Legionella pneumophila strain 130b evades macrophage cell death independent of the effector SidF in the absence of flagellin

International audienceThe human pathogen Legionella pneumophila must evade host cell death signaling to enable replication in lung macrophages and to cause disease. After bacterial growth, however, L. pneumophila is thought to induce apoptosis during egress from macrophages. The bacterial effector protein, SidF, has been shown to control host cell survival and death by inhibiting pro-apoptotic BNIP3 and BCL-RAMBO signaling. Using live-cell imaging to follow the L. pneumophila-macrophage interaction, we now demonstrate that L. pneumophila evades host cell apoptosis independent of SidF. In the absence of SidF, L. pneumophila was able to replicate, cause loss of mitochondria membrane potential, kill macrophages, and establish infections in lungs of mice. Consistent with this, deletion of BNIP3 and BCL-RAMBO did not affect intracellular L. pneumophila replication, macrophage death rates, and in vivo bacterial virulence. Abrogating mitochondrial cell death by genetic deletion of the effectors of intrinsic apoptosis, BAX, and BAK, or the regulator of mitochondrial permeability transition pore formation, cyclophilin-D, did not affect bacterial growth or the initial killing of macrophages. Loss of BAX and BAK only marginally limited the ability of L. pneumophila to efficiently kill all macrophages over extended periods. L. pneumophila induced killing of macrophages was delayed in the absence of capsase-11 mediated pyroptosis. Together, our data demonstrate that L. pneumophila evades host cell death responses independently of SidF during replication and can induce pyroptosis to kill macrophages in a timely manner

Environmental Conditions in Beef Deep-Bedded Mono-Slope Facilities

Ammonia (NH3), temperature, moisture content, pH, pack depth, nutrient composition and concentration of odorous volatile organic compounds (VOC) were measured at 56 locations in each of four pens in two commercial beef deep-bedded mono-slope facilities (BDMF). Areas of high NH3 concentration occurred randomly throughout the pens. Ammonia concentration increased as pack and ambient air temperature increased. Concentration of VOC was highest in transition areas between the bedded pack and the concrete floor. Depth, moisture content, and pH of the bedded pack did not influence concentration of NH3 and VOC. Nutrient composition of the manure/bedding material in BDMF is similar to manure in open feedlots, except that in BDMF the volatile solids content is much higher. E. coli concentrations can occur at high levels in BDMF and vary with differences in ambient temperature. Priority should be given to NH3 and E. coli mitigation during hot months. However, locationspecific NH3 mitigation will not be effective due to the random distribution of NH3 in the pen. Frequent cleaning of the area surrounding the bedded pack should reduce VOC concentration

Management of chronic lateral instability due to lateral collateral ligament deficiency after total knee arthroplasty: a case report

<p>Abstract</p> <p>Introduction</p> <p>Lateral instability following total knee arthroplasty (TKA) is a rare condition with limited report of treatment options. The objective of this case presentation is to demonstrate the outcomes of different surgical procedures performed in a single patient with lateral collateral ligament (LCL) deficiency.</p> <p>Case presentation</p> <p>We present a case of chronic lateral instability due to LCL deficiency after primary TKA in a 47-year-old Caucasian woman with an obesity problem. Multiple treatment options have been performed in order to manage this problem, including the following: ligament reconstruction; combined ligament reconstruction and constrained implant; and rotating-hinge knee prosthesis that was the most recent surgery. All ligament reconstruction procedures failed within one year. The varus-valgus constrained prosthesis provided stability for six years.</p> <p>Conclusions</p> <p>Ligament reconstruction alone cannot provide enough stability for the treatment of chronic lateral instability in patients with obesity problems and LCL deficiency. When the reconstruction fails, a salvage procedure with rotating-hinge knee is still available.</p

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1–TRAF2 complex to sensitize tumor cells to TNFα

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor κB (NF-κB) signaling, and sensitize cells to tumor necrosis factor α (TNFα). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1–Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1–TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-κB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFα-induced death occurs. TWEAK-induced loss of the cIAP1–TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFα-induced death, whereas primary cells remain resistant. Conversely, cIAP1–TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFα sensitization. Lysosomal degradation of cIAP1–TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells

Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.Wellcome Trus

Nitrite-derived nitric oxide reduces hypoxia-inducible factor 1α-mediated extracellular vesicle production by endothelial cells

Introduction Extracellular vesicles (EVs) are small, spherical particles enclosed by a phospholipid bilayer (∼30–1000 nm) released from multiple cell types, and have been shown to have pathophysiological roles in a plethora of disease states. The transcription factor hypoxia-inducible factor-1 (HIF-1) allows for adaptation of cellular physiology in hypoxia and may permit the enhanced release of EVs under such conditions. Nitric oxide (NO) plays a pivotal role in vascular homeostasis, and can modulate the cellular response to hypoxia by preventing HIF-1 accumulation. We aimed to selectively target HIF-1 via sodium nitrite (NaNO2) addition, and examine the effect on endothelial EV, size, concentration and function, and delineate the role of HIF-1 in EV biogenesis. Methods Endothelial (HECV) cells were exposed to hypoxic conditions (1% O2, 24 h) and compared to endothelial cells exposed to normoxia (21% O2) with and without the presence of sodium nitrite (NaNO2) (30 μM). Allopurinol (100 μM), an inhibitor of xanthine oxidoreductase, was added both alone and in combination with NaNO2 to cells exposed to hypoxia. EV and cell preparations were quantified by nanoparticle tracking analysis and confirmed by electron microscopy. Western blotting and siRNA were used to confirm the role of HIF-1α and HIF-2α in EV biogenesis. Flow cytometry and time-resolved fluorescence were used to assess the surface and intravesicular protein content. Results Endothelial (HECV) cells exposed to hypoxia (1% O2) produced higher levels of EVs compared to cells exposed to normoxia. This increase was confirmed using the hypoxia-mimetic agent desferrioxamine. Treatment of cells with sodium nitrite (NaNO2) reduced the hypoxic enhancement of EV production. Treatment of cells with the xanthine oxidoreductase inhibitor allopurinol, in addition to NaNO2 attenuated the NaNO2-attributed suppression of hypoxia-mediated EV release. Transfection of cells with HIF-1α siRNA, but not HIF-2α siRNA, prior to hypoxic exposure prevented the enhancement of EV release. Conclusion These data provide evidence that hypoxia enhances the release of EVs in endothelial cells, and that this is mediated by HIF-1α, but not HIF-2α. Furthermore, the reduction of NO2− to NO via xanthine oxidoreductase during hypoxia appears to inhibit HIF-1α-mediated EV production

Experiencing uncertainty – on the potential of groups and a group analytic approach for making management education more critical.

This document is the Accepted Manuscript. The final, definitive version of this paper has been published in Management Learning, November 2017, DOI: https://doi.org/10.1177/1350507617697868. Published by SAGE Publishing. All rights reserved.This article points to the potential of methods derived from group analytic practice for making management education more critical. It draws on the experience of running a professional doctorate for more experienced managers in a university in the UK over a 16 year period. Group analysis is informed by the highly social theories of S.H. Foulkes and draws heavily on psychoanalytic theory as well as sociology. First and foremost, though, it places our interdependence at the heart of the process of inquiry, and suggests that the most potent place for learning about groups, where we spend most of our lives, is in a group. The article prioritises three areas of management practice for which group analytic methods, as adapted for research environment, are most helpful: coping with uncertainty and the feelings of anxiety which this often arouses; thinking about leadership as a relational and negotiated activity, and encouraging reflexivity in managers. The article also points to some of the differences between the idea of the learning community and psychodynamic perspectives more generally and the limitations of group analytic methods in particular, which may pathologise resistance in the workplace.Peer reviewe

TAK1 Is Required for Survival of Mouse Fibroblasts Treated with TRAIL, and Does So by NF-κB Dependent Induction of cFLIPL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a “death ligand”—a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-κB and JNK signalling pathways. To determine the role of TGF-β-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1−/− MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-κB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-κB, protected TAK1−/− MEFs against TRAIL killing, suggesting that TAK1 activation of NF-κB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-κB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1−/− MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1–NF-κB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance

Ozone, aerosol, potential vorticity, and trace gas trends observed at high‐latitudes over North America from February to May 2000

Ozone (O3) and aerosol scattering ratio profiles were obtained from airborne lidar measurements on thirty‐eight flights over seven deployments covering the latitudes of 40°–85°N between 4 February and 23 May 2000 as part of the Tropospheric Ozone Production about the Spring Equinox (TOPSE) field experiment. Each deployment started from Broomfield, Colorado, with bases in Churchill, Canada, and on most deployments, Thule Air Base, Greenland. Nadir and zenith lidar O3 measurements were combined with in situ O3 measurements to produce vertically continuous O3 profiles from near the surface to above the tropopause. Potential vorticity (PV) distributions along the flight track were obtained from several different meteorological analyses. Ozone, aerosol, and PV distributions were used together to identify the presence of pollution plumes and stratospheric intrusions. Ozone was found to increase in the middle free troposphere (4–6 km) at high latitudes (60°–85°N) by an average of 4.6 ppbv/mo (parts per billion by volume per month) from about 54 ppbv in early February to over 72 ppbv in mid‐May. The average aerosol scattering ratios at 1064 nm in the same region increased rapidly at an average rate of 0.36/mo from about 0.38 to over 1.7. Ozone and aerosol scattering were highly correlated over the entire field experiment, and PV and beryllium (7Be) showed no significant positive trend over the same period. The primary cause of the observed O3 increase in the mid troposphere at high latitudes was determined to be the photochemical production of O3 in pollution plumes with less than 20% of the increase from stratospherically‐derived O3

Novel SMAC-mimetics synergistically stimulate melanoma cell death in combination with TRAIL and Bortezomib

BACKGROUND: XIAP (X-linked inhibitor of apoptosis protein) is an anti-apoptotic protein exerting its activity by binding and suppressing caspases. As XIAP is overexpressed in several tumours, in which it apparently contributes to chemoresistance, and because its activity in vivo is antagonised by second mitochondria-derived activator of caspase (SMAC)/direct inhibitor of apoptosis-binding protein with low pI, small molecules mimicking SMAC (so called SMAC-mimetics) can potentially overcome tumour resistance by promoting apoptosis. METHODS: Three homodimeric compounds were synthesised tethering a monomeric SMAC-mimetic with different linkers and their affinity binding for the baculoviral inhibitor repeats domains of XIAP measured by fluorescent polarisation assay. The apoptotic activity of these molecules, alone or in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and/or Bortezomib, was tested in melanoma cell lines by MTT viability assays and western blot analysis of activated caspases. RESULTS: We show that in melanoma cell lines, which are typically resistant to chemotherapeutic agents, XIAP knock-down sensitises cells to TRAIL treatment in vitro, also favouring the accumulation of cleaved caspase-8. We also describe a new series of 4-substituted azabicyclo[5.3.0] alkane monomeric and dimeric SMAC-mimetics that target various members of the IAP family and powerfully synergise at submicromolar concentrations with TRAIL in inducing cell death. Finally, we show that the simultaneous administration of newly developed SMAC-mimetics with Bortezomib potently triggers apoptosis in a melanoma cell line resistant to the combined effect of SMAC-mimetics and TRAIL. CONCLUSION: Hence, the newly developed SMAC-mimetics effectively synergise with TRAIL and Bortezomib in inducing cell death. These findings warrant further preclinical studies in vivo to verify the anticancer effectiveness of the combination of these agents