Organization of the genome into euchromatin and heterochromatin appears to be evolutionarily conserved and relatively stable during lineage differentiation. In an effort to unravel the basic principle underlying genome folding, here we focus on the genome itself and report a fundamental role for L1 (LINE1 or LINE-1) and B1/Alu retrotransposons, the most abundant subclasses of repetitive sequences, in chromatin compartmentalization. We find that homotypic clustering of L1 and B1/Alu demarcates the genome into grossly exclusive domains, and characterizes and predicts Hi-C compartments. Spatial segregation of L1-rich sequences in the nuclear and nucleolar peripheries and B1/Alu-rich sequences in the nuclear interior is conserved in mouse and human cells and occurs dynamically during the cell cycle. In addition, de novo establishment of L1 and B1 nuclear segregation is coincident with the formation of higher-order chromatin structures during early embryogenesis and appears to be critically regulated by L1 and B1 transcripts. Importantly, depletion of L1 transcripts in embryonic stem cells drastically weakens homotypic repeat contacts and compartmental strength, and disrupts the nuclear segregation of L1- or B1-rich chromosomal sequences at genome-wide and individual sites. Mechanistically, nuclear co-localization and liquid droplet formation of L1 repeat DNA and RNA with heterochromatin protein HP1α suggest a phase-separation mechanism by which L1 promotes heterochromatin compartmentalization. Taken together, we propose a genetically encoded model in which L1 and B1/Alu repeats blueprint chromatin macrostructure. Our model explains the robustness of genome folding into a common conserved core, on which dynamic gene regulation is overlaid across cells. Taken SUV39H H3K9 methyltransferases for example, SUV39H double-null cells still exhibit DAPI-dense heterochromatin foci despite the loss of pericentric H3K9me3 marks;39 and double knockout mice of SUV39H survive at birth with abnormalities.48 A phase-separation model of block copolymers with similar activity appears attractive in explaining compartmental formation.44 However, this hypothesis remains inconclusive, owing to a large void of identification and experimental validation of the molecular drivers that underlie compartmental segregation of euchromatin and heterochromatin.Evidence suggests that compartments and TADs may be formed by distinct mechanisms. TADs are thought to be formed by active extrusion of chromatin loops by the ring-shaped cohesin complex, which co-localizes with the insulator protein CTCF at the boundaries and anchor regions of contact domains and loops.51 Once regarded as genomic parasites,52 retrotransposons have been recently implicated in playing active roles in re-wiring the genome and gene expression programs in diverse biological processes.61 Long and short interspersed nuclear elements  are the two predominant subfamilies of retrotransposons in most mammals.62 L1  is the most abundant subclass of all repeats, making up to 19% and 17% (0.9–1.0 million copies) of the genome in mouse and human, respectively.63 B1 in mouse and its closely related, primate-specific Alu elements in human are the most abundant subclass of SINEs, constituting 3%–11% (0.6–1.3 million copies) of mouse and human genomes.65 L1 and B1/Alu have distinct nucleotide compositions and sequence lengths. L1 elements are 6–7 kb long and AT-rich, while Alu elements are ~300 bp long and rich in G and C nucleotides.66 Analysis of metaphase chromosome banding showed roughly inverse distributions of L1 and Alu elements in chromosomal regions with distinct biochemical properties.68 Initial studies suggested that Alu/B1 elements appear to be enriched in gene-rich, euchromatic A compartments, whereas L1 elements tend to be enriched in gene-poor, heterochromatic B compartments that interact with lamina-associated domains.71 However, evidence to pinpoint a role for L1 and B1/Alu repeats in organizing the genome has to our knowledge not been reported, albeit fragmented information about their localizations in scattered reports  significantly weakens spatial contacts of homotypic repeat DNA, disrupts the nuclear localization and segregation of L1- or B1-rich chromosomal sequences, and leads to attenuated compartmentalization of the higher-order chromatin structure. Moreover, we show that recombinant HP1α is able to bind RNA and to phase separate in the presence of RNA or DNA in vitro. Genome-wide co-localization of L1 and HP1α renders these repeat DNA and RNA sequences an advantage in promoting HP1α phase separation in heterochromatin contexts. Altogether, our findings suggest a genetically encoded mechanism by which L1 and B1/Alu repeats organize chromatin macrostructure at the compartmental level, providing an important clue to the conservation and robustness of the higher-order chromatin structure across mouse and human.We have postulated that the primary DNA sequences, particularly abundant repetitive elements embedded in the genome, may instruct genome folding.72 Dense L1 and B1 repeats appear to be enriched in distinct compartments across the mouse genome, and within a compartment they are evenly distributed without obvious bias towards the boundary  in the A compartments and L1 repeats  in the B compartments , as ~80% of the genome shows consistent compartmental labeling in at least 16 samples and ~40% is invariant in all 21 samples, in contrast to 7% and 0% to be expected by chance, respectively  and three B1-rich compartments  are alternately positioned along the linear DNA sequence  or B1-rich compartments , but not between these two compartments  or between c and e (ce) are much stronger than those of D or F with c or e , despite the fact that these regions are closer in the linear sequence. Note that L1- or B1-rich segments often span several adjacent TADs  across 2200 annotated TADs in mESCs  as an example to overlay L1 and B1 features on the Hi-C interaction matrix of mESCs. Interestingly, the plaid pattern of enriched and depleted interaction blocks in the Hi-C map is largely correlated with the compositions and distributions of B1 and L1 along the whole chr17 Fig. . In a 42nce Fig. . Strong nts Fig. . The intADs Fig. , consiste to u) and B1-rich segments (from F to T) exhibit high frequencies of homotypic contacts ] larger or smaller than 0 for B1-rich or L1-rich compartments, respectively. About 540 B1-rich and 648 L1-rich compartments were identified with a median size of 1.2 Mb across the mouse genome  using fluorescence-tagged oligonucleotide probes that specifically target the consensus sequences of B1 and L1 elements  at a density of 200–300 bp per probe. Indeed, three regions  annotated in the A compartment are colocalized with B1 FISH signals in the nuclear interior (205 out of 217 nuclei), whereas two B compartment-associated regions (g and q) are colocalized with L1 FISH signals in either LAD or NAD (248 out of 251 nuclei) . Indeed, L1 signals surround and partially overlap with the ring-shaped signals of NCL at the nucleolar periphery Fig. . The locei) Fig. .Moreover, to ask whether L1 and B1 localizations might vary with cell type, we analyzed four additional cell lines, including mouse neural stem cells (NSC), fibroblasts (NIH3T3), and myoblasts (C2C12), and human cervical cancer cells (HeLa) Fig. . SimilarWe then asked whether the nuclear segregation of L1- and B1-rich compartments could be re-constructed during mitosis when chromatin structure undergoes dynamic reorganization. DNA FISH analysis of synchronous mESCs showed that L1 and B1 localizations change dramatically at different cell cycle stages Fig. . S-phase79 In both cell types, G1-phase cells exhibit a classic plaid pattern of hierarchical interactions, with enriched and depleted interaction blocks outside of the diagonal region of the Hi-C interaction heatmap  cells of blastocysts  which inhibits Pol II transcription elongation, led to a partial loss of L1 perinucleolar localization and a gain of mixed nuclear L1 and B1 signals  in mouse embryos led to arrest at the 2-cell stage; and in mESCs, its depletion led to reduced proliferation and global de-repression of hundreds of L1-associated genes; however, it did not alter the expression of OCT4 and NANOG, two known master regulators of the pluripotency program, nor induced ESC differentiation.87 Using the same L1 AMO sequence, we depleted L1 RNA by 17.4% on average shown by RNA FISH  were arrested at the 2-cell stage in contrast to only 15.4% of embryos treated with scramble AMO that were arrested  into mouse zygotes. Two B1 ASOs significantly downregulated B1 RNA levels by 36% shown by RNA FISH (91 Treatment of mESCs with B1 ASO led to severe cell death within hours of transfection (data not shown), which precluded direct assessment of B1 RNA in chromatin organization. It has been suggested that nuclear organization is critically dependent on interactions within heterochromatin,92 where L1, the most abundant one of all repeat subclasses, is predominantly enriched. In subsequent analysis, we in-depth characterized the effects of depleting L1 RNA on chromatin organization.In order to dissect the effects independent of embryonic progression, we then tried to deplete B1 and L1 transcripts in mESCs. B1/Alu repeats have been broadly implicated in diverse processes, including transcription, RNA processing, and nuclear export.n = 30 cells)  showed significantly lower FISH segregation indexes . This indicates that the most majorities of L1 AMO-treated cells exhibit decreased L1/B1 segregation. In contrast, increases of 2-cell-like cells and G2/M-arrested cells occur in small populations, from 2% to 9% and from 13% to 31%, respectively.87 In addition, cells in M phase show a drastically different staining pattern of L1 and B1 from cells in S phase  or L1–L1 interactions  were downregulated in L1-depleted cells, whereas aberrant B1–L1 contacts  were increased. At the genome-wide level, L1-depleted cells show significantly lower Hi-C segregation indexes of L1 and B1 compared to control mESCs . Thus, consistent with DNA FISH, Hi-C analysis further revealed that depletion of L1 RNA causes abnormal increases in heterotypic contacts and genome-wide decreases in homotypic repeat contacts, L1/B1 segregation, and A/B compartmentalization. We noted that depletion of L1 did not alter TAD boundaries  to heterotypic (A–B or B–A) compartmental interactions.AMO Fig. . Comparee (L1-rich) and F (B1-rich), which are ~100-kb in length covered by 500 oligo probes  in the nuclei of control cells. L1 depletion significantly increased the nuclear distance between g and q to 1.81 ± 0.82 µm , implying heterochromatin decompaction  promote the phase separation of HP1α, which fails to phase separate on its own  show high activities compared to L1 sequences in the 5’ and 3’ ends. In mESCs, HP1α tends to bind the central region of L1 repeat DNA. These observations imply some degree of weak sequence-specificity for HP1α in recognizing its targets. This notion is congruent with several reports that HP1α preferentially binds nuclear RNA and rRNA rather than tRNA and randomly chosen RNA,105), or acting as a scaffold to anchor L1-rich chromosomal segments to the nucleolar and nuclear peripheries. Cases for RNA in organizing subnuclear domains have been reported. For example, Xist RNA binds the lamin B receptor in the inner nuclear membrane to anchor the inactive X chromosome.106 Transcription of satellite repeats precedes chromocenter formation and their transcripts help to recruit SUV39H to centromeric DNA sequences.108 In-depth investigation of the expression, function and mechanism of L1 RNA could be a subject of future studies.As a chromatin-bound noncoding RNA, L1 RNA may facilitate heterochromatin formation through a number of ways, such as promoting HP1α phase separation by providing a means of multivalency and recruiting RNA-binding proteins to increase local molecular mass, or stabilizing DNA-binding activities of heterochromatin-associated proteins  and satellite repeats, which promotes high-order assemblages of the nucleolus and pericentromeric domains, respectively.112We have shown that inhibition of Pol II in both embryos and mESCs led to delayed and incomplete formation of L1/B1 segregation and Hi-C plaid patterns without abolishing genome folding. Previously, the Solovei group reported clustering of exogenous sequences with genomic segments of the same repeat class.113 arguing against convergent evolution in driving retrotransposons in genome organization. Comparative genomics analysis across mammalian genomes has revealed that large-scale conserved patterns of retrotransposon accumulation follow similar evolutionary trajectories through conservation of synteny, gene regulation and nuclear organization, in spite of dissimilar retrotransposons.114 In addition, it has been reported that the landscape of endogenous L1 elements differs significantly from that of new L1 retrotransposon insertions, which broadly target all regions of the human genome, being insensitive to chromatin state.116 This suggests that purifying selection, rather than biased insertions, reshapes the genomic distributions of L1 and Alu/B1 post their integration.118 We speculate that the genetic marking of compartments with distinct activity is so important that during evolution it has imposed selective pressures on the most abundant subfamilies of LINE and SINE repeats for them to accumulate in specific compartments. In comparison, we find ERV retrotransposons to be randomly distributed, which is consistent with a previous report by the Ren group that no genome-wide enrichment of ERVs was found at TAD boundary.13 Recently, the Ren group reported that transcriptionally active HERV-H repeats, a subclass of ERVs, demarcate TADs in human hESCs.120 In fact, among > 1000 HERV-H repeats in human, < 50 show detectable expression in hESCs and only ~20 have a TAD boundary structure as indicated by directionality index. It is likely that a very small proportion of ERVs act at TAD and loop boundaries for more specific local chromatin regulation in a few genomic loci.Although different in nucleotide sequences and length, both primate-specific Alu and rodent-specific B1 elements belong to the same class of SINE repeats, which originate from a common ancient ancestor, 7SL RNA, prior to the primate-rodent split about 80 million years ago,122 Analogously, we propose that the primary DNA sequences, particularly L1 and B1/Alu elements, dictate how the genome folds and functions. We envision that genome folding occurs autonomously, through a process that is driven by homotypic clustering of regions containing L1 or B1/Alu repeat sequences, which could be further facilitated by transcription processes, transcripts produced at these repeat elements, regulatory proteins, and perhaps a combination of these factors that act above and beyond repeat DNA sequences to influence their chromatin states. The widespread yet conserved distribution of homologous repeats in mammalian genomes render them a unique advantage to perform such a task as the blueprint for genome organization and function, compared to histone marks and transcription activities. Structural information embedded in L1 and B1/Alu repeats may be universally recognizable, thus contributing to the high degree of stability and conservation in the compartmental organization that is observed across mouse and human cell types. We want to note that L1 and B1/Alu compartmental domains represent a structural and functional ground state of chromatin organization, on which subsequent regulatory features, such as dynamic enhancer–promoter interactions, are overlaid. Nevertheless, the same principle of homotypic clustering, phase separation and spatial segregation of chromosomal segments may be reiterated at different genomic scales, consequently folding the genome. Lastly, our study calls for more work towards a complete understanding how genome folding occurs, particularly, on revealing the causality and mechanisms how these repetitive sequences act.In summary, our study provides important initial evidence to unravel the fundamental principle of 3D genome organization. As discovered by Anfinsen in the late 1950s, the amino acid sequence of a protein determines its structure and function.123 We used the 10-kb bin in all repeat analyses unless otherwise indicated. For de novo compartment calling, the mouse genome was first segmented into 100-kb bins, and the densities of L1 and B1 repeats were normalized to their genome background (19% for L1 and 3% for B1), and log2 of the ratio of normalized B1 to L1 densities [log2(B1/L1)] was calculated. The adjacent regions with size >500 kb were kept (85% left) and assigned as B1-rich (540) or L1-rich (648) compartments with a median size of 1.1 Mb to 1.3 Mb  and ActD (1 μg/mL) for 3 h to inhibit transcription. For heterochromatin fractionation, sucrose gradient centrifugation of mESC nuclear extracts was performed as previously reported with modifications.126 For embryonic microinjection, ASO (5 μM) and AMO (1 mM) was injected into PN3 zygotes on a Leica DMI3000B microscope equipped with a Leica micromanipulator.For cell-cycle synchronization, mESC (J1) cells4 immuno-FISH,109 and RNA FISH127 in mESCs and embryos were performed as previously described with modifications. FISH Probes targeting consensus sequence of L1 and B1  is targeted by 4500 DNA probes. Probe sets are shown in Supplementary information, Table DNA FISH, B1 Fig.  were use B1 Fig. . For Oli87 To deplete B1 RNA, two antisense oligonucleotide (ASO) sequences targeting the B1 consensus sequence were synthesized by IDT (Integrated DNA technologies). Sequences are listed in Supplementary information, Table To deplete L1 RNA in mouse embryos and mESCs, we used the same morpholino antisense oligonucleotide (AMO or ASO) as Percharde et al. previously used.81 following two independent experiments of L1 or scramble (SCR) AMO transfection. The summary statistics for Hi-C quality control is shown in Supplementary information, Table 128 A and B compartments6 and compartmentalization strength94 were identified as described previously.Small-scale in situ Hi-C (sisHi-C) was performed as previously described,The FISH-based segregation index is defined as the negative value of Pearson’s correlation coefficient of L1 and B1 DNA signals in the nucleus. The Hi-C-based segregation index is defined as the ratio of homotypic versus heterotypic interaction frequencies between L1 and B1/Alu subfamilies.38 In phase separation assays, recombinant HP1α with DNA or RNA fragments for L1, 8× B1, or 8× SCR were incubated at 4 °C overnight.A series of eight 1-kb fragments, designated as F1 to F8, were produced to cover the full-length 6544-kb L1 sequence by PCR .Please also see Supplementary information, Data Supplementary information, Figure S1Supplementary information, Figure S2Supplementary information, Figure S3Supplementary information, Figure S4Supplementary information, Figure S5Supplementary information, Figure S6Supplementary information, Figure S7Supplementary information, Figure S8Supplementary information, Figure S9Supplementary information, Figure S10Supplementary information, Figure S11Supplementary information, Figure S12Supplementary information, Figure S13Supplementary information, Table S1Supplementary information, Table S2Supplementary information, Table S3Supplementary information, Table S4Supplementary information, Table S5Supplementary information, Text S1Supplementary information, Data S1


Deposition of amyloid-β (Aβ) in the brain is one of the important histopathological features of Alzheimer’s disease (AD). Previously, we reported a correlation between cell adhesion molecule L1 (L1) expression and the occurrence of AD, but its relationship was unclear. Here, we report that the expression of L1 and a 70 kDa cleavage product of L1 (L1-70) was reduced in the hippocampus of AD (APPswe) mice. Interestingly, upregulation of L1-70 expression in the hippocampus of 18-month-old APPswe mice, by parabiosis involving the joining of the circulatory system of an 18-month-old APPswe mouse with a 2-month-old wild-type C57BL/6 mouse, reduced amyloid plaque deposition. Furthermore, the reduction was accompanied by the appearance of a high number of activated microglia. Mechanistically, we observed that L1-70 could combine with topoisomerase 1 (Top1) to form a complex, L1-70/Top1, that was able to regulate expression of macrophage migration inhibitory factor (MIF), resulting in the activation of microglia and reduction of Aβ plaques. Also, transforming growth factor β1 (TGFβ-1) transferred from the blood of young wild-type C57BL/6 mice to the aged AD mice, was identified as a circulating factor that induces full-length L1 and L1-70 expression. All together, these findings suggest that L1-70 contributes to the clearance of Aβ in AD, thereby adding a novel perspective in understanding AD pathogenesis. APP gene [1-42 pathology in parallel with reducing protein kinase D1 (PKD1) function [1-42 formation and histone deacetylase 2 (HDAC2) expression, leading to downregulation of glucocorticoid receptor 1 activity, L1 can alleviate AD pathology [Alzheimer’s disease (AD) is an irreversible neurodegenerative disease and is the most common cause of dementia among older adults , 2. ClinAPP gene . In fronfunction . Throughathology . L1 can athology , 24. Furathology –28. TherWe here report that hippocampal L1-70 expression in AD mice decreases Aβ deposition by combining with topoisomerase 1 (Top1) to form a complex that enters the nucleus to regulate the expression of macrophage migration inhibitory factor (MIF), resulting in activation of microglia for clearance of amyloid plaques. The fact that L1-70 reduces levels of Aβ clearance not only promotes our understanding of Aβ metabolism but also provides a novel perspective on the study of AD pathogenesis.L1 is increased in the cerebrospinal fluid of AD patients , attenua+ leukocytes were recovered from the blood of the wild-type mouse (40%) and amounted to 45% in the CAG-EGFP mouse  APPswe mouse was joined to that of a healthy, young (2 months old) wild-type mouse, to assess whether circulatory components from the young mouse can affect L1/L1-70 expression and Aβ deposition in the aged APPswe mouse Fig. . ParabioTo evaluate whether increasing L1-70 levels could contribute directly to reduce Aβ levels, we explored how L1-70 in the nucleus could regulate transcription. First, co-immunoprecipitation and mass spectrometry was performed to find candidate proteins that might associate with L1 from the 5- to 7-day-old wild-type mouse brain. Among the many proteins found, we focused on topoisomerase 1 (Top1), identified by mass spectrometry.In order to further explore the L1-70/Top1 interaction, immunoprecipitation was performed with brain lysates using an antibody against the L1 cytoplasmic domain and probing the immunoprecipitate with an anti-Top1 antibody by western blot analysis Fig. . In the CMV-L1-70-Myc) and HA-tagged Top1 (CMV-Top1-HA), and the binding of L1-70 and Top1 was analyzed by ELISA using plates coated with antibodies against either Myc or HA. In this experiment, purified L1-70-Myc from transfected 293 T cells was added to the substrate-coated target molecules. Then, a series of dilutions of purified Top1-HA was added to allow binding to L1-70. The results showed that L1-70 binds to Top1 in a concentration-dependent manner  and Top1 (red) were observed to overlap in the nucleus Fig. , suggestAAV-L1-70-Myc and AAV-Top1-HA, Supplementary Fig. To verify the binding of exogenous L1-70-myc and Top-HA, cultured hippocampal cells were transduced with adeno-associated viruses expressing L1-70-Myc and Top-HA  because it had been reported to activate microglial cells and take part in the clearance of Aβ –33. To spMIF-EGFP). We also engineered two adeno-associated viruses to express L1-70-Myc and Top1-HA. Cultured human glioblastoma U87 cells, which normally do not express L1, were transduced with lentivirus, encoding the recombinant MIF-EGFP gene, for 48 h, then the cells were further transduced with adeno-associated virus mediating the expression of L1-70-Myc and Top1-HA for an additional 24 h. The EGFP gene was expressed strongly in U87 cells transduced with both AAV-L1-70-Myc and AAV-Top1-HA genes compared to no signal following transduction with AAV-Top1-HA only and a low signal transduced with AAV-L1-70-Myc only  in the blood from young mice are involved in this process. It has been shown that the expression of L1 in pancreatic duct cells and intestinal epithelial cells can be induced by TGFβ-1 –37. TherIn order to further investigate the relationship between circulating TGFβ-1, L1-70, and MIF expression, mouse neuroblastoma N2a cells were transfected with TGFβ-1 siRNA. After 48 h, decreased TGFβ-1 expression in the siRNA groups was accompanied by a decrease in L1-70 and MIF expression Fig. , furtherAlzheimer’s disease was recognized as early as in 1901. In the 1960s, the “amyloid cascade hypothesis” and “tau protein hyperphosphorylation hypothesis” were proposed to explain AD pathogenesis, promoting the rapid development of AD research. However, these efforts have been unsuccessful in developing effective drugs for AD remission or cure. This failure points to the complexity of AD pathogenesis, highlighting the limits of the current understanding of AD pathogenesis , 39.Cell adhesion molecule L1 has emerged to be interesting in dealing with the pathophysiology of AD since it has been shown to ameliorate the consequences in several mouse models of neurodegenerative diseases . It is iWe reported an increase of L1 in the cerebrospinal fluid of AD patients  and alsoWe have extended these studies to show the following: expression of L1 and L1-70 in the brain tissue of aged AD mice is lower in comparison to wild-type mice. Decreased L1-70 expression in these animals is accompanied by more amyloid plaques. In contrast, high levels of L1-70 in the wild-type hippocampus are accompanied by the absence of amyloid plaques. Using a parabiosis approach, in which the blood circulation of aged AD mice is joined with the blood circulation of young wild-type mice, we show that L1-70 expression increases within 3 days, and that amyloid plaques decrease within 14 days. These results suggest that L1-70 is involved in regulating the clearance of amyloid plaques. We also show that L1-70 forms a complex with Top1 in the nucleus where it may regulate transcription. As shown by ChIP with anti-L1 antibody and DNA sequencing to identify DNA fragments specifically binding to the L1-70/Top1 complex, and using MIF reporter gene assays, we showed that the L1-70/Top1 complex regulates MIF expression, which can activate microglia and promote clearance of Aβ in the brain , 32. FurIn pancreatic duct cells, TGFβ-1 had been shown to upregulate the expression of L1 . In our In conclusion, using a mouse model of AD, we found a correlation between L1-70 levels and reduction of amyloid plaques in a mouse model of AD. The data suggest that L1-70 is an intermediate molecule involved in Aβ clearance , APPswe transgenic mice were purchased from the Model Animal Research Center of Nanjing University . All animals were bred in the Center of Experimental Animals of Shantou University Medical College. Animal experiments were in accordance with the guidelines approved by the Institutional Committee of Shantou University according to international standards. All efforts and attention were given to minimize the pain and the number of mice used for the experiments. The mice were selected randomly for the experiments.® Gold Antifade reagent with DAPI  was used to stain cell nuclei. Cell culture dishes were coated with poly-d-lysine . Adeno-associated virus and lentivirus were constructed by VectorBuilder.The following antibodies and reagents were used in the experiments: mouse anti-L1 cytoplasmic domain , mouse anti-β-amyloid , mouse anti-MIF , rabbit anti-topoisomerase I , rabbit anti-Iba-1 , rabbit anti-TGFβ 1 , mouse anti-β-actin , mouse anti-Myc , and rabbit anti-HA . Secondary antibodies coupled to horseradish peroxidase or fluorescein were: goat anti-mouse , goat anti-rabbit , Alexa Fluor 488-conjugated donkey anti-mouse , Alexa Fluor 594-conjugated donkey anti-rabbit . Reagents and kits were radioimmunoprecipitation assay , phenylmethanesulfonyl fluoride , LDS sample buffer , sample reducing agent , BCA protein assay kit , PVDF membrane , enhanced chemiluminescence kit , TMB substrate solution for ELISA , Lipofectamine 2000 , Co-IP kit , Duolink kit , ChIP kit , protease inhibitor cocktail , tacrine . ProLongWestern blot analysis was performed as described by Sahu et al. (2016). Total proteins were collected by extracting brains tissues and cells in RIPA buffer supplemented with PMSF, mixed with 25% SDS-PAGE loading buffer, and then heated for 10 min at 100 °C. Forty micrograms of each protein sample was subjected to 10% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% BSA, the blots were probed with the following primary antibodies overnight at 4 °C: mouse anti-L1 cytoplasmic domain , rabbit anti-topoisomerase I , mouse anti-MIF , rabbit anti-TGFβ-1 , and mouse anti-β-actin . Membranes were then washed three times with PBST  for 5 min each wash, followed by incubation with secondary goat anti-mouse  and goat anti-rabbit  in 5% BSA for 2 h at room temperature (RT). Membranes were then washed three times in TBST for 5 min each wash at RT. Bands were visualized with an enhanced chemiluminescence kit . Signal intensity was detected and quantified using ImageJ software .d-lysine. Tissues were digested with trypsin in Hank’s balanced salt solution and gently triturated. After filtration and centrifugation, cells were resuspended in DMEM/F-12 culture medium  with 10% fetal bovine serum . Cell suspensions were seeded onto poly-d-lysine-coated 48-well or 24-well plates at 5 × 104 and 1 × 105 cells in DMEM/F-12  per well, respectively. After 4–6 h, the medium was removed, and Neurobasal-A medium (Thermo Fisher # 10888022) supplemented with 2% B-27 (Thermo Fisher # 17504044) and 1% penicillin-streptomycin solution (Solarbio # P1400) was added, and cells were maintained at 37 °C in a 5% CO2 atmosphere.Hippocampal neurons from 1-day-old wild-type C57BL/6 mice were cultured. In brief, cell culture dishes were coated with poly-® Gold Antifade reagent with DAPI . The intensity of immunopositivity in cultured neurons was collected using a fluorescence microscope . Fluorescence images from tissue sections were acquired with an Olympus laser confocal system .Cultures of hippocampal neurons were fixed with 4% formaldehyde solution for 10 min at RT and washed with phosphate-buffered saline, pH 7.3 (PBS) three times for 5 min each wash. Then, 0.1% Triton X-100 in PBS was used to permeabilize the plasma membrane. Hippocampal sections were selected randomly and fully dewaxed, and antigen retrieval was performed in citrate buffer  for 40 min at 99 °C. To block nonspecific binding sites, cells and sections were incubated with PBS containing 10% normal donkey serum for 1 h at RT. Samples were then incubated at 4 °C overnight with mouse anti-L1 cytoplasmic domain , rabbit anti-topoisomerase I , mouse anti-β-amyloid , or rabbit anti-Iba-1 . After washing with PBS three times for 5 min at RT, samples were incubated for 2 h at RT with donkey anti-mouse secondary antibody conjugated to DylightTM 488  or donkey anti-rabbit secondary antibody conjugated to DylightTM 594 . After three 5-min washes with PBS at RT, sections or cells were mounted using ProLongn = 6 mice per group). After partnering for 3 or 14 days, the hippocampus from the APPswe mice was prepared for immunofluorescence staining and western blotting at 3 and 14 days, respectively.Parabiosis is the surgical union of two related organisms  allowing sharing of the blood circulation and neovascularization without triggering an immunological reaction. The procedure was performed according to Paniz . Two-monCo-immunoprecipitation was performed following the company’s instructions . In brief, mouse anti-L1 cytoplasmic domain , or nonimmune mouse IgG antibody as control were incubated with beads for 2 h at RT. Wild-type C57BL/6 mouse (5–7 days old) brain lysates were incubated with antibody-bound beads overnight at 4 °C. After washing in IP buffer  and conditioning buffer , the beads were washed and the protein complexes bound to the antibody were eluted with elution buffer . Subsequent western blot analyses were performed as described.The same concentration of purified L1-70-Myc in PBS was used for substrate coating in 96-well plates at 4 °C overnight. After five 10-min washes in PBST  at RT, wells were blocked with 10% fetal bovine serum  in PBS for 3 h at 37 °C, then washed five times with PBST, 10 min each wash at RT. Several concentrations  of purified protein pCMV-Top1-HA were added to each well and incubated for 2 h at 37 °C. After washing with PBST, the wells were incubated with rabbit anti-HA for 1.5 h at 37 °C. OD values were determined using TMB  substrate solution for ELISA .In order to investigate the relationship between L1 and Top1, a proximity ligation assay (Duolink) was performed. Duolink  provides a sensitive method for in situ detection of proteins that co-localize at a distance of 40 nm or less. The experiment was performed according to the manufacturer’s instructions. Hippocampal neurons were prepared in the 24-well plates, and a blocking buffer was added to saturate nonspecific binding sites. Neurons were incubated with mouse anti-L1 cytoplasmic domain  and rabbit anti-Top1  antibodies at 4 °C overnight. Neurons incubated with only one antibody were taken as the negative control. After washing with Buffer A, the secondary antibodies with specific anti-mouse and anti-rabbit PLA probes were added and neurons were incubated at 37 °C for 1 h. Subsequently, the reaction solution for the ligase was added and incubated at 37 °C for 30 min, resulting in the circularization of the two adjacent PLA probes. Reaction amplifiers with polymerase were added and incubated at 37 °C for 100 min to visualize the red dots. Before imaging, slides were washed with Buffer A and mounted on a coverslip using Duolink In Situ Mounting Medium with DAPI. Confocal images were acquired with an Olympus laser confocal microscope .The adeno-associated virus and lentivirus were constructed by VectorBuilder  overnight at 4 °C. Nonimmune mouse IgG was used for control. After IP, cross-linking was reversed by boiling the sample with Chelex100  for 10 min. The supernatant containing DNA fragments was isolated by centrifugation at 12,000xg for 1 min.Hippocampal neurons were prepared for ChIP assay following the manufacturer’s protocol . Cross-linking between protein and chromatin was achieved by adding 1% formaldehyde in PBS for 15 min at RT, followed by quenching with 125 mM glycine for 5 min at RT. Cells were harvested in PBS and lysed with immunoprecipitation (IP) buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, Nonidet P-40, and Triton X-100, supplemented with protease inhibitor cocktail . Nuclear pellets were isolated by centrifugation at 12,000x6 cells per well for 24 h. Tacrine dissolved in Dulbecco’s modified Eagle’s medium F-12  at different concentrations  was added to the wells. After culturing for 48 h, cells were collected in RIPA buffer  supplemented with PMSF , mixed with 25% sample loading buffer , and heated at 100 °C for 15 min for western blot analysis.The L1 agonistic mimetic tacrine upregulates the expression of L1 in cultured L1-expressing cells . N2a celN2a cells were transfected with siRNA for TGFβ-1  in a 6-well-plate by application of 50 nM siRNA or 50 nM control siRNA, using a Lipofectamine 2000 kit . Dulbecco’s modified Eagle’s medium F-12  was changed after 4–6 h and then cells were incubated for 48–72 h. Total protein was harvested, and the concentration was determined by a bicinchoninic acid test  for western blot analysis.t-test was used to compare two treatment groups, and one-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for multiple group comparisons. The variance was similar between the groups. Values of *p < 0.05, **p < 0.01, ****p < 0.0001 were considered statistically significant.Statistical analyses were performed using SPSS 19.0 software . All values are presented as means ± standard deviation (SD). Student’s Legends of supplementary figure 1, 2 and 3Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3


Using the self-consistent-charge density-functional tight-binding (SCC-DFTB) method, we studied the effect of axial stretching on the electrical conductivity and quantum capacitance of hybrid films formed by AB-stacked bilayer graphene and horizontally oriented single-walled carbon nanotubes (SWCNTs) with indices chirality . The paper discusses several topological models of hybrid graphene/SWCNT  films, which differ in the width of the graphene layer in the supercell and in the value of the shift between the graphene layers. It is shown that axial stretching has a different effect on the electrical conductivity and quantum capacity of the hybrid graphene/SWCNT  film depending on the width of the graphene layer. For a topological model with a minimum width of the graphene layer (2 hexagons) under a 10% stretching strain, the transformation of bilayer graphene from planar to wave-like structures is characteristic. This transformation is accompanied by the appearance of the effect of anisotropy of electrical conductivity and a sharp decrease in the maximum of quantum capacitance. For a topological model with a graphene layer width of 4 hexagons, axial stretching, on the contrary, leads to a decrease in the effect of anisotropy of electrical conductivity and insignificant changes in the quantum capacitance. Based on the obtained results, the prospects for using hybrid graphene/SWCNT  films as a material for creating flexible electrodes of supercapacitors are predicted. In addition, heat-treated CNT-based film electrodes have twice the capacity than untreated electrodes (446 mAh g−1 at 0.5 C versus 165 mAh g−1). Sugiawati et al. found that pre-lithiated polymer-coated CNT anodes have a high capacity of 850 mAh g−1 at 1 C for 50 charge–discharge cycles [−1 at a current density of 60 mAg−1 [−1 and a low equivalent series resistance of 4.6 Ohm. Wang et al. presented a strong and flexible CNT film decorated with MnO2 nanosheets that exhibit superior Faraday pseudocapacitance when used as electrodes for flexible supercapacitors [−1 at a power density of 571.3 W kg−1 and the ability to retain capacity after 8000 galvanostatic charge/discharge cycles, as well as excellent flexibility and long service life. Currently, a rapidly developing field of modern electronics is flexible and stretchable electronics ,2. A keye cycles . CNT-bas60 mAg−1 . Gilshtepacitors . The pro2O5, MnO2, TiO2, and sulfur. to form a flexible electrode of batteries, graphene films demonstrate their unique properties, including excellent electrical conductivity, chemical stability, and high specific surface areas [3O4/graphene film for use as electrodes of lithium-ion batteries [−1 at 100 mA g−1) and an excellent cyclic stability . Qu et al. manufactured a supercapacitor electrode based on a graphene/g-C3N4 composite, which shows a specific capacity of 1500 mF cm−2, retaining 95% of the initial capacity after 5000 cycles and a maximum energy density of 0.075 mWh cm−2 [2O4 composite film that provides a high capacity of 874 mAh g−1 at a current density of 100 mA g−1 [−1 at 100 mA g−1 [−1 as compared to a supercapacitor based on CNT fibers (5.83 F g−1) [Flexible graphene electrodes can also serve as an anode for flexible lithium-ion batteries ,10,11,12ce areas . Wang etatteries . The fab0 mA g−1 . The mai0 mA g−1 . One of 0 mA g−1 . Sun el 3 F g−1) . A wide 3 F g−1) ,26 opens3 F g−1) , electro3 F g−1) , and tra3 F g−1)  of paral3 F g−1) ,31,32. A3 F g−1) , this to−5 eV/Å. The SCC tolerance was set at 10−5 a.u. All calculations were performed at a temperature of 300 K. The SCC-DFTB method was chosen due to the polyatomic nature of supercells of the studied atomic configurations of hybrid graphene/SWCNT structures containing several hundred atoms, which increases the complexity of quantum calculations of such objects. Using the atomic structure of graphene as an example, it has been shown that the SCC-DFTB method is able to successfully reproduce structural and energy characteristics comparable in accuracy with the results of calculations by the density-functional tight-binding (DFT) method, but at lower computational costs [The atomic structure of hybrid graphene/SWCNT films was carried out using the self-consistent-charge density-functional tight-binding (SCC-DFTB) method , implemeal costs . T(E) and static electrical conductivity G. The function T(E) is determined by the expression: FE is the Fermi energy of the material of the contacts, TF is the thermal broadening function; e is the electron charge, h is the Planck’s constant, and e2/h is the quantum of conduction .The electrical conductivity was calculated within the Landauer–Buttiker formalism . This foT(E) were carried out for supercells of hybrid graphene/SWCNT films consisting of 250–300 atoms. In view of the large dimension of the considered electronic system, the original method for the accelerated calculation of the transmission function T(E) [T(E) for a small number of k-points of the first Brillouin zone and then interpolating it for any k-point of the first Brillouin zone and recovering the full transmission function T(E).In this study, the calculations of the transmission function ion T(E)  was applion T(E) . This mem is the mass of the object, V is the displacement voltage, e is the elementary charge, D is the density of states (DOS) at the applied bias, and EF is the Fermi level. The calculation of the quantum capacity was carried out according to the following equation :(3)CQ(V)d was different for each model: for the V1 model, d = 0.48 nm; for the V2 model, d = 0.27 nm; and for the V3 model, d = 0.06 nm. The distance between graphene layers along the Z axis (perpendicular to the nanotube axis) was 0.34 nm for all three topological models. Previously, these models were tested for energy stability and were used by us to study the electronic properties of hybrid graphene/SWCNT films [This study was carried out for three topological models of two-dimensional (2D) hybrid graphene/SWCNT films formed by AB-stacked bilayer graphene and SWCNTs . The equilibrium configurations of the supercells of the hybrid graphene/SWCNT films are shown in NT films . The choNT films . In addiNT films . Since iNT films , which pNT films . Lx and Ly of the supercells of the hybrid graphene/SWCNT  films. Topological models of hybrid graphene/SWCNT films obtained by translating supercells in two directions (X and Y) are shown in Estrain on the strain value ΔL in relative units were constructed based on the results of the calculations. The strain energy was found from the difference between the total energy of the structure before and after axial stretching. Estrain value was also observed; however, at a 10% stretching strain, a sharp decrease in the strain energy occurred. As noted in the introduction, the main task of our study was to establish the effect of strain on the electrically conductive and electrochemical properties of hybrid films formed by bilayer graphene and SWCNTs . We chose the stretching along the X axis, i.e., along the zigzag edge of bilayer graphene. Since the nanotube  and bilayer graphene interacted by means of van der Waals forces in the supercell and the ribbon structure of graphene layers was taken into account, we chose such a variant of applying the straining force. The calculations were performed for the supercells of hybrid graphene/SWCNT films  shown in To explain this behavior of the strain energy curves, let us trace the changes occurring in the atomic structure of supercells of models V1, V2, and V3 under stretching. R for two directions of current transfer (Rx and Ry): along the X axis  and along the Y axis . We evaluated the electrochemical properties by the value of the quantum capacitance CQ. Rx and Ry for undeformed supercells and for selected cases of stretching strain , at which the most noticeable changes occurred in the atomic structure of the supercells of all three models.Let us proceed to study the effect of axial stretching on the electrically conductive and electrochemical properties of hybrid graphene/SWCNT  films from the standpoint of assessing their potential prospects as a material for flexible electrodes. The electrically conductive properties were estimated by the value of the electrical resistance Rx was observed, which increased by a factor of 30. In this case, the value of the resistance Ry increased only by ~3.5 kOhm. That is, for the case of the 10% stretching strain of model V1, the appearance of the effect of anisotropy of electrical conductivity and a significant deterioration of electrically conductive properties in the direction of current transfer along the X axis is characteristic. The resistance for model V2 for both the directions of current transfer turned out to be the least sensitive to stretching in the considered strain range. At a 10% stretching strain, the values of Rx and Ry decreased by only 0.2 kOhm in comparison with the resistance of the undeformed structure. For model V3, there was a clear improvement in the electrically conductive properties with increasing strain. If for an undeformed structure there was a pronounced anisotropy of electrical conductivity (Rx exceeded Ry by more than 10 times), then already at a 5% stretching strain Rx decreased by half, and at a 10% stretching strain it creased by about 4 times. The resistance Ry also decreased with increasing strain, and at a 10% stretching strain, it became half as much as in the Ry of the undeformed structure. The difference between the resistance values Rx and Ry was 5 times in the case of a 10% stretching strain, and it was ~8 times in the absence of deformation. It can be assumed that with the further stretching of the supercell of model V3, the tendency towards a decrease in the resistances Rx and Ry continue.After analyzing the data in the CQ of hybrid graphene/SWCNT  films changed under axial stretching. CQ for models V1, V2, and V3 in the absence of axial strain and under stretching strain. As in the analysis of the behavior of electrical resistance, the cases of 1%, 5%, and 10% stretching strains were selected for demonstration. Let us now discuss how the quantum capacity CQ on the bias voltage were similar: the curve had a pronounced peak near 0 V. When stretched, the height of this peak began to change. At a 5% stretching strain, it decreased by 100 F g−1 for model V1 and it increased by 200 F g−1 for model V2. At a 10% stretching strain, a sharp decrease in the peak height was observed for both models: for model V1, its height decreased to 200 F g−1 and for model V2, to 650 F g−1. In the case of model V1, such a significant change in the peak height can be explained by the transformation of the planar configuration of graphene layers into a wavy one. In the case of model V2, this transition did not occur, but this value of stretching strain for the given topology of the hybrid graphene/SWCNT  film was critical for the maximum value of the quantum capacitance. For model V3 (see CQ (by ~60 F g−1 at a 10% stretching strain) was observed near 0 V. Unlike models V1 and V2, the maximum value of the quantum capacitance CQ for model V3 was reached not near 0 V, but at 2.5 V. At the same time, the maximum value of CQ for model V3 (582 F g−1 at a 10% stretching strain) was noticeably lower as compared to the maxima of CQ for models V1 (939 F g−1 in the absence of strain) and V2 (1197 F g−1 at a 5% stretching strain). To explain the revealed features of the behavior of quantum capacitance, we calculated the DOS distribution for models V1, V2, and V3 for the same cases of stretching strain as in The graphs show that for models V1 see a and V2 l V3 see c, the quCQ according to Equation (3). For models V1 and V2, a high intensity of DOS peak was observed at the Fermi level (it shifted by 0 eV in the figure), which was also present in the graphs of CQ. As is known, the electronic states at the Fermi level make the decisive contribution to the conducting properties of a material. This explains the high values of the quantum capacitance at 0 V. For model V3, the DOS at the Fermi level was minimal, and the high-intensity DOS peaks were located far from the Fermi level. In this regard, the maximum of CQ for model V3 was much lower than the maximum values of CQ for models V1 and V2.The reasons for the significant differences in the behavior of the electrically conductive and electrochemical properties between models V1 and V2 and model V3 lie in the peculiarities of their atomic structure. Let us clarify them using the example of −1 among all the considered models. This maximum was achieved when stretched by 5%. Model V3 with a graphene nanoribbon width of 3 hexagons in the supercell turned out to be the least sensitive to strain in the considered stretching range. In general, based on the obtained simulation results, a preliminary conclusion can be drawn about the possible prospects for the use of hybrid graphene/SWCNT  films as a material for creating flexible electrodes for supercapacitors and batteries; however, it needs further experimental confirmation.Based on the SCC-DFTB calculation results, the regularities of changes in the atomic structure, as well as the electrically conductive and electrochemical characteristics of hybrid films formed by AB-stacked bilayer graphene and SWCNTs  under axial stretching by 1–10%, were revealed. It was found that elastic strain was observed in almost the entire considered stretching range for the topological models under study. The exception was model V1 with the minimum width of the zigzag graphene nanoribbon (2 hexagons) in the supercell. For this model, the transformation of the planar configuration of bilayer graphene into a wavy one was revealed at a 10% stretching strain. The rearrangement of the atomic structure of bilayer graphene caused the appearance of the effect of the anisotropy of electrical conductivity and a fivefold decrease in the maximum value of the quantum capacity for this case of stretching strain. Model V2 with a graphene nanoribbon width of 3 hexagons in the supercell exhibited the best ability to retain high electrically conductive properties under stretching. The electrical resistance for this model in both the directions of current transfer changed by several tenths of 1 kOhm in the course of stretching. The same model was characterized by the maximum quantum capacity of 1197 F g


Furthermore, the adhesion of the coating with 0.7 wt.% graphene was also the largest  and had the smallest diffusion coefficient , and the thermal stability improved by 18.6%. Finally, the corrosion resistance mechanism of the composite coating with different graphene contents at different soaking times was revealed based on the electrochemistry and morphology characteristics other than water absorption and contact angle.The working environment for tubing in oil and gas fields is becoming more and more serious due to the exploration of unconventional oil and gas resources, leading to the increasing need for a protective internal coating to be used in tubing. Therefore, a new mica–graphene/epoxy composite coating with different graphene contents  was prepared to improve the tubing resistance to a corrosive medium, an autoclave was used to simulate the working environment, and an electrochemical workstation assisted by three-electrodes was used to study the electrochemical characteristics of the coating. The results showed that the addition of a certain amount of graphene into the mica/epoxy coating significantly improved the corrosion resistance of the composite coating, and when the graphene content increased, the corrosion resistance of the mica/epoxy coating first increased and then decreased when the corrosion current density of a 35 wt.% 800 Metal pipelines suffer from the most common and serious corrosion in corrosive environments containing CO2. The leakage of oil and gas caused by the corrosion perforation of the metal pipes not only leads to the waste of resources and the pollution of the environment, but it also causes serious harm to personal safety [In the process of oil and gas exploitation, gathering and transportation, there are often many corrosive species in the produced fluid, such as Hl safety . The metl safety . Among tEpoxy resin (EP) is a thermosetting resin with the excellent comprehensive properties. EP has the characteristics of good adhesion, strong adhesion, outstanding corrosion resistance, excellent chemical resistance, and low shrinkage, and it is easy manufacture and of low cost. Therefore, EP is widely used in coatings, adhesives, and in other fields ,5,6,7,8.Nanomaterials have attracted the attention of many researchers because of their outstanding and unique properties , especiaTherefore, as a solid lubricating additive, graphene has shown good self-lubricating performance and has also become an ideal anti-corrosion material or filler. In recent years, researchers have found that the addition of graphene into epoxy resin effectively improves the shielding performance, strong acid and alkali resistance, and adhesion of coatings ,19. At tIn this paper, the mica and graphite power were used as fillers to prepare mica–graphene/epoxy composite coatings with different graphene contents. The effects of different graphene contents on the corrosion resistance of the composite coatings were investigated by electrochemical techniques and high–temperature and high–pressure tests. Combined with the water absorption characteristics and contact angle of the mica–graphene/epoxy, the corrosion resistance mechanism of the composite coatings with different graphene contents at different soaking times was revealed.2.In this experiment, epoxy resin (E-08)  was used as a base material; mica  and graphite power  were used as fillers; polyether amine (D230)  was used as the curing agent; and various additives were added to the mixture to prepare the coating . N80 carbon steel was used as the base metal material in the experiment and had a chemical composition (wt.%) of C: 0.34~0.38, Si: 0.20~0.35, Mn: 1.45~1.75, P: ≤0.15, S: ≤0.15, and Fe balance. The size of the test sample was 100 mm × 50 mm × 3 mm, and the size of the electrochemical test sample was ø20 mm × 3 mm. Among them, the sample used in the electrochemical tests was welded with copper wire and encapsulated with resin, and the reserved working area was 3.14 cm# (800 mesh) mica/epoxy coating, and the specific method was as follows: 35 wt.% 800# mica powder filler was mixed with epoxy resin (E-08), and stirred at 1200 r/min for 1 h, and it was then dispersed by ultrasonication for 1 h so that the filler was fully dispersed in the coating. It was then vacuumized for 30 min, and the mother liquor was prepared. The mother liquor was mixed with polyether amine (D230) in a ratio of 1:0.4, stirred again at 500 r/min for 3 min to ensure that the mixture was uniform, and it was then vacuumized again for 1 min. The second step was to prepare the mica–graphene/epoxy composite coating, and the specific method was as follows: different graphene  contents  were added to 35 wt.% 800# mica/epoxy coating, stirred at a rotational speed of 1200 r/min for 1 h, dispersed by ultrasonication for 30 min., and then vacuumized for 15 min. Each coating contained a different graphene content and were labeled as TWG-0.2, TWG-0.5, TWG-0.7, and TWG-1.0, respectively. The thickness of the coatings was controlled to 200 ± 10 µm.The preparation processes to develop the coating in this experiment were divided into two steps: The first step was to prepare a 35 wt.% 8002 partial pressure of 3 MPa) and soaking times of 60, 120, and 240 h, respectively. Before the test, N2 was introduced for 2 h to remove oxygen. After the testing system was stabilized in the open circuit potential test in the 10 wt.% NaCl solution, the AC impedance spectrum test was carried out with a sine wave disturbance signal value of 20 mV and a frequency test range of 105~10−2 Hz. The test results were fitted using the ZSimpWin software, ensuring that the overall coincidence value was less than 0.01, and the relative fitting error value of each circuit element in the equivalent circuit was less than 10%. During the polarization curve test, the potential scanning interval was −0.5~0.5 V, and the scanning rate was 0.5 mV·s−1. The polarization curve was fitted with the workstation software.The corrosion resistance behavior of the mica–graphene/epoxy composite coatings with different graphene contents was studied using a Princeton Electrochemical Workstation (P4000) with a three-electrode system, the auxiliary electrode was a platinum electrode, the reference electrode was a saturated calomel electrode (SCE), and the working electrode was the coating sample. The experiment was carried out under normal pressure. The working electrode was first soaked in 10 wt.% NaCl solution at 80 °C with a total pressure of 5 MPa . Then, the samples were soaked in a high-temperature autoclave with the setting parameters of 80 °C and total pressure of 5 MPa  for different times, and they were then taken out, and filter paper was used to dry any of the remaining solution on the surface of the sample. The weight was taken and was denoted as mt. Three parallel samples were measured in each group, and the mean value was taken as the final weight. The water absorption rate was calculated according to Equation (1):t represents the water absorption rate at time t; mt represents the weight of the sample at time t; and m0 represents the initial weight of the sample.An electronic balance with an accuracy of (0.0001 g) was used for the weighing procedures, and the test method was as follows: the samples before immersion were weighed and recorded as the initial weight , the values of  coating ,37,38,39 coating .Ecorr and self-corrosion current density icorr are shown in Ecorr and polarization resistance Rp of the composite coating first increased and then decreased, while the self-corrosion current density icorr showed an opposite trend. For example, after adding graphene (0.2~0.7 wt.%), the self-corrosion potential Ecorr of the composite coating increased by about 20 mV compared to that of the coating without graphene, indicating that the appropriate amount of graphene protected the matrix of the composite coating to some extent. Kumar [It can be seen from t. Kumar  et al. a−11 A·cm−2. As the graphene content increased, the self-corrosion potential of the coating increased, and the self-corrosion current density decreased. When the graphene content in the coating was 0.7 wt.%, the self-corrosion potential of the composite coating was 292 mV, which was higher than that (−332 mV) presented in reference [−13 A·cm−2, which was lower than that (1.15 × 10−7 A·cm−2) presented in Reference [−11 A·cm−2. The results after immersion for 120 and 240 h were basically consistent with those after immersion for 60 h. Generally, the lower the corrosion potential is, the smaller the corrosion current density is, and the better the corrosion resistance of the matrix is [At the same immersion time, for example, after immersion for 60 h, the self-corrosion potential of the coating without graphene was 46 mV, and the self-corrosion current density was 2.19 × 10eference , indicateference , indicatatrix is ,43,44.# mica–graphene/epoxy coating, its corrosion resistance was the best. However, when excessive graphene was added (1.0 wt.%) to the coating, the internal defects of the coating might have increased, resulting in a decrease in the anticorrosion performance of the coating. Therefore, the composite coating with 0.7 wt.% graphene obtained the best corrosion resistance, which is consistent with the results obtained by electrochemical impedance.At different immersion times, the coating with 0.7 wt.% graphene (TWG-0.7) had the lowest self-corrosion current density, the highest corrosion potential, and the largest polarization resistance, indicating that, when 0.7 wt.% graphene was added to the 35 wt.% 800The corrosion resistance of the composite coating was further analyzed by high-temperature autoclave, and the test parameters were set as shown in Graphene can effectively fill defects such as pores and cracks in the epoxy coating due to its small size , reduce 2 partial pressure). The test solution was 10% NaCl, and the sampling period was 24 h. It can be seen that the order of the final saturated water absorption of the coating was TWG-0.7 (1.153%) < TWG-0.5 (1.427%) < TWG-0.2 (1.576%) < TWG (2.03%) < TWG-1 (2.757%).# mica/epoxy coating, and when the amount of graphene was 0.7 wt.%, the water absorption of the composite coating was the smallest, and the water permeability of the composite coating was stronger against the corrosive medium. However, when the amount of graphene was 1.0 wt.%, the water absorption of the coating increased, further indicating that the ways through which the corrosive medium could be diffused into the coating increased.When the amount of graphene was 0.2~0.7 wt.%, the water permeability of the composite coating was higher than that of the 35 wt.% 800In order to investigate the influence of different amounts of graphene on the hydrophobic properties of the composite coating, the wetting contact angles of the mica–graphene/epoxy composite coatings before and after immersion were tested, as shown in 7 cm2·s−1. As the graphene content increased, the diffusion coefficient of the mica–graphene/epoxy composite coating first decreased and then increased. When 0.7 wt.% graphene was added, the diffusion coefficient of the coating reached its lowest value of 1.566 × 107 cm2·s−1. With the further addition of graphene (1.0 wt.%), the diffusion coefficient of the coating increased to 7.526 × 107 cm2·s−1. Furthermore, it was found that the diffusion coefficient of the composite coating to the aqueous solution was smaller when the amount of graphene was in the range of 0.2~0.7 wt.%, but it was larger when the amount of graphene was 1.0 wt.%. The smaller the diffusion coefficient is, the better the impermeability of the coating is. Therefore, the addition of the proper amount of graphene can effectively fill defects such as pores and cracks in the coating, enhance the impermeability of the coating, and delay the contact between the corrosive medium and the metal matrix, enhancing the corrosion resistance of the coating. The composite coating with 0.7 wt.% graphene had the best impermeability. However, after adding excessive graphene, the graphene was easy to agglomerate, and the internal defects of the composite coating increased, which provided favorable conditions for the penetration of the corrosive medium and the corrosion of metal matrix and reduced the impermeability of the composite coating. Finally, the corrosion resistance of the coating was reduced. At this time, the impermeability of mica–graphene/epoxy coating showed a negative growth trend, and it was easy for the corrosive medium to penetrate into the coating and react with the matrix, thus accelerating the corrosion of the metal matrix.It can be seen from The change rule of the adhesion of the composite coating with different graphene contents after immersion for different times was explored; It can be seen from The graphene particle size (graphite power: ~100 nm) was small, so the particles could fill the pores in mica and reduce the internal pore defects of the composite coating. Graphene was flexibly wound with the epoxy polymer chains in the composite coating, which increased the contact area between the composite coating and the substrate surface . AdditioIn addition, it can be seen from The adhesion of a coating treated with a corrosive medium can reflect the anti-corrosion performance of the coating because the corrosive medium will react with the metal matrix after passing through the coating, thus weakening the adhesion of the coating with the metal matrix. It can be seen from Based on the analysis mentioned above, the composite coating prepared by adding the appropriate amount of graphene into the mica/epoxy coating had a certain degree of hydrophobicity, and the surface of the coating had a small contact area with the corrosive medium, which reduced the infiltration of the corrosive medium into the coating to a certain extent. At the same time, a small amount of graphene was able to disperse itself well within the coating, and graphene was evenly distributed among the mica sheets, keeping the layers tightly stacked. At the same time, the advantage of the small size of the graphene was that the particles could fill in the internal porosity and defects of the coating, which made it more difficult for the corrosion medium to penetrate into the coating, resulting in the formation of a more complex anti−corrosion path.According to the results of the high temperature and high pressure immersion test, as shown in 2, which are generated by electrochemical reaction cannot diffuse through corrosion channels in time, thus resulting in gathering and bubbling in the composite coating. The water absorption performance of the coating was enhanced, and the coating was obviously loose when it was in contact with the metal matrix and accompanied by the generation of corrosion products, and the adhesion decreased. It is easy to accelerate the occurrence of corrosion for long-term immersion in a corrosion solution. According to the results of the electrochemical EIS test, the resistance of the mica–graphene/epoxy coating decreased from 1.872 × 1010 Ω·cm2 to 4.333 × 109 Ω·cm2 after immersion for 60 h; the resistance of the composite coating decreased from 1.537 × 109 to 4.621 × 108 Ω·cm2 after immersion for 120 h; and the resistance of the composite coating decreased from 4.130 × 108 to 1.321 × 108 Ω·cm2 after immersion for 240 h, but they were much higher than 106 Ω·cm2.When the amount of graphene was 0.7 wt.%, there was no bubbling at the contact interface between the coating and the corrosive medium, as shown in In the same immersion time, as the graphene content increased, the resistance of the coating first increased and then decreased. It can be seen that the addition of an appropriate amount of graphene into a mica/epoxy coating can not only play an excellent physical isolation role, but it can also increase the compactness of the coating, resulting in significant improvements in the anti-permeability performance of the composite coating, in prolonging the contact time between the corrosive medium and the metal substrate, and in achieving the effect of improving the corrosion resistance of the coating.From the test results obtained from the polarization curves, it can be also seen that with the extension of the immersion time, the self−corrosion potential of the coating decreased correspondingly, the corrosion current density increased, the polarization resistance decreased, and the corrosion resistance of the material decreased. However, as the graphene content in the composite coating increased at the same immersion time, the corrosion potential increased, the polarization resistance increased, the corrosion current density decreased, and the corrosion resistance of the coating increased.Therefore, when graphene and mica were incorporated into composite coatings, a chemical interaction took place between nano–silica (Mica) and graphene , and the(1)The addition of graphene effectively improved the anti-corrosive properties of the composite coatings based on corrosion thermodynamics and kinetics.(2)The addition of 0.7 wt.% graphene maximizes the impermeability of graphene to inhibit the diffusion of corrosive media and also result in good surface hydrophobicity.(3)The addition of the proper amount of graphene to the mica–epoxy coating served as a labyrinth physical barrier along with mica and reduced the pore defects in the organic coatings by reacting with epoxy and other filler, prolonging the corrosion initiation time and the development of the metallic matrix.


Previous evidence links the formation of extranuclear inclusions of transcription factors, such as ERK, Jun, TDP-43, and REST, with oxidative, endoplasmic-reticulum, proteasomal, and osmotic stress. To further characterize its extranuclear location, we performed a high-content screening based on confocal microscopy and automatized image analyses of an epithelial cell culture treated with hydrogen peroxide, thapsigargin, epoxomicin, or sorbitol at different concentrations and times to recreate the stresses mentioned above. We also performed a subcellular fractionation of the brain from transgenic mice overexpressing the Q331K-mutated TARDBP, and we analyzed the REST-regulated mRNAs. The results show that these nuclear proteins exhibit a mitochondrial location, together with significant nuclear/extranuclear ratio changes, in a protein and stress-specific manner. The presence of these proteins in enriched mitochondrial fractions in vivo confirmed the results of the image analyses. TDP-43 aggregation was associated with alterations in the mRNA levels of the REST target genes involved in calcium homeostasis, apoptosis, and metabolism. In conclusion, cell stress increased the mitochondrial translocation of nuclear proteins, increasing the chance of proteostasis alterations. Furthermore, TDP-43 aggregation impacts REST target genes, disclosing an exciting interaction between these two transcription factors in neurodegenerative processes. The presence of protein aggregates is a pathological hallmark for several neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease, and amyotrophic lateral sclerosis (ALS), to name a few . These pIn the case of ALS, TDP-43 is a primary component of these aggregates. TDP-43 is a ubiquitous protein that belongs to the heterogeneous nuclear ribonucleoprotein family and is encoded by the TARDBP gene. TDP-43 is mainly found in the nucleus of normal cells and is involved in RNA regulation, including transcriptional regulation, alternative splicing, and mRNA stabilization . TDP-43 In addition to TDP-43, other proteins with roles as transcriptional factors also show their cytoplasmic accumulation as aggregates . We inclThe dysfunction of the nuclear pore complex is linked to cytoplasmic mislocalization and TDP-43 aggregation, in addition to the dysregulation of stress granule formation . Indeed,Additionally, p-TDP-43, p-ERK , and RES2O2 in HMEC cells led to changes in the nuclear and non-nuclear distribution of p-TDP-43, while the nuclear intensity of p-TDP-43 was decreased in the milder oxidative conditions tested , followed by sex , but not the transgenesis , with sex interacting with the subcellular location also being a relevant factor .Concerning ERK, the findings in the spinal cord of that murine model reinforce the in vitro data. Thus, in line with the colocalization of p-ERK with complex V, the crude mitochondria showed a relevant concentration of p-ERK. However, the nuclear fractions showed a high concentration of p-ERK, while the total ERK levels were significantly lower in the nuclei than cytosol . Densitop < 0.0001), with sex also influencing the values . TDP-43 overexpression only explained 4% of the total variance in the sex and subcellular location  and sorbitol incubation  strongly influenced the TDP-43 immunostaining intensity. In this particular model, we explored the effect of REST targets on SHSY-5Y cells. The results of the RT-qPCR revealed that TDP-43 aggregation induced by sorbitol incubation was associated with significant decreases in the mRNA levels of SCN3B, FADD, PUMA, DAXX, SOD1, CAT, GAP43, 1433, MAPK11, MAPK12, and ARC, known REST targets. Sorbitol incubation also increased some REST targets such as NRX3 and ATP2B2 . Thus, after sorbitol incubation C, both td ATP2B2 D.To evaluate if TDP-43 aggregation can be related to mitochondrial dysfunction, we evaluated if sorbitol incubation in these conditions led to alterations in the cellular ATP production. The results  suggest p < 0001).We also examined if other ALS-related cell stressors, such as ER stress, could induce similar delocalizations of the evaluated proteins. We have previously shown that proteasomal and ER stress induce a cytosolic mislocalization of TDP-43 . Using sp < 0.0001). In p-Jun, there was a significant interaction between stress and the subcellular location, i.e., the effect on epoxomicin depended on the location. Therefore, epoxomicin treatment decreased the cytosolic levels of p-Jun  increase in the REST intensity in the cytosol was followed by a decrease in the nuclear amount at 4 h, A. The deThis work shows that several proteins implicated in ALS  and other neurodegenerative processes, such as AD , show a sensitivity to cell stress. Several neurodegenerative diseases exhibit protein aggregates where these proteins may be present . We evalIn the case of TDP-43, the results showed that, after oxidative stress or osmotic stress, the cytosolic levels of this protein increased both in the endothelial cell line and in the neuronal cell lines. Further, the degree of colocalization within the mitochondria also increased. Of note, TDP-43 may show differences in behavior with p-TDP-43. Thus, while TPD-43 is accumulated after sorbitol stress in the cytosol, it was nonsignificant in p-TDP-43. The in vivo evidence presented shows that this is also the case in the brain lysates, where p-TDP-43 and TDP-43 show differential responses to the overexpression of mutated TARDBP. Whatever the case, the subcellular fractionation data agree with the enrichment of both endogenous murine tdp-43 and hTDP-43 in mitochondrially enriched fractions. Both the functional and morphological analyses showed that the conditions linked to p-TDP-43 aggregation  were associated with major changes in the mitochondrial ATP production. We should indicate that sorbitol incubation is quite harsh, which could impair the mitochondrial function by several pathways.Regarding ERK, in response to increased oxidative stress, we noticed a decrease in the cytosolic and nuclear locations after a transient increase. Nonetheless, the degree of colocalization increased significantly, suggesting the close occurrence of p-ERK with mitochondrial epitopes again. In contrast with p-TDP-43, the slopes relating to nuclear and extranuclear p-ERK were inversely related to the oxidative stress intensity, suggesting the nuclear retention of this factor or rapid cytosolic clearance of it. The same phenomena (decreased both in the cytosol and in nuclei) was present after proteasome and ER stress. This later cell stress also decreased the colocalization within the mitochondrial epitopes. Interestingly, after the epoxomicin treatment, we observed an increase in the degree of mitochondrial colocalization. The subcellular fractionation in the TDP-43 model suggested that p-ERK could be significantly enriched with mitochondria, independently of the TARDBP overexpression.In the present work we show, concerning p-Jun, no clear accumulation in the extranuclear location was present after oxidative stress, though the mitochondrial colocalization increased. After proteasome inhibition and ER stress, the extranuclear levels of this factor were decreased with concomitant retention in the nuclei . In clear contrast with oxidative stress, both ER stress and proteasome inhibition decreased the mitochondrial colocalization. Indeed, the subcellular fractionation suggested that, while Jun accumulated with crude mitochondria, this was not the case with p-Jun.Regarding REST, its accumulation has been previously reported in a cytosolic location in neurodegenerative processes. Our data regarding the response to cell stress showed that its behavior was similar to p-Jun, showing an initial decrease in the cytosol followed by a slight increase in both the nuclei and cytosol. Epoxomicin treatment decreased the nuclear levels after the long exposure, while ER stress increased their values in the cytosol and nuclei. Indeed, the degree of colocalization increased after oxidative stress. Previous data also reported the relationship between changes in the REST expression and protein aggregates . Particu++ pump. Additionally, we noticed a decreased ARC mRNA expression in the cells, implicating altered mRNA traffic (one of the functions of TDP-43). We also detected an increased NRXN3 expression, encoding neurexin 3. This factor encodes a cell adhesion component whose paralogs have been recently implicated in synaptic strength, in close collaboration with the factors regulating RNA/aggregation toxicity [Interestingly, the in vivo data showed that the overexpression of mutated TARDBP increased the degree of REST enrichment in mitochondrial fractions. The interaction between TDP-43 alterations and REST is new, and it is reinforced by the fact that several REST-regulated genes appear affected under osmotic stress conditions, where TDP-43 is mislocalized. These genes included SCN3B, encoding a voltage-gated sodium channel , and twotoxicity . We alsotoxicity . This butoxicity . NoteworFoxo 1 mRNA. It is known that this key metabolic transcription factor promotes neuron death [We also detected a tendency for increased on death . TherefoAccounting for the fact that one of their primary functions is working as transcription factors  or interacting with RNA (in the case of TDP-43), their localization in the extranuclear placements  suggests the existence of pathogenic mechanisms operating in common. All these factors require nucleocytosolic transport; therefore, a loss in this cellular property’s homeostasis may partially explain their presence in the cytosol. Indeed, we have recently reported that the nuclear envelope (where the proteins responsible for nucleocytoplasmic shuttling reside) showed altered properties in ALS patients and models .Several reports have described the occurrence of TDP-43 in mitochondrial fractions ,28,29 anRegarding p-ERK, it is known that there are mitochondrial substrates for its kinase activity . Recent To the best of our knowledge, we have not found previous evidence of REST in the mitochondria. Previous evidence in human substantia nigra  showed aAs for the limitations of our work, we must remark that this evidence was present in an endothelial-like cell phenotype. Therefore, neuronal or glial cells could exhibit different dynamics. Nonetheless, some of the findings in the endothelial cell culture were replicated independently in lysates of the brain cortex of a murine model, and in N2A and SHSY-5Y cells, human neuronal lines further validated the potential findings. We shall also indicate that the subcellular fractionation was more enriching than a strict purification of the indicated compartment. However, we can exclude nuclear contamination of the mitochondrial fraction (based on purity markers).Cell stress related with ALS enhances the non-nuclear localization of transcription factors. These changes could be modeled in vitro with several cell types and may be related to impairments in nucleocytosolic traffic. These transcription factors, under cell stress, increase their interactions with the mitochondria and potentially influence their physiology (as shown for the TDP-43-REST interaction). In particular, TDP-43 aggregation was associated with alterations in the mRNA levels of the REST target genes involved in calcium homeostasis, apoptosis, and metabolism.n = 5 different mice from each sex) and from 90 days old, an age where the motor phenotype is not present. Housing and obtention of animals were as described [According to local laws and the Directive 2010/63/EU of the European Parliament, all experimental procedures were approved by the Institutional Animal Care Committee of the University of Lleida. The minimal number of animals was calculated according to the deviation of western-blot profiles in previous experiments . Both noescribed ,44. For g to obtain a nuclei-enriched fraction. The supernatant was washed twice by centrifuging for 10 min at 1500× g to eliminate any residual whole cells and cell debris. Supernatant obtained was centrifuged for 15 min at 10,000× g; the supernatant contained the ER/cytosol fraction, and the pellet contained the crude mitochondrial fraction. The ER/cytosol fraction was centrifuged for 1 h at 100,000× g in an ultracentrifuge and the supernatant was considered cytosol.Purification of nuclear, mitochondria and cytosol enriched fractions performed as described . Briefly2.Human mammary epithelial cells (HMEC) cell line  was grown in DMEM medium  supplemented with 10% fetal bovine serum heat inactivated (Invitrogen), 2-mM L-Glutamine (Invitrogen) and 20-U/mL penicillin and 20-µg/mL streptomycin (Invitrogen) as antibiotics. The cells were kept at 37 °C in humidify atmosphere with 5% of CO2.N2A and SHSY-5Y (ATCC) cell lines were grown in Advanced MEM medium (Invitrogen) supplemented with 10% fetal bovine serum heat inactivated (Invitrogen), 2-mM L-Glutamine (Invitrogen) and 20-U/mL penicillin and 20-µg/mL streptomycin (Invitrogen) as antibiotics. The cells were kept at 37 °C in humidify atmosphere with 5% of CO2O2 , 5-µM thapsigargin (Thp) (Millipore-Sigma), and 2.5-µM epoxomicin (Epox) (Millipore-Sigma), respectively, for 2 or 4 h or sorbitol (Millipore-Sigma) 0.4 M for 3 h. To avoid the influence of growth factors present in the fetal bovine serum on the results, the normal culture medium was replaced by Opti-MemTM (#31985062 Invitrogen) before 12 h before all assays, as described [To study the effects of oxidative stress, alteration of endoplasmic reticulum, inhibition of proteasome activity and osmotic stress, cells were treated with 10-µM Hescribed . Cell viTM, Invitrogen, Waltham, MA, USA) for 12 h were treated as indicated above. After incubation, cells were washed with PBS and then fixed with 3.7% paraformaldehyde for 10 min at room temperature. Cells were rinsed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 30 min and subsequently blocked with 5% normal goat serum at room temperature for 1 h. Cells were incubated with (1) the mouse anti-p-TDP-43 (pS409/410) monoclonal antibody (TIP-PTD-M01) ; (3) the rabbit anti-p-ERK 1/2 polyclonal antibody (4370) ; the rabbit anti-REST polyclonal antibody (ab21635) ; (4) the rabbit anti p-Jun polyclonal antibody ; and (5) the anti-ATP5A mouse monoclonal antibody (ab14748)  at 4 °C overnight. After 3 washes with 0.1% Triton X-100-PBS at RT for 10 min, cells were incubated with Alexa Fluor-488 goat anti-rabbit IgG or Alexa-Fluor-594 goat anti-mouse IgG -conjugated secondary antibody. Nuclei were stained with 4′,6′-diamino-2-phenylindole (DAPI) . The coverslips were mounted in Fluoromount-G (Southern Biotech) and images were taken with an Olympus FV10i laser scanning confocal microscope and an Olympus FV1000 confocal microscopy, 60× magnification. For the evaluation of nuclear and cytosolic protein intensity, we used a dedicated pipeline created on the open source CellProfiler software [Cells, seeded out on coverslips and incubated in serum and phenol red free medium . Gels were blotted onto PVDF membranes by transfer at a constant 100 volts, 1 h at RT. The membranes were then blocked by nonfat dry milk solution (5%) in 1 × TBS  and incubated in the desired primary antibody overnight.Protein homogenates were prepared in the presence of inhibitors of phosphatases (Sigma-Aldrich) and proteases . Protein concentration of the samples was measured using the Quick Start™ Bradford 1× Dye Reagent (Bio-Rad #5000205). Proteins were detected by immunoblotting using horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (Santa Cruz Biotechnology). Protein samples were run on SurePAGEMembranes were then washed in 1 × TBST with Tween 20 (0.05%) three times, 5 min each before incubating with the secondary antibody for 1 h at room temperature. Following the secondary antibody incubation, membranes were washed in 1 × TBST with Tween 20 (0.05%) three times, 5 min each and one time, 5 min, with TBS 1 ×. Blots were imaged in the Chemidoc MP Imaging System following incubation in the Immobilon ECL Ultra Western HRP Substrate .TM Precast gels . Cruz Marker™ molecular weight standards  were loaded in the gels. Gels were blotted onto low fluorescence PVDF membranes (Immobilon®-FL PVDF: sc-516541). Nonspecific binding was blocked in incubating membranes with UltraCruz® Blocking Reagent (sc-516214) for 1 h at room temperature, with shaking.For Jun/ p-c-Jun Westerns, protein samples were run on SurePAGE® conjugated primary antibodies (Anti-p-c-Jun Antibody (KM-1) Alexa Fluor® 790 and Anti c-Jun Antibody (G-4) Alexa Fluor® 680, from Santa Cruz) diluted 1:1000 in UltraCruz® Blocking Reagent. Cruz Marker™ MW Tag-Alexa Fluor® 680 (sc-516730) and Cruz Marker™ MW Tag-Alexa Fluor® 790 (sc-516731) at 1:1000 were added to obtain molecular weight distribution.The blocked membranes were incubated with the appropriates Alexa FluorMembranes were incubated in this mixture for 2 h at room temperature, in the dark, with shaking. Membranes were then washed three times for 5 min each with TBST and once for 5 min with TBS. Blots when then placed on the top of blotter paper and dried for 5–10 min. The western blot was imaged using the infrared (IR) laser-based instrumentation LI-COR Odyssey .RNA was extracted from cells and brain lysates using TRI Reagent  following the manufacturer’s instructions. RNA concentrations were measured using a NanoDrop ND-1000 (Thermo Fisher Scientific). One microgram of RNA was used for retrotranscription to cDNA employing TaqMan Reverse Transcription Reagent and random hexamers .RT-qPCR experiments were performed using a CFX96 instrument  with SYBR Select Master mix for CFX . Each 20 µL reaction mix contained 4-µL cDNA, 10-µL SYBR Select Master Mix, 0.2 nM of forward primer and 0.2 nM of reverse primer solutions and 4-µL PCR grade water. RT-qPCR run protocol was as follows: 50 °C for 2 min and 95 °C for 2 min, with the 95 °C for 15 s and 60 °C for 1 min steps repeated for 40 cycles, and a melting curve test from 65°C to 95 °C at a 0.1 °C/s measuring rate. Primers employed in these experiments, previously described in reference [Mitochondrial function was estimated employing the Seahorse XF HS Mini Analyzer (Agilent technologies). Briefly, to obtain an effect of the aggregation-prone conditions in the mitochondrial function we employed the Seahorse XFp Real-Time ATP Rate assay kit (Agilent#103591-100), according to the manufacturer’s instructions. 20,000 SHSY-5Y cells were plated in the microplates of the kit and incubated with sorbitol . After this incubation, oxygen consumption and extracellular acidification, were recorded, in basal conditions, and after oligomycin and rotenone/antimycin addition.t-tests, One-way, Two-way, and Three-way ANOVA analyses, with adequate post-hoc analyses, once normality of variables was tested by Kolmogorov–Smirnov test. The 0.05 level was selected as the point of minimal statistical significance in every comparison.All statistics were performed using the GraphPad Prism version9.1.2 for Windows software . Differences between groups were analyzed by the Student’s


Reactive oxygen species (ROS) encompasses a collection of complicated chemical entities characterized by individually specific biological reactivities and physicochemical properties. ROS detection is attracting tremendous attention. The reaction-based nanomaterials for ROS “turn-on” sensing represent novel and efficient tools for ROS detection. These nanomaterials have the advantages of high sensitivity, real-time sensing ability, and almost infinite contrast against background. This review focuses on appraising nanotechnologies with the ROS “turn-on” detection mechanism coupled with the ability for broad biological applications. In this review, we highlighted the weaknesses and advantages in prior sensor studies and raised some guidelines for the development of future nanoprobes. It mainly includes singlet oxygen (e (H2O2) . ROS hase (H2O2) , which ie (H2O2) . Therefoin vivo and live cell imaging  and good in vivo utility of this nanoparticle may support its eventual application in clinics (limit of detection (LOD) = 0.5 μM). Higher selectivity has been achieved for the nanoparticle for the detection of H2O2 over other ROS and biologically relevant species (2O2 (LOD = 3.33 μM). Moreover, they fabricated a H2O2-triggered drug release system for heart failure therapy. This system exhibits the potential for different variants of heart failure models to target theranostic treatment  MOF for “turn-on” sensing of H2O2 in live cells (LOD = 0.015 μM). However, it also has moderate response to some other ROS and biologically relevant species, indicating that the selectivity of this MOF material toward H2O2 is a major defect  mechanism was fabricated by Zhang’s group. In this nanoprobe, diphenylphosphine moiety is covalently attached to the surface of the carbon dot; they serve as the PET donor and acceptor, respectively. Subsequently, H2O2 can selectively oxidize the diphenylphosphine to produce the target oxide and prevent the PET mechanism; then the fluorescence will “turn on” (2O2 with a LOD of 84 nM (2O2 detection (LOD = 5 nM). Nanointegration of near-infrared carbon nanodots and peroxalate (P-CDs) with amphiphilic triblock copolymer as bridge can serve as “turn-on” sensors for the detection and imaging of H2O2 , in which, UCNPs and DNA-AgNPs serve as donors and quenchers, respectively. This design results in luminescence quenching of UCNPs using DNA-AgNPs by luminescence resonance energy transfer (LRET). Upon H2O2 introduction, AgNPs can be converted to Ag+, leading to the inhibition of the LRET process and inducing the recovery of upconversion luminescence -nanosheet-modified UCNPs for rapid detection of H2O2 (LOD = 0.9 μM). The MnO2 nanosheets on the surface of UCNPs serve as the quencher. Fluorescence of UCNPs will be recovered after the addition of H2O2, which can reduce MnO2 to Mn2+ and destroy the structure of the MnO2 quencher (2 probe (LOD = 0.87 μM) and a three-in-one stimulus-responsive nanoplatform , respectively, for H2O2 sensing with relatively improved selectivity or sensitivity , is the most deleterious and reactive species of ROS. The general reactivity of the main ROS in biological systems decreases in the order of •OH > 1O2 > H2O2 > O2•−   of the QDs. Consequently, the produced electron–hole pairs could emit strong luminescence through electron–hole recombination. This nanotechnology is demonstrated to have an application in detecting the endogenous release of •OH in living cells  acting as a functional group on the cytosensor is oxidized to ox TMB, a colored product, and can be monitored through colorimetric analysis. Coupled with a rational design, the nanotechnology has been constructed as a convenient method for the sensitive detection of MCF-7 cells (LOD = 20 cells ml−1) ( > O2•− . •OH canlifetime . Therefo 0.3 μM) . In thisng cells . Alternals ml−1) .1O2 has raised vital interest recently as a result of its significance in both chemical and biological systems. 1O2 is the lowest excited electronic state of molecular oxygen but is recognized to be highly reactive. Studies have demonstrated that 1O2 is highly toxic and destroys key biological molecules including proteins, DNA, and unsaturated lipids. Depending on a triphenylphosphonium derivative, the self-assembled nanomaterial has been fabricated for 1O2 “turn-on” detection (LOD = 33–56 μM)  through photonic luminescence enhancements has been developed  ion so as to induce these nanoprobes to become luminescent  in this system functions as a crucial moiety for the fluorescence “turn-on” sensing process. The presence of 1O2 in this system can result in change in the SOSG fluorescence quantum yield, which permits a stronger energy transfer from the SOSG probe to a proximal silver nanoparticle island film located in the near-electric field of the probe. This induces an increase in the target electric current flow, allowing for the sensing of the 1O2 (3–56 μM) . Howeverand ClO− . A strateveloped . The pSiinescent . Alterna the 1O2 .2•− is a by-product of ATP generation processes of the human body microenvironment, which plays a significant role in regulating biochemistry and organic pathology. Furthermore, exposure to excess O2•− would oxidize organisms, biological membranes, and tissues and cause diseases such as hepatitis, cancer, and diabetes (2•−. Silver nanoparticle (Ag NP)-coated carbon quantum dot (CQD) core–shell-structured nanocomposites (CQD@Ag NCs) have been developed for fluorescent sensing of intracellular O2•− (LOD = 0.3 μM) . The addition of O2•− can convert Fe3+ to Fe2+ and recover the fluorescence of carbon dots  nanoparticles with 4-hydroxy-tetramethylpiperidine at the protein tyrosine residue. The nanoparticles function as a metal-free magnetic resonance imaging (MRI) tool for O2•− monitoring . Enhancement of the fluorescence response of the MOF upon stepwise addition of O2•− has been recorded. The mechanism of the fluorescence “turn-on” procedure is recognized as follows: the structural collapse of the MOF in the presence of O2•− can result in the release of the linker (2-((pyridin-4-ylmethyl)amino)terephthalic acid) with the enhancement of the fluorescence intensity of the system  . In CQD@ecovered . Yue andbon dots  , which can elicit potent characteristic surface-enhanced Raman scattering (SERS) signals at 1,142, 1,386, and 1,432 cm−1 and directly enable the detection of ROS through a hemin-catalyzed Fenton reaction (LOD = 26 pM)  has been realized by the Au-PATP-Hemin nanoprobe. In two typical ROS-elevated mice models of allergic dermatitis and tumors, the Au-PATP-Hemin nanoprobe performed well in monitoring inflammation progression and tumor development in a sensitive and quantitative manner.In some conditions, the evaluation of cellular or system total ROS provides helpful information on cell proliferation, metabolism, and tumor detection. Distinct from the design of nanotechnologies for selective sensing of specific species of ROS, these nanomaterials can detect the combined species of ROS by one platform. Several nanomaterials, including PEGylated bilirubin-coated superparamagnetic iron oxide nanoparticles (PEG-BR@SPIONs) , UCNPs-Mnoflakes , multifunoflakes , para-amnoflakes , cyclotrnoflakes , ROS-resnoflakes , and thenoflakes , are desnoflakes . The “tu= 26 pM) . SimultaIn this short review, nanomaterials with the ability for ROS “turn-on” detection have been deciphered from the aspects of both nanotechnology and chemical reaction mechanisms. In general, “turn-on” nanotechnologies are powerful tools with a low detection limit, real-time sensing ability, and almost infinite contrast against background. Future studies for the design of ROS “turn-on” detection nanomaterials should make efforts to improve selectivity, detection limit, and biocompatibility. Another consideration is the accessibility of these nanomaterials. Reagents for the nanomaterial fabrication are commercially available or can be prepared in simple synthetic steps from commercially available building blocks, which will be greatly in vogue. Continuous efforts are poised to develop more powerful nanotechnologies in this promising field to shed light on critical information of ROS in biological systems.