Understanding the dynamics of Toll-like Receptor 5 response to flagellin and its regulation by estradiol

© 2017 The Author(s). Toll-like receptors (TLRs) are major players of the innate immune system. Once activated, they trigger a signalling cascade that leads to NF-ΰ B translocation from the cytoplasm to the nucleus. Single cell analysis shows that NF-ΰ B signalling dynamics are a critical determinant of transcriptional regulation. Moreover, the outcome of innate immune response is also affected by the cross-talk between TLRs and estrogen signalling. Here, we characterized the dynamics of TLR5 signalling, responsible for the recognition of flagellated bacteria, and those changes induced by estradiol in its signalling at the single cell level. TLR5 activation in MCF7 cells induced a single and sustained NF-k B translocation into the nucleus that resulted in high NF-k B transcription activity. The overall magnitude of NF-k B transcription activity was not influenced by the duration of the stimulus. No significant changes are observed in the dynamics of NF-k B translocation to the nucleus when MCF7 cells are incubated with estradiol. However, estradiol significantly decreased NF-k B transcriptional activity while increasing TLR5-mediated AP-1 transcription. The effect of estradiol on transcriptional activity was dependent on the estrogen receptor activated. This fine tuning seems to occur mainly in the nucleus at the transcription level rather than affecting the translocation of the NF-k B transcription factor

Role of Receptor-Interacting Protein 140 in human fat cells

<p>Abstract</p> <p>Background</p> <p>Mice lacking <it>Receptor-interacting protein 140 (RIP140) </it>have reduced body fat which at least partly is mediated through increased lipid and glucose metabolism in adipose tissue. In humans, <it>RIP140 </it>is lower expressed in visceral white adipose tissue (WAT) of obese versus lean subjects. We investigated the role of <it>RIP140 </it>in human subcutaneous WAT, which is the major fat depot of the body.</p> <p>Methods</p> <p>Messenger RNA levels of <it>RIP140 </it>were measured in samples of subcutaneous WAT from women with a wide variation in BMI and in different human WAT preparations. <it>RIP140 </it>mRNA was knocked down with siRNA in <it>in vitro </it>differentiated adipocytes and the impact on glucose transport and mRNA levels of target genes determined.</p> <p>Results</p> <p><it>RIP140 </it>mRNA levels in subcutaneous WAT were decreased among obese compared to lean women and increased by weight-loss, but did not associate with mitochondrial DNA copy number. <it>RIP140 </it>expression increased during adipocyte differentiation <it>in vitro </it>and was higher in isolated adipocytes compared to corresponding pieces of WAT. Knock down of <it>RIP140 </it>increased basal glucose transport and mRNA levels of <it>glucose transporter 4 </it>and <it>uncoupling protein-1</it>.</p> <p>Conclusions</p> <p>Human <it>RIP140 </it>inhibits glucose uptake and the expression of genes promoting energy expenditure in the same fashion as the murine orthologue. Increased levels of human <it>RIP140 </it>in subcutaneous WAT of lean subjects may contribute to economize on energy stores. By contrast, the function and expression pattern does not support that <it>RIP140 </it>regulate human obesity.</p

Does economic development contribute to sex differences in ischaemic heart disease mortality? Hong Kong as a natural experiment using a case-control study

<p>Abstract</p> <p>Background</p> <p>The male excess risk of premature ischemic heart disease (IHD) mortality may be partially due to an unknown macro-environmental influence associated with economic development. We examined whether excess male risk of IHD mortality was higher with birth in an economically developed environment.</p> <p>Methods</p> <p>We used multivariable logistic regression in a population-based case-control study of all adult deaths in Hong Kong Chinese in 1998 to compare sex differences in IHD mortality (1,189 deaths in men, 1,035 deaths in women and 20,842 controls) between Hong Kong residents born in economically developed Hong Kong or in contemporaneously undeveloped Guangdong province in China.</p> <p>Results</p> <p>Younger (35–64 years) native-born Hong Kong men had a higher risk of IHD death than such women (odds ratio 2.91, 95% confidence interval 1.66 to 5.13), adjusted for age, socio-economic status and lifestyle. There was no such sex difference in Hong Kong residents who had migrated from Guangdong. There were no sex differences in pneumonia deaths by birth place.</p> <p>Conclusion</p> <p>Most of these people migrated as young adults; we speculate that environmentally mediated differences in pubertal maturation (when the male disadvantage in lipids and fat patterning emerges) may contribute to excess male premature IHD mortality in developed environments.</p

Insights on Glucocorticoid Receptor Activity Modulation through the Binding of Rigid Steroids

Background: The glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity. Methodology/Principal Findings: Here, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GRDNA interaction dynamics rather than the receptor's ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different GR-ligand complexes. No correlation was found between GR intranuclear distribution, cofactor recruitment and the homodimerization process. Finally, Molecular determinants that support the observed experimental GR LBD-ligand/TIF2 interaction were found by Molecular Dynamics simulation. Conclusions/Significance: The data presented here sustain the idea that in vivo GR homodimerization inside the nucleus can be achieved in a DNA-independent fashion, without ruling out a dependent pathway as well. Moreover, since at least one GR-ligand complex is able to induce homodimer formation while preventing TIF2 coactivator interaction, results suggest that these two events might be independent from each other. Finally, 21-hydroxy-6,19-epoxyprogesterone arises as a selective glucocorticoid with potential pharmacological interest. Taking into account that GR homodimerization and cofactor recruitment are considered essential steps in the receptor activation pathway, results presented here contribute to understand how specific ligands influence GR behavior. © 2010 Presman et al.Fil:Presman, D.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Alvarez, L.D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Levi, V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Martí, M.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Veleiro, A.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Burton, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Pecci, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.Paul & Daisy Soros Fellowships for New Americans (New York, N.Y.)McGovern Institute for Brain Research at MIT (Friends of McGovern Institute Fellowship)Massachusetts Institute of Technology. Poitras Center for Affective Disorders ResearchUnited States. Department of Energy (Computational Science Graduate Fellowship)National Institute of Mental Health (U.S.) (5DP1-MH100706)National Institute of Mental Health (U.S.) (1R01-MH110049)New York Stem Cell FoundationPoitras FoundationSimons FoundationPaul G. Allen Family FoundationVallee FoundationTom HarrimanB. Metcalf

Functional crosstalk of PGC-1 coactivators and inflammation in skeletal muscle pathophysiology

Skeletal muscle is an organ involved in whole body movement and energy metabolism with the ability to dynamically adapt to different states of (dis-)use. At a molecular level, the peroxisome proliferator-activated receptor γ coactivators 1 (PGC-1s) are important mediators of oxidative metabolism in skeletal muscle and in other organs. Musculoskeletal disorders as well as obesity and its sequelae are associated with PGC-1 dysregulation in muscle with a concomitant local or systemic inflammatory reaction. In this review, we outline the function of PGC-1 coactivators in physiological and pathological conditions as well as the complex interplay of metabolic dysregulation and inflammation in obesity with special focus on skeletal muscle. We further put forward the hypothesis that, in this tissue, oxidative metabolism and inflammatory processes mutually antagonize each other. The nuclear factor κB (NF-κB) pathway thereby plays a key role in linking metabolic and inflammatory programs in muscle cells. We conclude this review with a perspective about the consequences of such a negative crosstalk on the immune system and the possibilities this opens for clinical applications