These results demonstrated that FOXC2-AS1 enhances FOXC2 mRNA stability to promote CRC proliferation, migration, and invasion by activation of Ca2+-FAK signaling, which implicates that FOXC2-AS1 may represent a latent effective therapeutic target for CRC progression.Long noncoding RNAs (lncRNAs) have been confirmed, which are involved in tumorigenesis and metastasis in colorectal cancer (CRC). FOXC2 antisense RNA 1 (FOXC2-AS1) was reported, facilitating the proliferation and progression in several cancers. However, the role of FOXC2-AS1 in CRC cell migration and metastasis is not unclear. In this study, we observed that lncRNA FOXC2-AS1 was upregulated in CRC tissues, and its high expression indicated the poor survival in CRC patients. Meanwhile, FOXC2-AS1 was higher in CRC tissues with metastasis than that of nonmetastatic tumor tissues. We found that FOXC2-AS1 was predominately expressed in the nucleus of tissues and cells. FOXC2-AS1 knockdown suppressed CRC cell growth, invasion, and metastasis in vitro and in vivo. Moreover, FOXC2-AS1 could positively regulate the neighboring gene FOXC2 and stabilized FOXC2 mRNA by forming a RNA duplex. Meanwhile, ectopic expression of FOXC2 could obviously alleviate the suppressed effects caused by silencing FOXC2-AS1. For the mechanism, FOXC2-AS1 knockdown could reduce intracellular Ca In recent years, remarkable progress has been made in CRC treatment, but over half of the patients with advanced CRC die from recurrence and metastasis, which led to the unfavorable prognosis3. Thus, a better illustration of the molecular mechanisms regarding CRC progression is urgently needed.Colorectal cancer (CRC) is a common digestive tract malignancy, accounting for about 40% new tumors of the digestive tract. According to the survey, there were nearly 1.3 million new CRC cases and more than 0.6 million deaths worldwide every year4. LncRNAs demonstrated widespread expression in animals and plants, which can exhibit function in cis-regulated expression of neighboring genes or in trans-regulation distal genes5. Numerous literature has confirmed that the dysregulation of lncRNAs is involved in the pathophysiology of numerous human disorders, especially in cancer7. lncRNA H19, MALAT1, HOTAIR, etc., were widely reported to be involved in human tumorigenesis and metastasis10. LncRNA LNMAT1 promotes bladder cancer metastasis through enhancing CCL2- dependent macrophage recruitment11; LncRNA MFI2-AS1 facilitates cell migration and invasion via acting as the ceRNA of miR-574 to regulate MYCBP in CRC12; HOXD-AS1 represses CRC growth and metastasis through suppressing HOXD3-activated integrin β3 transcription and MAPK/AKT pathway13. Thus, lncRNAs may play a key role in cancer metastastis; exploring the molecular mechanisms will be significant for cancer therapy.Long noncoding RNAs (lncRNAs) are a kind of RNA transcripts with lengths >200 nt, and limit or lack protein-coding capacity16. Besides, FOXC2-AS1 was reported to promote doxorubicin resistance in osteosarcoma17. Yet, FOXC2-AS1 is not well-characterized in cancers, including CRC. The expression, function, and mechanism of FOXC2-AS1 has not been reported in CRC.FOXC2-AS1 is an antisense RNA transcript of forkhead box protein C2 (FOXC2). Research has shown that FOXC2-AS1 was highly expressed in breast cancer, prostate cancer, and non-small- cell lung cancer (NSCLC); the upregulated expression of FOXC2-AS1 predicted poor survival, and facilitated proliferation and progression of these carcinomas2+ levels and promote FA formation via activation of Ca2+-FAK signaling pathway, ultimately contributing to CRC proliferation, migration, and invasion. Our investigation revealed the oncogenic role of FOXC2-AS1 through increased intracellular Ca2+ levels and promoted FA formation in CRC, and implied FOXC2-AS1 as a latent prognostic marker and therapeutic target for CRC.In this research, we found that FOXC2-AS1 was obviously upregulated in metastasis CRC tissue, and relates to the clinicopathologic features and poor prognosis in CRC patients. FOXC2-AS1 depletion significantly weakened CRC cell growth, invasion, and metastasis in vitro and in vivo. Moreover, FOXC2-AS1 could promote FOXC2 expression via enhancing its mRNA stability. Further investigation demonstrated that FOXC2-AS1 could increase intracellular Can = 38) and low group (n = 28)  Fig. . The assWe investigate the role of FOXC2-AS1 on CRC cells. First, we detected FOXC2-AS1 endogenous expression in CRC cell lines  and normal colonic cell line NCM460 by qRT-PCR. As shown in Fig. 18, and FOXC2 was the nearest gene, having some overlap sequences with FOXC2-AS1. Thus, we investigated whether FOXC2-AS1 regulates FOXC2 expression. First, FOXC2 expression was detected in CRC tissues and cell lines. It is found that FOXC2 was elevated in CRC specimens, which was consistent with the expression of FOXC2-AS1  in FOXC2-AS1-silenced and control cells. The results displayed that FOXC2-AS1 knockdown does not influence the level of nascent FOXC2 pre-mRNA  Fig. . Therefoown Fig. . Remarkaown Fig. , which i25. Immunofluorescence displayed that the number of FA was obviously reduced in FOXC2-AS1- silenced cells, but increasing FOXC2 expression could alleviate the inhibitory effects . Once activated by cellular signals, the concentrations increase nearly 100 times either through release from inter-Ca2+ stores or through extracellular Ca2+ influx, thus generating Ca2+ signals that activate downstream effectors, such as calmodulin, calmodulin-dependent protein kinase II, and calpain (CAPN)31. Disruption of Ca2+ homeostasis facilitates the formation of malignant phenotypes32. Increasing evidence has demonstrated that Ca2+ plays an important role in tumor proliferation, invasion, and metastasis. For example, PLPP4 depletion suppresses lung carcinoma cell proliferation and tumorigenesis via impeding the influx of intracellular Ca2+33. Gong et al. reported that P2RX6 might function via ATP-induced Ca2+ influx to promote renal carcinoma cell migration and invasion34. ABAT suppressed Basl-like breast cancer by downregulation of intracellular Ca2+ concentration and inactivation of Ca2+-NFAT1 axis35. Here, we found that FOXC2-AS1 knockdown decreases the level of Ca2+ via suppression of FOXC2; moreover, elevating the level of Ca2+ could mitigate the oncogenic effect by FOXC2-AS1 silencing. Consistent with previous reports, our data suggested that FOXC2-AS1/FOXC2 promotes CRC progression by elevating Ca2+ levels.Based on the GESA analysis in an expression-profiling data of FOXC2-overexpressing SW480 cells (GSE58980), we found that FOXC2 expression was positively associated with calcium signaling pathway and FA. As we know, Ca36. FA is recognized dominant in this process, which acts through signal-linkage complexes between the extracellular matrix (ECM) and the cytoskeleton37. The FA assembly and disassembly, named FA turnover, determine cell motility, and the speed of FA dynamic cycle is considered to be the critical step in tumor invasion38. FAK is a well-known nonreceptor protein tyrosine kinase involved in FA dynamics25. FAK contains six tyrosine phosphorylation sites ; FAK regulates cell adhesion and migration, mainly contributing to the autophosphorylation at Tyr397 site, which generates a high-affinity binding site for the SH2 domain of Src39. The interaction of FAK with Src subsequently phosphorylates Tyr407, 576, and 577, which lead to maximizing FAK activity39. Paxillin functions as a scaffolding protein that could recruit regulatory and structural proteins to regulate the dynamics of cell adhesion, modulating the turnover of FA complex and cytoskeletal remodeling, thus promoting cell migration and invasion40. When phosphorylating at Tyr118 by the FAK/Src complex, paxillin was activated; then multiple signaling pathways can coordinate to regulate cytoskeleton formation and promote cell migration41. In breast cancer, lung cancer, neuroblastoma, ovarian cancer, and hepatocellular carcinoma, studies have confirmed that FAK–Src–Paxillin cascade pathway modulates tumor cell proliferation, migration, and metastasis45. Our results proved that FOXC2 overexpression could reverse FOXC2-AS1 silencing-caused FA inhibition and FAK–Src–Paxillin cascade pathway inactivation. Notably, numerous evidence has demonstrated that Ca2+ signaling plays an important role in FA dynamics and FAK phosphorylation48. As shown in our results, elevating the level of Ca2+ mitigated FOXC2-AS1 silencing-induced FA inhibition and FAK–Src–Paxillin cascade pathway inactivation.Cell migration and invasion are a prerequisite for tumor metastasis. The migratory capacity facilitates cancer cells running away from the primary focus and disseminating through nearby circulations. The coordination of cell–substrate adhesion and detachment is necessary for cell migration2+/FAK signaling, and the new signal axis—Ca2+/FAK regulates cell FA and migration. These findings may provide a potentially effective therapeutic target for CRC progression.In summary, our research demonstrated that lncRNA FOXC2-AS1 promotes CRC progression via activation of CaSixty-eight cases of CRC specimens and 35 cases of adjacent tissues were diagnosed and collected in the second Xiangya hospital, Central South University between 2012 and 2016. No patient received chemotherapy and radiotherapy before surgery. The fresh specimens were snap-frozen in liquid nitrogen and then stored at −80 °C. All the patients were followed up at regular intervals after surgery. This research was approved by the Ethics Committee of The Second Xiangya Hospital of Central South University, and informed consent has been obtained by all the enrolled patients. The pathological information was obtained from patients’ medical records.CRC cell lines HT-29, HCT116, SW620, LoVo, and normal colonic epithelial cell line NCM460 were obtained from American Type Culture Collection (ATCC) and cultured following the manufacturer’s instructions.FOXC2-AS1 siRNAs that directly target the sequence were synthesized by Invitrogen ; scramble oligonucleotides were used as a negative control. The sequences of si-FOXC2-AS1 were as follows: si-FOXC2-AS1-1, 5′-GCGUGCCACUUAUUUCCAATA-3′; si-FOXC2-AS1-2, 5′-GCUGCGUAUUCGAUUCUCAGC-3′. FOXC2 cDNA ORF-overexpressed plasmid was purchased from Sino Biological . Plasmids and siRNAs were transfected into cells with Lipofectamine 2000  following the manufacturers’ instructions. Cells were collected 48 h after transfection.TM Kit  according to the product’s protocol. qRT-PCR assay was conducted using SYBR Green I  and performed on the LightCycler480 system . β-actin was applied as an internal control for normalizing. Each reaction was conducted in triplicate. The primer sequences were as follows: FOXC2-AS1 forward: 5′-CCTTCCTGGCTGTTCATCGG-3′, FOXC2-AS1 reverse: 5′-TGGAAATAAGTGGCACGCCC-3′; FOXC2 forward: 5′-CCTACCTGAGCGAGCAGAAT-3′, FOXC2 reverse: 5′-ACCTTGACGAAGCACTCGTT-3′; PDCD4-AS1 forward: 5′-CAGTCTAATGGGCAGAAGGGC-3′, PDCD4-AS1 reverse: 5′- AGGGGCACTGATCACATTCT-3′; β-actin forward: 5′-TTCCTTCCTGGGCATGGAGTC-3′, β-actin reverse: 5′-TCTTCATTGTGCTGGGTGCC-3′.Total RNAs from tissues and cell lines were isolated with Trizol reagent . RNA reverse transcription was performed using GoScript3 cells/well and cultured for 24 h. Next, in each well, we added 20 µl of MTT and incubated at 37 °C for 4 h. After removing the supernatant, each well was replenished with 150 µl of DMSO and incubated at 37 °C for 10 min. Finally, the absorbance was examined at 570 nm with a microplate reader . For the clone-formation experiment, 1 × 103 cells were seeded into a 35-mm dish and cultured for 2 weeks. Then cell colonies were fixed in 4% paraformaldehyde (PFA) and stained with Giemsa dye.For cell-proliferation examination, cells were plated into 96-well plates at the density of 5 × 10Cell migration was detected by wound-healing assay. Simply, cells were seeded in 12-well plates and grown to 100% confluence. Cell wounds were scratched with a 20-μl sterile pipette tip. Afterward, cells were washed with PBS 3 times and cultured with serum-free medium for 24 h. Wound closure was observed and measured the distance between the opposite edges of the wound.5) in serum-free medium were transferred into the upper chamber coated with Matrigel, while the lower chambers were loaded with medium containing 20% FBS. After 48 h, the noninvading cells were wiped using cotton swabs. The invasive cells were fixed by 4% formaldehyde and stained in 0.1% crystal violet for 15 min. Finally, the images were captured under the microscope.Invasion assay was assessed with the BioCoat Matrigel Invasion chamber . Cells  membrane . After blocking by 5% nonfat dry milk, the membrane was incubated with primary antibodies against FOXC2 , p-FAK (Try397) , p-FAK (Try407) , p-FAK (Try576/577) , FAK , p-Src (Try416) , Src , p-Paxillin (Try118) , and Paxillin , overnight at 4 °C. After washing with PBS, the membrane was incubated with HRP-conjugated secondary antibody; the immune signals were examined using enhanced chemiluminescence reagent . GAPDH was used as the loading control.The nuclear and cytosolic fractions of LoVo and SW620 cells were isolated with PARIS Kit  following the manufacturer’s protocol.The probe of FOXC2-AS1-1 was designed and synthesized by BersinBio , and its sequences were 5′-GAGAAUCGAAUACGCAGCCGAUGAACAGCCAG-3′ conjugated with CY3. The slides of cells were fixed by 4% PFA for 20 min, and incubated with protein K for 30 min at 37 °C. After washing with PBS and denaturing at 73 °C for 3 min, the slides were incubated with the hybridization reaction solution that contained 5 ng/μl probes. The hybridization was carried out in a moist chamber at 42 °C overnight. Subsequently, the slides were rinsed with 25% deionized formamide/2 × saline sodium citrate (SSC) at 50 °C and a descending series of SSC at 50 °C. Finally, the slides were stained with DAPI. The signal was observed and photographed under fluorescent microscopy .Fresh CRC samples were immediately fixed with 4% paraformaldehyde (PFA) for 48 h and dehydrated by graded ethanol. After vitrification in dimethylbenzene and embedding in paraffin, the tissues were dewaxed and hydrated. The paraffin-embedded tissues were subjected to FISH referrred to the cell slides.P < 0.05 and false-discovery rate (FDR) < 0.25 were the criterion for identifying statistically enriched genes.The GSEA was launched to analyze gene sets correlated with FOXC2 in CRC. Gene expression data of CRC were downloaded from TCGA database. FOXC2 expression was set into high and low categories based on the median expression value. The FOXC2-correlated gene sets and pathways were explored in the c2.cp.kegg.v7.0.symbols.gmt data set by GSEA v3 soft. 2+ concentration was examined with Fluo-3 AM . Cells were seeded in 12-well plates and incubated with 2 μM Fluo-3 AM diluted in Krebs–Ringer buffer  for 30 min. After removing the culture and washing with Krebs–Ringer buffer, the cells were collected and rinsed by PBS without Ca2+. Finally, the cells were measured by flow cytometry .Intracellular Ca3) were seeded in a 12-well plate paved with sterile slips. After 24 h, the cells were fixed, permeabilized, blocked, and then incubated with anti-paxillin antibody  at a dilution of 1:150 overnight at 4 °C. The next day, the slips were incubated with fluorescently labeled secondary antibody and phalloidin  for 2 h at room temperature. Subsequently, DAPI was used to stain nuclei. Finally, fluorescence was observed and imaged under the confocal laser-scanning microscope .Cells  according to the product’s instructions. Next, qRT-PCR was conducted to detect the level of FOXC2 nascent RNA as mentioned above.Cells were treated with the transcriptional inhibitor actinomycin D  for 0 h, 1 h, 2 h, 4 h, 6 h, and 8 h, respectively. Cells were harvested at different time points, and RNA was isolated. Then, qRT-PCR was used to detect the level of FOXC2 mRNA as mentioned above.Cells were lysed in lysis buffer. Then the lysate was filtrated with a needle and stood on ice for 10 min. Next, the final solution was added with DNase I (Thermo Fisher) 12.5 U/ml and 125 mM NaCl, and divided into two parts. One part was treated with RNase A , while the other was treated with RNase Inhibitor . After incubation at 37 °C for 30 min, the solutions were used for RNA isolation. qRT-PCR was used to detect the level of FOXC2-AS1 and FOXC2 as mentioned above.FOXC2-AS1 and its antisense RNA were in vitro transcribed from the recombinant plasmids pCMV3-FOXC2-AS1 to produce biotin-labeled RNA with the Biotin RNA Labeling Mix . Cell extracts were incubated with RNA probes, followed by streptavidin-mediated RNA pulldown. After RNA isolation, qRT-PCR was applied to investigate the interaction between FOXC2-AS1 and FOXC2.7 LoVo cells transfected with Lv-sh-FOXC2-AS1 , Lv-sh-FOXC2-AS1 + FOXC2, or Lv-NC were injected subcutaneously into the mice right flank. Tumor growth was observed and recorded every 5 days. After 25 days, animals were killed after they were anesthetized and stripped the tumors. Tumor volume was calculated by the formula: (length × width2)/2.The animal experiments were conducted according to the protocols authorized by the Animal Care Committee of the second Xiangya Hospital, Central South University, and were raised with in vivo tumorigenicity. BALB/c nude mice  were randomly divided into three groups (n = 5 for each group). In total, 1 × 106/0.2 ml PBS) were intrasplenically injected into each mice (n = 6 for each group). All mice were euthanized 8 weeks later, and the liver was surgically excised. The liver tissues were fixed in formalin and embedded in paraffin for hematoxylin and eosin (HE) examination. Metastatic nodules were analyzed under microscopy.For in vivo metastasis assays, Lv-sh-FOXC2-AS1, Lv-sh-FOXC2-AS1 + FOXC2, or control Lv-sh-NC lentivirus-transfected LoVo cells . The statistical analysis was conducted by SPSS 18.0 software . Student’s t test was applied to analyze the differential expression between the two groups. A chi-square test was applied to assess the relationship between FOXC2-AS1 expression and clinicopathological characteristics of CRC. Survival analysis was estimated by the Kaplan–Meier method and evaluated using the log-rank test. All the data used and analyzed in this study are included in this article.Figure S1Figure S2Supplementary figure legend


We propose a new method for regulating valley pseudomagnetoresistance in ballistic graphene-based valley field-effect transistors by taking into account the Y-shaped Kekulé lattice distortion and electric barrier. The device involves valley injection and valley detection by ferromagnetic-strain source and drain. The valley manipulation in the channel is achieved via the Y-shaped Kekulé lattice distortion and electric barrier. The central mechanism of these devices lies on Y-shaped Kekulé lattice distortion in graphene can induce a valley precession, thus controlling the valley orientation of channel electrons and hence the current collected at the drain. We found that the tuning external bias voltage makes the valley pseudomagnetoresistance oscillate between positive and negative values and colossal tunneling valley pseudomagnetoresistance of over 30,000% can be achieved. Our results suggest that the synergy of valleytronics and digital logics may provide new paradigms for valleytronic-based information processing and reversible computing. Graphene, being a two-dimensional sheet of carbon atoms, which have excellent carrier mobility and offers the thinnest possible channel for utilizing to design of metal-oxide-semiconductor field-effect transistors . SemenovOn the other hand, Dirac electrons in graphene possess extra valley degree of freedom besides the conventional charge and spin counterparts. Owing to the large momentum difference between the two valleys and the suppression of the intervalley scattering in clean graphene samples –7, the vδt to represent the energy modification to the electron’s hopping corresponding to these three bonds. We assume that the ferromagnetic graphene is made of the same FM metal stripe. The two magnetizations of the source and drain are directed along the current direction (the x axis), which can be in either the parallel (P) or the antiparallel (AP) alignment, with the help of an external in-plane magnetic field. In the Landau gauge, the magnetic vector potential arising from the fringe field has the form [Ay(x)=Ay[Θ(−x)±Θ(x−L)], where the plus(minus) sign corresponds to the P(AP) configuration of magnetizations, Θ(x) is the Heaviside step function. On the other hand, we assume that the same strain are applied on source and drain of the VFETs, which can be induced by a tension on the substrate of the graphene , where AS is the amplitude. In addition, a electic barrier are also applied in the Kek-Y lattice region, which can be tuned by external bias voltage.In this work, we propose a new type of valley field-effect transistors (VFETs) for graphene-based electron. The device design assumes a ferromagnetic-strain (FM-S) source/drain for valley polarized injection/detection, which resembles conventional spin transistor see Fig. a. Valleythe form , 17 \docgraphene . The elae y axis . For defThe low-energy excitation quasiparticles propagation in the VFETs with Kek-Y graphene superlattices can be described by thes following single particle Hamiltonian –121\docσ and τ are the Pauli matrices for the sublattice and the valley, respectively. P= is the momentum of massless Dirac electrons, τz=±1 for K and vF=106m/s is the velocity of Dirac electrons in the pristine graphene, and vτ≃vFδt/3t is the velocity modification term from the bond contraction effect in the Kek-Y lattice . In the graphene channel, the valley degenerate is also lift, but there is an important difference. In contrast to the lead case, where the phases of K and GP and GAP represent the conductance in P and AP configurations, respectively, and Before proceeding with the calculations, we discuss the band structure with lectrons . This in<E<AM+AS , 23. Simmentclasspt{minimaey space . And theey space , which iL=207nm, and restrict the Fermi energy 20 meV<E<140meV, assumed it satisfying |AM−AS|<E<AM+AS. Figure vt with Fermi energy E=80meV and rectangle potential barrier U=−10meV. We can find that GP and GAP have the same oscillation periods but the inverse phases. Therefore, the VPMR oscillates with increase of vt and the negative value VPMR can appear. Those phenomena are similar to the case of the magnetoresistance in ballistic graphene-based quantum tunneling junctions with the spin-orbit interaction [GP and GAP can be explained by the phase difference between the two valley components. When the incident angle ϕ0=0, the phase shift is given by: Δθ determines the orientation of valley polarization before the electron enters the drain, relative to that of the drain state [Δθ=±2nπ,n=1,2,3⋯, the two polarizations are aligned, leading to the conductance GP maximum and VPMR a high positive value . On the other hand, for Δθ=±(2n+1)π,n=0,1,2⋯, they are orthogonal to each other, leading to the conductance GAP minimum and VPMR negative .In the following, we present the numerical results for the FM-S/Kek-Y/FM-S junction in graphene. Throughout the paper, we set channel length Δθ scales are also linear with the Fermi energy and the potential barrier U. Figure E and U value, even when the effective barrier potential U is greater than Fermi energy E. The physical origin for such a phenomenon is related to the Klein tunneling [E and U, some differences can be also found. As E increases, the difference between GP and GAP conductance becomes smaller and smaller, which lead the oscillation amplitude of VPMR to becomes decreased with the increase of Fermi energy. While under the condition Δθ=±nπ is satisfied, the difference between GP and GAP is greater with increasing of U, especially in some location, the GP and GAP conductance presents switching characteristics. The characters are more desirable for the application of VPMR. Remarkably, the observed maximum value of VPMR is over 30,000% at small E. This value greatly exceeds MR of ~ 175% in the ballistic graphene-based quantum tunneling junctions with the spin-orbit interaction [~ 100% in bilayer graphene controlled by external gates [~ 10000% in a merging Dirac cones system [The conductance and VPMR are not only oscillation functions of the hopping-energy modification, they also oscillate with Fermi energy and the effective barrier potential since unneling . Althougeraction  and the al gates , which is system .Fig. 3CIn conclusion, we proposed a type of valley field-effect transistors for graphene-based electron and studied the valley pseudomagnetoresistance through it. We have shown that the oscillation feature of valley pseudomagnetoresistance not only related to the hopping-energy modification and Fermi energy, but also can be tuned largely by the effective barrier potential. The valley pseudomagnetoresistance tuned by external bias voltage benefits the valley field-effect transistor device, and we anticipate that the electric controlled valley quantum devices proposed here can play a role in quantum and quantum-classical hybrid computers.2, WS2, WSe2, etc.) analogs in graphene can also provide an interesting platform for other two-dimensional material-based valley field-effect transistors with Y-shaped Kekulé lattice distortion.Further research could involve the different strain  tunable the valley scattering of electrons and transport in our proposed graphene-based valley field-effect transistors since the stain is useful to control the degree of intervalley scattering in Kekulé patterns . Then, o


In the same sIBM cohort, comorbidity with T2DM was assessed by collecting anthropometric measures and performing an oral glucose tolerance test and insulin determinations. Results were compared to the standard population and other myositis (n = 7 dermatomyositis and n = 7 polymyositis). Mitochondrial contribution into disease was tested by measurement of oxidative/anaerobic and oxidant/antioxidant balances, respiration fluxes, and enzymatic activities in sIBM fibroblasts subjected to different glucose levels. Comorbidity of sIBM with AD was not detected. Clinically, sIBM patients showed signs of misbalanced glucose homeostasis, similar to other myositis. Such misbalance was further confirmed at the molecular level by the metabolic inability of sIBM fibroblasts to adapt to different glucose conditions. Under the standard condition, sIBM fibroblasts showed decreased respiration  and increased anaerobic metabolism . Moreover, when glucose conditions were changed, sIBM fibroblasts presented decreased fold change in mitochondrial enzymatic activities  and increased oxidative stress per mitochondrial activity , underlying scarce metabolic plasticity. These findings do not demonstrate higher prevalence of AD in sIBM patients, but evidences of prediabetogenic conditions were found. Glucose deregulation in myositis suggests the contribution of lifestyle conditions, such as restricted mobility. Additionally, molecular evidences from sIBM fibroblasts confirm that mitochondrial dysfunction may play a role. Monitoring T2DM development and mitochondrial contribution to disease in myositis patients could set a path for novel therapeutic options.Sporadic inclusion body myositis (sIBM) is an inflammatory myopathy associated, among others, with mitochondrial dysfunction. Similar molecular features are found in Alzheimer’s disease (AD) and Type 2 Diabetes Mellitus (T2DM), underlying potential comorbidity. This study aims to evaluate common clinical and molecular hallmarks among sIBM, AD, and T2DM. Comorbidity with AD was assessed in  Inflammatory myopathies are a heterogenic group of diseases characterized by a progressive symmetric weakness and inflammatory infiltrates within the skeletal muscle ,2. RegarThe former, sIBM, is the unique inflammatory myopathy associated with the accumulation of misfolded proteins and ageing . With a In stark contrast with other myositis, sIBM patients show asymmetrical wasting of proximal and distal muscles, in particular quadriceps and finger flexors, leading to difficulty in performing tasks requiring these muscles such as lifting objects or climbing stairs, among others. In addition, neck extensors and pharyngeal involvement may cause dropped head or dysphagia, respectively, affecting 60% of patients over time ,5.At the moment, muscle biopsy remains the definitive diagnostic procedure for sIBM . At the Interestingly, the presence of inflammation, protein depots, and mitochondrial failure seems not to be exclusive for sIBM and, in the last few years, growing evidence has supported the deregulation of these pathways in other chronic and age-related neurodegenerative or metabolic disorders. Among them, the deregulation of mitochondrial function and associated cell consequences has been speculated as the major molecular hallmark underlying a number of these disorders, since mitochondrial dysfunction can trigger, by its own, from bioenergetic failure and oxidative stress production to protein accumulation, inflammation, and cell death , featureIn physiologic conditions, mitochondria are essential for cell survival, playing a central role in calcium homeostasis, bioenergetics, metabolism, and cell respiration. In pathological conditions, they are indeed the main organelle involved in triggering apoptosis and producing reactive oxygen species (ROS).Thus, mitochondrial homeostasis and balance is fundamental for cell function, especially in highly energetic and dependent tissues , where its dysfunction may become evident much earlier.Despite the fact that pathological mitochondrial evidence is frequently observed in muscle biopsies from sIBM patients, mitochondrial abnormalities in this disease have scarcely been assessed at a molecular level ,11. Our Interestingly, mitochondrial abnormalities, energy failure, respiratory chain impairment, and generation of ROS have also been described in brain aging related to neural loss and synaptic alteration . MoreoveAlzheimer’s disease is the commonest cause of dementia, with huge implications for individuals and large sociosanitary and economic associated burdens currently affecting 50 million people. AD is the single biggest cause of dementia, accounting for 50%–75%, and is primarily a condition of later life, roughly doubling in prevalence every 5 years after age 65 . DementiSimilarly to sIBM and AD, the accumulation of proteins and inflammation has also been described as a trigger of type 2 diabetes mellitus (T2DM). In fact, the presence of amyloid deposits within the pancreatic islets is a pathophysiological hallmark of T2DM . MoreoveT2DM is one of the most prevalent and chronic metabolic disorders worldwide. It is characterized by the presence of hyperglycaemia caused by different alterations converging in the development of insulin resistance accompanied by relative to severe insulin deficiency secretion . This diThe link between AD and T2DM is clearly established in literature ,24. HoweIn this study, we aim to evaluate sIBM patients at two levels to assess clinical and molecular comorbidity between sIBM, AD, and T2DM. From a clinical point of view, we will evaluate the neurological and metabolic status in a cohort of sIBM patients to determine the comorbidity between sIBM and AD or T2DM, respectively. In addition, we aim to include a cohort of patients with inflammatory myopathies other than sIBM, such as DM and PM, to evaluate differential or common deregulation with diseases that clinically overlap with sIBM. From a molecular point of view, we aim to evaluate common molecular hallmarks between these degenerative and age-related diseases, focusing our interest on the mitochondrial homeostasis of sIBM fibroblasts that will be subjected to different pathophysiological conditions.The sIBM cases were diagnosed by clinical and pathological tests in the Department of Internal Medicine from the Hospital Clínic of Barcelona . All the patients fulfilled the criteria proposed by the European Neuromuscular Centre for sIBM diagnosis . ExclusiOn inclusion, sIBM patients completed the IBMFRS , a validated disease-specific functional rating scale to assess disease severity. Epidemiological and clinical data  were also recruited.Muscles biopsies were performed for diagnostic purposes indicated for muscle weakness or raised creatine kinase (CK) levels. Surgical muscle biopsies were obtained by trained physicians and the samples were processed routinely in the laboratory as described elsewhere .Fresh samples were frozen in cooled isopentane, sectioned by cryotome at −30 °C, and stained and reacted with different reagents for diagnostic purposes: haematoxylin and eosin, modified Gomori’s trichrome, non-specific esterase, periodic acid-Schiff stain, Oil Red O, acid and alkaline phosphatase, NADH, COX, SDH, and ATPase at pH 4.3, 4.6 and 9.4, as reported elsewhere . In addin = 7) and PM (n = 7). This second cohort was prospectively included in parallel to sIBM patients to evaluate differential or common deregulation with diseases that clinically overlap with sIBM. These patients were also diagnosed at the Department of Internal Medicine from the Hospital Clinic of Barcelona  following the current established clinical and pathological criteria [n = 5), who were prospectively included.According to the standards for diagnosis, results of clinical evaluation for T2DM and AD in sIBM patients were compared to age bracket control data from individuals free of disease or, alternatively, with a cohort of patients with inflammatory myopathies other than sIBM, such as DM , and apolipoprotein E (APOE) genotype.Participants were evaluated by a trained neurologist with a comprehensive neuropsychological battery of tests. Clinical diagnosis was established following the current clinical criteria for mild cognitive impairment (MCI)  and demeThe neuropsychological battery of tests encompassed five cognitive domains. The memory domain included the Free and Cued Selective Reminding Test , the lan3) was acquired at the IDIBAPS’s Imaging core facilities with a 3T whole-body MRI scanner .For each participant, a T1-weighted, high-resolution, MPRAGE structural MRI scan (echo time (TE) 2.98 ms, repetition time (TR) 2300 ms, inversion time 900 ms, flip angle 9°, bandwidth 240 Hz/pixel, matrix 256 × 256, 240 axial slices, isometric voxel size 1⁄4 1.0 mmFor all subjects, global cortical atrophy (GCA), medial temporal lobe atrophy (MTL), and white matter integrity (Fazekas) scores were obtained by a trained radiologist.Subjects underwent a lumbar puncture between 9 a.m. and 12 p.m. In the extraction, 10 mL of CSF was collected. The samples were centrifuged and stored in polypropylene tubes at –80 °C within the first hour after extraction. CSF Aß42 levels, total tau (tau), and phosphorylated tau at threonine-181 (ptau) were measured by enzyme-linked immunosorbent assay kits . The following cut off points were considered: (a) Aß42 ≤ 550 pg/mL, (b) tau ≥ 385 pg/mL, and (c) ptau ≥ 65 pg/mL.Genomic DNA was extracted from the peripheral blood of probands using the QIAamp DNAblood minikit . APOE genotyping was performed by polymerase chain reaction amplification and HhaI restriction enzyme digestion.Procedures for the 2h-OGTT required previous fasting after midnight, to obtain baseline fasting glucose level, and further administration of the oral glucose load (75 g) within a 5 min period. Blood specimens to determine the plasma glucose level were subsequently drawn at 30, 60, 90, and 120 min, timed from the beginning of the glucose load. Patients were established as having impaired fasting glucose  or having impaired glucose tolerance . Criteria for new-onset T2DM was a fasting plasma glucose level greater than 126 mg/dL or 120-min venous plasma glucose level greater than 200 mg/dL on the OGTT.The following data were collected from medical records: circulating baseline levels of insulin (mU/L) and glycated haemoglobin, HbA1c (%). Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated to determine the insulin resistance as: /22.5. Anthropometric and physical examination data concerning weight and height were also recruited to calculate body mass index .2 air incubator until 80% optimal confluence was reached. Fibroblasts were harvested with 2.5% trypsin  and centrifuged at 500 g for 8 min. All functional assays were performed in cells between passages 5 and 10.Fibroblasts were obtained by a skin punch biopsy. Cells were grown in 25 mM glucose Dulbecco’s Modified Eagle’s medium  supplemented with 10% heat-inactivated foetal bovine serum and 1% penicillin-streptomycin at 37 °C in a humidified 5% COTo test fibroblast response to pro-diabetogenic in vitro conditions, cells were cultured under different glucose conditions: high glucose (HG: 25 mM) and low glucose (LG: 5 mM) DMEM media 10 days prior the experiment, mimicking the glucose deregulation observed in T2DM.Expected response to 10 days glucose decrease (from HG to LG) should promote mitochondrial activation to enhance energetic output. To evaluate mitochondria activation in front of glucose reduction, we tested oxidative vs. anaerobic metabolism and oxidant/antioxidant balance, together with mitochondrial respiration and enzymatic activities.® technology  in non-permeabilized fibroblasts, following the manufacturer’s protocols [Mitochondrial respiration is the result of oxygen consumption by the MRC. It was measured in 1 million cells by high-resolution respirometry using Oroboros™ Oxygraph-2Krotocols . Two mea® Blood Analysis System, epoc System Software version 3.32.0 , normalized by cell count and expressed as mmols/L.Lactate measures were performed in 100 µL of fresh supernatant fibroblast culture using the epocTotal Antioxidant Capacity (TAC) was measured in 100 µL of cell supernatant using an OxiSelect™ Total Antioxidant Capacity Assay kit  by spectrophotometry (absorption maximum at 490 nm), normalized by cell count and expressed as µM CRE .Cytochrome c oxidase (COX) function was assessed in 40 µg of total protein from each fibroblast sample according to standardized protocols  and norm™ LPO-586™ kit  through the spectrophotometric measurement of malondialdehyde and 4-hidroxyalkenal, both of which are products of fatty acid peroxide decomposition. Lipid peroxidation was normalized by COX/CS activity as a measure of oxidative stress production per mitochondrial activity.Lipid peroxidation (an indicator of oxidative damage produced by ROS to cellular lipid compounds) was quantified in 150 µg of protein from a fibroblast sample using the OxysResearchp < 0.05.Results were expressed as mean ± standard error of the mean (SEM) or in fold change between conditions (HG vs. LG). Statistical analysis was performed after filtering for outliers to evaluate differential phenotypes depending on group-assignment (patients or controls) by using the non-parametric independent sample Mann-Whitney U test. The signification threshold was set at n = 14 subjects aged between 48 and 90 (mean age 67.70 ± 3.84 years), 9:5 male to female ratio, and all white Caucasian. According to the design of the study, no statistical differences were observed in regards to age, gender, or ethnicity between sIBM patients and the control cohorts. Specifically, PM patients presented a mean age of 69.57 ± 5.39 years and a 1:6 male to female ratio, DM subjects a mean age of 66.67 ± 3.20 years and a 2:5 male to female ratio, and healthy control volunteers aged 57.12 ± 5.38 years and presented a 1:4 male to female ratio, all white Caucasian, according to sIBM demographic characteristics.The sIBM patients included in this study corresponded to At the time of inclusion, the sIBM patient cohort scored 22.00 ± 2.37 out of 40 with the IBMFRS test, indicating moderate-to-advanced sIBM clinical severity.Ten sIBM patients underwent the neurological assessment. Education level ranged between 7 and 18 years and the cognitive reserve questionnaire scored 7 to 18 points. Demographics of the cognitive study are shown in Eight out of the ten subjects that underwent clinical and neuropsychological evaluation were cognitively impaired. From those, 5 were diagnosed with MCI and 3 with dementia (two of them were diagnosed as AD).Three subjects presented symptoms of anxiety and/or depression. MMSE ranged between 6 and 29  and the neuropsychological profile of the study subjects was heterogeneous, although most of them showed an amnestic pattern .Despite reported disturbances in the clinical and neuropsychological assessment, no higher incidence of dementia or AD was detected in sIBM patients when the results were compared to the prevalence of dementia in Europe . See cliEight sIBM subjects underwent the magnetic resonance scan. Medial temporal lobe atrophy (MTL score > 1) was found in 3 subjects  and all individuals presented different levels of white matter deterioration, including two subjects with a Fazekas score ≥ 2  see .However, MTL score > 1 was also found in controls in previous studies . Further42 with normal CSF tau and phosphor-tau levels  showed alterations in 42% (3/7) of the patients, while 14% (1/7) were in the prediabetic range (the plasma glucose level fell between 140 and 200 mg/dL) and the remaining 28% (2/7) felt into the T2DM category (glucose level greater than 200 mg/dL). Moreover, medical records in blood tests from the sIBM population (n = 8) showed increased HbA1c % in 50% (4/8) of these patients (HbA1c greater than 5.7). Finally, when HOMA-IR was assessed in the sIBM cohort (n = 7), 14% (1/7) of the patients were close to insulin resistance values  and PM (n = 7) showing prediabetic state values in 43% (3/7) of cases from both populations. The 2 h venous plasma glucose level in the DM population was greater in 43% (3/7) of patients, which is considered suggestive of a prediabetic state, while the PM cohort showed altered values in 71% (5/7) of patients, 28% (2/7) were considered as in a prediabetic state, and the other 43% (3/7) were consistent with T2DM. Medical data records from blood tests of DM and PM populations also showed altered HbA1c profiles in both cohorts. Particularly, 14% (1/7) of DM and 43% (3/7) of PM had greater values compared to age bracket control data (HbA1c < 5.7%) [Specifically, the basal glucose level was also determined in a cohort of patients with DM ( < 5.7%) . FinallyConsidering the T2DM definition stratified by age from the American College of Endocrinology , the altp = 0.024), increased anaerobic metabolism , and TAC levels , reflecting the mitochondrial dysfunction characteristic of the target tissue of the disease (muscle).Under standard conditions see , sIBM fiCells increase mitochondrial activity in response to the decrease of glucose levels, probably as an attempt to maximize the energetic output in case of glucose restriction. Such activation was observed in control fibroblasts when subjected to glucose changing conditions (HG vs. LG) in terms of respiration. Under high glucose concentration, the mitochondrial respiration rate was low, indicating decreased oxygen consumption and high anaerobic metabolism. When the glucose concentration was diminished, oxygen consumption increased, indicating the activation of the aerobic  metabolism .p = 0.017). Additionally, a lack of mitochondrial activation in sIBM fibroblasts was accompanied by increased fold change proton leak (92.29 ± 73.89 vs. 6.16 ± 83.05) and oxidative stress per mitochondrial activity   and enzymatic activities  .Glucose misbalance manifested at a clinical level in sIBM patients was further confirmed in vitro by the inability of sIBM fibroblasts to adapt to different glucose conditions, thus underlying the clinical and molecular comorbidity between sIBM and T2DM.Despite medical literature postulating potential comorbidity of sIBM with AD and T2DM, scarce data and epidemiological studies (if any) had been conducted to date to evaluate such overlap. Before our investigation, none of the sIBM patients had been diagnosed of T2DM or AD. In the present study, and regardless of the small sample size herein tested, we found null comorbidity between sIBM and AD, but trends towards comorbidity between sIBM and T2DM.Despite sIBM having been described as “Alzheimer in muscle” and both disorders sharing some pathophysiological mechanisms ,15,50,51Interestingly, partial comorbidity between sIBM and T2DM has been demonstrated in this work. Clinically, the metabolic deregulation in sIBM patients is characterized by the initial impairment of glucose metabolism compared with age bracket control data. Despite the fact that only a few of them could be concomitantly diagnosed by sIBM and T2DM, most of the sIBM patients showed clear signs of a prediabetogenic condition that underlie the comorbidity between both diseases.Such coexistence of T2DM in sIBM patients could be due to common etiological links between both disorders. Previous literature describes similar mitochondrial alterations in T2DM and sIBM ,20,52,53At a molecular level, the mitochondrial characterization of fibroblasts from sIBM patients under standard conditions of the present study demonstrate increased anaerobic metabolism and antioxidant capacity at the detriment of mitochondrial oxygen consumption. These results suggest that mitochondrial dysfunction is present in sIBM patients and is not only restricted to the target tissue of the disease (muscle), but may also extend to peripheric alternative tissues, including fibroblasts, as previously demonstrated in the case of blood cells . These fMoreover, we used these fibroblasts to evaluate mitochondrial contribution into sIBM-T2DM comorbidity by exposing sIBM fibroblasts to different glucose conditions. That way, we observed that control fibroblasts showed full mitochondrial plasticity to adapt to different glucose environments, whilst sIBM fibroblasts were mitochondrially unable to adapt its metabolism. Particularly, sIBM fibroblasts were unable to increase their mitochondrial function in terms of respiration and MRC enzymatic activities in front of changes in glucose bioavailability. Mitochondrial dysfunction in sIBM fibroblasts may lead to reduced bioenergetics and consequent cell compromise. In fact, such metabolic stiffness leads sIBM fibroblasts to increased proton leak and oxidative stress, frequently associated with cell damage ,59 and dTo our knowledge, there are no clinical or molecular evidences supporting the preferential affection of muscle tissue in sIBM or the particular targeting of certain muscles (greater in finger flexors and quadriceps), but there is probably a molecular rationale underlying such differential targeting and mitochondria (among others) may stand behind this.Overall, these data demonstrate that mitochondrial dysfunction limit sIBM cell adaption to changing glucose metabolism, being a source of cell damage and the potential pathophysiological link between sIBM and T2DM. These molecular data confirm clinical comorbidity between both disorders, pointing out sIBM as a new population of underdiagnosed T2DM patients. T2DM is a silent infradiagnosed disease and any clue for emerging groups of risks may account for the reduction of further associated consequences. Interestingly, partial deregulated glucose metabolism was also found in other myositis as PM and DM, thus suggesting common lifestyle cues’ contribution in observed comorbidity.Probably, both mitochondrial deregulation and lifestyle habits (such as restricted mobility) may be conditioning the manifestation of metabolic disorders such as T2DM in sIBM patients. Due to its idiosyncrasy, myositis are a group of disorders characterized by weakness and wasting of the muscle and consequently low physical activity or a sedentary lifestyle. This situation is associated with the development of several chronic diseases including T2DM. Additionally, due to skeletal muscle contribution to glucose homeostasis and muscle wasting on account of low patient activity, in a kind of vicious circle, glucose metabolism deregulation may be the final fate of a sedentary lifestyle, in this case, triggered by myositis and mitochondrial dysfunction.Consequently, some general recommendations may be added in the clinical follow-up of patients with myopathy to prevent or manage potential T2DM development in accordance with international standards . They arSome limitations must be acknowledged in the present work. The reduced number of patients included in the study may be the major downfall. Of note, due to the low prevalence of sIBM disorder (considered a rare disease), the study of bigger cohorts is often hampered but recommended. Additionally, further studies are warranted to provide more in-depth understanding of the deregulated pathways underlying glucose impairment in sIBM patients and whether the lipid profile and other features characteristic of the metabolic syndrome are affected in sIBM, even myositis in general, to establish a better correlation between these disorders and the metabolic syndrome. Similarly, the parallel inclusion of fibroblasts from PM or DM patients would have been of interest to deepen the knowledge of common and specific molecular patterns shared among these disorders that clinically overlap.In summary, the conclusions of the present work stand for null comorbidity between sIBM and AD but partial comorbidity between sIBM and T2DM through the impairment of glucose homeostasis, mitochondrial function, and probably lifestyle conditions. Although further investigation is needed to validate this pilot study, these findings provide better knowledge of sIBM complications.On account that effective treatment for sIBM is not yet available, this should be a red flag and the monitorization of the prediabetic state in myositis patients to avoid potential complications in clinical practice associated with the management of both diseases should be considered, thus contributing to improving the quality of life of patients with inflammatory myopathies.


Solid substrates often induce non-uniform strain and doping in graphene monolayer, therefore altering the intrinsic properties of graphene, reducing its charge carrier mobilities and, consequently, the overall electrical performance. Here, we exploit confocal Raman spectroscopy to study graphene directly free-floating on the surface of water, and show that liquid supports relief the preexisting strain, have negligible doping effect and restore the uniformity of the properties throughout the graphene sheet. Such an effect originates from the structural adaptability and flexibility, lesser contamination and weaker intermolecular bonding of liquids compared to solid supports, independently of the chemical nature of the liquid. Moreover, we demonstrate that water provides a platform to study and distinguish chemical defects from substrate-induced defects, in the particular case of hydrogenated graphene. Liquid supports, thus, are advantageous over solid supports for a range of applications, particularly for monitoring changes in the graphene structure upon chemical modification. Here, the authors report water as a superior platform to suspend graphene compared to solid substrates that induce non-uniformity and do not provide structural flexibility. They utilize confocal Raman spectroscopy to study graphene floating freely on the surface of water to show that a liquid support relieves the pre-existing strain. Use of supporting substrates facilitated its exploitability and prompted the development of the vast variety of graphene-based devices, such as field effect transistors4, transparent conducting electrodes5, gas and pressure sensors7, DNA single-molecule detectors, to name a few9. Although being widely adapted for the fabrication of current graphene-based devices and technologies, solid substrates largely affect graphene due to doping and induced strain, and thus hinder graphene’s intrinsic properties18. The effect is even more prominent for CVD -grown graphene samples, in which numerous inhomogeneities, inevitably caused by the growth and transfer processes, result in a wide variability in the band structure (and thus Raman signature)23 not only from sample to sample, but also from spot to spot within a single graphene sample. Here we study graphene supported by liquids, namely graphene at liquid/air and liquid/liquid interfaces, which provide well-defined interfacial boundaries, unlike solid/air interfaces.Graphene is typically perceived as a two dimensional film with outstanding electrical and optical characteristics, but impracticable as a fully self-standing material and, therefore, necessitating a solid support24. However, the exact effect of liquids on the morphology and the properties of large sheets of CVD graphene has not been ascertained yet. Although Raman spectroscopy has been recently successfully applied to characterize natural graphene flakes exfoliated in water26 in that case Raman spectrum is acquired as an average spectrum over all dispersed flakes and does not provide information about strain level and deviations in a single graphene flake. Moreover, no characterisation of CVD graphene at liquid–liquid interfaces has been reported so far.Graphene caged between two immiscible liquids displays strikingly higher charge carrier mobilities as compared to the same (e.g. grown under the same conditions) graphene supported by solid substrates, presumably attributed to fewer contamination and the absence of scattering from the substrate18. Additionally, statistical analysis of graphene Raman peaks showed that also the variations of strain and doping values across a graphene sheet are significantly smaller when supported by liquids, owing to more homogeneous and molecularly defined graphene-liquid interface, as opposed to a graphene-solid interface. We find that such exceptional stability of the Raman signature of graphene in a liquid environment can be used to characterize changes in the properties of graphene upon hydrogenation, and upon modifying the composition of liquid environment.In this work we demonstrate that liquids can be a standalone support for graphene and allow for important insights into intrinsic properties of graphene that are difficult to access otherwise. By applying confocal Raman spectroscopy to CVD-grown graphene free-floating at water/air and water/liquid interfaces, we found that graphene supported by liquid(s) undergoes very small to zero strain and doping effect, posing stark contrast with “conventional” solid-supported graphene, known to always be subjected to strain and doping28. Similarly, the use of a biphasic mixture of water (or aqueous etchant solution) with a nonpolar liquid causes the graphene/catalyst stack to float in between the two immiscible phases, which after the catalyst etching yields graphene free-floating at the liquid/liquid interface24. Such biphasic designs have been mostly employed for transferring CVD graphene and are advantageous over the conventional techniques in terms of preserving graphene’s intrinsic properties, particularly because graphene supported and protected by liquids on both of its sides is less subjected to contamination and mechanical perturbations29. However, up to now the only experimental insight into the graphene properties in situ at liquid/liquid and liquid/air interfaces has been the increase in charge carrier mobilities as compared to solid-supported graphene samples24, which indeed could be indicative of reduced contamination and strain.Graphene floats when placed on the surface of water due to the density and water surface tension. This property is routinely used when transferring CVD graphene from a catalyst substrate – the graphene/catalyst stack is placed on the surface of an aqueous solution of an etchant until the catalyst is fully dissolved and the graphene sheet remains free-floating at the water/air interface to be transferred further24) and graphene transferred onto SiO2/Si wafers . All graphene samples were grown according to the same growth protocol. The measurements were conducted at two excitation wavelengths, 457 nm and 532 nm respectively , see Fig. Here, we performed an extensive Raman study of a single-layer graphene at water/air, water/1-octanol and water/cyclohexane interfaces; graphene on copper (as-grown), free-standing graphene  or at ~2730 cm−1 (for 457 nm excitation wavelength). The G band originates from a first-order one-phonon scattering process and the 2D band from a second-order two-phonon intervalley scattering process, and are both typical of all sp2 carbon materials30. The frequencies, intensities and linewidths of the G and 2D bands of graphene are affected by the laser excitation energy (of the G band negligibly), number of graphene layers, strain and doping31. Presence of defects in graphene lattice  breaks the symmetry and activates the D band at ~1350 cm−1. The relative intensity of the D band with respect to the G band is commonly used to characterize the amount of defects and disorder in graphene materials.The two most intensive Raman bands of pristine graphene are the 2D band of graphene and one of the bands of 1-octanol at ~2730 cm−1 measured at 457 nm laser wavelength. But, given that the 2D band of graphene is at least three times as intensive, the overlap does not hinder the determination of the 2D band position , while no information about the location of the interface can be obtained by profiling the peaks of the liquids , and, in fact, can be quantified based on the shifts of the Raman bands31. Moreover, a correlative analysis of the G and 2D peaks frequencies allows for the disentanglement of the effects of strain and doping30. For that, the measured frequencies of G and 2D bands are plotted on a scatter plot with a non-orthogonal strain-doping framework . The correlation maps at 457 and 532 nm wavelengths were recalculated based on a 2D mode dispersion of 100 cm−1 eV−1 (ref. 35).The detailed analysis of Raman peaks shifts provide information about strain and doping in the graphene lattice, and, therefore, about the effect of the substrate and environment on graphene intrinsic properties. Strain and doping induced by the substrate and by the environment are known to alter the frequencies of the Remarkably, the correlation analysis displayed two critical distinctions of graphene supported by liquids from graphene supported by solid substrates and from free-standing graphene: very small absolute values and very small deviations of strain and doping induced by the liquids.15. In fact, the broadly scattered data points and wide frequency histograms in Fig. 2 and free-standing graphene indicate wide variations of strain and doping in these samples. We observed that graphene on copper (the growth substrate) undergoes a wide range of strain values – from −0.8% (compressive strain) till 0.7% (tensile strain) with no evident dominating values , in which the zero strain area is only in the centre of the suspended areaues Fig. . The wid37. Strain is known to vary significantly from spot to spot in suspended graphene films depending on the position of the spot towards the centre and the supported part36. Importantly, the measured strain values for free-standing CVD graphene are higher than those reported for an exfoliated free-standing graphene flake36, demonstrating that the CVD growth and possibly the transfer process induce the strain field that remains in graphene even when it is suspended, i.e. is in a theoretically zero- or low-strain geometry.Similarly, free-standing graphene exhibits very wide strain variations of −0.1–1% Fig. , c, indi2 substrate is known to have a strain relaxation effect on graphene21 and, agreeably, graphene transferred onto a Si/SiO2 wafer exhibits much narrower data scattering and strain variation from −0.3% to 0.2%  can also be used for monitoring strain and doping in graphene  than those of graphene on solid substrates, leading to less accurate determination of peak widths. The positions of peaks maximums, on the other hand, are not affected by increased noise level and can be determined accurately. Interestingly, the mean values of FWHM of the G bands of graphene on liquids are very close to that of graphene on copper and greater than those of graphene on Si/SiO2 wafer and free-standing graphene, indicating that the intrinsic charge density of the graphene is not affected by copper and the liquid supports  of the ene Fig. . A narroImportantly, the observed effect is stable in time, and the Raman signature and statistical distributions of the strain and doping values in graphene-on-water remained unchanged even after floating on the surface of water for eight days  results in an articulate relaxation of strain and reduction of doping level in graphene sheet. Additionally, the spot-to-spot deviations of strain and doping within the graphene sheet are significantly smaller for liquid-supported graphene, indicating more uniform properties of the graphene surface.G and 2D bands positions  and relative intensity (ID/IG ratio) of the Raman D band arising from the formation of C–H bonds and rehybridization of the graphene lattice46. Two types of samples with different hydrogenation degrees were prepared using a hydrogen plasma on as-grown graphene on copper (see Methods for details) – with plasma treatment duration of respectively 10 s and 60 s47. Raman spectra were recorded for a pristine graphene on copper and on water (same graphene sample before and after etching the copper and replacing the etchant with pure water) and for hydrogenated graphene samples on copper and water (same samples of hydrogenated graphene before and after etching the copper and replacing the etchant with pure water), Fig. Finally, we tested our technique for studying changes in the properties of graphene on the example of hydrogenation of graphene. Hydrogenated graphene (h-G) is typically characterized by the position , while in the case of h-G/Cu the multimodal distribution of wD could be caused by either presence of different types of defects (sp3 carbon atoms and other defects not related to hydrogenation) or by doping effect49. The samples of h-G/Cu and h-G/water are essentially the same sheet of graphene tested before and after copper etching, and, therefore, have identical types and amounts of defects. The main differences between the samples (accounting for the difference in D peaks) are the different levels of strain and doping induced by copper and water. In fact, although strain per se does not generate a D peak in graphene, the strain fields in graphene induced by copper can activate defects such as interstitials and vacancies along the grain boundaries of graphene 50, resulting in the shift and broadening of the D peak. This does not take place in the samples of h-G/water, because of lower strain induced by water .First, for the samples that underwent 10 s of hydrogenation, h-G floating on water have much narrower distributions of the ter Fig.  suggestster Fig. . AdditioD mode frequency and alter the D mode intensity51. As shown in Fig. D peak frequencies and intensities.In addition to strain-activated defects, doping is also known to cause shift in the D peak. Furthermore, not only the wD, but also the ID/IG ratio displays a lower variability across the h-G sheet when supported by water as opposed to copper  hydrogenation of the graphene sheet  of doping levels than h-G/Cu (within 15 × 1012 cm−2), which confirms that doping can be one of the reasons for the inhomogeneity of the D band position and intensity in the case of h-G/Cu. Similarly to pristine graphene, hydrogenated graphene is also subjected to greater and less uniform strain on copper, varying from −0.6% to 0.1%, than it is on water, where the strain varies from -0.2% to 0%  and is, therefore, a better indication of hydrogenation than the D peak of h-G/Cu. Interestingly, comparison between the ID/IG distributions of h-G/water in Fig. 53. Even more pronouncedly than in the case of 10 s hydrogenation  and possibly the presence of strain-activated defects , which adapts to and takes the shape of the surface the liquid is in contact with, i.e. graphene in our case62. Homogeneity, molecular smoothness, structural adaptability of liquid surfaces are direct consequences of the weak, soft intermolecular interactions and are universal properties of all liquids, independently of their chemical nature61. Remarkably, various liquid interfaces relax and unify strain in graphene down to similar values, indicating that the structural, rather than chemical, properties of liquids are responsible for the strain relaxation effect.A balance between attractive forces and thermal, repulsive forces enables free diffusion of molecules in fluidsD, G and 2D band remain unaffected by liquid substrates. Using liquids to support graphene, thus, can be beneficial over solid substrates whenever the properties of graphene need to be accurately monitored. Another big advantage of using liquid supports for graphene is that it does not require any handling or transfer of graphene. This essentially means that (1) obtaining and characterising graphene on water are technically easy and inexpensive (2) the results of the characterisation are highly reliable, because graphene is not contaminated (3) sample-to-sample variability is minimal due to the natural perfect uniformity of liquid surfaces. In contrast, properties of the solid substrates strongly depend on the fabrication and transfer methods, and may vary from manufacturer to manufacturer, and from lab to lab.In summary, we demonstrated that the functionality of confocal Raman spectroscopy for characterising CVD graphene can be amplified by studying graphene floating directly on a liquid or even buried in between two liquids. All three major Raman bands of graphene – the Finally, the advantages of a water support were showcased here through the example of hydrogenation of graphene, but can, in principle be applied for studying other effects on graphene structure.63).All graphene samples were grown on a copper foil with the thickness of 25 µm by chemical vapour deposition (CVD) method, after annealling the copper at 1035°  was added on top to form a biphasic system with graphene floating at the interface. During etching of the copper the samples were covered with lids to prevent evaporation of the top organic phase; more cyclohexane (or 1-octanol) was added during experiments to prevent full evaporation of the top phase. After copper removal the bottom phase underneath graphene was replaced with ultrapure water. As for Raman measurements it is important to minimise graphene movability on the surface of liquids, the size of graphene sample was to fit the size of the Petri dish.2 plasma in a computer controlled Diener plasma generator  for 10 and 60 s (ref. 47).Graphene on copper was hydrogenated using a HThe samples of hydrogenated graphene on copper were placed in a 0.1-M solution of APS for copper etching. After copper was etched away, the solution underneath hydrogenated graphene was replaced with ultrapure water.2 and free-standing graphene; ×70 immersion objective was used for graphene on liquid supports. Graphene/Cu samples were typically cut into 5 mm × 10 mm or 10 mm × 10 mm pieces, which were then studied by Raman spectroscopy directly on copper. Then graphene was transferred from copper to SiO2/Si substrate or TEM grid (for free-standing configuration) using the interfacial caging method24, or to liquid interfaces using the method described above. A comparative analysis showed no statistical difference between the samples of 5 mm × 10 mm and 10 mm × 10 mm  implemented in LAMMPS. The neighbour list was updated every single simulation step. The simulation cut-off was 10 Å for Lennard–Jones and 12 Å for short-range electrostatic interactions. Shifted cut-offs (charmm style) were used for Lennard-Jones interactions. In accordance with the Opls forcefield, 1–4 interactions were scaled by a factor 0.5. A time step of 0.0005 ps was used in all simulations. The dimensions of the simulation box were 60 × 60 × 160 Å3 . In all cases the graphene flake consisted of 678 atoms (~40 × 40 Å). The number of 1-octanol or cyclohexane molecules present was 490 and 512 respectively. All systems contained 4693 water molecules. Equilibration was inferred from systematic trends within the potential energy. The equilibration time of the simulated system was 0.5 ns, after which another additional 1 ns production run was performed. For the simulation of graphene in vacuum rigid body translational and angular momentum were removed in all dimensions. For the simulations of graphene on copper an additional simulated annealing was performed to prepare/equilibrate the system (the system was cooled from 1400 K to 300 K in 1 ps). All system were setup by stacking individual molecules into stacked layers, which were combined to compose the desired multi-layer system.All simulations where performed with the LAMMPS simulation package65. For 1-octanol and cyclohexane two recently developed models were used that are based on the Opls forcefield68. These two models were specifically refined on reproducing thermodynamic properties such as the bulk density. Water was modelled by the TIP3P model because of its compatibility with the Opls based n-alkane models. The non-bonded interactions between graphene and the different solvents were described by Lennard-Jones interactions. Here, Lorentz–Berthelot combining rules were used to calculate the interactions between the different atomic species  based on a graphene-graphene Lennard-Jones interaction of ε = 0.003035 eV and σ = 3.55 Å (graphene in Opls). For the interaction between graphene and TIP3P water we used Lennard-Jones interactions that were optimised to reproduce wetting angles via an evolutionary algorithm69 . Graphene was modelled as uncharged  to simulate the interaction of bulk graphene with the fluid interfaces (these are purely based on Lennard–Jones interactions). Finally, the simulations of graphene on copper on were performed using the reactive comb3 potential70 for both Cu–Cu, C–C and C–Cu interactions.Intra-molecular interactions within graphene flakes were described by the AIREBO potentialFurther information on research design is available in the Supplementary InformationReporting Summary


We present a novel strategy for designing a dual-band absorber based on graphene metasurface for terahertz frequencies. The absorber consists of a two-dimensional array of patches deposited on a metal-backed dielectric layer. Using an analytical circuit model, we obtain closed-form relatinos for the geometrical parameters of the absorber and the properties of the applied materials to achieve the dual-band absorber. Two absorption bands with perfect absorption at the preset frequencies of 0.5 and 1.5 THz are achieved. The results obtained by the analytical circuit model are compared to the simulations carried out by full-wave electromagnetic field analysis. The agreement between results is very good. We demonstrate that the graphene absorber remains as the dual band for a wide range of the chemical potential. Furthermore, the recommended dual band absorber is insensitive in terms of polarization and remain within various incident angles. Metamaterials are artificially-made media with uniquely engineered electric permittivities and magnetic permeabilities ,2 and enTo realize metamaterial-based absorbers in the low THz region, one can use the graphene patterned arrays. Graphene, consisting of a two-dimensional layer of carbon atoms arranged in a honeycomb lattice, has recently been demonstrated as one of the best materials for many applications in electrical and photonic devices due to its exotic properties ,21,22,23In this paper, a simple structure, composed of a period array of graphene patches deposited on a dielectric spacer terminated by a metallic back reflector, is used to design a dual-band THz absorber. We propose a circuit model to extract the geometrical parameters of the absorber and the properties of the material in the closed-form. Furthermore, we present a simple and effective approach for achieving perfect absorption at two preset frequencies. The input admittance of the absorber at the two frequencies is designed to match the admittance of free-space. Moreover, the imaginary part of the input admittance at the central frequency is set by applying an additional condition to be zero. To validate the approach, dual-band perfect THz absorbers have been investigated and designed. Then, full-wave simulations are used to confirm the design method. Excellent agreements have been achieved. It should be noted that the recommended absorber is polarization insensitive for normal incident electromagnetic waves owing to its symmetric structure. Besides, both dual-band and the high absorption effectiveness are affected slightly even at high angles of incidence for both TE and TMpolarizations.The paper is organized as follows. The equivalent circuit model of the proposed device is introduced, and then a design scheme leading to an dual-band absorber is proposed in D and gap g between the patches and the bottom layer of a reflective golden plate with finite conductivity of d. It is worth mentioning that in the fabrication process, the Si is used to support the graphene patches. The Au film is deposited on the Si bottom by gilding. Next, a continuous and uniform atomic monolayer of graphene can be grown by the chemical vapor deposition method, then patterned into closely packed square patch arrays using e-beam lithography.The geometry of the proposed absorber is schematically shown in x direction) is demonstrated in Based on transmission line theory, the whole structure can be modeled as circuit elements . The equS is the surface of a patch, An accurate circuit model has been proposed by Barzegar-Parizi et al. for a graphene patch array illuminated by a normally incident plane wave . The adme is the electron charge, ℏ is the reduced Planck’s constant. T is the temperature (fixed to 300 K) and It is known that graphene’s conductivity is derived using the Kubo formula, which is described with interband and intraband contributions ,33. For ntraband . TherefoDesigning the proposed absorber near the first resonance frequency of graphene patches, we can only consider the first mode and neglect the effect of higher-order modes. To avoid numerical calculation of the eigenvalues in the previous capacitance expression, we propose to replace the expression with the analytical capacitance admittance of metal patches proposed in . Therefoc is the speed of light) is the wavenumber of free space and the free-space admittance In the equivalent circuit model of Suppose perfect absorption can be achieved at two frequencies We consider a central frequency defined as Then, the admittance of the graphene array is seen as the input admittance of the structure, and it can be tuned to be matched to the free-space admittance near its resonant frequency . After sn. The relaxation time can be tuned by the chemical potential through the relation Choosing the two frequencies Equation  for give process ,39,40,41At the end, the geometrical parameters of the structure are computed by Equations –10) as as10) aWith the aid of the analytical equations presented in Equation , we havequations , the patThe absorption spectra of the structure are plotted in It should be pointed out that there are slight disagreements between the circuit model and FDTD simulation. These slight disagreements result from neglecting the effect of higher-order modes of graphene patches in the circuit model shown in Now, the tunability of the proposed device is investigated by changing the chemical potential of graphene. The polarization insensitive performance for the proposed absorber is important in practical applications. We plot the absorption spectra as a function of polarization angle and frequency in We have also investigated the omnidirectional characteristic for the proposed absorber. In summary, a dual-band perfect absorber based on graphene patches has been designed in the low terahertz regime by using impedance matching concept. First, we assigned an equivalent circuit to the absorber. We then adjusted the real part of the input admittance of the absorber to be close to the free space admittance and the imaginary part of the input admittance to be zero at two frequencies. In addition, the dielectric slab is considered as a quarter wavelength transmission line at the center of the two frequencies. This method resulted in closed-form relations for the geometry of the structure and the characteristics of the applied material. According to the results of simulation and analytical circuit model, the recommended absorber can operate with perfect absorption at 0.5 THz and 0.15 THz. Further, the device are insensitive to polarization and omnidirectional.