We describe how mechanical strain is able to control the flow of heat on a graphene sheet, since this material can either absorb or expel heat from/to a thermal reservoir, depending on the strain energy. In a similar fashion as the magneto- and electro-caloric effects, the present case considers the fact that a mechanical strain produces a pseudo-magnetic field that, on its turn, is responsible for the barocaloric effect. This result pushes graphene to the list of multicaloric materials. Just to name a few, we can cite its electrical, mechanical and thermal features, like a high electronic mobility2, strong mechanical resistance3 and, in addition, tunable thermal conductivity - from ultra-high (for heat-sinking applications), down to ultra-low values (for thermoelectric applications)4.Graphene has outstanding physical properties5, optoelectronics6, electro-7 and magneto-caloric9 effects. These last two are of great importance for the present article. Few years ago, one of us reported the interesting caloric properties of a graphene sheet due to a change of either a magnetic or an electric field; and the results were the oscillating magnetocaloric8 (OMCE) and electrocaloric7 (OECE) effects, respectively. Few years later, a follow-up of these papers appeared, discussing the properties of the OMCE of a multilayer graphene9 and applications into a six-stages thermodynamic cycle10.However, one special reason to justify the always fresh and renewed interest of the scientific community on graphene is the possibility of interplay of many of those outstanding, extraordinary properties. For instance, we can mention graphene plasmonics11), to exchange heat with a thermal reservoir ; or change its temperature  due to a change of an external parameter, say, either a magnetic or an electric field. The amount of heat exchanged with a thermal reservoir, considering an isothermal process, is given by 11; but, surprisingly, diamagnetic materials also present this effect (much reduced in magnitude in comparison to the former case), presenting, on the other hands, an oscillatory pattern13. These oscillations are based on the same physical mechanism as the de Haas-van Alphen14 and Shubnikov-de Haas effects15.In more details, those caloric effects are the ability of a material  stands for the electrons (holes) band; We have considered a graphene sheet on the 19. The remaining parameters above used were defined on the Methods section, when describing the Hamiltonian of the system. It is worth to note that equation th LL.However, the strain energy mentclass2pt{minimGrand potential 9. Thus, as described on the Methods section:From this energy spectra, one can obtain the 2 is the magnetic flux quantum; −2 is the density of charge carriers; It is worth to note that the strain contributes with an additional term to the Fermi energy. By contrast, the zero-strain relationship between the Fermi level and the number of charge carrier Grand potentials, it is possible to obtain the entropy due to the strain; that can be evaluated using a standard thermodynamic relationship: From these equation , is temp11, and can be achieved measuring the difference between field- and zero-field entropies: S = 0 and therefore the barocaloric effect on graphene reads as 8. For the sake of simplicity, as already done in other works8, we will only consider the The caloric effect can be defined by means of the entropy change, considering the isothermal process9 and the electrocaloric7 effects. In addition, this effect can be either normal or inverse, i.e., the material can either absorb or expel heat from/to a thermal reservoir, depending on the value of the pseudo-magnetic field, that, on its turn, depends on the strain energy. In other words, this (in/out) flow of heat can be managed by a simple handle of compression. In fact, the barocaloric effect largely overlaps the (zero-strain) oscillating magnetocaloric effect8 at low magnetic field, previously described by one of us, as can be seen on Fig. The barocaloric effect is drawn on Fig. mentclass2pt{minimmentclass2pt{minim18. One can say that the strain is able to both, either to mimic the magnetocaloric effect at low magnetic field and to produce a robust barocaloric effect. These properties can be regarded as unique advantages of strain gauge field. Based on this, more promising applications are thus eagerly expected to be realized in practice.From another perspective, the real magnetic field hardly achieves high value as c.a. 300 T at present, but the strain pseudo-magnetic field really does19. Although the degeneracy of the valleys in zero-An important point shall be addressed on Fig. Bs =  300 T at room temperature18.After a deeper looking on equation 9- and electro7-caloric effects previously proposed for graphene, moving therefore this material to the list of multicaloric substances - a fresh issue on the literature.We present the barocaloric effect (BCE) on graphene: the capacity of this material to exchange heat with a thermal reservoir due to a mechanical strain, like nanobubbles and even standard compression. In practice, strain produces a pseudo-magnetic field that, on its turn, changes the entropy of the material. Remarkable are the barocaloric oscillations, a pure quantum effect that allow to control the BCE as either normal or inverse, i.e., the material can either absorb or expel heat from/to a thermal reservoir, depending on the value of the pseudo-magnetic field. In other words, the flow of heat can be managed by a simple handle of compression. This effect is completely analogous to the oscillating magnetox–y plane and subject to a strain that gives rise to a pseudo-magnetic field et al.18. The 2D Dirac Hamiltonian for this model reads as:For the present work we have considered a graphene sheet on the et al.18: a value much higher than any other obtained in a laboratory, being therefore a unique opportunity to study a graphene sheet under an extremely high (pseudo-)magnetic field.The first term of the above equation represents the kinetic energy, where: Grand potential, represented on equations Grand potential calculation.The aim of this subsection is to provide further details on how to obtain the The total energy of the system at zero temperature reads as:20:Considering the generalised Zeta functionmentclasspt{minima2020The second contribution to the total energy, 20 it is known that:However, from ref.A comparison of these two equations leads to:20:20:The above term can be further developed. Considering now The term developed above has a cosine function and, for this reason, it is named as oscillatory contribution; while the other contribution (on equation 22. The procedure is to obtain the Grand potential following the equation:22:Above we presented our results on how to obtain the total energy, at zero temperature, of the system under consideration. From now on, let us consider the temperature effect, following the methodology of refsLet us first consider the non-oscillatory contribution, and, for this purpose, we need to insert equation Interestingly, the approximate result above for finite temperature is the same as equation 20:Grand potential can be obtained by inserting the above equation into equation 20:Analogously to above, let us now consider the oscillatory contribution, by considering the integral representation for Grand potential:Finally, we obtain the oscillatory term of the


Bone marrow sarcoidosis is extremely rare. The association between sarcoidosis and lymphoproliferative disorders has been previously speculated, although the diagnosis of sarcoidosis often precedes any hematological derangements.Here, we report for the first time, a case of a 57-year-old Caucasian woman with a previous diagnosis of monoclonal gammopathy of undetermined significance (MGUS) developing hypercalcemia and renal failure with workup notable for isolated bone marrow sarcoidosis and not multiple myeloma as expected. The patient was successfully managed with prednisone taper therapy with resolution of her hypercalcemia and repeat bone marrow biopsies demonstrating resolving granulomas.Our case illustrates the diagnostic challenges associated with bone marrow sarcoidosis and suggest that chronic immune stimulation in the bone marrow in the setting of MGUS may be associated with the development of localized sarcoidosis. The long term consequences of steroid therapy targeting sarcoidosis in this patient with underlying MGUS remain unknown. Greater surveillance and closer followup is planned in light of the increased risk of malignant transformation of MGUS into multiple myeloma in the setting of bone marrow sarcoidosis. Extrapulmonary manifestations of sarcoidosis are uncommon. Bone marrow sarcoidosis is infrequently clinically encountered. In the few cases reported, bone marrow involvement most often occurs in the setting of systemic sarcoidosis –3. IsolaA 57-year-old Caucasian woman with a four-year history of monoclonal gammopathy of undetermined significance (MGUS) complained of several weeks of dry mouth, polydipsia and episodes of confusion. Her serum calcium was 14.4 mg/dL when tested at the office of her primary care physician (PCP) and sent to the emergency department (ED) where her exam was remarkable only for dry mucous membranes. Initial labs were notable for Ca 14.4 mg/dL, albumin 5.0 g/dL, PTH 7 pg/mL, and Cr at 1.94. CBC was notable for mild pancytopenia with WBC 3.5 (th/cmm) with normal differential, Hgb 11.7 g/dL, Hct 34.2 (%), and PLT 145,000 (th/cmm). She received a bolus of normal saline and was subsequently admitted to inpatient service. Initially, the patient’s hypercalcemia was attributed to milk-alkali syndrome and her calcium supplements were discontinued. The patient was managed only with IV hydration. The patient’s serum calcium trended down to 10 mg/dL at the time of discharge.There was a concern for a neoplastic process, particularly myeloma, given her history of MGUS. The patient underwent bone marrow biopsy at the time of discharge, which was remarkable for non-necrotizing granulomas with no findings suggestive of plasma cell dyscrasia or other neoplasms. Fig.  Two weekThe patient underwent a rapid prednisone taper over 9 days. After discontinuation of prednisone, she developed hypercalcemia again. She was then treated with a slow prednisone taper for four months with normalization of serum calcium levels. A follow-up bone marrow biopsy 5 months later (1 month after 4-month course of slow prednisone taper) showed partial resolution of granulomas without malignancies. Fig.  At her fBone marrow sarcoidosis is rare and previously reported cases mostly involved patients with severe, chronic, and widespread sarcoidosis. Bone marrow biopsies are not routinely performed in the standard workup of sarcoidosis, thus the overall incidence of bone marrow involvement is unknown. One small cohort study  estimateOur case illustrates the diagnostic challenges associated with an atypical and rare manifestation of sarcoidosis. The clinical presentation of bone marrow sarcoidosis is variable, non-specific, and may overlap with those of systemic sarcoidosis which include fever, fatigue, malaise, weight loss, and night sweats. Patients with bone marrow involvement may have associated hematological abnormalities including anemia, leukopenia, and thrombocytopenia –5.Although the diagnosis of sarcoidosis seemed unlikely during the early workup of this patient, three lab abnormalities had some clinical utility and diagnostic value in support of sarcoidosis: elevated ACE serum level, hypercalcemia, and hypercalciuria.. ACE levels are elevated in 75% of untreated patients with sarcoidosis and levels tend to correlate with disease activity . One stuGiven the rare occurrence of bone marrow sarcoidosis, there are currently no randomized controlled trials comparing treatment modalities. However, a review of the relevant literature presents four case reports of patients with biopsy-proven bone marrow sarcoidosis demonstrating various approaches to treatment. One case report by Saliba et al.  involvedThese case reports highlight that the therapeutic approach to patients with bone marrow sarcoidosis depend on clinical presentation. Patients with symptomatic hypercalcemia appear to respond well to IV saline hydration and in severe cases may require steroid taper therapy. Ackermann et al.  recommenAn intriguing aspect of this case report involves the potential relationship with the patient’s underlying MGUS. The association between sarcoidosis and the increased risk of malignancy particularly lymphoproliferative disorders has been previously examined –8 and caSarcoidosis has been observed to develop in patients with hematologic malignancies with some investigators attributing this to the use of immunosuppressive therapy targeted at the underlying malignancy , 18. AsiIn conclusion, isolated bone marrow sarcoidosis is rare and can be a diagnostic challenge. The clinical suspicion for sarcoidosis should be raised in patients with underlying hematologic disorders or malignancies. Due to the rarity of the condition, long terms outcomes in such patients are unclear. Surveillance and regular follow-up is planned in view of the possibility of progression to multiple myeloma or another lymphoproliferative disorder.


The aqueous concentration can be as high as 5.0 mg mL−1, which is an order of magnitude higher than previously reports. The water soluble graphene can be directly spray-coated on various substrates, and the back-gated field effect transistor give hole and electron mobility of ~496 and ~676 cm2 V−1 s−1, respectively. These results achieved are expected to expedite various applications of graphene.Despite significant progresses made on mass production of chemically exfoliated graphene, the quality, cost and environmental friendliness remain major challenges for its market penetration. Here, we present a fast and green exfoliation strategy for large scale production of high quality water dispersible few layer graphene through a controllable edge oxidation and localized gas bubbling process. Mild edge oxidation guarantees that the pristine  Oxidation-reduction is the earliest wet process developed for the scalable fabrication of chemically exfoliated graphene, and it remains to be one of the most mature approaches ever developed, due to its versatility, scalability and controllability in terms of surface area, size and thickness1235et al. demonstrated high yield graphene production by electrochemical exfoliation of graphite with weak oxidationWeak oxidation or oxidation-free process is very helpful in preserving graphene’s lattice integrity, thus retaining its performance advantages. Extensive efforts were devoted towards this research direction, with many novel processes developed7891011et al. developed a method, using remnant structure defects after reduction of graphene oxide, to fabricate water-dispersable graphene−1, this method remains currently the most common method to obtain aqueous graphene dispersions.Water dispersibility is another key performance index for the development of graphene211−1, one order of magnitude higher than published results. The aqueous graphene can be directly spray-coated on flexible or rigid substrates, and the high quality was confirmed by carrier mobility measurements on back-gated field effect transistors (FETs). This process developed features good cost-effectiveness and environmental friendliness at the same time. It may be applicable for fabricating other two-dimensional materials with uncompromised quality and water solubility.The pursuit of high quality and environmental friendliness leads us to a novel strategy which is to mildly oxidize the graphite only at the edges and to realize exfoliation by bubbles generated by the catalytic reaction at the edges. Weak oxidation only at the edges minimizes lattice damage, while the oxidized edge brings in excellent solubility in water without any surfactants or additives. The water solubility reaches 5 mg mL4) facilitates the wetting of graphite in water and the edge opening of layered graphite sheets, while keeping most basal planes of graphite intact; Then, the exfoliation is accomplished by interlayer gas bubbling process of the precursor in the mixture of hydrogen peroxide (H2O2) and ammonia (NH3). The weak oxidation on the graphite edges was realized through controlling mass ratios between oxidant KMnO4 and natural graphite. The typical mass ratio of KMnO4 and natural graphite is 1:1, and the reaction temperature and time is 25 °C and 2 h, respectively. Compared with generally reported Hummers or modified Hummers, as shown in 4 for a graphite flake with 10 μm thickness, and the surface atomic layers will protect the inner layers from further oxidation while the edges have more defects as “weak” points to be attacked, and the space between graphite layers provides possibility for intercalation from the edges.Our approach is clearly illustrated in the schematic diagram of sp2 carbon structure)ID/IG, on the edge of the precursor is 0.8–1.5 while its most basal plane shows very low ID/IG , which proves that most parts of the sample has intact sp2 structure, and confirms the efficient and reliable edge oxidation on graphite. The edge oxidation can also be directly confirmed by SEM on the graphite edges before and after before oxidation. As shown in 4 addition. 4 and natural graphite. With the more KMnO4 addition, the weak oxidized area (ID/IG ~ 0) shrunk, and when the mass ratio of KMnO4 and natural graphite is 10:1, the oxidation became uniform for the whole flake, which further confirms that the oxidation of a graphite flake stars from the edges and goes into the inner parts gradually.The controllable edge oxidation of graphite was verified through Raman spectrum and scanning electron microscopy (SEM) measurements. 3 and H2O2. A lot of bubbles around precursor came into being, and as a result the graphite sheets floated on water although its density is much higher than that of water, as shown in −1 by just controlling the water amount during the exfoliation or re-dispersion. Especially, the aqueous concentration can be as high as 5.0 mg mL−1, which is an order of magnitude higher than previously reports2133 and H2O2 can be removed in the liquid before graphene collection, or by heating the dry products. The freeze-dried water re-dispersed graphene show excellent re-dispersibility in water, N,N-Dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP)  , keepingID/IG ratio (0.13) in Raman data on the central region of a graphene basal plane indicates the highly crystallized sp2 structure of the products. Compared with the ID/IG ratio of precursor (0.12), the slight change implies no additional oxygen-containing groups formed in exfoliation progress. Moreover, the symmetrical 2D band (ca. 2700 cm−1) suggests the sample is the complete few layer graphene rather than the multilayered graphite 15ca. 284.2 eV (83.75 at. %), along with an O 1s peak at ca. 532 eV (16.25 at. %), as shown in sp2-hybridised carbon , epoxy/hydroxyls  and carboxyl/carbonyl groups To evaluate the quality of bubbling-exfoliated graphene, the samples were characterized by SEM, transmission electron microscope (TEM), atomic force microscopy (AFM), X-Ray Diffraction (XRD), Raman, XPS and Fourier transform infrared spectroscopy (FT-IR). The SEM image of 1614cursor 0., the sliAccording to the synthesis procedure and measuring results, the bubbling exfoliated graphene should have good quality without the needs of reduction since most part of the basal plane is close to pristine graphene, differentiated from the uniform oxidation of graphite through the typical Hummers method. We further demonstrate the advantages of our preparing strategy through good water solubility, conductive film on polymer, and FET performance. 22232 (10 cm × 5 cm) polyethylene terephthalate (PET) substrate through a commercial liquid sprayer (−1 with a transmittance of 65.9%. SEM image (−1) and close to CVD graphene (500–1500 ohms ◻−1), as summarized in −1 when the transmittance is 65.9%, 42.9%, 32.5%, 29.3% and 5.56%, respectively, which means that the graphene can be easily spray coated onto polymer substrate, and can be used to construct the flexible conductive film. The inset in The water-dispersable graphene can be directly spray coated on several substrates. 1.0 mL of water-dispersable graphene ink can be spray coated on a 50 cm sprayer . After tEM image  shows th2/Si substrates. The inset of DS-VG) of the FETs measured at room temperature under ambient conditions. The typical IDS-VG curve measured at a VDS of 150 mV shows that the gate can cause either hole or electron conduction. The Dirac point of the FETs shifts slightly to a positive gate at VG ~ 25 V, demonstrating light p-type hole doping performance. According to the two slopes of the linear regions on both sides of the V-shaped curve, the hole mobility is μh ~ 496 cm2 V−1 s−1 and the electron mobility is μe ~ 676 cm2 V−1 s−1. As shown in 25262729303132333435363738To determine the transport properties of the synthesized graphene films, back-gated graphene field effect transistors FETs were fabricated on 300 nm SiO2O23+ as an example as shown in It is necessary to discuss the exfoliation mechanism to further understand this efficient approach. According to 4 as oxidant to avoid the Mn impurities. Then, we added graphite oxide, graphite oxide + MnSO4 (0.5 g + 10 mg), graphite oxide + MnO2 (0.5 g + 10 mg), graphite oxide + KMnO4 (0.5 g + 10 mg) into the 80 mL H2O2/NH3 solutions, respectively, as shown in 4 were added, as well as graphite oxide + MnO2 and graphite oxide + KMnO4, large amount of bubbles generated immediately, and the color of the mixture gradually changed to black. These results confirm that the Mn is a key factor for the bubbling exfoliation, no matter what its valence state.In fact, all kinds of Mn salts can catalytically accelerate the bubbling. To verify this, we fabricate graphite oxide through the Staudenmaire method using KClO2O2 requires the alkaline medium41423 also plays an important role in bubbling progress. NH3, not NaOH or KOH, was chosen since it can be easily removed in the obtained products. The exfoliation progressed in different mediums confirmed the importance of NH3 intuitively. As shown in 2O2 is 5, 4, 3, 2, 1, 1, 1, 1 and 0 units, respectively from left to right. At the same time, the volume of NH3 is 0, 1, 1, 1, 1, 2, 3, 4 and 5 units, respectively from left to right (1 unit volume is 10 mL). The drastic bubbling process can be observed only with the present of both H2O2 and NH3. However, no bubble can be observed without the present of NH3, which demonstrated its assisted catalysis effect. Thus, Mn ions, H2O2 and NH3 are the key factors for the bubbling exfoliation. The H2O2 is the most important factor, with the assistance of Mn ions and NH3. The O2 bubbles continually generate in the interlamination of graphite edges, which facilitates continuous and fast exfoliation of the precursor into water-dispersable graphene , the bubbles generated immediately at the edges and HOPG expanded lengthways, as shown in 2O2 with the help of NH3 to in-situ continuously generate bubbles, and that the bubbling exfoliation takes place between all the graphite layers from the edges, not a layer-by-layer model. It is the exfoliation model that helps the fast spontaneous exfoliation of edge-oxidized precursor into high quality graphene in 15 minutes.In order to further demonstrate the bubbling exfoliation mechanism intuitively, highly oriented pyrolytic graphite (HOPG) was selected as the raw material. HOPG was oxidized under the same reaction conditions as natural graphite to obtained edge oxidized HOPG. Before the bubbling exfoliation, the HOPG appearance had no obvious change after the weak oxidation. When it was put into the mixture of H4 and the in-situ continuous bubbling exfoliation with the catalytic H2O2 decomposition. The unique graphene, with edge oxidized and most basal plane intact, has excellent water solubility with extremely high concentration of 5 mg mL−1, good conductivity without the need of reduction, large lateral size of several microns and 1–6 atomic layer in thickness. This approach not only provides a new strategy for graphene synthesis, but also paves the way for large scale application of graphene in aqueous mediums, such as water-based green or functional coatings.For graphite to graphene, we employed a new, simple, mild, mild, fast and scalable spontaneous exfoliation approach. It is based on the weak edge oxidation with the controllable dosage of KMnO2SO4 (98%), H2O2 (30.0 wt. %) and NH3 (25 wt. %) were purchased from Lingfeng (Shanghai China). KMnO4 (99%) was purchased from Jianglaibio (Shanghai China). Highly oriented pyrolytic graphite (HOPG) was purchased from Shanghai NTI Co. Ltd (Shanghai China). The water used throughout all experiments was purified through a Millipore system.Natural graphite powder (45 μm) was purchased from Huatai Lubricant sealing S&T Co.Ltd (Qingdao China). H2SO4 for 30 minutes at 25 °C in 500 mL double-layer beaker with a cooling system based on Julabo F20. 10 g potassium permanganate (KMnO4) was added slowly to the mixture in 30 minutes. After the whole KMnO4 was added, the reaction was kept for 2 hours. Next, 100 mL deionized water was added and stirred continually for 2 hours. Finally, 10 mL (H2O2) was added to the mixture and reacted for 30 minutes. The final liquid was washed with the deionized water by a vacuum filtration and frozen drying in order to obtain the edge-oxidized graphite. The precursor was put into the mixing liquid of NH3:H2O2:H2O (1:1:5) for 30 minutes to finish the bubbling exfoliation. The exfoliated graphene was centrifuged, filtered and frozen dried. The yield was obtained by calculating the mass ratio of exfoliation graphene after frozen dried and the raw natural graphite.For the preparation of weak oxidized graphite, 10 g graphite was mixed and agitated with 200 mL concentrated H2SO4, KMnO4) with same dosages except various graphite additions . The mass ratio of KMnO4 and graphite is 10:1, 2:1 and 1:1, respectively.To control the edge oxidation, we used the same chemicals . Next, potassium permanganate (0.3 g) is added slowly to the mixture during 30 minutes (note: some ice is placed out of the beaker for ensuring the reaction in room temperature). After the whole KMnO4 is added, the reaction is kept for 2 hours and then deionized water (3.0 L) is added and stirred continually for 2 hours. Finally, H2O2 (0.6 mL) also is add to the mixture reacting for 30 minutes. The final liquid is washed with the deionized water by a vacuum filtration and frozen drying in order to obtain the dry product. The edge oxidized HOPG (0.3 g) is put into the mixing liquid of NH3:H2O2:H2O (1:1:5) and frozen drying. The high quality soluble graphene sheet would be obtained.HOPG (0.3 g) is mixed and agitated with concentrated H2SO4 . Next, potassium permanganate (500 g) is added slowly to the mixture during 30 minutes (note: some ice is placed out of the beaker for ensuring the reaction in room temperature). After the whole KMnO4 is added, the reaction is kept for 2 hours and then deionized water (5 L) is added and stirred continually for 2 hours. Finally, H2O2 (500 mL) also is add to the mixture reacting for 30 minutes. The final liquid is wash with the deionized water by a vacuum filtration and frozen drying in order to obtain the dry product. The 500 g edge oxidized graphite is put into the mixed solution of NH3:H2O2:H2O (1:1:5) to exfoliate the water soluble graphene. After 0.5 h, the homogeneous dispersion liquid of water soluble graphene is obtained. The yield is 52.2% .For the preparation of weak graphite oxide, the graphite (G) (500 g) is mixed and agitated with concentrated HAtomic Force Microscopy (AFM) data were obtained in a Bruker Dimension Icon with a Nanoscope 8.15 in tapping mode. Scanning electron microscopy  was used to image the morphology of the samples. Transmission electron microscopy (TEM) measurements were obtained on a Hitachi H-8100 EM  with an accelerating voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) measurements were carried out using a Thermo ESCALAB 250Xi spectrometer. Raman Spectroscopy with the excitation laser line of 532 nm was performed using a Thermo Fisher DXR Raman Microscope. X-ray diffraction (XRD) patterns were obtained from a X-Ray Diffractometer (Bruker D8 ADVANCE) with a monochromatized source of Cu Kα1 radiation (λ = 0.15405 nm) at 1.6 kW . The sheet resistances were measured by a Hall measurement system (Accent HL5500). Ultraviolet–visible spectra were obtained using a Varian Cary 300 Bio UV-visible spectrophotometer. Fourier transform infrared spectra were recorded on a PE Paragon 1000 spectromer (film or KBr disc).2 (10 sccm) at 300 °C for 8 h in a tube furnace. The back-gated GFETs were characterized under ambient conditions using the Agilent (B1500A) semiconductor parameter analyzer. The mobility was extracted using the following equation (S1):L and W are the channel length and width, respectively, Cox is the gate oxide capacitance (11 nF cm−2), VDS is the source drain voltage, IDS is the source drain current, and VG is the gate voltage. The linear regime of the transfer characteristics was used to obtain dIDS/dVG.The graphene films were spray coated to highly doped p-Si substrate with a 300 nm thick thermal oxide, followed by deposition of source and drain electrodes with Au/Ti (50/10 nm) by electron beam evaporation. Afterwards, another standard photolithographic step employing inductively coupled plasma (ICP) was used to pattern the graphene to form a field-effect transistor with a channel length of 8 μm and width of 2 μm. To impove the contact of the back-gated GFETs device, annealing was performed in Ar (500 sccm) and HHow to cite this article: Tian, S. et al. Controllable Edge Oxidation and Bubbling Exfoliation Enable the Fabrication of High Quality Water Dispersible Graphene. Sci. Rep.6, 34127; doi: 10.1038/srep34127 (2016).


We attribute variations in observed G peak asymmetry to a combination of a ‘stiffening’ of the E2g optical phonon where the graphene is supported by the underlying MRR waveguide structure, as a result of this increased hole concentration, and a lowering of the degeneracy of the same mode as a result of localized out-of-plane ‘wrinkling’ (curvature effect), where the graphene is suspended. Examination of graphene integrated with two different MRR devices, one with radii of curvature r = 10 μm and the other with r = 20 μm, indicates that the device geometry has no measureable effect on the level of doping.We present a Raman mapping study of monolayer graphene G and 2D bands, after integration on silicon strip-waveguide-based micro-ring resonators (MRRs) to characterize the effects of the graphene transfer processes on its structural and optoelectronic properties. Analysis of the Raman G and 2D peak positions and relative intensities reveal that the graphene is electrically intrinsic where it is suspended over the MRR but is moderately hole-doped where it sits on top of the waveguide structure. This is suggestive of Fermi level ‘pinning’ at the graphene-silicon heterogeneous interface, and we estimate that the Fermi level shifts  In factreported , 9–11.−1 [−1 [−1 [q = 0) specifically by an electron-phonon DR scattering process, whereas the D peak requires highly localized electron-defect scattering to conserve momentum [Graphene exhibits three principal Raman scattering peaks, each with a distinct physical origin: the doubly resonant (DR) D peak appears around 1350 cm−1  and is r−1 [−1 , 12, andmomentum –16. The momentum , 13.Graphene integration with the silicon photonics platform is interesting from a number of device applications perspectives, e.g. for demonstrating enhanced biochemical sensors in which the graphene acts as a high affinity surface functional layer for adsorbed species that may be probed by evanescent optical fields in the underlying silicon photonics device. The two-dimensional nature of graphene also leads to an optoelectronic band structure, the charge filling of which can be tuned by very low power electrostatic gating. In this case, the ‘Pauli blocking’ effect can alter the opacity of the material to incoming photons, providing the possibility of very fast (GHz) optical modulation or switching, which is likely to be of use in telecoms applications. Previous reports –20 of thThe Si MRR devices in this study were fabricated in a commercial Si foundry  and comprise of strip-waveguides of 335-nm width, lithographically formed from commercial 220-nm silicon-on-insulator with a 2-μm-thick buried oxide layer. These waveguide dimensions, specifically, the relatively narrow waveguide width , were selected to ensure good modal overlap with surface integrated graphene, post-transfer. Two ‘racetrack’-type MRR devices are studied, one in which the radial component is 10 μm and the other is 20 μm and both having identical 20-μm-long linear sections. Prior to graphene transfer, the devices were washed with acetone, isopropyl alcohol (IPA), de-ionized water and resist stripper (NMP: 1-methyl-2-pyrrolidone). This was followed by an oxygen plasma etch (for 40 s) immediately before transfer. The graphene was grown by chemical vapour deposition (CVD) on copper foils  and then transferred onto the pre-cleaned waveguides using a polymer-mediated wet transfer procedure . The grar = 10 μm and 20 μm. The maps were obtained from a 120 × 120 point array with a step size between each point of 0.25 μm, and the precise frequency, intensity and width of the Raman G and 2D peaks were determined by fitting with Lorentzian line-shapes to the spectral peaks. From measurements of a single crystal silicon sample using the same instrument configuration , we estimate a spectral resolution from the bandwidth of the main Si scattering peak of 4.6 cm−1 or better.The Raman spectral mapping was performed at room temperature in back scattering configuration, using a Horiba LabRAM HR Evolution Spectrometer with a 600 g/mm grating. The scattering signal was collected confocally and detected with an integrated Peltier cooled charge coupled device (CCD) camera. The samples were excited by a 633-nm helium neon laser light, and mechanical movement of the sample during mapping was provided by a Marzhauser motorized microscope XYZ stage. The incident laser light was focused on the sample surface using a × 50 objective lens with numerical aperture of 0.75. In order to avoid laser heating, the laser power density on the sample was kept below 2 mW . Raman m−1. This combination of a relatively intense, symmetric 2D scattering peak and a G peak frequency close to the predicted value, ωG (n) = 1581.6 + 11/(1 + n1.6) where n is the layer number [r = 10 μm) is presented in Fig. −1, respectively) where the graphene sits on top of the MRR waveguide structure relative to where it is suspended.In order to check if we had transferred the single layer graphene, prior to the Raman mapping study, we also measured the single point Raman scattering signal, Fig. r number , confirmr number . The optωD2/∂ε) is approximately six times that of the G peak (∂ωG/∂ε) [n) and hole (p) doping, the frequency of the G peak always increases from the intrinsic value, meaning that a plot of G peak position with Fermi level is nearly symmetric about zero. However, for the 2D peak, whilst the frequency is up-shifted considerably for a moderate increase in p-doping level (~ 15 cm−1 for 3 × 1013 cm−2), it remains virtually unchanged from its intrinsic position up to an electron concentration of ~ 3 × 1013 cm−2, above which it down-shifts rapidly. This leads to a highly asymmetric curve for the 2D peak position with the Fermi level about zero. That we observe shifts that are both similar in magnitude and in the same direction for the G and 2D peaks strongly suggests that the graphene is moderately p-doped, where it sits on the waveguide, compared to where it is suspended. In order to quantify this effect, we used the following empirical relations , the strain-related shift of the 2D peak (∂∂ωG/∂ε) . That we∂ωG/∂ε) . For botω0(G) (=1580 cm−1 [ω0(2D) (=2640 cm−1 [ω(G) and ω(2D) are the G and 2D peak positions we have determined for each point in our maps and EF is the Fermi level in units of eV. In Fig. EF ~ 0) but that the hole concentration is increased  where the graphene sits atop the waveguide structure. A similar analysis of an MRR with radius r = 20 μm (not shown here) gave a very similar result, indicating that the effect is not dependent on the waveguide geometry, rather that it is purely a material-dependent (substrate) doping effect. The source of this doping is almost certainly the result of locally trapped, static ad-charges at the interface between the silicon/SiO2 and graphene. The density of these ad-charges is known to be increased in samples which have received more aggressive cleaning treatments (such as the O2 plasma etch we have employed) [where 580 cm−1 ) and ω0640 cm−1 ) are thecm−1 [ω0D (=2640 mployed) . AlthougRepresentative Raman scattering spectra (from the mapping) are shown in Fig. R2 = 0.993) by a single, symmetric Lorentzian line-shape, a signature of single layer graphene [The 2D peak is well described (graphene . We notegraphene .R2 > 0.995) by a double Lorentzian line-shape, indicative of two distinct scattering processes. We note that the width of the main (G+) peak decreases by ~  25%  going from the suspended graphene to where it is supported by the MRR waveguide structure. This is consistent with current understanding and prior observations of the ‘stiffening’ of the graphene E2g optical phonon, as a result of doping [−), responsible for the asymmetry, also exhibits a significant decrease in width of ~ 35%  going from the suspended graphene to where it is supported by the MRR waveguide structure. Asymmetry in the graphene Raman G peak has previously been attributed to highly localized charge inhomogeneity within the laser probe area [E2g optical mode can be split between phonons along the nanotube axis, −) scattering mode itself can be broadened and even split when nanotubes buckle and collapse under high pressure loading. We note from fitting the graphene G band spectra here that both the frequency difference ΔωG and the line-width of the G− mode  doping level, principally because of a quenching of the 2D mode with increasing carrier-phonon scattering [I2D/IG, from ~ 3 where the graphene was suspended to ~ 2.5 on the waveguide structure, we note that this change is small relative to the degree of G peak shift we observe, when compared with other reports [I2D/IG as a function G peak position, which appears to increase with excitation wavelength suggesting this alone may not be the most reliable indicator of absolute doping level, especially in the low doping limit.We also examined the ratio of peak intensities, attering , 32. How reports  for the  reports , there iAG/A2D, which takes account of the peak widths as well as the variations in peak heights can be used to obtain the carrier concentration directly from Eq.  ~ 0.069 when νf is taken to be the electron velocity, 1.17 × 108 cm/s. Our measurements indicate that where mined in  to be ~ ed in [2 , which in through Eq.  in a so-called vector decomposition plot, introduced by Lee et al. , Fig. 5.ωD2/∆ωG ~ 2.2) are very different for those associated with doping (∆ωD2/∆ωG ~ 0.7) [p-type doping vectors. With increasing tensile strain or p-doping, the ωG, ωD2 coordinate values will move from the origin , either along the doping-free (strain) or strain-free (p-doping) lines, respectively. The G-2D coordinate space is divided into four quadrants, Q1–Q4 by these strain and doping vectors, and so any significant deviation of the coordinate data from these lines, say into region Q1 (Q4), would indicate that the peak shifts are the result of a combination of compressive (tensile) strain and p-doping. Scattering of data within Q2 and Q3 is forbidden because both n- and p-doping manifests only in increases in the G peak position.Representing the data in this type of plot enables us to determine to what degree the peak shifts may be influenced by strain. This is based on the fact that the rates of variation in peak position ratios for strain (∆G ~ 0.7) . Any coop-doping) vector (dashed line) and doping-free (strain) vector (solid line), after [p-doping) vector for the ON-ring with the average ON-ring coordinate value (purple dot) being . The increased scatter for the ON-ring data along the strain-free line indicates a greater range of doping levels detected from the relative peak shifts, likely because of the uncertainty in probing a highly localized substrate doping effect produced by the underlying, sub-micron waveguide width, compared with the probe laser spot size (> 1 μm). Despite the apparent scatter in the data, into both Q4 and Q1, we discount any significant global strain effects because the average ON-ring coordinate lies so close to the strain-free line. We suggest that the peak shifts we observe are only due to silicon substrate induced hole-doping and the average ON-ring G-2D coordinate confirms this to be in the range of (2 to 3) × 1012 cm−2.We define the intrinsic, unstrained graphene peak frequency coordinate as the origin (red dot) , 26 and ), after . Data fo2 waveguide (substrate doping effect). The data for the suspended region reveals no measurable distinction from intrinsic graphene, but for the supported region, a maximum down-shift of the Fermi level of ~ 0.2 eV is determined, which corresponds to a peak hole concentration of ~ 3×1012 cm−2. An asymmetry in the Raman G peak, which varies according to whether the graphene is suspended or supported, indicates a combination of doping-induced ‘stiffening’ and lifting of the degeneracy of the E2g optical mode. These effects should be taken into account when graphene is combined with silicon photonics platforms, certainly when attempting to use such platforms to determine graphene’s characteristic properties and for optimization of future graphene-integrated silicon photonics devices, such as optical modulators and sensors.In summary, monolayer CVD graphene was integrated with silicon waveguide-based MRR photonic devices. Frequency shifts and integrated intensities of the characteristic graphene Raman G and 2D peaks were determined for mapped regions, and these indicate a Fermi level ‘pinning’ where the graphene sits on the Si MRR structure as a result of unintentional hole-doping from the underlying silicon/SiO


The 43-kDa trans-activating response region DNA-binding protein 43 (TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS). Despite of accumulating evidence that mitochondrial dysfunction underlies the pathogenesis of TDP-43–related ALS, the roles of wild-type TDP-43 in mitochondria are unknown. Here, we show that the small TDP-43 population present in mitochondria binds directly to a subset of mitochondrial tRNAs and precursor RNA encoded in L-strand mtDNA. Upregulated expression of TDP-43 stabilised the processing intermediates of mitochondrial polycistronic transcripts and their products including the components of electron transport and 16S mt-rRNA, similar to the phenotype observed in cells deficient for mitochondrial RNase P. Conversely, TDP-43 deficiency reduced the population of processing intermediates and impaired mitochondrial function. We propose that TDP-43 has a novel role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts. TDP-43 binds tightly the (UG)n motif4 and helps regulate several aspects of mRNA biogenesis including transcription, pre-mRNA splicing and export, and mRNA stability5. TDP-43 is one of the causative gene products of amyotrophic lateral sclerosis (ALS) that is an adult-onset neurodegenerative disorder characterised by progressive degeneration of upper and lower motor neurons The majority of TDP-43 mutations that cause ALS are found in the C-terminal region, although some are found in the RRMs10. ALS mutant TDP-43 proteins, including those with mutation of the RRMs have an increased half-life in cell models12.The trans-activating response region DNA-binding protein 43 (TDP-43) is a member of the family of heterogeneous nuclear ribonucleoproteins and contains two highly conserved RNA recognition motifs (RRMs) and a non-conserved C-terminal region that mediates protein-protein interactions13. TDP-43 is cleared from the nuclear compartment and is incorporated into cytoplasmic ubiquitinated and/or hyperphosphorylated inclusions in patients with familial or sporadic ALS and in patients with frontotemporal lobar degeneration16. Several animal models over-expressing wild type TDP-43 leads to neurodegeneration similar to that observed in ALS24, accumulate mitochondria in cytoplasmic inclusions in motor neurons23, lack mitochondria in motor axon terminals24, or have abnormal juxtanuclear aggregates of mitochondria in spinal motor neurons22, suggesting that wild type TDP-43 affects mitochondrial function. Supporting this, TDP-43 localises to mitochondria of NSC34 cells, a hybrid cell line that has several motor neuron features25. TDP-43–induced cytotoxicity also strongly correlates with mitochondrial functions, including respiratory capacity and respiratory chain activity, in yeast cells expressing human TDP-4326. In addition, TDP-43 co-localises with mitochondria in motor neurons, and ALS-associated mutants enhance its localisation in mitochondria28. The enhanced localisation of mutant TDP-43 to mitochondria also disrupts mitochondrial dynamics and function28. The mitochondrial fusion protein mitofusin 2 (Mfn2) has also been demonstrated to be involved in mitochondrial impairment, but the functional relationship between TDP-43 and Mfn2 is unknown27. In this regard, TDP-43 mutants also disrupt the vesicle-associated membrane protein-associated protein B (VAPB)– protein tyrosine phosphatase-interacting protein-51 (PTPIP51) interaction and cellular Ca2+ homeostasis, which disrupts mitochondria–endoplasmic reticulum interactions that are implicated in several physiological processes including ATP production, mitochondrial biogenesis, and apoptosis29; however, TDP-43 is involved only indirectly in these processes through its ability to activate glycogen synthase kinase-3β29. In addition, it was recently proposed that TDP-43 and its mutants impair the assembly of electron transfer complex I by binding to mitochondrial mRNAs encoding NADH-ubiquinone oxidoreductase chain 3 (ND3) and ND6 and inhibiting their translation28. Despite the obvious involvement of TDP-43 in mitochondrial function, the direct mitochondrial target of TDP-43 remains elusive.Although TDP-43 shuttles between the nucleus and the cytoplasm, it mainly resides in the nucleus under physiological conditions32. We established human Flp-InTM T-RExTM 293 (T-REx 293) cell line expressing doxycycline-inducible wild-type human TDP-43 carrying a triple affinity-purification tag  sequence replacements were made corresponding to a specific region of mt-tRNALeu  as well as mt-tRNALeu as a negative control , we first investigated the association of RNA with exogenously expressed TDP-43 using mass spectrometry (MS) and the Ariadne search engineAGE Fig. . The MS-nts Fig.  and are nts Fig. , and theion Fig. . To deteion Fig. , and sho-43 Fig. .Figure 1Asn, mt-tRNAGln, and mt-tRNAPro present within the mitochondria of T-REx 293 cells.To assess localisation of TDP-43 in mitochondria further, we treated a mitochondrial fraction with proteinase K and detected both endogenous and exogenous TDP-43; TDP-43 was not detected, however, if the mitochondrial membrane was permeabilised with Triton X-100 prior to proteinase K treatment Fig. . Further33. The L-strand encodes one oxidative phosphorylation system subunit (ND6) and eight mt-tRNAs , Tyr, Cys, Asn, Ala, and Gln)  to inhibit mtDNA transcription and first examined the effect of EtBr on stability of L-strand coded mt-RNAs in T-REx 293 cells. EtBr reduced the levels of all of the L-strand-encoded mt-tRNAs with time but it did not affect a genomic tRNAMet   and mt-12S rRNA by inhibiting mtDNA transcription with EtBr before and after doxycycline induction of TDP-43. Overexpression of TDP-43 slowed the degradation of mt-tRNAAsn and mt-tRNAGln , whereas it had only a negligible effect on the degradation of mt-12S rRNA  upon overexpression of TDP-43 cannot be attributed to stabilisation mediated by direct binding of TDP-43.In human mitochondria, large polycistronic RNA transcripts are generated from the L- and H-strand promoters of mtDNAGln Fig. . MoreoveRNA Fig. . These rMet, U1 snRNA, and 5.8S rRNA) as the references. Overexpression of TDP-43 significantly increased the cellular levels of all L-strand-encoded mt-tRNAs  , and mt-tRNALeu(UUR) appeared slightly larger than the mature transcripts upon overexpression of TDP-43  Fig. . In addi-43 Fig. . These dTyr (Y)-mt-tRNAAla (A), the region complementary to COXI mRNA (mirrorCOXI)-Y, mirrorND2-mt-tRNAGln (Q), and mirrorCYB-mt-tRNAGlu (E) to test for cleavage within these boundary regions. All boundaries were found to increase in abundance with time after doxycycline induction compared with GAPDH mRNA  and confirmed the direct binding of the precursor RNA to recombinant TDP-43 . The RT-PCR primers were designed to amplify the boundaries among mt-tRNAs or between mt-tRNAs and mt-mRNAs in the region containing mt-tRNARNA Fig. . In addi-43 Fig.  and the -43 Fig. . These rLeu(UUR)  Fig. , we alsoUR) Fig. . To deteUR) Fig.  and showUR) Fig. , W-COXII34. Therefore, accumulation of large precursor transcripts was also investigated by northern blotting after knockdown of MRPP3 or ELAC2 , and W-COXII (detected by probes 6–9) relative to 28S rRNA staining compared with a mock knockdown with a sequence-scrambled RNA -mediated cleavage of precursor molecules at the 5′ end, and tRNase Z (ELAC2)-mediated cleavage at the 3′ endAsn and mt-tRNAGln  did not inhibit cell proliferation  of this L-strand transcription unit, although we cannot exclude the possibility that the entire L-strand transcript is stabilised by the binding of TDP-43 because the region containing mt-tRNAPro is located in the first part of the L-strand mtDNA that is transcribed, and the region containing mt-tRNAAsn/mt-tRNAGln is located at the 3′ end. It is also likely that TDP-43 binds to regions of mt-transcripts other than the regions corresponding to the three mt-tRNAs because of its general RNA-binding properties2. We also propose that the increase in the L-strand transcript and/or its processing products induced by TDP-43 overexpression may stabilise all or part of the H-strand transcript and/or its processing products through complementary association, resulting in increased levels of these processing products  seems to be corresponded to RNA 19 that is increased in diseases associated with mt-tRNALeu(UUR) gene mutations39.In human mtDNA, the genes are located in three different transcription units (one in the L-strand and two in the H-strand) that are transcribed to different extents. The one H-strand transcription unit that includes only the mtcts Fig. . Rackham4, our findings provide important insight into the physiological roles of this negative feedback control and indicate that the strict control of TDP-43 expression is critical for maintaining mitochondrial homeostasis. In this context, mutations in TDP-43 that affect its stability or expression are expected to reduce mitochondrial function. Our present data provides a new mechanism underlying disruption of mitochondrial function by TDP-43 that differs from the mechanisms that involve perturbation of Mfn227, the VAPB–PTPIP51 interaction29, or translation of ND3 and ND628.Because TDP-43 strictly controls its own expression through a negative feedback loop in which TDP-43 binds to the 3′ untranslated region in its own mRNAg for 30 min at 4 °C, the supernatants were used as cell lysate for the following immunoprecipitation. Cell lysate (5 mg) prepared from 2 × 107 cells were incubated with 15 µl of anti-FLAG M2 magnetic beads (Sigma-Aldrich) for 2 h at 4 °C. The beads were washed five times with 1 ml of lysis buffer, and proteins and RNA were eluted with 150 µl of Protein-RNA extraction buffer , 10 mM EDTA, and 2% 2-mercaptoethanol) for 5 min. The eluted proteins and RNA were subjected to SDS-PAGE or denaturing (8 M) urea PAGE, respectively, as described previously42.Confluent cells were washed with PBS and suspended in five packed cell volumes of lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.5% (w/v) IGEPAL CA-630) containing 2 mM ribonucleoside-vanadyl complex, 1 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml pepstatin A, and 2 µg/ml leupeptin for 30 min on ice. After removal of any insoluble residue by centrifugation at 20,000 × 31. LC-MS analysis of RNA, and database search and interpretation of MS/MS spectra of RNA were performed as described41.In-gel RNA digestion was performed as described previously2. For mt-mRNAs analysis, the membranes were stained with methylene blue. Hybridizatioin with biotin-labeled DNA probes and detection of RNA signals were performed as described previously41. The oligonucleotides used as probes are shown in Supplementary Table Total RNA was isolated using the RNAgents Total RNA Isolation System (Promega). For analysis of mt-mRNAs, 5 µg of total RNA was subjected to 2% agarose/formaldehyde gel electrophoresis in 3-(N-morpholino)propanesulfonic acid running buffer. For analysis of mt-tRNAs, 1 µg of total RNA was subjected to denaturing (8 M) urea PAGE in Tris-borate-EDTA running buffer, stained with SYBR Gold (Invitrogen) for 10 min, and visualised using the LAS4000 Luminescent Image Analyzer System (Fujifilm). The separated total RNAs were transferred to a Hybond N + membrane . The membrane was dried and subsequently cross-linked using an ultraviolet cross linker  at 120 mJ/cm2, and DAP-TDP-43-associated complexes were immunoprecipitated with anti-FLAG M2 magnetic beads. After heating for 5 min at 65 °C in SDS sample buffer, DAP-TDP-43-associated complexes were released from the anti-FLAG M2 magnetic beads, and separated by SDS-PAGE using a Bis-Tris gel (Running gel: 360 mM Bis-Tis-HCl (pH 6.8), 8% acrylamide, 0.1% tetramethylethylenediamine, 1% ammonium persulfate; Stacking gel: 360 mM Bis-Tis-HCl (pH6.8), 5% acrylamide, 0.1% tetramethylethylenediamine, 1% ammonium persulfate) and Bis-Tris running buffer propanesulfonic acid, 5 mM EDTA, 0.5% SDS). The separated proteins were electrophoretically transferred to a Protran BA 85 Nitrocellulose membrane . The membrane was blocked with 5% non-fat dried skim milk in TBST for 30 min, washed with TBST, and then incubated with HRP-conjugated streptavidin  for 30 min. After washing three times with TBST for 10 min, the Chemiluminescent Nucleic Acid Detection Module kit (Thermo Fisher Scientific) was used for detecting biotin of DAP-TDP-43. Stained parts of the membrane were excised and incubated in 250 µl of Proteinase K buffer  at 37 °C for 30 min. After addition of 250 µl of Protein-RNA extraction buffer, RNA was isolated by phenol-chloroform extraction and isopropanol precipitation and subjected to northern blot analysis.T-REx 293 cells expressing DAP-TDP-43 were cross-linked by ultraviolet irradiation at 400 mJ/cmin vitro transcription of mt-tRNA(Asn), AC-stem(Leu), D-Loop(Leu), pAntiCdn(Leu), Var-R(Leu), T-Loop(Leu), and Leu(UUR) RNA were generated by annealing the primer sets shown in Supplementary table 2, respectively. In vitro transcription was performed with 0.2 pmol of the template DNA using CUGA® 7 in vitro transcription kit (NIPPON GENE) according to the manufacturer’s instructions. Transcripts were collected by isopropanol precipitation, and purified using reverse-phase liquid chromatography. To synthesize biotinylated mirrorCOXI-YCNA RNA, template DNA for in vitro transcription was prepared. We amplified DNA fragments corresponding to mtDNA between mirror mt-tRNAAsn to COX I with the primer set (mirrorANCY-COXI-for/ mirrorANCY-COXI-for) using KOD neo plus (TOYOBO), and inserted them into the BamHI/PstI site of the pSPT19 vector (Roche) (mirrorANCY-COXI pSPT19). mirrorANCY-COXI pSPT19 linearlized by digesting with SmaI was used as template DNA for in vitro transcription. Biotin-labeled mirrorCOXI-YCNA was in vitro transcribed from the DNA template (0.06 pmol) in the presence of 7.5 mM of ATP, 7.5 mM of CTP, 7.5 mM of UTP, 7.5 mM GTP, 0.5 mM of biotin-UTP (Roche) using CUGA® 7 in vitro transcription kit , and purified by 1% agarose gel electrophoresis and the subsequent gel extraction using Zymoclean Gel RNA Recovery Kit (ZYMO RESEARCH).The template DNAs for 2, 0.01% IGEPAL CA-630, 1 mM DTT, 10 µg of yeast tRNA (Ambion), 10 µg BSA, 10 ng of synthetic RNA, 200 ng of recombinant protein), and electrophoresed on a 6% non-denaturing polyacrylamide gel at 100 V for 50 min in 0.5× Tris borate/EDTA buffer (44.5 mM Tris-borate and 1 mM EDTA). The separated RNA-protein complexes were transferred to a Hybond N+ membrane that was dried and UV-crosslinked using the FS-1500 crosslinker (Funakoshi) at 120 mJ/cm2. The synthetic RNA was detected by northern blot analysis using the indicated probes.Each binding reaction was performed at room temperature for 20 min. Each reaction was performed in 10 µl binding buffer (40 mM Tris-HCl (pH 7.4), 30 mM KCl, 1 mM MgCl7 cells expressing DAP-TDP-43 was used for this assay. Synthetic RNA (sRNA) (1 pmol) was added to the total cell lysate and subjected to immunoprecipitation using anti-FLAG magnetic beads as described above. RNA isolated from DAP-TDP-43 complex was subjected to denaturing urea-PAGE and northern blot analysis.Total cell lysate from 1.0 × 102 dishes. Cells washed twice with PBS were sedimented (800 × g for 5 min at 4 °C), and resuspended in 9 ml of ice-cold Solution A . Cells were homogenized by passing ten times with a 25 G needle. The nuclei from this suspension were sedimented (800 × g for 10 min at 4 °C), and the centrifugation step was repeated for the supernatant once more. Mitochondria were sedimented from the supernatant by centrifugation , and washed once with 1 ml of Solution A. Protein concentration was measured using NanoDrop. For Proteinase K protection assay, the isolated mitochonaria was diluted into 1 mg/ml with Solution B . 100 µg of mitochondria was treated with the indicated concentration of Proteinase K  for 20 min on ice. Mitochondria were treated with 0.5% (w/v) of Triton X-100 along with the treatment of Proteinase K for a control of Proteinase K protection assay. Proteinase K reaction was stopped with the addition of 1 mM PMSF, and the mitochondria were centrifuged , resuspended with SDS sample buffer, and subjected to western blot analysis.Mitochondria were prepared from T-REx 293 cells or 293 T cells grown in 5 × 15 cm100 µg of Mitochondria isolated from 293 T cells were resuspended with 500 µl of 20 mM Hepes-KOH pH 7.4, and incubated for 20 min on ice. After sedimenting mitochondria by centrifugation , pellet were washed once with Solution B, resuspended with 50 µl of 20 mM Hepes-KOH pH 7.4, and disrupted twice for 30 sec using Bioruptor UCD-250 (Cosmo Bio). After sedimenting them by centrifugation , the supernatants were used as a mitoplast extraction. Protein concentration was measured using BCA assay.41. The eluted proteins and RNA were subjected to SDS-PAGE or northern blot analysis using 1.5% denaturing agarose gel. Biotinylated RNA was detected using HRP-conjugated Streptavidine.mtRNA degradation assay was performed at 25 °C for 5, 10 or 30 min. Each reaction was performed in 20 µl of reaction buffer (20 mM Tris-HCl (pH 7.4), 60 mM KCl, 12.5 mM MgCl2, 0.1 EDTA, 2 mM DTT, 10% Glycerol, 10 µg mitoplast extract, 10 fmol biotinylated mirrorCOXI-YCNA RNA, 600 ng TF-fused protein). Reaction was stopped by the addition of 150 µl Protein-RNA extraction buffer , 10 mM EDTA, and 2% 2-mercaptoethanol), and protein or RNA in the reaction was purified as described previouslySupplementary Information


Mage = 23.35; SDage = 4.93), leading to a nine-item scale (Study 1). Third, in Study 2, the internal factor structure, temporal stability, and concurrent validity were examined through a series of structural hypothetical modelisation in a sample of 129 male athletes . The results indicated that the scale has good psychometric properties. Specifically, the PCA, and the series of structural hypothetical modelisation, suggested two theoretical factors , and more, with a bi-factor model in a SEM. The results also indicated sufficient concurrent validity with the Male Body Dissatisfaction Scale (MBDS) and adequate internal consistency . The findings overall suggest good reliability and construct validity for this French version of the DMS (DMS-FR), which will be useful for future research and clinical practice in French-speaking countries.The purpose of this paper was to adapt and examine the psychometric properties of a French-language Drive for Muscularity Scale (DMS). First, a preliminary version of the scale, based on both the English-language version of the DMS and the literature on the drive for muscularity, was developed following a committee validation process. Second, the factor structure of the DMS-FR was investigated with principal component analysis (PCA) in a sample of 114 male athletes ( Social values have changed in the past few decades, and appearance issues now affect both men and women . StudiesSeveral questionnaires have been developed to measure DM or its associated constructs. Some scales assess DM, such as the Drive for Muscularity Scale (DMS) , the DriThe most extensively used questionnaire, with good levels of reliability and validity, is the DMS . In the I think about taking anabolic steroids") but other recent studies have found that this item loads onto the MB subscale [In line with the recommendations of McCreary et al. , some stsubscale .The two-factor model of DMS scores of the parent study has been confirmed by McCreary et al.  and suppEscoto et al.  first foA good factorial structure was found in the study of McCreary et al. , revealiThe DMS has been translated into several languages: Brazilian Portuguese , ItalianAnother research avenue concerns the construct validity of the scale. Despite its extensive use and the finding of Tod et al.  that theThis study is not just a transcultural validation of the DMS. In link with previous transcultural studies of the DMS and observed limitations, we have chosen to adapt a short version of the questionnaire with two subscales based on the English version of the DMS. The authors of the present study have removed some items to realize validation of a questionnaire only on two concepts: body dissatisfaction and muscularity. The purpose of this study was to adapt and validate a French-language measure of the drive for muscularity to establish a more theoretically based scale for future research. In the first step, a preliminary version of the DMS was developed in French; in the second step (Study 1), the factor structure of the DMS-FR was investigated using principal component analysis; and in the third step (Study 2), the internal factor structure was examined through a series of hypothetical equation modelisation and the temporal stability and concurrent validity were assessed.These three steps were conducted following the validation procedure of Vallerand  and MyerThe purpose of this step was to adapt a preliminary short version of the DMS in French and assess the clarity of its items.The development of the preliminary version followed the usual recommendations . Five stThe DMS scale was initially translated from English into French-language by the first and third authors, both of whom are fluent French-speakers. Two criteria guided the translation process: (a) conformity with the original questionnaire intentions and (b) clarity of the items in French.The two translated versions were compared and the two translators agreed on the preliminary version of the DSM-FR.Based on this preliminary version, a back-translation was performed by a translator unaffiliated with the study and with no prior knowledge of the original instruments. The first and third authors compared the original English version of the DSM, the back-translation, and the preliminary version of the DSM-FR and worked together to ensure a clear final French-language version, equivalent to the English one in terms of semantics and concept. The two other authors did a back-translation of this final preliminary version. The back-translation was satisfactory since the back-translated questionnaire was identical to the original English version.I wish that I were more muscular"; "I think that my arms are not muscular enough"; "I think that my chest is not muscular enough"; "I think that my legs are not muscular enough") and muscularity behaviors . Items related to other constructs such as self-efficacy  or subjective norms/social approval  were removed for conceptual clarity. After the committee validation process, the final preliminary version of the DSM-FR was composed of 10 items classed into two subscales: Body Dissatisfaction Muscularity (5 items) and Muscularity Behaviors (5 items).A bilingual review committee was set up with three sport and health psychology researchers and one PhD student (the four authors). The committee first worked on the 15 basic items of the English version of the DSM and selected items which were clearly related to muscularity body dissatisfaction  recruited through a social network. A 6-point Likert scale ranging from 1  to 6 (item quite clear) was chosen to remove the neutral option at the mid-point. Questionnaire completion was carried out under standardized conditions  and did not exceed more than 10 minutes. Then, meetings were held to elicit feedback on problems understanding the questionnaire items or questions about them .The clarity of the items of the preliminary version of the DMS-FR was assessed by 20 males ranging in age from 21 to 41 years . The clarity of each item was satisfactory for the two subscales of Muscularity Body Dissatisfaction and Muscularity Behaviors . A few minor changes were made on the basis of comments. The final 10 translated items of the DMS-FR are presented in The descriptive statistics related to clarity assessment showed that the preliminary version of the questionnaire was very clear for the participants  using principal component analysis (PCA).Mage = 23.35; SDage = 4.93) and had been practicing sport from 1 to 30 hours per week for 9.76 years on average (SDnb of year = 5.44). All were French-speaking athletes practicing mostly bodybuilding or strength training. In this sample, 25 male athletes were not competitors and 89 male athletes competed at different levels. All participants were from France or the French-speaking part of Switzerland. A coding system was adapted to ensure no duplicate data.The exploration of the factorial structure of the DMS-FR was conducted with 114 men, all of whom were eligible to participate in the study because they met the following criteria: (a) a minimum age of 16 years, (b) more than one hour of physical training per week, and (c) more than one year of sports experience. Participants were 18–48 years old  and that all responses would be used for research purposes only. Participation was entirely voluntary and full confidentiality was guaranteed. The authors collected information about nationality in order to ensure that the participants were French-speakers.At the beginning of the questionnaire, participants were first invited to give personal information before responding to the DMS-FR: their age and main sport .Drive for Muscularity Scale (DMS-FR): At this stage, the DSM-FR was composed of 10 items. Participants answered each item with a Likert scale ranging from 1  to 6 (absolutely).Parallel Analysis: The factorial structure was examined by principal axis factor analysis (Promax-type rotation). In order to extract the most appropriate factors, parallel analysis [analysis  was usedanalysis . The iteI lift weights to build up muscle"). The nine remaining items all showed item-total factorial weights greater than .40. Cronbach’s alphas were .87 for the Muscularity Body Dissatisfaction subscale and .85 for the Muscularity Behaviors subscale, demonstrating high levels of internal consistency of the subscales [Based on several indicators from this analysis, including the scree plot, eigenvalues, and proportion of variance, a two-factor solution accounting for 69.2% of the common variance was found. The first factor was labeled Muscularity Body Dissatisfaction and the second factor was labeled Muscularity Behaviors . The facubscales .The aim of study 1 was to explore the factorial structure of the DMS-FR. The PCA produced a two-factor model that replicated the original DMS subscales  and thosThe aim of study 2 was to examine the psychometric properties of the DMS-FR by conducting a series of structural hypothetical modelisation according the four steps of Myers et al. . We thenMage = 27.03; SDage = 7.81) and had been practicing sport from 1 to 20 hours per week for 7.76 years on average (SDnumber of years = 6.82). The participants were French and Swiss male athletes practicing mostly bodybuilding or strength training. In this sample, 91 male athletes were not competitors and 38 male athletes were competitors at different levels. Correlations between age, sport experience and the DMS did not show significant results. When considered as covariates, the results of the exploratory structural equation modelling (ESEM) did not change. This yielded no significant effect for these two variables. This sample size was considered appropriate according to the 1:10 subjects-to-variables ratio generally adopted in the literature for CFA or SEM sample size calculations [The 129 selected participants were 16–57 years old  from the total sample were contacted four weeks after responding to the first DMS-FR and agreed to complete it a second time under the same conditions.To test the temporal stability, 61 male athletes : At this stage, the DMS-FR consisted of nine items and two factors: Muscularity Body Dissatisfaction  and Muscularity Behaviors . The items were answered with a Likert scale from 1  to 6 (absolutely).Male Body Dissatisfaction Scale (MBDS): The MBDS [The MBDS  was valiThe MBDS . It is cFor the factorial validity, because of the significant multivariate non-normality of the data , the analysis were performed with the AMOS 7.0  using thp < .001; TLI = .99; CFI = .99; RMSEA = .01. Comparison of acceptable models  revealed that model d provided the best goodness of fit indices, as well as the lowest ECVI and AIC indices [The results are presented in  indices . Satisfar = .86, p < .01, and for Muscularity Body Dissatisfaction, and Muscularity Behaviors, the correlation coefficients were .83, p < .01 and r = .86, p < .01, respectively. Estimates of internal consistency  in Time 2 were .81 and .74 for Muscularity Body Dissatisfaction and Muscularity Behaviors, respectively.The DMS-FR demonstrated adequate test–retest reliability. The Pearson product-moment correlation coefficient between the two sets of total scores (Time 1 and Time 2) was r = .35, p < .01. The two subscales of the DMS-FR (Muscularity Body Dissatisfaction and Muscularity Behaviors) were significantly and positively correlated with the Muscularity Body Dissatisfaction subscale of the MBDS . The Muscularity Body Dissatisfaction subscale of the DMS-FR and the General Body Appearance Dissatisfaction subscale of the MBDS were negatively related in the expected directions . However, the General Body Appearance Dissatisfaction subscale of the MBDS and the Muscularity Behaviors subscale of the DMS were not significantly related  with a view to establishing a more theoretically based scale for future research. Two studies were carried out following the validation procedure of Vallerand . The valContrary to studies where participants’ age had a significant effect on the overall DMS and younger men are more likely to desire a more muscular body , age didOur results indicated a two-factor structure that mirrored the model proposed by McCreary et al.  and agreThe DMS has been previously used in many populations: males , male adThis study presents some limitations and points to directions for future research. First, the data of studies 1 and 2 were collected online, which may have limited participation to those with access to a computer, smartphone, and/or stable internet connection. Second, a typical limitation of research based on self-report measures is the potential for social desirability bias. Third, our sample included only male athletes, and it is thus now necessary to test this survey in non-athlete males to generalize our results. An interesting question is whether the DMS scores are multi- or one-dimensional. Only two previous transcultural studies have examined fit of the higher-order dimensionality of DMS scores and have found equivocal results. Nerini et al.  reportedThe drive for muscularity has been linked to significant physical and psychological problems in males . The avaTo conclude, the drive for muscularity is an important component of men's self-image. Theorists and researchers are currently at the beginning of the long path that will lead to a better understanding of the individual differences in men's body image perceptions and body-related behaviors. The DMS-FR can be used to measure the drive for muscularity in French-speaking male athletes and this should facilitate future research on the antecedents, consequences, and correlates of DM in this population. Greater insight into the drive for muscularity should also aid in the development of better prevention strategies for coaches and male athletes.S1 DatasetData used for the realization of the different analyses.(XLSX)Click here for additional data file.