Autophagy drives fibroblast senescence through MTORC2 regulation

Sustained macroautophagy/autophagy favors the differentiation of fibroblasts into myofibroblasts. Cellular senescence, another means of responding to long-term cellular stress, has also been linked to myofibroblast differentiation and fibrosis. Here, we evaluate the relationship between senescence and myofibroblast differentiation in the context of sustained autophagy. We analyzed markers of cell cycle arrest/senescence in fibroblasts in vitro, where autophagy was triggered by serum starvation (SS). Autophagic fibroblasts expressed the senescence biomarkers CDKN1A/p21 and CDKN2A/p16 and exhibited increased senescenceassociated GLB1/beta-galactosidase activity. Inhibition of autophagy in serum-starved fibroblasts with 3-methyladenine, LY294002, or ATG7 (autophagy related 7) silencing prevented the expression of senescence-associated markers. Similarly, suppressing MTORC2 activation using rapamycin or by silencing RICTOR also prevented senescence hallmarks. Immunofluorescence microscopy showed that senescence and myofibroblast differentiation were induced in different cells, suggesting mutually exclusive activation of senescence and myofibroblast differentiation. Reactive oxygen species (ROS) are known inducers of senescence and exposing fibroblasts to ROS scavengers decreased ROS production during SS, inhibited autophagy, and significantly reduced the expression of senescence and myofibroblast differentiation markers. ROS scavengers also curbed the AKT1 phosphorylation at Ser473, an MTORC2 target, establishing the importance of ROS in fuelling MTORC2 activation. Inhibition of senescence by shRNA to TP53/p53 and shRNA CDKN2A/p16 increased myofibroblast differentiation, suggesting a negative feedback loop of senescence on autophagy-induced myofibroblast differentiation. Collectively, our results identify ROS as central inducers of MTORC2 activation during chronic autophagy, which in turn fuels senescence activation and myofibroblast differentiation in distinct cellular subpopulations

Detection of human neutrophil elastase (HNE) on wound dressings as marker of inflammation

Chronic wound fluids have elevated concentration of human neutrophil elastase (HNE) which can be used as inflammation/infection marker. Our goal is to develop functional materials for fast diagnosis of wound inflammation/infection by using HNE as a specific marker. For that, fluorogenic peptides with a HNE-specific cleavage sequence were incorporated into traditional textile dressings, to allow real-time detection of the wound status. Two different fluorogenic approaches were studied in terms of intensity of the signal generated upon HNE addition: a fluorophore 7-amino-4-trifluormethylcoumarin (AFC) conjugated to a HNE-specific peptide and two fluorophore/quencher pairs (FAM/Dabcyl and EDANS/Dabcyl) coupled to a similar peptide as a Förster resonance energy transfer (FRET) strategy. Also, two immobilization methods were tested: sonochemistry immobilization onto a cotton bandage and glutaraldehyde (GTA)-assisted chemical crosslinking onto a polyamide dressing. The immobilized fluorogenic AFC peptide showed an intense fluorescence emission in the presence of HNE. HNE also induced an enhanced fluorescent signal with the EDANS/Dabcyl FRET peptide which showed to be a more sensitive and effective strategy than the AFC peptide. However, its chemical immobilization onto the polyamide dressing greatly decreased its detection, mainly due to the more difficult access of the enzyme to the cleavage sequence of the immobilized peptide. After optimization of the in situ immobilization, it will be possible to use these fluorescence-functionalized dressings for an effective and specific monitoring of chronic wounds by simply using a portable ultraviolet (UV) light source. We envision that the development of this point-of-care medical device for wound control will have a great impact on patients life quality and reduction of costs on health care system.This study was funded by the European project InFact-Functional materials for fast diagnosis of wound infection (FP7-NMP-2013-SME-7-grant agreement no. 604278). The work done at Centre of Biological Engineering (CEB) was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte 2020-Programa Operacional Regional do Norte

First Dark Matter Search Results from the LUX-ZEPLIN (LZ) Experiment

The LUX-ZEPLIN (LZ) experiment is a dark matter detector centered on a dual-phase xenon time projection chamber operating at the Sanford Underground Research Facility in Lead, South Dakota, USA. This Letter reports results from LZ's first search for Weakly Interacting Massive Particles (WIMPs) with an exposure of 60 live days using a fiducial mass of 5.5 t. A profile-likelihood ratio analysis shows the data to be consistent with a background-only hypothesis, setting new limits on spin-independent WIMP-nucleon, spin-dependent WIMP-neutron, and spin-dependent WIMP-proton cross-sections for WIMP masses above 9 GeV/c$^2$. The most stringent limit is set at 30 GeV/c$^2$, excluding cross sections above 5.9$\times 10^{-48}$ cm$^2$ at the 90\% confidence level.Comment: 9 pages, 6 figures. See https://tinyurl.com/LZDataReleaseRun1 for a data release related to this pape

The LUX-ZEPLIN (LZ) Experiment

We describe the design and assembly of the LUX-ZEPLIN experiment, a direct detection search for cosmic WIMP dark matter particles. The centerpiece of the experiment is a large liquid xenon time projection chamber sensitive to low energy nuclear recoils. Rejection of backgrounds is enhanced by a Xe skin veto detector and by a liquid scintillator Outer Detector loaded with gadolinium for efficient neutron capture and tagging. LZ is located in the Davis Cavern at the 4850' level of the Sanford Underground Research Facility in Lead, South Dakota, USA. We describe the major subsystems of the experiment and its key design features and requirements

The LUX-ZEPLIN (LZ) experiment

We describe the design and assembly of the LUX-ZEPLIN experiment, a direct detection search for cosmic WIMP dark matter particles. The centerpiece of the experiment is a large liquid xenon time projection chamber sensitive to low energy nuclear recoils. Rejection of backgrounds is enhanced by a Xe skin veto detector and by a liquid scintillator Outer Detector loaded with gadolinium for efficient neutron capture and tagging. LZ is located in the Davis Cavern at the 4850’ level of the Sanford Underground Research Facility in Lead, South Dakota, USA. We describe the major subsystems of the experiment and its key design features and requirements

The LUX-ZEPLIN (LZ) radioactivity and cleanliness control programs

LUX-ZEPLIN (LZ) is a second-generation direct dark matter experiment with spin-independent WIMP-nucleon scattering sensitivity above 1.4×10−48cm2 for a WIMP mass of 40GeV/c2 and a 1000days exposure. LZ achieves this sensitivity through a combination of a large 5.6t fiducial volume, active inner and outer veto systems, and radio-pure construction using materials with inherently low radioactivity content. The LZ collaboration performed an extensive radioassay campaign over a period of six years to inform material selection for construction and provide an input to the experimental background model against which any possible signal excess may be evaluated. The campaign and its results are described in this paper. We present assays of dust and radon daughters depositing on the surface of components as well as cleanliness controls necessary to maintain background expectations through detector construction and assembly. Finally, examples from the campaign to highlight fixed contaminant radioassays for the LZ photomultiplier tubes, quality control and quality assurance procedures through fabrication, radon emanation measurements of major sub-systems, and bespoke detector systems to assay scintillator are presented