In this study, a basic design of area-arrayed graphene nanoribbon (GNR) strain sensors was proposed to realize the next generation of strain sensors. To fabricate the area-arrayed GNRs, a top-down approach was employed, in which GNRs were cut out from a large graphene sheet using an electron beam lithography technique. GNRs with widths of 400 nm, 300 nm, 200 nm, and 50 nm were fabricated, and their current-voltage characteristics were evaluated. The current values of GNRs with widths of 200 nm and above increased linearly with increasing applied voltage, indicating that these GNRs were metallic conductors and a good ohmic junction was formed between graphene and the electrode. There were two types of GNRs with a width of 50 nm, one with a linear current–voltage relationship and the other with a nonlinear one. We evaluated the strain sensitivity of the 50 nm GNR exhibiting metallic conduction by applying a four-point bending test, and found that the gauge factor of this GNR was about 50. Thus, GNRs with a width of about 50 nm can be used to realize a highly sensitive strain sensor. In an aging society with a declining birthrate, the development of structural health-monitoring systems is extremely important in order to maintain a secure and reliable society for human beings through early detection of material damage. Especially in basic infrastructures such as energy plants and transportation systems, online damage-monitoring technology is strongly required due to the complexity of the systems and the shortage of skilled engineers due to the declining birthrate and aging population. In order to quantitatively evaluate material damage and prevent various failures, it is necessary to monitor the stress and strain fields of components in operation with high sensitivity and low cost; in other words, there is a strong demand for highly sensitive strain sensors. For these needs, strain sensors based on carbon nanoparticles, such as carbon nanotubes and graphene, have attracted attention as a viable alternative to conventional sensors based on metallic or semiconducting materials, mainly due to their superior electrical properties. Various sensors based on carbon nanomaterials have been proposed and practically used, and most of them are made by adding carbon fillers to a polymer matrix and forming an electrical network between the carbon fillers to provide high piezoresistivity ,2,3,4,5.5 cm2 V−1 s−1 [Recently, much attention has been focused on graphene as an available material for strain sensors with high sensitivity and large deformability . Graphen V−1 s−1 ,8,9,10.  V−1 s−1 . ZGNRs f V−1 s−1 . On the  V−1 s−1 ,14,15,16In this paper, we propose a basic design of an area-array-type, GNR-based strain sensor that can measure micro-strains with high sensitivity and high resolution. Each GNR arranged in an area array works as a strain gauge, and thus the distribution of strain in the area to be measured can be evaluated. In order to realize a GNR-based high-sensitivity strain sensor, it is essential to fabricate a high-quality GNR stably. High-quality GNRs can be fabricated by two methods: the bottom-up method and the top-down method. The bottom-up method is a direct synthesis of GNRs using synthetic chemical methods. The remarkable advantage of the bottom-up approach is the accuracy in controlling the ribbon width, edge structure, and film thickness when forming thin films at the atomic level, enabling accurate GNR structures with angstrom-level resolution and obtaining physically smooth and chemically clean edges ,18,19. HIn order to establish a stable and reliable fabrication process of the device using GNRs, the synthesis of a high-quality, single-layer graphene sheet is one of the important factors. In this study, graphene was grown on the inner surface of a Cu foil pocket structure by applying thermal LPCVD  using acetylene . Since t3. After washing the etched sample with dilute HCl and pure water, the cleaned sample was placed on a silicon substrate with a 300 nm thick thermal oxide film, and the PMMA layer was removed with acetone. An example of the transferred graphene is shown in In order to fabricate a GNR structure, the graphene synthesized on the Cu foil was transferred onto an oxidized silicon substrate. In this study, a wet transfer process was applied using PMMA (polymethyl methacrylate). This transfer process is also known as the PMMA-assisted transfer process . At firs2 plasma. The etching rate of PMMA with O2 plasma was 30 to 40 times larger than that of HSQ, and thus the HSQ pattern could be transferred to the graphene layer [The patterning process using EB lithography is described as follows. After the graphene was transferred onto the Si substrate, a Pt layer with a 5 nm thickness and a Au layer with an 80 nm thickness were deposited continuously by EB deposition using a stencil mask to form metal electrodes. After deposition of the metal electrodes, GNRs were patterned between the electrodes using the EB lithography technique. First, PMMA was coated and cured on graphene, and then HSQ (hydrogen silsesquioxane: XR1541-006 Toray Dow Corning) was spin-coated as a negative resist to form an HSQ/PMMA laminated resist layer. As PMMA has a high affinity for graphene, sufficient adhesion can be achieved with low-temperature curing compared to the case of a single layer of HSQ. Next, line patterns ranging from 50 nm to 400 nm in width and 18 μm in length were irradiated onto HSQ and developed with TMAH (tetramethylammonium hydroxide) to form the patterns. During the development, the PMMA layer was not dissolved in TMAH, and only HSQ was developed. Finally, PMMA was removed by RIE with One layer . An exam−1 and corresponds to carbon–carbon bonds; the second peak is called the D band, which appears at 1350 cm−1 and indicates the presence of disordered carbon atoms, or defects. The last peak, called the 2D band, appears at 2700 cm−1 and is related to the total number of layers in the graphene sheet. The relative intensity of the D and G bands (ID/IG) corresponds to the defect density in the observed region, and the relative ratio of I2D to IG corresponds to the number of layers in the graphene sheet. Therefore, the quality of graphene sheets improves with decreasing ID/IG values, and if the I2D/IG value is greater than 2.0, the graphene is considered to be composed of a single layer. For the fabricated GNRs, Raman spectroscopy was used to verify whether graphene existed under the resist layer and whether the etching removed the unused graphene. As shown in 2D/IG ratio was approximately 2.6, which indicated the presence of single layer of graphene. On the other hand, the intensity of the D band increased compared to graphene before the GNR fabrication process, with the ID/IG ratio increasing from 0.09 [The quality of the fabricated GNRs was evaluated by Raman spectroscopy. When a laser beam is irradiated on the surface of graphene, three typical Raman shifts are known to appear in the resulting reflection spectrum. One is called the G band, which appears at 1590 cmrom 0.09  to 0.4. D/IG < 0.05 sold by BGT Material Limited is 300–600 Ω/square [Ω/square . The sheSince it has been theoretically shown that AGNRs exhibit piezoresistive effects regardless of whether their initial electrical conduction properties are metallic or semiconducting , it was The strain dependence of the measured resistance of GNR is shown in In this study, we fabricated area-arrayed GNRs and evaluated the quality and strain dependence of their electrical conduction properties in order to realize a GNR-based strain sensor applicable in structural health monitoring. GNRs with widths ranging from 400 to 50 nm were fabricated by EB lithography between metal electrodes with 18 μm spacing. The I–V characteristics were evaluated for the fabricated GNRs. The current values of GNRs with widths of 400, 300, and 200 nm increased linearly with increasing applied voltage. It was confirmed that fabricated GNRs with a width of 200 nm or more exhibited metallic properties, and good ohmic contact was obtained between graphene and metal electrodes. The sheet resistance of GNRs was also evaluated based on the measurement results. The sheet resistances of GNRs with widths of 400 and 300 nm were 510–750 Ω/square, and the sheet resistance of GNRs with width of 200 nm was about 1000 Ω/square. The sheet resistance of GNRs with a width of 200 nm increased, but the GNR fabrication process developed in this study did not cause significant damage. On the other hand, for GNRs with a width of 50 nm, there were two types of GNRs: one showing metallic conduction in which the current value increased linearly with increasing applied voltage, and the other in which the current value increased nonlinearly. Theoretically, GNRs with ribbon widths of 100 nm or less are known to exhibit piezoresistive effects. Therefore, GNRs with a width of 50 nm were subjected to strain in a four-point bending test, and the strain dependence of electrical resistance was evaluated. As a result, a maximum gauge factor of about 50 was obtained in a strain range of 0.08% for GNRs showing metallic conductivity. This result indicated that GNRs could be applied to the development of highly sensitive strain sensors. Thus, we have successfully fabricated low-damage GNRs and demonstrated the possibility of developing highly sensitive strain sensors by applying GNRs.


Combining graphene with other nanomaterials can further tune the properties of graphene, and also can enhance the characteristics of the hybrid materials produced by the addition of this monoatomic-thick carbon layer. The range of possible applications of such hybrid materials is quite vast, including but not limited to energy storage, sensing, biomedicine, photocatalysis, or optoelectronics. This Special Issue presents several of the latest advances in the synthesis and characterization of graphene-based hybrid materials and nanostructures, with relevance for the development of sodium-ion batteries , NO2 sen sensors , or the  sensors . The fab sensors .0.2Ni0.25Mn0.75Oδ to form a sodium-ion cell. The so-obtained SIB was cycled at various rates and its performance was evaluated by monitoring the capacity as a function of the discharge rate and cycle number. It was confirmed that the battery was able to reversibly cycle sodium ions. The cell’s power response was good, and the cell cycled for over 100 cycles with capacities ranging from 120 up to 160 mA·h·g−1.In recent years, several intercalation materials have been investigated as potential electrodes for sodium-ion batteries (SIB). In the paper by Prosini et al. , the aut2 at room and elevated temperatures. It was shown that the FG particle fraction affects the relative response via the efficiency of charge transfer between the NO2 molecules and FG layers. The authors demonstrated that highly fluorinated graphite films provided a greater increase in the sensor response as compared to less fluorinated films.Graphene-based materials have been widely studied as sensitive layers of resistive gas sensors, as the electrical conductivity of graphene is strongly sensitive to surface adsorbates. Significant efforts were conducted to improve the response and recovery time of these sensors by controlling the functionalization of graphene. Sysoev et al.  reported−2, while higher fluences yielded web-like surface structures. The sheet resistance and the wettability of both pristine and graphene-doped PS were significantly affected by the applied laser treatment. The construction of different LIPSS structures is very important, for example, in studies involving cell-substrate interaction, cell guidance, or tailoring protein adsorption on biomimetic surfaces.Fajstavr et al.  reported2S on its surface. Preserving the high LSPR intensity of silver nanoparticles is of key importance in potential applications. Our group [Sensors based on the localized surface plasmon resonance (LSPR) of silver or gold nanoparticles are sensitive analytical tools which can be applied in chemical and biosensing. However, silver has poor stability under ambient conditions, forming Agur group  reportedIn a review by Barshutina et al. , researc


We compare the polarizability valuecomputed using density functional theory with the output from previouslypublished experimental data on the electrostatically controlled interlayerasymmetry potential in BLG and data on the on-layer density distributionin tBLG. We show that monolayers in tBLG are described well by polarizabilityα The manifestations of the qualitative change ofelectronic characteristics of both Bernal (BLG) and twisted (tBLG)bilayer graphene, produced by electrostatic gating3 and interlayer misalignment,5 were observedin numerous experimental studies of the electronic transport in graphene-basedfield-effect transistor (FET) devices. These versatile electronicproperties make FETs based on BLG and tBLG an attractive hardwareplatform for applications tailored8 for various quantum technologies.While, over the recent years, the fundamental electronic propertiesof bilayer graphene have been intensively studied, a mundane but practicalcharacteristic of this material related to the out-of-plane dielectricsusceptibility of graphene layers largely escaped attention of thoseinvestigations, despite several already recorded indications13 of its relevance for the quantitative modeling of the operationof graphene-based FET devices.Bilayer grapheneab initio density functional theory (DFT). We use that toestimate the effective dielectric susceptibility, ϵz, of graphene and to design a recipe for implementingit in the self-consistent description of electrostatics in bilayers,both twisted and with Bernal stacking. For tBLG with twist anglesoutside the magic angle range,8 we perform a mesoscaleanalysis of the on-layer carrier densities, finding a good agreementwith the earlier observations.13 Then, we implement the same recipe in the analysis of the interlayerLandau level pinning in strongly twisted bilayers. In this case, wealso find an excellent agreement between the theoretical results andthe measurements performed on a newly fabricated FET with a 30°-twistedtBLG. Finally, we take into account the out-of-plane dielectric susceptibilityof a single graphene layer in the self-consistent analysis of theinterlayer asymmetry gap in Bernal bilayer graphene,1 improving on the earlier calculations15 and successfully comparing the computedgap dependence on the vertical displacement field, Δ(D), with the earlier-measured interlayer exciton energiesin gapped BLG.16The out-of-plane dielectric susceptibilityof a single graphenelayer stems from the polarizability of its carbon orbitals, that is,from the mixing of π and σ bands by an electric fieldoriented perpendicular to the 2D crystal. Hence, we start by computingthe out-of-plane polarizability of a graphene monolayer using 17 with ultrasoft pseudopotentials.We use a 53 × 53 × 1 k-point grid, a largeplane-wave cutoff of 566 eV, and a variety of interlayer distances c along the z-axis to compute the totalenergy, Dz/ϵ0, centered on the carbon sites ofthe graphene layer (D being the displacement fieldand −c/2 < z < c/2). Then we determine (c using the relation δα(c) ∝ c–1, we fit the obtainedDFT data with α(c) = α∞ + a/c + b/c2 and find αDFT ≡ α(c → ∞) = 11Å3 per unit cell of graphene with the Perdew–Burke–Ernzerhof(PBE) functional and αDFT = 10.8Å3 with the local density approximation (LDA).To determine thetheoretical value of the out-of-plane dielectricpolarizability of a graphene monolayer, we employ the CASTEP plane-wave-basisDFT codeetermine  the pola3 = 10.9 Å3, and when recalculated into an effective “electronic thickness” 20 We also compared thecomputed DFT values with the polarizability computed using the variational(VMC) and diffusion (DMC) quantum Monte Carlo methods26 implemented in the CASINO code.27 Inthese calculations, we used the DFT-PBE orbitals generated using theCASTEP plane-wave DFT code28 and the orbitalsbeing rerepresented in a localized B-spline “blip” basis.The localized basis improves the scaling of the quantum Monte Carlo(QMC) calculations and allows the use of aperiodic boundary conditionsin the z-direction. The Jastrow correlation factorcontained isotropic electron–electron, electron–nucleus,and electron–electron–nucleus terms as well as 2D plane-waveelectron–electron terms,29 all optimizedusing VMC energy minimization.30 The DMCpart of the calculations was executed with a time step of 0.01 Ha–1 and a target population of 4096 walkers. The resultingQMC out-of-plane polarizability of graphene is α ≈ 10.5± 0.2 Å3, which is also close to the above-quotedDFT-LDA value, so that, in the analysis below, we will use αDFT = 10.8 Å3 for the polarizabilityof the graphene monolayer.These DFT values are close to the DFT-PBE polarizability reportedin ref , α= 0.8DFT todescribe the on-layer potentials andcharges in bilayers, as a function of doping and vertical displacementfield, D. For this, we note that z-polarization of carbon orbitals in each monolayer is decoupled fromthe charges hosted by its own π-bands because of mirror-symmetriccharge and field distributions produced by the latter, see in u, in the top and bottom layers of a bilayer, each with the electrondensity nb/t, has the formz is the effective out-of-plane dielectricsusceptibility, and d is the distance between thecarbon planes in the bilayer.For the analysis below, we use d = 3.35 Å, resultingin ϵz = 2.6 for BLG, and d = 3.44 Å (as in turbostratic graphite31), leading to ϵz ≈ 2.5 for tBLG. This expression is applicable to the descriptionof both BLG and tBLG in a FET, improving on the earlier-publishedstudies15 where theout-of-plane dielectric susceptibility of graphene layers was missedout in the self-consistent band structure analysis.Now, we will use the microscopicallycomputed polarizability α5v =10.2 · 106 m/s is Dirac velocity in monolayer graphene. 4 characteristic low-energy bands, illustrated in 1/ℏvK ≡2° . This spectrumfeatures two Dirac minivalleys at κ and κ′  multiterminaltBLG FET shown in the inset in T = 2 K) tBLG resistivity at zero (B = 0) and quantizing magnetic field. In the experimentally studieddevice, tBLG was encapsulated between hBN films on the top and bottom,thus providing both a precise electrostatic control of tBLG for B = 0 measurements and its high mobility, enabling us toobserve the quantum Hall effect at a magnetic field as low as B = 2 T. The measured displacement field and densitydependence of resistivity is shown in the form of color maps on theright-hand side panels in B = 0 and B = 2 T, respectively, where the form of “bright spots”of Rxx that appear ineach of these two cases is affected by the interlayer charge transfer,controlled by tBLG electrostatics in In fact, the weakest interlayerhybridization, λ →0, appears in “maximally” misaligned layers in a tBLGwith θ = 30°. In that case, the comparison between thetheory an experiment is simplified by that B = 0, we assumed elasticscattering of carriers from residual Coulomb impurities in the encapsulatingenvironment with a dielectric constant ϵ (ϵ ≈ 5for hBN), with an areal density nc, screened jointly by the carriers in the top and bottom layers.The screening determines32 the Fourierform factor of the scatterers3232 the density of states, γt/b, cancels out from each xx = ρtρb/[ρt +ρb], dependent on the carrier densityonly through the wavenumber transfer, 2kFt/b sin(φ/2), and screening.This produces ridge-like resistance maxima at kFt = 0 or kFb = 0, that is, whenFor a quantitative comparison of the measured andmodeled tBLGtransport characteristics at Lines corresponding to the above relationare laid over the experimentallymeasured resistivity map for a direct comparison.Nt and Nb, to each other and to their commonchemical potential, μ. As a result, we find that B = 2 T and Γ ≈ 0.5 meV is mapped in tot = hn/eB (n = nt + nb). Here, the “bright”high-DoS spots indicate the interlayer LL pinning conditions, whereasthe “dark” low-DoS streaks mark conditions for incompressiblestates in a tBLG. We compare this DoS map with ρxx measured in the quantum Hall effect regime (similar to theones observed earlier10 in other tBLG devices), wherethe high values of Rxx manifest mutual pinning of partially filled LLs,and the minima correspond to the incompressible states. To mention,the computed pattern broadly varies upon changing ϵz, whereas the value of ϵz = 2.5 gives an excellent match between the computed and measuredmaps in For comparisonbetween the theory and experiment at quantizingmagnetic fields, we studied the Landau level pinning between the twographene monolayers. In a magnetic field, graphene spectrum splitsinto Landau levels (LLs) with energies 1 in BLG, taking into account out-of-planepolarizability of its constituent monolayers. In this case, we use DFT using d = 3.35 Å,and the BLG Hamiltonian1ξ ≡ ξkx + iky, k = is the electron wave vector in thevalleys Kξ = ξ, ξ = ±. The computed sublattice amplitudes, Finally, we analyze the electrostaticallycontrolled asymmetrygapu, anda band gap, Δ, in the BLG spectrum  largely overestimate their values. At the sametime, the values of u and Δ obtained usingϵz = 2.6 appear to be less thanthe exciton energy measured in optics, for interlayer coupling acrossthe whole range 0.35  < γ1 <  0.38 eVcovered in the previous literature.42 This discrepancy may be related to that the interaction terms inthe electron self-energy are only partially canceled by the excitonbinding energy.37 It may also signal thatthe out-of-plane monolayer polarizability, α, is reduced by∼10% when it is part of BLG, as the values of Δ computedwith ϵz = 2.35 and γ1 = 0.35 eV agree very well with the measured optical gap values.The on-layer potentialenergy difference,  in refs , 14), a, au, and in refs ) and wit3, accounts verywell for all details of the electrostatics of twisted bilayers, includingthe on-layer electron density distribution at zero magnetic fieldand the interlayer Landau level pinning at quantizing magnetic fields.For practical applications in modeling of FET devices based on twistedbilayers, the polarizability of monolayer graphene can be convertedto its effective dielectric susceptibility, ϵz ≈ 2.5, which should be used for the self-consistentelectrostatic analysis of tBLG using In summary, the reported analysisof the out-of-plane dielectricsusceptibility of monolayer graphene shows that the latter plays animportant role in determining the electrostatics of both Bernal andtwisted bilayer graphene. We found that the DFT-computed polarizabilityof the monolayer, α = 10.8 Å


Contractile activity is a fundamental property of skeletal muscles. We describe the establishment of a “feeder-supported in vitro exercise model” using human-origin primary satellite cells, allowing highly-developed contractile myotubes to readily be generated by applying electrical pulse stimulation (EPS). The use of murine fibroblasts as the feeder cells allows biological responses to EPS in contractile human myotubes to be selectively evaluated with species-specific analyses such as RT-PCR. We successfully applied this feeder-supported co-culture system to myotubes derived from primary satellite cells obtained from sporadic inclusion body myositis (sIBM) patients who are incapable of strenuous exercise testing. Our results demonstrated that sIBM myotubes possess essentially normal muscle functions, including contractility development, de novo sarcomere formation, and contraction-dependent myokine upregulation, upon EPS treatment. However, we found that some of sIBM myotubes, but not healthy control myotubes, often exhibit abnormal cytoplasmic TDP-43 accumulation upon EPS-evoked contraction, suggesting potential pathogenic involvement of the contraction-inducible TDP-43 distribution peculiar to sIBM. Thus, our “feeder-supported in vitro exercise model” enables us to obtain contractile human-origin myotubes, potentially utilizable for evaluating exercise-dependent intrinsic and pathogenic properties of patient muscle cells. Our approach, using feeder layers, further expands the usefulness of the “in vitro exercise model”. However, this widely used model has limitations, especially as regards human myotubes derived from primary satellite cells. For example, given that human myotubes generally display a very flat and spread-out morphology, attaching firmly to the culture substratum, human myotubes usually have much poorer contractile activity than murine myotubes originating from the C2C12 muscle cell line under conventional culture conditions even with EPS treatment8. Moreover, while inoculation with a very high cell-density of human-origin myoblasts (e.g. more than five-fold higher than murine C2C12 cells) slightly improves their contractility development and myokine upregulations upon EPS7, it is usually difficult to obtain sufficient numbers of human satellite cells from biopsy samples, particularly from those of patients suffering from rare muscle diseases such as sporadic inclusion body myositis (sIBM)9. Thus, a useful and simple method allowing the generation of human myotubes capable of vigorously contracting in the culture system, which would markedly boost not only basic muscle research but also the diagnostic use of muscle cells obtained from patient biopsy samples, a very limited resource, is eagerly anticipated.Myotubes obtained under conventional culture conditions normally exhibit little or no contractile activity due to a lack of excitation stimuli from motor neurons. Recently, “in vitro exercise models” with contractile myotubes generated by applying electric pulse stimulation (EPS) in culture11 and is pathologically characterized by sarcoplasmic rimmed vacuole formation along with protein aggregates of p6212 and mis-localized cytoplasmic TAR DNA-binding protein of 43 kDa (TDP-43)13 as well as mitochondrial abnormalities in affected muscle fibers15. This disease results in slowly progressive and often asymmetric weakness with finger flexors and knee extensors being predominantly affected16. To date, the basic cellular functions of muscles from sIBM patients have not been well investigated, though one study demonstrated that primary sIBM myoblasts show no apparent morphological abnormalities and are fully capable of forming normal myotubes that can be properly innervated with rat embryo spinal cord in an in vitro co-culture system17. Since the outcome of this degenerative muscle disease is progressive loss of muscle contractile activity, analysis of sIBM muscle cells, ideally under contractile (exercising) conditions, is necessary for gaining meaningful insights into the pathogenic mechanisms underlying this disease.sIBM is an acquired progressive muscle disease that typically affects patients older than 50 years of ageWe herein succeeded in establishing a new “in vitro exercise model” applicable to human-origin muscle cells, which is based on a co-culture system with species-different (murine) fibroblasts as feeder cells efficiently supporting the contractility development of human myotubes upon appropriate EPS treatment. We applied this “feeder-supported in vitro exercise model” to sIBM myotubes derived from the primary satellite cells of sIBM patients and examined their functional muscle properties in response to EPS-evoked contraction by comparing them with those of myotubes derived from healthy human subjects. Our results indicate sIBM myotubes to have basically the same muscular properties upon EPS-evoked contraction as normal myotubes, though some of sIBM myotubes often exhibited cytoplasmic TDP-43 accumulations in response to EPS-evoked contraction, which was not detected in healthy controls. While such an exercise-loading test would not be feasible for patients suffering from severe diseases of muscular dysfunction, such as sIBM, the culture system developed in this study should allow us to better understand a patient’s muscle cell condition, particularly responses to actual contraction loading, potentially providing important diagnostic information allowing for customized therapies.7, myotubes derived from human skeletal muscle myoblasts (HSMM) displayed a flattened myotubular morphology reflecting their firm adhesion to the substratum. In the absence of 3T3L1 feeder cells, though human myotubes appeared morphologically well-differentiated, they exhibited minimal contraction in response to EPS . Their contraction activity, as assessed by the movement index during the final ~ 15 min. of the EPS treatment, was dependent on an EPS intensity of at least until 20 V  confirmed that nuclei of the myotubes were all positive for anti-HNA in the co-presence of murine 3T3L1 fibroblast feeders in the same culture Fig. A. This rity Fig. B.Figure Taking these data together, we can reasonably assume that although human myotubes alone without feeders exhibited poor or little contractile activity even when subjected to EPS treatment, feeder layers of fibroblasts provide a favorable substratum for human myotubes, resulting in better development of their contractile activity and de novo sarcomere structure formation upon EPS treatment.panels e–h) on feeder cells was equivalent to that of control satellite cells (panels a–d), but no spontaneous contractile activity was detected in either sIBM (panels e and f) or control myotubes (panels a and b) when EPS treatment was lacking. However, EPS treatment efficiently endowed vigorous contractile ability in both control (panels c and d) (Supplementary Movie panels g and h) (Supplementary Movie panels c and d). These results indicate that the sIBM myotubes fully retained EPS-dependent de novo sarcomere formation and the consequent vigorous contractile activity lasted for at least the duration of the 24-h EPS treatment.Taking advantage of the in vitro exercise model co-cultured with feeder fibroblasts (feeder-supported in vitro exercise model), we next examined the contractile activity of myotubes derived from primary satellite cells obtained from biopsy samples of healthy subjects or sIBM patients. As shown in Fig. 2 and in vitro7. We therefore examined CXCL1 mRNA expression levels in human myotubes of the feeder-supported in vitro exercise model . Hence, consistent with the movement index results, sIBM myotubes on the feeders have similar responsiveness to EPS-evoked contraction in terms of contraction-inducible CXCL1 upregulation.CXCL1 has been recognized as a myokine that is upregulated in response to muscle contractile activity in vivodel Fig. C. Specie20, and immunostaining analysis using anti-phospho-TDP-43 and anti-p62 antibodies confirmed that biopsy samples of all three of our sIBM patients possessed myofibers containing pTDP-43-positive and p62-positive granular structures . Unlike healthy control myotubes, some of the sIBM myotubes exhibited, especially after EPS-dependent contraction, obvious TDP-43 accumulations in cytoplasm , though such accumulation of TDP-43 was not evident in sIBM myotubes without EPS treatment. It should be noted that nuclear TDP-43 localization was also observed in 3T3L1 fibroblasts. In this set of experiments, we further examined a total of 6 individual human myotubes originating from 3 sIBM patients and 3 healthy control subjects and evaluated the TDP-43 deposits by counting numbers of myotubes possessing intracellular TDP-43 accumulations. All three healthy control myotubes only rarely displayed TDP-43 deposits, regardless of EPS-evoked contraction. In contrast, some populations of sIBM myotubes had TDP-43 deposits after EPS-evoked contraction, especially those derived from sIBM patients #4 and #6, while there were fewer deposits in those from patient #5  or healthy control subjects , and the total p62-positive areas in myotubes also did not differ among the experimental conditions .In contrast to EPS-induced alterations in TDP-43 localization in sIBM myotubes, the p62 localization pattern was not influenced by EPS-evoked contraction in any of the 6 myotubes studied, i.e., regardless of whether they were derived from sIBM patients . It should be also noted that all three sIBM patients similarly exhibited the typical sIBM phenotype of rimmed vacuoles. Thus, future studies are warranted to elucidate potential pathogenic contributions of abnormal TDP-43 accumulation in sIBM myotubes as well as to clarify the mechanism underlying the abnormal TDP-43 accumulation triggered by EPS-evoked contraction in some, but not all, sIBM myotubes. Nevertheless, our data strongly suggest the existence of intrinsic vulnerability, affecting the sIBM myotube itself, that emerges only upon actual contractile activity but not under routine culture conditions17.Taking our present in vitro data and the aforementioned clinical in vivo data together, abnormal extracellular TDP-43 accumulations are indeed sporadic and such TDP-43 abnormalities might occasionally be induced even under cell culture conditions wherein there are no other cell types such as immune cells that may directly and/or indirectly be related to the pathogenesis of sIBM40 and abnormal p62 accumulation has thus been implicated in the pathogenesis of sIBM41, due to a mechanism involving impaired autophagic clearance of aggregation-prone proteins including amyloid precursor protein43. However, unlike the very distinctly different TDP-43 localizations in sIBM and healthy control myotubes, the p62-positive small granule-like (puncta) structures were similar, being scattered throughout the cytoplasm in both sIBM and healthy control myotubes regardless of whether or not of EPS treatment had been applied  culture conditions45, we can only speculate that our culture conditions during EPS treatment likely influenced the autophagic responses in which p62 might participate. In addition, we examined only one EPS treatment condition because this EPS protocol consistently provides highly reliable and reproducible outcomes from in vitro exercise models7. Nonetheless, the current results do not allow us to assert the significance of the aforementioned TDP-43 abnormality observed in some sIBM myotubes in this study. Moreover, the underlying mechanism of abnormal TDP-43 accumulation triggered by EPS-evoked contraction in some, but not all, sIBM myotubes, remains unclear. These crucial issues thus need to be addressed in future studies by examining large numbers of myotubes originated from sIBM patients. As the limitations of this study, it should also be noted that control and sIBM samples were taken from different muscle tissues, i.e., the biceps brachii or rectus femoris muscles from two male and one female sIBM patient, all 69–86 years of age, while the subscapularis muscles were sampled in three healthy male subjects 60–68 years of age. Thus, while their ages are relatively well matched, with all being categorized as elderly, background features, i.e., sex and tissue type differences, might have influenced the phenotypes of myotubes observed in the present study. This issue needs to be carefully evaluated in a future study. Accordingly, further improvements of the feeder-supported in vitro exercise model, aimed at appropriately mimicking the pathogenic processes of myopathic diseases, are essential. Prospectively eliciting such processes in response to a contractile burden under certain experimental culture conditions such as various nutritional states, different exercise intensities/modalities, in the presence of other cell types, and so on, would likely provide valuable clinical and research insights.p62 serves as a cargo receptor in the degradation of ubiquitinated proteins and organelles during autophagic responsesied Fig. A,B. In bied Fig. C, though27 as it can regenerate and mimic muscle tissue-like environments that contain numerous fibroblastic cells. In this case, co-culture with fibroblastic cells from the same individual with sIBM, instead of mouse fibroblasts, as a feeder, may establish situations more pathologically similar to the disease state by allowing cells to communicate with each other as observed in the samples derived from patient muscle biopsy specimens.In the present study, we focused on contractility development in human-origin myotubes, and its impacts, in response to actual contraction, on intrinsic muscle properties. For sIBM myotubes particularly, this co-culture system may also be useful for elucidating higher-order functional interplays between contracting muscle cells and surrounding fibroblastic cellsIn summary, we established a “feeder-supported in vitro exercise model” applicable to sIBM myotubes derived from the primary satellite cells of sIBM patients. This newly established “feeder-supported in vitro exercise model” enables us to readily produce contractile human-origin myotubes that can be subjected to further experimental and diagnostic analyses to gain details regarding exercise-evoked biological responses in vitro. Thus, our approach, using feeder cells, further expands the usefulness of the “in vitro exercise model”.Dulbecco’s modified Eagle’s medium (DMEM), and Ham’s F-10 medium were purchased from Fujifilm Wako Pure Chemical Corp. . Penicillin/streptomycin, and trypsin–EDTA were obtained from Thermo Fischer Scientific . Cell culture equipment and rectangular 8-well plates were obtained from BD Biosciences  and Thermo Fisher Scientific, respectively. SUMILON Cell-disk LF1 was from Sumitomo Bakelite Co. LTD . Calf serum (CS) and fetal bovine serum (FBS) were obtained from BioWest . Matrigel was obtained from Corning . Unless otherwise noted, all chemicals were of the purest grade available from Sigma Chemical  or Fujifilm Wako Pure Chemical Corp.2 as previously reported46. Primary satellite cells were cultured in a growth medium containing DMEM/Ham’s F10 mixture supplemented with 20% FBS, 1% penicillin–streptomycin, 1% chicken embryonic extract , and 2.5-ng/ml basic fibroblast growth factor  at 37 °C under a 5% CO2 atmosphere. Mouse 3T3L1 fibroblasts were maintained in DMEM containing 4.5 g/l glucose supplemented with 10% CS, 4 mM l-glutamine, 30 μg/ml penicillin, and 100 μg/ml streptomycin (growth medium) at 37 °C under a 5% CO2 atmosphere.Human skeletal muscle myoblasts (HSMM) (Cat.# CC-2580) were purchased from Lonza  and were cultured following the vendor’s instructions. Primary human satellite cells were obtained by fluorescent-activated cell sorting (FACS) from muscle biopsy tissues at Tohoku University Hospital under the approval of the Ethics Committee of Tohoku University, and written informed consent was obtained from all subjects (see below). The primary human satellite cells were amplified and stocked in liquid N5 cells onto an 8-well plate. Three hours after seeding, human myoblasts (activated satellite cells) were seeded onto the feeder layer of 3T3L1 fibroblasts at a density of 3 × 105 cells in each well of an 8-well plate in 3 ml of growth medium. For immunofluorescent analysis, 3T3L1 fibroblasts were seeded onto a Cell-Disc LF in a 24-well plate at a density of 1 × 104 cells. Three hours after seeding, human myoblasts (activated satellite cells) were seeded onto the feeder 3T3L1 fibroblasts on the Cell-Disc LF in 1 ml of growth medium at a density of ~ 6 × 104 cells. Two days after plating, differentiation was induced by switching the growth medium to DMEM supplemented with 5% horse serum, 30 μg/ml penicillin, and 100 μg/ml streptomycin (differentiation medium). The differentiation medium was changed every 24–48 h during the 7–8 days of differentiation.For the feeder layer, 3T3L1 fibroblasts were seeded at a density of 0.5 × 102. In the present study, 24 h in total of EPS  were applied and the cells were then harvested for analyses.The differentiated human myotubes on feeder 3T3L1 fibroblasts in 8-well plates or on Cell-Disc LF being transferred into an 8-well plate, were placed in a chamber for EPS . EPS  was applied to the cells in the C-Dish using a C-Pace 100 pulse generator (IonOptix). DMEM containing 2% CS supplemented with 200% amino acids (Sigma) was used during the EPS treatments, as previously reported7. Briefly, high-quality images of cells were taken sequentially with a CCD camera  and a dissection microscope (Olympus CKX41) equipped with a 20× objective lens during EPS. We obtained the movies at completion  of the total 24 h of EPS treatment applied for the contractile activity evaluations. The differential images were obtained by subtracting the relaxation image from the contraction image. Given that the difference in pixel intensity between the first (contraction) and successive (relaxation) images is due to changes in the movement of scattering objects, the overlaid images of these subtracted images indicate moving parts. The calculated average intensity of the differential image indicates the amount of myotube movement (movement index). Three different fields under each culture condition were used for evaluation of the movement index.The index of movement was calculated by the differential image subtraction method with a slight modification, as we reported previouslyAfter the experimental treatments, cells were washed with PBS and fixed for 20 min with 2% paraformaldehyde in PBS containing 0.1% Triton X-100, then washed and blocked in PBS containing 5% CS and 1% BSA at room temperature. For immunofluorescence analysis, we used anti-human nuclear antigen (HNA) antibody , anti-caveolin 3 antibody , anti-sarcomeric α-actinin , anti-TDP-43 , and anti-p62/SQSTM1  as the first antibodies, and Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Flour 594-conjugated anti-rabbit IgG as the secondary antibodies (Thermo Fischer Scientific) at 1:100 and 1:1000 dilutions, respectively, in a solution of 1% BSA in PBS. The samples were mounted on glass slides with Vectashield  and observed with a confocal fluorescence microscope  with an oil-immersion objective lens  and ASW v.1.3 software (Olympus). The fluorescence of DAPI, Alexa 488, and Alexa 555 was excited at 405, 488, and 543 nm laser wavelengths and detected through BA430-470, BA505-525, and BA560-600 nm bandpass filters, respectively. The pinhole diameters are set automatically according to the selected dyeing method, and imaging was performed at 8.0–10 μs/pixel (Scan speed) for 1024 × 768 (4:3) or 1024 × 1024 (1:1) pixels applying a sequential scan mode with Kalman filtering. The number of myotubes displaying cytoplasmic TDP-43 aggregations and the total number of myotubes, as assessed by the sarcomeric α-actinin staining profile for discriminating feeder 3T3L1 fibroblasts, were counted in each field, and the ratio of aggregated TDP-43-positive myotubes vs. the total number of myotubes was evaluated. Images were imported into Adobe Photoshop 6.0  for processing. Three independent experiments were performed under each condition.For evaluating TDP-43-positive nuclei and intracellular TDP-43 accumulations, we used Fiji ImageJ software . Briefly, we first defined the myotube boundary according to the anti-sarcomeric-α-actinin immunofluorescent signal, and then obtained all the nuclei (by DAPI) and TDP-43-positive nuclei according to their respective immunofluorescent signals. The number of all nuclei in the myotube was used as the denominator, and the number of all TDP-43-positive nuclei was used as the numerator to obtain the ratio of TDP-43-positive nuclei in the myotube. For evaluating cytoplasmic TDP-43 accumulation, we used the Image calculator function of Fiji ImageJ software. Briefly, we first determined the entire TDP-43-positive area and the TDP-43-positive nuclear area of the myotubes by delineating sarcomeric-α-actinin staining and DAPI staining, respectively. Then, we subtracted the TDP-positive nuclear area from the total TDP-43 area to obtain the fluorescent intensity of the TDP-43 accumulated in cytoplasmic regions. Finally, we counted numbers of total myotubes and the myotubes exhibiting cytoplasmic TDP-43 accumulation to determine the ratio of cytoplasmic TDP-43-positive myotubes. For the p62 area, we calculated the p62-positive puncta area to the total myotube area using Fiji ImageJ software.−∆CT method with reference genes. The following primers for qRT-PCR analyses were employed; for human CXCL1, 5ʹ-GCT TGC CTC AAT CCT GCA TC-3ʹ and 5ʹ-GGT CAG TTG GAT TTG TCA CTG T-3ʹ; for human IL6, 5ʹ-ATC TGG ATT CAA TGA GGA GAC T-3ʹ and 5ʹ-TGT TCC TCA CTA CTC TCA AAT CTG-3ʹ; for human TDP-43, 5ʹ-GAG AAG TTC TTA TGG TGC AGG TC-3ʹ; 5ʹ-GCT CAT CTT GGC TTT GCT TAG-3ʹ; for human p62/SQSTM1, 5ʹ-GCG TAG AAT TGC AGG TCT CTG T-3ʹ and 5ʹ-TCA CTT GTT TTG CTG CCC TAA ATG-3ʹ; for human RPLP0, 5ʹ-GGA AAC TCT GCA TTC TCG CT-3ʹ and 5ʹ-GCA AGT GGG AAG GTG TAA TCC-3ʹ.Total RNA was extracted from cells employing TRI reagent  and cDNA was then synthesized using a Transcriptor First Strand cDNA synthesis kit with oligo-dT primers . Next, qRT-PCR was performed with SsoAdvanced Universal SYBR Green Supermix, and detected with a Bio-Rad CFX Connect Systems . The relative expression levels of the target genes were calculated using the 215. As the healthy controls, we used intact subscapularis muscle specimens from three male patients, ranging in age from 60 to 68 years, who had agreed to undergo biopsy during surgical repair of arthroscopic rotator cuff tears46.The three patients, who had been clinically diagnosed with sIBM at Tohoku University Hospital, ranged in age from 69 to 86 years and had no family history of muscle diseases. The basis of the sIBM diagnosis was the presence of rimmed vacuoles in muscle biopsy tissue. The distribution of muscle weakness, creatin kinase activity, and the results of electrophysiological studies were evaluated during the disease course. Clinical data obtained from all sIBM patients, two males and one female, who had been hospitalized between 2015 and 2016 in the Department of Neurology of Tohoku University hospital, were evaluated47, with mouse monoclonal antibodies (diluted to 1:200) raised against p62  and mouse monoclonal antibodies  raised against phosphor-TDP-43 .Immunohistochemical staining was carried out using the avidin–biotin complex immunostaining method as previously described46. This study was approved by the Tohoku University Hospital Institutional Review Board , and written informed consent was obtained from all participants. All experiments were performed in accordance with relevant guidelines and regulations.Human satellite cells of both the three sIBM patients and the three healthy subjects were isolated from muscle tissue biopsy specimens, as previously reportedα (clone 16A1). The negative set included blood markers CD11b and CD45, and endothelial markers CD31 and CD34. While CD34 is known to be expressed by the majority of mouse satellite cells48, human muscle-derived CD34+ cells are myogenic and adipogenic, whereas CD34− cells are myogenic but not adipogenic49. We therefore used CD34 as a negative selection marker. Human satellite cells were defined as single live mononuclear CD11b−CD31−CD34−CD45−CD56+ cells. FACS was performed on a FACS ARIA II flow cytometer (BD Biosciences). Cells were cultured in a growth medium and cultured at 37 °C in a 5% CO2 atmosphere. When cells reached 60–80% confluence, adherent cells were split onto a new Matrigel-coated 15-cm dish to expand the activated satellite cells. Activated satellite cells (myoblasts) were suspended in Cell Banker  and stored in liquid nitrogen.Briefly, the muscle tissue was minced and digested with collagenase, filtered through a 70-μm cell strainer  and then subjected to immunostaining for FACS. Cells were incubated with an Fc receptor blocking solution and then labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD45 (clone HI30), FITC-conjugated anti-CD11b (clone ICRF444), FITC-conjugated anti-CD31 (clone WM59), phycoerythrin (PE)/Cy7-conjugated anti-CD34 (clone 581), allophycocyanin (APC)-conjugated anti-CD56 (clone MEM-188), and PE-conjugated anti-PDGFRt-test or ANOVA with Tukey’s multiple comparison test, and p values < 0.05 were considered to indicate a statistically significant difference unless otherwise specified. Data are expressed as means ± SE unless otherwise specified.Statistical analyses were performed using Student’s Supplementary Figures.Supplementary Video 1.Supplementary Video 2.


As COVID-19 took the world by storm, primary care clinicians (PCCs) played a critical role in identification and management of this disease. Yet, knowledge around COVID-19 is constantly evolving, leaving clinicians with many unanswered questions. We sought to examine what questions PCCs had about COVID-19 and whether there were any trends over time. We sought to examine what questions PCCs had about COVID-19 and whether there were any trends over time. We collected questions from PCCs during 4 live virtual 60-minute continuing medical education (CME) panel discussions on COVID-19 led by infectious disease experts from November 2020 to February 2021. Questions were independently sorted and analyzed by 2 MDs using constant-comparison and tie-break methodology. A total of 600 questions pertaining to COVID-19 were collected across 4 sessions. Top questions asked by PCCs ranked in descending order related to the following topics, with most common themes listed in parentheses: 1. Vaccines  2. Medication-Specific Treatment  3. Testing  4. Other Management  5. Personal Protective Equipment . [Table 1] The percentage of questions around vaccination increased from 5% of total questions in October 2020 to 67% in February 2021. Questions related to Treatment declined from 20% to 6%, Testing declined from 21% to 3%, Other Management declined from 6% to 1% and PPE increased from 3% to 8% during this period. Table 1. Top 5 topics of questions listed in descending order of frequency across all 4 COVID-19 panel sessions.Table 2. Percentage of questions in the top 5 topics for each of the 4 COVID-19 panel sessions, with associated trendline.PCCs nationally have gaps in knowledge around COVID-19 which can impact clinical decision-making. Based on our analysis of questions submitted by PCCs to infectious disease experts in a CME setting, the greatest gaps in knowledge were around vaccination, treatment, and testing with vaccination showing the greatest shift in interest over time.All Authors: No reported disclosures


Background: Recurrent or persistently active sarcoidosis is a risk factor for permanent organ damage. Whether this damage is due to accumulated focal injuries or progressive disease extent is not known, as the natural history of chronic inflammation in sarcoidosis is poorly characterized. The objective of this study is to determine the pattern of disease in recurrently active sarcoidosis.Methods: We identified patients with recurrent cardiac sarcoidosis (N = 21) retrospectively from an imaging database, and with recurrent cutaneous sarcoidosis (N = 17) from a prospective registry. The longitudinal patterns of cardiac sarcoidosis were established by findings on cardiac positron emission tomography scans, and of cutaneous sarcoidosis by the validated Cutaneous Sarcoidosis Activity and Morphology Instrument clinical scoring system. Patterns of recurrent disease were compared to baseline findings.Results: Recurrent sarcoidosis occurred in a nearly identical pattern and distribution as baseline disease, and spread of disease was rarely observed for both cardiac and cutaneous sarcoidosis: 97% of heart segments positive on recurrence scans were positive on baseline scans, and only one new region of facial disease was observed. In some cases, recurrence followed years of apparent remission.Discussion: Across phenotypes, and across a long period of follow-up, the extent of sarcoidosis was stable in spite of fluctuations in disease activity. For patients with a demonstrated history of recurrent disease affecting critical organs, our findings support the need for long-term follow-up. Sarcoidosis is an inflammatory disease of unknown etiology, marked by the formation of granulomas in affected tissues. The diagnosis is established by compatible clinical and histopathology findings, following the exclusion of other causes of granulomatous inflammation . While tKey immunopathologic events in sarcoidosis include an influx of CD4+ T cells, the release of inflammatory cytokines, and the formation of epithelioid granulomas at sites of disease. The oligoclonal expansion of CD4+ T cells and granuloma features support the fundamental assumption that sarcoidosis is antigen driven , 7. Yet,Treatment with immunosuppression can improve symptoms and the function of organs with active sarcoid inflammation. However, for patients with chronically active disease, improvements are often tenuous and several studies have demonstrated a high rate of recurrence following withdrawal of immunosuppression –12. Whet18F]fluorodeoxyglucose positron emission tomography (FDG-PET) imaging and expert visual inspection, respectively.With the hypothesis that the extent of recurrently active sarcoidosis is stable, we serially examined patients with chronic cardiac sarcoidosis and characterized longitudinal disease patterns. To test whether our observations are restricted to a single organ phenotype, we also measured the pattern of recurrently active lymph node disease and, in a separate cohort with cutaneous involvement, of recurrently active cutaneous sarcoidosis. These phenotypes were chosen as they are amenable to anatomical disease tracking via serial assessments of disease activity with [18F]FDG-PET studies for suspected active myocardial disease from 2006-2018 . Subjects were accrued from two sources: (1) a clinical database of patients with sarcoidosis who underwent three or more thoracic FDG, a glucose analog  at each visit, with disease activity mapped via the cutaneous sarcoidosis activity and morphology instrument (CSAMI) . For thiCalculations were performed using Graphpad Prism 8.0 . Where indicated, data were expressed as means +/-SD.From the 39 subjects with cardiac sarcoidosis who had undergone at least three valid cardiac PET scans, 21 had evidence of recurrent disease. Nearly all subjects (18/21) were White, and 71% (15/21) were male . The diaAcross the 21 subjects with recurrent cardiac sarcoidosis, a combined 163 diagnostic PET scans were available for review. At baseline, cardiac sarcoidosis was highly prevalent throughout the left ventricle (LV). On average, 9.6 segments  on baseline scans demonstrated increased FDG uptake. Certain regions of the LV were more likely to be affected than others . IncreasThe first negative PET scan following a positive baseline or recurrent PET scan defined a resolution event. For over half of these events , resolution was evident on the first scan following the initiation of treatment. However, 18 resolution events followed multiple sequential positive PET scans. For most (16/18) of these events of delayed resolution, the presence of persistently positive PET scans was associated with failure to initiate treatment, or initiation of an insufficient regimen as clearance was eventually achieved following an increase in treatment. Subject 13 , was an For the majority of subjects (17/21), the distribution of recurrent sarcoid inflammation occurred at anatomical locations that were grossly identical to the location of original lesions . Of the Of the 104 patients in the cutaneous sarcoidosis database, 38 had three or more visits. Of those, 17 had a CSAMI score decrease of >5 at a follow-up visit, followed by recurrent disease involving the face. Facial sub-compartments assessed on serial skin exams included the nose, cheeks, perioral regions, and ears. Patients had on average 2 regions affected by sarcoidosis. Peri-nasal disease  was most common, followed by disease involving the cheek . Perioral disease was observed in 9/17 subjects at baseline, 7 of whom had disease recurrence at this site. Ear involvement was less common (3/17 at baseline). For 94% (16/17) of cases, recurrently active lesions occurred in the same region as initial disease activity . Direct A total of 16 subjects had PET scan findings consistent with recurrent lymph node inflammation; 12 were from the recurrent cardiac sarcoidosis cohort and an additional four subjects were identified from the 37 subjects with cardiac sarcoidosis screened for recurrent disease . While rWe found that recurrent cardiac sarcoidosis has a strong predilection to recur in a distribution similar to previously active disease. The same finding was observed for patients with recurrently active cutaneous sarcoidosis. The finding that the extent of disease remains remarkably stable over time was observed over many years of follow-up. Our data suggest that, for a given organ affected by sarcoidosis, persistent or recurrent inflammation, with the potential transition to irreversible fibrosis, are more concerning risks than local expansion of disease. For approximately half of our cardiac sarcoidosis cohort, recurrence events either did not trigger initiation of treatment or were followed by an insufficient regimen such that multiple serial cardiac PET scans were positive, in some cases resulting in active disease spanning several years. For these cases, disease extent still remained stable. Therefore, even in the setting of persistently active inflammation, cardiac sarcoidosis does not appear to have a high propensity to progress beyond the initial extent of disease. This novel finding has profound implications for understanding and treating chronic sarcoidosis.Lymph node involvement is common in sarcoidosis, and many of our patients had their diagnosis established by lymph node biopsy. However, the specificity of PET imaging for nodal sarcoidosis activity is unknown, and increased nodal FDG uptake may be seen in a variety of inflammatory conditions. In contrast to the findings for cardiac and cutaneous sarcoidosis, the distribution of thoracic lymph node activity was more variable over time, with a higher rate of new FDG avid lymph node stations observed on recurrently positive PET scans. This finding may be due to a lower specificity of PET imaging for nodal sarcoidosis activity, with the possibility of false positive findings on follow-up scans. Alternatively, the lymph node network truly may be more prone to a progressive extent of disease in patients with chronic sarcoidosis.The stability of lesional boundaries in cardiac and cutaneous sarcoidosis contrasts with the possibility that the systemic burden of sarcoidosis may increase over time, which has been observed in previous studies of patients followed longitudinally . An incrWhile immunosuppression therapy is often successful in suppressing inflammation, disease recurrence following cessation is a vexing outcome in both cardiac and cutaneous sarcoidosis . The optThe mechanism of recurrent sarcoidosis is unknown. In a study on recurrent Lofgren's disease, repeat exposure to antigen was invoked to account for episodes of recurrence occurring remote from the time of initial disease . In contOne cautious interpretation of our findings is that the containment of antigen is not compromised over time. Altered immunity has been implicated in antigenic spread in tuberculosis, another granulomatous disease , 29. YetThere are several important limitations of this study. The ability to anatomically localize inflammatory lesions by imaging techniques, such as PET, is inherently imprecise. We cannot rule out that minor or microscopic changes in the extent of disease occurred. We found that the average extent of disease was lower at disease recurrence compared to baseline scans. However, treatment was often started or increased upon disease recurrence, which may chemically constrain the extent of inflammation. This single site study has a modest cohort size; larger studies are needed to more robustly establish the patterns of chronically active disease. For two subjects with cardiac sarcoidosis, biopsy was deferred after a careful risk-benefit assessment, and the diagnosis was established on other clinical grounds. While a common situation, the diagnosis is less certain in such cases. Finally, we recognize the stark differences in demographics between our cohorts of cardiac and cutaneous sarcoidosis. These differences raise concern that identification of cardiac sarcoidosis, which is often done through screening studies initiated by health care providers, may be influenced by demographics. Differences in access to health care may also play a role.We focused on the evaluation of organs which tend to have countable and localizable lesions, and our findings may not be generalizable to all phenotypes of sarcoidosis. An especially important question un-answered by our study is the nature of recurrent lung sarcoidosis. Tracking individual lung lesions, which are often tiny, clustered, or indistinct, is inherently less precise than tracking the more distinct cardiac and cutaneous lesions. Yet, a better understanding of how pulmonary sarcoidosis progresses is critically important as chronic lung disease, along with cardiac involvement, accounts for the majority of fatal outcomes in sarcoidosis . While wIn conclusion, the evolution of sarcoidosis has been a key unknown of the disease process, and a challenge for clinical management. In our cohorts of cardiac and cutaneous sarcoidosis, the extent of inflammation in recurrently active disease was strikingly stable. The anatomical fidelity of recurrent disease activity raises the possibility that the sarcoid antigen is contained but not cleared. A paradigm shift away from sole reliance on long-term immunosuppression to the development of therapeutics which target immune clearance or halt fibrotic transformation is needed. Our findings also indicate that, for patients with demonstrated disease recurrence or with high-stakes organ involvement, long-term follow-up is warranted even for those who appear to be in clinical remission soon after stopping treatment.Upon request, the raw data supporting the conclusions of this article will be available by the authors.The studies involving human participants were reviewed and approved by University of Pennsylvania Institutional Review Board. Written informed consent for participation was obtained for patients in the cutaneous sarcoidosis cohort, but was not required for the cardiac sarcoidosis cohort in accordance with institutional requirements. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images included in this article.KP had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors have made substantial contributions to the work reported in this manuscript, including study design, data collection and analysis, and writing and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Graphene nanoribbons are a type of graphene characterized by remarkable electrical and mechanical properties. This review considers the prospects for the application of graphene ribbons in biomedicine, taking into account safety aspects. According to the analysis of the recent studies, the topical areas of using graphene nanoribbons include mechanical, chemical, photo- and acoustic sensors, devices for the direct sequencing of biological macromolecules, including DNA, gene and drug delivery vehicles, and tissue engineering. There is evidence of good biocompatibility of graphene nanoribbons with human cell lines, but a number of researchers have revealed toxic effects, including cytotoxicity and genotoxicity. Moreover, the damaging effects of nanoribbons are often higher than those of chemical analogs, for instance, graphene oxide nanoplates. The possible mechanism of toxicity is the ability of graphene nanoribbons to damage the cell membrane mechanically, stimulate reactive oxidative stress (ROS) production, autophagy, and inhibition of proliferation, as well as apoptosis induction, DNA fragmentation, and the formation of chromosomal aberrations. At the same time, the biodegradability of graphene nanoribbons under the environmental factors has been proven. In general, this review allows us to conclude that graphene nanoribbons, as components of high-precision nanodevices and therapeutic agents, have significant potential for biomedical applications; however, additional studies of their safety are needed. Particular emphasis should be placed on the lack of information about the effect of graphene nanoribbons on the organism as a whole obtained from in vivo experiments, as well as about their ecological toxicity, accumulation, migration, and destruction within ecosystems. Graphene nanoribbons (GNRs) are narrow strips of graphene composed of repeating hexagonal carbon cells, up to 50 nm wide and up to several dozens of micrometers long, depending on the synthesis method . GraphenMoreover, the structure and physical properties of GNRs vary significantly, depending on the synthesis method. In fact, GNRs obtained via different methods are very different and have little in common. Today, three main approaches to obtaining GNRs exist :1. Electron-beam lithography and photolithography: This method makes it possible to obtain single-layer GNRs on the substrate surface; however, the fields of application of lithographically obtained GNRs are very limited. In addition, the ribbons obtained by the lithographic method have jagged edges ,13,14.2. Bottom-up synthesis from polycyclic molecules: This method includes multistage organic synthesis based on the cyclization of previously synthesized polymer chains. This method provides very narrow ribbons with an atomically precise edge configuration ,16,17,183. Unwrapping carbon nanotubes (CNTs): The third approach is based on longitudinal opening or cutting of multiwalled carbon nanotubes (MCNTs) ,20. ThisTypical characteristics of GNRs are determined by transmission electron microscopy (TEM), atomic force microscopy (AFM), Raman-scattering spectroscopy, and X-ray photoelectron spectroscopy (XPS).As previously noted, GNRs can be considered as quasi-one-dimensional graphene strips. This makes them similar to a large class of conjugated polymers, for which the synthesis conditions and the method of film formation determine the performance characteristics ,23. The GNRs with armchair-type and zigzag-type edges are the most common ones. AGNRs are characterized by a wide band gap that varies with width, while ZGNRs are predicted to have smaller bandgaps with localized edge states that are magnetic and show great potential for spintronic applications ,28,29,30Thus, to take full advantage of the exceptional characteristics of GNRs for practical applications, it is necessary to take into account the fabrication method and the type of spatial structure.Remarkable electrical and mechanical properties of GNRs make them a promising material for biological and medical applications ,34,35,36According to the available information, GNRs in biomedicine are mainly used to create various ultra-small devices such as molecular sensors, photo-, thermo-, and acoustic detectors, sequencers, drug and gene delivery vehicles, and tissue engineering constructions .The research works aimed at the integration of GNRs into devices are going in several directions, but all of these devices require connections and heterojunctions .8 V/m at the heterojunction, these materials are applicable for the photovoltaic industry and electronics 85], the The analysis of the cytotoxicity of films consisting of chitosan, alginate, and 2.5 wt % GNRs (CHI/f–GNR–ALG) obtained by unzipping MWCNTs on the mouse fibroblasts (L929) showed the sample cytocompatibility. In particular, L929 is able to proliferate on the surface of CHI/f–GNR–ALG (200 µL) . On thisForeman et al.  analyzed50 values for chemo-photothermal therapy toward U87 glioma cells than the IC50 values in traditional chemotherapy. They found that PL–PEG–O-GNRs was excreted from the body via the renal pathway in the urine, and the hematological analysis showed that this nanomaterial was not toxic. The authors consider PL-PEG-O-GNRs as potential nanocarriers of drugs to develop an effective cancer treatment strategy that will not only increase the effectiveness of therapy, but also reduce the risk of side effects of the nanocarrier in the body.The authors of reference  investigThus, there is evidence that the cytotoxicity of GNRs can be significantly reduced by their edge or surface chemical modification. For example, doping with nitrogen, oxidation, coating with biocompatible polymers such as PEG or chitosan give an obvious increase in biocompatibility. The hydrophilization of carbon nanomaterials is a versatile strategy to provide their bioavailability and applicability in biomedicine. This is especially important to take into account when developing bioengineering constructs based on GNR, which should have minimal cytotoxicity.According to Mbeh et al. , O-GNRs The results of the study  showed tThe same authors showed that O-GNR–PEG-DSPE at concentrations of 20, 80, and 160 μg/mL caused a low concentration-dependent deformation of erythrocytes, which did not lead to hemolysis . There wIn the study , the cytChowdhury et al.  demonstrA comparative analysis of the toxicity of single-layer reduced O-GNRs (rO-GNRs) and reduced graphene oxide sheets (rGOS) in relation to human mesenchymal stem cells (hMSCs) showed significant cytotoxic effects of rO-GNR at a concentration of 10 μg/mL of after 1 h of exposure, while rOGS showed the same degree of toxicity at a concentration of 100 μg/mL after 96 h. The main mechanism of rGOS action was assumed to be oxidative stress, which causes minor damage to the cell membrane. While the outflow of RNA from hMSCs showed that neither the formation of reactive oxygen nor significant damage of the membranes cells can explain the destruction of cells caused by rO-GNRs. The results also showed that rO-GNRs could penetrate cells and cause DNA fragmentation and chromosomal aberrations even at a low concentration of 1.0 μg/mL, after a short exposure time of 1 h ,95.Talukdar et al.  investigFor the imaging and phototherapy of human glioblastoma (U87MG), Akhavan et al. functionalized rO-GNRs with polyethylene glycol (rO-GNR–PEG). Cytotoxic and genotoxic effects on the cells depended on the concentration of rO-GNR–PEG. When the cells were incubated in the dark with 100 μg/mL of rO-GNR–PEG for 24 h, more than 72% of cell death and more than 29% of DNA fragmentation were observed. At a lower concentration (1 μg/mL), cell death and DNA fragmentation decreased to about 11% and 7%, respectively ,98.According to the considered researches on cyto- and genotoxicity, the possible mechanisms of the toxic action of GNRs can be suggested mechanical damage to cell membranes; ROS production, which leads to the inhibition of proliferation, induction of apoptosis, autophagy, and DNA fragmentation; chromosomal aberrations .It should be noted that the studies of the toxicity of GNRs for the whole organism have not yet been carried out, excluding the work of Lu et al. . HoweverSimilar to other carbon nanomaterials, GNRs can enter the environment not only as part of the nanoindustry products, but also during natural processes, for example, during the combustion of organic matter. There is no information on the environmental toxicity of GNRs for ecosystems in the analyzed researches. An exception is the work of Lalwani et al., which investigates the oxidative biodegradation of O-GNRs and rO-GNRs by lignin peroxidase (LiP) . LiP is The studies that are concerned with the closest chemical and structural analogues of GNRs–CNTs and graphene indicate their significant effects on various types of living organisms. A large number of studies demonstrate the toxic effects of carbon nanomaterials in relation to bacteria ,107,108,Based on this, it can be assumed that GNRs can also have a significant impact on components of ecosystems, especially microorganisms, and it can be bioaccumulated and migrate within ecosystems. These issues certainly warrant further study.GNRs demonstrate the greatest prospects in the field of biomedicine, particularly in creating nanodevices for the biomolecules detection and single-molecular techniques. In addition, GNRs are of interest as gene and drug delivery vehicles. There are researches that prove the possibility of using GNRs in tissue engineering.Practical applications of GNRs in direct contact with the human body are limited by their potential toxicity. A number of researchers have noted the damaging effects of GNRs, including cytotoxicity and genotoxicity. Moreover, the toxicity of nanoribbons is higher than those of such chemical analogs as O-GNPs. According to several studies, the possible mechanisms of toxicity are as follows: the ability to induce ROS production and autophagy; inhibition of proliferation; induction of apoptosis; DNA fragmentation; chromosomal aberrations. However, other authors have not confirmed any toxic effects of GNRs in experiments with human cell lines. The discrepancy in the observed results can be explained by differences in the methods of synthesis and structure of GNRs, their functionalization by different groups, and the use of different concentrations. In addition, different cell lines exhibit individual responses to exposure to nanomaterials. Furthermore, in order to obtain consistent results, the unification of the nanotoxicological experimental conditions is needed.The chemical modification of the surface of GNRs with hydrophilic groups can significantly increase their bioavailability and biocompatibility.There is an evidence of the biodegradability of GNRs in the environment. By analogy with other carbon nanomaterials, GNRs can be toxic to living organisms of various species. They can be bioaccumulated and migrate through ecosystems, but there are currently no data on this.In general, this review allows concluding that GNRs, as components of high-precision nanodevices and therapeutic agents, have good prospects for application in biomedicine, but the limiting factors for the use of GNRs are their high hydrophobicity and insufficiently studied toxicity. There is a lack of information on the effect of GNRs on the whole organism using in vivo experiments, as well as on environmental toxicity, accumulation, migration, and destruction within ecosystems.


Microaggressions, a concept originally introduced for ethnic minorities, represent subtle day‐to‐day discrimination, damaging the psychological health and well‐being of lesbian, gay, bisexual, and transgender individuals as well. This study aimed to assess whether microaggressions occur in psychotherapists’ assessments of clients who identify as either lesbian or transgender woman when compared with those identifying as heterosexual woman.N = 5) and microaggressive (N = 5), used to determine a clinical impression of the client. A repeated measure analysis of variance was conducted to understand the likelihood of clients of different gender identity and sexual orientation receiving microaggressions.The study included a sample of 135 licensed psychotherapists (110 cisgender women and 25 cisgender men). Participants were presented with an audio file of a woman introducing herself during her first therapy session. Three versions were presented: a transgender, a lesbian, and a heterosexual client. Participants were asked to assess the clinical relevance of 10 questions defined as neutral (Participants were more prone to consider microaggressive questions relevant where the client identified as either lesbian or transgender.The findings highlighted a bias against lesbian and trans women in evaluating the relevance and usefulness of clinical information while making a psychological assessment of a case. In an eTransgender people also experience situations in which they necessarily have to come out , which can expose them to an increased risk of discrimination and its consequences  link of the patient's gender identity or sexual orientation to the problematic or symptomatic aspects presented. We also expect that the self‐assessed emotional arousal generated by a patient belonging to a marginalized group will moderate the relevance attributed to microaggressive questions for the transgender and lesbian condition. Moreover, starting from the evidence of the relation between emotional arousal and automatic responses, we hypothesized highly activated clinicians to be more prone to use microaggressions toward sexual and gender minorities than clinicians at moderate levels of arousal.2To test whether psychotherapists could enact microaggressions in the context of the first consultation with a client, we had to create ad hoc questions that were relevant to the presented case. For this purpose, we asked 84 early career psychologists to listen to the audio files, that were then used in the main study, and produce up to 10 questions they would have asked the client to have a better understanding of the clinical case.Participants generated 575 questions in total, with an average of 6.8 questions each. Three different raters (blind to the study's hypotheses) independently classified each of the items as nonmicroaggressive, possibly microaggressive, or microaggressive. The raters were trained to recognize microaggressions; they were provided with the definition of microaggressions several examples of microaggressions targeting sexual and gender minorities. Approximately 30.9% of the sample produced at least one microaggressive question (rated as microaggression by two out of three raters). Only the questions from the original pool of items on which all three raters agreed upon were used to construct the questions for the following study.33.13.1.1η2partial = 0.25) suggested a sample size of N = 120 for a 3 between‐participants × 2 within‐participants analysis of variance (ANOVA). In total, 135 licensed psychotherapists took part in the study, all of whom self‐identified as cisgender, White Italian citizens, with 110 (81.5%) identifying as female. Other demographic characteristics of the sample are summarized in Table An a priori power analysis was conducted to estimate the required sample size using G*Power  introducing herself and describing her problems (related to panic) during the first session with the participant  to 7 (extremely relevant). The questions reflected both microaggressive and neutral themes. The questions generated from the pilot study were used to build five microaggressive (Cronbach's α = 0.90) and five neutral questions (Cronbach's α = 0.71) for the current study  Hayes, . Finally44.1To compare the relevance of microaggressive and neutral questions depending on the sexual identity of the client, we computed a 3  × 2  ANOVA with the first‐factor as between‐participants and the second‐factor as within‐participants.F  = 119.36, p < 0.001, ηp2 = 0.47. Independent from the experimental condition, neutral questions were considered significantly more relevant  than microaggressive questions . The ANOVA did not yield a significant main effect of condition: F  = 1.76, p = 0.18, ηp2 = 0.03. However, in line with the hypothesis, a significant interaction effect between condition and type of questions emerged: F  = 14.56, p < 0.001, ηp2 = 0.18. As displayed in Figure M = 5.70, SD = 0.75), than in the transgender client condition , t(86) = 2.22, p = 0.03, d = 0.48, 95% confidence interval [CI] [0.05–0.90]. The analysis revealed no differences between cisgender lesbian  and transgender conditions, t(95) = 0.92, p = 0.36, and between cisgender straight and lesbian conditions, t(83) = 1.36, p = 0.18. The post hoc comparisons of the relevance of microaggressive questions showed no difference between the cisgender lesbian  and transgender conditions , t(95) = 1.68, p = 0.09. Crucially, microaggressive questions were rated as significantly less relevant for the cisgender straight  than for the cisgender lesbian client, t(83) = 2.26, p = 0.03, d = 0.49, 95% CI [0.06–0.93], and the transgender client, t(86) = 3.94, p < 0.001, d = 0.85, 95% CI [0.41–1.29].The analysis suggested a main effect for the type of questions: F , 132 = 14.2α = 0.78), suggesting that both positive and negative items of the scale measured the same construct. A more theoretical reason is based on the argument that the positive affect and negative affect subscales of the PANAS both measure the latent construct of emotional arousal . At a high level of arousal ( = 2.55), therapists rated microaggressive questions more relevant for cisgender lesbian and transgender women than for cisgender straight women . At low levels of arousal ( = 1.85), the difference between cisgender straight and cisgender lesbian/transgender conditions was not significant .To ascertain whether emotional arousal influenced the relevance attributed to microaggressive questions, a moderation model was carried out through PROCESS  Hayes, . In this4.3F  = 0.001, p = 0.97, or any interaction effects with gender . The same results were obtained introducing sexual orientation as an independent variable . We also controlled for the effect of the therapists’ theoretical background  and years of practice on relevance evaluations. No interaction or main effects of theoretical background were identified , or for years of practice .To ascertain whether the therapists’ individual differences influenced their responses, we controlled for the effect of the therapists’ gender and sexual orientation. The first ANOVA yielded no significant main effect for gender, 5The aim of this study was to investigate the presence of microaggressive responses when psychotherapists work with clients of a marginalized sexual orientation or gender identity . The findings highlighted a bias in evaluating the relevance and usefulness of information while making a psychological assessment of a case. Data showed the effect of the client's gender identity and sexual orientation in rating the relevance of microaggressive questions by the therapists. Such questions were considered less important for the normative straight cisgender client and more important for the less conforming clients (cisgender lesbian and transgender). Conversely, asking general questions about symptoms, life events, and mental health history was considered less relevant for the assessment in the transgender client condition compared to the cisgender straight client condition.Results indicated that both cisgender lesbian and transgender women were equally targets of microaggressions. In addition, neutral questions were considered significantly less relevant only in the transgender condition compared with the cisgender straight condition. This result could be explained by considering the long history of pathologization of transgender identities. Once a client is labeled as transgender, they are immediately “diagnosed” by some of the psychotherapists that probably still rely on a pathologizing understanding of transgender identities. Thus, the main issues and complaints they bring to the consultation fade into the background as the therapist no longer needs additional information. Such bias relies on specific assumptions based on cisgenderism and heteronormativity .6In spite of these limitations, the study presents numerous strengths, including investigating microaggressions directed toward gender/sexual minorities “in vivo,” with a sample of licensed and experienced psychotherapists. This approach, rather than relying on the reported experiences of LGBT patients in therapy, allows observing directly the therapists' response to the client's disclosure of information. The use of an audio file in which the client introduces herself identifying as lesbian, trans, or heterosexual, allows the understanding of the intrinsic and implicit functioning of microaggressions through the approach of the therapist. Furthermore, the inclusion of emotional arousal among the study variables allows spreading lights on other dimensions that can potentially be targeted in the training of mental health professionals on LGBT issues.https://publons.com/publon/10.1002/jclp.23140The peer review history for this article is available at


The relative free volume fraction rf gives a good explanation of the variation for surface resistivity, melting temperature, and thermal stability, and the variation of tensile strength and thermal conductivity agree well with the results of interfacial interaction intensity β. The results showed that rf and β have a significant effect on the properties of the obtained graphene/PE composites, and the effect on the properties was revealed.The quantitative characterization of microstructure is most desirable for the establishment of structure-property relationships in polymer nanocomposites. In this work, the effects of graphene on the microstructure, mechanical, electrical, and thermal properties of the obtained graphene/polyethylene (PE) composites were investigated. In order to reveal the structure-performance relationship of graphene/PE composites, especially for the effects of the relative free volume fraction ( Polyethylene (PE) is one of the most important commercial polymer materials. In the PE family, low density PE has a wide application in our daily life due to its low cost and good flexibility ,2. When rf) of the obtained composites, and the interfacial interaction (β) between graphene and the polymer matrix. As β determines the dispersion state of graphene, it has a dramatic influence on graphene’s stress dispersion and phonon scattering. In addition, the rf determines the transport performance, such as electrical conductivity, thermal stability, and gas/liquid barrier properties [rf and β), and the correlation between these parameters and properties for graphene/PE nanocomposites.The key factor that influences the properties of graphene/polymer composites is the microstructure, including the relative free volume fraction . Positrons are preferentially localized in the cavity of the free volume hole. According to the free volume hole model, the pick-off annihilation lifetime τ3 is related to the cavity size. When the cavity size of the free volume hole increases, the opportunity of electron seen by o-Ps decreases, leading the lifetime of o-Ps to become longer [Positron annihilation lifetime spectroscopy (PALS) is a useful tool in determining the atomic scale defects and free volume hole for a wide variety of materials ,29. The e longer .τ2 is assigned to the positron annihilation in various vacancies [I2) is related to the interfacial interaction in the polymer composites. According to the simple mixture rule, if there is no interfacial interaction between polymer matrix and filler, I2 only comes from the annihilation in the matrix and filler, respectively. In this case, I2 is the linear correlation with the content of filler. Actually, the interfacial interaction always exists in polymer composites, thus deviations occurred between the theoretical results and the experimental results [β was introduced to investigate the interface between the polymer matrix and the filler [Recently, the PALS technique can not only be used for the probe of the free volume hole, but also for the characterization of the interfacial interaction. The second lifetime acancies . The cha results ,32. Conse filler ,34. Thisrf and β had a significant effect on the mechanical, electrical, and thermal properties of the obtained graphene/PE composites, and their effect on the properties was revealed.In this work, the PE incorporated with different contents of graphene were prepared by premixing followed by hot pressing. Firstly, the interfacial interaction of the obtained graphene/PE composites was qualitatively characterized with traditional infrared spectroscopy. In order to give a deep insight into the microstructure, PALS was used to determine the free volume hole characteristics and interfacial interaction intensity of graphene/PE composites. The results showed that the Graphene was purchased from Suzhou TanFeng Graphene Tech Co., Ltd. . Low density polyethylene  was purchased from Aladdin Chemical Reagent Co., Ltd. . n-Hexane was obtained from Sinopharm Chemical Reagent Co., Ltd.  without purification.Graphene/PE composites with different graphene concentration (0~2.0 wt%) were prepared by premixing followed by hot pressing. Following typical procedure, the graphene was dispersed into 40 mL n-Hexane using a bath sonicator for 1 h. Then, 8 g of PE powder was added into the dispersion with vigorous stirring for 2 h and the mixture was sonicated for 1 h. The obtained mixture was centrifuged at 4000 r/min for 0.5 h and the precipitate was dried at 60 °C under vacuum to obtain a constant weight. Finally, the obtained mixture was pressed at 140 °C, 20 MPa for 10 min with a d = 0.5 mm steel mould.Fourier transform infrared (FTIR) spectra were recorded on an Avatar 360 Nicolet instrument  by measuring the powder mixture. The mechanical properties of the graphene/PE composites were measured by a tensile testing machine . Five pieces of each sample were tested to obtain average values. The surface resistance of the obtained composites was measured with an ultra-high resistance micro-current tester . The thermal conductivity of the composites was measured using thermal conductivity test equipment . The Differential scanning calorimeter (DSC) curves were recorded with a DSC-200-F3  from 30 to 160 °C, with a heating rate of 10 °C/min. Thermogravimeter (TG) analysis was conducted with a TG-209-F3  under nitrogen atmosphere with a heating rate of 10 °C/min.−1, the C-H bending vibration signal at 1471 cm−1, and the C-H rocking vibration signal at 721 cm−1—are present in the spectrum of PE [−1 observed in the composite, and this change in the C-H bending vibration signal is evidence supporting the occurrence of CH–π interactions between PE and graphene sheets [To demonstrate the chemical structure of pure PE and reveal the interfacial interaction between PE and graphene, the FTIR spectra of PE and 1 wt% graphene/PE composites were shown in um of PE . All sete sheets ,35. Acco22Na (15 μCi) was sealed in a 7 μm thick Kapton foil and sandwiched between two identical pieces of sample for measurement. One million counts were recorded for each spectrum, which was resolved into three components by a PATFIT fitting program. The long lifetime component τ3 was used to calculate the volume of free volume hole (fV) according to the following equations:To quantitatively reveal the effects of graphene on the microstructure of graphene/PE composites, the PALS was recorded. The PALS was carried out by a conventional fast–fast coincidence spectrometer at room temperature. The positron source R represents the radius of the cavity, and ΔR = 1.656 Å [o-Ps (I3) is related to the concentration of the free volume hole. The fraction (f) of free volume hole was calculated as: 1.656 Å ,33. The C can be approximately regarded as a constant [−3; the relative free volume fraction (rf) is defined as:constant ,36, withτ3), free volume hole size (fV), the formation probability of o-Ps (I3) and the relative fraction (rf) of the free volume hole as a function of graphene content were shown in τ3 than that of pure PE, suggesting that the obtained composites have a lower mean size of free volume hole, which is consistent with the results of PVA/rGO composites [τ3 and fV for the composites were observed containing 0.25 wt% graphene. This can be ascribed to the highly exfoliated and well dispersed graphene at low content, where the large surface area provided by the graphene is beneficial to the formation of CH-π interaction  is opposite to the o-Ps lifetime, which has also been observed in the PVA/rGO composites reported by Wang and co-workers [I3. However, with a further increase of graphene content, this disruption of graphene gets weak due to the agglomeration of graphene, which results in the decrease of free volume hole concentration and I3 [As show in -workers . This can and I3 ,33.rf) is present in rf decreases with the increase of graphene content (31.63% to 28.38%), meaning that graphene sheets limit the moving space of PE chains [Based on the analysis of the free volume hole size and the free volume hole concentration, the relative free volume fraction (E chains . These rI2) change of the second lifetime can be used to analyse the interfacial interaction intensity in the composites. The interfacial interaction determines the dispersion state of graphene; thus, it has a dramatic influence on its stress dispersion. For the description of the interfacial interaction, PALS is more useful than FTIR spectra. The FTIR can only determine the type of interfacial interaction , while PI2 only originated from positron annihilation in graphene and PE, which should be a linear correlation to the content of graphene. Actually, interfacial interaction exists in most polymer composites, and variation with the change of nanofiller content. Consequently, the interfacial interaction parameter (β) was introduced to reveal the interaction intensity between graphene and the PE matrix, and the β can be calculated according the following equation [According to the simple mixture rule, if no interfacial interaction exists between the graphene and the PE matrix, equation ,38,39,40G and P represent the graphene and PE, respectively; W is the graphene weight fraction; the I2 of graphene here is 94.78% [β as a function of the graphene content is shown in β (1.99), indicating the strongest interfacial interaction. With the increase of graphene content, the β decreased gradually, which is due to the aggregation and poor dispersion of graphene sheets at high loading [fV, which will have a significant effect on the mechanical property and thermal conductivity of the obtained composites.The superscripts s 94.78% . The int loading ,37, Thisβ against graphene content  of the obtained composites [rf can produce a high-density interfacial region, restricting the mobility and relaxation of PE chains, which is of benefit for the formation of a conductive network, thus lower surface resistivity composites were obtained with a lower rf. Similar phenomena have also been observed in the PC/rGO composites [According to electrical percolation theory, graphene sheets provide percolated pathways for electron transfer, which impart electrical conductivity to the composites. However, the improved efficiency of graphene sheets dramatically depends on the relative free volume fraction  characterization of PE and graphene/PE composites . The results of FTIR absorption spectra revealed that the CH-π interaction between PE and graphene can be formed in the processing. The CH-π interaction can serve as a sacrificial bond to dissipate stress, leading to the interaction intensity β having the same trend as the tensile strength. The highest values of β (1.99) and tensile strength (11.35 MPa) were found for the composites containing 0.25 wt% graphene. The decreased rf is beneficial for the formation of a conductive network, thus the lowest log(surface resistivity) (9.65) was obtained for those composites possessing the lowest rf (28.38%). The strong interaction intensity β for the 0.25 wt% graphene/PE composites can scatter phonons and damp the vibration amplitude, inducing a higher thermal resistance. As the content of graphene increased from 0 to 2.0 wt%, the rf decreased from 31.63% to 28.38%, making movement of the polymer chain more difficult. This leads to an increase of mT from 109 °C to 112 °C. As the low rf can prevent the heat transfer and the diffusion of pyrolysis products, the greatest thermal stability was obtained for the composites possessing the lowest rf (2.0 wt% graphene/PE). These results give deep insight into the relationship between structure and performance for polymer composites; future work will focus on the establishment of quantitative relationships.In summary, the graphene/PE composites were prepared by premixing followed by hot pressing; the microstructure of the obtained composites was determined simultaneously by using the qualitative (FTIR) and quantitative (PALS) methods. Enhanced properties were obtained for the graphene/PE composites, and the structure–performance relationship was established through the quantitative characterization of microstructure (


Bioengineered have retracted the following article:We, the authors, Editors and Publisher of the journal Bioengineered, Volume 11, 2020, Pages 958-971.“Upregulation of oxidative stress-responsive 1(OXSR1) predicts poor prognosis and promotes hepatocellular carcinoma progression,” Jianhui Chen, Jiangfan Zhou, Haotian Fu, Xiaofeng Ni & Yufeng Shan. Since publication, the authors raised the following concerns:Recently, in order to further explore the molecular mechanism of oxidative stress-responsive1(OXSR1) promoting the malignant progression of hepatocellular carcinoma (HCC), we repeated the previous research results. However, when we repeated the proliferation experiments, we found that compared with the result of the control group, there was no statistically significant difference in proliferation after knocking down OXSR1. Therefore, we continued to repeat the proliferation experiments several times. Unfortunately, the results of our repeated experiments did not show statistical differences. This indicates that the OXSR1 that the previous study proved to promote the proliferation of HCC is an unreliable result. Actually, OXSR1 does not promote the proliferation of HCC. Considering that in our previous study, OXSR1 promotes the proliferation of HCC is a very important conclusion, we decided to retract this article.The authors alerted the issue to the Editor and Publisher and all have agreed to retract the article to ensure the integrity of the scholarly record.We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions.The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.