology and Strains of Microorganisms (SSCIBSM) and 46 Salmonella strains isolated from poultry. E. coli ATCC 25922, Bacillus cereus ATCC 11778, Listeria monocytogenes ATCC 19112 from UNCMS collection were used to check the specificity of the primers as heterologous samples. Bacterial DNA was extracted using a DNA Sorb B (Amplisens) kit, and realtime PCR was accomplished with the "Real-time PCR kit" (Syntol) on Bio-rad CFX. A series of 10-fold S. Typhimurium and S. Enteritidis DNA dilutions were studied to evaluate the sensitivity of the primers: 10-1-10-5. The analytical sensitivity of primers for detection of the genus Salmonella is: for S. Typhimurium - 0.25 ng/sample (Typhimurium) and S. Enteritidis - 0.27 ng/ sample (Enteritidis). The results of the studies confirmed the specificity of the primer set and the high sensitivity. No hybridization of primers with DNA samples of other bacteria found, in particular, the nonspecific reaction products were absent. The primer sets for the detection of DNA of Enteritidis and Typhimurium serovars also has high specificity. If necessary, this set of primers can be used to perform a multiplex qPCR, that can simultaneously identify bacteria of the Salmonella genus and differentiate Enteritidis and Typhimurium serovars. Keywords: Salmonella, bacteria, polymerasechainreaction, DNA, qPCR.
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Dyer, Clare E. F., Naomi E. Clarke, Dinh Ng Nguyen, H. M. P. Dilrukshi Herath, Sze Fui Hii, Russell Pickford, Rebecca J. Traub, and Susana Vaz Nery. "Assessing the efficacy of albendazole against hookworm in Vietnam using quantitative PCR and sodium nitrate flotation." PLOS Neglected Tropical Diseases 16, no. 10 (October 31, 2022): e0010767. http://dx.doi.org/10.1371/journal.pntd.0010767.
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Preventive chemotherapy (PC), consisting of the regular distribution of anthelmintics to populations or groups of populations at risk, is the primary tool used to control soil-transmitted helminth (STH) infections. This strategy, whilst cost-effective, raises the concern of potential emergence of drug resistance. The efficacy of anthelmintics against STH infections is measured using cure rate (CR) and egg reduction rate (ERR), using microscopy-based techniques such as the Kato-Katz thick smear. However, Kato-Katz has low sensitivity, especially for low-intensity infections, and requires fresh samples that need to be processed quickly. Realtime quantitative PCR (qPCR), which is more sensitive, is emerging as a “gold standard” for STH diagnostics given its higher sensitivity (important in low prevalence settings) and ability to differentiate hookworm species, while sodium nitrate flotation (SNF) may provide a low-cost more sensitive and practical alternative to Kato-Katz in the field. In this study, we examined the efficacy of a locally manufactured brand of albendazole 400 mg (“Alzental”) against hookworm in Đắk Lắk province, Vietnam, using both qPCR and SNF. For qPCR, formulae to convert qPCR cycle threshold (Ct) values into eggs per gram of faeces (EPG) were utilised to determine efficacy calculations, and these values directly compared with efficacy values generated using SNF. Factors associated with CR and ERR were examined, and Alzental tablet quality was assessed by comparing with an Australian TGA-approved equivalent “Eskazole” tablet. We observed a CR and ERR of 64.9% and 87.5% respectively using qPCR, and 68.4% and 67.6% respectively using SNF. The tablet composition of Alzental was comparable to Eskazole in terms of active albendazole drug concentration with no evidence of impurities. This study demonstrates that the efficacy of Alzental against hookworm is within the range of previously reported studies for albendazole 400 mg. The study also demonstrates the value of qPCR and SNF as alternatives to standard Kato-Katz methodology for assessment of anthelmintic efficacy.
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Rasmussen, S., A. J. Parsons, Q. Liu, H. Xue, and J. A. Newman. "High nutrient supply and carbohydrate content reduce endophyte and alkaloid concentration." NZGA: Research and Practice Series 13 (January 1, 2007): 135–38. http://dx.doi.org/10.33584/rps.13.2006.3103.
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Two controlled environment experiments were performed to test the effects of nitrogen, phosphorus and carbohydrates on endophyte (Neotyphodium lolii) and alkaloid concentrations in ryegrass (Lolium perenne). Three perennial ryegrass cultivars ('high sugar grasses' AberDove and AberDart; control Fennema) that differ in carbohydrate content were infected with three strains of N. lolii (common strain, CS; AR1; AR37). Infected and uninfected plants were grown under high (9 mM) and low (2.25 mM) nitrogen (AberDove, Fennema; CS, AR1, AR37) or under high (2 mM KH2PO4) and low (0.05 mM KH2PO4) phosphorus (AberDart, Fennema; CS, AR1). Quantitative realtime Polymerase Chain Reaction (qPCR) was used to estimate endophyte concentrations in harvested leaf tissues. High N and P supply as well as high carbohydrate content of the host grass reduced endophyte concentrations. Alkaloid production was also reduced under both increased N supply and in the high sugar cultivar, and was linearly related to endophyte concentration (except ergovaline). The results stress the need for wider quantification of fungal endophytes in the grassland/ foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes, or increasing nutrient inputs, affect the role of endophytes in grassland ecosystems. Keywords: Neotyphodium lolii, foliar endophyte, Lolium perenne, perennial ryegrass, qPCR, high sugar ryegrass, nitrogen, phosphate, carbohydrate, AR1, AR37, alkaloids
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Karolak, Justyna A., Barbara Ginter-Matuszewska, Katarzyna Tomela, Michal Kabza, Dorota M. Nowak-Malczewska, Malgorzata Rydzanicz, Piotr Polakowski, Jacek P. Szaflik, and Marzena Gajecka. "Further evaluation of differential expression of keratoconus candidate genes in human corneas." PeerJ 8 (August 20, 2020): e9793. http://dx.doi.org/10.7717/peerj.9793.
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Background Keratoconus (KTCN) is a progressive eye disease, characterized by changes in the shape and thickness of the cornea that results in loss of visual acuity. While numerous KTCN candidate genes have been identified, the genetic etiology of the disease remains undetermined. To further investigate and verify the contribution of particular genetic factors to KTCN, we assessed 45 candidate genes previously indicated as involved in KTCN etiology based on transcriptomic and genomic data. Methods The RealTime ready Custom Panel, covering 45 KTCN candidate genes and two reference transcripts, has been designed. Then, the expression profiles have been assessed using the RT-qPCR assay in six KTCN and six non-KTCN human corneas, obtained from individuals undergoing a penetrating keratoplasty procedure. Results In total, 35 genes exhibiting differential expression between KTCN and non-KTCN corneas have been identified. Among these genes were ones linked to the extracellular matrix formation, including collagen synthesis or the TGF-β, Hippo, and Wnt signaling pathways. The most downregulated transcripts in KTCN corneas were CTGF, TGFB3, ZNF469, COL5A2, SMAD7, and SPARC, while TGFBI and SLC4A11 were the most upregulated ones. Hierarchical clustering of expression profiles demonstrated almost clear separation between KTCN and non-KTCN corneas. The gene expression levels determined using RT-qPCR showed a strong correlation with previous RNA sequencing (RNA-Seq) results. Conclusions A strong correlation between RT-qPCR and earlier RNA-Seq data confirms the possible involvement of genes from collagen synthesis and the TGF-β, Hippo, and Wnt signaling pathways in KTCN etiology. Our data also revealed altered expression of several genes, such as LOX, SPARC, and ZNF469, in which single nucleotide variants have been frequently identified in KTCN. These findings further highlight the heterogeneous nature of KTCN.
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Weng, Wenfeng, Xiang Lu, Meiliang Zhou, Anjing Gao, Xin Yao, Yong Tang, Weijiao Wu, et al. "FtbZIP12 Positively Regulates Responses to Osmotic Stress in Tartary Buckwheat." International Journal of Molecular Sciences 23, no. 21 (October 28, 2022): 13072. http://dx.doi.org/10.3390/ijms232113072.
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ABFs play a key role in regulating plant osmotic stress. However, in Tartary buckwheat, data on the role of ABF genes in osmotic stress remain limited and its associated mechanism in osmoregulation remain nebulous. Herein, a novel ABF family in Tartary buckwheat, FtbZIP12, was cloned and characterized. FtbZIP12 is a transcriptional activator located in the nucleus; its expression is induced by NaCl, mannitol, and abscisic acid (ABA). Atopic expression of FtbZIP12 in Arabidopsis promoted seed germination, reduced damage to primary roots, and improved the tolerance of seedlings to osmotic stress. The quantitative realtime polymerase chain reaction (RT-qPCR) results showed that the expressions of the typical genes related to stress, the SOS pathway, and the proline synthesis pathway in Arabidopsis were significantly (p < 0.05) upregulated under osmotic stress. FtbZIP12 improved the osmotic pressure resistance by reducing the damage caused by reactive oxygen species to plants and maintained plant homeostasis by upregulating the expression of genes related to stress, osmotic regulation, and ion homeostasis. This study identified a key candidate gene for understanding the mechanism underlying osmotic-stress-regulated function in Tartary buckwheat, thereby providing a theoretical basis for improving its yield and quality.
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Abdelwahab, Siddig Ibrahim, Loiy Elsir Ahmed Hassan, Amin M. S. Abdul Majid, Sakina M. Ahmed Yagi, Syam Mohan, Manal Mohamed Elhassan Taha, Syahida Ahmad, et al. "Cucurbitacin L 2-O-β-Glucoside Demonstrates Apoptogenesis in Colon Adenocarcinoma Cells (HT-29): Involvement of Reactive Oxygen and Nitrogen Species Regulation." Evidence-Based Complementary and Alternative Medicine 2012 (2012): 1–8. http://dx.doi.org/10.1155/2012/490136.
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Emerging evidence suggests that reactive oxygen (ROS) and nitrogen (RNS) species can contribute to diverse signalling pathways of inflammatory and tumour cells. Cucurbitacins are a group of highly oxygenated triterpenes. Many plants used in folk medicine to treat cancer have been found to contain cucurbitacins displaying potentially important anti-inflammatory actions. The current study was designed to investigate the anti-ROS and -RNS effects of cucurbitacin L 2-O-β-glucoside (CLG) and the role of these signaling factors in the apoptogenic effects of CLG on human colon cancer cells (HT-29). This natural cucurbitacin was isolated purely fromCitrullus lanatusvar.citroides(Cucurbitaceae). The results revealed that CLG was cytotoxic to HT-29. CLG increased significantly (P<0.05) RNA and protein levels of caspase-3 in HT-29 cells when verified using a colorimetric assay and realtime qPCR, respectively. The results showed that lipopolysaccharide/interferon-gamma (LPS/INF-γ) increased nitrous oxide (NO) production inR AW264.7macrophages, whereas N(G)-nitro-L-argininemethyl ester (L-NAME) and CLG curtailed it. This compound did not reveal any cytotoxicity on RAW264.7 macrophages and human normal liver cells (WRL-68) when tested using the MTT assay. Findings of ferric reducing antioxidant power (FRAP) and oxygen radical absorption capacity (ORAC) assays demonstrate the antioxidant properties of CLG. The apoptogenic property of CLG on HT-29 cells is thus related to inhibition of reactive nitrogen and oxygen reactive species and the triggering of caspase-3-regulated apoptosis.
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Ysrafil, Ysrafil, Indwiani Astuti, Sumadi Lukman Anwar, Ronny Martien, Firasti Agung Nugrahening Sumadi, Tirta Wardhana, and Sofia Mubarika Haryana. "MicroRNA-155-5p Diminishes in Vitro Ovarian Cancer Cell Viability by Targeting HIF1α Expression." Advanced Pharmaceutical Bulletin 10, no. 4 (August 9, 2020): 630–37. http://dx.doi.org/10.34172/apb.2020.076.
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Purpose : Ovarian cancer is the most lethal of gynecological malignancies. Recently, the development of microRNA (miRNA) -based therapeutics that could impact broad cellular programs, leading to inhibition of cancer cell viability, is gaining attention in the therapeutic landscape. The therapy is based on the presence of aberrant expressions of miRNA in cancer cells. Decreasing of tumor suppressor miRNA expression causes upregulation of oncoprotein, which worsens the prognosis of the ovarian cancer. Methods: miR-155-5p mimics were carried by chitosan nanoparticles using new nanotechnology methods. Cellular uptake of miRNA was assessed by fluorescence microscope while MTT and qPCR assay were used to determine miRNA profile and the effect of CS-NP/miRNA on SKOV3 cells. Results: Results of profiling validated using quantitative realtime-polymerase chain reaction (PCR) found one of the most altered tumor suppressor miRNAs, miR-155-5p was downregulated 892.15-fold. According to bioinformatic analysis we identified the miRNA could recognize and regulate HIF1α expression. Transfection of mimics for miR-155-5p showed significantly increased miR-155-5p endogen SKOV3 expression level compared to the control group. We found differences after transfection mimics for miR-155-5p 31.5 and 63 nanoMolar. Increasing of miR-155-5p endogen lead to diminished SKOV3 viability (by 30%; <0.05 at concentration 80 nanoMolar). These mimics may cause an increase in upregulated miR-155-5p endogen that can reduce HIF1α expression. Here we found 2-fold and 2.8-fold reduction of HIF1α expression level after transfection compared to the control group. Conclusion: According to these findings, the mimics miR-155-5p can inhibit ovarian cancer cell proliferation by regulating HIF1α expression.
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Wang, Jijun, Xiaolong Wang, Tong Chen, Liyu Jiang, and Qifeng Yang. "Huaier Extract Inhibits Breast Cancer Progression Through a LncRNA-H19/MiR-675-5p Pathway." Cellular Physiology and Biochemistry 44, no. 2 (2017): 581–93. http://dx.doi.org/10.1159/000485093.
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Background/Aims: Increasing evidence indicates that Huaier extract has promising therapeutic effects against cancer. However, the mechanisms that underlie its anti-tumor effects remain unclear. In recent years, various studies have shown that long noncoding RNAs (lncRNAs) play a critical role in the regulation of cancer development and progression. Here, we explored the role of lncRNAs in Huaier-induced tumor suppression. Methods: Microarray profiling was performed to identify the candidate lncRNAs affected by Huaier extract. Quantitative realtime PCR (qPCR) was used to evaluate the transfection efficiency and the influence of Huaier extract on H19 expression. The effect of Huaier extract on the cell viability was examined by MTT. Moreover, the rates of apoptotic cells were detected using flow-cytometric analysis. Western blot analysis was applied to show the protein levels of CBL. Results: Microarray data derived from Huaier-treated breast cancer cells identified H19 as a potential target. Huaier extract reduced the expression of H19. The over-expression of H19 inhibited the cytotoxic effects of Huaier extract; in contrast, reduced H19 expression enhanced the function of Huaier extract. MiR-675-5p was identified as a mature product of H19. Moreover, Huaier extract reduced the miR-675-5p expression. Upregulating miR-675-5p reversed the inhibitory effects of Huaier extract, whereas downregulating miR-675-5p sensitized breast cancer cells to the effect of Huaier extract. In addition, Huaier extract increased the expression of CBL protein, a direct target of miR-675-5p. Conclusion: Collectively, the data demonstrate that Huaier extract reduces viability and induces apoptosis in breast cancer cells via H19-miR-675-5p-CBL axis regulation.
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Ni, Dongjiao, Xiang Huang, Zhibo Wang, Lin Deng, Li Zeng, Yiwei Zhang, Dongdong Lu, and Xinhua Zou. "Expression characterization and transcription regulation analysis of porcine Yip1 domain family member 3 gene." Asian-Australasian Journal of Animal Sciences 33, no. 3 (March 1, 2020): 398–407. http://dx.doi.org/10.5713/ajas.19.0076.
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Objective: The Yip1 domain family (YIPF) proteins were proposed to function in endoplasmic reticulum (ER) to Golgi transport and maintenance of the morphology of the Golgi, which were homologues of yeast Yip1p and Yif1p. YIPF3, the member 3 of YIPF family was a homolog of Yif1p. The aim of present study was to investigate the expression and regulation mechanism of porcine YIPF3.Methods: Quantitative realtime polymerase chain reaction (qPCR) was used to analyze porcine YIPF3 mRNA expression pattern in different tissues and pig kidney epithelial (PK15) cells stimulated by polyinosine-polycytidylic acid (poly [I:C]). Site-directed mutations combined with dual luciferase reporter assays and electrophoretic mobility shift assay (EMSA) were employed to reveal transcription regulation mechanism of porcine YIPF3.Results: Results showed that the mRNA of porcine YIPF3 (pYIPF3) was widely expressed with the highest levels in lymph and lung followed by spleen and liver, while weak in heart and skeletal muscle. Subcellular localization results indicated that it expressed in Golgi apparatus and plasma membranes. Upon stimulation with poly (I:C), the level of this gene was dramatically up-regulated in a time- and concentration-dependent manner. pYIPF3 core promoter region harbored three cis-acting elements which were bound by ETS proto-oncogene 2 (ETS2), zinc finger and BTB domain containing 4 (ZBTB4), and zinc finger and BTB domain containing 14 (ZBTB14), respectively. In which, ETS2 and ZBTB4 both promoted pYIPF3 transcription activity while ZBTB14 inhibited it, and these three transcription factors all played important regulation roles in tumorigenesis and apoptosis.Conclusion: The pYIPF3 mRNA expression was regulated by ETS2, ZBTB4, and ZBTB14, and its higher expression in immune organs might contribute to enhancing ER to Golgi transport of proteins, thus adapting to the immune response.
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Sandbichler, Adolf M., Bianca Jansen, Bettina A. Peer, Monika Paulitsch, Bernd Pelster, and Margit Egg. "Metabolic Plasticity Enables Circadian Adaptation to Acute Hypoxia in Zebrafish Cells." Cellular Physiology and Biochemistry 46, no. 3 (2018): 1159–74. http://dx.doi.org/10.1159/000489058.
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Background/Aims: Reduced oxygen availability, hypoxia, is frequently encountered by organisms, tissues and cells, in aquatic environments as well as in high altitude or under pathological conditions such as infarct, stroke or cancer. The hypoxic signaling pathway was found to be mutually intertwined with circadian timekeeping in vertebrates and, as reported recently, also in mammals. However, the impact of hypoxia on intracellular metabolic oscillations is still unknown. Methods: For determination of metabolites we used Multilabel Reader based fluorescence and luminescence assays, circadian levels of Hypoxia Inducible Factor 1 alpha and oxidized peroxiredoxins were semi quantified by Western blotting and ratiometric quantification of cytosolic and mitochondrial H2O2 was achieved with stable transfections of a redox sensitive green fluorescent protein sensor into zebrafish fibroblasts. Circadian oscillations of core clock gene mRNA´s were assessed using realtime qPCR with subsequent cosine wave fit analysis. Results: Here we show that under normoxia primary metabolic activity of cells predominately occurs during day time and that after acute hypoxia of two hours, administrated immediately before each sampling point, steady state concentrations of glycolytic key metabolites such as glucose and lactate reveal to be highly rhythmic, following a circadian pattern with highest levels during the night periods and reflecting the circadian variation of the cellular response to hypoxia. Remarkably, rhythms in glycolysis are transferred to cellular energy states under normoxic conditions, so that ADP/ATP ratios oscillate as well, which is the first evidence for cycling ADP/ATP pools in a metazoan cell line to our knowledge. Furthermore, the hypoxia induced alterations in rhythms of glycolysis lead to the alignment of three major cellular redox systems, namely the circadian oscillations of NAD+/NADH and NADP+/NADPH ratios and of increased nocturnal levels of oxidized peroxiredoxins, resulting in a highly oxidized nocturnal cellular environment. Of note, circadian rhythms of cytosolic H2O2 remain unaltered, while the transcriptional clock is already attenuated, as it is known to occur also under chronic hypoxia. Conclusion: We therefor propose that the realignment of metabolic redox oscillations might initiate the observed hypoxia induced attenuation of the transcriptional clock, based on the reduced binding affinity of the CLOCK/BMAL complex to the DNA in an oxidized environment.
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Gaudette, Brian, Neal N. Iwakoshi, and Lawrence H. Boise. "Bcl-xL Protects From UPR-Associated Apoptosis During Plasma Cell Differentiation." Blood 120, no. 21 (November 16, 2012): 3288. http://dx.doi.org/10.1182/blood.v120.21.3288.3288.
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Abstract Abstract 3288 Understanding factors that control plasma cell survival is important for the development of therapeutic approaches to diseases including multiple myeloma and autoimmune disorders. As part of the program that allows for B cell differentiation to a plasma cell, a required signal includes the activation of an unfolded protein response (UPR). However unlike stress-induced activation of the UPR, induction of apoptosis does not occur, suggesting that compensatory survival signals are also activated during plasma cell differentiation. The compensatory survival pathways are less defined and require further research. Therefore we employed a model of plasma cell differentiation to better define the survival signaling during this process. The murine B cell lymphoma cell line, Bcl1 can be stimulated to secrete immunoglobulin using IL-5 and LPS. To determine the effects of exogenous ER stress on plasma cell differentiation, we treated the cells with the inhibitor of N-linked glycosylation, tunicamycin, for 5 hours prior to the differentiation signal. The 5 hour pulse of tunicamycin was sufficient to induce significant apoptosis in undifferentiated cells or cells treated with IL-5, resulting in 78% and 74% cell death respectively by 24 hours post treatment. However, if LPS was included in the differentiation stimulus the cells were able to differentiate into IgM-secreting plasma cells with similar kinetics as cells differentiated in the absence of tunicamycin pretreatment. Thus LPS-induced differentiation is sufficient to block ER stress-induced cell death. Since these cells also activate a UPR during differentiation, we hypothesized that part of the differentiation program included protection from UPR-associated cell death. To investigate this effect, we first examined the levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1 during plasma cell differentiation. We found that differentiation induced Bcl-xL and caused the loss of Mcl-1. From this data we hypothesized that the differentiation of these cells resulted in Bcl-xL dependence during plasma cell differentiation. To test this we used ABT-737, which selectively blocks the binding pocket of Bcl-xL and Bcl-2 but not Mcl-1 and kills cells that are dependent on Bcl-2 or Bcl-xL. Undifferentiated Bcl1 cells were insensitive to ABT-737 with an IC50 > 2μM. However ABT-737 sensitized LPS-treated Bcl1 cells to tunicamycin pretreatment resulting in 89% death in 24 h compared to 23% in untreated cells. These data suggest that the induction of Bcl-xL is responsible for the survival of cells undergoing ER stress. Most importantly, cells treated with LPS and IL-5 for differentiation became sensitive to ABT-737 with 59% cell death versus 26% in untreated cells, thus demonstrating that during plasma cell differentiation, cells switch to a Bcl-xL-dependent state. To determine the molecular basis for these findings we investigated the effects of ABT-737 on the expression levels of Bcl-2 proteins as well as the effects of differentiation on their interactions. ABT-737 did not induce changes in the expression of Bcl-2 family proteins. However, co-immunoprecipitation demonstrated a shift in Bim binding from Mcl-1 in untreated cells to Bcl-xL in differentiating cells. This latter finding is consistent with a shift from Mcl-1 dependence to Bcl-xL during plasma cell differentiation. To validate these data, primary C57BL/6 splenocytes were isolated, depleted of non-B cells and subsequently stimulated with IL-4 and LPS to differentiate into plasmablasts. Realtime qPCR showed an increase in Bcl-xL mRNA and loss of Mcl-1 and Bcl-2 mRNA in both the primary B cells and the Bcl1 cell line. Western blotting of primary B cell lysates also showed an increase in Bcl-xL protein and loss of Bcl-2 and Mcl-1 protein. Together these data indicate that during plasma cell differentiation the cell enters a Bcl-xL-dependent state that protects against differentiation-induced apoptosis. Disclosures: No relevant conflicts of interest to declare.
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Ogweno Obuya, Clifford, Amolo Stephen Asito, V. Ann Stewart, and John N.Waitumbi. "EVALUATION OF PERFORMANCE OF TWO MONOPLEX QUANTITATIVE REAL TIME PCR ASSAYS IN COMPARISON TO MICROSCOPY FOR IDENTIFICATION OF MALARIA PARASITES." PARIPEX INDIAN JOURNAL OF RESEARCH, March 15, 2022, 32–38. http://dx.doi.org/10.36106/paripex/8706762.
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Background: Microscopy is the gold standard for Malaria diagnosis with shortcomings such as false positives, false negatives,errors in species identification,and errors in enumeration of parasites.Quantitative real-time PCR (qPCR) has improved submicroscopic malaria diagnosis. This study evaluated the performance, concordance, correlation and methods agreement of two monoplex qPCR assays against expert malaria microscopy for the detection and enumeration of malaria parasites. Methods: This was a cross sectional study utilizing 127 archived blood samples collected from five provinces in Kenya. Malaria microscopy was conducted by two independent microscopists then 18S-rRNA-qPCR and non-18S-rRNA-qPCR assays were done to identify and quantify the infecting species.The sensitivity,specificity,and predictive values.Cohen Kappa value was used to quantify the method agreement and Bland Altman test was used to assess the bias and limits of agreement.Correlation between microscopy and qPCR parasite densities was determined by the Spearman's rank test. Statistical significance was taken at p<0.05. Results: A higher sensitivity and a lower specificity were observed in all the three plasmodium species in non 18SrRNA-qPCR compared to 18S-rRNA-qPCR. The sensitivity and specificity of 18S-rRNA-qPCR was 91.3% and 75% in detection of P.falciparum,67.6% and 88.1% in detection of P.malariae,and 55.8% and 91.4% in detection of P.ovale.The sensitivity and specificity of non 18S-rRNA-qPCR was 99.1% and 66.7% in detection of P.falciparum,77.9% and 88.1% in detection of P. malariae, and 79.4% and 90.3% in detection of P. ovale. All the positive and negative predictive values were above 70% except the negative predictive value for 18S-rRNA-qPCR (47.4%).Kappa of more than 0.5 was observed between microscopy and both18S-rRNA-qPCR and non-18S-rRNA-qPCR in the detection of all three malaria parasites. The non-18S-rRNA-qPCR method had higher kappa > 0.65,in all the three species compared to 18S-rRNA-qPCR method (kappa < 0.55).There was a clear positive correlation between microscopy parasite density and the parasite densities estimated by the 18S-rRNA-qPCR and Non-18S-rRNA-qPCR (P<0.001). Conclusion: The results showed that both monoplex realtime PCR methods demonstrated a high performance compared to microscopy proving to be better methods in the identification and speciation of malaria parasites especially of low parasitemia.The realtime PCR methods also had a positive correlation with parasite density and hence can be used in accurate determination of parasite densities when compared to microscopy. Therefore, this study recommends the utilization of realtime PCR methods in the detection,speciation and quantification of both microscopic and submicroscopic malaria parasites.
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Herdina, Anna Nele, Franz Ratzinger, Monika Breuer, Julia Schellnegger, Rui Qiang Chen, Thomas Watkins-Riedel, Nicole Perkmann-Nagele, and Robert Strassl. "Performance Evaluation of the Fully Automated NeuMoDx RT-PCR Platform for the Quantification of CMV and EBV DNA in EDTA Plasma: Implications for Clinical Management and Establishment of a Conversion Formula." Microbiology Spectrum, November 7, 2022. http://dx.doi.org/10.1128/spectrum.02157-22.
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Clinical management of solid organ transplant (SOT) patients requires the careful monitoring of immunosuppression and viral infection or reactivation. qPCR is the gold standard for the detection and quantification of very small amounts of viral DNA and allows for an early assessment of viral load kinetics. The tested NeuMoDx 96 platform provides faster results than the previously used RT-PCR workflows for CMV (Abbott m2000 and RealTime CMV assay) and EBV (LightCycler 480 II, Roche high pure extraction, and Altona RealStar EBV assay) DNA detection.
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Micalessi, Isabel M., Gaëlle A. V. Boulet, Johannes J. Bogers, Ina H. Benoy, and Christophe E. Depuydt. "High-throughput detection, genotyping and quantification of the human papillomavirus using real-time PCR." Clinical Chemistry and Laboratory Medicine 50, no. 4 (January 1, 2012). http://dx.doi.org/10.1515/cclm.2011.835.
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AbstractThe establishment of the causal relationship between high-risk human papillomavirus (HR-HPV) infection and cervical cancer and its precursors has resulted in the development of HPV DNA detection systems. Currently, real-time PCR assays for the detection of HPV, such as the RealTime High Risk (HR) HPV assay (Abbott) and the cobas® 4800 HPV Test (Roche Molecular Diagnostics) are commercially available. However, none of them enables the detection and typing of all HR-HPV types in a clinical high-throughput setting. This paper describes the laboratory workflow and the validation of a type-specific real-time quantitative PCR (qPCR) assay for high-throughput HPV detection, genotyping and quantification. This assay is routinely applied in a liquid-based cytology screening setting (700 samples in 24 h) and was used in many epidemiological and clinical studies.The TaqMan-based qPCR assay enables the detection of 17 HPV genotypes and β-globin in seven multiplex reactions. These HPV types include all 12 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), three probably high-risk types (HPV53, 66 and 68), one low-risk type (HPV6) and one undetermined risk type (HPV67).An analytical sensitivity of ≤100 copies was obtained for all the HPV types. The analytical specificity of each primer pair was 100% and an intra- and inter-run variability of <6.4% was observed.The type-specific real-time PCR approach enables detection of 17 HPV types, identification of the HPV type and determination of the viral load in a single sensitive assay suitable for high-throughput screening.
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Zhang, Guoli, Jian Li, Hongtao Wei, Yuhuan Yue, Guangmou Wu, Yuan Tian, Yuling Liu, et al. "Inhibitory Effect of GnRH and Its Analog on Tongue Cancer Cells Might Be Related to Downregulation of GnRHR mRNA." Case Reports In Cancer, August 27, 2020, 1–5. http://dx.doi.org/10.31487/nl.crc.2020.01.02.
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Aim: We investigated the expression of type-I gonadotropin-releasing hormone receptor (GnRHR-I) in human tongue cancer SCC-25 cells and the effects of gonadotropin-releasing hormone (GnRH)-I and its analog triptorelin on SCC-25 cells and the related mechanisms of action. Methods: We used RT-PCR, realtime PCR (qPCR) and western blotting to detect the expression of GnRHR-I mRNA and protein in SCC-25 cells, We also used a Cell Counting Kit-8 (CCK-8) assay to calculate cell viability, after treatment with GnRH-I or triptorelin. Results: Results of agarose gel electrophoresis and DNA sequencing showed that the GnRHR-I mRNA of SCC-25 cells were amplified correctly. Western blotting showed that the protein samples of SCC-25 cells had a specific reaction at approximately 60 kDa. GnRH or triptorelin inhibited the proliferation of SCC-25 cells in a dose-dependent manner. GnRH-I and triptorelin at the same concentrations did not significantly differ in inhibiting the proliferation of SCC-25 cells (10 µg/ml: P=0.44,20 µg/ml: P=0.57, 40µg/ml: P=0.28,80 µg/ml: P=0.051). Results of qPCR showed that triptorelin treatment led to stronger inhibition of GnRHR-I mRNA expression in SCC-25 cells than did GnRH-I treatment. (P<0.05). Triptorelin also decreased GnRHR mRNA expression time-dependently. Conclusion: The SCC-25 cell line expresses GnRHR protein and mRNA. The inhibitory effect of GnRH-I and triptorelin on tongue cancer cells might be related to the downregulation of GnRHR-I mRNA.
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Zhu, Deqing, Xuan Li, Hao Gong, Jing Li, Xike Lu, Honggang Xia, Xia Chen, et al. "Effect and Mechanism of Transthyretin Over-Expression on Proliferation and Cell Cycle of Lung Cancer A549 Cells." Iranian Journal of Public Health, April 14, 2021. http://dx.doi.org/10.18502/ijph.v50i4.5995.
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Background: The effects of transthyretin (TTR) over-expression on the proliferation and cell cycle of nonsmall cell lung cancer (NSCLC) A549 cells and its possible mechanism were verified.
Methods: A total of 196 LC patients and 20 healthy controls were enrolled at Tianjin Hospital, Tianjin, China between Apr 2017 and Oct 2017. The serum TTR content was detected by ELISA. Through lentiviral transfection method, NSCLC cells were divided into non-transfected group (group A), negative control group (group B) transfected with empty vector and experimental group (group C) transfected with TTR overexpression. Cell proliferation was detected by CCK-8 method, TTR mRNA expression was detected by realtime quantitative polymerase chain reaction (RT-qPCR), and TTR protein expression was tested by Western blot (WB). Cell cycle was detected by flow cytometry, Wnt3a/β-catenin protein expression was detected by WB, and mRNA expression was detected by RT-qPCR.
Results: The serum TTR content in early, middle and late LC group was remarkably lower than that in healthy group (P<0.05). Compared with late stage, TTR content in early and middle stages of LC group was higher, and the difference was statistically marked (P < 0.05). The absorbance value of group C was lower than that of groups A and B, indicating that the cell proliferation activity dramatically decreased, with statistically marked difference (P<0.05). LC A549 cells in group C were obviously blocked in G2M, with statistical significance (P<0.05).
Conclusion: TTR over-expression can inhibit the proliferation of NSCLC A549 cells, and the expression is related to Wnt3a/β-catenin pathway. TTR in serum of patients was helpful for diagnosing LC and has certain clinical value.
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Xie, Yushuai, Shuangshuang Gao, Yiwen Cao, Yuexin Ji, Qihuan Zhang, Youchuan Wei, and Zhitao Qi. "Molecular characterization and functional analysis of DIGIRR from golden pompano (Trachinotus ovatus)." Frontiers in Immunology 13 (August 24, 2022). http://dx.doi.org/10.3389/fimmu.2022.974310.
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Mammalian single immunoglobulin (Ig) interleukin-1 receptor related molecule (SIGIRR), an important member of the Toll/interleukin-1 receptor (TIR) family, plays important balancing roles in the inflammatory responses. In the present study, the double Ig interleukin-1 receptor related molecule (DIGIRR), the homologous of SIGIRR, was characterized in golden pompano (Trachinotus ovatus) (termed as trDIGIRR). The full-length cDNA of trDIGIRR was 2,167 bp with an open reading frame (ORF) of 1,572 bp encoding 523 amino acids. The trDIGIRR contained several conserved domains including a signal peptide, two Ig domains, a transmembrane domain and a TIR domain, and shared high sequence identities with its teleost counterparts. Realtime qPCR analysis revealed that the trDIGIRR was distributed in all tissues examined, with high expressions in intestine, liver and head kidney. The expressions of trDIGIRR were induced by Vibrio alginolyticus, lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C) challenge. Further analysis revealed that trDIGIRR was mainly located in the cytoplasm. In addition, the co-immunoprecipitation (co-IP) assay identified that trDIGIRR could interact with myeloid differentiation factor 88 (MyD88), but not interact with TIR domain containing adaptor protein inducing interferon-β (TRIF). Our results provide basis for studying the immune role of fish DIGIRR.
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H, Gou, Yu R, Luo J, Liu L, Sun X, and Xu C. "Circular RNA Circrna_0072387 Promotes Hepatoblastoma Progression by Regulating Hsa-Mir-490/Histone Deacetylase 2 Axis." Thrombosis & Haemostasis: Research 6, no. 1 (March 25, 2022). http://dx.doi.org/10.26420/thrombhaemostres.2022.1074.
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Circular RNAs have been reported to be associated with the occurrence and progress of various cancers. However, its biological roles in hepatoblastoma (HB) remain undefined. This study intended to explain a possible mechanism of hsa_circ_0072387 (circ72387) in hepatoblastoma progression. The expression of circ72387, hsa-miR-490 (miR490) and histone deacetylase 2 (HDAC2) in HB tissues and cell lines (HepG2/Huh6) were determined by quantitative realtime polymerase chain reaction (qPCR) or Western blot. Cell Counting Kit-8 (CCK-8) and colony formation assay were used to detect the proliferation of HB cell lines. The interaction between miR490 with circ72387 was predicted by CircInteractome and was verified by Dual-Luciferase reporter assay while the interaction between miR490 with HDAC2 was predicted by Targetscan and was verified by western blotting assay. Results showed that circ72387 was upregulated in both HB tissues and cells, and miR490 was downregulated in HB cells. Furthermore, the silence of circ72387 suppressed the proliferation of hepatoblastoma cells. Circ72387 promoted the proliferation of HB cells through competitively binding to HDAC2 against miR490 acting as a Sponge of miR490 in HB. Circ72387 played an essential role in HB development through the circ72387-miR490-HDAC2 regulatory axis, which may pave the way for potential diagnostic markers and therapeutic targets research in HB.
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Yadav, Rajeev Kumar, Madhavi Reddy Kambham, Saravan Kumar Parepally, Meenal Vyas, Krishna Reddy Manem, and Pagadala Damodaram Kamala Jayanthi. "Encounter With a Selfish Virus Sabotages Its Vector to Orient Toward Requisite Host Plant: A Case Study With Chili Leaf Curl Virus-Whitefly." Frontiers in Ecology and Evolution 10 (April 6, 2022). http://dx.doi.org/10.3389/fevo.2022.819023.
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Interactions of a virus with its vector and host plant have challenged entomologists, pathologists and biologists alike. Phytophagous insects depend on specific host volatile cues to locate suitable host plants for feeding and oviposition. Several studies have revealed that plant viruses modify their insect vector’s orientation toward specific host plants to facilitate their spread and survival. The ecological and molecular basis of this vector behavior modification remains largely unknown and was therefore explored in this study. Interestingly, host volatile preference for non-viruliferous female whiteflies [Bemisia tabaci (Genn.)] was found to be preferentially oriented toward infected chili plant [with chili leaf curl (ChLCV)] volatiles, while viruliferous whiteflies preferred healthy chili plant (Capsicum annum L.) volatiles in olfactometer. The electrophysiological studies involving electroantennogram (EAG) assays exhibited similar trend in EAG response amplitudes. Gas Chromatography linked electroantennodetection (GC EAD) revealed specific plant volatile cues responsible for altered host orientation behavior of the vector. Transcriptome profiling of the viruliferous and non-viruliferous whiteflies and Realtime qPCR validation showed differential expression of certain odorant binding proteins (OBPs) in viruliferous whiteflies. Our results suggest that there is a plant virus mediated altered chemoecological behavior in the vector with respect to orientation toward its host plant. Based on the findings we speculate that the virus mediates such change in the vector for a continued transmission success to the host.
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Jain, Sumit, Claudia Rodrigues, Huijun Yuan, Jing Liu, Keith A. Webster, and Nanette H. Bishopric. "Abstract 3385: Cardiac Stem Cell Survival During DNA Damage Is Regulated By Modulation Of Histone Acetyl Transferase p300." Circulation 118, suppl_18 (October 28, 2008). http://dx.doi.org/10.1161/circ.118.suppl_18.s_416-a.
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Background: Many sources of oxidative stress, including ischemia/reperfusion and chemotherapy, can induce DNA damage in the heart, leading to loss of cardiomyocytes and development of heart failure. Cardiac stem cells (CSCs) may serve to regenerate injured myocardium. However, ischemic injury has been associated with senescence and apoptosis in CSCs, potentially limiting their contribution to tissue repair. The histone acetyltransferase p300 has been shown to be an important regulator of genome stability and is involved in cell survival in response to DNA damage. We hypothesized that p300 could play a role in CSC response to oxidant stress. Methods: c-kit+, sca-1+ CSCs were isolated and cloned by serial dilution from adult mouse hearts. CSC clones were stably transfected with lentivirus vectors carrying shRNAmir against p300 or a non-silencing RNA (NS) and maintained under puromycin selection prior to treatment. CSCs were exposed to doxorubicin (DOX), a DNA-damaging agent, for defined intervals. Levels of p300 were monitored by Western analysis and expression of DNA damage-regulated genes was assessed using multiplex realtime QPCR. CSCs were treated with 1 uM DOX and survival was assessed over a period of 72h. Results: Levels of p300 increased rapidly in CSCs within 4h after exposure to doxorubicin and were sustained for at least 24h. CSCs expressing p300 shRNAmir (p300 KD) contained ~20% of the amount of p300 in CSCs transfected with the NS sequence (NS CSCs). QPCR analysis of DNA repair and cell cycle control genes indicated that 44 were significantly expressed in CSCs; 13 of these were up-regulated by p300 loss, and 5 were down-regulated, indicative of a DNA damage response to p300 loss. Both cell proliferation rates and progression through the cell cycle were slower in p300 KD vs. NS CSCs. Surprisingly, following 48h treatment with DOX, p300 KD CSCs had 32% improved survival vs. NS CSCs. Conclusions: Under acute genotoxic stress, p300 accumulates in CSCs. Reducing p300 levels induces the expression of genes involved in the DNA damage response, probably as a compensatory response, and slows CSC proliferation. Loss or inhibition of p300 is likely to cause multifactorial growth arrest in CSCs and thereby reduce their vulnerability to genotoxic agents. This research has received full or partial funding support from the American Heart Association, AHA National Center.
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Jiang, Ziwei, Lixia Pei, Ying Xie, Qun Ye, Xiaoqiang Liang, Yiyi Ye, and Sheng Liu. "Ruyiping formula inhibits metastasis via the microRNA-134-SLUG axis in breast cancer." BMC Complementary Medicine and Therapies 21, no. 1 (July 5, 2021). http://dx.doi.org/10.1186/s12906-021-03365-4.
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Abstract Background Metastasis is the leading cause of death among breast cancer patients. MicroRNA-134 has been reported to have a tumor-suppressive role in breast cancer. Ruyiping (RYP), a traditional Chinese formula, has been shown with the ability to reduce breast cancer metastasis in pre-clinical studies. This present study was designed to examine whether miR-134 was involved in RYP-inhibited breast cancer metastasis. Methods The expression of SLUG, E-Cadherin, N-Cadherin and miR-134 in MDA-MB-231 and 4 T1 cells treated with RYP or vehicle control were determined by quantitative realtime-PCR and western blot. Invasiveness determined by transwell assay as well as SLUG gene expression determined by qPCR were detected in cells transfected with chemically synthesized miR-134 mimics or inhibitors. BALB/c mice were injected with 4 T1 cells orthotopically and fed with RYP through gavage. Breast tumor growth, metastasis and tumor expression of EMT markers were detected. Results Compared with the control, Ruyiping formula significantly inhibited SLUG-regulated breast cancer cells invasion. MiR-134 was induced by RYP in vitro and in vivo and was able to suppress SLUG by targeting its 3’UTR. RYP suppressed SLUG expression and cell invasion through miR-134. In 4 T1 tumor-bearing mice, RYP significantly inhibited 4 T1 tumor growth and lung metastasis, increased the levels of miR-134 and epithelial marker while decreased the levels of SLUG and mesenchymal marker. Conclusion Our data uncovered that Ruyiping formula exerts an anti-metastatic activity against breast cancer cells by regulating SLUG through miR-134. MiR-134-SLUG axis might be a promising strategy in breast cancer therapy.
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Zijie, Wang, Jiang Anan, Xiao Hongmei, Yuan Xiaofan, Zhang Shaoru, and Qin Xinyue. "Exploring the potential mechanism of Fritiliariae Irrhosae Bulbus on ischemic stroke based on network pharmacology and experimental validation." Frontiers in Pharmacology 13 (November 17, 2022). http://dx.doi.org/10.3389/fphar.2022.1049586.
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Objective: To study the potential targets and molecular mechanisms of Fritiliariae Irrhosae Bulbus (FIB) in the treatment of ischemic strokes based on a network pharmacology strategy, with a combination of molecular docking and animal experiments.Methods: The active components and targets of FIB were screened by TCMSP database and TCMIP database, and the related targets of ischemic strokes were screened by GeneCards, OMIM, CTD, and DrugBank, then the intersection targets of the two were taken. The protein interaction network was constructed by STRING, the PPI network diagram was drawn by using Cytoscape software, and the key targets of FIB treatment of ischemic strokes were analyzed by MCODE. The DAVID database was used for GO and KEGG enrichment analysis, and the potential pathway of FIB against ischemic strokes was obtained. Molecular docking was performed by using AutoDock Tools 1.5.6 software. Finally, a mouse model of ischemic stroke was established, and the results of network pharmacology were verified by in vivo experiments. Realtime Polymerase Chain Reaction was used to detect the expression levels of relevant mRNAs in the mouse brain tissue. Western blot was used to detect the expression levels of related proteins in the mouse brain tissue.Results: 13 kinds of active components of FIB were screened, 31 targets were found in the intersection of FIB and ischemic strokes, 10 key targets were obtained by MCODE analysis, 236 biological processes were involved in GO enrichment analysis, and key targets of KEGG enrichment analysis were mainly concentrated in Neuroactive light receptor interaction, Calcium signaling pathway, Cholinergic synapse, Hepatitis B, Apoptosis—multiple specifications, Pathways in cancer and other significantly related pathways. There was good binding activity between the screened main active components and target proteins when molecular docking was performed. Animal experiments showed that the infarct volume of brain tissue in the FIB treatment group was considerably reduced. RT-qPCR and the results of Western Blot showed that FIB could inhibit the expression of active-Caspase3, HSP90AA1, phosphorylated C-JUN, and COX2.Conclusion: Based on network pharmacology, the effect of FIB in the treatment of ischemic strokes was discussed through the multi-component-multi-target-multi-pathway. The therapeutic effect and potential mechanisms of FIB on ischemic strokes were preliminarily explored, which provided a ground work for further researches on the pharmacodynamic material basis, mechanism of action and clinical application.
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Demine, Stéphane, Andrea Alex Schiavo, Sandra Marín-Cañas, Piero Marchetti, Miriam Cnop, and Decio L. Eizirik. "Pro-inflammatory cytokines induce cell death, inflammatory responses, and endoplasmic reticulum stress in human iPSC-derived beta cells." Stem Cell Research & Therapy 11, no. 1 (January 3, 2020). http://dx.doi.org/10.1186/s13287-019-1523-3.
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Abstract Background Adult human pancreatic beta cells are the “gold standard” for studies on diabetes pathogenesis, but their use is limited by insufficient availability and variable quality. An important effort has recently taken place to differentiate beta cells from human induced pluripotent stem cells (iPSCs) and validate their use for diabetes research. We presently used a 7-stage protocol to generate beta cells from human iPSC and evaluated whether these cells are responsive to the pro-inflammatory cytokines (IFNγ, IL-1β, or IFNα) that play a role in type 1 diabetes. Methods The iPSC-derived islet-like cell clusters contained 40–50% beta and 10–15% alpha cells and expressed the receptors for IFNγ, IL-1β, or IFNα. Cells were exposed to either IFNγ (1000 U/mL) + IL-1β (50 U/mL) or IFNα alone (2000 U/mL) for 24/48 h. Apoptosis was quantified using Hoechst/propidium iodide staining or the RealTime Glo Apoptosis Kit (Promega). After treatment, CXCL10 secretion was quantified by ELISA. The expression of multiples genes (Ins, Gcg, Nkx2.2, Nkx6.1, Pdx1, Mafa, BiP, Chop, Atf3, CXCL10, CXCL9, CCL5, and HLA-ABC) was quantified by RT-qPCR. Phosphorylation state and total expression of STAT1/STAT2, as well as expression of PDL1 and of the ER chaperone BiP, were quantified by Western blotting. The co-localization of HLA-ABC or cleaved caspase-3 and Ins/Gcg expression was assessed by immunohistochemistry. The presence of HLA-ABC at the plasma membrane was measured by flow cytometry. Results IFNγ + IL-1β and IFNα induced apoptosis of the cells after 48 h of exposure. Cleaved caspase-3 co-localized mostly but not exclusively with Ins+ cells. Exposure to IFNγ + IL-1β induced a pro-inflammatory phenotype, including increased CXCL10, CXCL9, and CCL5 expression; CXCL10 secretion; and HLA-ABC expression. HLA overexpression was confirmed at the protein level by Western blotting and flow cytometry. Exposure to IFNγ + IL-1β (but not IFNα) also induced beta cell dedifferentiation and endoplasmic reticulum stress (increase in BiP, Chop, and Atf3 mRNA expression). Phosphorylation of STAT1 was stimulated already after 1 h by IFNγ + IL-1β and IFNα, while phosphorylation of STAT2 was only activated by IFNα at 1–4 h. PDL1 expression was increased by both IFNγ + IL-1β and IFNα. Conclusions Our data show that human iPSC-derived beta cells respond to pro-inflammatory cytokines IL-1β + IFNγ and IFNα, by activating the same pathogenic processes as adult human primary beta cells. These cells thus represent a valuable tool for future research on the pathogenesis of type 1 diabetes.