SIRT1 regulates macrophage self-renewal.

Mature differentiated macrophages can self-maintain by local proliferation in tissues and can be extensively expanded in culture under specific conditions, but the mechanisms of this phenomenon remain only partially defined. Here, we show that SIRT1, an evolutionary conserved regulator of life span, positively affects macrophage self-renewal ability in vitro and in vivo Overexpression of SIRT1 during bone marrow-derived macrophage differentiation increased their proliferative capacity. Conversely, decrease of SIRT1 expression by shRNA inactivation, CRISPR/Cas9 mediated deletion and pharmacological inhibition restricted macrophage self-renewal in culture. Furthermore, pharmacological SIRT1 inhibition in vivo reduced steady state and cytokine-induced proliferation of alveolar and peritoneal macrophages. Mechanistically, SIRT1 inhibition negatively regulated G1/S transition, cell cycle progression and a network of self-renewal genes. This included inhibition of E2F1 and Myc and concomitant activation of FoxO1, SIRT1 targets mediating cell cycle progression and stress response, respectively. Our findings indicate that SIRT1 is a key regulator of macrophage self-renewal that integrates cell cycle and longevity pathways. This suggests that macrophage self-renewal might be a relevant parameter of ageing.

A Comprehensive Analytical Strategy To Identify Malondialdehyde-Modified Proteins and Peptides.

Mass spectrometric-based proteomics is a powerful tool to analyze post-translationally modified proteins. Carbonylation modifications that result from oxidative lipid breakdown are a class of post-translational modifications that are poorly characterized with respect to protein targets and function. This is partly due to the lack of dedicated mass spectrometry-based technologies to facilitate the analysis of these modifications. Here, we present a comprehensive approach to identify malondialdehyde-modified proteins and peptides. Malondialdehyde is among the most abundant of the lipid peroxidation products; and malondialdehyde-derived adducts on proteins have been implicated in cardiovascular diseases, neurodegenerative disorders, and other clinical conditions. Our integrated approach targets three levels of the overall proteomic workflow: (i) sample preparation, by employing a targeted enrichment strategy; (ii) high-performance liquid chromatography, by using a gradient optimized for the separation of the modified peptides; and (iii) tandem mass spectrometry, by improving the spectral quality of very low-abundance peptides. By applying the optimized procedure to a whole cell lysate spiked with a low amount of malondialdehyde-modified proteins, we were able to identify up to 350 different modified peptides and localize the modification to a specific lysine residue. This methodology allows the comprehensive analysis of malondialdehyde-modified proteins.

Malondialdehyde epitopes as mediators of sterile inflammation.

Enhanced lipid peroxidation occurs during oxidative stress and results in the generation of lipid peroxidation end products such as malondialdehyde (MDA), which can attach to autologous biomolecules, thereby generating neo-self epitopes capable of inducing potentially undesired biological responses. Therefore, the immune system has developed mechanisms to protect from MDA epitopes by binding and neutralizing them through both cellular and soluble effectors. Here, we briefly discuss innate immune responses targeting MDA epitopes and their pro-inflammatory properties, followed by a review of physiological carriers of MDA epitopes that are relevant in homeostasis and disease. Then we discuss in detail the evidence for cellular responses towards MDA epitopes mainly in lung, liver and the circulation as well as signal transduction mechanisms and receptors implicated in the response to MDA epitopes. Last, we hypothesize on the role of MDA epitopes as mediators of inflammation in diseases and speculate on their contribution to disease pathogenesis. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.

Evaluation of a rapid dipstick (Crystal VC) for the diagnosis of cholera in Zanzibar and a comparison with previous studies.

BACKGROUND: The gold standard for the diagnosis of cholera is stool culture, but this requires laboratory facilities and takes at least 24 hours. A rapid diagnostic test (RDT) that can be used by minimally trained staff at treatment centers could potentially improve the reporting and management of cholera outbreaks. METHODS: We evaluated the Crystal VC™ RDT under field conditions in Zanzibar in 2009. Patients presenting to treatment centers with watery diarrhea provided a stool sample for rapid diagnostic testing. Results were compared to stool culture performed in a reference laboratory. We assessed the overall performance of the RDT and evaluated whether previous intake of antibiotics, intravenous fluids, location of testing, and skill level of the technician affected the RDT results. RESULTS: We included stool samples from 624 patients. Compared to culture, the overall sensitivity of the RDT was 93.1% (95%CI: 88.7 to 96.2%), specificity was 49.2% (95%CI: 44.3 to 54.1%), the positive predictive value was 47.0% (95%CI: 42.1 to 52.0%) and the negative predictive value was 93.6% (95%CI: 89.6 to 96.5%). The overall false positivity rate was 50.8% (213/419); fieldworkers frequently misread very faint test lines as positive. CONCLUSION: The observed sensitivity of the Crystal VC RDT evaluated was similar compared to earlier versions, while specificity was poorer. The current version of the RDT could potentially be used as a screening tool in the field. Because of the high proportion of false positive results when field workers test stool specimens, positive results will need to be confirmed with stool culture.

A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis.

The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. Here we identify specific miRNAs that function at synapses to control dendritic spine structure by performing a functional screen. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the alpha(13) subunits of G proteins (Galpha(13)). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Galpha(13) both suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Galpha(13) might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis.