75 research outputs found
Role of Polypyrimidine Tract Binding Protein in Mediating Internal Initiation of Translation of Interferon Regulatory Factor 2 RNA
BACKGROUND: Earlier we have reported translational control of interferon regulatory factor 2 (IRF2) by internal initiation (Dhar et al, Nucleic Acids Res, 2007). The results implied possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general. Here we have investigated the secondary structure of the Internal Ribosome Entry Site of IRF2 RNA and demonstrated the role of PTB protein in ribosome assembly to facilitate internal initiation. METHODOLOGY/PRINCIPAL FINDINGS: We have probed the putative secondary structure of the IRF2 5'UTR RNA using various enzymatic and chemical modification agents to constrain the secondary structure predicted from RNA folding algorithm Mfold. The IRES activity was found to be influenced by the interaction of trans-acting factor, polypyrimidine tract binding protein (PTB). Deletion of 25 nts from the 3'terminus of the 5'untranslated region resulted in reduced binding with PTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putative contact points of PTB on the IRF2-5'UTR using primer extension inhibition assay. Majority of the PTB toe-prints were found to be restricted to the 3'end of the IRES. Additionally, Circular Dichroism (CD) spectra analysis suggested change in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silenced for PTB gene expression, resulted in reduced binding by other trans-acting factors. Finally, we have demonstrated that addition of recombinant PTB enhances ribosome assembly on IRF2 IRES suggesting possible role of PTB in mediating internal initiation of translation of IRF2 RNA. CONCLUSION/SIGNIFICANCE: It appears that PTB binding to multiple sites within IRF2 5'UTR leads to a conformational change in the RNA that facilitate binding of other trans-acting factors to mediate internal initiation of translation
Effects of PPARÎł ligands on TGF-ÎČ1-induced epithelial-mesenchymal transition in alveolar epithelial cells
BACKGROUND: Transforming growth factor beta1 (TGF-beta1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPAR gamma ligands have been shown to inhibit fibroblast activation by TGF-beta1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-beta1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1 alpha 1 (COL1A1), CTGF and MMP-2 mRNA. METHODS: Serum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 microM) in the absence or presence of the PPAR gamma antagonist GW9662 (10 microM) before TGFbeta-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR. RESULTS: TGFbeta-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGF beta 1-induced changes in cell morphology, and PPAR gamma-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-beta1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-beta1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-beta1 was not inhibited by RGZ or CGZ. CONCLUSIONS: RGZ and CGZ inhibited profibrotic changes in TGF-beta1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPAR gamma-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-beta1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis
Characterisation of Clostridium difficile Hospital WardâBased Transmission Using Extensive Epidemiological Data and Molecular Typing
A population-based study in Oxfordshire (UK) hospitals by Sarah Walker and colleagues finds that in an endemic setting with good infection control, ward-based contact cannot account for most new cases of Clostridium difficile infection
Host Genes Related to Paneth Cells and Xenobiotic Metabolism Are Associated with Shifts in Human Ileum-Associated Microbial Composition
The aim of this study was to integrate human clinical, genotype, mRNA microarray and 16 S rRNA sequence data collected on 84 subjects with ileal Crohnâs disease, ulcerative colitis or control patients without inflammatory bowel diseases in order to interrogate how host-microbial interactions are perturbed in inflammatory bowel diseases (IBD). Ex-vivo ileal mucosal biopsies were collected from the disease unaffected proximal margin of the ileum resected from patients who were undergoing initial intestinal surgery. Both RNA and DNA were extracted from the mucosal biopsy samples. Patients were genotyped for the three major NOD2 variants (Leufs1007, R702W, and G908R) and the ATG16L1T300A variant. Whole human genome mRNA expression profiles were generated using Agilent microarrays. Microbial composition profiles were determined by 454 pyrosequencing of the V3âV5 hypervariable region of the bacterial 16 S rRNA gene. The results of permutation based multivariate analysis of variance and covariance (MANCOVA) support the hypothesis that host mucosal Paneth cell and xenobiotic metabolism genes play an important role in host microbial interactions
Genome Wide Expression Profiling Reveals Suppression of Host Defence Responses during Colonisation by Neisseria meningitides but not N. lactamica
Both Neisseria meningitidis and the closely related bacterium Neisseria lactamica colonise human nasopharyngeal mucosal surface, but only N. meningitidis invades the bloodstream to cause potentially life-threatening meningitis and septicaemia. We have hypothesised that the two neisserial species differentially modulate host respiratory epithelial cell gene expression reflecting their disease potential. Confluent monolayers of 16HBE14 human bronchial epithelial cells were exposed to live and/or dead N. meningitidis (including capsule and pili mutants) and N. lactamica, and their transcriptomes were compared using whole genome microarrays. Changes in expression of selected genes were subsequently validated using Q-RT-PCR and ELISAs. Live N. meningitidis and N. lactamica induced genes involved in host energy production processes suggesting that both bacterial species utilise host resources. N. meningitidis infection was associated with down-regulation of host defence genes. N. lactamica, relative to N. meningitidis, initiates up-regulation of proinflammatory genes. Bacterial secreted proteins alone induced some of the changes observed. The results suggest N. meningitidis and N. lactamica differentially regulate host respiratory epithelial cell gene expression through colonisation and/or protein secretion, and that this may contribute to subsequent clinical outcomes associated with these bacteria
Uncoordinated Transcription and Compromised Muscle Function in the Lmna-Null Mouse Model of Emery-Dreifuss Muscular Dystrophy
LMNA encodes both lamin A and C: major components of the nuclear lamina. Mutations in LMNA underlie a range of tissue-specific degenerative diseases, including those that affect skeletal muscle, such as autosomal-Emery-Dreifuss muscular dystrophy (A-EDMD) and limb girdle muscular dystrophy 1B. Here, we examine the morphology and transcriptional activity of myonuclei, the structure of the myotendinous junction and the muscle contraction dynamics in the lmna-null mouse model of A-EDMD. We found that there were fewer myonuclei in lmna-null mice, of which âŒ50% had morphological abnormalities. Assaying transcriptional activity by examining acetylated histone H3 and PABPN1 levels indicated that there was a lack of coordinated transcription between myonuclei lacking lamin A/C. Myonuclei with abnormal morphology and transcriptional activity were distributed along the length of the myofibre, but accumulated at the myotendinous junction. Indeed, in addition to the presence of abnormal myonuclei, the structure of the myotendinous junction was perturbed, with disorganised sarcomeres and reduced interdigitation with the tendon, together with lipid and collagen deposition. Functionally, muscle contraction became severely affected within weeks of birth, with specific force generation dropping as low as âŒ65% and âŒ27% of control values in the extensor digitorum longus and soleus muscles respectively. These observations illustrate the importance of lamin A/C for correct myonuclear function, which likely acts synergistically with myotendinous junction disorganisation in the development of A-EDMD, and the consequential reduction in force generation and muscle wasting
Omega-3 fatty acids and vitamin D in immobilisation: Part B- modulation of muscle functional, vascular and activation profiles
Abstract Objectives: This study set out to determine whether two potential protein-sparing modulators (eicosapentaenoic acid and vitamin D) would modulate the anticipated muscle functional and related blood vessels function deleterious effects of immobilisation. Design: The study used a randomised, double-blind, placebo-controlled design. Setting: The study took part in a laboratory setting. Participants: Twenty-four male and female healthy participants, aged 23.0±5.8 years. Intervention: The non-dominant arm was immobilised in a sling for a period of nine waking hours a day over two continuous weeks. Participants were randomly assigned to one of three groups: placebo (n=8, Lecithin, 2400 mg daily), omega-3 (-3) fatty acids (n=8, eicosapentaenoic acid (EPA); 1770 mg, and docosahexaenoic acid (DHA); 390 mg DHA, daily) or vitamin D (n=8, 1,000 IU daily). Measurements: Isometric and isokinetic torque, antagonist muscle co-contraction (activation profile), muscle fatigability indices, and arterial resting blood flow were measured before, at the end of the immobilisation period, and two weeks after re-mobilisation. Results: Muscle elbow flexion and extension isometric and isokinetic torque decreased significantly with limb immobilisation in the placebo group (P0.05) towards attenuating the decreases observed in the placebo group. There was no significant change in muscle fatigue parameters or co-contraction values with immobilisation and no effect of supplementation group (P>0.05). Similarly, this immobilisation model had no impact on the assessed blood flow kinetics. All parameters had returned to baseline values at the re-mobilisation phase of the study. Conclusion: Overall, at the current doses, neither -3 nor vitamin D supplementation significantly attenuated declines in torque associated with immobilisation. It would appear that muscle function (described here in Part B) might not be as useful a marker of the effectiveness of a supplement against the impact of immobilisation compared to tissue composition changes (described in Part A)
International Consensus Statement on Rhinology and Allergy: Rhinosinusitis
Background: The 5 years since the publication of the first International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICARâRS) has witnessed foundational progress in our understanding and treatment of rhinologic disease. These advances are reflected within the more than 40 new topics covered within the ICARâRSâ2021 as well as updates to the original 140 topics. This executive summary consolidates the evidenceâbased findings of the document. Methods: ICARâRS presents over 180 topics in the forms of evidenceâbased reviews with recommendations (EBRRs), evidenceâbased reviews, and literature reviews. The highest grade structured recommendations of the EBRR sections are summarized in this executive summary. Results: ICARâRSâ2021 covers 22 topics regarding the medical management of RS, which are grade A/B and are presented in the executive summary. Additionally, 4 topics regarding the surgical management of RS are grade A/B and are presented in the executive summary. Finally, a comprehensive evidenceâbased management algorithm is provided. Conclusion: This ICARâRSâ2021 executive summary provides a compilation of the evidenceâbased recommendations for medical and surgical treatment of the most common forms of RS
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