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This protocol details about immunoprecipitation using anti-HA magnetic beads.

This protocol describes how to assess mitophagy using Halo assay developed by Mizushima lab (DOI: 10.7554/eLife.78923).

Activated carbon fiber (ACF) is widely used as an adsorption fiber in air purification systems. In this study, MgO and CuO nanoparticles were immobilized on ACF with enhancement of (3-aminopropyl)triethoxysilane (APTES). The obtained fibers’ coating efficiency, structural deformation, and antimicrobial activities were investigated. The MgO–CuO/APTES/ACF fiber (DA-MC) sample showed high antimicrobial activity (Escherichia coli and Staphylococcus aureus after 24-hour treatment. DA-MC also showed the highest coating efficiency, with no observed structural deformation. The presence of APTES and curing step at high temperature is believed to increase the coating efficiency and thus result in the high antimicrobial activity and also protect the ACF from deformation.

Helicobacter pylori infection is the main cause of gastric cancer. Vacuolating cytotoxin A (VacA) is a H. pylori pore-forming toxin and a key determinant of gastric cancer risk. VacA is secreted as an 88-kDa polypeptide (p88) that upon interaction with host cells induces cytotoxic effects, including cell vacuolation and mitochondrial dysfunction. These effects are currently believed to be due to VacA p88 accumulating inside host cells and forming oligomeric anion-specific channels in membranes of intracellular compartments. However, the molecular nature of intracellular VacA channels in host cells remains undefined. Here we show that VacA p88 does not accumulate inside human epithelial cells, but instead is rapidly processed in endosomes into smaller p31/p28 and p37 products in a manner that precedes VacA-induced vacuolation. VacA processing requires endosomal acidification and concerted cleavage by multiple endo-lysosomal proteases including cathepsins. In situ structural mapping reveals that upon processing, the toxin’s central hydrophilic linker and globular C-terminus are excised, whereas oligomerization determinants are retained. Congruently, the processed products are constituents of a high-molecular-weight complex inside the host cell ─ which we propose is the intracellular, mature and active VacA pore. These findings suggest that VacA exploits human endosomes for proteolytic processing and intracellular activation.Significance StatementHelicobacter pylori is a cancer-causing bacterium that infects the stomach of billions of people worldwide. Vacuolating cytotoxin A (VacA) is an important H. pylori virulence factor and its activity directly correlates with gastric carcinogenesis. Yet despite decades of intense research, the mechanisms underlying VacA activity in human cells remain incompletely understood. Here, we present evidence suggesting that VacA is activated inside human cells by multi-step proteolytic processing involving endo-lysosomal proteases including cathepsins. We also track and identify the functional processed VacA isoforms in host cells. These results revolutionize our understanding of the mechanism of VacA activation in human cells, whilst expanding our knowledge of the diversity of microbial virulence factors that exploit human endo-lysosomes for pathogenesis.

Cargo sequestration is a fundamental step of selective autophagy in which cells generate a double membrane structure termed an autophagosome on the surface of cargoes. NDP52, TAX1BP1 and p62 bind FIP200 which recruits the ULK1/2 complex to initiate autophagosome formation on cargoes. How OPTN initiates autophagosome formation during selective autophagy remains unknown despite its importance in neurodegeneration. Here, we uncover an unconventional path of PINK1/Parkin mitophagy initiation by OPTN that does not begin with FIP200 binding nor require the ULK1/2 kinases. Using gene-edited cell lines andin vitroreconstitutions, we show that OPTN utilizes the kinase TBK1 which binds directly to the class III phosphatidylinositol 3-kinase complex I to initiate mitophagy. During NDP52 mitophagy initiation, TBK1 is functionally redundant with ULK1/2, classifying TBK1’s role as a selective autophagy initiating kinase. Overall, this work reveals that OPTN mitophagy initiation is mechanistically distinct and highlights the mechanistic plasticity of selective autophagy pathways.

This protocol details the procedure of determination of edits in CRISPR-edited cell lines by sequencing.

This protocol details the procedure of generation of CRISPR constructs.

This protocol details the procedure of mito-keima assay to assess mitophagy.

This protocol details the procedure of immunofluorescence assay (IFA).

This protocol details the procedure to assess mitophagy by analysing COXII degradation via Western blotting.

The Vegetarian Festival in Tam Vu Town, Chau Thanh District, Long An Province was born more than 100 years ago. The festival is organized by the local community to commemorate the heroic heroes, pray for thedead spirits and for the peace of local residents. With the spirit of chiseling and opening up, the Vegetarian Festival maintains and develops beautiful cultural values that demonstrate the tradition of “being grateful to ancestors”, the patriotism, the solidarity, the community connection and the noble humanistic spirit, contributing to cultural goals for the socio-economic development of the locality. By combining two basic methods: (1) synthesizing, analyzing the written documents of relevant predecessors and (2) filed surveys,expert and community interviews, this article focuses on evaluating the nature and value of the Vegetarian Festival from 2014 to present. The research initially shows that, despite the strong effects of the current industrialization and urbanization, the Vegetarian Festival in Tam Vu basically retains the tradition of decades ago, becoming a "cultural dialogue" channel between the current Tam Vu community and past traditions, between the people and local authorities, and between the local people. It is the value of “cultural dialogue” that is the resource and driving force to help this festival be preserved and developed until today.

The continuous fresh consumption or juice production of orange fruits (citrus nobilis) has discarded a vast number of orange peels, which has caused significant impacts on environmental issues. This study attempted to utilize discarded orange peels to extract essential oils (EOs) and evaluated their physiochemical properties, antibacterial, antifungal, and antioxidant activities. EOs were extracted via a distillation system using a Clevenger apparatus and gas chromatography - mass spectrometry analysis was employed to characterize their chemical components. The antibacterial and antifungal test were evaluated using a well diffusion method, and antioxidant activity was determined based on DPPH radicals scavenging effect and ferric reducing antioxidant power (FRAP). The obtained EOs with the yield of 3.29 ± 0.24% in which limonene was found to be the most abundant compound in the EOs (90.42%) followed by β-myrcene (4.7%) and α-pinene (1.22%). The result showed that Gram-positive bacterium (Bacillus cereus) was susceptible to the 50% EOs than Gram-negative bacterium (Escherichia coli) with respect to inhibitory zone diameter of 15.00 ± 0.58 mm and 11.33 ± 0.58 mm. The 50% EOs also inhibited nearly 70% of the mycelial growth of Aspergillus flavus as well as exhibiting antioxidant activity with IC50 values of 0.15 ± 0.01 mg/mL and 18.29 ± 0.13 mg/mL for DPPH and FRAP assay, respectively. The orange peel EOs could be a promising alternative to synthetic preservatives in food industry due to their antimicrobial and antifungal activity as well as their antioxidant activity.

Titanium dioxide (TiO2) is a semiconductor photocatalyst widely applied in numerous fields due to possessing prominent photocatalytic properties. However, its practical applications in the form of nanoparticles or powders still have remained several limitations. Recently, novel photocatalytic porous composites have been discovered to be potential alternative approaches. In the present study, nanostructured magnesium-aminoclay-based TiO2 (MgAC–TiO2) was successfully deposited on an activated carbon fiber (ACF) matrix using the sol–gel approach followed by calcination at 350°C in an air atmosphere. The structure and photocatalytic activity of this as-prepared photocatalyst composite were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), the Brunauer-Emmett-Teller (BET), and UV-vis diffuse reflectance spectral analysis. The photocatalytic activity of MgAC–TiO2/ACF was investigated under batch conditions for the removal of methylene blue (MB) in solution under UV irradiation and dark conditions. The results revealed that MB is absorbed by MgAC–TiO2/ACF and that its photodecomposition occurs under UV irradiation. The addition of MgAC can prevent the sintering of TiO2 act as a dispersing agent to create a high specific surface area, and thus enhance photocatalytic efficiency. In addition, ACF in the MgAC–TiO2/ACF composite can additionally improve the photocatalytic activity by hindering electron-hole recombination, which is known as a synergetic effect, and thereby enhancing the photodegradation and removal efficiency of MB.

The formation of autophagosomes and their fusion with lysosomes are key events that underpin autophagic degradation of cargoes. The core ATG8 system, which consists of the ATG8 family of ubiquitin-like proteins and the machineries that conjugate them onto autophagosomal membranes, are among the most-studied autophagy components. Despite the research focus on the core ATG8 system, there are conflicting reports regarding its essential roles in autophagy. Here, we reconcile prior observations of the core ATG8 system into a unifying model of their function that aims to consider apparently conflicting discoveries. Bypass pathways of autophagy that function independently of the core ATG8 system are also discussed.

Macrophages can prevent infections from intracellular pathogens by restricting access to essential nutrients, termed nutritional immunity. With the exception of tryptophan depletion, it is unclear if other amino acids are similarly regulated in infected macrophages. Here, we show that the expression of nutrient transporters in Legionella-infected macrophages is modulated by the short chain fatty acid butyrate. Butyrate prevented the upregulation of the cystine/glutamate exchanger, Slc7a11, in macrophages infected with L. pneumophila, which decreased cellular cysteine levels. Butyrate and the Slc7a11 inhibitor erastin impaired intracellular Legionella replication in macrophages in vitro, with these being restored by exogenous supplementation with cysteine. Butyrate caused increased histone acetylation in infected macrophages, and pan- and class II HDAC inhibitors also restricted intracellular Legionella growth in a cysteine-dependent manner. Intranasal administration of butyrate reduced L. pneumophila lung burdens in mice. Our data suggest that butyrate alters the metabolism of macrophages to promote nutritional immunity by decreasing cysteine levels and that this can be harnessed to treat bacterial lung infections.

The sole proteases of the macroautophagy/autophagy machinery, the ATG4s, contribute to autophagosome formation by cleaving Atg8-family protein members (LC3/GABARAPs) which enables Atg8-family protein lipidation and de-lipidation. Our recent work reveals that ATG4s can also promote phagophore growth independently of their protease activity and of Atg8-family proteins. ATG4s and their proximity partners including ARFIP2 and LRBA function to promote trafficking of ATG9A to mitochondria during PINK1-PRKN mitophagy. Through the development of a 3D electron microscopy framework utilizing FIB-SEM and artificial intelligence (termed AIVE: Artificial Intelligence-directed Voxel Extraction), we show that ATG4s promote ER-phagophore contacts during the lipid-transfer phase of autophagosome biogenesis, which requires ATG2B and ATG9A to support phagophore growth. We also discovered that ATG4s are not essential for removal of Atg8-family proteins from autolysosomes, but they can function as deubiquitinase-like enzymes to counteract the conjugation of Atg8-family proteins to other proteins, a process that we have termed ATG8ylation (also known as LC3ylation). These discoveries demonstrate the duality of the ATG4 family in driving autophagosome formation by functioning as both autophagy proteases and trafficking factors, while simultaneously raising questions about the putative roles of ATG8ylation in cell biology.

Cleansing the cytosol Most human cells, not just those belonging to the immune system, mount protective responses to infection when activated by the immune cytokine interferon-gamma (IFN-γ). How IFN-γ confers this function in nonimmune cells and tissues is poorly understood. Gaudet et al. used genome-scale CRISPR/Cas9 gene editing to identify apolipoprotein L-3 (APOL3) as an IFN-γ–induced bactericidal protein that protects human epithelium, endothelium, and fibroblasts against infection (see the Perspective by Nathan). APOL3 directly targets bacteria in the host cell cytosol and kills them by dissolving their anionic membranes into lipoprotein complexes. This work reveals a detergent-like mechanism enlisted during human cell-autonomous immunity to combat intracellular pathogens. Science , abf8113, this issue p. eabf8113 ; see also abj5637, p. 276

Silver nanoparticles have attracted great interests widely in medicine due to its great characteristics of antibacterial activity. In this research, the antibacterial activity and biocompatibility of a topical gel synthesized from polyvinyl alcohol, chitosan, and silver nanoparticles were studied. Hydrogels with different concentrations of silver nanoparticles (15 ppm, 30 ppm, and 60 ppm) were evaluated to compare their antibacterial activity, nanoparticles’ sizes, and in vivo behaviors. The resulted silver nanoparticles in the hydrogel were characterized by TEM showing the nanoparticles’ sizes less than 22 nm. The in vitro results prove that the antibacterial effects of all of the samples are satisfied. However, the in vivo results demonstrate the significant difference among different hydrogels in wound healing, where hydrogel with 30 ppm shows the best healing rate.

Current models of selective autophagy dictate that autophagy receptors, including Optineurin and NDP52, link cargo to autophagosomal membranes. This is thought to occur via autophagy receptor binding to Atg8 homologs (LC3/GABARAPs) through an LC3 interacting region (LIR). The LIR motif within autophagy receptors is therefore widely recognised as being essential for selective sequestration of cargo. Here we show that the LIR motif within OPTN and NDP52 is dispensable for Atg8 recruitment and selectivity during PINK1/Parkin mitophagy. Instead, Atg8s play a critical role in mediating ubiquitin-independent recruitment of OPTN and NDP52 to growing phagophore membranes via the LIR motif. The additional recruitment of OPTN and NDP52 amplifies mitophagy through an Atg8-dependent positive feedback loop. Rather than functioning in selectivity, our discovery of a role for the LIR motif in mitophagy amplification points toward a general mechanism by which Atg8s can recruit autophagy factors to drive autophagosome growth and amplify selective autophagy., Selective autophagy receptors are thought to selectively recruit Atg8 positive membranes to cargo via their LIR motif. Here, the authors show the LIR motifs in OPTN and NDP52 are dispensable for selectivity, functioning instead to recruit additional receptors and amplify mitophagy.

Summary Mitophagy is a selective garbage disposal pathway that rids cells of excess or damaged mitochondria. In this issue, Ma et al. uncover a role for Friendly in driving depolarisation-induced mitophagy in plants and highlight a physiological role for mitophagy during plant development.

CuO nanoparticles (NPs) have been used for the antimicrobial agent against different pathogenic microorganisms. In this study, CuO NPs are immobilized on the surface of activated carbon fiber (ACF) with the enhancement of (3-aminopropyl)triethoxysilane (APTES) as an organic binder. The obtained fibers are evaluated by coating efficiency, structural deformation, and antimicrobial activities. In the results, APTES can improve the immobilization of CuO on the surface of ACF. Also, the curing of silane layers at high temperature leads to the high coating efficiencies as well as structural reinforcement. The samples with drying step after APTES coating step (denoted as DA-CuO) have the highest antimicrobial activity against both Escherichia coli and Staphylococcus aureus after 24 hours treatment, respectively.

Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome–lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended “shepherd’s staff” architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome–lysosome fusion. The hEPG5–GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5’s overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5., Nam and Cheung et al. describe the structural and biochemical characterization of human autophagy factor EPG5 that functions in autophagosome–lysosome tethering. They show that hEPG5 adopts an extended shepherd’s staff architecture, binds preferentially to GABARAP proteins, and is recruited to mitochondria during mitophagy.

The ATG8 family of proteins regulates autophagy in a variety of ways. Recently, ATG8s were demonstrated to conjugate directly to cellular proteins in a process termed “ATG8ylation,” which is amplified by mitochondrial damage and antagonized by ATG4 proteases. ATG8s may have an emerging role as small protein modifiers. Refereed/Peer-reviewed

SummaryThe sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The Atg4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like Atg8 family (LC3/GABARAPs). We make the unexpected discovery that human Atg4s can directly promote autophagosome formation independently of their protease activity and of Atg8 family processing. High resolution structures of phagophores generated with artificial intelligence-directed 3D electron microscopy reveal a role for the Atg4 family in promoting phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. Atg4 proximity interaction networks stimulated by PINK1/Parkin mitophagy are consistent with roles for Atg4s in protein/vesicle transport and lipid modification. We also show that Atg8 removal during autophagosome maturation does not depend on Atg4 de-lipidation activity as previously thought. Instead, we find that Atg4s can disassemble Atg8-protein conjugates, revealing a role for Atg4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the Atg4 family beyond the Atg8 lipidation axis and provide an AI driven framework for high-throughput 3D electron microscopy.

LC3/GABARAP (hereafter ATG8) conjugation machineries have long been thought to play an essential role in autophagy by driving ATG8 lipidation on autophagosomal membranes. In this issue, Ohnstad et al (2020) describe an ATG8 lipidation bypass pathway which governs autophagy-dependent turnover of NBR1, highlighting that there is more than one road to autophagic degradation.

Titanium dioxide (TiO₂) is a semiconductor photocatalyst widely applied in numerous fields due to possessing prominent photocatalytic properties. However, its practical applications in the form of nanoparticles or powders still have remained several limitations. Recently, novel photocatalytic porous composites have been discovered to be potential alternative approaches. In the present study, nanostructured magnesium-aminoclay-based TiO₂ (MgAC-TiO₂) was successfully deposited on an activated carbon fiber (ACF) matrix using the sol-gel approach followed by calcination at 350°C in an air atmosphere. The structure and photocatalytic activity of this as-prepared photocatalyst composite were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), the Brunauer-Emmett-Teller (BET), and UV-vis diffuse reflectance spectral analysis. The photocatalytic activity of MgAC-TiO₂/ACF was investigated under batch conditions for the removal of methylene blue (MB) in solution under UV irradiation and dark conditions. The results revealed that MB is absorbed by MgAC-TiO₂/ACF and that its photodecomposition occurs under UV irradiation. The addition of MgAC can prevent the sintering of TiO₂ act as a dispersing agent to create a high specific surface area, and thus enhance photocatalytic efficiency. In addition, ACF in the MgAC-TiO₂/ACF composite can additionally improve the photocatalytic activity by hindering electron-hole recombination, which is known as a synergetic effect, and thereby enhancing the photodegradation and removal efficiency of MB.

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.

We report a detailed investigation on the second harmonic generation (SHG) emission from single 150 nm diameter non-centrosymmetric gold nanoparticles. Polarization-resolved analysis together with scanning electron microscopy images shows that these nanostructures exhibit a unique polarization-sensitive SHG that depends strongly on the particle's shape. An analytical approach based on multipolar analysis is introduced to link SHG properties to the nanoparticles' shape. Those multipolar modes can be probed using polarization-resolved SHG. This multipolar analysis offers a physical picture of the relation between shape (size, symmetries, defects, etc.) and nonlinear polarized optical efficiency.

Although photocatalytic materials have been under extensive investigation for several decades, attempts to improve the current well-studied photocatalysts and develop novel photocatalysts for indoor air purification are still ongoing. The present work examines the progress made over the past three years in the application of photocatalysts for degrading gas-phase air pollutants. Three main issues are considered: (1) the basic principles of using photocatalytic degradation for air purification are briefly elucidated, including mass transfer, photocatalytic reaction mechanisms, degradation pathways, and strategies to enhance the photocatalytic efficiency; (2) novel photocatalytic systems with improved photocatalytic performance for the removal of air pollutants are discussed, focusing on state-of-the-art photocatalysts; and (3) future trends in the development of photocatalytic materials for commercialization are considered.