The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration. Among modulators of autophagy, only vacuolar-ATPase inhibitors, such as bafilomycin A1 (Baf), increased the levels of the full-length and cleaved forms of TDP-43 and the autophagosome marker LC3-II (microtubule-associated proteins 1A/1B light chain 3B) in extracellular vesicle fractions prepared from the culture media of HeLa, SH-SY5Y, or NSC-34 cells, whereas vacuolin-1, MG132, chloroquine, rapamycin, and serum starvation did not. The C-terminal fragment of TDP-43 was required for Baf-induced TDP-43 secretion. The Baf treatment induced the translocation of the aggregate-prone GFP-tagged C-terminal fragment of TDP-43 and mCherry-tagged LC3 to the plasma membrane. The Baf-induced secretion of TDP-43 was attenuated in autophagy-deficient ATG16L1 knockout HeLa cells. The knockdown of PGRN induced the secretion of cleaved TDP-43 in an autophagy-dependent manner in HeLa cells. The KO of PGRN in mouse embryonic fibroblasts increased the secretion of the cleaved forms of TDP-43 and LC3-II. The treatment inducing TDP-43 secretion increased the nuclear translocation of GFP-tagged transcription factor EB, a master regulator of the autophagy-lysosomal pathway in SH-SY5Y cells. These results suggest that the secretion of TDP-43 is promoted by dysregulation of the PGRN-driven autophagy-lysosomal pathway. The abnormal aggregation of the nuclear protein, transactive response DNA-binding protein 43 kDa (TDP-43) in the cytoplasm is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP) , 2. TDP-de novo pathology of cytoplasmic accumulation of TDP-43 in a mouse brain, which progressively spread throughout the brain in a time-dependent manner via the neuroanatomic connectome  haploinsufficiency because of a loss-of-function mutation in the PGRN gene causes FTLD-TDP , 33. Altvia EV. To elucidate the mechanisms underlying the release of TDP-43 into the extracellular space and the role of PGRN in this process, we examined the secretion of TDP-43 using different classes of modulators for autophagy. We used the v-ATPase inhibitors, Baf and concanamycin A (Con), the PIKfyve  inhibitor, vacuolin-1 (Vac), and the autophagosome–lysosome fusion inhibitor, chloroquine (CQ), the autophagy inducer, rapamycin (Rapa), and serum starvation (SS) as well as the knockdown of siRNA.In the present study, we aimed to examine whether the autophagy-lysosome pathway regulates extracellular release of TDP-43 A). HeLa cells expressing the fluorescence autophagy marker were treated with 100 nM Baf, an inhibitor of v-ATPase and autophagosome–lysosome fusion, 1 μM MG, an inhibitor of proteasome degradation and an inducer of autophagy, or 100 nM Vac, an inhibitor of PIKfyve and autophagosome–lysosome fusion, for 24 h . LC3-II-positive autophagosomes were identified as yellow puncta because of merged GFP and RFP signals and autolysosomes as red puncta because of the loss of the GFP signal under low pH (or acidic) conditions. Baf, MG, and Vac significantly increased yellow puncta, indicating a higher number of autophagosomes . On the other hand, the formation of autolysosomes was eliminated by Baf and Vac but not by MG . These results suggest that the increase in the number of autophagosomes induced by Baf and Vac was due to the suppression of autophagosome–lysosome fusion, whereas MG accelerated the formation of autophagosomes. Autophagic flux was assessed using HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG (A and B). On the other hand, MG decreased the GFP/RFP ratio, indicating its facilitation of autophagic flux (C and D). The GFP/RFP ratio of Vac-treated cells was similar to that of vehicle-treated cells (C and D). Immunostaining of the phagophore marker WIPI2 (WD repeat domain phosphoinositide interacting 2) showed a significant increase in signal intensity with both Baf and MG but not with Vac (A and B). These results suggest that Baf and MG increased the formation of autophagosomes. The inhibition of lysosomal degradation using E64d and pepstatin A increased LC3-II levels in MG- or Vac-treated cells but not in Baf-treated cells (A–D), suggesting that Baf suppressed lysosomal degradation. Lysosomal acidity, assayed using AcidiFluor ORANGE staining, was elevated in cells treated with MG and Vac but was decreased by Baf (D and E). These results suggest the distinguishable effects of Baf and Vac on the inhibition of autolysosome formation.To elucidate the relationship between TDP-43 secretion and autophagy, we investigated whether stimulators of TDP-43 secretion, such as Baf, MG132 (MG), and Vac , 44, indFig. 1for 24 h , 46  for the collection of larger vesicles, followed by 110,000g (the P2 EV fraction) for the collection of smaller vesicles. The expression levels of cellular proteins were quantified using the lysates of adhered cells, and the loading volumes of samples from the P1/P2 EV fractions were normalized by protein levels in the cell lysates. Immunoblotting results showed that the cleaved form of TDP-43 was detected by the anti-C-terminal TDP-43 antibody, but not by the anti-N-terminal TDP-43 antibody, suggesting that full-length and C-terminal TDP-43 proteins were both released by the Baf treatment (F). To differentiate the cleaved variants of TDP-43 present in the cell, P1, and P2 fractions, we subjected each fraction to immunoprecipitation using the polyclonal rabbit anti-C-terminal TDP-43 antibody. Samples were then immunoblotted with the monoclonal mouse anti-TDP-43 antibody. Immunoprecipitation analysis revealed the presence of multiple variants of cleaved TDP-43 proteins in the cell fraction as well as the P1 and P2 EV fractions (A). Four bands  were detected in the cell fraction. Bands b and c were the predominant bands in the P1 EV fraction, whereas blurry band d was dominant in the P2 EV fraction (B). Densitometric data obtained from immunoblotting results on HeLa, SH-SY5Y neuroblastoma, and NSC-34 motor neuron–like cells showed that Baf, but not MG or Vac, significantly increased full-length and cleaved TDP-43 levels in the P1 and P2 EV fractions without affecting full-length TDP-43 expression levels in the cell fraction . The levels of the exosome marker TSG101 (tumor susceptibility gene 101 protein) in the P1 and P2 EV fractions were markedly increased by Baf but not by MG or Vac without any changes in the cellular expression levels . Baf, MG, and Vac increased the level of LC3-II, an autophagosome marker, in the cell fraction, whereas only Baf markedly increased LC3-II levels in the P1 and P2 EV fractions . These results suggest that the inhibition of autophagosome–lysosome fusion by Baf increased the secretion of full-length and C-terminal TDP-43 via vesicles associated with autophagosomes or exosomes.Cells release EV with the different size and intracellular origin, and the proteins enriched within these vesicles differ between the small and large sizes . To quanreatment F. To difractions A. Four bfraction B. DensitG and H, , A–C. ThA). Full-length TDP-43 levels in the cell fraction were higher in cells treated with Baf plus SS than in those treated with Baf only. On the other hand, cleaved TDP-43, TSG101, and LC3-II levels remained unchanged in the cell fractions of cells treated with Baf, Baf plus Rapa, and Baf plus SS . In the presence of Baf, the levels of full-length and cleaved TDP-43 and LC3-II were higher in the P1 EV fraction from cells treated with Rapa or SS compared with the untreated cells. The treatment with Baf plus SS increased TSG101 levels in the P1 EV fraction more than Baf alone or the Baf plus Rapa treatment . The levels of full-length and cleaved TDP-43, TSG101, and LC3-II in the P2 EV fraction were similar in cells treated with Baf, Baf plus Rapa, and Baf plus SS. SS significantly decreased cleaved TDP-43 levels in the P2 fraction . The autophagic flux assay using HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG showed no change in the GFP/RFP ratio in response to the Rapa treatment and a decrease in response to SS, suggesting that SS, but not Rapa, facilitated autophagic flux (A and B). WIPI2 immunostaining revealed that both Rapa and SS, similar to Baf, increased WIPI2 signal intensity. Moreover, the combination of Baf plus Rapa or Baf plus SS induced a larger increase in WIPI2 signal intensity than the single treatment with Baf, Rapa, or SS (A and B). The inhibition of lysosomal degradation using E64d and pepstatin A increased LC3-II levels in cells treated with Rapa or SS, indicating that Rapa or SS did not suppress lysosomal degradation as effectively as Baf (A and B). These results suggest a positive correlation between the levels of secreted TDP-43 and the secretion of autophagy-associated vesicles triggered by the inhibition of autophagosome–lysosome fusion.Baf increased the formation of autophagosomes under the inhibition of autophagosome–lysosome fusion. To investigate the relationship between EV-mediated TDP-43 secretion and autophagy, we examined the effects of autophagy inducers on TDP-43 secretion, phagophore formation, autophagic flux, and lysosomal degradation in the presence or the absence of Baf. HeLa cells were treated with Baf, Rapa, SS, Baf plus Rapa, or Baf plus SS for 24 h and then subjected to assays A. Full-lreatment , B–E, P1fraction , B–E, P2gic flux , A and Ba, or SS , A and By as Baf , A and BA). Vesicles in the P1 EV fraction exhibited a wide range of sizes (approximately 50–1000 nm in diameter), whereas those in the P2 EV fraction were smaller (approximately 50–200 nm in diameter). Immunolabeling with the anti-C-terminal TDP-43 antibody confirmed the presence of both the full-length and cleaved forms of TDP-43. However, the TDP-43 fibril-like structures reported in a previous study  (C–E), indicating that EV derived from cells exposed to Baf shared some of the properties with previously reported EV , and the p62 band in the P2 EV fraction displayed a somewhat diffuse pattern   indicaterf.gov/) . Moreoverf.gov/) , ACTN4 arf.gov/) . The Baforted EV . The aut pattern . These rTARDBP) was silenced in HeLa cells using siRNA, and the Baf-induced secretion of autophagy-associated vesicles was then examined. As shown in A and B, full-length TDP-43 levels were significantly decreased in the cell, P1, and P2 fractions upon the knockdown of TARDBP (A and B). The knockdown of TARDBP significantly decreased cleaved TDP-43 levels in the cell and P1 EV fractions but not in the P2 EV fraction (C). TSG101 levels in the cell and EV fractions were not affected by the loss of TARDBP (D). The levels of LC3-II were significantly increased not only in the cell fraction, as previously reported (E). These results suggest that the loss of function of TDP-43 enhanced the cellular accumulation and the extracellular release of autophagy-associated vesicles induced by Baf.The accumulation of the nuclear protein TDP-43 in the cytoplasm of an affected region in FTLD-TDP or ALS patients causes not only the toxic gain of function of TDP-43 in the cytoplasm but also the loss of function of TDP-43 in the nucleus . Confocal live-cell images showed the prominent nuclear localization of TDP-43 amino acids 1 to 414 and TDP-43 amino acids 1 to 273. In contrast, TDP-43 amino acids 162 to 414 colocalized with mCherry-LC3 . Sequential centrifugation was conducted to identify which regions of TDP-43 were required for the Baf-induced secretion of TDP-43 . Equivalent levels of the TDP-43 amino acids 1 to 414, TDP-43 amino acids 1 to 273, and TDP-43 amino acids 162 to 414 proteins were detected in the cell fraction. The P1 EV fraction had lower levels of TDP-43 amino acids 1 to 273 than those of TDP-43 amino acids 1 to 414 and TDP-43 amino acids 162 to 414 (D). Only TDP-43 amino acids 162 to 414 were detected in the P2 EV fraction . These results suggest that the C-terminal 162 to 414 region of TDP-43 was required for the Baf-induced secretion of TDP-43.We examined the structural element of TDP-43 that was required for incorporation into EV using already characterized GFP-tagged full-length TDP-43 (amino acids 1–414), GFP-tagged N-terminal TDP-43 (amino acids 1–273), or GFP-tagged C-terminal TDP-43 (amino acids 162–414) . HeLa ceFig. 5A and C). In contrast, when cells were treated with Baf, TDP-43 amino acids 162 to 414 inclusions larger than 0.2 μm in diameter (B and D). These results suggest that the Baf treatment facilitated the fusion of autophagosomes incorporating TDP-43 with the plasma membrane.The Baf-induced incorporation of TDP-43 in autophagosomes with the plasma membrane was monitored using confocal live-cell imaging. Without the Baf treatment, TDP-43 amino acids 162 to 414 and mCherry-LC3 were detected near the plasma membrane but clearly distal , A and Cdiameter  increasediameter , B and DA and B). A deficiency of autophagosomes was confirmed by the loss of LC3-II production in ATG16L1 KO cells (C). Densitometric data revealed that ATG16L1 KO attenuated the incorporation of full-length TDP-43 in P1 vesicles and cleaved TDP-43 in P1 and P2 vesicles (D and E). On the other hand, ATG16L1 KO did not affect TSG101 levels in the cell or EV fraction (F). These results suggest that the formation of autophagosomes was necessary for the Baf-induced secretion of both the full-length and cleaved TDP-43 proteins.The loss of cellular ATG16L1 expression has been shown to suppress the formation of autophagosomes . TherefoKO cells C. Densitvesicles , D and Efraction F. These A–D). The formation of autolysosomes indicated by DALGreen (DALG) was also significantly suppressed by 100 nM Con, 100 μM CQ, or 100 nM Baf (A and B). These results suggest that both Con and CQ suppressed autophagosome–lysosome fusion, similar to Baf. On the other hand, the Con and CQ treatments increased WIPI2 signal intensity, which suggested that they promoted autophagosome formation, similar to Baf (A and B). In parallel, lysosomal acidity was suppressed by the treatment with 100 nM Con, 100 μM CQ, or 100 nM Baf as assessed by AcidiFluor ORANGE (C and D). The treatment with 100 nM Con increased the levels of both full-length and cleaved TDP-43 proteins in the P1 and P2 EV fractions, as did treatment with 100 nM Baf, but 10 or 100 μM CQ failed to induce the secretion of TDP-43 , which encodes a SNARE (soluble NSF attachment protein receptor) protein that regulates autophagosome–lysosome fusion  and lysosomal acidification indicated by the signal of AcidiFluor ORANGE (C and D), consistent with previous reports . Similarly, the knockdown of STX17 significantly increased LC3-II levels in the cell fraction but not in the P1 or P2 EV fraction (E and H). These results suggest that the suppression of autophagosome–lysosome fusion did not contribute to increases in the secretion of TDP-43.The roles of autophagosome–lysosome fusion in the secretion of TDP-43 were further examined in HeLa cells by the siRNA knockdown of the syntaxin 17 gene  in HeLa cells or NSC-34 motor neuron–like cells. The knockdown of GRN significantly decreased DALG (A and B) and AcidiFluor ORANGE (C and D) signal intensities, indicating the suppression of autolysosome formation and lysosomal acidification. The knockdown of GRN increased the levels of the cleaved TDP-43 protein in the P1 and P2 EV fractions, whereas full-length TDP-43 levels remained unchanged in all three fractions of HeLa cells (E–G). Furthermore, the knockdown of GRN increased TSG101 levels in the P1 and P2 EV fractions but decreased them in the cell fraction (E and H). In addition, the knockdown of GRN increased LC3-II levels in the cell and EV fractions (E and I). Importantly, in NSC-34 cells, which exhibit a lower abundance of autophagosome-like particles (F), the knockdown of Grn increased cleaved TDP-43 levels in the P2 EV fraction, mimicking the effects observed in HeLa cells (E and F). To examine the role of autophagy in the GRN knockdown–induced secretion of TDP-43, we conducted assays using ATG16L1 KO HeLa cells (J). Without the expression of ATG16L1, the knockdown of GRN no longer induced the secretion of the full-length and cleaved TDP-43 proteins into the P1 and P2 EV fractions. On the other hand, it significantly increased TSG101 levels in the EV fraction, even in ATG16L1-deficient cells (J–M).PGRN regulates the autophagy-lysosomal pathway, and its insufficiency causes FTLD-TDP , 40, 55.sed DALG , A and Br ORANGE , C and DLa cells , E–G. Fufraction , E and Hractions , E and Iarticles F, the knLa cells . TherefoLa cells , E and FLa cells J. Withount cells , J–M.FigA). In WT MEFs, PGRN localized at vesicle-like structures  and was detected in the cell and EV fractions . Notably, the molecular weight of PGRN was higher in the P1 and P2 EV fractions than in the cell fraction . The deglycosylation of the P1 and P2 EV fractions using PNGase F resulted in a downward shift of the PGRN band to a similar position to that of the cell fraction . Therefore, PGRN included in EV was highly glycosylated. The loss of PGRN expression significantly reduced full-length TDP-43 levels in the cell fraction and increased cleaved TDP-43 levels in the P2 EV fraction . The level of LC3-II, but not TSG101, was elevated in all three fractions of PGRN-null cells . These results suggest that the PGRN insufficiency increased the secretion of TDP-43 in an autophagy-dependent manner.To further validate the role of PGRN in the secretion of cleaved TDP-43 and LC3-II, we used MEFs derived from PGRN WT or KO mice A. In WT A and B) or the knockdown of TARDBP or GRN (C and D), TFEB-GFP translocated to the nucleus. In contrast, treatments that did not promote TDP-43 secretion, such as 100 μM CQ, 100 nM Vac, or the knockdown of STX17, did not lead to the nuclear localization of TFEB-GFP (A–D). Therefore, TDP-43 secretion appeared to correlate with the nuclear localization of TFEB. Conversely, we investigated whether the knockdown of TFEB interfered with the Baf-induced secretion of TDP-43. The knockdown of TFEB using siRNA decreased the levels of both full-length and cleaved TDP-43 in the P1 and P2 EV fractions, whereas it increased the levels of full-length TDP-43 in the cell fraction. Consistently, the knockdown of TFEB decreased LC3-II levels in the cell and EV fractions (E–I). These results suggest that an increase in the nuclear translocation of TFEB is a key event for the secretion of TDP-43 via EV release.The present results suggest that the inhibition of autolysosome formation followed by lysosomal acidification plays a role in the secretion of TDP-43. On the other hand, the treatment with CQ and knockdown of STX17, each of which also suppressed autolysosome formation, were insufficient to promote TDP-43 secretion, suggesting the involvement of other factors in TDP-43 secretion. We previously reported that a treatment with Baf increased the nuclear localization of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy , 56. FurP or GRN , C and DTFEB-GFP , A–D. Thractions , E–I. ThIn the brains of ALS and FTLD-TDP patients, the pathological accumulation of TDP-43 is initially deposited in specific areas and then spreads widely throughout the brain with disease progression . The C-tvia EV was regulated by autophagy. Inhibitors of v-ATPase, such as Baf and Con, suppressed autophagy but facilitated the secretion of endogenous TDP-43. On the other hand, MG, an inhibitor of proteasome degradation, facilitated autophagy and did not affect the secretion of endogenous TDP-43. In contrast, Iguchi et al. , polyclonal rabbit anti-C-terminal TDP-43 antibody , polyclonal rabbit anti-N-terminal TDP-43 antibody , polyclonal rabbit anti-PGRN antibody , polyclonal rabbit anti-TSG101 antibody , polyclonal rabbit anti-ACTN4 antibody , polyclonal rabbit anti-ADAM10 antibody , and polyclonal rabbit anti-TFEB antibody  were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody  and polyclonal rabbit anti-GFP antibody  from BioAcademia; a monoclonal mouse anti-LC3 antibody , normal rabbit immunoglobulin G (IgG) , polyclonal rabbit anti-Syntaxin-17 (human) antibody , and polyclonal rabbit anti-ATG16L1 antibody  from MBL; a monoclonal mouse anti-p62 antibody  from BD Biosciences; a polyclonal sheep antimouse PGRN antibody  from R&D Systems; a monoclonal mouse anti-WIPI2 antibody  from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody , biotin-conjugated goat anti-rabbit IgG antibody , and biotin-conjugated rabbit antisheep IgG antibody  from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG , and Alexa Fluor 488–conjugated donkey antisheep IgG  from Abcam.A monoclonal mouse anti-TDP-43 antibody , DALG , PlasMem Bright Green , and PlasMem Bright Red  were purchased from Dojindo; Baf , Con , and Rapa  were purchased from Adipogen; 4′,6-diamidino-2-phenylindole (DAPI) solution  and dimethyl sulfoxide (DMSO)  were purchased from Nacalai Tesque; E64d (4321-v) and pepstatin A (4397-v) were purchased from the Peptide Institute; AcidiFluor ORANGE (GC301) was purchased from Goryo Chemical; CQ diphosphate  was purchased from Fujifilm Wako; Vac  was purchased from Cayman Chemical; MG  was purchased from Enzo Life Sciences; and PNGase F  was purchased from New England Biolabs.2, and the number of passages was within 20 because a new vial was thawed. Mycoplasma contamination was tested using EZ-PCR (Sartorius).HeLa cells , HeLa cells stably expressing mRFP-GFP-LC3 , 72, ATGHeLa cells stably expressing mRFP-GFP-LC3 , 72 on c5 cells on 12-well plates cultured under the indicated conditions for 24 h were lysed in 1% (w/v) Sarkosyl in A68 -N,N,N,N-tetraacetic acid). Protein concentrations in the lysates were estimated using a BCA Protein Assay Kit (Thermo Fisher Scientific), and SDS-sample buffer was added to give a final concentration of 1% SDS. Samples were boiled for 10 min and subjected to SDS-PAGE followed by an immunoblot analysis to quantify GFP and RFP levels.HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG seeded at a density of 0.5 × 102O , and 0.3% (v/v) H2O2 (FUJIFILM Wako). Images were digitized by a scanner and subjected to densitometry using ImageJ. To quantify cleaved TDP-43 bands, four cleaved TDP-43 bands surrounded by the dashed square were subjected to densitometry. The specificity of each antibody used in immunoblot analysis was validated based on the molecular weight.Immunoblotting was performed as previously reported , 67. SamImmunocytochemistry was carried out as shown previously . Cells g5 cells on coverslips (Matsunami Glass) and cultured under the indicated conditions for 24 h were fixed for 20 min with 4% paraformaldehyde in PBS and subjected to WIPI2 immunostaining to assess phagophore formation. WIPI2-positive puncta were monitored using the LSM780 confocal laser microscope equipped with a 60× objective lens. The signal intensity per cell was quantified using ImageJ and the Analyse Particles plugin . At least 33 cells in each of the three independent experiments were subjected to counting.As described in a previous study , HeLa ce5 cells on a 12-well plate cultured under the indicated conditions with or without 10 μg pepstatin A and E64d for 24 h were lysed in 1% (w/v) Sarkosyl in A68. Protein concentrations in the lysates were estimated using a BCA Protein Assay Kit, and SDS-sample buffer was added to give a final concentration of 1% SDS. Samples were boiled for 10 min and subjected to SDS-PAGE followed by immunoblotting to quantify LC3-II levels.As described in a previous study , HeLa ceHeLa cells in 12-well plates were incubated with AcidiFluor ORANGE for 2 h under the indicated conditions and subjected to live-cell imaging using the BZ-X710 fluorescence microscope (Keyence) equipped with a 20× objective lens. Five different frames of images (>335 cells) were captured, and the AcidiFluor ORANGE signal intensity per image was quantified using ImageJ with the Analyse Particles plugin . The signal intensity per cell was calculated from the total value of the signal intensity divided by the total number of cells counted from Hoechst 33342 staining. Three independent experiments were performed for quantification.et al.  Sarkosyl in A68 by sonication. Protein concentrations in cell lysates were estimated using a BCA Protein Assay Kit, and SDS-sample buffer was added to give a final concentration of 1% SDS. Regarding the fractionation of EV in culture medium, culture medium collected from the 10 cm dish was precleared by centrifugation at 800g at 4 °C for 10 min to remove nuclei and cell debris. The precleared medium was pooled in a centrifugation tube  and centrifuged at 20,000g at 4 °C for 1 h. The pellet was collected as the P1 fraction and lysed with 50 μl 2% SDS-sample buffer. Supernatants were recollected in the centrifugation tube  and centrifuged at 110,000g at 4 °C for 1 h. The pellet was collected as the P2 fraction and lysed with 50 μl of 2% SDS-sample buffer. Samples were boiled for 10 min and subjected to an immunoblot analysis.Cellular and EV fractions were prepared using the method reported by Tripisciano et al. . To prepImmunoprecipitation was performed as previously described . BrieflyImmunoelectron microscopy was conducted as previously described . The P1 m/z was employed in the positive ion mode, followed by data-dependent MS/MS using the higher-energy collisional dissociation operating mode on the top 15 ions in order of abundance. Data were analyzed with Proteome Discoverer , Mascot software , and Scaffold software . The SwissProt database (https://www.uniprot.org/) updated on July 15, 2022 and GenBank database (https://www.ncbi.nlm.nih.gov/) updated on February 27, 2020 were searched with the following parameters: monoisotopic mass, peptide mass tolerance of 10 ppm, fragment ion mass tolerance of 0.05 Da, complete tryptic digestion allowing three missed cleavages, static modification of beta-methylthiolation, variable modifications of methionine oxidation, deamidation, and pyroglutamination. A scaffold was used for label-free quantification. Peptide and protein probability thresholds of 99 and 99%, respectively, were applied. Using a target decoy approach, a peptide false discovery rate of 0.1% was selected. Data are presented in LC–MS/MS was performed according to our previously reported protocol . The P1 Proteins detected by the LC–MS/MS analysis were divided into three groups: proteins detected in the P1 fraction only, in the P2 fraction only, and in both the P1 and P2 fractions. The cellular components of proteins in these groups were examined using DAVID software .mCherry-hLC3B-pcDNA3.1  was purchased from Addgene. Full-length TDP-43 (amino acids 1–414) in EGFPC1 (Clontech), the C-terminal fragment of TDP-43 (amino acids 162–414) in EGFPC1, and the N-terminal fragment of TDP-43 (amino acids 1–273) in EGFPC1 were previously characterized , 67.To generate HeLa cells stably expressing mCherry-LC3, HeLa cells seeded on a 6 cm dish were transfected with 1 μg of a mCherry-LC3 construct and 3 μl of TransIT2020 reagent (Mirus) according to the manufacturer’s protocol. G418 -resistant clones stably expressing mCherry-LC3 were obtained by limiting dilution cloning in 96-well plates. Steady-state expression levels were confirmed by immunoblotting.TARDBP , human STX17 , human TFEB , mouse Grn , were used for the knockdown of human TARDBP, STX17, TFEB, and mouse Grn, respectively. Predesigned stealth siRNA duplexes to target human GRN  were used for the knockdown of human GRN, as previously reported (Transfection was performed as previously reported . In tranreported . The steGFP-tagged expression vectors of full-length (amino acids 1–414), the N-terminal fragment (amino acids 1–273), and C-terminal fragment (amino acids 162–414) of TDP-43 were previously prepared , 67. HeLHeLa cells were transfected with TDP-43 amino acids 162 to 414 or mCherry-LC3 for 24 h and then seeded on μ-Dish, 35 mm high (ibidi). After a 24 h stimulation with 100 nM Baf, the plasma membrane was visualized with PlasMem Bright Green or PlasMem Bright Red according to the manufacturer’s protocol. Live-cell imaging was conducted using the LSM780 confocal laser microscope equipped with a 60× objective lens.As shown in previous studies , 67, autvia Oviductal Nucleic Acids Delivery (iGONAD) , transactivating CRISPR RNA , and 1 mg/ml Cas9-NLS protein (NIPPON GENE), and electroporated using the NEPA21 electroporator (Nepagene) on embryonic day 0.7 (approximately 15:00). Newborn mice were weaned at 3 weeks old, and their genome was extracted from their tails. The alignment of the genome-edited site was identified with Sanger sequencing (Azent). Mice with the mutation p.Ala24GlyfsTer22 were selected and backcrossed with ordinary C57BL/6 mice three times. Mice were bred in the laboratory animal facility (Imabari) in accordance with the guidelines of the Institute for Laboratory Animal Research under controlled light , temperature (23 ± 1 °C), and humidity (55 ± 10%), and were given free access to food and water. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Okayama University of Science and were approved by the Institutional Animal Care and Use Committee of Okayama University of Science (permit number: 2019-047).PGRN CRISPR KO mice were generated by improved-Genome editing (iGONAD) , 78. BriThe preparation of MEFs was described in a previous study . Embryos2O. In the PNGase F-positive group, pellets were mixed with 2.5 μl GlycoBuffer 2, 20 μl H2O, and 2.5 μl PNGase F. After a 24 h incubation at 37 °C, samples were mixed with 25 μl SDS-sample buffer  and boiled for 10 min for immunoblotting.According to the manufacturer’s protocol, the N-linked glycans of proteins in the P1 and P2 fractions of WT MEFs were deglycosylated with PNGase F . Briefly, P1 and P2 pellets were prepared as shown previously. In the PNGase F-negative group (control), pellets were mixed with 2.5 μl GlycoBuffer 2  and 22.5 μl HSH-SY5Y cells stably expressing TFEB-GFP  seeded ot test, Mann–Whitney U test, or Tukey’s post hoc test were performed using Excel (Microsoft Office) or EZR  or EZR . Data arAll data are contained in the figures, figure legends, or This article contains The authors declare that they have no conflicts of interest with the contents of this article.


This review provides an overview of recent advancements in applying graphene-based materials as sorbents for liquid chromatography (LC) analysis. Graphene-based materials are promising for analytical chemistry, including applications as sorbents in liquid chromatography. These sorbents can be functionalized to produce unique extraction or stationary phases. Additionally, graphene-based sorbents can be supported in various materials and have consequently been applied to produce various devices for sample preparation. Graphene-based sorbents are employed in diverse applications, including food and environmental LC analysis. This review summarizes the application of graphene-based materials in food and environmental water analysis in the last five years (2019 to 2023). Offline and online sample preparation methods, such as dispersive solid phase microextraction, stir bar sorptive extraction, pipette tip solid phase extraction, in-tube solid-phase microextraction, and others, are reviewed. The review also summarizes the application of the columns produced with graphene-based materials in separating food and water components and contaminants. Graphene-based materials have been reported as stationary phases for LC columns. Graphene-based stationary phases have been reported in packed, monolithic, and open tubular columns and have been used in LC and capillary electrochromatography modes. Graphene and graphene derivates are emerging materials in research and development , includiDespite the potential for graphene and its derivates as a highly suitable material, the manufacturing obstacle hinders its adoption on an industrial scale. The mechanical exfoliation of graphite through adhesive tape is a practical way to produce graphene-based materials ,10. ThisThe diversity of potential functionalizations of graphene-based materials allows for a wide range of applications in separation science. These applications include the use of techniques such as stir bar sorptive extraction (SBSE) ,26, dispThe present review investigates the applications of graphene-based materials in food and water analysis, including their utilization as sorbents for sample preparation and analytical columns for LC, in a time frame from 2019 until 2023. The escalating necessity for enhanced analytical processes in food and water analysis has stimulated interest in using graphene-based materials as efficient and trustworthy solutions. Therefore, this review article aims to illuminate the current state and potential prospects for graphene-based sorbents in food and water analysis.Offline sample preparation techniques are the most common strategy in the analytical chemistry field. They consist of preparing the sample for analysis while disconnected from the instrument or application. Some advantages of this strategy are the simple procedure, which is usually not dependent on complex instrumentation set-up, and the possibility of analyzing sample fractions prepared with different analytical techniques. This section summarizes offline extraction techniques in which graphene-based sorbents have been employed recently.Dispersive solid phase extraction (d-SPE) and microextraction (d-SPME) are examples of sample preparation techniques that involve graphene-based materials as dispersive sorbents . This sa3O4/TBA) nanocomposite was successfully applied in the determination of herbicides in vegetables and water samples [3O4/TBA sorbent was prepared by co-precipitating GO and Fe3O4 particles. After the obtention of the GO/Fe3O4 particles, they were functionalized with TBA using ethylene glycol to form covalent bonds. A d-SPME method using the GO/Fe3O4/TBA to quantify 2,4-dichlorophenoxyacetic acid presented limit of detection (LOD) and limit of quantitation (LOQ) values lower than 0.02 μg·mL−1, and the recovery range was between 88.0 and 94.0%. Another graphene-based magnetic phase that has been successfully applied to the analysis of contaminants in vegetables is the GO-Fe3O4 nanoparticles functionalized with maltodextrin (MD) and β-cyclodextrin (β-CD), which was used for the investigation of triazole and triazine pesticides in corn, tomato, and potato [3O4 magnetic particles produced this magnetic extraction phase. After the obtention of the GO-Fe3O4, this material was functionalized with MD and β-CD using epichlorohydrin as a linker. The LODs obtained for determining the pesticides in the vegetables ranged from 0.01 to 0.08 μg·L−1 and recovery was between 88.4 and 112.0%. Metal-organic frameworks (MOF) have also been used to functionalize graphene-based sorbents. MOFs are porous crystalline materials formed by assembling metal ions and organic ligands with large surface areas, high porosity, and controllable pore structures. As a recent example, in d-SPME, Fe3O4 particles functionalized with GO sheets and UiO-66 (a Zr-based MOF) have been used as a sorbent for the determination of food dyes in soft drinks, candies, and pastilles [3O4, followed by the addition of ZrOCl2·8H2O and terephthalic acid into a GO-Fe3O4 suspension in order to produce the magnetic GO/UIO-66 sorbent. The s-SPME method employing the GO/UIO-66 sorbent presented LODs less than 0.218 ng·mL−1, LOQs lower than 9.368 ng·mL−1, and recovery ranging from 95.01 to 106.33%. The interaction mechanism between the MOF-based sorbent and the food dye analytes was attributed to multiple mechanisms, including n–π/π–π interactions, Yoshida/dipole–dipole hydrogen bonds, and electrostatic interactions. Though magnetic particles are the primary support for sample preparation in d-SPME and d-SPE in food and water analysis, no magnetic materials have been used. For example, GO-coating polystyrene (PS) microspheres were applied to extract bisphenol endocrine disruptors in environmental water samples [−1 and LOQs of 0.07–0.37 μg·L−1 and recovering in a range from 71.1% to 104.8%. The retention mechanism of this GO-PS extraction phase was attributed to the π–π interactions and hydrogen interactions between the sorbent and the analytes. Another interesting variation of the d-SPME using graphene-based sorbents applied in food analysis is the “rotating flat surface solid phase microextraction (RFS-SPME)” method. This strategy consists of suspending and stirring foams of the stationary phase, instead of powder material, in the matrix, and after the end of the stirring the foam can be removed with forceps [−1, LOQs lower than 0.475 µg·L−1, and recoveries over 90.0% The evaluated 3D GO/La Ni foams’ extraction phase presented an extraction mechanism sorbent–sorbate combining π–π interaction, hydrogen bonding, and electrostatic interaction.Graphene-based materials have been employed in d-SPE and d-SPME, and mainly supported in magnetic nanoparticles . Magneti samples . The GO/d potato . Co-precastilles . This in samples . To prod forceps . The RFS forceps . To prodCoating techniques involve sample preparation strategies where the sorbent is coated over, typically, a non-sorptive surface. For example, stir bar sorptive extraction (SBSE) and fiber solid phase microextraction (fiber-SPME) are techniques in which the sorbent is fixed over an inert surface. Usually, the sample preparation process using these techniques involves (i) immersing the device coated with sorbent in a matrix solution, (ii) stirring the solution for a specific time, (iii) removing and washing the device to remove the matrix, and finally (iv) desorbing the analyst in an appropriated solvent before analysis.−1 and recovery in a range of 83% to 112%. The rGO-Ni SBSE bar presented an attributed extraction mechanism based on π–π interaction and hydrophobic interactions [−1, LOQs of 0.5 to 1.0 ng·mL−1, and a recovery range of 89.38–108.54%. To produce this device, it was first necessary to activate the SS wire surface to allow the chemical binding of the GO. To do that, the SS wire was submitted to a sequence of reactions until the polydopamine-SS wire was formed. The GO was suspended in a solution and then reacted with the polydopamine-SS wire forming the GOF-SS wire SBSE bar [3O4 particles’ sorbent (G-Fe3O4) in the determination of seven pesticides in water [3O4 particles were produced by the co-precipitation method, similar to that previously described in the d-SPME section. This SBSDμE method presented a recovery range between 20% and 75%, with a LOQ between 5 ng·mL−1 and 9.5 ng·mL−1. Another interesting sample preparation technique based on the sorbent phase coating a surface was fiber solid phase microextraction (fiber-SPME). Fiber-SPME consists of the sorbent coating a capillary’s inner or outer surface, forming the extraction fiber. Commonly, fiber-SPME devices are filled with monolithic extraction phases, including the ones modified with graphene-based materials. Graphene-based materials as fiber-SPME sorbents have also been explored for food and water LC analysis in the last five years. For example, a composite prepared with GO, MOF zeolitic imidazolate framework 8 (ZIF-8), and molecularly imprinted polymers (MIP) deposited in fiber was used to evaluate sterol and steroid hormones in white meat, egg yolks, and vegetables [−1 and recovery ranging from 95.0% to 101.0%. This sorbent presented a mixed interaction mechanism, primarily attributed to the MIP efficient imprinting effect and the differences in size, functional group type, and position of the analytes and polar interactions, hydrogen bonds, and electrostatic interactions [−1 and LOQs lower than 2.27 μg·L−1 were obtained, and a recovery of range between 70.0 and 117% was reported using this sample preparation method.SBSE and related techniques are simple and effective ways to extract analytes from the matrix. SBSE devices are usually produced by chemically or physically adhering the sorbent to an inert material . Graphenractions . Moreoveractions , presentSBSE bar . AnotherSBSE bar . This baSBSE bar . A recenin water . The maggetables . The fibractions . Anotherractions . Using mractions . LODs lo−1 and LOQs lower than 0.91 μg·L−1. This extraction phase presented an interesting proposed interaction mechanism in which hydrogen bonding participates. However, the adsorption process also influences the interaction of π-electrons of the analyte with the Cr and back donation from the sorbent material into the molecular anti-bonding π∗ orbital [Hollow fiber solid phase microextraction (HF-SPME) is a variation of the fiber-SPME in which the sorbent coats the inner wall of a capillary, forming an open channel in the device’s core . Unfortu orbital .Packed sorbent techniques are sample preparation techniques that use a solid adsorbent material packed in a cartridge, a column, or a pipette. The sample preparation procedure of these techniques comprises the following steps: (i) equilibrate the sorbent by flushing a suitable solvent through the cartridge, (ii) load the sorbent with the sample by flushing it through the cartridge (for SPE), (iii) wash the sorbent to remove the matrix, and (iv) elute and collect the analytes using a proper solvent.−1 and a recovery range from about 55 to 90%. This stationary phase presents a complex interaction mechanism between the sorbent and the analytes due to the participation of π–π stacking, hydrophobic interaction, and size complementarity interactions. Another interesting graphene-based material packed in an SPE sample preparation cartridge is a zwitterionic sorbent modified with graphene (G) and fixed over Si particles [−1 and a recoveries range from 48 to 85%. Though graphene-based materials are usually fixed over support, SPE cartridges can be packed with graphene sheets that are not fixed in support. As mentioned above, this kind of packing presents limitations, such as blocking the frits caused by the accumulation of the graphene sheets, but it is still possible. For example, it was reported that rGO sheets packed on SPE cartridges were used to extract aflatoxins from food samples [−1, LOQs lower than 2.83 ng·g−1, and relative recoveries ranging from 70 to 113% using this cartridge. Another interesting example of an SPE sorbent not supported in particles is 3D reduced graphene oxide (3D-rGO), a self-supported material [Sorbents packed on cartridges are a usual approach for producing SPE devices. Silica particles are frequently used as a support for graphene-based sorbents due to their capacity to provide a stable structure and prevent blockages in cartridges . Additioarticles . This soarticles , present samples . It was material . In thismaterial . A sorbe−1 for juice and 5.0–25.0 ng·g−1 for candy and syrup. Additionally, recoveries ranging from 48 to 69%, 34 to 89%, and 28 to 76% were obtained for juice, candy, and syrup, respectively. MOF materials modified with graphene-base materials have also been reported as sorbents for PT-SPME. Si microspheres covalently bonded to fluorinated graphene (FG) and functionalized with ZIF-8 nanocrystals were packed in a PT-SPME device and applied to determine chlorophenols in tap water, honey, and black tea [−1 and LOQs of 15.9 µg·kg−1. This extraction phase presented multi-interaction mechanisms, including π-stacking and hydrophobic and hydrogen-bonding interactions between the adsorbent and the compounds. Though Si particles are standard, other types of particles can be used as supports for graphene-based materials in PT-SPME and have been applied in LC analyses of foods. For example, the nanocomposite GO-starch-polyacrylamide was packed in PT-SPME devices and used to determine antibiotic residues in cow’s milk [3 to form the porous material. The PT-SPME method presented LODs of 2.7–5.0 μg·kg−1 and a recovery range of 88–102%. This extraction phase presented an adsorption mechanism mainly based on hydrogen bond interactions between the analytes and the sorbent [Pipet tip SPME (PT-SPME) can be considered a variation of the SPE because the sorbent packed on a pipet tip forms a device similar to a cartridge. Si particles have also been used as a sorbent support for PT-SPME sample preparation. As a recent example, GO sheets fixed on Si particles have reportedly been packed in PT-SPME devices and applied to determine herbicides in sugarcane-derived foods . The SiGlack tea . About 8w’s milk . The sor sorbent .In the last five years, graphene-based sorbents have successfully been employed in diverse sample preparation techniques for food and water analysis. Water, milk, vegetables, cereals, and meat are examples of the diversity of samples that graphene-based sorbents could prepare recently . The divThe online sample preparation strategy presents a significant advantage over offline techniques; the automation process minimizes the chance of human error, one of the most common error sources in analytical methods ,62. Thou−1 and recovery between 70.4 and 91.6%. In-tube SPME also utilized SiGO particles’ functionalization with other bindings. For example, SiGO particles functionalized with C18 and end-capped were used for the online sample preparation of coffee samples, targeting the determination of xanthines [−1 and recoveries between 73 and 109% were obtained. Both works above employed a similar route to prepare the SiGO particle or its functionalized derivate with C18 and end-capping. Briefly, GO sheets, produced by Hummer’s method, were covalently bonded to the Si particle’s surface. For the functionalization with C18 and end-capping, the SiGO particles formed were reacted with chlorodimethyl-n-octadecylsilane and trimethylchlorosilane in separate steps [−1 for milk and 0.6 μg·kg−1 in chicken muscle and recoveries of about 70.3 to 98.5% and 79.0 to 108.0% for each matrix, respectively, were obtained [−1.Graphene-based materials fixed over silica particles are an interesting sorbent for this application. For example, GO supported into the Si particles’ (SiGO) sorbent was packed inside a fused silica capillary to produce a miniaturized extraction column employed in the extraction of β-lactam antibiotics from environmental water samples . This meanthines . LOQs ofte steps . Monolitte steps . After pte steps . The in obtained . Coated obtained . An inteobtained . This ITobtained  This IT-−1, a LOQ of 0.3 ng·mL−1, and a recovery range between 90.0 and 106.5%.Headspace in-tube solid phase microextraction (HS-SPME) using a graphene-based sorbents is another sample preparation strategy that has been applied recently, hyphenated to LC. This strategy consists of introducing the sample in a vail and inserting the HS-SPME device into the space above the liquid surface; after the extraction procedure, the HS-SPME system is connected online to LC equipment. A recently reported application of graphene-based sorbents in the HS-SPME technique for food analysis was performed, utilizing a stainless steel tubing coated with a composite formed by GO and ionic liquid (IL) for the analyses of the headspace of naphthalene, a volatile PAH, in honey samples . The GO-In short, a trend exists to employ GO as the graphene-based sorbent in online sample preparation . The fixGraphene-based materials have also been used as stationary phases for liquid chromatography columns. These sorbents have been employed in packed, monolithic, and open tubular (OT) columns, the three most common types of LC columns . These sPacked columns are the standard types of columns in LC. They comprise particles of the stationary phase packed inside a tubing (or capillary tubing). This type of column is easy to produce given its production procedures demand only a pump that allows the packing of the stationary phase in the column hardware . The simGraphene-based packed LC columns are still underexplored for food and water analysis. However, some applications reported in the last five years are described in the literature. In seed samples, a capillary column packed with a SiGO-C18ec stationary phase, produced by the functionalization of SiGO particles with C18 followed by end-caping (as previously mentioned), was employed to separate lecithin, a common phospholipid component in foods. This study evaluated the viability of applying an online liquid extraction (OLE) system coupled with an LC-MS to evaluate the hyphenated extraction and separation of lecithin from the pounder of seeds . The SiGMonolithic columns are another popular type of LC column composed of a monolith of the stationary phase, usually inside a capillary tubing. These columns are, conventionally, produced by the in situ polymerization of polymer-based  and siliSchisandra, a purple-red berry typically used in traditional Chinese medicine [Graphene-based monolithic columns have been used in LC and CEC modes, but recent food and water analysis applications present few reported examples. In LC, a monolithic column prepared by the polymerization of 3-chloro-2-hydroxypropylmethacrylate (HPMA-Cl) and ethylene dimethacrylate (EDMA), followed by the fixation of GO, was used recently for the determination of chloramphenicol (CAP), a veterinarian antibiotic, and chloramphenicol glucuronide (CAPG), a metabolite from CAP, in honey and milk samples . Anothermedicine . For thimedicine . The CECmedicine . These cmedicine .Open tubular columns have gained popularity in liquid chromatography due to their intrinsic advantages over packed columns, such as low pressures, and their theoretically higher performance ,93,94. TNevertheless, the separation of small molecules on OT columns has also been described ,104,105.2) as stationary phases for determining sulfonamides in environmental water [2 dispersion into a fused-silica capillary and leaving it overnight for the GO-MoS2 to bond in the fused-silica capillary wall [Graphene-based OT-LC columns have not received much attention for food and environmental water analysis. Very few works explore graphene-based OT columns in food and water analysis. An example reported in the last five years is the separation of casein protein variants from milk samples using an OT column produced with Poly-L-Lysine (PLL) grafted on a methacryloyl graphene oxide nanoparticles (MGONPs) stationary phase . In CEC,al water . The colary wall . The separy wall . For exaary wall . The enaary wall .Graphene sorbents have been employed in diverse analytical column configurations in the last five years. Packed, monolithic, and open tubular are the three most common column types, and all present examples are of graphene-based materials as stationary phases . In foodGraphene-based sorbents are becoming more popular year after year in diverse applications. In food and environmental water LC analysis, graphene-based materials have been underexplored in the last five years. However, a trend in the diversification of the application of those materials is also being observed. In offline sample preparation, dispersive solid phase extraction receives the most attention for the application of graphene-based sorbents in food and environmental water LC analysis, but other techniques, such as SBSE, Fiber-SPME, SPE, HF-SPME, and others, could employ graphene-based sorbents with success. Hence, graphene-based sorbents are in an early stages of development, and the number and variety of sample preparation techniques using these materials is tending to increase. In online sample preparation methods, IT-SPME is the most common application of graphene sorbents in food and environmental water analysis. This kind of online sample preparation allows an easy connection to LC analysis, which has been a great advantage of these techniques.Additionally, the extraction column might be packed with a wide variety of graphene-based sorbents and can deal with a high diversity of samples. Graphene-based sorbents have also been used for analytical LC columns. These sorbents have been utilized in packed, monolithic, and open tubular columns. Graphene oxide bonded to Si particles is the most common graphene-based sorbent used as stationary phases. Graphene-based sorbents have been used as stationary phases in the analysis of food components such as caseins, lecithin, and schizandrols. Graphene-based sorbents have also been employed in separating water contaminants such as alkylbenzenes and antibiotics. The future of graphene-based stationary phases in food and water analyses is promising, with packed columns becoming more frequent for LC and OT columns for CEC analysis. GO should remain the most preferred graphene-based material due to its molecular characteristics, allowing it to be functionalized. Functionalizing these materials with different bindings should be encouraged to find solutions to specific problems.Graphene-based sorbents present advantages and disadvantages in analyzing food and water samples through liquid chromatography. One significant advantage is the versatility of these sorbents in extracting various analytes while being compatible with different sample preparation techniques. Furthermore, functionalizing GO, and other graphene-based materials, offers a range of possibilities for online approaches. In contrast, as its main disadvantage, these phases are relatively new and may present a lower scope of applications than conventional commercially available extraction phases. In other words, graphene-based materials are not multipurpose, which means that for some applications it is crucial to select an adequate material to extract specific analytes. Another advantage of graphene-based materials is that they can be used as stationary phases in packed, monolithic, and open tubular chromatographic columns. Conversely, detachment followed by the accumulation of graphene sheets in the column outlet can again hinder effectiveness and limit the column’s lifetime. Nonetheless, the functionalization of graphene-based materials with different bindings for specific applications presents intriguing opportunities for improving the diversity of applications for food and water analysis.


The ferroelectric domains are visualized through the imaging of the plasmonic response in a graphene monolayer adjacent to the moiré WSe2 bilayers. Specifically, we find that the ferroelectric polarization in moiré domains is imprinted on the plasmonic response of the graphene. Complementary nano-photocurrent measurements demonstrate that the optoelectronic properties of graphene are also modulated by the proximal ferroelectric domains. Our approach represents an alternative strategy for studying moiré ferroelectricity at native length scales and opens promising prospects for (opto)electronic devices.Ferroelectricity, a spontaneous and reversible electric polarization, is found in certain classes of van der Waals (vdW) materials. The discovery of ferroelectricity in twisted vdW layers provides new opportunities to engineer spatially dependent electric and optical properties associated with the configuration of moiré superlattice domains and the network of domain walls. Here, we employ near-field infrared nano-imaging and nano-photocurrent measurements to study ferroelectricity in minimally twisted WSe 2 by visualizing the plasmonic and photo-thermoelectric response of an adjacent graphene monolayer.Recent experiments have shown the formation of ferroelectric domains in twisted van der Waals bilayers. Here, the authors report near-field infrared nano-imaging and nano-photocurrent measurements to investigate ferroelectricity in minimally twisted WSe Moiré superlattices not only inherit characteristics of the constituent layers but also exhibit new emergent phenomena. Among these emergent effects, interfacial ferroelectricity was recently discovered in marginally twisted hexagonal boron nitride (h-BN)7 and transition metal dichalcogenides (TMDs)11. In minimally twisted bilayers, lattice reconstruction results in triangular domains with periodically alternating AB and BA stacking registries14. The charge transfer between the two layers creates moiré patterns with alternating out-of-plane polarization16. Moiré ferroelectricity in twisted TMDs and h-BN has been confirmed by local Kelvin probe force microscopy (KPFM) imaging17 and by area-averaged transport measurements8.Moiré superlattices of two-dimensional van der Waals materials have emerged as a capable platform to explore exotic electric and optical properties that can be controlled by selecting building blocks for the assembled atomic layers and manipulating the twist angles between them18 and tune photo-excited carrier dynamics19, among other effects. In transport experiments, modulation of the doping of graphene is detected and employed as a sensor for the ferroelectric polarizations20. However, in transport experiments, the ferroelectrically induced doping density was inferred from area-averaged analysis by assuming that the entire sample reveals a uniform polarization under an applied electric field in a gatable device8. In practice, such devices may possess domains with the opposite polarization even at high electric fields, as was recently revealed by backscattering electron imaging9 and KPFM5. This observation indicates that in stacked vdW devices, unlike conventional ferroelectric materials, aligning all the polarization in the same direction may require substantial energy for bending and eventually merging all the adjacent domain walls22. Therefore, in order to read out the doping from the ferroelectric potential, it is imperative to measure the carrier density in each moiré domain. Furthermore, novel optical and optoelectronic properties in devices integrated with ferroelectric have been investigated using far-field optical spectroscopy26. These measurements are diffraction-limited and thus cannot probe the rich optical or optoelectronic properties at the underlying moiré domain scale. Therefore, all existing results call for measurements capable of spatially resolving ferroelectric domains. Piezoresponse force microscopy (PFM) and KPFM can directly measure the domain structures of ferroelectric via electromechanical surface deformation and electrostatic force, respectively. Whereas the ferroelectric domains can be clearly visualized by PFM27, PFM results directly reflect the piezoelectric response, making it challenging to quantitatively evaluate ferroelectric properties. Usually, KPFM can provide a quantitative characterization of a ferroelectric by measuring the work function7. But KPFM has difficulty in quantitatively characterizing ferroelectric devices that include multiple materials because disentangling the electrostatic force from individual layers is a formidable task.Creating ferroelectricity by stacking vdW layers provides new opportunities to engineer materials and control their optoelectronic properties, as the ferroelectric polarization is expected to modulate the doping of an adjacent material2 (t-WSe2) revealing ferroelectricity. We first demonstrate that the plasmonic response of graphene is altered by the proximate twisted ferroelectric domains. Notably, by investigating the local plasmonic contrast in graphene residing underneath the ferroelectric domains, we obtain a reading of the local ferroelectric polarization. Moreover, we show that the proximity to the ferroelectric bilayer breaks the inversion symmetry and modulates the Seebeck coefficient of graphene, thereby allowing the generation of photocurrent. The nano-photocurrent mapping displays moiré patterns and further confirms the notion of the local modulation of the carrier density in graphene prompted by ferroelectric domains. These results uncover alternative approaches to controlling the optoelectronic response of graphene integrated with ferroelectric materials.Here, we utilize near-field infrared nano-imaging and nano-photocurrent to investigate the optical and optoelectronic properties of back-gated graphene encapsulated with a minimally twisted bilayer of WSe2 devices with the same general architecture. These devices are based on back-gated graphene structures, with graphene encapsulated by a minimally twisted bilayer of WSe2 on the top and h-BN at the bottom  the magnitude of the near-field scattering signal35 and (ii) the periodic oscillating patterns (fringes) arising from the propagating plasmon polaritons37. In principle, both observables are governed by the carrier density in graphene. In practice, for samples with moiré super-structures, all domain boundaries are potentially capable of launching and reflecting propagating plasmons38, leading to complex patterns that make it difficult to extract the local carrier density. Therefore, we will primarily focus on an analysis of the evolution of the near-field amplitude The experimental concept is outlined in Fig. er8 Fig. . The fermentclass2pt{minim−1 is shown in Fig. 39. The period of the moiré patterns is inhomogeneous as the twist angle between the WSe2 layers varies over the field of view due to strain and wrinkles. Indeed, across the wrinkles, the observed moiré periods prominently change. Intriguingly, the nearest-neighbor domains reveal clear contrast, while the next-nearest-neighbor domains show almost the same near-field intensity. We performed measurements for several devices, all of which consistently produced these moiré features  and graphene, a process governed by the Fermi energy43.  experiments45. Nano-photocurrent imaging has been extensively used to investigate domains and domain walls in twisted bilayer graphene46. The formation of photocurrent requires broken inversion symmetry, which can be accomplished by introducing p-n or n-n+ junctions in the graphene layer. In our devices, the staggered ferroelectric potential breaks the inversion symmetry, thereby allowing photocurrent formation.Now we use nano-photocurrent imaging to study how the moiré ferroelectricity of t-WSe2 displays moiré patterns marked by prominent sign flipping: red and blue colors denote positive and negative currents, respectively. In addition to the moiré regions, the photocurrent flips sign across wrinkles and terrace edges, as shown in Fig. 48.The nano-photocurrent mapping in Fig. 49, Now we elucidate how the PTE generates the photocurrent and interpret the above photocurrent features. In our experiment, when the tip is scanned over the devices, the tip-focused electric field increases the electron temperature of graphene. As a result, local thermoelectric current The first term on the right-hand side of Eq.  denotes The Seebeck coefficient of graphene depends on the Fermi energy Fig. . As the With the photocurrent mechanism in mind, we can gain a deeper understanding of the spatial features of the photocurrent. When graphene is locally heated, the heat spreads on a characteristic length referred to as the cooling length, which is hundreds of nanometers  and the use of a Bruker atomic force microscope (AFM). Monolayer WSe2 was first cut into two halves using a laser cauterization method and then underwent a dry stacking process in which an h-BN/graphene stack was used to pick up the two pieces of identical WSe2 without any intentional rotation. The final stack was flipped upside down to expose the WSe2/graphene upward using a flipped chip method55. Finally, we used low-temperature, high-vacuum annealing to remove the buried polypropylene carbonate (PPC). The final device geometry was defined by electron-beam lithography and reactive ion etching , followed by electron-beam evaporation (EBE) to deposit Cr/Au = 5/150 nm as the surface contacts. Piezoresponse force microscopy (PFM) imaging was achieved with the Bruker atomic force microscope.Monolayer WSeThe nano-infrared scattering experiments were performed using a home-built scattering-type scanning near-field optical microscope (s-SNOM) housed in an ultra-high vacuum chamber with a base pressure of ~The s-SNOM works by scattering tightly focused light from a sharp AFM tip. The spatial resolution of the s-SNOM is predominantly set by the tip radius of curvature The tapping amplitude is used as the z feedback. The tip excitation phase is controlled by a phase lock loop. The AFM also has the functions of a side-band Kelvin probe force microscope, and the feedback bias voltage is applied onto the AFM tip to compensate for the potential difference between the tip and the sample. This applied bias can ensure that the tip vibration is not influenced by the ferroelectric potential. Thus, the observed near-field contrast is the genuine near-field scattering signal, which is generated purely from the sample conductivity. During the near-field mapping, the z feedback, phase lock loop, and Kelvin probe force microscope are turned on simultaneously. We note that for a tip with a resonance frequency of 285 kHz, the large force constant of about 42 N/m makes it insensitive to the potential difference between the sample and the tip, so no discernible potential response is detected by the tip. Therefore, the KPFM function is not required when using tips with a stiff cantilever.-1 was used to avoid phonon resonances from substrate h-BN and SiO2. The laser beam was focused onto the metallized AFM tip using a parabolic mirror with a 12-mm focal length. The back-scattered light was registered by a mercury cadmium telluride (MCT) detector and demodulated following a pseudoheterodyne scheme. The signal was demodulated at the nth harmonic of the tapping frequency, yielding background-free images. To eliminate the far-field background, we chose n = 3 and 4 in this work.The laser source is a tunable quantum cascade laser (QCL) from Daylight Solutions. Photon energy of 860 ~ 920 cm7 and corresponding bandwidth >1 MHz. In order to isolate the photocurrent generated by the near fields underneath the tip, the photocurrent was sent to a lock-in amplifier and demodulated at the nth harmonic of the tip tapping frequency. In this work, n = 2 was used.The nano-photocurrent measurements were performed in a home-built s-SNOM housed in an ultra-high vacuum chamber with a base pressure of ~Supplementary InformationPeer Review File


Fixed dose combination (FDC) dolutegravir (DTG) plus rilpivirine (RPV) is an approved antiretroviral treatment regimen for people with HIV. The steady-state PK of FDC DTG+RPV in those requiring hemodialysis (HD) has not been previously studied.We performed a single-center, prospective evaluation of the steady-state PK of FDC DTG (50mg)+RPV(25mg) in HIV-negative adults either requiring HD  or with normal renal function, defined as CrCl ≥ 75mL/min . All participants received DTG+RPV daily for 10-14 days with food before undergoing an intensive 24-hour PK evaluation . Plasma drug and metabolite concentrations were measured using a validated LC/MS/MS assay method (QTRAP 6500+LC-MS/MS system) with turboelectrospray source operating in both positive (confirmation) and negative (quantification) modes. We did not evaluate dialysis extraction of DTG+RPV. Descriptive PK parameters were calculated.2). The Table shows the PK parameters assessed in the study population for circulating plasma DTG, DTG-glucuronide (DTG’s primary metabolite), and RPV.No participant experienced serious or grade 3-4 adverse events; there were no study discontinuations. The 4 HD and 2 normal renal function participants were of similar ages  and BMI  and RPV (12ng/mL). These data suggest no dosing modifications are needed for the FDC DTG+RPV regimen in HD.In this study, HD did not lead to clinically appreciable differential exposures to DTG and RPV; the markedly increased exposure to DTG-glucoranide (which is considered inert) in HD suggests increased UGT1A1 activation. All participants maintained exposures throughout the dosing interval greater than the reported ICSamir K. Gupta, MD, Gilead Sciences: Advisor/Consultant|ViiV Healthcare: Advisor/Consultant|ViiV Healthcare: Grant/Research Support Allon Friedman, MD, Eli Lilly: Advisor/Consultant|Eli Lilly: Stocks/Bonds|GI Dynamics: Advisor/Consultant|Gila Therapeutics: Advisor/Consultant


Previous research has indicated altered attentional processing in individuals with experiences of maltreatment or victimization in childhood and adolescence. The present study examined the impact of child and adolescent experiences of relational peer victimization on attentional processes in adulthood when confronted with emotional facial expressions.As part of an online study, a community sample of adults completed a facial dot-probe task. In the present task, pictures of facial expressions displaying four different emotions  were used.The results of the hierarchical regression analyses showed that retrospective reports of peer victimization made a significant contribution to the prediction of facilitated orienting processes for sad facial expressions. Experiences of emotional child maltreatment, on the other hand, made a significant contribution to the prediction of attentional biases for angry facial expressions.Our results emphasize the relevance of experiences of emotional and relational maltreatment in childhood and in adolescence for the processing of social stimuli in adulthood. The findings regarding emotional child maltreatment are more indicative of attentional biases in the context of threat detection, whereas the altered attentional processes in peer victimization are more indicative of mood-congruent biases. These altered processes may be active in social situations and may therefore influence future social situations, behavior, feelings, and thus mental health. There are a variety of studies demonstrating the negative impact of maltreatment experiences in a peer context in childhood and adolescence on psychosocial adjustment and particularly mental health  and Psychotherapeutische Ambulanz der Universität Bielefeld (PAdUB)] were recruited for participation. The flyer contained information about the aims and methods of the study as well as a notice about the anonymity of the participation. At the beginning of the experiment information on general sociodemographic variables such as age, gender, educational level, and family status were requested. Furthermore, the instrument assessed the presence of mental illnesses, the use of medication, and other physical and psychological health questions in addition to the questionnaires used in this study. The sociodemographic and psychopathological characteristics of the 90 participants who were included in the analyses can be found in Participants were recruited through the distribution of the participation link or QR code with access to the study Questionnaires were administered using the Qualtrics survey platform. For the experiment, the web version of the program Inquisit 6 (Millisecond software) was used. At the beginning of the study, participants were informed that participation was voluntary and that it was possible to quit the study at any time without penalty. They were also informed that participation would not be remunerated and that confidential information about mental health symptoms and stressful life experiences would be collected. Participation was only possible after participants had given their consent to participate by clicking on a box. After answering the questionnaires, participants were redirected to the Inquisit homepage, from where the Inquisit application could be downloaded. The experiment was designed in such a way that it could be carried out on both computers and mobile devices. The procedure was approved by the Ethics Committee of Bielefeld University.To assess general symptoms of psychopathology the Symptom Check List-27 . In addition, individual trials were removed in which the reaction time deviated more or less than 2 SD from the individual mean reaction time . For measuring the attentional bias scores for each trial type  the overall attentional bias score was calculated by subtracting the reaction times for congruent trials  from the reaction time for incongruent trials . Attention biases are reflected in shorter reaction times for the dot when attention was focused on this area and longer reaction times for the dot when attention was not focused there. Based on the calculation of the score, positive values for the attentional bias score indicated that the attention was on the emotional faces, whereas negative values indicated that the attention of the subjects was not on the emotional face, but the neutral face. To specify altered attentional processes more precisely with respect to the different types of attentional biases, the orientation score and the disengaging score were calculated in addition to the attentional bias score to capture processes of facilitated attention or difficulties of disengagement . Thus, with α = 0.05, power = 0.95, and the initially planned inclusion of seven predictors  the required sample size was N = 86.For sample size estimation a statistical power analysis was calculated for the multiple regression analyses using G Power 3.1  correction  and the STAI scores , only the SCL-27 was included in the regression analyses. In a second step, the sum scores of the CTQ subscales were included in the model . In a final step, the FBS sum score  was included as the last predictor in the model. These regression analyses were conducted separately for the individual bias indices and the respective emotion presented. Participants who scored on all three items of the minimization/denial subscale of the CTQ (n = 3) were excluded from the analyses . For all analyses, a significance level of t-tests of the absolute index scores for the presentation of one emotion each are shown in t-tests showed that the orienting scores for disgusted and happy faces differed significantly from zero and were negative, indicating attentional avoidance of happy and disgusted facial expressions.A detailed description of the attentional bias index scores for the different emotions, the mean values, standard deviations, and one-sample p's > 0.05). The analyses showed a positive correlation between the emotional abuse score and the disengaging score for sad-neutral trials as well as a positive correlation between the emotional neglect score and the disengaging score for sad-neutral trials.The bivariate Pearson correlation coefficients between maltreatment experiences and the different index scores for each trial type can be found in F = 2.34, adjusted R2 = 0.08, p = 0.039]. There was no significant relationship between the index scores and the level of peer victimization experiences for angry faces  = 0.91, adjusted R2 = –0.01, p = 0.494], the orienting score  and the disengaging score . In addition, there were no significant effects for trials in which happy facial expressions were presented for the attentional bias score, the orienting score, and the disengaging score  = 3.08, adjusted R2 = 0.12, p = 0.009]. The associations with the attentional bias score behaved in opposite ways. Higher scores on the emotional abuse subscale were associated with higher attentional bias scores, whereas higher scores on emotional neglect were associated with lower attentional bias scores on angry faces. For the orienting score, emotional abuse was a significant predictor, with an overall non-significant final model . Associations between emotional maltreatment experiences and the orienting score for happy faces could also be found in happy-neutral trials  = 1.86, adjusted R2 = 0.06, p = 0.098].Regarding the prediction of attentional biases in angry-neutral trials see , the regials see . This reGiven the ambiguous findings on attentional biases in the context of peer victimization, the present work provided new insights into the relationship between relational peer victimization and attentional biases beyond the influence of child maltreatment experiences. In this context, the present study was designed to provide differentiated accounts of attentional biases in the context of maltreatment and peer victimization experiences, thus extending previous research. Consistent with our hypothesis we found altered attentional processes in individuals reporting higher levels of victimization experiences in the present sample. However, this influence was found in sad faces and not, as previously hypothesized, in emotions that were expected to be relevant as threatening stimuli in the context of peer victimization. Furthermore, altered attention processes were found in individuals reporting experiences of emotional maltreatment when confronted with angry facial expressions.In the present study, the results indicated evidence for facilitated attention to sad facial expressions in individuals with higher levels of relational peer victimization experiences beyond the influence of experiences of child maltreatment. This effect was seen even when controlling for symptoms of psychopathology. These findings were consistent with the results of Günther et al. , who alst-tests suggest that participants generally showed significant avoidance of happy and disgusted faces. Therefore, the findings for the overall sample are not reflected in the analyses for peer victimization. The present results contradict the findings of Iffland et al.  had an influence on the results. In addition, the online study could not control the situational conditions under which the performance took place. However, by adjusting the experimental data, we were able to minimize the influence that the behavior would have had if the instructions had not been followed or if the subjects had been unfocused or distracted. Moreover, results were consistent with the findings of several studies that have used reaction times, and also neural measures (Günther et al., Limitations of the present study must be considered when interpreting the results. One-third of the participants stated that they were currently suffering from a mental disorder. For reasons of anonymity, no information could be collected on whether participants were patients of the outpatient clinics. It cannot be excluded that treatment or current medication influenced the attentional processes or reaction times. However, by including psychopathological symptom severity in the regression models, and by adjusting the reaction time data, we were able to reduce the potential influence on our results. In addition, the data did not allow us to assert causal relationships due to the cross-sectional design of our study. Longitudinal studies for analyzing the relationship to mental health should be addressed in the future. The retrospective assessment of experiences of child maltreatment and peer victimization as self-reports also limits the interpretability of the results as they may be affected by distortions (Baldwin et al., In line with previous results, our study showed that experiences of relational peer victimization and emotional child maltreatment in childhood and adolescence influence attentional processes in adulthood. Higher levels of peer victimization were associated with facilitated attention to sad facial expressions in our sample. The results are thus indicative of mood-congruent attentional biases in individuals who have experienced relational peer violence. In addition, altered attentional processes for angry faces were present in participants with higher levels of emotional child maltreatment experiences. Adverse childhood experiences, particularly experiences of emotional maltreatment and relational peer victimization, can thus be considered relevant to the development of cognitive schemata that continue to be activated in adulthood, and therefore can potentially influence new experiences, feelings, thoughts in social situations, and thus presumably mental health.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of Bielefeld University. Written informed consent was not provided because of the design as an online study. The participants gave their informed consent by clicking on a box.KB contributed to the conception and design of the work, the acquisition, analysis, interpretation of the data, drafted, revised, approved the manuscript, and ensures the accuracy and integrity of any part of the work. BI was the chief investigator for this study, contributed to the conception of the study, supervised data analyses, participated in the interpretation of the data, and critically revised the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.