Podcasts about methods expression

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Best podcasts about methods expression

Latest podcast episodes about methods expression

PaperPlayer biorxiv neuroscience
Bidirectional transcription at the PPP2R2B gene locus in spinocerebellar ataxia type 12

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Apr 3, 2023


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.02.535298v1?rss=1 Authors: Zhou, C., Liu, H. B., Bakhsh, F. J., Wu, B., Ying, M., Margolis, R. L., Li, P. P. Abstract: OBJECTIVE: Spinocerebellar ataxia type 12 (SCA12) is a neurodegenerative disease caused by expansion of a CAG repeat in the PPP2R2B gene. Here we tested the hypothesis that the PPP2R2B antisense (PPP2R2B-AS1) transcript containing a CUG repeat is expressed and contributes to SCA12 pathogenesis. METHODS: Expression of PPP2R2B-AS1 transcript was detected in SCA12 human induced pluripotent stem cells (iPSCs), iPSC-derived NGN2 neurons, and SCA12 knock-in mouse brains using strand-specific RT-PCR (SS-RT-PCR). The tendency of expanded PPP2R2B-AS1 (expPPP2R2B-AS1) RNA to form foci, a marker of toxic processes involving mutant RNAs, was examined in SCA12 cell models by fluorescence in situ hybridization. The toxic effect of expPPP2R2B-AS1 transcripts on SK-N-MC neuroblastoma cells was evaluated by caspase 3/7 activity. Western blot was used to examine the expression of repeat associated non-ATG-initiated (RAN) translation of expPPP2R2B-AS1 transcript in SK-N-MC cells. RESULTS: The repeat region in PPP2R2B gene locus is bidirectionally transcribed in SCA12 iPSCs, iPSC-derived NGN2 neurons, and SCA12 mouse brains. Transfected expPPP2R2B-AS1 transcripts are toxic to SK-N-MC cells, and the toxicity may be mediated, at least in part, by the RNA secondary structure. The expPPP2R2B-AS1 transcripts form CUG RNA foci in SK-N-MC cells. expPPP2R2B-AS1 transcript is translated in the Alanine ORF via repeat-associated non-ATG (RAN) translation, which is diminished by single nucleotide interruptions within the CUG repeat, and MBNL1 overexpression. INTERPRETATION: These findings suggest that PPP2R2B-AS1 contributes to SCA12 pathogenesis, and may therefore provide a novel therapeutic target for the disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Medizin - Open Access LMU - Teil 15/22
The role of the novel Th17 cytokine IL-26 in intestinal inflammation

Medizin - Open Access LMU - Teil 15/22

Play Episode Listen Later Jan 1, 2009


Background and aims: Interleukin 26 (IL-26), a novel IL-10-like cytokine without a murine homologue, is expressed in T helper 1 (Th1) and Th17 cells. Currently, its function in human disease is completely unknown. The aim of this study was to analyse its role in intestinal inflammation.Methods: Expression studies were performed by reverse transcription-PCR (RT-PCR), quantitative PCR, western blot and immunohistochemistry. Signal transduction was analysed by western blot experiments and ELISA. Cell proliferation was measured by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. IL-26 serum levels were determined by an immunoluminometric assay (ILMA).Results: All examined intestinal epithelial cell (IEC) lines express both IL-26 receptor subunits IL-20R1 and IL-10R2. IL-26 activates extracellular signal-related kinase (ERK)-1/2 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) mitogen-activated protein (MAP) kinases, Akt and signal transducers and activators of transcription (STAT) 1/3. IL-26 stimulation increases the mRNA expression of proinflammatory cytokines but decreases cell proliferation. In inflamed colonic lesions of patients with Crohn's disease, an elevated IL-26 mRNA expression was found that correlated highly with the IL-8 and IL-22 expression. Immunohistochemical analysis demonstrated IL-26 protein expression in colonic T cells including Th17 cells expressing the orphan nuclear receptor RORtextgreekgt, with an increased number of colonic IL-26-expressing cells in active Crohn's disease.Conclusion: Intestinal cells express the functional IL-26 receptor complex. IL-26 modulates IEC proliferation and proinflammatory gene expression and its expression is upregulated in active Crohn's disease, indicating a role for this cytokine system in the innate host cell response during intestinal inflammation. For the first time, IL-26 expression is demonstrated in colonic RORtextgreekgt-expressing Th17 cells in situ, supporting a role for this cell type in the pathogenesis of Crohn's disease.

Medizin - Open Access LMU - Teil 16/22
Effect of dsRNA on Mesangial Cell Synthesis of Plasminogen Activator Inhibitor Type 1 and Tissue Plasminogen Activator

Medizin - Open Access LMU - Teil 16/22

Play Episode Listen Later Jan 1, 2009


Background/Aims: Viral infections are a major problem worldwide and many of them are complicated by virally induced glomerulonephritides. Progression of kidney disease to renal failure is mainly attributed to the development of renal fibrosis characterized by the accumulation of extracellular matrix components in the mesangial cell compartment and the glomerular basement membrane. Plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (t-PA) are major regulators of plasmin generation and play an important role in generation and degradation of glomerular extracellular matrix components. Viral receptors expressed by mesangial cells (MC) are known to be key mediators in immune-mediated glomerulonephritis. We investigated the effect of stimulation of the viral receptors toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I) on the expression of PAI-1 and t-PA. Methods: Expression of PAI-1 and t-PA in immortalized human MC stimulated with polyriboinosinic: polyribocytidylic acid {[}poly(I:C)] RNA and cytokines were analyzed by real-time RT-PCR and ELISA. Results: Incubation of MC with poly(I:C) RNA to activate the viral receptors TLR3 and RIG-I upregulates the expression of PAI-1 and t-PA. Knockdown of viral receptors with specific siRNA abolishes the induction of PAI-1 and t-PA. Conclusion: For the first time a link between the activation of viral receptors on MC and potentially causative agents in the development of glomerulosclerosis and tubulointerstitial fibrosis is shown. The progression of inflammatory processes to glomerulosclerosis can be postulated to be directly enhanced by viral infection. Copyright (C) 2009 S. Karger AG, Basel

Medizin - Open Access LMU - Teil 14/22
Interleukin 31 mediates MAP kinase and STAT1/3 activation in intestinal epithelial cells and its expression is upregulated in inflammatory bowel disease

Medizin - Open Access LMU - Teil 14/22

Play Episode Listen Later Jan 1, 2007


Background/aim: Interleukin 31 (IL31), primarily expressed in activated lymphocytes, signals through a heterodimeric receptor complex consisting of the IL31 receptor alpha (IL31Rtextgreeka) and the oncostatin M receptor (OSMR). The aim of this study was to analyse IL31 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal inflammation.Methods: Expression studies were performed by RT-PCR, quantitative PCR, western blotting, and immunohistochemistry. Signal transduction was analysed by western blotting. Cell proliferation was measured by MTS assays, cell migration by restitution assays.Results: Colorectal cancer derived intestinal epithelial cell (IEC) lines express both IL31 receptor subunits, while their expression in unstimulated primary murine IEC was low. LPS and the proinflammatory cytokines TNF-textgreeka, IL1textgreekb, IFN-textgreekg, and sodium butyrate stimulation increased IL31, IL31Rtextgreeka, and OSMR mRNA expression, while IL31 itself enhanced IL8 expression in IEC. IL31 mediates ERK-1/2, Akt, STAT1, and STAT3 activation in IEC resulting in enhanced IEC migration. However, at low cell density, IL31 had significant antiproliferative capacities (p

Medizin - Open Access LMU - Teil 14/22
Helicobacter pylori outer membrane proteins and gastroduodenal disease

Medizin - Open Access LMU - Teil 14/22

Play Episode Listen Later Jan 1, 2006


Background and aims: A number of Helicobacter pylori outer membrane proteins (OMPs) undergo phase variations. This study examined the relation between OMP phase variations and clinical outcome.Methods: Expression of H pylori BabA, BabB, SabA, and OipA proteins was determined by immunoblot. Multiple regression analysis was performed to determine the relation among OMP expression, clinical outcome, and mucosal histology.Results:H pylori were cultured from 200 patients (80 with gastritis, 80 with duodenal ulcer (DU), and 40 with gastric cancer). The most reliable results were obtained using cultures from single colonies of low passage number. Stability of expression with passage varied with OipA > BabA > BabB > SabA. OipA positive status was significantly associated with the presence of DU and gastric cancer, high H pylori density, and severe neutrophil infiltration. SabA positive status was associated with gastric cancer, intestinal metaplasia, and corpus atrophy, and negatively associated with DU and neutrophil infiltration. The Sydney system underestimated the prevalence of intestinal metaplasia/atrophy compared with systems using proximal and distal corpus biopsies. SabA expression dramatically decreased following exposure of H pylori to pH 5.0 for two hours.Conclusions: SabA expression frequently switched on or off, suggesting that SabA expression can rapidly respond to changing conditions in the stomach or in different regions of the stomach. SabA positive status was inversely related to the ability of the stomach to secrete acid, suggesting that its expression may be regulated by changes in acid secretion and/or in antigens expressed by the atrophic mucosa.