These results suggest that a misfolding-specific intrabody prone to synergistic proteolysis by proteasomal and autophagic pathways is a promising strategy for mitigation of TDP-43 proteinopathy in ALS.Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is implicated in the pathogenesis of sporadic and certain familial forms of amyotrophic lateral sclerosis (ALS), suggesting elimination of TDP-43 aggregates as a possible therapeutic strategy. Here we generated and investigated a single-chain variable fragment (scFv) derived from the 3B12A monoclonal antibody (MAb) that recognises D247 of the TDP-43 nuclear export signal, an epitope masked in the physiological state. In transfected HEK293A cells, 3B12A scFv recapitulated the affinity of the full-length MAb to mislocalised TDP-43 with a defective nuclear localising signal and to a TDP-43 inclusion mimic with cysteine-to-serine substitution at RRM1. Moreover, 3B12A scFv accelerated proteasome-mediated degradation of aggregated TDP-43, likely due to an endogenous PEST-like proteolytic signal sequence in the VH domain CDR2 region. Addition of the chaperone-mediated autophagy (CMA)-related signal to 3B12A scFv induced HSP70 transcription, further enhancing TDP-43 aggregate clearance and cell viability. The 3B12A scFv also reduced TDP-43 aggregates in embryonic mouse brain following  Emerging evidence suggests that the accumulation of various disease-related misfolded proteins may underlie these pathogenic processes. Despite advances in understanding ALS pathogenesis, however, there is no curative or effective control therapy.Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterised by progressive muscle wasting and weakness, leading ultimately to lethal respiratory failure. Multiple pathogenic pathways have been implicated in the motor neuron degeneration of ALS, such as excitotoxicity, neuroinflammation, proteolysis impairment, oligodendrocyte dysfunction, mitochondrial dysfunction and disruption of RNA homeostasis9. TDP-43 contains a nuclear localising signal (NLS) as well as a nuclear export signal (NES)10, which enables the physiological shuttling of TDP-43 between nucleus and cytosol. Under stress-free conditions, TDP-43 interacts with mRNAs on which ribosomes are located separately, forming polysomes. Various stresses induce clustering of ribosomes into a ‘stalled’ state, resulting in the formation of stress granules (SG) containing TIA-1, G3BP, ataxin-2 and eIF4G1/2. In the stalled state, transcription is inhibited as a homeostatic response. However, sustained stress and ensuing TDP-43 misfolding creates aberrant SGs and pathogenic TDP-43 aggregates12. Moreover, membrane-less organelles in the cytosol formed by the liquid−liquid phase separation of RNPs4 and RNA13 are implicated in TDP-43 proteinopathy. Misfolding and cytosolic mislocalisation also lead directly to a loss of normal TDP-43 function, and the resultant disruption of protein and RNA homeostasis is considered another likely pathogenic mechanism14 in addition to the toxicity of inclusions. TDP-43 inclusions can be identified in the majority of ALS patients with the exception of patients with superoxide dismutase 1 (SOD1) mutations15, suggesting that these aggregates are a core pathology of sporadic ALS. Aberrant TDP-43 inclusions are also observed in familial ALS (FALS) cases associated with various mutations such as C9orf7216, MATR317, hnRNP118, UBQLN219, SQSTM120, VCP21 and OPTN22, as well as in cases with mutations of the TDP-43 gene (TARDBP) itself, indicating that misfolded TDP-43 is a common pathogenic molecule in familial as well as sporadic ALS. These lines of evidence suggest that targeting pathogenic TDP-43 species is a possible therapeutic strategy against ALS.The TAR DNA-binding protein of 43 kDa (TDP-43) is a DNA/RNA-binding protein predominantly located in the nucleus under physiological conditions. However, hyperphosphorylated, fragmented and ubiquitinated forms of TDP-43 were identified as core components of cytosolic inclusions in sporadic ALS and frontotemporal lobar degeneration (FTLD)23. Unexpectedly, the suppression of cytoplasmic human TDP-43 expression after disease onset (DOX-ON) reversed TDP-43 pathology and rescued the motor deficits23. These results indicate that mislocalised TDP-43 is directly toxic and further suggest that removal of misfolded TDP-43 is a promising therapeutic strategy for TDP-43-linked ALS, even after formation of TDP-43 inclusions. Ubiquitination and phosphorylation of TDP-43 are pathological hallmarks of sporadic ALS. However, they are relatively late phenomena in ALS progression26, and thus targeting the early conformational changes in pathogenic TDP-43 is required for pre-emptive therapy. We previously identified E246 and D247 as molecular epitopes of the mislocalised/misfolded TDP-4327. These residues are located at the assembly interface of the RNA Recognition Motif 2 (RRM2) domain formed upon interaction with single-stranded DNA28. Using these residues as epitopes, we successfully generated a misfolding-specific monoclonal antibody (MAb), 3B12A, which recognises mislocalised or misfolded TDP-43 in cultured cells and spinal cord sections from ALS patients, but not wild-type (WT) TDP-43 in the nucleus27.A transgenic mouse model with doxycycline (DOX)-dependent suppression of mutant human TDP-43 harbouring a defective nuclear localising signal (NLS) exhibited accumulation of cytoplasmic TDP-43 aggregates, loss of endogenous nuclear TDP-43 and progressive motor impairments in the DOX-OFF statein vitro and in vivo. This effect was mediated by dual proteolytic signals, an endogenous PEST-like signal and a chaperone-mediated autophagy (CMA)-localising signal. Moreover, aggregate binding to CMA-fused 3B12A scFv unexpectedly induced heat shock protein (HSP) 70, which further enhanced TDP-43 clearance by promoting protein refolding.Here we report the development of self-degradative intrabodies derived from 3B12A MAb, and demonstrate effective elimination of misfolded TDP-43 both 29. To apply this antibody inside cells as a potential scavenger of intracellular TDP-43 aggregates, we generated a single-chain variable fragment (scFv) to be used as an intrabody. The cDNAs encoding variable fragments of the heavy chain (VH) and light chain (VL) were obtained from hybridoma 3B12A mRNA, and domain profiles were investigated. Unexpectedly, DNA sequence analysis of these 3B12A intrabodies showed that the VH domain contains a RIDPEDGETK sequence with PEST score as high as 9.02 at the complementarity determining region 2  and VL-linker-VH (VL_VH) as scFv forms and VH and VL as nanobodies tagged with Myc at the C-terminus  as a model for misfolded and aggregated TDP-43, and TDP-43 with an NLS mutation (TDP-43mNLS)34 as a TDP-43 cytosolic aggregate mimic in the present study , but not with TDP-43WT. Moreover, VH_VL preferentially colocalised with TDP-43 aggregates over TDP-43WT whether in the nucleus or cytosol  leads to the formation of TDP-43 aggregates sharing pathological and functional features of the inclusions observed in sporadic ALSsol Fig. . Likewissol Fig. . These rsol Fig. . These 3mNLS, TDP-43C173S/C175S and TDP-43mNLS,C173S/C175S over TDP-43WT  chase study. For this purpose, we constructed a VH_VL intrabody containing two protein disassembling signals, CL1 for proteasome localisationy39 Fig. , based oy39 Fig. . The inty39 Fig. . Furthery39 Fig. . Consisty39 Fig. . We confy39 Fig. .Figure 3WT or misfolded TDP-43 species  but did not significantly accelerate clearance of the cytoplasmic TDP-43 mutant (TDP-43mNLS) , and cell lysates were analysed at several time points post-labelling. The HaloTag pulse chase assay demonstrated that 3B12A scFv co-expression markedly enhanced the degradation of the cytoplasmic aggregation-prone TDP-43 mutant TDP-433WT Fig. . Moreove3WT Fig. . Co-exprLS) Fig. , confirmLS) Fig. , indicatmNLS,C173S/C175S demonstrated a gradual increase in the number and size of cytoplasmic aggregates over 48-h post-transfection. Conversely, Hoechst counterstaining demonstrated a concomitant decline in live cells, indicating that these cytoplasmic TDP-43 aggregates are cytotoxic  cells at 48-h post-transfection, indicating reduced aggregation and thus rescued from cell death  induced the highest cytotoxicity, consistent with our previous studies in the NSC-34 cell line33. This cytotoxicity was significantly reduced in the presence of 3B12A scFv (VH_VL), and the inclusion of the CMA signal significantly enhanced this protective effect  TDP-43 by the distribution of GFP fluorescence45. Coronal brain sections exhibited GFP-TDP-43 mutant protein in both the lateral and medial neocortices, and the cellular distribution was grossly similar to that reported previously43 , a marker of mature neurons  . Similar to mice electroporated with the empty vector, those expressing 3B12A scFv-CMA were safely born at term, grew normally and exhibited age-appropriate behaviours until P21  was not degraded by 3B12A scFv in cultured cells /cullin-2 ubiquitin ligase complex for misfolded TDP-4329. This explains why 3B12A scFv specifically interacted with mislocalised/misfolded TDP-43 but not with TDP-43WT in cells  in RRM133. The sustained mislocalisation of TDP-43 in the cytosol by the NLS mutation induces cell death and occasionally forms inclusions both in vitro and in vivo54. However, these aggregates are relatively small in number and rarely phosphorylated or ubiquitinated54; thus TDP-43mNLS is an insufficient model for TDP-43 proteinopathy. Transgenic mice overexpressing human TDP-43WT and several known FALS mutants such as A315T and Q331K under native promoters develop similar mild phenotypes for FTLD-motor neuron diseases, but nuclear exclusion and cytosolic aggregates are prominent only in the FALS mutant models55. We reported that both WT and NLS mutant TDP-43 induce cell toxicity in immortalised cultured cells34. It should be noted that in the current work and our previous study33, aggregate-prone TDP-43mNLS,C173S/C175S induced greater cell toxicity than TDP-43WT or TDP-43mNLS, irrespective of mislocalisation. The pathologies induced by this TDP-43 mutant share several crucial properties with TDP-43 proteinopathy in addition to the strong trend for aggregation, including the involvement of prion-like domains, impaired RNA splicing, ubiquitination and phosphorylation, as well as motor neuron toxicity33. Based on the extent of aggregate toxicity, the TDP-43mNLS,C173S/C175S mutant may be the most robust form for modelling ALS-associated TDP-43 proteinopathy, especially for studies on therapeutic strategies and efficacy.There is yet no model TDP-43 species replicating all pathological features of sporadic ALS. In this study, we used the C173S/C175S artificial mutant as a TDP-43 inclusion mimic based on evidence of structural similarity to aggregated TDP-43 inclusions in ALSin utero electroporation did not show aberrant/ectopic neuronal cells, gliosis or microglial activation compared with vector controls. Of course, the restricted distributions of exogenous human TDP-43 and 3B12A scFv-CMA protein in mice following in utero electroporation limit future experiments on mitigation of neurological behaviour phenotypes, and the environment of the pup brain is distinct from the aged human brain. However, the successful elimination of TDP-43 aggregates in vivo validates the basic principle and justifies further analyses in older disease model animals. In the clinical setting, our intrabody may be effectively delivered by a virus vector, such as an adeno-associated virus (AAV). Indeed, AAV9-mediated intrathecal delivery of a scFv derived from the D3H5 monoclonal antibody against misfolded SOD1 reduced misfolded SOD1 in the spinal cord, delayed disease onset and enhanced the survival of SOD1G93A transgenic mice56. A recent study demonstrated that suppression of human TDP-43 mutant expression after disease onset in a mouse model decreased TDP-43 pathology, rescued motor deficits and prolonged survival23. These results provide strong support for our approach of pathological TDP-43 removal using a 3B12A intrabody, even after disease onset and formation of pathological TDP-43 aggregates. Further investigations evaluating the potential of our degradative 3B12A intrabody against pathogenic TDP-43 using appropriate animal models are required to advance this novel molecular targeting therapy for ALS.Most promising for clinical applicability, 3B12A scFv-CMA reduced cytoplasmic TDP-43 aggregates in the lateral CP of murine embryos Fig.  without 34. The TDP-43 substitution mutant with serine at Cys173 and Cys175 , familial ALS-linked mutations (A315T and Q331K) and mNLS (R82L/K83Q) were generated by site-specific mutagenesis34. A plasmid for Halo-tagged TDP-43  was generated by subcloning TDP-43 cDNA into pFC14A HaloTag® CMV Flexi® Vector . cDNA for variable fragments of the heavy chain (VH) and light chain (VL) were obtained using mRNA from a hybridoma producing 3B12A monoclonal antibody, which recognises mislocalised/misfolded TDP-4327. For in utero electroporation studies, target protein expression was driven by the CAGGS promoter57. Detailed protocols regarding construction and domain maps of Halo-tagged TDP-43 , CAGGS promoter-driven TDP-43, and single-chain variable fragment (scFv) for VH-VL-Myc, VL-VH-Myc, VH-Myc, VL-Myc, VH-VL-CL1-Myc and VH-VL-CMA-Myc are described in Supplementary Materials and Methods and Figure In cell culture studies, mammalian expression plasmids for TDP-43 tagged with FLAG (pcDNA3-TDP-43-FLAG) or EGFP (pEGFP-N3-TDP-43) were constructed using a conventional PCR technique as described previouslyAntibodies used in this work are described in the Supplementary Table.2 and 100% humidity. HEK293A  and N2a cells  were maintained in Dulbecco’s modified Eagle’s medium  containing 10% foetal bovine serum (FBS) and penicillin/streptomycin . The FuGene HD Transfection Reagent (Promega) was used for plasmid transfection. At 48 h after transfection, cells were treated for various analyses as indicated.All cultured cells were maintained at 37 °C under 5% CO58.Cultured cells were lysed in sodium dodecyl sulfate (SDS) buffer containing 2-mercaptoethanol for Western blotting or in immunoprecipitation assay (RIPA) buffer  containing protease inhibitor cocktail  for immunoprecipitation. Ten percent of each cell lysate volume was analysed as the total cell lysate, and the remaining 90% was incubated with anti-FLAG M2 affinity gel  or anti-Myc affinity beads  at 4 °C overnight. The affinity beads were subsequently washed with RIPA buffer five times, and the immunoprecipitates were eluted in 2% SDS sampling buffer for 5 min at 95 °C. The eluates were separated on polyacrylamide gels  and proteins transferred onto PVDF membranes . Proteins were detected using an enhanced chemiluminescence system . Densitometric analysis of protein bands was performed using ImageJ softwareCultured cells were fixed in 4% paraformaldehyde (PFA)/PBS (pH 7.2) and permeabilised with 0.1% Triton-X100/PBS containing 5% normal goat serum as a blocking agent. Cells were reacted with primary antibody  and subsequently with a fluorophore-tagged secondary antibody  for 1 h at room temperature. Cells were counterstained with 4′-6 diamidino-2-phenylindole (DAPI). Fluorescence images were obtained using a confocal laser microscope .Target FLAG antibody in coating buffer (Roche) was coated onto ELISA plates  at a 1:100 dilution. Coated FLAG antibody was then reacted with cell lysates from transfected HEK293A cells (prepared in RIPA buffer) for 1 h at room temperature and subsequently with anti-Myc antibody at a 1:500 dilution . Then, the coated antibody was reacted with the peroxidase-conjugated secondary antibody . Finally, reaction buffer containing 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonate (Roche) was applied, and the absorbance was measured by a spectrometer at 405 nm with a reference at 490 nm.At 48 h after cotransfection of HEK293A cells, the HaloTag-fused protein was labelled with 1 μM diAcFAM ligand (Promega) in FBS-free culture medium for 15 min in a cell culture incubator. Cells were then washed three times with warm PBS and cell lysates prepared in SDS sample buffer at 0, 12 and 24 h after ligand treatment. All lysate samples were separated on polyacrylamide gels (Wako) by SDS-PAGE, and gels were subsequently analysed on a fluorescence scanner .2 as aggregates. Intensity is expressed in arbitrary units.At 24 h after cotransfection, HEK293A cells were counterstained with Hoechst 33342 . Stained cells were observed at 30-min intervals over 48 h using a fluorescent microscope . For quantification of GFP-positive TDP-43 aggregates with ImageJ software, we removed background noise and set fluorescence thresholds of GFP fluorescence at 2.5% to distinguish TDP-43 aggregates from diffusely expressing cytoplasmic non-aggregated species and subsequently counted GFP-positive areas over 10 µmProtein half-life in HEK293A cells was estimated starting 48 h after transfection by chronological chase analysis. Cells were treated with CHX (100 μg/ml) to inhibit protein synthesis. In several experiments, lactacystin (10 μM) or bafilomycin (0.1 μM) was added with CHX to inhibit proteasome or autophagy–lysosome activity, respectively. Dimethyl sulfoxide (DMSO) was used as a vehicle control.We estimated the cytotoxicity and viability of transfected N2a cells using the MultiTox-Fluor multiplex cytotoxicity assay kit (Promega). Briefly, N2a cells were seeded onto 96-well black culture plates and plasmids encoding FLAG-tagged WT or mutant TDP-43 and 3B12A scFv were co-transfected at 0.1 µg each per well. At 48 h after transfection, cell-permeant glycyl-phenylalanylamino fluorocoumarin (GF-AFC) and cell-impermeant bisalanyl-alanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110), fluorescent indicators for live cells and dead cells, respectively, were applied. After a 2-h incubation, relative fluorescence emission was measured on a multi-plate reader (Perkin Elmer) with excitation/emission of 400/505 nm for live cells and 485/520 nm for dead cells.HSP70 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were analysed using a real-time PCR Detection Systems  and SYBER quantitative PCR kit  with the following primer pairs: HSP70, 5′-CAA GAT CAC CAT CAC CAA CG-3′ and 5′-TCG TCC TCC GCT TTG TAC TT-3′; GAPDH, 5′-GCA CCG TCA AGG CTG AGA AC-3′; and 5′-TGG TGG TGA AGA CGC CAG TGG A-3′. GAPDH was used as an internal standard, and the relative mRNA expression levels were calculated by the ΔΔCT method according to the manufacturer’s protocol using the included software (BIO-RAD).Total RNA samples were purified from HEK293A cells using a commercially available kit (Invitrogen) and converted to cDNA with reverse transcriptase (Invitrogen). The mRNA expression levels of WT-GFP or pCAGGS-TDP-43mNLS,C173S/C175S-GFP], 1 μg/μl of effector plasmids [pCAGGS-3B12A-VH_VL-Myc-CMA or empty plasmid], and 0.5 μg/μl of normalisation plasmid [pCAGGS-mCherry]) were mixed and diluted in PBS (−) containing 0.05% Fast Green, and 1–2 μl of the solution was injected into the lateral ventricles of embryos. After plasmid injection, embryo heads were held using a tweezer-type electrode  with the anode on the forebrain side in which the plasmid was injected, and five cycles of electric pulses  were delivered by an electroporator . The uterus was repositioned in the abdominal cavity, the skin sutured and pregnancy allowed to continue.Timed-pregnant ICR mice (E13.5) were anaesthetised by inhalation of isoflurane  and intraperitoneal injection of sodium pentobarbital . The uterine horns were then exposed in the abdominal cavity. The expression vectors . The slides were washed with 0.1 M PBS/0.1% Triton X-100 (PBS-T) and blocked with 3% bovine serum albumin  in PBS-T at room temperature for 30 min. After subsequent incubation with primary antibody at 4 °C overnight, slides were incubated in Alexa Fluor-conjugated antibody (Invitrogen) at room temperature for 1 h, washed with PBS-T, and placed onto cover glass (Matsunami) with mounting medium containing DAPI for nuclear counterstaining . For quantification of GFP-positive TDP-43 aggregates in the lateral cortical plates with ImageJ software, we removed background noise and set fluorescence thresholds of GFP fluorescence at 1% to distinguish TDP-43 aggregates from diffusely expressing cytoplasmic non-aggregates, and subsequently counted GFP-positive areas over 20 µm2 as aggregates. Intensity is expressed in arbitrary units.Embryos were anaesthetised on ice and postnatal mice by intraperitoneal injection of sodium pentobarbital and isoflurane inhalation. Brains were fixed by transcardial perfusion with 4% paraformaldehyde (PFA) in 0.1 M PBS. The brains were immersed in 4% PFA at 4 °C overnight, incubated in 20% sucrose/0.1 M PBS at 4 °C overnight, embedded in OCT compound , and frozen in liquid nitrogen. The restricted distribution of the plasmids in the dorsolateral cortex of the pup mice by p < 0.05 was considered statistically significant.Multiple group means were compared by one-way ANOVA with post hoc Tukey’s multiple comparison tests for pair-wise comparisons. Factor estimation in the two chronological data groups was evaluated by two-way ANOVA using Prism software . A The protocols for both genetic transformation experiments and animal experiments were approved by and performed under the guidelines of the Kyoto University Graduate School of Medicine ethics committee (#130181 for the genetic transformation experiments) and the Shiga University of Medical Science .Supplementary Movie 1aSupplementary Movie 1bSupplementary Movie 2Supplementary information


Azadirachta indica) in India suggests its potential efficacy in periodontal diseases. The objective of this study is to explore the antibacterial effects of Neem leaf extract on the periodontophatic bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and its antioxidant capacities alone and in combination with bacteria and polycationic peptides that may be at the site of inflammation.The common usage of chewing sticks prepared from Neem tree  of Neem leaf extract against each bacterial strain. The effect of Neem leaf extract on the coaggregation of the bacteria was assessed by a visual semi-quantitative assay. The antioxidant capacities of Neem leaf extract alone and in combination with bacteria, with the addition of red blood cells or the polycationic peptides chlorhexidine and lisozyme, were determined using a chemiluminescence assay.Neem leaf extract was prepared by ethanol extraction. The growth kinetics of P. gingivalis, however, had no effect on the growth of F. nucleatum nor on the coaggregation of the two bacteria. Yet, it showed intense antioxidant activity, which was amplified following adherence to bacteria and with the addition of red blood cells or the polycationic peptides.Neem leaf extract showed prominent dose-dependent antibacterial activity against Neem leaf extract, containing polyphenols that adhere to oral surfaces, have the potential to provide long-lasting antibacterial as well as synergic antioxidant activities when in complex with bacteria, red blood cells and lisozyme. Thus, it might be especially effective in periodontal diseases. Porphyromonas gingivalis and Fusobacterium nucleatum. The adherence of bacteria to mucosal cells is a critical step in the development of periodontal infection. P. gingivalis is able to adhere to cellular and acellular surfaces  (100 μM) and 10 μM Co [II] , 36. TheNeem leaf extract may bind to mammalian and microbial surfaces , 37–39, p < 0.05.The results were expressed as the mean ± SEM. One or two-way ANOVA with the Newman-Keuls test for post-hoc comparisons were applied. Differences were considered statistically significant at P. gingivalis growth. Whereas approximately 50% growth inhibition was induced by 5 μM GAE of Neem leaf extract, higher concentrations (10 and 20 μM GAE) markedly inhibited P. gingivalis growth . Significant growth inhibition was noted already after 18 h . In contrast, Neem leaf extract had no effect on the growth of F. nucleatum even at the highest concentrations  [P. gingivalis and F. nucleatum.Coaggregation of t shown) . This inP. gingivalis pre-coated with Neem leaf extract. It should be noted that P. gingivalis alone did not possess any OSA.As shown in Fig. P. gingivalis coated with Neem leaf extract was further markedly enhanced by small amounts of RBC.As shown in Fig. F. nucleatum-Neem-lysozyme complex exerts greater OSA, with about 85% reduction of luminescence, as compared to F. nucleatum-Neem complex. However, the addition of the polycationic peptide chlorhexidene to F. nucleatum-Neem complex did not intensify the antioxidant effect of F. nucleatum-Neem complex, alone.Similarly, as shown in Fig. Azadirachta indica) leaves has antibacterial properties against the periodontophatic bacterium P. gingivalis and potent antioxidant abilities. Our findings also show that small numbers of RBC further enhance the OSA of P. gingivalis pre-coated with agents present in Neem leaf extract, and that lysozyme also amplifies the OSA of bacteria pre-coated with Neem leaf extract.Our results show that an ethanol extract derived from Neem tree (P. gingivalis and F. nucleatum. Also, surprisingly, it has no effect on the growth of F. nucleatum, as opposed to its antibacterial effect on P. gingivalis, when tested under similar experimental conditions (Fig. P. gingivalis and F. nucleatum exhibited diverse effects, such as antibacterial activities against both bacteria by propolis extracts [Neem leaf extract has no effect on the coaggregation of ons Fig. , maybe dextracts , and no extracts . On the extracts , 44. Theextracts , 46.F. nucleatum-Neem complex, as compared to the antioxidant capacity of this complex alone deserves further evaluation.Neem leaves are extremely rich in a large variety of polyphenol agents, possessing antioxidant properties that can modulate inflammation –18. FurtTaken together, Neem extract, adsorbed by bacteria, in addition to its antibacterial properties, provides profound antioxidant activity, intensified in the presence of RBC or lysozyme. However, the potent antioxidant activity in Neem extracts might also act as a double-edged sword. On one hand scavenging ROS might lower inflammatory damage to the gum, but on the other hand it may also protect periopathogenic catalase-negative bacteria against oxidants generated by inflammation or cariogenic streptococci.In addition, our report has two major limitations: First, the tested species in this study were evaluated in a planktonic state, whereas, in vivo they are part of a multi-specious biofilm, which has a profound impact in the effectiveness of antimicrobial agents. Therefore, further clinical studies using periodontal parameters are needed to shed more light on the potential clinical implication of Neem extracts as a modulator of periodontal diseases. Second, we studied fresh leaves obtained from Neem trees grown in Israel, applying a specific extraction protocol. Further experiments are needed to extend our findings to Neem extracts obtained from other parts of this plant and to explore comparable extracts produced from trees grown abroad. Furthermore, we did not label and quantify the numerous compounds plausibly extracted from the plant, and we did not identify components responsible for the antimicrobial properties of the extract. Nor did we compare the characteristics of our product with extracts obtained by other methods. Additional studies are needed to address these important limitations, and safety evaluation is required preceding clinical trials.P. gingivalis but not against F. nucleatum. It possesses strong anti-oxidant activity, which is amplified following adherence to bacteria, to red blood cells and to the polycationic peptide lysozyme. The clinical relevance of these findings is based on the co-existence of bacteria, RBCs and polycationic compounds in the oral cavity. Conceivably, Neem extract could adhere to these compounds, and provide a long lasting bacteriostatic as well as anti-oxidant activity at the site of inflammation in periodontal diseases. Prospective randomized trials in-vivo are needed to strengthen these in-vitro findings, suggesting potential clinical implication of the use of Neem leaf extract in periodontal diseases.We report that Neem extracts possess prominent dose-dependent bacteriostatic activity against


X-ray photoelectron spectroscopy and electron paramagnetic resonance revealed the changes in surface states, composition, changes in Ce4+ to Ce3+ ratio, and other defects. Transmission electron microscopy (TEM) and high resolution TEM revealed that the fabricated CeO2 NPs to be spherical with particle size of ~10–12 nm. Combination of defects in CeO2 NPs with optimal amount of two-dimensional graphene sheets had a significant effect on the properties of the resulting hybrid CeO2-Graphene nanostructures, such as improved optical, photocatalytic, and photocapacitive performance. The excellent photocatalytic degradation performances were examined by monitoring their ability to degrade Congo red ~94.5% and methylene blue dye ~98% under visible light irradiation. The photoelectrode performance had a maximum photocapacitance of 177.54 Fg−1 and exhibited regular capacitive behavior. Therefore, the Ce3+-ion, surface-oxygen-vacancies, and defects-induced behavior can be attributed to the suppression of the recombination of photo-generated electron–hole pairs due to the rapid charge transfer between the CeO2 NPs and graphene sheets. These findings will have a profound effect on the use of CeO2-Graphene nanostructures for future energy and environment-related applications.Cerium oxide nanoparticles (CeO Since the innovation of water splitting under UV light irradiation by Honda and Fujishima2, semiconductor photocatalytic technology using solar energy has been studied widely to understand the purification of polluted water, production of renewable hydrogen energy from water splitting, and the removal of harmful microorganisms3. The TiO2 semiconductor is one of the most widespread photocatalysts owing to its low cost, non-toxicity, relatively high chemical stability, and strong oxidizing power9. On the other hand, the wide band gap (3.2 eV) of TiO2, which means that it can only utilize ultraviolet light that makes up about 4% of solar energy, and low quantum efficiency limits its use in large scale applied applications10. To resolve these drawbacks of TiO2, considerable efforts have been made to improve its photocatalytic ability and to extend its light-response range, such as doping12, and cocatalyst loading13. Nevertheless, there are still many problems to solve and it is urgent and critical to develop an efficient, sustainable, and stable photocatalyst that is driven by visible light17, which accounts for approximately 45% of solar energy18. Over the past few years, considerable efforts have been made towards the development of highly effective and robust photocatalysts for the oxidative degradation of organic dyes to harmless end-products, such as CO2, and H2O18. Nanosized metal oxides  and their composites with carbon based materials have been reported to have electronic properties with excellent recyclability owing to their extended surface areas with enhanced photocatalytic activities24. The finding of graphene, which is a flat single layer of carbon atoms with a perfect sp2-hybridized two-dimensional structure, has led researchers to investigate composites of graphene with other nanomaterials, such as noble metals, complex oxides, and metal oxides25. Among them, graphene-metal oxide nanostructures have attracted particular attention as a viable way to boost the efficiency of various photocatalytic and photocapacitive applications27.Visible light-induced photocatalysis using harmless, sustainable, and inexhaustible solar energy is used widely in water purification to remove toxic organic pollutants, such as harmful organic dyes, herbicides, and pesticides2) is a favorable rare earth metal oxide material that has been assessed for many technological applications30. CeO2 NPs are a favorable redox supercapacitor material because it is the most abundant and least expensive rare earth metal oxide with excellent redox characteristics32. In addition, the spherical size of nanoparticles with a higher surface area has a prominent effect on the pseudo-capacitance32. CeO2 is a wide band gap semiconductor (~3.4 eV), which limits its use as an electronic material and as a photocatalyst28. The chemical functionalization of CeO2 is an excellent method for tuning the band structure and the majority carrier type for electronic and optical applications30. In particular, defects, such as oxygen vacancies, in CeO2 play a crucial role in its photocatalytic properties because these oxygen vacancies can be formed easily and removed rapidly32. The oxygen vacancies in CeO2 determine the Ce4+ to Ce3+ ratio, and an increased level of Ce3+ leads to enhanced visible light-induced photocatalytic activity by narrowing the band gap of CeO2. Oxygen vacancies in CeO2 can attach to graphene sheets, which promotes the formation of stable CeO2-Graphene nanostructure33. A simple strategy is used to improve the visible light photocatalytic properties of CeO2 to combine it with conductive carbon-based materials, which include noble metal particles, carbon nanotubes, and graphene matrix34. Graphene, which is a two-dimensional matrix with excellent electrical mobility (200000 cm2V−1s−1), extremely large theoretical specific surface area (~2600 m2g−1), high thermal conductivity (~5000 Wm−1K−1), and outstanding mechanical properties (high Young’s modulus), has been applied widely as a remarkable support and electron transport material37. Graphene sheets can be used/act as an efficient electron acceptor to improve photoinduced charge transfer and hinder the backward reaction by separating the evolution sites of hydrogen and oxygen for improved photocatalytic and photocapacitive performance19. On the other side, the photocatalytic and photocapacitive properties of graphene nanostructures with rare earth oxides have barely been investigated.Ceria  and electrochemical impedance spectroscopy (EIS) under visible light irradiation in an aqueous solution at room temperature43. These investigations can promote the further development of CeO2–Graphene-based devices for future energy or environment-related applications.To the best of the author’s knowledge, there has been no research on both the photocatalytic degradation of organic model dye pollutants and photoelectrode studies using Ce2 NPs which was indexed to the fluorite cubic phase of CeO2 (JCPDS 34-03494). The XRD peaks of bare CeO2 NPs were observed at 28.54°, 33.08°, 47.48°, 56.33°, 59.08°, 69.69° and 79.06° 2θ, which were assigned to (111), (200), (220), (311), (222), (400), (331), and (420) planes respectively44. The mean crystallite size of the pure-CeO2 NPs was calculated to be 11.8 nm using the Scherer’s formula, D = κλ/β cos θ, where κ is the shape factor that has a typical value of ~0.9, λ is the wavelength (Cu Kα = 0.15405 nm), β is the full width at half maximum of the most intense peak (in radians), and θ is the main peak of pure-CeO2 NPs at 28.54° 2θ. The crystallite size of the most intense peak was 11.8 nm 45. The XRD pattern of CeO2-Graphene nanostructures in Fig. 2 NPs nanostructure demonstrating that CeO2 NPs has been anchored successfully to the graphene matrix. Finally, the two prominent peaks, 26.53° (002) and 54.70° (100), were also present in the XRD pattern of the CeO2-Graphene nanostructure, which further confirmed the existence and purity of the graphene matrix  for the defect-induced band gap narrowing of different metal oxides for the applications, such as the photodegradation of organic dyes and photoelectrodes46.Table 2 NPs were anchored/decorated to its surface. The Raman spectrum of graphene consisted of three peaks for the D and G bands at 1350 cm−1 and 1590 cm−1, respectively, and for the 2D band peak at 2729 cm−1 46. Similar characteristic peaks of the D, G bands at approximately 1350 cm−1 and 1590 cm−1 and a broad 2D band 2729 cm−1 were observed for the CeO2-Graphene nanostructure, as shown in Fig. 48. The intensity of the D (ID) band is related to the vibrational mode of the k-point phonons of A1g symmetry. The G band (ID) corresponds to the tangential stretching mode of the E2g phonon of the sp2 carbon atoms, whereas the 2D peak is the secondary D peak that it has the largest intensity in a single/few layer of graphene sheet49. In Fig. 2 NPs anchored onto graphene sheets. This further confirms the successful decoration of CeO2 NPs onto the graphene sheets  at room temperature over the wavelength range, 200–800 nm. In Fig. 46. After the anchoring/decoration of CeO2 NPs onto the graphene matrix, the optical absorption of the 1 mM and 3 mM CeO2-Graphene nanostructures increased with a hump (~265–365 nm) and the absorption edge was red shifted considerably compared to that of pure-graphene  is the K-M function or re-emission function; R∞ is the diffuse reflectance of an infinitely thick sample; K(λ) is the absorption coefficient; s(λ) is the scattering coefficient; hν is the photon energy. The optical band gap (Eg) was determined by extrapolating the linear portion hν)1/2] versus hν. The calculated band gap of pure-CeO2 and as-synthesized 1 mM and 3 mM CeO2-Graphene nanostructure were 3.16, 2.89, and 2.69 eV, respectively. This further shows that the changes in the %R  of pure-CeO5. The photoluminescence was observed at 570 to 650 nm after the irradiation of pure-CeO2 NPs with a He-Cd laser (λex = 325 nm). In this case, however, under the experimental conditions for the as-synthesized CeO2-Graphene nanostructures, a lower PL intensity graph recorded indicates a lower recombination rate of electrons. This is due to the presence of surface defects or oxygen vacancies in the CeO2 NPs48. At higher concentrations of Ce3+ states and a corresponding high concentration of oxygen vacancy states, a higher percentage of valence electrons can be excited to the defect state or oxygen vacancy states, which would lead to a lower PL signal intensity as observed in the case of the 3 mM CeO2-Graphene nanostructure. The presence of these oxygen vacancies was also confirmed by XPS  was carried out for the pure-CeO eV Fig. .2 NPs, in which two major peaks were assigned to Ce and O and a small peak of carbon (C 1s), which might be due to instrumental error. In addition, no contaminant peak was detected within the sensitivity of the technique. The binding energy (BE) of Ce 3d5 and O 1 s was recorded at 882.8 and 530.2, respectively52. Figure 2 NPs, in which four major peaks can be seen clearly. The Cd 3d peaks were confirmed by XPS analysis: Ce4+ 3d5/2, Ce3+ 3d3/2, Ce4+ 3d3/2 and. Ce3+ 3d5/2. The main characteristic peaks of Ce4+ 3d5/2 and Ce4+ 3d3/2 were observed at 916.7 and 898.4 eV, respectively, whereas the peaks located at 901.1 and 882.6 eV were assigned to Ce3+ 3d3/2 and Ce3+ 3d5/2, respectively. These spectral data are fully consistent with previous reports53. Figure 2-Graphene nanostructure, which displays a small peak for Ce 3d5 and O 1 s at a binding energy of 882.8, and 530.2 eV, which confirms the successful formation and anchoring of the CeO2 NPs to the graphene matrix. The sample yielded a high intensity peak of C1s at 284.6 eV, which was assigned to sp2-bonded carbon (C-C), indicating the successful formation of 1 mM of CeO2-Graphene nanostructure19 54. Figure 46. Figure 2 precursor. The C 1s peak confirmed the presence of sp2 carbon atoms related to the graphene matrix. Figure 4+ 3d5/2, Ce3+ 3d3/2, Ce4+ 3d3/2 and Ce3+ 3d5/2 at binding energies of 916.7, 901.1, 899.2, and 882.6.4 eV, respectively. The main focus is the concentration and intensity of Ce3+ 3d3/2 and Ce4+ 3d3/2. As shown from the Ce3+ 3d3/2 peak, the intensity of Ce3+ increased with increasing concentration (3 mM compared to the 1 mM CeO2-Graphene nanostructure). As indicated by XPS, the 1 mM and 3 mM CeO2-Graphene nanostructures contained a large number of defects and Ce3+ oxidation states54, which might lead to the formation of a surface state energy band of oxygen. Figure 46. The oxygen adsorption, desorption, and diffusion processes might occur easily on the surface of the 1 mM and 3 mM of CeO2-Graphene nanostructures, which can greatly improve their optical properties and impart visible light-induced photocatalytic degradation of organic dyes and photoelectrodes response44  of pure-CeO2 NPs and 1 mm and 3 mM CeO2-Graphene nanostructures as the powder samples exhibit weak room temperature (RT) ferromagnetism  spectroscopy is well suited to examining the nature of the oxidation state  plane of the CeO2 phase calculated from the XRD pattern. The SAED pattern of the nanostructure exhibited a series of bright rings, suggesting that the sample is polycrystalline  displays a sequence of bright rings, which suggests that the sample is polycrystalline in nature. Figure 2 NPs decorated over the graphene sheets. Figure 2-Graphene nanostructure. Figure 2-graphene nanostructures indicating visible lattice fringes and particle size distribution graph of CeO2 NPs for 1 mM and 3 mM CeO2-Graphene nanostructures.The two dimensional (2D) sheet-like surface of the graphene matrix was decorated/anchored by CeO2-BET (Nitrogen adsorption Brunauer-Emmett-Teller) was used. The BET specific surface area analysis of pure-graphene, 1 mM and 3 mM CeO2-Graphene nanostructures were measured to be 35.0134 ± 0.0223, 37.1380 ± 0.0999 and 40.8460 ± 0.1068 m2/g, respectively. The single point surface area at P/Po was also recorded for pure-graphene, 1 mM and 3 mM CeO2-Graphene nanostructures as shown in Table 2 samples was increased as the precursor amount increased. This is because of smaller particles having higher surface areas. In the photocatalytic study, higher surface areas and small particles are favored because more active sites are available for molecules that can provide the surface for dye molecules for oxidation or reduction56. These results suggest that the visible light photocatalytic performance of CeO2-Graphene nanostructures could be improved greatly due to the high concentration of surface donor defects, such as oxygen vacancies, Ce3+ centers and increased surface area  reactions under the visible light illuminated semiconductors18. When the semiconductor catalyst was exposed with photons, whose energy is equal to or greater than their band-gap energy (Eg), an electron (e-cb) is promoted from the valence band (VB) into the conduction band (CB), leaving a hole (h+-vb)19. Furthermore, the excited electrons and holes transfer to the surface. The rate of recombination is often inhibited by a scavenger or crystalline surface defects, which can easily trap electrons or holes. Therefore, better crystallinity with surface defects can generally decrease the trapping states and recombination sites, subsequent in increased effectiveness in the procedure of the photogenerated carriers for the preferred photoreactions46. For higher photocatalytic efficiency, the electron–hole pairs should be separated efficiently, and charges should be transferred rapidly across the surface/interface to restrain the recombination rate52. The present photocatalytic degradation of CR and MB were carried out to evaluate the photocatalytic degradation performance of the pure-CeO2 and 1 mM and 3 mM CeO2-Graphene nanostructures under visible light irradiation using a 400 W lamp (λ > 500 nm). Figure 0) vs. time (t). The photocatalytic degradation rate of anionic CR dye was negligible in the absence of CeO2 NPs, which is shown by an analysis of the control  industries rol Fig. . The pur2-Graphene nanostructures under visible light irradiation. A small decrease in the rate of degradation of MB was observed in the presence of pure-graphene after visible light irradiation, which suggests that MB is quite stable under visible light57. The 1 mM and 3 mM CeO2-Graphene nanostructures showed high photodegradation efficiency up to ~85.2 and ~98%, respectively, after 180 min under visible light irradiation. This was attributed to the high surface-to-volume ratio of the small spherical shaped CeO2 NPs decorated over the graphene sheets, which helped increase the photocatalytic sites and graphene helps reduce the recombination rate.Figure 58. This also proves that graphene is highly efficient for enhancing the catalytic performance of CeO2 NPs and behaves as an electron sink, which increases the separation of the photogenerated electron–hole pairs significantly and inhibits their recombination in the presence of the CeO2-Graphene nanostructure as a photocatalyst. The variation in the photocatalytic activity of 1 mM and 3 mM CeO2-Graphene nanostructures was also supported by DRS  is a widely used technique for examining the redox behavior and heterogeneous electron-transfer (HET) rate kinetics as well as defining the electron stoichiometry of any systems61 Fig. .Figure 12-Graphene nanostructures photoelectrodes. The photocapacitive performance of the pure-graphene and CeO2-Graphene nanostructures were examined systemically and typical CV curves are shown in Fig. 2-Graphene nanostructures, however, the first cathodic peak was recorded at the oxidation peak area of 8.0844 and 23.668, whereas for the anodic sweep, the peak area was 15.466 and 26.632 in the dark and under visible light irradiation, respectively. Pure-graphene did not show significant enhancement in the oxidation (cathodic sweep) and reduction peaks (anodic sweep) at scan rates of 0.05 mV/s, whereas the as-synthesized 1 mM and 3 mM CeO2-Graphene nanostructures showed well-determined oxidation and reduction peaks. The large peak separation (ΔEp) was observed in the case of the CeO2-Graphene nanostructures, which was higher than that of pure-graphene. This specifies the improved redox behavior, which could be due to the oxygen vacancy/defects induced CeO2 NPs present on the graphene surface. These findings confirm the improved photocapacitive behavior of the CeO2-Graphene nanostructures, which is probably due to the small, spherical and uniform decoration of CeO2 NPs on the graphene surfaces, which has various defects and Ce3+ ions.Figure 2-Graphene nanostructures showed the upgraded positive and negative sweep, specifying their pseudo capacitive behavior. The peak current of the CeO2-Graphene nanostructures increased linearly in the dark and under visible light illumination with a positive shift of the cathodic peak and a negative shift of the anodic peak. From the CV graph, the specific capacitances (Csp) of the photoelectrodes were determined using the following equation:The CV plot Fig.  of the C−1 and 10.93 F g−1 at a current density of 5 mA g−1, while the Csp for the 1 mM CeO2-Graphene nanostructures was 31.00 F g−1 and 59.40 F g−1 in the dark and under visible light irradiation. In contrast, for 3 mM, the calculated capacitance was 78.89 F g−1 and 177.54 F g−1 in the dark and under visible light irradiation. The as-synthesized nanocomposite exhibited enhanced Csp compared to pure-graphene. This was attributed to the highly conductive nature of the graphene sheets, which facilitated the charge transfer of CeO2-Graphene nanostructures, and ensured high electrochemical utilization of graphene with Ce3+-ions/ defect-induced CeO2 NPs61. Figure 2-Graphene nanostructures. The plots in Fig. ct), linking to the deprived electrical conductivity of the materials, whereas the more vertical line is related to an electrode that is closer to an ideal capacitor62. On the other hand, as shown in the Fig. ct occurs due to the formation of defects in the CeO2 NPs as a combination with pure-graphene for the electrode material. In contrast, pure-graphene combined with CeO2 NPs shows a smaller Rct because of its good conductivity63. This confirms the catalytic effect of CeO2, or the synergistic effects of the defect induced CeO2 NPs and pure-graphene. In addition, the considerable decrease in the Rct of the electrodes may be related to the improvement in the charge transfer rate at the CeO2–Graphene nanostructure.From Fig. 2-Graphene nanostructures as a photocatalyst and pure-graphene as a control under visible light irradiation. When the CR and MB dye solution comprising the CeO2-Graphene nanostructure is exposed to visible light, electron–hole pairs are generated because of the ejection of an electron from the VB, which makes a hole in the VB. For the duration of the photoexcitation process, some of the ejected electrons transfer to the CB and some electrons are trapped by the localized defect (defects and oxygen vacancies as confirmed by the XPS and EPR results) levels lying below the Ce 4f band64. The photogenerated CB electrons are transferred rapidly to the conducting graphene sheets and react with the oxygen dissolved in the dye solution making superoxide radicals (O2·). During this process, graphene sheets act as a trapping center/electron sink and as a co-catalyst. A speedy transfer of the photogenerated electrons from the excited CeO2 NPs is the result. Therefore, due to the transfer of electrons from CeO2 NPs to the graphene sheets, the recombination rate of photo-generated electron–hole pairs in CeO2 NPs is suppressed64  radicals. These OH· radicals interact with the dye molecules and degrade them. After degradation of the dye, these molecules present in the solution can immediately diffuse to the graphene surface via π–π conjugation for further degradation. The O2· and OH· radicals can attack the C–S+=C functional group of the dye, which is attached to the surface of the catalysts through Coulomb interactions, and degrade the dye to the final products, such as CO2, SO42−, NO3−, H2O, and H+. The complete photodegradation process can be summarized by the following reaction steps:XPS and EPR analysis confirms that Ce2 NPs were grown uniformly on graphene sheets using a simple and low-cost hydrothermal method without a surfactant or supporting material. All the synthesized samples were characterized by XRD, Raman, UV-Vis, PL, XPS, EPR, and TEM. TEM and HR-TEM revealed uniform and spherical particles of CeO2 NPs, which were decorated uniformly over the graphene sheets. UV-Vis spectroscopy showed that the oxygen vacancy/defects in the CeO2-Graphene nanostructures provide a suitable material for the absorption of visible light by decreasing the band gap. XPS and EPR confirmed the defects/oxygen vacancy and paramagnetic behavior of the CeO2-Graphene nanostructure. Interestingly, excitation-dependent tunable luminescence behavior could be perceived, which might be reasonably useful for applications in designing new optical devices. Moreover, the photocatalytic performance test of the as-synthesized CeO2-Graphene nanostructures showed that the CeO2-Graphene nanostructure displayed excellent photocatalytic activity compared to pure-graphene. Within 180 minutes, ~94.5% and ~98% of the CR and MB dye, respectively, had degraded under visible light irradiation. This excellent photocatalytic activity may be due to the synergistic effects of graphene sheets and CeO2 NPs (Ce3+-ions/defects induced), which are responsible for the smaller optical band-gap values. The photocapacitance investigation indicated that the CeO2-Graphene nanostructure, as a photoelectrode, had a maximum photocapacitance of 177.54 F g−1 and influenced the regular capacitive behavior. Therefore, the as-synthesized material shows excellent photocatalytic and photocapacitive performance for water treatment and energy storage applications. Based on the improved optical, photocatalytic, and photocapacitive performance, this type of material can be used for real applications, such as real water treatment devices and supercapacitors.CeO3)3·6H2O (≥99.99%), cerium oxide (nano CeO2) nanostructure, and ammonia (NH3) solution (≥99.99%)) were purchased from Sigma Aldrich. Pure-graphene sheets were purchased from Iljin Nano Tech, Seoul, Korea (7–8 layer graphene sheets with a mean length of 500 nm). Sodium acetate (CH3COONa) and sodium sulfate (Na2SO4) were supplied by Duksan Pure Chemicals Co. Ltd., South Korea. Ethyl cellulose and α-terpineol (C10H18O) were acquired from KANTO Chemical Co., Japan. Fluorine-doped transparent conducting oxide glass  was supplied by Pilkington, USA. All the above reagents used in this study were of analytical grade and used as received. All solutions were prepared from DI water obtained using a PURE ROUP 30 water purification system.Cerium nitrate hexahydrate (Ce(NOin situ growth), while in the second procedure, the as-synthesized nanostructures are anchored/deposited onto the surface of the graphene matrix (self-assembly approach)65  hydrothermal method was used for the controlled growth of CeO2 nanoparticles on graphene sheets. Scheme 1 presents a schematic diagram of the mechanism for the fabrication of CeO2-Graphene nanostructure. The CeO2-Graphene nanostructures were synthesized using a simplistic hydrothermal method. Briefly, in the first step, 100 mg of optimal pure-graphene nanopowder was dispersed in 200 mL of de-ionized water and subjected to a low sonication system  for 1 h. Subsequently, 0.4322 g (Ce(NO3)3·6H2O) of 1 mM aqueous solution was added slowly to the above graphene dispersion. 1 mL of aqueous ammonia solution was added drop wise to the above reaction mixture with constant stirring to maintain the pH value of reaction ~9. The resulting reaction mixture was stirred vigorously for 1 h, poured into a 100 mL of Teflon-lined stainless steel autoclave tube and kept in an electric oven for 24 h at 180 °C to perform the hydrothermal treatment. Once the reaction was accomplished, the autoclave was cooled naturally to room temperature. The product was washed several times with de-ionized water and absolute ethanol, and dried overnight in a vacuum at 60 °C. Finally, a black color powder was obtained. A 3 mM CeO2-Graphene nanostructure sample was also synthesized using the same amount of precursor as the above stated procedure.Herein, a simple, low cost and single step  and each solution was sonicated 10 min for homogeneous mixing. The dye solutions were stirred in the dark for 30 min to complete the adsorption and desorption equilibrium of the specific substrate on the CeO2-Graphene nanostructure and pure-graphene. Visible light irradiation of the solutions was performed using a 400 W lamp (λ > 500 nm). Three sets of experiments were observed over a 120 min period. The rate of dye degradation was monitored by taking 2 mL of the samples from each set every 30 min, centrifuging, removing the catalyst, and recording the UV–vis spectrum. As a control experiment, pure-graphene was used and considered as the reference photocatalyst. Each experiment was performed in triplicate to ensure the photocatalytic activities of the CeO2-Graphene nanostructure.The photocatalytic degradation ability of the as-synthesized 1 mM and 3 mM CeO2-Graphene nanostructures powder in water and sonicating it for 1 h. The CeO2 NPs leaching in the solution, if any, were observed using a UV–vis spectrophotometer, which confirmed the constancy of the CeO2-Graphene nanostructure and the possibility of using them as a reusable catalyst  with frequencies ranging from 1 to 104 Hz at 0.0 V vs. Ag/AgCl in potentiostatic mode.The photocapacitive performance of the pure-graphene, 1 mM and 3 mM CeO2-Graphene nanostructure. The optical properties of the CeO2-Graphene nanostructure were examined by UV-VIS-NIR diffuse absorbance/reflectance spectrophotometry . Electron paramagnetic resonance (EPR) measurements were performed using a Bruker EMX system with a microwave frequency of 9.64 GHz and a microwave power of 1 mW at room temperature. Microstructural analysis was observed by field emission transmission electron microscopy  operating at an accelerating voltage of 200 kV. Selected-area electron diffraction (SAED) was carried out by TEM. The elemental mapping of the sample containing the phases with different valences was obtained by TEM. Quantitative analysis was performed by energy dispersive spectrometry (EDS). The specimens for TEM and HRTEM measurements were prepared by drop-casting a droplet of ethanol suspension onto a copper grid coated with a thin amorphous carbon film, and dried in air. The BET specific surface area of the samples was measured using a Belsorp II-mini . X-ray photoelectron spectroscopy  was conducted using the following X-ray source: monochromatic Al Kα, hν = 1486.6 eV, X-ray binding energy (BE) of 15 kV, 150 W and spot size of 500 μm; take-off angle of 90°; Pass energy of 20 eV; and BE resolution of 0.6 eV. All the BE values were calibrated using the BE of C 1s (284.6 eV). XPS fitting was performed using “AVANTAGE” software with a Shirley subtraction and the shape of the peaks used for deconvolution was Gaussian–Lorentzian. XPS was conducted at the Korea Basic Science Institute (KBSI), South Korea. The photocatalytic and photoelectrochemical experiments were carried out using a 400 W lamp  with λ > 500 nm and an intensity of 31 mW cm−2.The structure of the as-prepared samples and their crystallite size were confirmed by X-ray diffraction  using Cu Kα radiation (λ = 0.15405 nm). The peaks of the crystalline phases were matched to the standard compounds reported in the JCPDS data file, Raman spectroscopy  was performed to confirm the synthesis of the CeO2-Graphene nanostructures used as the reference, counter, and working photoelectrodes, respectively. EIS was performed in a 0.2 M Na2SO4 solution; CV was performed in a 0.2 M phosphate buffer solution  as the supporting electrolyte at room temperature. The projection area of the photoelectrode was 1 cm2.The photocatalytic degradation of the organic pollutants process was monitored by measuring the absorption of the organic model pollutants by UV–vis spectrophotometry (OPTIZEN 2120UV). The photoelectrochemical studies, such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), were performed using a potentiostat  comprised of a standard three-electrode system. Ag/AgCl (3 M KCl), a Pt gauge and FTO glass coated with pure-graphene and CeOThe working electrodes were prepared as follows. A 100 mg mass of each sample was mixed thoroughly by adding ethyl cellulose as a binder and α-terpineol as the solvent. The mixture was stirred instantaneously and heated at hot plate magnetic stirrer until a thick paste was obtained. The obtained paste was then coated on carbon paper using the doctor-blade method and kept drying under a 60 W lamp overnight and the electrode was later used as a photoelectrode.Supplementary Information


Glioblastoma (GB), the most-common cancer in the adult brain, despite surgery and radio/ chemotherapy, is to date almost incurable. Many hypoxic tumors, including GB, show metabolic reprogramming to sustain uncontrolled proliferation, hypoxic conditions and angiogenesis. Peroxisome Proliferator-activated Receptors (PPAR), particularly the α isotype, have been involved in the control of energetic metabolism. Herein, we characterized patient-derived GB neurospheres focusing on their energetic metabolism and PPARα expression. Moreover, we used a specific PPARα antagonist and studied its effects on the energetic metabolism and cell proliferation/survival of GB stem cells. The results obtained demonstrate that tumor neurospheres are metabolically reprogrammed up-regulating glucose transporter, glucose uptake and glycogen and lipid storage, mainly under hypoxic culture conditions. Treatment with the PPARα antagonist GW6471 resulted in decreased cell proliferation and neurospheres formation. Therefore, PPARα antagonism arises as a potent new strategy as adjuvant to gold standard therapies for GB for counteracting recurrences and opening the way for pre-clinical trials for this class of compounds. When tumor neurospheres were grown in hypoxic conditions in the presence of different glucose concentrations, the most diluted one (0.25g/L) mimicking the real concentration present in the neurosphere core, PPARα increase/PPARγ decrease, increased proliferation and cholesterol content, decreased glycogen particles and LDs were observed. All these responses were reverted by the 72 h treatment with the PPARα antagonist. Mevalonate genes expression profiles were evaluated with specific primer sets (Table t method .Neurospheres treated or not with 8µM GW6471 were extracted with chloroform:isopropanol:NP40 (7:11:0.1) and the organic phase was dried at 50C° and put under vacuum for 30 min. Dried lipids were dissolved with Cholesterol Assay Buffer and assayed according to manufacturer’s instructions . The fluorescence of the samples was measured at Ex/Em = 535/590 nm with a microplate reader .Cell proliferation is evaluated by the CellTrace™ CFSE cell proliferation kit , according to manufacturer’s protocol.Neurospheres were allowed to adhere on poly-L-lysine coated glass coverslips and fixed in 4% paraformaldehyde in PBS, for 10 minutes, at RT. Coverslips were mounted with Vectashield Mounting Medium and examined at a Leica TCS SP5 confocal microscope.Neurospheres grown in 0.25g/L glucose,and 16µM GW6471 treated were homogenized with ice cold glycogen hydrolysis buffer, for 10 min, centrifuged at 12000 rpm, for 5 min and supernatants were assayed according to manufacturer’s instructions . The product was revealed at 450nm.P < 0.05; **P < 0.005, ***P < 0.0005. All data are mean ± SD of three separate experiments run in triplicate..For statistical analysis samples were processed by SPSS software and analyzed by ANOVA test followed by Scheffe’s post hoc test” analysis. *


Currently, four immunohistochemical assays are registered with the US Food and Drug Administration to detect the expression of PD-L1. We investigated the PD-L1 expression in thymic carcinomas using these four diagnostic assays. The cases of 53 patients were reviewed and their specimens were subjected to four PD-L1 assays with different antibodies . The PD-L1 expression in tumor cells (TCs) and immune cells (ICs) was evaluated. In TCs, the four assays showed similar scores in each case. Histopathologically, high TC scores were observed in squamous cell carcinomas (SqCCs). Meanwhile, there were no significant relationships among the IC scores in the four assays. In SqCCs, the high expression of PD-L1 (defined as ≥50% TC score) in TCs tended to be associated with early stage cancer. The patients with high expression levels of PD-L1 tended to show longer overall survival in the 22C3 assays (p=0.0200). In thymic carcinomas, the staining pattern showed high concordance among the four assays when TCs – rather than ICs – were stained. High PD-L1 positivity in TCs, especially in SqCCs, indicated that PD-1/PD-L1 targeted therapy may be a promising therapeutic approach. There is currently no standardized treatment for thymic epithelial tumors because of their low incidence, histological heterogeneity, and unknown molecular pathogenesis –3. In paRecently, immunotherapy targeting programmed death 1 /programmed death ligand 1  has been shown to be clinically effective and thus represents a promising therapeutic alternative in some oncologic cases . The binCurrently, three agents  have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of PD-L1-positive NSCLC. Meanwhile, durvalumab  is still under clinical development for use in NSCLC. Several companies have developed different primary antibodies, which have been used to detect PD-L1 proteins in immunohistochemical analyses; these use different staining protocols, scoring algorithms, and threshold criteria. Each FDA-approved agent has its corresponding immunohistochemical assay as a companion or complementary diagnostic test; thus, there is currently a one drug–one diagnostic test co-development approach. Four studies have been performed to compare the companion diagnostic tests for NSCLC, with the aim of better understanding the similarities and differences among the four assays –21.PD-1/PD-L1 targeted therapy has not yet been established for thymic carcinoma. However, the comparison of different assays is essential for selecting appropriate therapies, for achieving a satisfactory clinical outcome in cases of thymic carcinoma, and for promoting the appropriate arrangement of PD-L1 assays in clinical studies. Thus, it is necessary to set up a standard highly reproducible assessment for PD-L1 immunostaining because a scoring system affects the initiation of treatment. The aim of this study is to establish a highly reproducible standard assessment for each companion or complementary PD-L1 antibody in thymic carcinoma and to elucidate the association between the expression of PD-L1 and the clinicopathological features.Patients’ characteristics are shown in Table In the 5 thymic control cases, the percentage of PD-L1 positive cells ranged from 0 to 25% in the four assays; the SP142 assay showed a slightly higher expression rate than the other three assays . Figure The distribution of the PD-L1 expression, as detected by the four antibodies, is shown in The PD-L1 expression status (above or below the assay threshold) in all samples was compared using the cutoffs applied to NSCLC. We determined the concordance among the four assays when classifying cases according to the assay and the selected cutoff combination and specified each drug and assay combination. The heat map in Figure The clinicopathological features in all thymic carcinoma patients and the PD-L1 expression are shown in Table PD-L1 is a transmembranous protein that downregulates immune responses by binding to its two receptors, PD-1 and B7.1 , 9, 23. Six previous studies have addressed the immunohistochemical expression of PD-L1 in TCs in the context of the patients with thymic carcinomas. In 2015, Padda et al. evaluated the PD-L1 expression in four patients using clone 15  and reported that the expression of PD-L1 was confirmed in three patients (75.0%) . KatsuyaOne aspect of the present study that was superior to previous studies: the present study clarified the differences in the expression of PD-L1 according to the tissue type in thymic carcinoma. We found that the expression of PD-L1 in the TCs in patients with SqCCs and LECs was significantly high in comparison to adenocarcinomas and neuroendocrine tumors. Similar results were reported in patients with NSCLC and uterine cervical cancer , 31. Yu Several recent studies using different assays compared the expression of PD-L1 in NSCLC and showed the slight superiority of the assay using clone SP263, which showed the highest concordance rates for TC scoring, while 22C3 and 28-8 showed comparable yields; SP142 showed the lowest concordance rates for TCs –21. ThisIn IC staining, no significant correlations were observed among the four assays. Tumor-infiltrating lymphocytes (TILs) are measured morphologically, and there are no established thresholds for TILs at present . HoweverDifferent results regarding the relationship between the PD-L1 expression and the patient's prognosis have been reported in various studies of various carcinomas , 9. In tA large number of companies and academic laboratories are attempting to develop antibodies to detect PD-L1 as a potential predictive marker for therapies that interfere with the interaction of one or both of the PD-1 ligands and the PD-1 receptor. The best PD-L1 antibody and the appropriate cutoff expression level for determining the PD-L1 expression remain controversial. The cutoff PD-L1 expression levels that were used in this study ranged from 1% to 50%, depending on the previous studies and assays that were used for lung cancer patients. The KEYNOTE-010 study compared pembrolizumab to docetaxel in previously treated patients with NSCLC, using 1% and 50% cutoff values for the expression of PD-L1 . PembrolTo the best of our knowledge, this is the largest study ever carried out on thymic carcinomas, and it is also the first validation trial conducted on patients except for lung cancer patients using four companion or complementary diagnostic tests. We believe that this research gives us very realistic information that will be useful in the clinical administration of therapeutic drugs. The present study was associated with several limitations. First, the specimens that were used for the immunohistochemical examinations were not all from surgically resected tissues. Second, the auto-stainer that was used for immunohistochemistry for SP142 and SP263 was not the Ventana Benchmark Ultra, which is an optimized platform. Third, despite the fact that the present study included 53 cases of thymic carcinomas and thymic neuroendocrine tumors, which was a relatively large sample size, further prospective studies with more cases and a longer follow-up period are needed. Further studies are required, including prospective analyses of large cohorts and biological analyses to investigate genomic abnormalities or PD-L1 transcriptional levels in thymic carcinomas.In conclusion, we evaluated the PD-L1 expression in TCs and ICs in patients with thymic carcinomas, with the aim of achieving the harmonization of the four immunohistochemical assays. This study revealed that patients with thymic carcinomas, especially SqCC patients, showed high PD-L1 positivity and tended to have a favorable prognosis. Furthermore, high concordance in the four assays—which showed greater utility in TC staining than in IC staining—was observed. Our results suggest that the PD-1/PD-L1 pathway is a potential immunotherapeutic target in thymic carcinoma.We reviewed FFPE tissue specimens from 53 patients with thymic carcinoma or neuroendocrine tumors and five controls of non-neoplastic thymic epithelial cells adjacent to thymomas. All of the specimens were obtained from Nagoya City University Hospital, Toyota Memorial Hospital, and Kariya Toyota General Hospital from 1984 to 2016. There were 39 surgical specimens and 14 needle biopsy specimens. All of the cases were microscopically reviewed and diagnosed by two expert pathologists (TM and HI). No specimens associated with any therapeutic trial or immune checkpoint inhibitor therapy were included in this study. The relevant clinical data were collected from the patients’ medical records. This study was approved by the Institutional Review Boards of the abovementioned institutions and was carried out in accordance with the Declaration of Helsinki.The H&E-stained specimens in each case were microscopically reviewed and each case was pathologically diagnosed according to the 2015 WHO classification [The positivity of TCs and ICs was assessed by two expert pathologists (TM and HI). TCs in which the membrane was immunostained at any intensity were considered to be positive for PD-L1. The ratios of PD-L1-positive TCs in all of the carcinoma cells were evaluated by microscopic observation (TC scores). Meanwhile, ICs in which the membrane or cytoplasm was immunostained at any intensity were considered to be positive for PD-L1, because the stained membrane and cytoplasm could not be distinguished in lymphocytes due to their small size. Most PD-L1-positive ICs were macrophages and lymphocytes. ICs were quantified by evaluating the ratio of the area covered by stained ICs in the tumor area (IC scores), as described in previous reports , 45, 46.The correlations between the PD-L1 expression and the clinicopathological features were evaluated by Wilcoxon rank-sum test for continuous variables  and by Fisher's exact test for categorical variables . Bonferroni correction was used to account for multiple comparisons as appropriate. Spearman's correlation coefficient was calculated to evaluate the correlation regarding PD-L1 positivity in each assay. Survival curves were generated using the Kaplan-Meier method, and the log-rank test was used to assess the statistical significance of differences between groups. A Cox proportional hazards model was used to estimate the hazard ratios and 95% confidence intervals. The prognostic variables identified by a univariate analysis were further analyzed in a multivariate Cox model, where we evaluated the variance inflation factors to assess the multicollinearity for the proposed model. A two-sided p value of <0.05 was considered to indicate a statistically significant difference. The statistical significance level was adjusted for multiple analyses according to Bonferroni correction. All of the statistical analyses in this study were performed using the JMP software program .


Charge carriers in the quantum Hall regime propagate via one-dimensional conducting channels that form along the edges of a two-dimensional electron gas. Controlling their transmission through a gate-tunable constriction, also called quantum point contact, is fundamental for many coherent transport experiments. However, in graphene, tailoring a constriction with electrostatic gates remains challenging due to the formation of p–n junctions below gate electrodes along which electron and hole edge channels co-propagate and mix, short circuiting the constriction. Here we show that this electron–hole mixing is drastically reduced in high-mobility graphene van der Waals heterostructures thanks to the full degeneracy lifting of the Landau levels, enabling quantum point contact operation with full channel pinch-off. We demonstrate gate-tunable selective transmission of integer and fractional quantum Hall edge channels through the quantum point contact. This gate control of edge channels opens the door to quantum Hall interferometry and electron quantum optics experiments in the integer and fractional quantum Hall regimes of graphene. Quantum point contacts are gate-tunable constrictions allowing for control of charge carrier transmission in 2D electron gases. Here, the authors fabricate a hBN/graphene/hBN van der Waals heterojunction to enable quantum point contact devices in the integer and fractional quantum Hall regimes. In two-dimensional electron gases formed in semiconductor heterostructures, confinement of electron transport through nano-patterned constrictions has led to tremendous advances in quantum transport experiments2345681012Yet, in monolayer graphene, demonstration of QPC operation in split-gate geometry remains challenging. The major hurdle precluding engineering split-gated constrictions stems from the gapless graphene electronic band structureThe alternative route that consists in confining electron transport through etched constrictions has long been difficult due to the fact that physically etched constrictions in low-mobility devices are subject to electron localization by disorder and charging effects151617In the QH regime, tunability of the QPC constriction with split-gate electrodes is mandatory to control the transmission of QH edge channels and the tunnelling between counter-propagating QH edge channels. However, the p–n junction formed along gate electrodes also poses problems as electron-type QH edge channels co-propagate along the junction with hole-type edge channels, as illustrated in 21222527In this work we employ high-mobility hBN/graphene/hBN van der Waals heterostructures29High-mobility samples were fabricated following the recent tour de force in graphene device fabrication techniques using van der Waals pick-up2(285 nm)/Si++ substrate serves as a back gate. The six contacts enable measurement of three voltages  of the bulk graphene—the resistance peak independent of Vsg—is located at RL indicates charge neutrality in the split-gated region of graphene. Its slope corresponds to the ratio of capacitances between back-gated and split-gated regions (Csg/Cbg=7) and hence provides a way to assess quantitatively for the charge carrier density beneath the split gates (using −2).Vsg=0 V leads to a mean free path of 1.8 μm that coincides with the width of the Hall bar, and a bulk mobility superior to 200 000 cm2 V−1 s−1 at a charge carrier density of n∼1012 cm−2. These, together with signatures of negative nonlocal resistance . RL versus Vbg and B taken at Vsg=0 V  signal the presence of QH states. In addition to the usual graphene sequence νb=4, these broken symmetry states are furthermore accompanied by fractional QH plateaux that are pronounced in the Hall conductance . At positive Vsg, these diagonal strips break up into rhombi that are horizontally delimited by the width of the bulk QH plateaux, centred at integer values of νb. Diagonal grey dotted lines index the expected filling factor beneath the split gates, νg, related to the local charge carrier density .Let us now investigate the control of integer QH edge channels by split-gate electrodes defining the QPC. We begin with a set of data taken at Vsg regime where the charge carrier density below the split gates is lower than the graphene bulk density, as is required for confining bulk QH edge channels into the QPC. Inspecting the conductance strips we note that their slope does not match the lines of constant νg. The shallower slope rather indicates a smaller capacitive coupling to the split-gate electrodes and hence a region of the graphene device with a charge carrier density in between those of the bulk and the split gates. This region is nothing but the QPC saddle-point constriction that is capacitively coupled to both the back-gate and the split-gate electrodes. We thus introduce a third filling factor νQPC related to the charge carrier density in the QPC  with the conductance value of each strip , and between νb=2 and νg=−1 edge channels (upward spin polarization), gives e2/h, as measured on the νQPC=0 strip. For νQPC=−1, a hole state connects the split gates as sketched in N=0 LL provides a full explanation of the anomalous conductance values of the strips when the filling factor in the QPC is reduced to 1, 0 or −1.As a result, for nnel see  leads toVsg, the filling factor configuration changes to νb<νQPC<νg. Extra electron-type QH edge channels can thus connect left and right edges of the graphene device  over a large range of gate values, indicating that equilibration is less efficient at 14 T than at 7 T, most likely due to the larger energy gaps.We complete this study of the QPC operation in the integer QH regime by addressing the pinch-off that occurs for en at  T . The conνb±1/3 develop at a relatively low magnetic field of 14 T . Such conjectures that are based on electrostatics of QH edge channels call for further theoretical works including edge channel reconstruction and full degeneracy lifting of graphene QH states.Interestingly, in our high-mobility graphene devices, the regime of partial equilibration at To conclude, our overall understanding of the precise edge channel configurations allows us to demonstrate gate-tunable and equilibration-free transmission of both integer and fractional QH edge channels through QPCs in graphene. Such a control of edge channel transmission enables future investigations of the equilibration processes at play that limit adiabatic transport45810495034323336The data that support the findings of this study are available from the corresponding author on request.How to cite this article: Zimmermann, K. et al. Tunable transmission of quantum Hall edge channels with full degeneracy lifting in split-gated graphene devices. Nat. Commun.8, 14983 doi: 10.1038/ncomms14983 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Notes and Supplementary References


Adsorption of gas molecules on the surface of atomically layered two-dimensional (2D) materials, including graphene and transition metal dichalcogenides, can significantly affect their electrical and optical properties. Therefore, a microscopic and quantitative understanding of the mechanism and dynamics of molecular adsorption and desorption has to be achieved in order to advance device applications based on these materials. However, recent theoretical calculations have yielded contradictory results, particularly on the magnitude of the adsorption energy. Here, we have experimentally determined the adsorption energy of oxygen molecules on graphene and 2D tungsten disulfide using temperature-programmed terahertz (THz) emission microscopy (TPTEM). The temperature dependence of THz emission from InP surfaces covered with 2D materials reflects the change in oxygen concentration due to thermal desorption, which we used to estimate the adsorption energy of oxygen molecules on graphene (~0.15 eV) and tungsten disulphide (~0.24 eV). Furthermore, we used TPTEM to visualize relative changes in the spatial distribution of oxygen molecules on monolayer graphene during adsorption and desorption. Our results provide much insight into the mechanism of molecular adsorption on the surface of 2D materials, while introducing TPTEM as a novel and powerful tool for molecular surface science. Many other 2D materials have since been discovered with a wide range of characteristics, from metallic to semiconducting to insulating, opening up exciting new opportunities for the development of devices based on monolayers, bilayers, and heterostructures of 2D materials8. However, since these materials typically consist of one or a few atomic layers, their properties are extremely susceptible to perturbations from their environment. Exposure to gases, for example, has been shown to drastically affect their electrical and optical properties15, which means that in order to realize 2D-materials-based devices, it is crucial to understand and control the influence of gas adsorption and desorption dynamics on their properties.The successful isolation of monolayer graphene in 2004 and its remarkable properties found subsequently have paved the way for a new research field of two-dimensional (2D) atomic layer materials2) is one of the most important because not only it significantly alters the properties through doping, it is also the second most abundant gas in the atmosphere and is therefore highly likely to affect the performance of devices in practical applications. Though theoretical simulations proved to be useful in understanding the interaction of O2 molecules and/or O atoms with 2D materials, conflicting results for the adsorption energies were obtained due to the inability of the approximation functionals used to properly describe the dispersion forces23. Knowing the correct value of the adsorption energy is important since it describes the strength of the interaction of the adsorbate with the 2D material, as well as the extent to which its properties are altered. Experimental measurements using thermal desorption spectroscopy (TDS) [also known as temperature-programmed desorption (TPD)] were done as a complementary investigation to the theoretical results26. However, these measurements were done only on highly-oriented pyrolytic graphite (HOPG), which is the bulk form of graphene. Another drawback is that in TDS/TPD, the desorption of molecules is usually measured from a large sample area, failing to provide information on local adsorption and desorption dynamics. This is a significant disadvantage because the large surface-to-volume ratio enhances the importance of surface interactions in 2D materials.Of the possible gas adsorbates/contaminants, oxygen (O2 adsorption and desorption dynamics in graphene and tungsten disulfide (WS2) with micrometer resolution. THz spectroscopy has been proven to be a powerful tool in studying the chemical and physical processes in a variety of systems28. In our work, we used the THz emission from the surface of InP coated with monolayer graphene to observe the relative concentration of O2 adsorbates30. In this non-contact and non-destructive method, femtosecond infrared (IR) laser pulses are used to generate THz waves from a spot on the substrate surface. Absorption of both the incident IR and the generated THz radiation by graphene  is very small compared to the effects of adsorption of O2 molecules on the 2D materials to the THz emission, which allows us to use THz emission as a probe in studying O2 adsorption.Here, we used temperature-programmed terahertz (THz) emission microscopy (TPTEM) to probe local O2 adsorbates are present on the graphene surface because the electric dipoles induced by the adsorbates modify the surface depletion field in InP, which changes both the polarity and magnitude of the emitted THz wave. By correlating the changes in the waveform of THz emission and the relative concentration of O2 molecules on the sample surface as a function of temperature, we were able to obtain the first experimental value of the adsorption energy of locally physisorbed O2 molecules on graphene and WS2. Furthermore, the method can be used to directly visualize the relative amount of O2 molecules on the sample surface, allowing us to monitor the spatial distribution of O2 adsorbates in monolayer graphene under controlled conditions. This capability allowed us to observed the influence of sample surface non-uniformity on O2 adsorption and desorption, as well as the reported UV-illumination-induced enhancement of O2 adsorption33.The waveform of the THz radiation changes dramatically when O2 adsorbates from the graphene surface as shown in Fig. 2 desorption occurs upon vacuum pumping, and the change in the THz waveforms reflects the influence of the decrease in adsorbate concentration on the photoexcited current in InP. Both features in the THz waveform gradually vanish, followed by the appearance of a positive peak at ~2 ps. Irradiation with femtosecond IR pulses also promotes desorption of O2, with the rate of desorption dependent on the laser fluence29. With further removal of adsorbates by annealing, the photoexcited current flows towards the bulk, and the THz emission from graphene/InP becomes similar to that of the bare InP substrate. O2 adsorption by exposure to air reverts the THz waveform back to the initially observed waveform, and this adsorption is dramatically enhanced under UV light illumination. The change in the THz emission from graphene/InP (due to O2 adsorption/desorption) shown in Fig. x (0 ≥ x ≥ 1) is their relative weight which is proportional to the concentration of adsorbed O2 on graphene29.The time-domain waveforms of the THz emission from graphene/InP changes with the removal of Omentclass2pt{minim2 nanosheets prepared by LPE. This is done to determine how sensitive is the change in the THz emission from 2D-coated InP due to O2 adsorption/desorption, and if TPTEM can be used to observe changes in adsorption/desorption dynamics at different conditions. Differences in the temporal evolution of the THz emission peak (at ~2 ps) from the samples can be observed under continuous IR irradiation at various conditions as shown in Fig. 2 molecules; for instance, 2D materials prepared by LPE usually contain more structural defects than those prepared by CVD, and these defects can act as adsorption sites for molecules36.With the TPTEM setup, we monitored the THz emission from InP coated with graphene prepared by chemical vapor deposition (CVD) and liquid phase exfoliation (LPE), as well as with WS2 in graphene and WS2. The experiment was designed to be similar to TDS/TPD, wherein the sample of interest is pre-exposed to a gas adsorbate and then heated in a controlled fashion while monitoring the partial pressures of the evolved molecular species by using, for example, a mass spectrometer38. The main difference of TPTEM from TDS/TPD is that it can be used to monitor the relative concentration of the remaining adsorbates within the spot on the sample illuminated by the femtosecond laser. This means that it is possible to probe not only desorption dynamics but also adsorption dynamics in 2D materials within the excitation spot.We then used TPTEM to measure the adsorption energy of O2/InP in Fig. 2 adsorbates on the surface decreases with increasing temperature, and we can separate its effect on the measured THz waveforms by simply subtracting the THz emission of annealed samples from the unannealed samples, or Eannealed waveforms represent emission from samples without adsorbates. Annealing at this temperature under vacuum conditions should completely remove physically adsorbed O2 molecules from the surface of monolayer graphene based on optical conductivity measurements , while in Fig. 2, the absolute peak value reaching zero at 413 K could mean either (a) complete O2 desorption at this temperature, or (b) no further desorption happened during the annealing from 413 K to 445 K. Considering the relatively larger adsorption energy in WS2 and the high possibility of the existence of chemisorbed species22, the latter is more likely the case.The temperature dependence of THz radiation from CVD graphene/InP before and after annealing at 445 K for 1 hour are shown in Fig. 40. In the far-field approximation, the amplitude of this radiation is proportional to the time derivative of the surge current, given by41,THz emission from InP primarily occurs through the surge current effectJ(t), is due to the transient photocarriers generated by IR excitation. One of the simplest approximations of this surge current (density) is given by the expression42,q is the electron/hole charge, μ(t) is the electron/hole mobility, n(t) is the carrier density, and Ed(t) is the driving electric field, which in this case is the built-in surface field in InP. The parameters μ(t), n(t), and Ed(t) determine the observed THz field through equation  (see Supporting Information for more details), which in turn is caused by the electric field on the surface of InP due to the dipoles induced by the O2 adsorbates in graphene (Edipole)29. Simulations show that charge transfer occurs between the O2 molecules and carbon atoms in graphene19, and similarly for other 2D materials22, supporting the possibility of the existence of these electric dipoles. If we denote the modified driving field due to the presence of adsorbates as Ed,2 = Ed,1 + Edipole, then the change in the THz emission from graphene/InP due to O2 adsorbates can be obtained using equations  with a linear fit for each sample are shown in Fig. x = 1) or non-dissociative desorption in our data analyses since a large energy barrier would have to be overcome for O2 molecules to dissociate into O atoms once adsorbed in graphene and MoS2 19. In this case, the activation energy of desorption obtained is approximately equal to the adsorption energy37. We then calculated Edes from the slope of the linear fits in Fig. 2 molecules on WS222 and on graphene and HOPG26. The chemistry and morphology of the supporting substrate significantly influence molecular interaction in graphene44 and possibly in other 2D materials as well. This influence becomes weaker with increasing number of layers47. In our work, similar adsorption energy values were obtained for CVD monolayer graphene and LPE graphene multilayer  nanosheets. This leads us to believe that these values are good approximation of the actual adsorption energy of oxygen molecules on pristine graphene.Plots of mentclass2pt{minim2 molecules were removed in the temperature range considered in measuring Edes. However, it is clear that for LPE graphene a significant number of adsorbates remain at T > 400 K, which represent the O2 molecules (or O atoms) attached to graphene at higher adsorption energies. The inherent defect sites in LPE graphene could lead to spontaneous chemisorption of oxygen, and increasing the temperature range in this experiment should allow us to probe the desorption dynamics of these chemisorbed species.Only the physisorbed O2 adsorption/desorption dynamics at different sites in the sample surface, we adjusted the excitation spot size to ~0.2 mm and used the imaging capability of TPTEM to study the spatial distribution of adsorbed O2 molecules in CVD graphene at various controlled conditions. This can be done by placing the sample chamber on a computer-controlled stage. First, we checked the distribution of adsorbed O2 molecules within a ~1.65 × 1.65 mm2 surface area of the monolayer graphene deposited on InP, after exposing the sample in air for a long time. The acquired image . Structural defects and doping in graphene have significant effects on its reactivity towards adsorbates48.The adsorption energies presented in Table 2 molecules after exposure to air for a few minutes  and after air exposure is taken as the measure of the adsorbed O2 molecules. Figure 2 adsorbates after exposure to air, which shows the “natural” affinity to O2 molecules, i.e., O2 adsorption process without external influence like UV light, of different regions in the sample surface. Regions 1 and 2 (5) in Fig. 2 adsorption, and this dramatic effect can be seen in Fig. The non-uniformity of the surface and its influence on adsorption/desorption dynamics can be clearly seen by taking THz emission images under controlled conditions. First, we removed the adsorbates in the sample by annealing it at 445 K in vacuum for at least 5 minutes, and then we monitored the spatial distribution of the relative amount of adsorbed Osee Fig.  and withsee Fig.  UV illum2 molecules in graphene and WS2 with micrometer spatial resolution. Thermal annealing at 445 K shows complete removal of the physisorbed O2 molecules in graphene. We estimate the adsorption energy of O2 molecules physisorbed on graphene and WS2 to be ~0.15 eV and ~0.24 eV, respectively. Spatial mapping of adsorbed O2 molecules in monolayer graphene under controlled conditions also reveals the effects of sample non-uniformity on O2 adsorption and desorption dynamics. The regions in graphene which are identified to have a higher affinity to O2 (and thus had more adsorbed molecules upon exposure to air) also indicate a lower adsorption energy between the O2 molecules and the graphene surface. In addition, we show that the imaging capability of TPTEM combined with its feasibility in doing quantitative measurements makes it a valuable tool in studying O2 adsorption and desorption dynamics in 2D materials in a non-destructive manner. Since the primary requirement for this technique to work is that the gas adsorbates should induce local electric dipoles that will change the THz emission from InP, TPTEM is expected to be useful for studying the adsorption and desorption dynamics of other types of gas molecules in any 2D material, such as NH3, as long as charge transfer occurs between the adsorbate and the 2D material in question. All these results promise the possibility of doing local spatiotemporal studies on the molecular adsorption and desorption on a variety of 2D materials.In conclusion, we have developed the temperature-programmed THz emission microscope (TPTEM) system and studied the adsorption and desorption dynamics of O49. Thin poly(methyl methacrylate) (PMMA) film was deposited on top of graphene to act as supporting layer during the wet transfer method. Diluted ammonium persulfate solution was used to dissolve the copper foil and then washed several times in deionized water. The floating PMMA/graphene film was carefully transferred onto InP (100) substrate to produce the CVD graphene/InP sample and air-dried before removing the PMMA film using acetone. Bulk graphite and WS2 (Sigma Aldrich) were used as starting materials to produce nanosheets using the liquid phase exfoliation (LPE) technique as previously described50. After annealing, each powder sample is dispersed in isopropyl alcohol and then exfoliated by applying ultrasound using a horn-tip sonicator, followed by centrifugation and decantation of the supernatant to produce the final nanosheets dispersions. 2D materials produced this way are typically few-layered nanosheets with varying laterial dimensions. The dispersions were then drop-casted onto mildly heated semi-insulating InP (100) substrate to make the LPE graphene/InP and LPE WS2/InP samples.Using a chemical vapor deposition (CVD) technique, methane was used as precursor to synthesize monolayer graphene on a copper foil52. At this range of laser fluence, THz radiation is generated mainly by ultrafast surface surge-currents in the semi-insulating InP substrates40. THz radiation emitted by samples were focused onto a dipole-shaped low-temperature-grown GaAs photoconductive switch using a pair of off-axis parabolic mirrors. The output current from the switch was fed into a lock-in amplifier which was referenced to a 2.0 kHz optical chopper signal. During the measurements, the samples were placed in a vacuum chamber equipped with a PID temperature controller. The vacuum chamber containing the sample was mounted on a computer-controlled sample stage for THz imaging.The samples were optically excited using near-infrared pulses with a duration of ~100 fs and photon energy of 1.55 eV from a Ti:sapphire laser. The excitation beam was incident on the sample surface at an angle of 45°. The beam diameter was adjusted by changing the relative distance between the sample and the focusing lens nearest to it. In the experiments for determining the adsorption energy, the excitation beam power used was 30 mW and the beam diameter was ~1.35 mm, while in the imaging measurements, the power was 20 mW and the diameter was ~0.2 mm. In all experiments, the laser fluence was a few orders of magnitude lower than the damage threshold reported for grapheneAdsorption energy of oxygen molecules on graphene and two-dimensional tungsten disulfide