S6K regulates inflammageing, immunosenescence and lifespan through the endolysosomal system

Suppression of Target of Rapamycin complex 1 (TORC1) induces longevity and healthspan in diverse species. Suppression of S6 kinase (S6K) is an essential downstream mediator of the effect of TORC1 on ageing in Drosophila, but the mechanisms at work remain largely obscure. In this thesis, the role of S6K activity in ageing was investigated in Drosophila. I showed that reducing S6K activity ubiquitously in adult flies, using the GeneSwitch system, increased lifespan. Tissue-specific analysis revealed that the lifespan-extending effect was only observed when S6K activity was suppressed specifically in the fat body, the tissue that is functionally equivalent to the liver and white adipose tissue in mammals. I also found that expression of a constitutively active S6K protein in the fat body was sufficient to block the lifespan-extending effect of the TOR inhibitor rapamycin, suggesting that the activity of S6K specifically in the Drosophila fat body is essential for rapamycin-mediated longevity. Using proteomics profiling and network propagation analysis, I found that in young adults, the proteins directly affected by TORC1-S6K signalling were endosome/lysosome-, oogenesis-, and translation-related, while at later age immune-, translation-, and lipid-related processes were altered. Therefore, I tested the effect of TORC1-S6K signalling on fecundity, global translation and triacylglycerol homeostasis, but none of these were altered upon TORC1-S6K manipulation. Although the Drosophila intestine has recently been shown to play a crucial role in TORC1-dependent longevity, I found that S6K activity in the fat body does not affect gut health. By using electron microscopy, lysosome-specific live cell staining and genetic reporter flies, I found that TORC1 inhibition by rapamycin treatment repressed enlarged multilamellar lysosomes in the Drosophila fat body, and that this effect was blocked by elevating S6K activity. Inhibition of autophagy by Atg5 RNAi did not affect lysosomal size, but impairment of late endosomes by expressing a dominant negative form of Rab7 induced lysosomal enlargement. By proteomic analysis, I found that the protein level of Syntaxin 13 (Syx13), a SNARE family protein, was elevated by the inhibition of TORC1-S6K signalling. Furthermore, repressing Syx13 induced enlarged lysosomes in the fat body and overexpressing Syx13 attenuated the S6K-induced enlarged multilamellar lysosomes. Thus, Syx13 is a downstream effector of the TORC1-S6K pathway that mediates the lysosomal structural changes in Drosophila. During ageing in Drosophila chronic inflammation (termed “inflammageing” and mainly represented by the activation of the immune deficiency (IMD) pathway) increases, and pathogen clearance decreases (termed “immunosenescence”). Activation of the IMD pathway, represented by elevated nuclear translocalisation of the NF-κB like transcription factor Relish and expression of Diptericin A, and decline of pathogen clearance in old flies, were both suppressed by reduced S6K activity or rapamycin treatment, whereas activation of S6K in the fat tissue blocked this effect. Syx13 inhibition also blocked the rapamycin-related effect on IMD activation. S6K activation-related IMD activation and immune dysfunction were blocked by Syx13 overexpression. Furthermore, I found that ameliorating inflammageing by knocking down Relish level in the fat body from middle-adulthood on significantly improved bacterial clearance and extended fly lifespan. These results suggest that the TORC1-S6K-Syx13 signalling plays a crucial role in regulating the endolysosomal system and the aged immune system in the fat body of Drosophila. To assess if the regulation of Syx13 by rapamycin is also conserved in mammals, I used mice as a model system. I found that the chronic rapamycin treatment significantly increased Syntaxin 12/13 (Stx12) protein levels in the liver. The impact of reduced TORC1-S6K signalling on immunoageing was assessed in mice by proteomics profiling and network propagation analysis of liver samples from old mice treated with rapamycin and integrating these data with previous published transcriptome datasets of livers of aged mice with rapamycin treatment or S6K1 deficiency. I revealed that immune-related processes, such as inflammation and leukocyte proliferation, were commonly down-regulated by S6K1 deficiency or rapamycin treatment. These findings suggest that the regulation of Syntaxin 12/13 expression and its effect on immune ageing by TORC1-S6K signalling are conserved between flies and mammals. In summary, my work established that suppression of the TORC1-S6K-Syx13 axis ameliorates both inflammageing and immunosenescence in hepatic tissues through the endolysosomal system, and thereby extends longevity in Drosophila, providing a mechanistic explanation for the effects of rapamycin and suppression of S6K on immune function and lifespan in model organisms and, potentially, humans

Toxicity of C9orf72-associated dipeptide repeat peptides is modified by commonly used protein tags

Hexanucleotide repeat expansions in the C9orf72 gene are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts of the expansions are translated into toxic dipeptide repeat (DPR) proteins. Most preclinical studies in cell and animal models have used protein-tagged polyDPR constructs to investigate DPR toxicity but the effects of tags on DPR toxicity have not been systematically explored. Here, we used Drosophila to assess the influence of protein tags on DPR toxicity. Tagging of 36 but not 100 arginine-rich DPRs with mCherry increased toxicity, whereas adding mCherry or GFP to GA100 completely abolished toxicity. FLAG tagging also reduced GA100 toxicity but less than the longer fluorescent tags. Expression of untagged but not GFP- or mCherry-tagged GA100 caused DNA damage and increased p62 levels. Fluorescent tags also affected GA100 stability and degradation. In summary, protein tags affect DPR toxicity in a tag- and DPR-dependent manner, and GA toxicity might be underestimated in studies using tagged GA proteins. Thus, including untagged DPRs as controls is important when assessing DPR toxicity in preclinical models

Repeat length of C9orf72-associated glycine–alanine polypeptides affects their toxicity

G4C2 hexanucleotide repeat expansions in a non-coding region of the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). G4C2 insertion length is variable, and patients can carry up to several thousand repeats. Dipeptide repeat proteins (DPRs) translated from G4C2 transcripts are thought to be a main driver of toxicity. Experiments in model organisms with relatively short DPRs have shown that arginine-rich DPRs are most toxic, while polyGlycine–Alanine (GA) DPRs cause only mild toxicity. However, GA is the most abundant DPR in patient brains, and experimental work in animals has generally relied on the use of low numbers of repeats, with DPRs often tagged for in vivo tracking. Whether repeat length or tagging affect the toxicity of GA has not been systematically assessed. Therefore, we generated Drosophila fly lines expressing GA100, GA200 or GA400 specifically in adult neurons. Consistent with previous studies, expression of GA100 and GA200 caused only mild toxicity. In contrast, neuronal expression of GA400 drastically reduced climbing ability and survival of flies, indicating that long GA DPRs can be highly toxic in vivo. This toxicity could be abolished by tagging GA400. Proteomics analysis of fly brains showed a repeat-length-dependent modulation of the brain proteome, with GA400 causing earlier and stronger changes than shorter GA proteins. PolyGA expression up-regulated proteins involved in ER to Golgi trafficking, and down-regulated proteins involved in insulin signalling. Experimental down-regulation of Tango1, a highly conserved regulator of ER-to Golgi transport, partially rescued GA400 toxicity, suggesting that misregulation of this process contributes to polyGA toxicity. Experimentally increasing insulin signaling also rescued GA toxicity. In summary, our data show that long polyGA proteins can be highly toxic in vivo, and that they may therefore contribute to ALS/FTD pathogenesis in patients

Numerical simulation of the solidification process of Cu-0.45% Sn alloy in upward continuous casting

Upward continuous casting is the key process in the production of contact wire for electric railway. The stability of the process and the quality of the produced billet are directly related to the performance of the contact wire and ultimately the safety of the railway operation. To ensure the quality of continuous-casting billet, the optimal process conditions need to be experimentally determined, which is not only costly but also time-consuming. To facilitate this optimisation process, the simulation of the solidification process of Cu-0.45%Sn alloy in upward continuous casting is described in this paper to assess the influence of casting temperature, upward continuous casting speed, the time of stop-pull, and primary cooling water flow rate on the liquid core length. The results show that the speed of upward continuous casting exhibits a great influence on the liquid core length, while the casting temperature has only little influence. In a certain range of the ratio of stopping time to pulling time, the quality of updraft Cu-0.45%Sn alloy billet is improved; exceeding a certain ratio results in a decrease of the surface quality and an increase in internal and external defects. The liquid core length of the continuous casting rod decreases with the increase of the cold water flow rate, and properties are stable when the flow rate reaches 0.45 m ^3 ·h ^−1 . For a billet with a diameter of 20 mm, the appropriate upward continuous casting process parameters are determined as a casting temperature of 1175 °C, an upward continuous casting speed not exceeding 25 cm·min ^−1 , a ratio of stopping time to pulling time not exceeding 2.13, and a cooling water flow rate of 0.45 m ^3 ·h ^−1

Impact Wear Behavior of the Valve Cone Surface after Plasma Alloying Treatment

Valves are prone to wear under harsh environments, such as high temperatures and reciprocating impacts, which has become one of the most severe factors reducing the service life of engines. As a lightweight ceramic, CrN is considered an excellent protective material with high-temperature strength and resistance to wear. In this study, a CrN coating was applied onto the valve cone surface via double-layer glow plasma surface metallurgy technology. The formation process, microstructure, phase composition, hardness, and adhesion strength were analyzed in detail. Impact wear tests were conducted on the valve using a bench test device. The SEM and EDS results showed that the CrN coating evolved from an island-like form to a dense, cell-shaped surface structure. The thickness of the coating was approximately 46 μm and could be divided into a deposition layer and a diffusion layer, from the outer to the inner sections. The presence of element gradients within the diffusion layer proved that the coating and substrate were metallurgically bonded. The adhesion strength of the CrN coating measured via scratch method was as high as 72 N. The average Vickers hardness of the valve cone surface increased from 377.1 HV0.5 to 903.1 HV0.5 following the plasma alloying treatment. After 2 million impacts at 12,000 N and 650 °C, adhesive wear emerged as the primary wear mode of the CrN coating, with an average wear depth of 42.93 μm and a wear amount of 23.49 mg. Meanwhile, the valve substrate exhibited a mixed wear mode of adhesive wear and abrasive wear, with an average wear depth of 118.23 μm and a wear amount of 92.66 mg, being 63.7% and 74.6% higher than those of the coating. Thus, the CrN coating showed excellent impact wear resistance, which contributed to the enhancement of the service life of the valve in harsh environments

Effect of MEVVA ion implantation on fatigue properties of TC18 titanium alloy

International audienceTo evaluate the effects of implantation on fatigue performance of TC18 alloy, Cr and Zr were implanted into specimens using metal vapor vacuum arc plasma source (MEVVA) with a dose of 1 × l017 ions/cm2. The test results show that the fatigue resistance decreased after Cr implantation and Zr implantation, compared to no implanted specimen. The increase in surface roughness after implantation has been found. To analyze the effects of surface roughness produced by ion implantation, the related surface parameters and estimated effective fatigue stress concentration factors was discussed using A&R model. Apart from the residual surface stress and other factors, the surface roughness may have an influence on the fatigue limits of the implanted samples

Corrosion and Tribocorrosion Behaviors for TA3 in Ringer’s Solution after Implantation of Nb Ions

Ti alloys are prone to corrosion and wear due to the hostile environment in bodily fluids, but the Ti-45Nb alloy is considered to be a promising titanium alloy with excellent biocompatibility and resistance to physiological corrosion. In this study, Nb ions were implanted into a TA3 alloy and the effect on the biological corrosion as well as tribocorrosion behavior of TA3 in Ringer’s solution was systematically investigated. The surface microstructure and XRD results revealed that the implanted samples showed a smoother surface due to the sputtering and radiation damages, and the Nb ions mainly existed in the alloy as the solid solution element. The electrochemical polarization tests showed that the implantation of Nb ions can increase the corrosion potential of the samples, showing a better thermodynamic stability. The tribocorrosion tests showed that the implanted samples exhibited a better thermodynamic stability in a corrosive environment accompanied by wear behavior, and the worn surface showed fewer pitting pits, indicating a better corrosion resistance. However, the abrasive wear and oxidation wear degree of the sample increased because of partial softening of the surface and brittle passivation film

Microstructure, Mechanical and Antibacterial Properties of TiNb-Based Alloy Implanted by Silver Ions

In this study, in order to obtain an antibacterial property for the TiNb-based alloy, the metal vapor vacuum arc (MEVVA) ion implantation technology was applied to implant the silver on the surface of TiNb-based alloy, which brought the change of the microstructures and mechanical properties for the surface of substrate. It was found that the diffusely distributed silver nanoparticles generated on the outermost surface of the implanted layer and the Ag element exist as a solid-solution state in the implanted layer. Meanwhile, the region of the implanted layer mainly constituted nanocrystalline structures based on the analyses of microstructures. Hence, the nanocrystalline strengthening effect formed by high-energy ion bombardment and the solid solution strengthening effect of silver atoms made contributions to the increase of surface comprehensive mechanical properties, including the surface hardness and elastic modulus. Finally, the suitable Ag-implanted specimen can obtain excellent antibacterial ability. Except for the antibacterial mechanism of silver ions release, the dispersed silver nanoparticles on the surface also provide the contact antimicrobial mechanism, which is the Schottky barrier–dependent antimicrobial efficacy of silver nanoparticles

Double Glow Plasma Surface Metallurgy Technology Fabricated Fe-Al-Cr Coatings with Excellent Corrosion Resistance

Double glow plasma surface metallurgy (DGPSM) technology was applied to obtain a Fe-Al-Cr coating on the surface of Q235 carbon steel. The influence of the sample temperature, gas pressure, the distance between the substrate, and the source electrode on the quality of the obtained Fe-Al-Cr coatings was systematically investigated. The results showed that the parameters for DGPSM have a significant effect on the uniformity, particle size, compactness, and thickness of the coating. Under the optimized parameters (sample temperature: 800 °C, gas pressure: 35 Pa, and electrode distance: 15 mm), the obtained Fe-Al-Cr coating contains Fe2AlCr, Fe3Al(Cr), FeAl(Cr), Fe(Cr) solid solution, Cr23C6, and α-Fe(Al), exhibiting excellent corrosion resistance in a 0.5 mol/L H2SO4 solution, which is even better than that of the 304 stainless steel

Tribocorrosion behavior of Nb coating deposited by double-glow plasma alloying

Titanium and titanium alloys are considered as good implant materials, but their poor tribocorrosion resistance limits further development. In this study, niobium coating (DG-Nb) was deposited on TA3 pure titanium (Pure Ti) by double-glow plasma alloying aiming to improve the tribocorrosion resistance of Pure Ti. The properties of the samples were characterized by using X-ray diffraction (XRD), scanning electron microscope (SEM). Open circuit potential (OCP) and coefficient of friction (COF) tests were used to examine the tribocorrosion performance. The results showed that DG-Nb can largely enhance the tribocorrosion resistance of Pure Ti in Ringer’s physiological solution