Rolling enables the directional alignment of the reinforcements in graphene/Cu composites while achieving uniform graphene dispersion and matrix grain refinement. This is expected to achieve a breakthrough in composite performance. In this paper, the process parameters of rolling are investigated, and the defects, thickness variations of graphene and property changes of the composite under different parameters are analyzed. High-temperature rolling is beneficial to avoid the damage of graphene during rolling, and the prepared composites have higher electrical conductivity. The properties of graphene were investigated. Low-temperature rolling is more favorable to the thinning and dispersion of graphene; meanwhile, the relative density of the composites is higher in the low-temperature rolling process. With the increase of rolling deformation, the graphene defects slightly increased and the number of layers decreased. In this paper, the defect states of graphene and the electrical conductivity with different rolling parameters is comprehensively investigated to provide a reference for the rolling process of graphene/copper composites with different demands. Cu metals, alloys, and composites are widely used in industrial production. Cu has excellent electrical and thermal conductivity, as well as good processability and corrosion resistance, second only to Ag ,2. They However, the current development of graphene/Cu composite faces the following problems. Since graphene is a nano reinforcement, it is very easy to agglomerate after being prepared into a composite, which causes the two-dimensional strengthening characteristics of the graphene to fail to fully play, which reduces the performance ,17. In a4C3 was observed at the interface.The uniform dispersion of graphene in the metal matrix can be achieved through the deformation of the composites. Shao et al. ,21 foundWang et al.  reportedThe parameters of the deformation have an important influence on the dispersion and deformation of graphene in the matrix, but the rules are still unclear. However, the research on the specific effects of deformation temperature and deformation on the structure and properties of the composite is not comprehensive enough.In this work, the microstructural evolution behavior of graphene under different rolling parameters was studied. Due to the stable chemical properties of Cu and no interfacial reaction with graphene, it was convenient to observe the deformation behavior of graphene. Therefore, Cu was selected as the matrix for preparing composites. In order to clarify the deformation behavior of graphene in the matrix, this paper comprehensively studied the effects of deformation temperature and rolling deformation on graphene/Cu composites. The organization evolution of graphene was studied through Raman spectroscopy, and the mechanical and electrical properties were tested, which provide a reference for subsequent deformation of composite materials.−1, the G peak at 1570 cm−1, and the 2D peak at 2700 cm−1. The D peak reflects the asymmetric lattice vibrations, and the G peak reflects the symmetrical lattice vibrations [D/IG) is often used to reflect the change of graphene defects. 2D peaks appearing at approximately 2700 cm−1 is related to the number of graphene layers. And the peak position, peak shape, and intensity of 2D peaks can be used to judge the number of graphene layers [The graphene used in this experiment was supplied by the Sixth Element Changzhou Materials Technology Co. Ltd. China. The pure Cu powder used in this study was purchased from Beijing Xingrongyuan Co., Ltd., Beijing, China, with a purity of 99.9% and an initial particle size of about 3 μm. The characterization of graphene and Cu powder is shown in brations ,30. The e layers ,32,33. Te layers ,35.In this study, a planetary ball mill  was used to disperse the graphene, and the ball-milling speed of 120 r/min for 2 h was used. The content of graphene is 0.6 wt.%. The mixed graphene-Cu mixed powder is sintered through SPS. The powder mixture of GNPs and Cu was put into a 40 mm diameter mold and pressed into preforms at a pressure of 40 MPa. The pressure was maintained for 5 min. The mold with the preforms was then placed into the SPS equipment and heated to 1020 °C at 5 °C/min and kept at high temperature for 10 min. The current-to-time ratio (ton:toff) during sintering was 2:1. After the sintering was completed, the sample was cooled to room temperature in the mold with the furnace. The prepared 0.6 wt.% GNPs/Cu composite was subjected to subsequent rolling research.In this work, a double-roll mill  is used to optimize the deformation of the prepared composite. Under the action of the stresses generated during the rolling process, the Cu matrix and graphene undergo shear deformation. The total thickness reduction (%) is counted as the amount of down-compression deformation. The 0.6 wt.% graphene/Cu composite material was cut into 55 mm × 10 mm × 4 mm rectangular parallelepiped and rolled from the original thickness of 4 mm. The prepared composite is rolled at 20 °C, 300 °C, 500 °C, 700 °C. The deformation amount of each rolling is 10%, and the samples with large deformation amounts are prepared by the method of multiple rolling. The total reduction of multiple rolling is 20–80%. The hardness and electrical conductivity of the composites were subsequently investigated at different rolling temperatures and deformation amounts.mρ and Hρ2O are the densities of the composite and the sample, respectively, and m1 and m2 are the masses of the composite in air and water, respectively. The relative density is the ratio of the experimental density to the theoretical density of the sample.Morphologies of the GNPs/Cu powders and the composites were observed by FEI Quanta 200FEG . Raman analysis was performed on a JY-HR800 laser Raman spectrometer  using a 532 nm solid-state laser as an excitation source. The image analysis microhardness of HV-1000IS  was used to test the Vickers hardness of the graphene/Cu composites. In the hardness test, the experimental parameter load is 200 gf, the pressure time is 15 s. An FD 102 digital eddy current conductivity meter  was used for the conductivity test. Each sample is randomly measured 5 times at a certain distance, and the average value is obtained. The density of the material was measured by Archimedes’ drainage method. The mass of the sample in air and the mass in water are tested separately and the density of the material is calculated according to the formula:D/IG of graphene in the composite before rolling is 0.20, and the I2D/IG is 0.43. The state of the graphene in the sintered composite is close to that of the raw graphene because the graphene is less damaged during the preparation processing. The defect content of graphene only increased slightly, and the number of layers did not change significantly.The GNPs/Cu composite material was rolled and deformed at rolling temperatures of 20 °C, 300 °C, 500 °C, and 700 °C, and the total deformation was 80%. Raman characterization of the composite material, the results are shown in After rolling at different temperatures, the graphene changes significantly. Comparing the positions of the characteristic peaks of the Raman spectrum curve at different rolling temperatures, it can be seen that the positions of the D peak and the G peak are basically the same. The peak shape of the 2D peak changes slightly due to the change in the number of graphene layers. The ratio of the intensity of change of the graphene D peak, the G peak, and the 2D peak is quantified, as shown in D/IG of graphene increased significantly compared to the unrolled graphene/Cu composites, indicating a significant increase in the defect content in graphene. Graphene suffers huge damage during the rolling process. I2D/IG also increased slightly, indicating that the number of graphene layers decreased during the rolling process.It can be seen that when the composites were rolled, the ID/IG and I2D/IG drops slightly. It shows that after the rolling temperature is increased, the rolling damage to the graphene is less, and at the same time, the peeling effect of the graphene is reduced. This is because when the rolling temperature is lower, the composite is subjected to greater stress during cold rolling. Graphene shears, bends, and twists as the substrate deforms, creating more edge and hole defects. At the same time, the large shear stress also causes the multi-layer graphene layer to slip, which reduces the number of graphene layers. To further characterize the changes in the number of graphene layers at different rolling temperatures, the deviation of the graphene G peak and 2D peak is observed, as shown in the As the rolling temperature rises, I−1. Based on the results reported in other papers [Based on the research results of Malard et al. , as the r papers ,33, it cAt 700 °C, under the condition of 10% deformation in a single press, the composite is subjected to multiple rolling treatments. The total rolling deformation is 4.0 mm → 10% (3.6 mm) → 20% (3.2 mm) → 30% (2.8 mm) → 60% (2.4 mm) → 50% (2.0 mm) → 60 (1.6 mm) → 70% (1.2 mm) → 80% (0.8 mm). The Raman characterization of rolled composites with different deformations is performed, and the results are shown in D/IG ratio of different total deformations has shown an upward trend from 20% to 80%, indicating that the defects of graphene are gradually increasing during the rolling process. At the beginning of rolling, the value of ID/IG has a large standard deviation value, indicating that the structure is not uniform in the initial stage of deformation. When the total deformation is 40%, the standard deviation values of the ID/IG ratio begin to decrease, indicating that the change of graphene begins to stabilize. As the rolling deformation continues, when the total deformation reaches 70%, the ID/IG ratio once again greatly increases. The graphene defects increase significantly, and the variance of the ID/IG ratio continues to increase. It showed that in the later stage of rolling deformation, under the action of large deformation, the Cu matrix began to crack, and stress concentration was formed at the crack during the deformation, which caused more serious damage to the graphene near the crack.The intensity ratio of the D peak and the G peak is quantitatively calculated to determine the state of graphene defects, as shown in The shift of graphene G and 2D peaks in rolled composites with different deformations was observed, as shown in To further study the change law of the composite microstructure and explain the abnormal rise of graphene defects after the deformation exceeds 70% during cold rolling, the relative density and microstructure of the composite are characterized.The relative density of the composite rolled at different temperatures and different deformation amounts are shown in SEM observation is performed on the 0.6 wt.% graphene/Cu composite rolled at 20 °C, and the reason for the decrease in density is analyzed, as shown in The hardness test of the composite after rolling treatment at different temperatures and deformations is shown in Combining the density and the Raman characterization, the reasons for the performance changes can be analyzed. When the amount of rolling deformation is low, as the amount of deformation increases, the agglomerated multilayer graphene is thinned, and the matrix defects are reduced, increasing the hardness of the composite. When the deformation exceeds 70%, the Cu matrix will crack and the graphene will be severely broken, which will degrade the properties of composites. The performance after rolling shows an overall downward trend as the temperature increases. Combined with the Raman characterization, it can be seen that when the rolling temperature is lower, the matrix has greater stress, so the graphene has a more obvious thinning effect. At the same time, the graphene is more uniformly dispersed, and the strengthening ability is more easily exerted. Therefore, the material has greater hardness when rolled at low-temperature. The change in the properties of the composites was also related to the change in Cu grain size. It was reported that the Cu matrix grains were found to be coarse at higher rolling temperatures and the material properties decreased substantially ,37,38. IAfterward, the electrical conductivity of the composites rolled at different temperatures and deformations is tested, and the results are shown in As the rolling temperature increases, the conductivity of the composite increases. Based on previous studies, it can be seen that the higher the rolling temperature, the less damage the rolling will cause to graphene. Low-damage graphene has more contribution to electrical conductivity, so the electrical conductivity of composites prepared by high-temperature rolling is higher. The conductivity is also related to the grain size of the Cu matrix, the larger the grain size and the less grain boundary content, the higher the conductivity . The graIn addition, rolling can produce an oriented arrangement of graphene in the composite. Guo et al.  investigIn this work, the microstructure and properties under different rolling temperatures and rolling deformation were systematically investigated. When the rolling temperature decreases and the deformation increases, the thinning effect of graphene during rolling is enhanced, and more damage to graphene is also produced. At rolling temperatures below 300 °C, rolling facilitates the uniform dispersion of few-layer graphene in the Cu matrix. When the temperature is higher than 300 °C, the damaging effect of rolling on graphene can be reduced.The hardness and electrical conductivity of the composites increased with the increase of rolling deformation. When the deformation of low-temperature rolling exceeds 70%, its properties are weakened. The hardness of the composites decreases with increasing rolling temperature, while the electrical conductivity increases with increasing rolling temperature. In this paper, the structural and property changes of the composites under different rolling processes are comprehensively studied to provide a reference for the rolling process of graphene/copper composites with different demands.


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.