Neuroinflammation, a major hallmark of Alzheimer’s disease (AD), has recently emerged as a potential therapeutic target. The important role of TNF-α, a master pro-inflammatory cytokine, in AD is well known. However, anti–TNF-α therapies have failed to treat AD, possibly due to the opposing functions of its receptors TNFR1 (neurodegeneration) and TNFR2 (neuroprotection). Thus, in this study, we investigated the effects of activating TNFR2 with a TNFR2 agonist in an AD mouse model. We show that stimulation of TNFR2 mitigates neuropathological features in AD mice by drastically decreasing the production rate of amyloid β and increasing its clearance by glial cells, resulting in an improvement of memory functions. Therefore, TNFR2 activation may be valuable as potential therapy for AD. Tumor necrosis factor-α (TNF-α) is a pleiotropic, proinflammatory cytokine related to different neurodegenerative diseases, including Alzheimer’s disease (AD). Although the linkage between increased TNF-α levels and AD is widely recognized, TNF-α–neutralizing therapies have failed to treat AD. Previous research has associated this with the antithetic functions of the two TNF receptors, TNF receptor 1, associated with inflammation and apoptosis, and TNF receptor 2 (TNFR2), associated with neuroprotection. In our study, we investigated the effects of specifically stimulating TNFR2 with a TNFR2 agonist (NewStar2) in a transgenic Aβ-overexpressing mouse model of AD by administering NewStar2 in two different ways: centrally, via implantation of osmotic pumps, or systemically by intraperitoneal injections. We found that both centrally and systemically administered NewStar2 resulted in a drastic reduction in amyloid β deposition and β-secretase 1 expression levels. Moreover, activation of TNFR2 increased microglial and astrocytic activation and promoted the uptake and degradation of Aβ. Finally, cognitive functions were also improved after NewStar2 treatment. Our results demonstrate that activation of TNFR2 mitigates Aβ-induced cognitive deficits and neuropathology in an AD mouse model and indicates that TNFR2 stimulation might be a potential treatment for AD. Tumor necrosis factor-α (TNF-α) is a master proinflammatory cytokine involved in the regulation of innate and adaptive immunity . TNF-α p5As a consequence, several studies aimed at selective targeting of TNFR1 or TNFR2, instead of completely inhibiting TNF-α. First, targeting TNFR1 by using specific TNFR1 antagonists or sTNF inhibitors resulted in amelioration of inflammation and apoptosis in various in vivo neurodegenerative disease models, such as models of multiple sclerosis , 12, ParHowever, clinical and preclinical studies on the effect of specific therapeutic targeting of TNFR2 in AD are lacking. It is acknowledged that during AD, the deposition of Aβ plaques, one of the main hallmarks of AD pathogenesis, occurs as a consequence of an imbalance between Aβ production and clearance . The β-sPrevious studies have proven that TNFR2 activation is neuroprotective in different disease models; however, it remains to be established whether TNFR2 stimulation has a protective effect against Aβ-mediated neuropathology and amyloid deposition and if this could improve cognitive functions. Therefore, in this study, we tested the hypothesis that selective stimulation of TNFR2 is able to abrogate Aβ-associated neuropathology and cognitive impairments.SI Appendix, Fig. S1 B and C).To assess the potential effects of the TNFR2 agonist NewStar2 in J20 mice on cognition, NewStar2 was administered either centrally via osmotic pumps or systemically via intraperitoneal (IP) injections. After 6 wk of treatment, we had the mice perform the elevated-plus maze (EPM), spontaneous alternation Y-maze, and Morris water maze (MWM) tests A. In theIn the MWM training phase, mice treated IP with NewStar2 had a significantly lower escape latency compared with the animals in the IP PBS control group. No significant difference in the escape latency was found when comparing both osmotic-pump groups. However, the escape latency of the mice of both osmotic-pump groups was equivalent to that of the animals IP treated with NewStar2 D, which SI Appendix, Fig. S1A).Moreover, changes in body weight were evaluated. Animals carrying osmotic pumps had an overall increase in body weight growth compared with animals that received peripheral injections . These results confirm that NewStar2 remains bioactive at 37 °C for at least 6 wk.Additionally, since NewStar2 was administered to the animals via an osmotic pump for a period of 6 wk, we investigated whether NewStar2 would remain stable during this period of time at the animal’s body temperature (∼37 °C). To this end, a cytotoxicity assay using kym-1 cells was performed prior to the start of the animal experiments. Pretreatment of kym-1 cells with 500 ng/mL NewStar2 for 24 h resulted in a significant decrease in cell viability for both freshly conserved NewStar2 and 6-wk preincubated NewStar2  threefold increase in Foxp3-positive Tregs in the hippocampus compared with PBS-treated control mice , a specific marker for regulatory T cells (Tregs), a cell type which plays a key role in modulating immune responses . Mice trrol mice A and D. rol mice B and D. rol mice  C.Next, we examined the differences in the ionized calcium–binding adapter molecule 1 (Iba1) in cornu ammonis sector 1 (CA1), cornu ammonis sector (CA3), and dentate gyrus (DG) hippocampal areas after NewStar2 administration. Iba1 is a broadly used immunohistochemical marker for activated microglia and macrophages . We founSI Appendix, Fig. S3 A and C). In contrast, animals in the osmotic-pump groups did not show any significant differences when comparing treatments, although a tendency toward higher P2RY12 total coverage was observed in the CA3 and DG areas of NewStar2-treated animals .To evaluate the effect of NewStar2 on the inflammatory status of microglia, we investigated the changes in the purinergic receptor P2Y12 (P2RY12) in CA1, CA3, and DG hippocampal areas after NewStar2 administration. P2RY12 is expressed in microglia and platelets and has been identified as a specific marker for resting or nonactivated microglia, which enables the distinction between homeostatic (resting) and activated microglia , 28. We Finally, to explore the effect of NewStar2 on astrocytic activity, we investigated the levels of glial fibrillary acidic protein (GFAP), an astrocyte activity marker. We found that IP administration of NewStar2 resulted in significantly higher GFAP total coverage in all areas of the hippocampus  compared with IP PBS-treated control mice K and L. To further investigate the effect of NewStar2 on microglia, we evaluated the association of Aβ plaques with CD68, a marker for phagocytic microglia. The presence of CD68-positive microglia in the vicinity of Aβ plaques has been related to clearance and degradation of Aβ plaques , 30. We SI Appendix, Fig. S4 A, B and E). Significantly higher PKB/Akt phosphorylation was observed in NewStar2-treated groups at 10, 25, and 50 µM glutamate concentrations compared with control groups . Furthermore, to assess whether the neuroprotective effect of NewStar2 is glutamate dependent, we treated neurons with NewStar2 for 24 h, followed by incubation with a glutamate receptor antagonist (MK-801) prior to glutamate exposure. We observed that incubation with MK-801 prevented the decrease in PKB/Akt phosphorylation in the control groups . However, treatment with NewStar2 resulted in a significantly higher increase in PKB/Akt phosphorylation at the 25 and 50 µM glutamate concentrations . Overall, these results indicate that NewStar2 treatment has a neuroprotective effect in primary cortical neurons, which is not glutamate-receptor dependent.The neuroprotective effect mediated by TNFR2 activation is dependent on the stimulation of the PI3K-PKB/Akt pathway , 31. IncSI Appendix, Fig. S5).Moreover, we tested whether different concentrations of NewStar2 labeled with fluorescein isothiocyanate (FITC) were able to cross the blood-brain barrier (BBB) in an in vitro transcytosis BBB model composed of human cerebral microvascular endothelial (hCMEC)/D3 cells. Addition of both 25 µg and 50 µg of FITC-NewStar2 showed a positive transcytosis passage of ∼3% across the BBB model -HC:sc(mu)TNF80 , which iTo test the stability of NewStar2, a cytotoxicity assay using crystal violet was performed in Kym-1 cells. NewStar2 was placed in an incubator for 6 wk at 37 °C and its bioactivity was compared with that of NewStar2 stored at 4 °C.l-glutamine. Kym-1 cells (1.5 × 104 cells/well) were transferred to 96-well cell-culture plates and treated with 500 ng/mL NewStar2 for 24 h at 37 °C. Cells were washed with PBS and incubated with crystal violet  for 20 min. Crystal violet was removed by intensively rinsing with water and cells were air-dried overnight. Methanol was added to resolve the crystal violet and optical density at 550 nm was measured. Each sample was analyzed in six replicates, using Microsoft Excel and IBM SPSS Statistics 26.0.In brief, Kym-1 cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 5% penicillin/streptomycin, and 5% 4 cells/well) were plated in a 96-well polyethyleneimine-precoated plate. Primary neurons were cultured using neurobasal medium (Life Technologies) supplemented with 2% B-27 supplement, 0.2% penicillin/streptomycin, and 0.25% l-glutamine (Life Technologies). After 48 h, 10 µM cytosine arabinoside (Sigma) was added to the cells for 24 h to prevent nonneuronal cell growth, and the medium was subsequently completely refreshed. After 7 d of culture, neurons were preincubated with 1 µg/mL NewStar2 for 6 or 24 h and were subsequently treated with different concentrations of glutamate  for 1 h. When necessary for the experiment, cells were incubated with a glutamate-receptor antagonist, MK-801 (Sigma), for 1 h prior to glutamate exposure. Then, the medium was refreshed, and neurons were allowed to recover for 24 h. Next, cells were collected for Western blot analyses.Cortical neurons were treated with NewStar2 to assess the potential neuroprotective effect of TNFR2 activation on glutamate-induced neuronal cell death. Primary cortical neurons were prepared as previously described . Primaryg), and the protein concentration of supernatants was measured by the Bradford assay and adjusted to 1 µg/µL. Twenty micrograms of total protein were denatured in Laemmli buffer for 10 min at 70 °C and separated by 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis . Proteins were then transferred to polyvinylidene difluoride membranes and blocked with 5% skim-milk powder solution in Tris-buffered saline (TBS) containing 0.1% Tween 20 for 1 h. Subsequently, membranes were incubated overnight at 4 °C with primary antibodies of the specificity of interest: anti–phospho-Akt Ser-473 ; anti-Akt . After that, membranes were washed with TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase–conjugated secondary antibody goat anti-rabbit  in TBS containing 0.1% Tween 20 for 2 h at room temperature. Immunoreactivity was detected using enhanced chemiluminescence (Pierce Biotechnology) with a ChemiDoc XRS system (Bio-Rad Laboratories). GAPDH expression was analyzed as loading control.The neuroprotective effects of TNFR2 stimulation were evaluated by measuring protein expression levels of PKB/Akt. Cells were washed twice with PBS and directly lysed by addition of 250 µL lysis buffer . Cell lysates were centrifuged at 4 °C . Mice had access to food and water ad libitum and were on a 12:12 light/dark cycle. All animal experiments performed in this study were approved by the animal ethics committee of the University of Groningen .To investigate the effects of NewStar2 at the behavioral and pathological levels, the J20 AD mouse model was used. J20 mice overexpress human APP with two mutations (Swedish and Indiana), and Aβ plaques can be observed in the brain between 5 and 7 mo of age . J20 micn = 12; PBS: n = 11) or systemically via IP injections . The treatment started at 6 mo of age and lasted for 6 wk. After that, animals were subjected to different behavioral tests and, finally, mice were euthanized to collect brain tissue, blood, and cerebrospinal fluid  connected to a cannula into the lateral ventricle, which allows constant infusion of the compound. Pumps delivered either PBS or NewStar2 (10 µg/wk) dissolved in PBS. Before the surgical procedure, pumps were filled with the corresponding solution and incubated in PBS at 37 °C for at least 60 h to ensure immediate pumping at the moment of implantation. Mice were anesthetized with 5% isoflurane and fixed in a Kopf stereotactic frame (Kopf Instruments Model 900). The body temperature of the mice was kept at 37 °C, using a heating blanket, during the whole surgical procedure. One small hole was drilled into the skull for the placement of the cannula in the lateral ventricle , and a second hole was drilled for the placement of an anchoring screw. A subcutaneous pocket was made in the back of the animal to accommodate the pump. The osmotic pump was inserted in the subcutaneous pocket and the cannula fixed on a cannula holder. The cannula was slowly introduced into the first hole at a depth of 2.5 mm. Subsequently, dental cement was added to the cannula, as was an anchoring screw, to properly fix the cannula to the skull. After the dental cement dried, the incision was sutured and mice were returned to their cage. Mice were monitored until total recovery from anesthesia.NewStar2 (2.5 mg/kg in 200 µL of PBS) or PBS were administered systemically twice a week for a period of 6 wk via IP injections.After the completion of the 6-wk treatment, mice were subjected to different behavioral tests to investigate their cognitive function. Tests were performed in the following order: EPM, Y-maze spontaneous alternation, and MWM to minimize the effects of stressful events on subsequent testing procedures. All behavioral tests were performed in a separate experimental room and were conducted by the same experimenter, who was blinded to treatments.Prior to the start of the behavioral tests, mice were habituated to handling by the researcher and to the experimental room. Each mouse was separately handled in the experimental room for 2 min during 5 consecutive days before the start of the behavioral tests.To evaluate anxiety-like behavior, mice were tested in an EPM . The mazTo assess spatial working memory, mice were tested in a Y maze to measure spontaneous arm alternation . The mazTo investigate hippocampus-dependent spatial memory, mice were tested in the MWM . Mice weAfter the last behavioral test was completed, mice were anesthetized with sodium pentobarbital and transcardially perfused with 0.9% heparin saline and 4% paraformaldehyde. Brains, blood and cerebrospinal fluid were collected from each animal. Brains were postfixed for 24 h in 4% paraformaldehyde and then extensively washed with 0.01 M phosphate buffer, after which brains were cryoprotected and dehydrated with 30% sucrose, frozen, and cut in 20 μm–thick coronal sections using a cryostat at −20 °C.2O2) in 0.01 M TBS for 30 min to inactivate endogenous peroxidases. Sections were washed 3 × 5 min in 0.01 M TBS, preincubated with blocking buffer  for 1 h and incubated with primary antibodies (in blocking buffer) at 4 °C for 48 h (anti-Aβ) or 72 h . Next, sections were washed 6 × 5 min in 0.01 M TBS and incubated with Biotin-SP–conjugated secondary antibodies  for 2 h. After washing 6 × 5 min in 0.01 M TBS, sections were incubated in avidin-biotin complex  for 1 h. Subsequently, the staining was developed using 3,3′-diaminobenzidine (DAB) at a concentration of 0.7 mg/mL . The DAB reaction was initiated by the addition of 0.0033% H2O2 and stopped by three quick washes in 0.01 M TBS. Sections were left in TBS overnight at 4 °C. Afterward, sections were mounted onto Menzel Superfrost glass microscope slides (Thermo Scientific), using 1% gelatin, and left to dry overnight. Next, mounted sections were dehydrated through an ethanol to xylol series  and coverslipped using DPX mounting medium (Sigma). The following primary antibodies were used: mouse anti-Aβ1–16 , mouse anti-GFAP , rabbit anti-Iba1 , and rabbit anti-P2RY12 . Images were taken using an Olympus BH2 microscope (Leica QWin Software) at a ×100 , ×200 (Iba1) or ×400 (P2RY12) magnification.To assess the neuropathological effects of NewStar2, several immunohistochemical analyses were completed. All procedures were performed on hippocampal free-floating sections. Sections were washed 3 × 5 min in 0.01 M TBS and then incubated in 0.3% hydrogen peroxide , rabbit anti-BACE-1 , rat anti-CD68 , and rat anti-Foxp3 . Fluorescent images were obtained using the Leica DMI6000 B microscope (Leica Microsystems) at a ×400 magnification (Aβ + CD68) or ×200 (Aβ + BACE-1 and Foxp3).Free-floating hippocampal sections (20 μm) were washed 3 × 5 min in 0.01 M TBS, preincubated with blocking solution  for 1 h, and incubated with primary antibodies (in blocking buffer) at 4 °C for 24 h. Next, sections were washed 6 × 5 min in 0.01 M TBS and incubated with corresponding Alexa Fluor–conjugated secondary antibodies  for 2 h in the dark. Subsequently, sections were washed 3 × 5 min in 0.01 M TBS, mounted onto Menzel Superfrost glass microscope slides (Thermo Scientific) in TBS, and coverslipped using Mowiol (Sigma) as a mounting medium. The following primary antibodies were used: mouse anti-AβFor quantification of Aβ, CD68, GFAP, BACE-1, and P2RY12, ×100 or ×400 magnification images were analyzed using ImageJ. The percentage of coverage was quantified by automatically dividing positively stained structures from the selected regions of interest  by the area of the image field. For quantification of Iba1, both the percentages of coverage and microglial activation were determined in ×200-magnification images. Microglial activation was quantified based on the protocol by Hovens et al.  by calcuNewStar2 was brought to a sodium bicarbonate buffer (pH 9) at a protein concentration of 2 mg/mL using a 10-kDa molecular weight–cutoff dialysis cassette (Thermo Scientific #66380) with three buffer changes over a 24-h period. FITC (Sigma Aldrich #7250) was dissolved in dimethyl sulfoxide at a concentration of 2 mg/mL; 2 mg of NewStar2 and 30 µg of FITC were mixed and incubated for 2 h at room temperature. Unbound FITC was removed by dialysis against PBS (pH 7.4) with three buffer changes over a 24-h period using a 10-kDa molecular weight–cutoff dialysis cassette (Thermo Scientific #66380).2 flasks precoated with 150 μg/mL rat-tail collagen type-I (Enzo LifeSciences) in endothelial basal medium (EBM) 2 (Lonza) supplemented with 1 ng/mL human basic fibroblast growth factor (Peprotech), 5 μg/mL ascorbic acid (Sigma-Aldrich), 1.4 μM hydrocortisone (Sigma-Aldrich), 10 mM HEPES (Gibco), 1% (volume per volume) chemically defined lipid concentrate (Gibco), 5% (volume per volume) FBS, 100 units/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. hCMEC/D3 cells were plated at 50,000 cells/cm2 on 1-μm pore–size polycarbonate membrane Transwell inserts (Corning) and allowed to grow until complete confluency within 5 d. Transwell inserts were coated with 150 μg/mL rat-tail collagen type I. Medium was changed every 48 h. On day 5, cells were washed with prewarmed Hanks' Balanced Salt Solution (HBSS), and 25 μg or 50 μg FITC-NewStar2 was added apically in EBM + 5% FBS, and incubated for 4 h at 37 °C. We added 1,000 μL EBM + 5% FBS medium basally. Apical and basal media were collected in labeled Eppendorf tubes after 4 h. Then, filters were washed once with 500 μL of HBSS, which was added to the apically collected 500 μL of medium. Subsequently, filters were cut out and soaked in 500 μL fH2O for 10 min to osmotically burst cells, and 500 μL EBM + 5% FBS medium was added to the solution. The fluorescence intensity in apical, cellular, and basal media was measured using a FluoStar Optima plate reader (BMG Labtech). Background fluorescence was subtracted from each reading before the relative signal intensity was calculated for each compartment. Relative fluorescence per compartment was calculated by dividing the fluorescence per compartment by the sum fluorescence per well.As previously described , hCMEC/Dt test when comparing two normally distributed groups or Mann-Whitney test when data were not normally distributed. One-way ANOVA followed by Tukey’s multiple comparison post hoc test or two-way ANOVA followed by Bonferroni multiple-comparison post hoc test were used to analyze multiple groups. All statistical tests performed were two-tailed. All data are reported as mean ± SEM and statistically significant differences were considered when P < 0.05. Statistical analyses were performed using GraphPad Prism 8.0 for Windows.Results were analyzed by unpaired Student’s Supplementary File


These are biological activities that are reported for a POT for the first time. Quorum sensing and biofilm facilitate the bacterial colonization, antibiotic resistance and persistence in both the environment and host, and its impairment by POTs can greatly contribute to the control of bacterial infections, such as those caused by multiresistant bacteria. Moreover, antiviral activity was also observed using the enterovirus Qβ. NMR stability studies of P5W30 demonstrate that it remains intact, suggesting its responsibility in the described biological activities. Taken together, our results emphasize the potential biomedical use of POTs, particularly the Preyssler-type POT, to fight antibiotic-resistant MRSA strains and their ability to form biofilm, besides being a promising antiviral agent.Besides showing the antibacterial activity of the Preyssler-type polyoxotungstate (POT) P2W18, P2W17, P2W15, and Preyssler P5W30 type structures to differently affect Gram-negative and Gram-positive microorganisms, either susceptible or resistant to antibiotics. The compound P5W30 showed the highest activity against the majority of the tested bacterial strains in comparison with the other tested POTs  that did not show inhibition zones for the Gram-negative bacteria, A. baumanii I73775, E. coli DSM 1077, E. coli I73194, K. pneumoniae I7092374, and P. aeruginosa C46281). Generally, the results evidenced that Gram-positive bacteria are more susceptible to the POTs tested. The compound P5W30 was the one most active against S. aureus ATCC 6538 and MRSA16, reaching <0.83 mg·mL−1 (100 μM) and 4.96 mg·mL−1 (600 μM), respectively. Moreover, it was verified by NMR spectroscopy that the most promising POT, P5W30, remains intact under all the experimental conditions, after 24 h at 37 °C. This prompted us to further evaluate the anti-quorum sensing activity of P5W30 using the biosensor Chromobacterium violaceum CV026, as well as its antibiofilm activity both individually and in combination with the antibiotic cefoxitin against the methicillin-resistant Staphylococcus aureus 16 (MRSA16). P5W30 showed a synergistic antibacterial effect with the antibiotic cefoxitin and chloramphenicol against MRSA16. Moreover, the antibiofilm activity of P5W30 was more pronounced when used individually, in comparison with the combination with the antibiotic cefoxitin. Finally, the antiviral activity of P5W30 was tested using the coliphage Qβ, showing a dose-dependent response. The maximum inactivation was observed at 750 μM (6.23 mg·mL−1). In sum, P5W30 shows anti-quorum sensing and antibiofilm activities besides being a potent antibacterial agent against S. aureus and to exhibit antiviral activities against enteric viruses.The increase in bacterial resistance to antibiotics has led researchers to find new compounds or find combinations between different compounds with potential antibacterial action and with the ability to prevent the development of antibiotic resistance. Polyoxotungstates (POTs) are inorganic clusters that may fulfill that need, either individually or in combination with antibiotics. Herein, we report the ability of the polyoxotungstates (POTs) with Wells-Dawson P In the group of critical and high-priority ones are the designated ESKAPE bacterial pathogens . The ESKAPE pathogens are deadly, infectious bacteria that are brilliant at developing multiresistant patterns. Apart from the fact that they are genetically different, their mechanisms of resistance consist of the previously mentioned antibiotic target change, inactivation, decreased uptake, and drug efflux pumps (15−n)−  anion, have shown great potential as antibacterial ) at −80 °C, except Chromobacterium violaceum CV026, which was maintained in Luria Broth Base (LB) . The culture media Mueller–Hinton broth (MHB) and Gelose Mueller–Hinton (GMH) were purchased from Biokar Diagnostics (Beauvais). Prior to use, bacteria were transferred to fresh BHI agar plates and incubated at 37 °C. The recovery of Streptococcus pneumoniae D39 was performed in BHI supplemented with 5% sheep blood  at 37 °C under microaerophilic conditions using an anaerobic jar.The tested microorganisms are indicated in 6[α-PV2WVI18O62]·14H2O (abbreviated P2W18) mono-lacunary Wells-Dawson K10[α2-PV2WVI17O61]·20H2O (P2W17), tri-lacunary Wells-Dawson K12[α-PV2WVI15O56]·24H2O (P2W15), and Preyssler (NH4)14[NaPV5WVI30O110]·31H2O (P5W30) (31P and 183W NMR spectroscopy (The POTs used in this study are the intact Wells-Dawson K (P5W30) . The POTtroscopy . Stock stroscopy .183W NMR and 31P NMR spectra were recorded with a Bruker FT-NMR spectrometer Avance Neo 500 MHz  at 25 °C. Chemical shifts were measured relative to 1 M Na2WO4 and 85% H3PO4. 183W NMR samples were prepared in 2.7 mL solvent with a POT concentration of 10 mM and measured in 10 mm tubes. The experimental time for 183W NMR was ca. 60 h, with a standard pulse program at 20.836 MHz and a 63° flip angle with 1 s relaxation delay. Subsequently, 31P NMR spectra were measured at 202.53 MHz in standard 5 mm tubes.600nm = 0.8–1.0) was transferred into 40 mL of liquefied MH agar medium. The mixture was poured into a sterile Petri dish, which was kept at room temperature, in a laminar flow cabinet, until solidification of the inoculated culture medium. Then, using an inverted sterile Pasteur pipette, 6 mm diameter wells were made into which 40 µL of each compound at the concentrations to be tested were dispensed: for P5W30, 0.83 mg·mL-1 (100 µM), 1.65 mg·mL-1 (200 µM), 2.48 mg·mL-1 (300 µM), 3.31 mg·mL-1 (400 µM), and 4.13 mg·mL-1 (500 µM). Incubation was conducted at 37 ± 1 °C for 24 h. The assay with the S. pneumoniae D39 bacteria was carried out in Columbia agar  supplemented with 5% sheep blood  and under microaerophilic conditions. Three biological replicates and two technical replicates were performed. After 24 h incubation, the inhibition zone was determined , 1.65 mg·mL−1 (200 μM), 3.31 mg·mL−1 (400 μM), 4.96 mg·mL−1 (600 μM), 6.61 mg·mL−1 (800 μM), and 8.26 mg·mL−1 (1000 μM). The antibiotic chloramphenicol (30 μg·mL−1) was used as control. A set of wells containing only the culture medium was included as the negative control. Three biological and three technical replicates for each strain were used. The incubation of the microplate was performed at 37 °C for 24 h. The bacterial growth was followed by spectrophotometry (OD600 nm) in a microplate reader . The MIC value was considered the lowest concentration of the compound that caused the inhibition of the bacterial growth (95–100%). The lowest concentration that did not allow the recovery of cells in GMH plates was considered the minimum bactericidal concentration (MBC). The MIC values for the MRSA16 strain for the antibiotics erythromycin, vancomycin, cefoxitin, and chloramphenicol were determined and the resistant profile was established according to the conventional breakpoints 10-  POT is [α2-PV2WVI17O61]10−. It is worth noting that solutions of P2W18 , mono-lacunary Wells-Dawson K10[α2-PV2WVI17O61]·20H2O (P2W17), tri-lacunary Wells-Dawson K12[α-PV2WVI15O56]·24H2O (P2W15), and Preyssler-type (NH4)14[NaPV5WVI30O110]·31H2O (P5W30), on different Gram-negative and Gram-positive strains, either susceptible or resistant to antibiotics  that did not show inhibition zones for the Gram-negative bacteria, A. baumanii I73775, E. coli DSM 1077, E. coli I73194, K. pneumoniae I7092374, and P. aeruginosa C46281). S. aureus ATCC 6538 was the most susceptible, followed by S. pneumoniae D39 to the four polyoxotungstates tested. No inhibition zone was observed against E. coli DSM 1077 by the action of P5W30, in contrast to the multiresistant strain E. coli I73194, which showed a slight susceptibility that increased with the concentration , and P2W18 (>2.5×), in comparison with MRSA15, and the lowest MIC value difference was observed for P5W30 (>1.5×). It is important to highlight that the strain MRSA16 showed resistance to erythromycin that, according to EUCAST , 200 μM [1.65 mg·mL−1], 300 μM [2.48 mg·mL−1], and 400 μM [3.31 mg·mL−1]  and with the combination of P5W30 with the antibiotic cefoxitin at concentrations of 85 μM + 470 μM (0.7 mg·mL−1 + 2 μg·mL−1). The 2× MIC value of P5W30 and the combination of P5W30 with the antibiotic was selected, taking into account the higher resistance of sessile cells that is not affected either by P5W30 at the MIC value of or by the antibiotic cefoxitin at the MIC value. The results are represented in p < 0.0001) both from the treatment with P5W30 individually or in combination with the antibiotic cefoxitin after 6 h of treatment. However, the exposure of sessile cells to P5W30 alone was more detrimental (p < 0.05) in comparison with the combination  in comparison with the control  in the disruption of sessile cells in comparison with the combination (5W30 to penetrate the matrix of exopolysaccharides that impregnates the aggregated cells in the biofilm. It is possible that the combination of P5W30 with the antibiotic cefoxitin at concentrations of 85 μM + 470 μM (0.7 mg·mL−1 + 2 μg·mL−1), although in a more limited way, can disrupt the aggregated cells by interfering with the exopolysaccharide layer. It is important to stress that the antibiotic cefoxitin acts by inhibiting the biosynthesis of the bacterial cell wall by binding to transpeptidases , and it is resistant to the action of extended-spectrum β-lactamases [For the evaluation of the antibiofilm properties, the MRSA16 cells were allowed to form biofilms in coverslips over 24 h, and afterwards, were treated for 6 h and 24 h with the compound Pbination A. The ex control B. Howevebination B. As expAs for the anti-quorum-sensing activity, to the best of our knowledge, the current study is the first that describes the antibiofilm action of a POT compound.5W30 was evaluated against the enterovirus Qβ at concentrations of 250 μM (2.08 mg·mL−1), 500 μM (4.15 mg·mL−1), and 750 μM (6.23 mg·mL−1). The impact of the compound on the phage infectivity is represented in 5W30 tested. The lowest reduction value (p < 0.0001) was observed at a concentration of 250 μM, achieving the maximum reduction at the highest concentration tested of 750 μM (reduction of 8.11± 0.14 Log10 PFU·mL−1)  . The col viruses . The abi viruses  and the  viruses . It is p5W30 was also able to impair quorum sensing and biofilm formation, as illustrated in 5W30 action and in general for POMs, several potential mechanisms have been evoked and recently summarized [In sum, in addition to the antibacterial and antiviral activities, we showed that Pmmarized ,37,39,725W30 with the ability to affect MRSA cells, we demonstrated that P5W30 also displays other properties, such as anti-quorum sensing and antibiofilm properties. These are biological activities that are reported for a polyoxotungstate for the first time. Quorum sensing and biofilms facilitate bacterial colonization, antibiotic resistance, and persistence in both the environment and host, and its impairment by POT can greatly contribute to the control of bacterial infections, such as those caused by multiresistant bacteria. Moreover, antiviral activity was also observed using the enterovirus Qβ. NMR stability studies of P5W30 demonstrate that it remains intact, suggesting that it is an active species in the described biological activities. Taken together, our results emphasize the potential biomedical use of POTs, particularly Preyssler-type, to combat antibiotic-resistant MRSA strains and their ability to form biofilms, in addition to being a promising antiviral agent. The molecular mechanisms of action of the various biological activities described still need to be elucidated, which will lead to a new understanding on a molecular basis.In addition to showing the antibacterial activity of P


The Pab1 RRM2 domain interacts with eIF4G1 BOX3, the canonical interaction site, but also with BOX2, a conserved element of unknown function to date. The RNA1 region interacts with RNA through a new RNA interaction motif and with the Pub1 RRM3 domain. This later also interacts with eIF4G1 BOX1 modulating its intrinsic self-assembly properties. The description of the biomolecular interactions involving eIF4G1 to the residue detail increases our knowledge about biological processes involving this key translation initiation factor.Yeast eIF4G1 interacts with RNA binding proteins (RBPs) like Pab1 and Pub1 affecting its function in translation initiation and stress granules formation. We present an NMR and SAXS study of the N-terminal intrinsically disordered region of eIF4G1 (residues 1–249) and its interactions with Pub1, Pab1 and RNA. The conformational ensemble of eIF4G1 The eukaryotic translation initiation factor eIF4G is a central player in the regulation of protein expression. First, it enhances translation initiation, the rate-limiting step, by mRNA 3′/5′-end circularization . StructuSaccharomyces cerevisiae that have similar domain architecture (https://mobidb.bio.unipd.it/P39935): in the N-terminus , middle (residues 481–591) and C-terminus (residues 870–952). The remaining regions contains the domains for interaction with eIF4A , arranged as bead-on-string, and various low complexity domains (LCD). These proteins are constituents of biological condensates and, in response to temperature increase or acidic pH, undergo ticipate . Pab1, Pticipate .Knowing the structural details of the protein/RNA network involving eIF4G1, Pab1, and Pub1 will help to propose integrative models of translation regulation that include activation and repression pathways. However, because these interactions involve IDRs their study is difficult with X-ray crystallography or Cryo-EM. Therefore, we performed an NMR/SAXS structural study of the eIF4G1 N-terminal IDR and generate an “all-atoms” ensemble stabilized by cation-π and π-π transient contacts. Furthermore, we mapped the binding sites of Pab1, Pub1, and RNA, and show the simultaneous binding of the two RNA Binding Proteins (RBP), reconfigure the conformation of eIF4G1 IDR inducing self-assembly through its conserved element BOX1. The implications of these findings for translation regulation and biomolecular condensation are discussed.Saccharomyces cerevisiae eIF4G1 contains three boxes of about 15–20 conserved residues each, and a conserved RNA binding region (RNA1), within the first 249 amino acids  with low dispersion in the proton dimension and sharp signals  that are not compatible with a fully disordered state. We identified several minor species  that correspond to cisPro conformers, and two uncommon chemical isomerization forms at positions 41 and 76 that were assigned to isoaspartates  revealed the presence of an α-helix within BOX3 (1H-15N-HSQC-NOESY-1H-15N HSQC spectrum (187-234)  indicates long-range PREs for S200C and Q109C mutants  . No furt2 values . The exi mutants . In a prribed in  and Supp15N-labeled eIF4G11-249, and then measured PRE. The PRE fingerprint shows that eIF4G11-249 can self-interact through contacts involving BOX1 and RNA1, as these elements “sense” the presence of the spin label in trans  NOE-derived distance restraints and 13C-derived φ/ψ dihedral restraints for the parts of the protein that are well-folded  and 2) ambiguous restraints for the cation-π and π-π interactions (involving Arg and Tyr) that we propose as dominant transient residue-residue contacts. We refer to these latter types of restraints as “knowledge-based” and used cautions to avoid biases in their selection . We calculated 80,000 eIF4G11-249 structures and sorted them using our own greedy algorithm that optimized the fitting to experimental PRE data stepwise. As a seed for the ensemble the protocol chooses the structure with minimum PRE violations and then continues building up the ensemble stepwise using the same criteria , minimizes violations). The target function reached a minimum value relatively quickly and increased slowly afterwards  genetic algorithm . As expected, the back-calculated PREs from the collection of EOM ensembles showed poorer agreement with the experimental data, but neatly reflected the overall trends regarding the long-range contacts in eIF4G11-249  and/or computational approaches . Here weterwards . We arbiterwards . Because4G11-249 . To valilgorithm  to modellgorithm . Importa4G11-249 .1-249 ensemble of conformers showed a flexible α-helix in BOX3 and, despite the presence of long-range contacts, no predominant tertiary fold was found. The average Cα-Cα distance maps revealed that local and long-range contacts were prevalent between BOX1 and RNA1-1/RNA1-2 boxes, and between these three elements and BOX3 , in which BOX1 plays the chief role.In summary, these data showed that eIF4G11-249 by analysis of changes on the 1H-15N HSQC spectra, which indicated three putative binding sites for RRM3 in eIF4G11-249 , which shed light on the key elements required for molecular recognition  interact with a shallow cleft in Pub1 RRM3, defined by the contact between helix α1 and strand β2. The eIF4G1 Y41 is inserted into a small cavity and contacts I358, I367 and F370 of Pub1, whereas the N42 and N43 of eIF4G1 are more exposed but contact L368 and F366 of Pub1. The latter residue was previously shown to be important for eIF4G1-Pub1 interaction  for calculation of the structure of the complex. To overcome this technical problem, we constructed recombinant chimeras of eIF4G1ub1 RRM3 . The NMRub1 RRM3  right, wub1 RRM3 , left, sognition . The streraction . The int15N-eIF4G11-249  are transferred to free Pub1 RRM3.During the course of the study, we found that Pub1 RRM3 interacts differently with different eIF4G1 constructs. Titration with eIF4G1peptides  left. Hoosspeaks  right. T1-184 BOX1 mutants , whereas deletion of BOX2 (ΔBOX2) still caused the Pub1 aggregation-like pattern. Thus, BOX1 is the element causing the differential binding mode of Pub1 RRM3, but how. Our hypothesis is that Pub1 RRM3 binding causes conformational rearrangements on eIF4G11-184 exposing aggregation-prone BOX1 (1-184 experiences a complex degradation (1-184 (and eIF4G11-184 ΔBOX1) samples age to hydrogels that bind Congo-red  trigger its aggregation-prone properties.We further investigate the cause of Pub1 RRM3 behavior and discovered that it recover the weak-binding pattern with several eIF4G1one BOX1 . Interesradation  that we radation . New C-tongo-red , a dye uongo-red , but eIF1-249 is more stable . Consistent with this hypothesis, titration of Pab1 RRM12 into the eIF4G11-249/Pub1 RRM3 complex retrieve the Pub1 RRM3 broad spectrum, suggesting that the Pab1 RRM12 binding to BOX2 and BOX3 acts as a second switch that releases the BOX1 oligomers. NMR data showed that Pub1 RRM3 did not interact with Pab1 RRM12  and Ficoll 70 (200 g/L) to simulate crowding in the cellular environment. Dynamic light scattering (DLS) indicated that large aggregates were formed in some protein combinations  revealed the presence of discrete rounded particles . We titrate the eIF4G11-82 construct with three poly (A) probes and found that the strength of the interaction is higher with longer oligos  , a well-known RNA binding motif  . These sng motif , is lessDuring the last 40 years stress granules have been described in multiple organisms and upon different stressors, and have been extensively characterized both morphologically and in their composition . However1-249 at the residue level, its complex conformational landscape and its interactions with other SG principal components: the RBPs Pub1 and Pab1. The N-terminus of eIF4G1 contains several short segments (BOXes) that are conserved in the Saccharomycetales family and act as short linear interaction motifs (SLiMs) for various purposes: 1) intramolecular and intermolecular self-recognition; 2) specific recognition of RBPs; and 3) RNA recognition.Along this line, our work provides an extensive NMR characterization of eIF4G11-249 conformational ensemble, among the few atomistic models of an IDP  . In the e system . Under ne system . We showe system . Now ourSaccharomyces cerevisiae genomic DNA using the DNA polymerases KOD or Pfu. These DNA fragments were cloned into a pET28-modified vector that contains an N-terminal thioredoxin A fusion tag, an internal 6xHis tag and a TEV protease site. eIF4G1 mutants were obtained using the Quick-change Lightning Kit and specific DNA primers. Plasmids corresponding to mutant and wild-type proteins were transformed into E. coli BL21 (DE3) competent cells and expressed in kanamycin-containing (30 μg/L) LB medium.Plasmids and proteins used in this work are described in the 15NH4Cl (1 g/L) and/or 13C-glucose (4 g/L). Cultures of eIF4G1 and its mutants were grown at 37°C until OD600nm = 0.6–0.8, when they were induced with 0.5 µM IPTG for 4 h. Pab1 and Pub1 cultures, after reaching OD600nm = 0.6, were transferred to 25°C for induction with IPTG overnight (12–16 h).For isotope labelling of samples, a K-MOPS derived minimal medium  was suppFor purification of all recombinant proteins, cell pellets were resuspended in lysis buffer , lysed by sonication and cleared by ultracentrifugation. The supernatant was purified by metal affinity chromatography using a HiTrap™ 5 ml column and elution with 25 mM potassium phosphate buffer pH 8.0, containing 300 mM NaCl and 300 mM imidazole. The samples containing the fusion protein were exchanged into 20 mM Tris pH 8.0 (in the case of the Pab1 construct this buffer was supplemented with 1 mM DTT), and digested overnight at 4°C with homemade TEV protease. In the case of the Pub1 and Pab1 constructs, the samples were re-loaded onto the HiTrap nickel column to capture the protease, the cleaved N-terminal part of the fusion protein and the undigested protein. The flow through was further purified by ion exchange using an anion exchanger column (Q 5 ml) for all proteins except for Pub1 RRM3 that was purified with a cation exchange column (SP 5 ml). In either case, proteins were eluted with a linear salt gradient (to 1 M NaCl). In the case of the eIF4G1 construct, we observed that the second nickel column negatively affected protein stability and aggregation; we therefore purified the protein away from the uncleaved protein, thioredoxin A and TEV using a cation exchange column (SP 5 ml). Finally, the purified proteins were concentrated and the buffer was exchanged according to their intended use.1-249 were 15 mg/ml, 10 mg/ml, 8 mg/ml, 5 mg/ml, 3 mg/ml, and 1 mg/ml. The final SAXS curves were generated using PRIMUS and deposited in the SASBDB under the code SASDP88 (https://www.sasbdb.org/data/SASDP88/678c641ui8/).SAXS experiments were performed using the P12-EMBL beamline at the DESY synchrotron in Hamburg and were analysed with ATSAS software. All data were collected in batch using 25 mM potassium phosphate pH 6.5 and 25 mM NaCl. The concentrations used for analysis of eIF4G12O) and experimental data were acquired at 25°C on a cryoprobe-equipped Bruker AV800 MHz spectrometer. Assignment of the backbone 1H, 15N and 13C atoms was achieved by following the standard methodology. The 3D HNCA, HNCO, HN(CO)CA, CBCA(CO)NH and CBCANH experiments were used for backbone assignment and 3D (H)CCH-TOCSY were recorded to assign side chain resonances [(1-249) and 34,517 (eIF4G135-49-Pub1 RRM3). The 15N backbone amide relaxation T1 and T2 parameters were measured with series of 1H-15N spectra of standard inversion-recovery and Carr-Purcell-Meiboom-Gill sequences (CPMG). NMR spectra were processed using TOPSPIN v4.1 (Bruker) and NMRPipe, and analyses were done with CcpNmr Analysis.All samples were prepared in NMR buffer . NMR experiments were carried out at 298 K in a Bruker Avance III 800 MHz spectrometer equipped with a cryogenic triple resonance probe. Two samples (isotropic and anisotropic) were prepared and couplings (J and J + D) were measured with 15N-HSQC-DSSE (In Phase Anti Phase IPAP). Experiments were processed using TOPSPIN v2.1 and NMRPipe, and were analyzed with the program CcpNmr Analysis.The filamentous phage Pf1 was used at a final concentration of 20 mg/ml to induce weak alignment of eIF4G11-249 mutants (S200C and Q109C) were chemically modified using the following protocol. Mutant protein samples (600–700 µM) were pre-treated with 5 mM DTT for 2 hours at room temperature. The DTT was then eliminated by fast buffer exchange into 25 mM Tris pH 9.0 and 25 mM NaCl using a Nap-5 desalting column. Labelling with 4-(2-Iodoacetamido)-TEMPO was initiated immediately after column elution by adding a tenfold molar excess of the probe dissolved in ethanol (25 mM spin label stock). The reaction was allowed to proceed for 30 min at room temperature in the dark. The excess iodoacetamide label was quenched with 10 mM 2-mercaptoethanol for 10 min, and afterwards the protein adduct was exchanged into 25 mM potassium phosphate pH 6.5, 25 mM NaCl and 1 mM DTT for later use. The NMR samples were prepared in 5 mm tubes sealed in an N2 atmosphere to avoid reduction by air, and high resolution 1H-15N HSQC spectra were recorded for the oxidized state (active spin label). Subsequently, the spin label was reduced with 10 μM ascorbate  and angular restraints  using the program Cyana. Protein assignments were obtained by comparison with other eIF4G1 constructs and confirmed by triple resonance 3D spectra: CBCA(CO)NH, HNCACB and HNCO.The NMR structure of the eIF4G37-51 were constructed, with eIF4G135-49 fused either to the N- or C- terminus of Pub1 RRM3. Of these constructs, only the N-terminal fusion (eIF4G135-49) proved to have the right topology and the structure was determined using a similar protocol to the analysis of the structure of Pub1 RRM3  were calculated with the program Cyana 3.0 using: 1) experimental NOE-derived distance restraints for the BOX3 region; 2) π- π interactions between Tyr, Phe and Trp; and 3) π-cation interactions between Arg and Tyr/Phe/Trp. The latter two interaction types were referred to as knowledge-based constraints (K-BC) and were included, given the importance of these types of contacts for IDP interactions (see main text for references). For each individual structure calculation, the origin residue (Arg/Tyr/Phe/Trp) was randomly selected (80% probability) and ambiguous restraints were generated for the other interaction partner (Arg/Tyr/Phe/Trp). In this way, each of the individual structure calculations contains a unique set of knowledge-based distance restraints. This protocol ensures high variability by avoiding biases of specific pairwise iterations. Thus, the final interactions present on each conformer are freely selected during the calculations. A similar protocol was followed to calculate the structures  of eIF4G11-249 dimers, using the same experimental restraints and ambiguous Tyr-Tyr contacts as dimerization driving interactions.eIF4G11-249 monomers and dimers. Correlation time τc was estimated from the averaged T1/T2 and d was estimated from the distances between amide backbones (N) and Q109/S200 side-chains (Cβ). We next used a home-made greedy algorithm to select the ensembles that better reproduced the PRE profiles. The algorithm calculates the residual to the experimental data (Q109 and S200) and chooses, as a seed, the conformer that better agrees  with the data. In the next steps, the residuals were computed across 2, 3,.., n structures always choosing the combination with minimal violations. The procedure was repeated until the ensemble size reached 2,000 conformers . The first 500 structures were included in the final eIF4G11-249 monomer and eIF4G11-249 dimer ensembles (PRE-derived).The theoretical PRE-derived intensity ratios were calculated using equations in  Supplem for the 1-249 monomers  and dimers . These two pools were combined with the genetic algorithm in the EOM program to model the curve with a fixed size ensemble (50 structures). The percentages of each pool were freely selected by the algorithm and the procedure. The procedure was repeated 10 times, obtaining a 500-member set. It should be noted that some of the structures are repeated between individual EOM calculations. The theoretical values of the PREs, and other structural properties, were calculated as averages across the different ensembles using home-made perl scripts.We used the EOM protocol to fit the SAXS data. Pools of theoretical SAXS curves were constructed for the eIF4G11-249, eIF4G11-249 ∆BOX1, Pub1 RRM123 and Pab1 RRM12) were prepared from extensively centrifuged stocks , and were filtered (0.22 μm) in PBS buffer and 200 g/L Ficoll 70 stock. Samples were mixed thoroughly and placed in a plastic cuvette (Eppendorf) for measurements. Individual correlation curves were recorded (10 acquisitions of 10s) every 15 min over a 5 h period.The DLS measurements were carried out at 25°C in a DynaPro Titan (Wyatt Technologies) instrument and were analysed with Dynamics V6 software. Protein mixtures , using protein to probe ratios of 1:3. The coupling reaction was carried out in the dark in PBS (pH 7.4) buffer for 30 min on ice and the unreacted probe was removed by size exclusion chromatography using a Nap-5 column. Samples for visualization were prepared by mixing eIF4G11-249 with Pub1 RRM123 and Pab1 RRM12 in different combinations and at concentrations of 1, 5 and 20 μM respectively, in PBS, 0.1 mM DTT (pH 7.4) buffer and 200 g/L Ficoll 70. Samples, that contain 1 μM of fluorescently labelled protein (Alexa Fluor 488-labelled eIF4G1 and/or Alexa Fluor 647-labelled Pub1 or Pab1) for visualization, were placed in silicone chambers that were glued to coverslips and were visualized with Leica TCS SP2 or TCS-SP5 inverted confocal microscopes with a HCX PL APO ×63 oil immersion objective . Alexa 488 and Alexa 467 were excited using 488 and 633 nm laser excitation lines, respectively. The concentration of the various Alexa-labelled proteins was kept at 1 μM and the solution was supplemented with unlabeled proteins to reach the above-mentioned concentrations. Various images were registered for each sample, corresponding to different observation fields.eIF4G1


Legionella pneumophila uses hundreds of effector proteins to manipulate multiple processes of the host cells to establish a replicative niche known as Legionella-containing vacuole (LCV). Biogenesis of the LCV has been known to depend on host small guanosine triphosphatases (GTPases), but whether bacterial effector GTPases are also involved remains unknown. Here, we show that an ankyrin repeat containing effector LegA15 localizes directly in host lipid droplets (LDs), leading to Golgi apparatus fragmentation of the host cells by hijacking the host vesicular transport factor p115. LegA15 is a GTPase with a unique catalytic mechanism, unlike any eukaryotic small GTPases. Moreover, the effector LegA15 co-opts p115 to modulate homeostasis of the host LDs in its GTPase-dependent manner. Together, our data reveal that an atypical GTPase effector regulates the host LDs through impeding the vesicle secretion system of the host cells for intracellular life cycle of Legionella.The intracellular bacterial pathogen  Legionella effector LegA15 as a GTPase hijacks host p115 to regulate lipid droplets and subvert the host secretory pathway. Legionella pneumophila, a Gram-negative bacterial pathogen, causes an atypical pneumonia known as legionnaires’ disease (Legionella invades macrophages and generates a Legionella-containing vacuole (LCV), a unique compartment for bacterial replication to take place. The LCV is successively diverted away from the host endosomal-lysosomal degradation pathway as a result of the bacterial Dot/Icm type IV secretion system (T4SS), which translocates more than 300 effectors into the host cell  are key regulators of intracellular membrane trafficking and the fusion with target membranes  domain that is known to mediate protein-protein interactions in eukaryotic cells (L. pneumophila wild-type (WT) strain containing a LegA15–overexpressing plasmid  containing its ANK domain and C-terminal unknown domain, respectively, in HEK293T cells, and found that the ANK domain but not the C terminus was colocalized with the LDs . TogetheWe next sought to identify host proteins that interact with LegA15. We expressed Flag-LegA15 in HEK293T cells and treated the cells with formaldehyde and performed an immunoprecipitation (IP) using the anti-Flag antibody, followed by mass spectrometric analysis . More thS-transferase (GST) pulldown experiment. The ND but not the CD strongly interacted with LegA15 , a coiled-coil region, and a highly acidic C-terminal tail. To map their interaction regions, we produced proteins of two separate regions 1 to 651 (ND) and 651 to 962 (CD) of p115 in addition to its full length for a glutathione h LegA15 , suggesth LegA15 .p115 interfered with cell proliferation, we found that ectopic expression of LegA15 in this cell line did not induce cell death as seriously as it did in WT cells . This experiment thus suggested that the cytotoxicity of LegA15 is dependent on p115.Because ectopic expression of LegA15 was toxic to mammalian cells, we next asked whether its cytotoxicity depends on p115. We established a stable line from HEK293T cells with its p115 substantially knocked down by short hairpin RNA (shRNA) and further transiently expressed LegA15 in this cell line . AlthougWT cells . ApproxiLegionella effector AnkX (The p115 protein as a biomarker for Golgi plays a key role in vesicular transport between the ER and the Golgi apparatus by tethering the coat-protein complex I (COPI) . In prestor AnkX . Howeverp115 knockdown cell line built above  (+ (Kd) for binding by GTP and GDP are 1.38 and 20.5 μM, respectively.To further understand the molecular mechanism of LegA15, we solved its crystal structure at a resolution of 2.59 Å (table S2). LegA15 contains an N-terminal ANK and a C-terminal domain (CTD) . The CTD+)  of 1.0 Å]  mutant combined with Arg291, Arg408, and Lys367 as well as the K367A mutant almost abolished the GTP binding. Together, these data strongly supported that LegA15 is a GTP-binding protein.We next solved a binary structure of LegA15 bound with an unhydrolyzable GTP analog 5′-guanylyl imidodiphosphate (GDPNP) at a resolution of 2.27 Å . GDPNP if 1.0 Å] . GDPNP af 1.0 Å] . The guakcat/Km was about 1.0 × 10−3 μM−1 s−1 and the maximum velocity (Vmax) was about 8.0 × 10−3 μM s−1 for LegA15 on GTP . However, the deletion did not affect bacterial growth in mouse BMDM cells (fig. S10B). Consistently, LegA15 was previously shown not important for the bacterium to grow in Drosophila cells . The overexpressed LegA15 was still associated with LDs, but their sizes seemed smaller in the p115 depletion cells  or GTPase-activating proteins (GAPs) to regulate host small GTPases, such as RalF  2-aminoethanesulfonic acid (ACES)–buffered yeast extract (AYE) broth with additional thymidine (200 μg/ml) for the thymidine auxotrophic mutant and its derivatives.All Escherichia coli strains DH5α and BL21 Rosetta DE3 were grown in lysogeny broth (LB) shaking at 37°C. Antibiotics were added to culture media at the following concentrations: ampicillin (100 μg/ml), kanamycin (30 μg/ml), and streptomycin (100 μg/ml), unless specified. All strains were stored at −80°C in LB supplemented with 10% (v/v) glycerol.legA15-deleted L. pneumophila strain, the legA15 polymerase chain reaction (PCR) products using primers PL1/PL2 and PL3/PL4 were digested with Sal I/BamH I and BamH I/Sac I and inserted at Sal I/Sac I sites of pSR47s , kanamycin (20 μg/ml), and streptomycin (100 μg/ml). Bacterial mutants were screened by PCR from those grown on the medium containing 5% sucrose, as described previously , 3.2 to 4.0] and bacterial motility.The 5 and 2 × 105 per well for intracellular growth and immunofluorescence assays, respectively, for 1 day before infection. U937 macrophage cells were differentiated in fresh medium by phorbol myristate acetate (10 ng/ml) for 36 hours as described previously (L. pneumophila at a multiplicity of infection (MOI) of 0.05 were synchronized at 2 hours by washing three times with fresh medium to remove all extracellular bacteria. Infected macrophages were continued to culture at 37°C in the presence of 5% CO2. To measure the bacterial growth, the macrophages were lysed with 0.2% saponin. Colony forming unit of infected bacteria was determined by plating a series of lysate dilutions on solid CYE medium.Mouse BMDMs were seeded into Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum in 24-well plates at 4 × 106 cells per 100-mm dish and transfected with plasmid pCMV-3×Flag-LegA15 using a liposomal transfection reagent (Yeasan). The cells were collected at 24 hours of post-transfection and lysed on ice for 10 min with an IP buffer  by a sonication for 30 s. The cell lysis supernatant was collected by a centrifugation at 13,000 rpm for 10 min and incubated with Flag affinity resins at 4°C overnight followed by three washes with the IP buffer. The captured proteins were eluted with 50 μl of 3×Flag peptide (200 μg/ml) for 30 min on ice. The elution was boiled with an SDS loading buffer and separated on 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with appropriate antibodies.HEK293T cells were plated at a density of 2 × 104 cells per well were transfected with pCMV-LegA15-mCherry for 18 hours. Cells were then fixed with 4% (w/v) paraformaldehyde and permeabilized with 0.1% Triton X-100 for 10 min. Organelles were stained by their specific protein antibodies (1:200). LDs were stained using dye BODIPY. Nuclei were stained by dye 4′,6-diamidino-2-phenylindole (DAPI).For intracellular LegA15 localization, HEK293T cells seeded on coverslips in a 12-well plate at a density of 1.0 × 10For the LegA15 and p115 colocalization, HEK293T cells were cotransfected with pCMV-LegA15-mCherry and pEGFP-p115 for 18 hours. The endogenous p115 was shown by anti-p115 antibody followed by Alexa 594–conjugated secondary antibody.5 per well were seeded on coverslips in 24-well plates and were infected with L. pneumophila at 0.1 MOI. After 12 hours, the cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. The extracellular bacteria were stained with anti-Legionella antibody before permeabilization. Total bacteria and LegA15 were stained with the anti-Legionella antibody and an anti-LegA15 antibody, respectively, after the cells were permeabilized by 0.2% Triton X-100 for 10 min. The polyclonal anti-LegA15 antibody was produced by immunizing a rabbit with 6×His-LegA15 protein purified as described below. Images were acquired by a laser scanning confocal microscope . To analyze number and size of lipid droplets in cells, the cells were randomly selected and calculated in ImageJ (https://imagej.nih.gov/ij).For bacterial infections, Raw264.7 cells at density of 2 × 10E. coli strain BL21/DE3 transformed with each expression construct was cultivated in LB medium at 37°C until its optical density OD600= 0.8 and induced by 400 μM isopropyl β-d-1-thiogalactopyranoside for 16 hours at 18°C. To prepare selenomethionine-substituted LegA15 protein, the E. coli expression strain grew in M9 medium supplemented with 2 mM MgSO4, 2 mM CaCl2, vitamin B (0.25 mg/ml), ampicillin (100 mg/ml), d-glucose (2 g/liter), NH4Cl (1 g/liter), selenomethionine (40 mg/liter), and other 19 amino acids (40 mg/liter).6×His-tagged proteins were purified using a following protocol. The cell pellets were suspended in a lysis buffer containing 50 mM tris (pH 8), 500 mM NaCl, 10% (v/v) glycerol, 5 mM β-mercaptoethanol, protease inhibitors, and additional 20 mM imidazole and lysed using sonication on ice. The cell lysate was centrifuged for 30 min at 18,000 rpm, and its supernatant was collected and incubated with nickel sepharose beads (Sangon) for 2 hours at 4°C. The beads were washed by the lysis buffer and eluted by the lysis buffer with an additional gradient of imidazole: 50, 100, 200, and 500 mM. The best fractions that contained our protein of interest were pooled, concentrated, and further purified using a Superdex 200 size exclusion column  in 20 mM tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM dithiothreitol (DTT). The best fractions of protein peaks were pooled, concentrated to 15 mg/ml with an Amicon Centrifugal filter (Millipore), flash-frozen in liquid nitrogen, and stored at −80°C.A280) and calculated using their theoretical extinction coefficients.GST-tagged p115 and its fragments were expressed using their pET-GST constructs and purified similarly as His-tagged proteins except using glutathione sepharose beads (Thermo Fisher Scientific) and captured proteins were eluted with the lysis buffer and additional 20 mM reduced glutathione. Final protein concentration was measured at absorbance at 280 nm , and 25% (v/v) polyethylene glycol (PEG) 3350 at 25°C in about 4 days. After optimization, the best crystallization buffer was 2 mM MgCl2, 0.1 M bis-tris (pH 6.2), and 22% PEG 3350. The crystals of SeMet-LegA15 were obtained under similar conditions. Complex of LegA15 and GDPNP was prepared by mixing LegA15 (10 mg/ml) and 10 mM GDPNP, and their crystals grew in 0.17 M ammonium acetate, 0.085 M sodium citrate (pH 5.6), 25.5% (v/v) PEG 4000, and 15% glycerol at 25°C in 1 week. The LegA15 crystals were cryoprotected by brief soaking in the crystallization buffer with additional 15% (v/v) PEG 200 and flash-frozen in liquid nitrogen. The LegA15/GDPNP crystals were cryoprotected by brief soaking in their crystallization buffer added with 15% glycerol and 5 mM GDPNP and flash-frozen in liquid nitrogen. Diffractions of native and SeMet-substituted protein crystals were collected at Shanghai Synchrotron Radiation Facility (SSRF) . Datasets were indexed, integrated, and scaled with HKL2000 software suite (http://pymol.org).Crystallization of 6×His-LegA15 was performed at 25°C using a sitting drop diffusion method by mixing 0.4 μl of protein (10 mg/ml) with equal volume of reservoir solution. The crystals initially grew in 0.2 M MgCl6 cells per dish, six of which were transfected with pCMV-3×Flag-LegA15 plasmid, while the other three were transfected with empty pCMV-3×Flag vector as a control. The cells after 18 hours of transfection were washed twice by PBS, cross-linked with 1% (v/v) formaldehyde for 10 min at 37°C, and terminated by 125 mM glycine for 10 min at room temperature. The cells were then harvested by centrifugation at 2000 rpm for 3 min, washed twice by PBS, and lysed by sonication for 1 min in a lysis buffer . The cell lysate was rocked for another 4 hours at 4°C and centrifuged at 13,000 rpm for 30 min. Supernatant of the lysate was collected into a new tube and incubated with 50 μl of Flag affinity resin at 4°C overnight. The affinity resin was then collected by a centrifugation at 2000 rpm for 5 min, washed in lysis buffer three times, and boiled for 10 min with 1× SDS loading buffer. All coimmunoprecipitated proteins were separated on 12% SDS-PAGE gel and further identified by mass spectrometrical analyses.HEK293T cells were plated in nine 10-cm dishes at a density of 2 × 10GST-p115 fusion proteins of 100 μg each were incubated for 30 min with 30 μl of GST beads in a binding buffer . After extensive washing, the beads were further rocked overnight at 4°C with 200 μg of 6×His-LegA15 in 1 ml of the binding buffer. Unbound proteins were washed off using the buffer, and trapped proteins were eluted from the beads by 1× SDS loading buffer and resolved by 12% SDS-PAGE followed by Western blot using appropriate antibodies.The impact of GTP/GDP on the p115 and LegA15 interaction was examined with 1 mM GTP or GDP added in the binding and washing buffers. After extensive washing in the pulldown buffer, the captured proteins were released by 1× SDS loading buffer, resolved by 12% SDS-PAGE, stained with Coomassie brilliant blue and analyzed by Western blot using appropriate antibodies.The following sequences were designed for USO1/p115 targeting shRNA: forward, 5′-GCAGCTTTGTACTATCCTAAT-CTCGAG-ATTAGGATAGTACAAAGCTGC-TTTTTGTAGAATTCTCGACCTCG-3′; and reverse: 5′-GCAGCTTTGTACTATCCTAAT-CTCGAG-ATTAGGATAGTACAAAGC-TGCCCGGTGTTTCGTCCTTTCC-3′.6 cells each well by liposome transfection reagent Turbofect (Thermo Fisher Scientific). The cells were changed into fresh medium after 8 to 12 hours and continued to culture for another 24 to 36 hours. The viral particles were then harvested by a centrifugation at 3000 rpm for 5 min at room temperature. A new batch of HEK293T or U937 cells were infected by the lentivirus in 60-mm dishes, and continued to culture for several generations with puromycin (10 μg/ml) until a cell line was obtained. The efficiency of p115 knockdown was evaluated by Western blot using anti-p115 antibody.The shRNA sequences were inserted into pLKO.1 lentiviral vector by “Quickgene” method (+ was prepared at a concentration of 50 mM in 100 mM tris-HCl (pH 7.5) and further neutralized by 1 M NaOH. All nucleotides were diluted in the binding buffer to a working concentration of 20 mM. Titrations of these nucleotides to LegA15 were performed at room temperature using a Cary Eclipse fluorescence spectrophotometer . The wavelength interval (Δλ) between the excitation and emission wavelengths was fixed for 15 nm. Both slit widths of excitation and emission were set to 5 nm. The ligands were titrated up to 20 μM. Relative fluorescence intensity was calculated by dividing Ft (fluorescence intensity of each titrate) to F0 .Synchronous fluorescence titration was performed in a binding buffer [50 mM tris-HCl (pH 7.5) and 50 mM NaCl]. LegA15 protein purified for crystallization experiments above was placed in a 2-ml quartz cuvette (10 mm by 10 mm). GTP/ATP/CTP were prepared at a concentration of 100 mM in 100 mM tris-HCl (pH 7.5) and further neutralized by 1 M NaOH. NAD+ stocks were prepared above and diluted in the same buffer at concentration of 1 mM. GDP was prepared as the same as GTP and used at concentration of 1 mM. Titrations were set for 20 injections of 2 μl each with 200-s intervals. Baseline subtraction and data analysis were performed using NanoAnayze. Heat spikes were integrated and fit with 1:1 binding model. The first injection of each experiment was excluded from the analysis.The ITC experiments were performed using a microcalorimeter Affinity ITC  at 20°C. The LegA15 protein purified above was diluted into a buffer of 50 mM tris-HCl (pH 8.0) and 50 mM NaCl to a final concentration of 0.1 mM. GTP and NADet al. , 50 mM NaCl, and 10 mM MgCl2] for 30 min at 35°C using 2.5 μM nucleotide-bound LegA15, and released phosphate was measured by a colorimetric ATPase/GTPase activity assay kit (Expedeon) according to manufacturer’s manual.The GTP hydrolysis assay followed Mishra E. coli phosphate binding protein  was expressed, purified by its N-terminal His tag, and labeled with 7-diethylamino-3-[N- carbamoyl] coumarin  to produce a sensitive phosphate probe PBP-MDCC. The reactions were set with 1 μM LegA15 purified above, 4 μM PBP-MDCC, and a GTP gradient in a buffer  at 35°C. The release of inorganic phosphate was monitored continuously for 30 min at an excitation wavelength of 425 nm and an emission wavelength of 457 nm. The GTP hydrolysis background was measured without LegA15 under the same condition and substracted in the final analysis. The absolute phosphate concentrations were derived from a linear curve of fluorescence using 4 μM PBP-MDCC with a concentration gradient of standard phosphate. Initial velocity (V0) was calculated by fitting to exponential model function as described by Shutes and Der . Mammalian cells were plated in a 96-well plate at a density of 5 × 10et al.  on ice for 30 min and centrifuged at 10,000 rpm for 5 min. In addition, their supernatant was collected into a new tube and also heated at 65°C for 10 min. The SEAP activity was detected at 410 nm according to the manufacturer’s manual and repeated at least three times.SEAP assay was performed by an SEAP reporter assay kit (Invivogen). HEK293T cells were cultured in 96-well plates at a density of 1 × 10r) for colocalization analysis and significant difference analysis of one-way analysis of variance (ANOVA) and Student’s t test were calculated all in GraphPad Prism 8 software . P < 0.05 was considered as significant difference.Pearson correlation coefficient (Resources of key materials in this study are listed in table S5.


The specific surface area and pore volume of the best OPAC sample was found to be 3150 m2 g−1 and 1.64 cm3 g−1, respectively. The hydrogen uptake of all the OPAC samples was established to be above 3 wt% (at 77 K and 1 bar) with the highest being 3.67 wt% (800 °C). Additionally, the OPAC-800 °C achieved a specific capacitance of 169 F g−1 at a specific current of 0.5 A g−1 and retained a capacitance of 149 F g−1 at 5 A g−1 in a three electrode system using 3 M KNO3. A symmetric supercapacitor based on the OPAC-800 °C//OPAC-800 °C electrode provided a capacitance of 158 F g−1 at 0.5 A g−1. The maximum specific energy and power was found to be 14 W h kg−1 and 400 W kg−1, respectively. Moreover, the device exhibited a high coulombic efficiency of 99.85% at 5 A g−1 after 10 000 cycles. The results suggested that the high surface area graphene-like carbon nanostructures are excellent materials for enhanced hydrogen storage and supercapacitor applications.High surface area activated carbons (ACs) were prepared from a hydrochar derived from waste onion peels. The resulting ACs had a unique graphene-like nanosheet morphology. The presence of N (0.7%) and O content (8.1%) in the OPAC-800 °C was indicative of  Graphene-like activated carbons (ACs), with excellent properties for enhanced hydrogen storage and supercapacitor applications, were prepared from waste onion peels. The type of carbonaceous feedstock and the activation process play critical roles in determining the properties of the resulting ACs.3,8 For example, a recent study based on cellulose acetate-derived ACs with oxygen functional groups achieved a record-breaking hydrogen uptake value i.e. gravimetric capacity of 8.9 wt% at −196 °C and 30 bar.9 On the other hand, N-doped ACs have also received great interest in electrochemical energy storage applications since N presence leads to enhancement of the specific capacitance.10,11 The carbonization of N-containing biomass is also found to be cheaper and presents a straightforward approach for obtaining N-doped carbons devoid of using nitrogen-containing chemical precursors.Carbon-based porous materials have widely been used for hydrogen storage as well as in electrochemical energy storage applications. Even though these materials can be derived from many different feedstocks,Allium cepa L.) are known to contain high oxygen functionalities due to the flavour precursors such as (+)-S-alk(en)yl cysteine sulphoxides (CSOs), trans-S-1-propenyl cysteine sulphoxide (PeCSO) and S-propyl cysteine sulphoxide (PCSO).12 The lachrymatory effect of onions is caused by presence of volatile propanthial S-oxide.13 A study by Venkateswarlu et al.14 confirmed that oxygen richness of carbonised onion peel aided As(iii) adsorption from contaminated water. On the other hand, the presence of N and S functionalities in ACs have also been reported to enhance hydrogen storage.15–17 Even though there are some studies that have reported the use of onion to derive ACs, these studies either used discarded green onion leaves18 or used un-optimised activation procedures19 which masked their true potential. Concerning the use of green onion leaves versus onion bulb scales, a study conducted by Lancaster et al.20 reported that the flavour precursors moves from the leaf blades towards the bulb scales which means that the concentration of oxygen-rich functionalities are often higher in onion bulb scales than in the leaves.Owing to the need to identify a carbon precursor with high concentrations of oxygen and nitrogen functional groups, waste onion peels were considered as an attractive feedstock. In this case, onions  and an attractive pore volume (0.94–1.64 cm3 g−1). The OPAC material obtained at 800 °C (OPAC-800 °C) had the highest hydrogen uptake capacity of 3.67 wt% at 1 bar and 77 K. When used as the electrode material for supercapacitors in 3 M KNO3, the OPAC-800 °C achieved a specific capacitance of 169 F g−1 at 0.5 A g−1 in a three-electrode system. A fabricated symmetric device using the OPAC-800 °C displayed a capacitance of 158 F g−1 with corresponding specific energy and power of 14 W h kg−1 and 400 W kg−1 at 0.5 A g−1.Herein, we report on the utilization of onion peels as the carbonaceous feedstock and KOH as the activating agent to produce nanosheet-like porous carbons. The effect of activation temperature on the structural and textural properties of the onion peel derived activated carbons (OPAC) was investigated. The obtained ACs exhibited a high specific surface area , argon, Ar  and hydrochloric acid, HCl  were used during the preparation of the onion derived ACs.2.2via hydrothermal carbonization which involved the heating of an aqueous mixture of the onion waste (300 g L−1) in an autoclave at 200 °C for 4 h. The resulting hydrochar was recovered by filtration, washed several times using deionised water and thereafter dried at 90 °C for 12 h.In this work, red onion skin wastes (peels) were collected, washed to remove excess dirt and then dried at 60 °C for 12 h. Thereafter, the onion-based hydrochar was obtained 2.3−1. The pre-carbonized hydrochar was then mixed thoroughly with KOH at a mass ratio of 1 : 4 (hydrochar : KOH) and a few drops of water added to form a slurry which was then allowed to dry at 70 °C for 12 h. The mixture was then heated up to a pre-activation temperature of 400 °C for 30 min and then to an activation temperature of 600 °C for 1 h in a chemical vapour deposition reactor. The product was mixed with 10 wt% HCl for 24 h followed by washing with deionised water to a neutral pH and thereafter dried in an oven at 100 °C for 12 h to give the AC denoted as OPACs-600 °C. A similar procedure was used at activation temperatures of 700 °C and 800 °C to produce OPACs-700 °C and OPACs-800 °C, respectively.Pre-carbonization of the onion hydrochar was performed at 500 °C for 1 h under argon atmosphere with a ramp rate of 5 °C min2.42.4.1−1 under a mixture of air (60 mL min−1) and N2 (40 mL min−1). Micrometrics ASAP 2020 HD analyser was used for N2 and H2 sorption measurements at liquid N2 temperature (77 K) and up to 1 bar. The samples were firstly degassed for 8 h at 200 °C, under vacuum conductions (down to 10−7 bar), to remove any traces of adsorbed water and/or other physisorbed gases. X-ray photoelectron spectroscopy (XPS) analysis was conducted using a Physical Electronics Quantum 2000 and the data analysed using the CasaXPS software.The ACs morphology was determined using a Zeiss Ultra Plus 55 field emission scanning electron microscope (FE-SEM) and a JEOL JEM-2100F transmission electron microscope operated at working voltage of 2 kV and 200 kV, respectively. The degree of graphitization of the ACs was investigated using a T64000 micro-Raman spectrometer (532 nm laser excitation wavelength and power of 5 mW). A PANalytical X'Pert Pro powder diffractometer was used for X-ray diffraction analysis. A Mettler, Toledo, TGA/SDTA 851e instrument was used for thermogravimetric analysis (TGA) which was conducted upto ∼1000 °C at a ramping rate of 10 °C min2.4.2N-methyl-2-pyrrolidone (NMP). The resulting mixture was then coated onto a 1 cm2 nickel foam and dried under vacuum at 80 °C for 6 h. Electrochemical measurements were conducted using the Bio-Logic VMP300 potentiostat . Varying scan rates were employed during the collection of cyclic voltammetry (CV) data. Also, different specific current values were used for galvanostatic charge–discharge (GCD) measurements. A frequency range of 10 mHz to 100 kHz was used during the electrochemical impedance spectroscopy (EIS) measurements. A three-electrode conformation that consisted of the as-prepared OPAC material (as working electrode), glassy carbon counter electrode and Ag/AgCl reference electrode was assembled for electrochemical measurements in a 3 M KNO3 electrolyte solution. A coin cell configuration employing a Whatman separator and 3 M KNO3 was used for a symmetric device. The discharge curve of the GCD plot was used to calculate the specific capacitance for a half cell using m depicts the total mass of the electrode material, I is the current applied, Δt being the discharge duration, Cel is the specific capacitance of a single electrode, and ΔV refers to the device voltage window.The onion-derived activated carbon electrode material, acetylene carbon black and polyvinylidene fluoride (PVDF) were mixed following a mass ratio of 80 : 10 : 10 and made into a slurry by adding a few drops of 3.3.114 reported the generation of graphene-oxide like 2D carbon sheets after the pyrolysis of waste onion sheaths at 700 °C for 2 h in N2. Since the peels were obtained from mature onions, Turnbull et al.22 had earlier reported that the bulb cells of mature onions are often larger and thin walled. Therefore, we can postulate that upon the pre-carbonization of the onion hydrochar, the carbon wall tends to expand and this process can be accelerated by the expulsion of carbon dioxide during the activation step to lead to the creation of the observed nano-sheets that resembles graphene sheets.−1 characteristic of the breathing mode of sp2 carbon atoms as well as the presence of sp3 hybridized amorphous carbon domains within the AC structure.23,24 A G peak attributed to the Raman active E2g in-plane vibration mode of sp2 atoms in graphite was observed at 1586 cm−1.25ID/IG ratios relative to the activation temperature can be ascribed to the creation of more defects within the carbon matrix  diffraction plane of graphite structure.26 However, for the OPAC 800 °C sample, this peak is barely visible and implies that graphitization decreased with the increase in the activation temperature. This observation correlates well with the Raman analysis presented in θ ∼ 24° was noted. This could suggest that a possible rearrangement of the structure and the creation of layer structure defects occurred due to K intercalation into the carbon structure.27,28 Fig. S2The upshift in the D band position as well as the observed increase in n matrix . Besides3.22 sorption isotherms of the three ACs generated from the three temperature variations are presented in 29 The sharp gas uptake in all the samples at low partial pressure region is a confirmation that most of the exposed surface resides mostly inside the micropores. This observation is further confirmed by the pore size distribution curves presented in i.e. 1.05 and 1.08 nm, respectively). However, OPAC 800 °C sample exhibited a trimodal pore size distribution having pores positioned at 1.08, 1.48 and 2.31 nm. The presence of pores above 2 nm is an indication of the presence of mesopores which is further corroborated by the occurrence of the slight hysteresis loop in the isotherm.The N2 g−1) whereas the samples from 600 °C and 700 °C activation temperatures had 2706 m2 g−1 and 2241 m2 g−1, respectively. The temperature-dependent increase in the surface area can be attributed to the creation of more pores at higher temperatures. Typically, the KOH reacts with the carbon during the activation process to give K2CO3 and metallic K which intercalates into the carbon matrix.30 The K2CO3 then reacts with carbon resulting in a partial etching of carbon atoms, emission of CO gas and the intercalation of more K thus, creating additional pores on the carbon surface at higher activation temperatures (700 °C and 800 °C). Finally, the removal of the intercalated potassium by the washing of the final product with HCL increases the porosity of the carbon nanosheet. The unprecedented high surface areas are exceptional for biomass-derived carbons considering that the activating agent used was the conventional KOH without the inclusion of other special activation procedures. When compactivation strategy was applied by Mokaya's group,31 the compaction approach lead to ACs with 25% higher specific surface area. It is believed that the unexpected high specific surface areas obtained in our case could be due to the nature of the obtained morphological features that had sheet-like surface structures. Almost similar morphological features were obtained when activation of glucose was done using potassium oxalate.3 However, the specific surface areas were found to be relatively lower (1270–1690 m2 g−1) compared to our current study except in the case when a mixture of melamine and potassium oxalate was utilised. From The surface areas of the obtained ACs are presented in 3.3322 C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C , sp3 C–C/C–N  and CO bonds , respectively.33,34 The higher percentage of the sp2-hybridized carbons as compared to the sp3 carbon can be ascribed to the restoration of the graphitic carbons within the AC matrix at the high activation temperature (800 °C). From O), 532 eV (O–C) and 533.5 eV (O–C–O). 35,36 The presence of pyridinic-N and pyrrolic-N is known to readily promote carbon surface wettability, which consequently enhances the electrochemical capacitive properties.35,37,38The elemental composition of the OPAC-800 °C sample was determined using X-ray photoelectron spectroscopy. The integral areas of the deconvoluted C 1s, O 1s and N 1s peaks were used to calculate the atomic percentages present in the material. After pre-carbonization and KOH activation at 800 °C, the oxygen, carbon and nitrogen contents were 8.1 at%, 91.2 at% and 0.7 at%, respectively. The low nitrogen content can be ascribed to the loss of nitrogenous products at the high activation temperature.3.42 uptake that was above 3 wt% at 77 K and 1 bar  was comparatively higher than values often reported for typical porous carbons which are usually within the range of 2–3 wt%.9 Most importantly, the best hydrogen uptake value of 3.67 wt% up to 1 bar at 77 K obtained using the onion peels derived activated carbons is superior to most reported data for other related materials28,39–44, as listed in 2 adsorption capacity45–48 and hence the observed enhancements of the performance of the activated carbon in this study.The hydrogen sorption isotherms are presented in 2 gravimetric capacity with increasing surface area commonly observed in nanoporous materials.49 It is also thought that the presence of oxygen functionality (as confirmed by XPS) could have also played a role in the observed high H2 adsorption capacity. In this regard, an earlier study by Blankenship II et al.9 had reported that a combined effect of high surface area, oxygen-rich functionality and high microporosity contributed to the enhancement of gravimetric hydrogen storage capacity. The researchers further alluded that the oxygen beneficial effect is more pronounced at low pressure (∼1 bar) region owing to the fact that the interaction between the hydrogen and the material surface is more intimate when compared to the higher pressure ranges where hydrogen uptake is most likely to happen by space filling mechanisms. Even though high H2 uptake values are often reported for analyses conducted at cryogenic temperatures, doping the sorbent materials with heteroatoms or inclusion of metal particles (such as Pt) can enhance hydrogen storage capacities at room temperature.50 These strategies could be applied to the OPACs materials obtained in this study.As shown in 3.53 electrolyte solution. −1. The operating cell potential of 0.8 V was chosen as no overpotential peak as observed in Fig. S3a51 However, the CV curve for the OPAC-600 °C was slightly distorted suggesting that the material has more oxygenated functional groups.52−1 specific current in an operating cell potential of 0.0 to 0.8 V. The calculated specific capacitance values at 1 A g−1 were 61, 67 and 77 F g−1 for the OPAC-600 °C, OPAC-700 °C and OPAC-800 °C, respectively. It was found that the OPAC-800 °C gave the highest current response as well as the longest discharge time when compared to the others. From 3. Fig. S3b–dversus specific current displaying a similar trend to what was observed in the positive potential range. From Fig. S3d,−1 when measured at a specific current value of 1 A g−1. The higher specific capacitance of the OPAC-800 °C can be ascribed to its higher surface area that provided better electrolyte ion accessibility to the pores within the carbon matrix. Good rate capability was observed for all the electrode materials.The evaluation of electrochemical performance of the as-synthesized OPAC electrode materials was based on a three-electrode configuration using a 3 M KNO53–55 The ESR was measured to be 0.90, 0.91 and 1.14 Ω for OPAC-600 °C, OPAC-700 °C and OPAC-800 °C, respectively. The OPAC-600 °C depicted a large semicircle with a charge transfer resistance value of 1.02 Ω. In contrast, the OPAC-800 °C had the smallest semicircle corresponding to a charge transfer resistance value of 0.03 Ω and the shortest diffusion length, which are responsible for the improved electrochemical performance.The impedance properties of the OPAC materials at an operating frequency of 10 mHz to 100 kHz  was stud−1) both in the negative and positive operating potentials, ranging from −0.8 to 0 V and 0 to 0.8 V, respectively. The figures show that the materials could operate well even within a reversible potential range.−1 depicting the fast ion diffusion kinetics on the reversal of potential.56−1 to 6 A g−1 in 3 M KNO3 electrolyte solution. The calculated specific capacitance values were 169, 157, 151, 150, 149 and 149 F g−1 at 0.5, 1, 2, 3, 4 and 5 A g−1 in the potential range of −0.8 to 0.0 V. This shows that a good rate capability of 88% was maintained at higher specific current of 5 A g−1. At positive potential ranges of 0.0 to 0.8 V, the specific capacitance values were 83, 77, 74, 71, 70 and 69 F g−1 at 0.5, 1, 2, 3, 4 and 5 A g−1, respectively. It is observed that the OPAC-800 °C showed good rate capability at the distinct specific currents. As a result of the significant stability of the OPAC-800 °C electrode material, an assembly of a symmetric supercapacitor was deemed worthwhile.A quasi-rectangular shape had been maintained even at higher scan rates of 100 mV s3 electrolyte was used to investigate the electrochemical performance of the symmetric device because of the inherent potential that was exhibited from the half-cell analysis. −1. The associated GCD profiles of the full device at various specific current are presented in 57 The Nyquist plot for the symmetric device with an equivalent series resistance of 0.44 Ω and a charge transfer resistance of 0.31 Ω is presented in 58,59−1 at a specific current of 0.5 A g−1. −1, specific energy of 14.0 W h kg−1 corresponding to specific power of 400 W kg−1 was achieved. While at a specific current of 1 A g−1, a specific energy of 13.4 W h kg−1 corresponding to a specific energy of 800 W kg−1 was obtained. At 10 A g−1, specific energy of 10.4 W h kg−1 and corresponding specific power of 7655 W kg−1 was maintained. The symmetric device exhibited a coulombic efficiency of 99.9% and conserved 77% of the initial capacitance after 10 000 charge and discharge cycles at a specific current of 5 A g−1  and pore volume (1.64 cm3 g−1) was achieved when activation was conducted at 800 °C. Noteworthy, hydrogen sorption (at 77 K and 1 bar) for the 3 activated samples was above 3 wt% with the highest being 3.67 wt% when activation was conducted at 800 °C. These values were comparatively higher than those often reported for typical porous carbons which usually range from 2 to 3 wt%. Furthermore, the obtained ACs samples also exhibited commendable electrochemical performance, by having a specific capacitance of 158 F g−1 (at a specific current of 0.5 A g−1), specific energy of 14 W h kg−1, power of 400 W kg−1 and with a capacitance retention of 77% at 5 A g−1 even after 10 000 charge–discharge cycles. The results demonstrated that the onion derived ACs could be considered as promising candidates for hydrogen storage and electrochemical energy storage applications.This study showed that activated carbons prepared from waste onion sheaths using KOH, as the activating reagent, exhibited exceptional properties that made them desirable for hydrogen storage and electrochemical applications. The resulting ACs were found to have a nano-sheet type of morphology and had a moderate concentration of nitrogen and oxygen functional groups. The highest specific surface area (3150 mThere are no conflicts of interest to declare.RA-010-D0RA04556J-s001