The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for reasons that are not entirely clear. Here we demonstrate that Nef and Eed complex with the integrin ...effector paxillin to recruit and activate TNFα converting enzyme (TACE alias ADAM 17) and its close relative ADAM10. The activated proteases cleaved proTNFα and were shuttled into extracellular vesicles (EVs). Peripheral blood mononuclear cells that ingested these EVs released TNFα. Analyzing the mechanism, we found that Pak2, an established host cell effector of Nef, phosphorylated paxillin on Ser272/274 to induce TACE-paxillin association and shuttling into EVs via lipid rafts. Conversely, Pak1 phosphorylated paxillin on Ser258, which inhibited TACE association and lipid raft transfer. Interestingly, melanoma cells used an identical mechanism to shuttle predominantly ADAM10 into EVs. We conclude that HIV-1 and cancer cells exploit a paxillin/integrin-controlled mechanism to release TACE/ADAM10-containing vesicles, ensuring better proliferation/growth conditions in their microenvironment.
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► HIV-Nef translocates activated TACE/ADAM17 into extracellular vesicles (EVs) ► The mechanism is initiated by interaction of Eed and paxillin with TACE ► Translocation of TACE into EVs is regulated by Pak2 ► Melanoma cells activate the same mechanism to upload activated ADAM10 into EVs
Membrane surveillance and repair is of utmost importance to maintain cellular integrity and allow cellular life. Several systems detect cell envelope stress caused by antimicrobial compounds and ...abiotic stresses such as solvents, pH-changes and temperature in bacteria. Proteins containing an Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH)-domain, including bacterial flotillins have been shown to be involved in membrane protection and membrane fluidity regulation. Here, we characterize a bacterial SPFH-domain protein, YdjI that is part of a stress induced complex in
. We show that YdjI is required to localize the ESCRT-III homolog PspA to the membrane with the help of two membrane integral proteins, YdjG/H. In contrast to classical flotillins, YdjI resides in fluid membrane regions and does not enrich in detergent resistant membrane fractions. However, similarly to FloA and FloT from
, deletion of YdjI decreases membrane fluidity. Our data reveal a hardwired connection between phage shock response and SPFH proteins.
Extracellular vesicles (EVs) are increasingly gaining interest as biomarkers and therapeutics. Accurate sizing and quantification of EVs remain problematic, given their nanometre size range and small ...scattering cross‐sections. This is compounded by the fact that common EV isolation methods result in co‐isolation of particles with comparable features. Especially in blood plasma, similarly‐sized lipoproteins outnumber EVs to a great extent. Recently, interferometric nanoparticle tracking analysis (iNTA) was introduced as a particle analysis method that enables determining the size and refractive index of nanoparticles with high sensitivity and precision. In this work, we apply iNTA to differentiate between EVs and lipoproteins, and compare its performance to conventional nanoparticle tracking analysis (NTA). We show that iNTA can accurately quantify EVs in artificial EV‐lipoprotein mixtures and in plasma‐derived EV samples of varying complexity. Conventional NTA could not report on EV numbers, as it was not able to distinguish EVs from lipoproteins. iNTA has the potential to become a new standard for label‐free EV characterization in suspension.
Keloid is an aberrant scarring process of the skin, characterized by excessive extracellular matrix synthesis and deposition. The pathogenesis of this prevalent cutaneous disorder is not fully ...understood; however, a persistent inflammatory process is observed. To obtain more insight into this process, we analyzed lesional, perilesional and healthy tissue using multi-antigen-analysis (MAA) in conjunction with a data mining approach. Here, we demonstrate that monocyte-derived inflammatory dendritic cells (CD1a+, CD11c+, CD14+) and activated CD4+ T lymphocytes (CD45 RO+) dominated the immune infiltration in keloids while associating with fibroblasts. In perilesional tissue, precursor immune cells were dominant in the perivascular area, suggesting that they were attracted by an immune process, potentially in the lesional area. Supporting this hypothesis, only in keloid lesions, high levels of ADAM10/17 and Neprilysin (CD10) were observed in both fibroblasts and leukocytes. The spatial proximity of these two cell types, which could be confirmed by image analysis only in lesional tissue, could be a potential factor leading to the activation of fibroblasts. Our findings provide new insight into the pathogenesis of keloid formation and reveal metalloproteinases as a target for therapeutical intervention.
Entanglement between stationary systems at remote locations is a key resource for quantum networks. We report on the experimental generation of remote entanglement between a single atom inside an ...optical cavity and a Bose-Einstein condensate (BEC). To produce this, a single photon is created in the atom-cavity system, thereby generating atom-photon entanglement. The photon is transported to the BEC and converted into a collective excitation in the BEC, thus establishing matter-matter entanglement. After a variable delay, this entanglement is converted into photon-photon entanglement. The matter-matter entanglement lifetime of 100 μs exceeds the photon duration by 2 orders of magnitude. The total fidelity of all concatenated operations is 95%. This hybrid system opens up promising perspectives in the field of quantum information.
BACKGROUND
The efficacy of fertility awareness based (FAB) methods of family planning is critically reviewed. The objective was to investigate the efficacy and the acceptability of the symptothermal ...method (STM), an FAB method that uses two indicators of fertility, temperature and cervical secretions observation. This paper will recommend a more suitable approach to measure the efficacy.
METHODS
Since 1985, an ongoing prospective observational longitudinal cohort study has been conducted in Germany. Women are asked to submit their menstrual cycle charts that record daily basal body temperature, cervical secretion observations and sexual behaviour. A cohort of 900 women contributed 17 638 cycles that met the inclusion criteria for the effectiveness study. The overall rates of unintended pregnancies and dropout rates have been estimated with survival curves according to the Kaplan–Meier method. In order to estimate the true method effectiveness, the pregnancy rates have been calculated in relation to sexual behaviour using the ‘perfect/imperfect-use’ model of Trussell and Grummer-Strawn.
RESULTS
After 13 cycles, 1.8 per 100 women of the cohort experienced an unintended pregnancy; 9.2 per 100 women dropped out because of dissatisfaction with the method; the pregnancy rate was 0.6 per 100 women and per 13 cycles when there was no unprotected intercourse in the fertile time.
CONCLUSIONS
The STM is a highly effective family planning method, provided the appropriate guidelines are consistently adhered to.
A highly conserved signaling property of Nef proteins encoded by human or simian immunodeficiency virus is the binding and activation of a PAK kinase whose function is unclear. Here we show that ...Nef-mediated p21-activated kinase (PAK) activation involves phosphatidylinositol 3-kinase, which acts upstream of PAK and is bound and activated by Nef similar to the manner of Polyoma virus middle T antigen. The Nef-associated phosphatidylinositol-3-PAK complex phosphorylated the pro-apoptotic Bad protein without involving the protein kinase B-Akt kinase, which is generally believed to inactivate Bad by serine phosphorylation. Consequently, Nef, but not a Nef mutant incapable of activating PAK, blocked apoptosis in T cells induced by serum starvation or HIV replication. Nef anti-apoptotic effects are likely a crucial mechanism for viral replication in the host and thus in AIDS pathogenesis.
The viral Nef protein is important for the progression of the human and simian immunodeficiency virus (HIV/SIV) infection. So far, experimental evidence has suggested that Nef enhances viral ...replication and infectivity through a combination of different effects. Recent insights, however, indicate that its functions are more complex than previously anticipated. By targeting the T cell receptor, Nef may not only prime viral replication but, more importantly, ensure viral survival through distinct mechanisms of immune evasion and antiapoptosis.
Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25+ cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used ...experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen–specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator–dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25+ cell killing that may affect their clinical use and could be tested for novel indications. This trial was registered at www.clinicaltrials.gov, #NCT00056134.
•ONTAK blocks DC maturation by coreceptor downmodulation and inhibition of Stat3 phosphorylation to induce a tolerogenic phenotype.•ONTAK kills activated CD4 T cells but stimulates antiapoptosis in resting Treg by engagement and stimulation through CD25.
Before and after surgery melanoma patients harbor elevated levels of extracellular vesicles in plasma (pEV), suppressing tumor cell activity. However, due to technical reasons and lack of ...cell-specific biomarkers, their cellular origin remains obscure.
We mimicked the interaction of tumor cells with liver cells and PBMC in vitro, and compared newly secreted EV-associated miRNAs and protein factors with those detected in melanoma patient`s pEV.
Our results suggest that pEV from melanoma patients are secreted in part by residual or relapsing tumor cells, but also by liver and peripheral blood mononuclear cells (PBMC). Our approach identified factors that were seemingly associated either with tumor cell activity, or the counteracting immune system, including liver cells. Notably, the presence/absence of these factors correlated with the clinical stage and tumor relapse.
Our study may provide new insights into the innate immune defense against tumor cells and implies that residual tumor cells could be more active than previously thought. In addition we provide some preliminary evidence that pEV marker patterns could be used to predict cancer relapse.
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