Proinflammatory cytokines inhibit osteogenic differentiation from stem cells: implications for bone repair during inflammation

SummaryObjectiveThe effects of inflammation on bone development from mesenchymal stem cells (MSC) are unclear due to the difficulty in isolating MSC. The aim of this study was to develop a MSC isolation method and to determine the in vitro effects of interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) on their osteogenic differentiation.MethodsMurine MSC were isolated from the limbs of C57/Bl6 mice through collagenase digestion of bone and enriched as the Stem cell antigen (Sca-1)+ CD31− CD45− population, using lineage immunodepletion, followed by fluorescence-activated cell sorting (FACS). They were differentiated along the osteoblast linage in the presence or absence of IL-1β and TNFα. Mineralization was measured as was the expression of a number of osteogenic genes by quantitative polymerase chain reaction (PCR).ResultsWe show that osteogenic differentiation from the MSC population is suppressed by IL-1β and TNFα. In addition to suppression of bone mineralization, both cytokines inhibited the differentiation-associated increases in alkaline phosphatase (ALP) activity and the gene expression for ALP, α1(I) procollagen, runt-related transcription factor 2 (Runx2) and osterix. However, only TNFα inhibited osteonectin and osteopontin mRNA expression and only IL-1β reduced cell proliferation.ConclusionsThe convenient isolation technique enables the easy generation of sufficient MSC to permit the molecular analysis of their differentiation. We were thus able to show that the proinflammatory cytokines, IL-1β and TNFα, can compromise bone development from this primary MSC population, although with some significant differences. The potential involvement of specific inflammatory mediators needs to be taken into account if optimal bone repair and presumably that of other tissues are to be achieved with MSC

Pathway-Based High-Throughput Chemical Screen Identifies Compounds That Decouple Heterochromatin Transformations

Heterochromatin protein 1 (HP1) facilitates the formation of repressive heterochromatin domains by recruiting histone lysine methyltransferase enzymes to chromatin, resulting in increased levels of histone H3K9me3. To identify chemical inhibitors of the HP1-heterochromatin gene repression pathway, we combined a cell-based assay that utilized chemical-mediated recruitment of HP1 to an endogenous active gene with high-throughput flow cytometry. Here we characterized small molecule inhibitors that block HP1-mediated heterochromatin formation. Our lead compounds demonstrated dose-dependent inhibition of HP1-stimulated gene repression and were validated in an orthogonal cell-based system. One lead inhibitor was improved by a change in stereochemistry, resulting in compound 2, which was further used to decouple the inverse relationship between H3K9 and H3K4 methylation states. We identified molecular components that bound compound 2, either directly or indirectly, by chemical affinity purification with a biotin-tagged derivative, followed by quantitative proteomic techniques. In summary, our pathway-based chemical screening approach resulted in the discovery of new inhibitors of HP1-mediated heterochromatin formation while identifying exciting new molecular interactions in the pathway to explore in the future. This modular platform can be expanded to test a wide range of chromatin modification pathways yielding inhibitors that are cell permeable and function in a physiologically relevant setting

Fusion product losses due to fishbone instabilities in deuterium JET plasmas

During development of a high-performance hybrid scenario for future deuterium–tritium experiments on the Joint European Torus, an increased level of fast ion losses in the MeV energy range was observed during the instability of high-frequency n  =  1 fishbones. The fishbones are excited during deuterium neutral beam injection combined with ion cyclotron heating. The frequency range of the fishbones, 10–25 kHz, indicates that they are driven by a resonant interaction with the NBI-produced deuterium beam ions in the energy range  ≤120 keV. The fast particle losses in a much higher energy range are measured with a fast ion loss detector, and the data show an expulsion of deuterium plasma fusion products, 1 MeV tritons and 3 MeV protons, during the fishbone bursts. An MHD mode analysis with the MISHKA code combined with the nonlinear wave-particle interaction code HAGIS shows that the loss of toroidal symmetry caused by the n  =  1 fishbones affects strongly the confinement of non-resonant high energy fusion-born tritons and protons by perturbing their orbits and expelling them. This modelling is in a good agreement with the experimental data.This work has been carried out within the framework of the EUROfusion Consortium and has received funding from the Euratom research and training programme 2014-2018 under grant agreement No 633053 and from the RCUK Energy Programme [grant No EP/P012450/1]. To obtain further information on the data and models underlying this paper please contact [email protected] . The views and opinions expressed herein do not necessarily reflect those of the European CommissionPeer ReviewedPostprint (author's final draft

SIRT1-NOX4 Signaling Axis Regulates Cancer Cachexia

Approximately one third of cancer patients die due to complexities related to cachexia. However, the mechanisms of cachexia and the potential therapeutic interventions remain poorly studied. We observed a significant positive correlation between SIRT1 expression and muscle fiber cross-sectional area in pancreatic cancer patients. Rescuing Sirt1 expression by exogenous expression or pharmacological agents reverted cancer cell-induced myotube wasting in culture conditions and mouse models. RNA-seq and follow-up analyses showed cancer cell-mediated SIRT1 loss induced NF-κB signaling in cachectic muscles that enhanced the expression of FOXO transcription factors and NADPH oxidase 4 (Nox4), a key regulator of reactive oxygen species production. Additionally, we observed a negative correlation between NOX4 expression and skeletal muscle fiber cross-sectional area in pancreatic cancer patients. Knocking out Nox4 in skeletal muscles or pharmacological blockade of Nox4 activity abrogated tumor-induced cachexia in mice. Thus, we conclude that targeting the Sirt1-Nox4 axis in muscles is an effective therapeutic intervention for mitigating pancreatic cancer-induced cachexia

International variation in distribution of ASA class in patients undergoing total hip arthroplasty and its influence on mortality: data from an international consortium of arthroplasty registries

Background and purpose — A challenge comparing outcomes from total hip arthroplasty between countries is variation in preoperative characteristics, particularly comorbidity. Therefore, we investigated between-country variation in comorbidity in patients based on ASA class distribution, and determined any variation of ASA class to mortality risk between countries. Patients and methods — All arthroplasty registries collecting ASA class and mortality data in patients with elective primary THAs performed 2012–2016 were identified. Survival analyses of the influence of ASA class on 1-year mortality were performed by individual registries, followed by meta-analysis of aggregated data. Results — 6 national registries and 1 US healthcare organization registry with 418,916 THAs were included. There was substantial variation in the proportion of ASA class III/IV, ranging from 14% in the Netherlands to 39% in Finland. Overall, 1-year mortality was 0.93% (95% CI 0.87–1.01) and increased from 0.2% in ASA class I to 8.9% in class IV. The association between ASA class and mortality measured by hazard ratios (HR) was strong in all registries even after adjustment for age and sex, which reduced them by half in all registries. Combined adjusted HRs were 2.0, 6.1, and 22 for ASA class II–IV vs. I, respectively. Associations were moderately heterogeneous across registries. Interpretation — We observed large variation in ASA class distribution between registries, possibly explained by differences in background morbidity and/or international variation in access to surgery. The similar, strong mortality trends by ASA class between countries enhance the relevance of its use as an indicator of comorbidity in international registry studies

On the mechanisms governing gas penetration into a tokamak plasma during a massive gas injection

A new 1D radial fluid code, IMAGINE, is used to simulate the penetration of gas into a tokamak plasma during a massive gas injection (MGI). The main result is that the gas is in general strongly braked as it reaches the plasma, due to mechanisms related to charge exchange and (to a smaller extent) recombination. As a result, only a fraction of the gas penetrates into the plasma. Also, a shock wave is created in the gas which propagates away from the plasma, braking and compressing the incoming gas. Simulation results are quantitatively consistent, at least in terms of orders of magnitude, with experimental data for a D 2 MGI into a JET Ohmic plasma. Simulations of MGI into the background plasma surrounding a runaway electron beam show that if the background electron density is too high, the gas may not penetrate, suggesting a possible explanation for the recent results of Reux et al in JET (2015 Nucl. Fusion 55 093013)

Velocity-space sensitivity of the time-of-flight neutron spectrometer at JET

The velocity-space sensitivities of fast-ion diagnostics are often described by so-called weight functions. Recently, we formulated weight functions showing the velocity-space sensitivity of the often dominant beam-target part of neutron energy spectra. These weight functions for neutron emission spectrometry (NES) are independent of the particular NES diagnostic. Here we apply these NES weight functions to the time-of-flight spectrometer TOFOR at JET. By taking the instrumental response function of TOFOR into account, we calculate time-of-flight NES weight functions that enable us to directly determine the velocity-space sensitivity of a given part of a measured time-of-flight spectrum from TOFOR