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There is accumulating evidence to indicate that long non-coding RNAs (lncRNAs) are important regulators of the inflammatory response. In this report, we have employed next generation sequencing to identify 14 lncRNAs that are differentially expressed in human lung fibroblasts following the induction of inflammation using interleukin-1β (IL-1β). Knockdown of the two most highly expressed lncRNAs, IL7AS, and MIR3142HG, showed that IL7AS negatively regulated IL-6 release whilst MIR3142HG was a positive regulator of IL-8 and CCL2 release. Parallel studies in fibroblasts derived from patients with idiopathic pulmonary fibrosis showed similar increases in IL7AS levels, that also negatively regulate IL-6 release. In contrast, IL-1β-induced MIR3142HG expression, and its metabolism to miR-146a, was reduced by 4- and 9-fold in IPF fibroblasts, respectively. This correlated with a reduced expression of inflammatory mediators whilst MIR3142HG knockdown showed no effect upon IL-8 and CCL2 release. Pharmacological studies showed that IL-1β-induced IL7AS and MIR3142HG production and release of IL-6, IL-8, and CCL2 in both control and IPF fibroblasts were mediated via an NF-κB-mediated pathway. In summary, we have cataloged those lncRNAs that are differentially expressed following IL-1β-activation of human lung fibroblasts, shown that IL7AS and MIR3142HG regulate the inflammatory response and demonstrated that the reduced inflammatory response in IPF fibroblast is correlated with attenuated expression of MIR3142HG/miR-146a.

Phenotypic changes in lung fibroblasts are believed to contribute to the development of Idiopathic Pulmonary Fibrosis (IPF), a progressive and fatal lung disease. Long intergenic non-coding RNAs (lincRNAs) have been identified as novel regulators of gene expression and protein activity. In non-stimulated cells, we observed reduced proliferation and inflammation but no difference in the fibrotic response of IPF fibroblasts. These functional changes in non-stimulated cells were associated with changes in the expression of the histone marks, H3K4me1, H3K4me3 and H3K27ac indicating a possible involvement of epigenetics. Following activation with TGF-β1 and IL-1β, we demonstrated an increased fibrotic but reduced inflammatory response in IPF fibroblasts. There was no significant difference in proliferation following PDGF exposure. The lincRNAs, LINC00960 and LINC01140 were upregulated in IPF fibroblasts. Knockdown studies showed that LINC00960 and LINC01140 were positive regulators of proliferation in both control and IPF fibroblasts but had no effect upon the fibrotic response. Knockdown of LINC01140 but not LINC00960 increased the inflammatory response, which was greater in IPF compared to control fibroblasts. Overall, these studies demonstrate for the first time that lincRNAs are important regulators of proliferation and inflammation in human lung fibroblasts and that these might mediate the reduced inflammatory response observed in IPF-derived fibroblasts.

Despite increasing evidence to indicate that long non-coding RNAs (lncRNAs) are novel regulators of immunity, there has been no systematic attempt to identify and characterize the lncRNAs whose expression is changed following the induction of the innate immune response. To address this issue, we have employed next-generation sequencing data to determine the changes in the lncRNA profile in four human (monocytes, macrophages, epithelium, and chondrocytes) and four mouse cell types (RAW 264.7 macrophages, bone marrow-derived macrophages, peritoneal macrophages, and splenic dendritic cells) following exposure to the pro-inflammatory mediators, lipopolysaccharides (LPS), or interleukin-1β. We show differential expression of 204 human and 210 mouse lncRNAs, with positional analysis demonstrating correlation with immune-related genes. These lncRNAs are predominantly cell-type specific, composed of large regions of repeat sequences, and show poor evolutionary conservation. Comparison within the human and mouse sequences showed less than 1% sequence conservation, although we identified multiple conserved motifs. Of the 204 human lncRNAs, 21 overlapped with syntenic mouse lncRNAs, of which five were differentially expressed in both species. Among these syntenic lncRNA was (antisense), which was induced in multiple cell types and shown to regulate the production of the pro-inflammatory mediator interleukin-6 in both human and mouse cells. In summary, we have identified and characterized those lncRNAs that are differentially expressed following activation of the human and mouse innate immune responses and believe that these catalogs will provide the foundation for the future analysis of the role of lncRNAs in immune and inflammatory responses.

Myositis is characterised by muscle inflammation and weakness. Although generally thought to be driven by a systemic autoimmune response, increasing evidence suggests that intrinsic changes in the muscle might also contribute to the pathogenesis. Long non-coding RNAs (lncRNAs) are a family of novel genes that regulate gene transcription and translation. To determine the potential role of lncRNAs, we employed next generation sequencing to examine the transcriptome in muscle biopsies obtained from two histologically distinct patient populations, inclusion body myositis (IBM) and anti-Jo-1-associated myositis (Jo-1). 1287 mRNAs and 1068 mRNAs were differentially expressed in the muscle from Jo-1 and IBM patients, respectively. Pathway analysis showed the top canonical pathway in both Jo-1 and IBM was oxidative phosphorylation and mitochondrial dysfunction. We identified 731 known and 325 novel lncRNAs in the muscles biopsies. Comparison with controls showed 55 and 46 lncRNAs were differentially expressed in IBM and Jo-1 myositis, respectively. Of these, 16 lncRNAs were differentially expressed in both IBM and Jo-1 myositis and included upregulated H19 , lncMyoD and MALAT1 . Given that these are known to regulate muscle proliferation and differentiation, we speculate that changes in lncRNAs might contribute to the phenotypic changes in Jo-1 and IBM myositis.

G protein-coupled receptors (GPCRs) are important cell signaling mediators, involved in essential physiological processes. GPCRs respond to a wide variety of ligands from light to large macromolecules, including hormones and small peptides. Unfortunately, mutations and dysregulation of GPCRs that induce a loss of function or alter expression can lead to disorders that are sometimes lethal. Therefore, the expression, trafficking, signaling and desensitization of GPCRs must be tightly regulated by different cellular systems to prevent disease. Although there is substantial knowledge regarding the mechanisms that regulate the desensitization and down-regulation of GPCRs, less is known about the mechanisms that regulate the trafficking and cell-surface expression of newly synthesized GPCRs. More recently, there is accumulating evidence that suggests certain GPCRs are able to interact with specific proteins that can completely change their fate and function. These interactions add on another level of regulation and flexibility between different tissue/cell-types. Here, we review some of the main interacting proteins of GPCRs. A greater understanding of the mechanisms regulating their interactions may lead to the discovery of new drug targets for therapy.

Sequencing studies have provided a comprehensive catalog of the expression of intergenic long noncoding RNAs (lincRNAs) in 13 subsets of human T cells and B cells. Subtype-selective lincRNAs are among those identified, including linc-MAF-4, that might regulate T cell differentiation.

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 2 (RAMP2) comprise a receptor for adrenomedullin (AM). Although it is known that AM induces internalization of CLR•RAMP2, little is known about the molecular mechanisms that regulate the trafficking of CLR•RAMP2. Using HEK and HMEC-1 cells, we observed that AM-induced activation of CLR•RAMP2 promoted ubiquitination of CLR. A mutant (CLRΔ9KR), lacking all intracellular lysine residues was functional and trafficked similar to the wild-type receptor, but was not ubiquitinated. Degradation of CLR•RAMP2 and CLRΔ9KR•RAMP2 was not dependent on the duration of AM stimulation or ubiquitination and occurred via a mechanism that was partially prevented by peptidase inhibitors. Degradation of CLR•RAMP2 was sensitive to overexpression of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), but not to HRS knockdown, whereas CLRΔ9KR•RAMP2 degradation was unaffected. Overexpression, but not knockdown of HRS, promoted hyperubiquitination of CLR under basal conditions. Thus, we propose a role for ubiquitin and HRS in the regulation of AM-induced degradation of CLR•RAMP2.

A theoretical framework is elaborated to account for the effect of a transmembrane potential in computer simulations. It is shown that a simulation with a constant external electric field applied in the direction normal to the membrane is equivalent to the influence of surrounding infinite baths maintained to a voltage difference via ion-exchanging electrodes connected to an electromotive force. It is also shown that the linearly-weighted displacement charge within the simulation system tracks the net flow of charge through the external circuit comprising the electromotive force and the electrodes. Using a statistical mechanical reduction of the degrees of freedom of the external system, three distinct theoretical routes are formulated and examined for the purpose of characterizing the free energy of a protein embedded in a membrane that is submitted to a voltage difference. The W-route is constructed from the variations in the voltage-dependent potential of mean force along a reaction path connecting two conformations of the protein. The Q-route is based on the average displacement charge as a function of the conformation of the protein. Finally, the G-route considers the relative charging free energy of specific residues, with and without applied membrane potentials. The theoretical formulation is illustrated with a simple model of an ion crossing a vacuum slab surrounded by two aqueous bulk phases and with a fragment of the voltage-sensor of the KvAP potassium channel.

Background The MyPeBS study is an ongoing randomised controlled trial testing whether a risk-stratified breast cancer screening strategy is non-inferior, or eventually superior, to standard age-based screening at reducing incidence of stage 2 or more cancers. This large European Commission-funded initiative aims to include 85,000 women aged 40 to 70 years, without prior breast cancer and not previously identified at high risk in six countries (Belgium, France, Italy, Israel, Spain, UK). A specific work package within MyPeBS examines psychological, socio-economic and ethical aspects of this new screening strategy. It compares women’s reported data and outcomes in both trial arms on the following issues: general anxiety, cancer-related worry, understanding of breast cancer screening strategy and information-seeking behaviour, socio-demographic and economic characteristics, quality of life, risk perception, intention to change health-related behaviours, satisfaction with the trial. Methods At inclusion, 3-months, 1-year and 4-years, each woman participating in MyPeBS is asked to fill online questionnaires. Descriptive statistics, bivariate analyses, subgroup comparisons and analysis of variations over time will be performed with appropriate tests to assess differences between arms. Multivariate regression models will allow modelling of different patient reported data and outcomes such as comprehension of the information provided, general anxiety or cancer worry, and information seeking behaviour. In addition, a qualitative study (48 semi-structured interviews conducted in France and in the UK with women randomised in the risk-stratified arm), will help further understand participants’ acceptability and comprehension of the trial, and their experience of risk assessment. Discussion Beyond the scientific and medical objectives of this clinical study, it is critical to acknowledge the consequences of such a paradigm shift for women. Indeed, introducing a risk-based screening relying on individual biological differences also implies addressing non-biological differences (e.g. social status or health literacy) from an ethical perspective, to ensure equal access to healthcare. The results of the present study will facilitate making recommendations on implementation at the end of the trial to accompany any potential change in screening strategy. Trial registration Study sponsor: UNICANCER. My personalised breast screening (MyPeBS). Clinicaltrials.gov (2018) available at: https://clinicaltrials.gov/ct2/show/NCT03672331 Contact: Cécile VISSAC SABATIER, PhD, + 33 (0)1 73 79 77 58 ext + 330,142,114,293, contact@mypebs.eu.

Unregulated activation of Src kinases leads to aberrant signalling, uncontrolled growth and differentiation of cancerous cells. Reaching a complete mechanistic understanding of large-scale conformational transformations underlying the activation of kinases could greatly help in the development of therapeutic drugs for the treatment of these pathologies. In principle, the nature of conformational transition could be modelled in silico via atomistic molecular dynamics simulations, although this is very challenging because of the long activation timescales. Here we employ a computational paradigm that couples transition pathway techniques and Markov state model-based massively distributed simulations for mapping the conformational landscape of c-src tyrosine kinase. The computations provide the thermodynamics and kinetics of kinase activation for the first time, and help identify key structural intermediates. Furthermore, the presence of a novel allosteric site in an intermediate state of c-src that could be potentially used for drug design is predicted. Activation of c-src kinase is associated with uncontrolled growth and metastasis of tumour cells. Shukla et al. model conformational changes in c-src during activation, and identify an allosteric site in an intermediate state that may provide a target for small molecule therapeutics.