The feasibility of realizing largermembranes with diameters of 300 μm and even higher compliancesis shown, although these have lower yields. We present a process forlocally growing graphene on a silicon wafer and realizing suspendedmembranes of patterned graphene across through-silicon holes by bulkmicromachining and sacrificial layer etching, such that no transferis required. This transfer-free method results in a 100% yield formembranes with diameters up to 155 μm on 132 fabricated drums.The device-to-device variations in the mechanical compliance in theaudible range (20–20000 Hz) are significantly smaller thanthose in transferred membranes. With this work, we demonstrate a transfer-freemethod for realizing wafer-scale multilayer graphene membranes thatis compatible with high-volume manufacturing. Thus, limitations oftransfer-based methods for graphene microphone fabrication such aspolymer contamination, crack formation, wrinkling, folding, delamination,and low-tension reproducibility are largely circumvented, settinga significant step on the route toward high-volume production of graphenemicrophones.During the past decadesmicro-electromechanical microphones havelargely taken over the market for portable devices, being producedin volumes of billions yearly. Because performance of current devicesis near the physical limits, further miniaturization and improvementof microphones for mobile devices poses a major challenge that requiresbreakthrough device concepts, geometries, and materials. Grapheneis an attractive material for enabling these breakthroughs due toits flexibility, strength, nanometer thinness, and high electricalconductivity. Here, we demonstrate that transfer-free 7 nm thick multilayergraphene (MLGr) membranes with diameters ranging from 85–155to 300 μm can be used to detect sound and show a mechanicalcompliance up to 92 nm Pa Todorović et al. demonstrated a membrane witha 5 mm membrane diameter composed of 300 layers of chemical vapordeposition (CVD) graphene with a 10 dB higher sensitivity than commercialnickel-based microphones.4 Woo et al. realizeda high-sensitivity microphone for hearing aids with a heterostructuremembrane composed of 0.6 μm of graphene and 3 μm of PMMAon a 2.65 mm diameter membrane.5 Wittmannet al. reached almost similar performance as commercial silicon-basedcapacitive microphones using 15× smaller membranes (diameter2r = 40 μm) made by only few-layer graphene.6 The high performance is mainly due to graphene’shigh flexibility, low tension, and low out-of-plane stiffness, whichresults in large displacements in response to sound pressure. Significanteffort has been made to improve the process to realize free-standinggraphene by transferring the material to the target substrate by acarrier polymer with wet, dry, or semidry methods.7 The introduction of the inverted floating method (IFM)by Lee et al. and Akbari et al. helped fabricate large free-standingCVD graphene up to 2r = 500–750 μm.9 A combination of hydrogen bubbling transfer with thermal annealingby Chen et al.10 resulted in a suspendedfive-layer graphene membrane with a diameter of 1.5 mm. Carvalho etal. developed an anthracene sublimation assisted process achievingten layers of CVD graphene suspended over 4 mm openings.11 Recently, six-layer graphene and 450 nm of PMMAwere suspended over a closed cavity by HF vapor release (VHF) of thesacrificial layer after the graphene transfer.12 The dry release avoids liquids that usually introduce capillaryforces that pull down the suspended part introducing ruptures or breaks.13 Moreover, exposing the graphene to wet HF mightalso lead to unwanted delamination of the graphene from the substrate.14 However, despite the very high aspect ratiosand high crystallinity of the demonstrated free-standing membranes,the mentioned works do not provide a clear route toward industrializationof the devices because the transfer-based methods employed are noteasily scalable toward high-volume wafer-level fabrication since nocommercial equipment for transferring suspended graphene is available,and current methods often suffer from low yield, polymer contamination,cracks, and folding, leading to adhesion issues, especially for nonplanarizedtarget substrates.7 This work proposesan alternative approach to overcome part of the previous limitationswith a wafer-scale transferless method where multilayer graphene (MLGr)drums are grown and released on the same substrate. The method ispotentially less prone to contamination and degradation of the membranesand less sensitive to topography, allowing it to be applied on nonplanarizedsurfaces. The realized multilayer graphene drums made by using thisapproach reach a peak mechanical compliance of ≈92 nm Pa–1 for 300 μm membranes with a yield of 18% andof ≈9 nm Pa–1 with a 100% yield of functionaldevices with diameters ranging between 85 and 155 μm. A studyof the relation between the membrane diameter (2r = 85–300 μm) and the mechanical compliance, for morethan 50 drums, demonstrates a good tension uniformity since it followsthe expected quadratic dependency.15Inthe past 10 years, suspended graphene has attracted the interestof scientific and engineering communities due to the possibility ofusing its unique properties for sensor devices with novel functionalityand improved performance like microphones, pressure, gas and Hallsensors.17 ML-graphene (MLGr) is preferred here due to its higher mechanicalstrength that is required to realize membranes of sufficiently largediameter for microphone applications. Because the synthesis of thegraphene is performed at 935 °C, all the materials are selectedto have a sufficiently high melting point and mechanical stabilityduring graphene CVD. Thermal SiO2 and LPCVD silicon-richlow-stress SiNx are stable at these hightemperature and show an etch selectivity of 40:1 to the vapor HF basedon preliminary results and previous works on Si MEMS.19 The SiNx layer is used as clamping supportlayer all around the edge of the graphene membrane and is also usedto protect certain parts of the SiO2 from being etchedduring the silicon backside deep reactive ion etch (DRIE) and vaporHF sacrificial layer SiO2 release etch (The deviceprocess design utilizes multilayer (ML) transfer-free graphene andvapor HF compatible materials to ensure the structures survive thesacrificial layer etch. Prepatterned Mo is used as a catalyst seedlayer to locally synthesize the graphene on Si substrates along thesame procedure described in earlier studies.ase etch 1.2Cl2 315 sccm/NH3 85sccm) is deposited at 850 °C on top of 1 μm thermal oxideon a 100 mm silicon p-type wafer. The SiNx is dry-etched in defined regions on the topside and entirely removedon the backside where a PECVD TEOS-based 5 μm is then depositedto work as etching mask for the silicon DRIE as shown 2 chemistry. Because of thenature of the sputtering process, Mo covers all the steps in the topography. Inthis way, the CVD-grown graphene will not suffer from any discontinuities.The positive photoresist is then removed by O2 plasma,and all the remaining residuals are washed in N-methyl-2-pyrrolidone(NMP) with subsequent DI water washing . No polymersor tapes are involved during the final isotropic etching step sincethey might trap HF molecules due to their porous nature, and theycan usually be only removed by aggressive methods such as O2 plasma or other dry-etching chemistry that can damage or removethe suspended graphene. With this proposed approach, a lower temperatureof T > 110 °C can be used to clean all theresidualsthat originate from the LPCVD SiNx growthand vapor HF reaction,21 which is low comparedto the T > 250 °C cleaning step typicallyneededto remove the polymer used to transfer the graphene on prepatternedholes. The low temperature in the proposed approach is advantageousbecause thermal removal of the transfer polymer residuals generallyresults in a lower number of surviving membranes due to thermal expansioncoefficient mismatch of the suspended graphene and the substrate,especially for larger membranes.9A layer of 110 nm LPCVD silicon-richlow-stress (SiH22 Acoustic actuation at a sound pressure level of 1 Pa (≈ 94dB SPL) is used to test the fabricated membranes.The input sound pressurefrom a speaker is measured with a reference microphone (SonarworksXREF20) placed next to the sample. A Moku:Lab hardware platform fromLiquid Instruments records the signal detected by the reference microphone,the mechanical frequency response of the graphene membrane as detectedby using a Polytec vibrometer focused at the center of the membrane,and the output signal of the speaker. After proper correction forthe corresponding sensitivities of the vibrometer controller and referencemicrophone, the mechanical compliance is obtained from the ratio ofthe two signals received by the Moku:Lab.2/MLGr heterostructure (before HF etch of theSiO2) is optically inspected by a 3D laser scanning confocalmicroscope on more than 100 drums. The graphene is observed to displayout-of-plane deformation  is relatively far from theory, which is attributed to thepresence of higher buckling modes.26 Oncethe SiO2 is etched, the graphene restores its originalflat shape in the suspended region by adhesion to the unetched siliconsubstrate along its circumference . We verify its flatness by measuring the step height betweenthe surface of the Cr/Au electrode and the Si surface of the suspendedMLGr . They may negatively affect the grapheneintegrity creating randomly distributed tears and localized stress.During the vapor HF, handling, and storing they might be a sourceof crack propagations leading to final membrane breakage. Gas pressureon the membrane during handling and storage might also play a roleand account for the yield reduction in the larger membranes. Thisyield reduction might be mitigated in future processes by tuning thestress and thickness of the oxide layer or by using strain compensationmeasures like suspending the MLGr from a SiNx frame that generates tensile stress.27Whereas this model captures the deflection of the small membranesquite well, the deflection mference S1b. We vmference S1b. We v+ laser maintained at 5 mW to minimizeany possible damages by laser heating. A 100× objective witha numerical aperture of 0.9 is used, giving a spot size of about 696nm. The graphene properties are mainly characterized by two Ramanbands: The first is the G band, which is characteristic of all thegraphitic sp2-type structures and typically centers at1580 cm–1 when the material is in a stress and doping-freestate. The second band is the 2D band, which is centered at 2700 cm–1 and mainly gives information about the number andstacking order of the layers. In 17 This increased defectivitysource is mainly attributed to the lift-off step where N-methylpyrrolidone (NMP) is used for the Cr/Au electrode fabrication.The graphene on Mo is exposed for 40 min at 65 °C with a finallow-power ultrasonic bath of 90 s to strip the cross-linked photoresist(PR). Intercalation of NMP into the stacked layers and the short ultrasonicbath have probably negatively affected the quality of the material,as already reported in other works.30 We exclude that theDRIE is invasive since the graphene is not exposed to the plasma etchfrom the backside of the wafer and instead faces the tool’schuck. VHF is also not considered a possible defective source becausethe ID/IG ratiodoes not show any significant change after the release  from Cypher Asylum Research is used in semicontactmode for the graphene thickness measurement, and a value of ≈7± 2 nm is found, where the standard deviation of 2 nm representsits nonuniformity as shown by the variations in the line scans in x, the reported AFM thicknessmeasurements are obtained on graphene which is processed with allthe reported steps except the VHF and then wet-transferred in DI wateron a clean thermally oxidized silicon chip. The graphene needs tobe transferred because SiNx is partiallyetched during the Mo patterning and the VHF exposure and can thereforenot be regarded as a flat reference point. The small wrinkles observedin 35x/Si interfaceacts as a clamping support for the suspended graphene.The crystallinity of theMLGr is investigated through a Horiba HR800 Raman spectrometer equippedwith a 514.5 nm Ar release 3b. Upon ded MLGr 3d. These–4 kPa to reduce air damping effects. The samples are actuated by shakingthem with a piezoelectric element below the chip that is actuatedby using a voltage-controlled stage with a 0.5 V sine wave in thefrequency range f = 50–350 kHz where the fundamentalresonance frequency is given by 36n0 is thepretension (N/m) of the graphene, ρ the mass density, and t the thickness of the graphene. In this relation, the contributionof the bending rigidity is neglected since its is estimated to besmall.37 The inspected membranes show fundamentalfrequencies over a range of ≈156–218 kHz for diametersof 2r = 120–155 μm .The firstmode of the resonance frequency is measured by a digital holographicmicroscope (LynceeTec) using a laser with a wavelength of 666 nm anda 10× objective lens. The interference between the reflectedlaser beam from the sample and the reference path provides the intensityand phase of each pixel defining the 3D topography. The measurementsare performed in a chamber connected to a vacuum pump at 105 μm see 4a and ≈9 N/m see 4a. The d3 Far below the resonancefrequency in the linear regime (such that cubic terms in z can be neglected), the membrane displacement z isrelated3 to the sound pressure level ΔP by the equationr for a constantsound pressure level ΔP. The reported mechanicalcompliances of 76 drums shown in –1 for the smaller membranes with 2r = 85–155 μm and ≈43–92 nm Pa–1 for large membranes with 2r = 300 μm. Sm0 = 3 nm Pa–1.Finally, we characterize the acousticmembrane displacement inthe presence of sound at a frequency of 1 kHz at a sound pressurelevel of 94 dB. The compliance or sensitivity in nm/Pa of the fabricatedgraphene membranes is determined optically by using a single pointlaser Doppler vibrometer (LDV) (OFV-5000 Polytec GmbH) at the centerof the membrane. Details of the experimental setup and procedure canbe found in ref  and the n0 are needed to fit the large (2r = 300μm) and small membranes. Large membranes are closer to the analyticalresults obtained with lower pretension n0 = 0.1 N/m, whereas the smaller membranes yield n0 = 0.2 N/m. This could be attributed to a lower pretensiondue to the larger suspended region where membrane sagging might bemore profound. Moreover, the tension is also affected by the grapheneclamping geometry, where for the same diameter of 300 μm thetrampoline shows an even higher mechanical compliance (by a factor1.4) due to lower tension of n0 = 0.07N/m compared to the fully clamped geometry.2 The mechanical compliance of drums with different sizes and geometries,over the entire audible range, is also reported . When comparing the pretension extracted from n0 are obtained as in r = 300 μm by monitoring the motion ofthe graphene membrane using the Polytec vibrometer22 . The output signal from the polytec in response to an arbitrarysound file is recorded with a sampling frequency of 20 kHz to detectthe sound waveform, and the measured trace is then reconverted toan audio file with Python.The graphene data are fit by rge 2r = 00μm andreported S3. When S, defined as open-circuit sensitivity,38 is measured by an electrical read-out. Theoretically, it is equalto the product between the electrical sensitivity Se and the mechanical sensitivity Sm0 contribution by the equation3822 where the input values Se (m V Pa–1 or dB),pretension (N/m), resonance frequency (Hz), the distance membrane–bottomelectrode g0 (m), and Vb (V) are obtained from the reportedworks.43 For most of the presented data , the mechanicalcompliance is indirectly calculated from Vb (bias voltage),resulting in an underestimation of the extrapolated reported mechanicalcompliance.The proposed wafer-scale transfer-freemultilayer graphene performancesare visually compared in r = 85 to 300 μm. Large arrays of graphene drums withdiameters up to ∼155 μm are fabricated with a high yieldof 100% by using a CMOS-compatible process flow without any transfersteps. These graphene membranes are shown to operate as microphones,detecting sound with mechanical compliances as high as 92 nm Pa–1, which is much higher than that of commercial MEMSmicrophones that typically have a compliance of 3 nm Pa–1. The graphene attains this high sensitivity by using a membranearea that is 10 times smaller than that of the MEMS device. This demonstratesthe great potential of graphene for microphone applications. The fabricationroute for trampoline designs offers the possibility to invent andengineer new suspended graphene geometries for realizing very highmechanical compliances at the wafer scale. When integrated with electrodesand read-out electronics, it can enable next-generation, high volume,wafer-scale graphene microphone technologies.With this work, we present an efficient transferlessmethod tofabricate wafer-scale graphene drums with diameters from 2


Bony union is a primary predictor of outcome after surgical fixation of long bone fractures. Murine models offer many advantages in assessing bony healing due to their low costs and small size. However, current fracture recovery investigations in mice frequently rely on animal sacrifice and costly analyses. The modified Radiographic Union Score for Tibia fractures (mRUST) scoring system is a validated metric for evaluating bony healing in humans utilizing plain radiographs, which are relatively inexpensive and do not require animal sacrifice. However, its use has not been well established in murine models. The aim of this study was to characterize the longitudinal course of mRUST and compare mRUST to other conventional murine fracture analyses. 158 mice underwent surgically created midshaft femur fractures. Mice were evaluated after fracture creation and at 7, 10, 14, 17, 21, 24, 28, 35, and 42 days post-injury. mRUST scoring of plain radiographs was performed by three orthopaedic surgeons in a randomized, blinded fashion. Interrater correlations were calculated. Micro-computed tomography (μCT) was analyzed for tissue mineral density (TMD), total callus volume (TV), bone volume (BV), trabecular thickness, trabecular number, and trabecular separation. Histomorphometry measures of total callus area, cartilage area, fibrous tissue area, and bone area were performed in a blinded fashion. Ultimate torque, stiffness, toughness, and twist to failure were calculated from torque-twist curves. A sigmoidal log-logistic curve fit was generated for mRUST scores over time which shows mRUST scores of 4 to 6 at 7 days post-injury that improve to plateaus of 14 to 16 by 24 days post-injury. mRUST interrater correlations at each timepoint ranged from 0.51 to 0.86, indicating substantial agreement. mRUST scores correlated well with biomechanical, histomorphometry, and μCT parameters, such as ultimate torque , manual stiffness , bone percentage based on histomorphometry , cartilage percentage , tissue mineral density , BV/TV based on μCT , and trabecular thickness , among others. These data demonstrate that mRUST is reliable, trends temporally, and correlates to standard measures of murine fracture healing. Compared to other measures, mRUST is more cost-effective and non-terminal. The mRUST log-logistic curve could be used to characterize differences in fracture healing trajectory between experimental groups, enabling high-throughput analysis. Approximately 1.1 million human bone fractures are treated annually in the United States (US). In fact, long bone fractures comprise 10% of all non-fatal injuries and incur an estimated inpatient expenditure of $10.4 billion annually in the US . These aIn vivo μCT allows for the generation of longitudinal data, and studies have identified protocols for non-invasive monitoring of rodent fracture healing  scanning provides quantitative, three-dimensional measurements of both the structure and mineralization of the fracture callus. In fact, numerous studies have demonstrated that CT-based measurements directly correlate with callus stiffness and strength , 4. In t healing –8. Howev healing  found th healing  develope healing , 12. AddDespite the success of mRUST in human studies, there is no accepted methodology for utilizing plain radiographs to assess fracture healing in mouse models. The standard measures of fracture healing in mice include biomechanical testing, μCT analysis, and histomorphometric analysis, which are associated with significant time and costs . Should All animal procedures were performed with prior approval from Indiana University School of Medicine Institutional Animal Care and Use Committee. All experiments were performed in compliance with NIH guidelines on the use and care of laboratory and experimental animals. Three month old male, C57BL6/J mice were purchased from Jackson Laboratories  and housed in the Indiana University School of Medicine Laboratory Animal Resource Center under a 12-h light, 12-h dark cycle for 6 weeks. Food and water were provided ad libitum.Mice were anesthetized using 1.0% isoflurane for induction and 0.6-0.8% for maintenance, at 1000 mL/minute utilizing a SomnoSuite Low-Flow Anesthesia System (Kent Scientific). Analgesia was provided by 1.0mg/kg of buprenorphine sustained-release subcuticular fashion pre-operatively. The right leg was shaved and scrubbed with betadine followed by ethanol (triplicate scrubs). All instruments and implants were autoclaved before use. A surgically created mid-shaft femur fracture was utilized as previously described . In brieAnteroposterior (AP) and lateral X-ray imaging at the aforementioned time points were obtained using a Kubtec Xpert80 X-ray system . All images were taken using a set of standardized imaging parameters to ensure direct comparisons within and across specimens. AP radiographs were obtained by placing the mouse on its left  hip. A small piece of tape was used to gently pull the surgical leg laterally and downwards while ensuring that the tail was not overlapping the femur  and lateral (anterior and posterior cortices) X-rays as follows: 1 = no healing; 2 = callus present, no bridging; 3 = bridging callus, fracture line visible; 4 = bridging callus with no fracture line visible. The scores for all 4 cortices were summed to provide a final score ranging from 4  to 16 . Mean mRUST scores were calculated for each specimen at each time point, and interrater correlations were calculated.Mice were euthanized at the indicated time points using carbon dioxide gas and cervical dislocation. Both the fractured and unfractured femora were harvested and cleaned of subcutaneous tissue and muscle before analysis. The intramedullary needle was removed using a needle driver to facilitate histologic sectioning and limit metal artifact during μCT analysis. Femora designated for histomorphometry and μCT were fixed in 10% neutral buffered formalin for 48 hours and then rinsed and stored in 70% ethanol at 4°C. Femora designated for μCT and biomechanical analysis were wrapped with gauze soaked in deionized water and stored at -20°C.3. The long axis of each femur was aligned with the vertical axis of the scanner. Femurs were imaged using a Bruker SkyScan 1172 desktop imaging system with X-ray tube voltage 59kV, intensity 167μA, rotation step 0.7°, integration time 885ms, and voxel size 9.83μm. Scanned images were then reconstructed into 3D image stacks  with smoothing 2, beam hardening 20%, ring artifact 5, and a dynamic image range with minimum and maximum attenuation coefficients of 0 and 0.75. They were then analyzed using CT Analyzer software . The region of interest was the entire fracture callus determined by manual and automated techniques. The proximal and distal boundaries of the callus were identified manually. Next, a custom script was utilized to trace the fracture callus excluding the cortical bone and marrow areas. Bone was differentiated from non-bone by a single global threshold grayscale index of 90. Recorded measures included: tissue mineral density (TMD), bone volume (BV), tissue volume (TV), bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N).Analyses were conducted after all mice were euthanized and all specimens were treated at the same time to minimize batch to batch variation. For μCT analyses, the scanner was calibrated using two phantom rods 2mm in diameter, at concentrations of 0.25 and 0.75 g/cmFemora harvested for histomorphometric analysis were decalcified in Immunocal™ Decalcifier  at pH 7.2-7.4 for 48 hours. Longitudinal paraffin sections of 5 μm thickness were prepared and the center-most section from each femur was stained with picrosirius red and alcian blue. Brightfield and polarized images were obtained from a Leica DM2700M microscope. Histomorphometric measures of total callus area, cartilage area, fibrous tissue area, and bone area were performed in a blinded fashion using an image manipulation program  .For femora undergoing biomechanical analyses, frozen femora were rehydrated in Dulbecco’s phosphate-buffered saline (DPBS) with calcium and magnesium at room temperature for 16-24 hours. Femora underwent immediate biomechanical torsion analysis after μCT to prevent repeated freeze-thaw cycles and to maintain structural integrity of the bone. Femora were potted into 0.38 bullet casings and held in place with orthodontic resin . The resin was allowed to harden for 5 minutes prior to testing. Biomechanical testing was then performed in torsion at 1 degree/second until failure using a Mark-10 advanced torque-testing system . Ultimate torque, stiffness, toughness, and twist to failure were calculated from the resultant torque-twist curves. Ultimate torque was defined as the maximum torque sustained, manual stiffness was defined as the slope of the torque-twist curve over 50-75% of the ultimate torque, maximal stiffness was defined as the greatest slope over a 5 degree segment of twist, toughness was calculated as the area under the torque-twist curve, and twist to failure was defined as the twist to failure after 25% of the ultimate torque was reached on the torque-twist curve (Continuous variables were summarized by means and standard deviations (SDs). Gwet’s AC2 was calculated to quantify the interrater reliabilities of mRUST scores. The average mRUST scores across raters were then used for subsequent analysis. Spearman’s correlation coefficients were calculated between the mRUST scores and other measures because these associations appeared to be monotonic but nonlinear. For the temporal trend of mRUST scores, a nonlinear mixed effects model was fit with a log-logistic curve as the mean function as follows:Y(0) a known initial value at day zero (mRUST=4 by definition), Y∞ is the upper asymptote at the end of the fracture healing, k is the healing rate, and X0.5 is when half the healing is complete. The log-logistic curve also has an inflection point where the fastest healing happens. Random effects of INLINE, k, and X0.5 were included. All analyses were performed using SAS 9.4 , and p<0.05 was considered statistically significant.where ∞) as 15.38, the inflection point (X0.5) as 12.08 days, and the rate (k) as 4.43. Additionally, this curve has approximate exponential growth, transitional, and plateau phases beginning at 7-, 17-, and 28-days post injury, respectively, which reflects the expected timeline of fibrocartilaginous callus, bony callus, and remodeling phases of fracture healing in mice.For mRUST to be a viable metric of murine fracture healing, it should correlate well with traditional measures. Biomechanical testing offers the most direct quantification of bone’s ability to support efficient locomotion and is thus pivotal for determining the functionality of the end product of fracture repair. As observed in Histological staining followed by histomorphometry offers another avenue for assessment of the fracture callus that is best for differentiating between tissue types . As detailed in Micro-computed tomography enables high‐resolution visualization of fracture callus architecture, and resolution of fracture related deficits in μCT parameters often correlates with restoration of mechanical function. As shown in The goal of this study was to assess the validity of mRUST X-ray scoring as a metric of fracture repair outcomes in mice. X-ray evaluation is pivotal in gauging fracture repair success clinically. In fact, the FDA defines non-union solely based on radiographic criteria and definitions of non-union in orthopaedic research include radiographic criteria more often than clinical criteria (62% vs 45%) . SeveralDespite the known predictive value and reliability of RUST/mRUST scoring of fracture repair success, there is minimal evidence supporting its use in animal models , 19–22. In the study by Cooke and colleagues , the autFiset et al.  performeTo our knowledge, RUST and mRUST have been used as metrics to examine non-RUST and mRUST related hypotheses in few rodent studies. Meeson et al.  implemen∞), an increased or unchanged rate (k), and an earlier inflection point (X0.5). Additionally, although mRUST appears to correlate well with other modalities of fracture repair assessment, if and how these correlations may be disrupted in various disease states and following experimental interventions is unclear. For instance, Cooke et al. . We chose radiographic assessment, as this is the most commonly used clinical modality and is less technically demanding/time intensive compared with other protocols for rodent non-invasive longitudinal monitoring of fracture healing. Our purpose is not to supplant other modalities, but rather to investigate whether quantitative evaluation of mRUST would at all be of use when applied longitudinally to radiographic evaluation. We demonstrate that it is a feasible technique, has reasonable correlations to standard measures of rodent fracture healing, and we uniquely provide a “mathematical” description of routine fracture healing based on mRUST scoring over time that could be valuable for future studies. X-rays may also be useful in cases where the more advanced imaging modalities are not available.Several limitations to our study exist. We had only three raters and do not know how this would fair with a larger number of raters with varying degrees of experience. Another limitation is that we intended to house five mice per cage. Over the course of the study, some were split due to aggression or other behavior based on veterinarian and/or study personnel observation. This may have contributed to variation in measured parameters as previously described . AdditioIn conclusion, mRUST is a reliable, cost-effective, and non-terminal measure to follow fracture healing longitudinally in murine models. It follows a consistent trend that reflects the phases of fracture repair and correlates well with traditional metrics of fracture repair. We predict that implementation of the log-logistic mRUST curve will enable accurate and meaningful representation of various alterations in fracture repair following experimental intervention. This will facilitate characterization of healing trajectory in endocrine disease states, such as menopausal osteoporosis, glucocorticoid use, renal osteodystrophy, pituitary disease, and anorexia nervosa, as well as assess the efficacy of potential treatments. For large exploratory studies , a shift in parameters and phases of the log-logistic curve could be used to gauge general impact on fracture healing. Upon finding a change in mRUST healing trajectory, more detailed experiments involving μCT, histomorphometry, biomechanics and other assessments could be undertaken at targeted time points. In this way, longitudinal mRUST trajectories could expedite discovery of new fracture healing interventions for further investigation.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by Indiana University School of Medicine Institutional Animal Care and Use Committee.RN, MK, CC and T-MC conceived of the idea, designed the experiments and assisted with data interpretation and manuscript preparation. MK provided equipment, funding and other resources. VA, AK, CS, AM, JC, SM, MS, RB, UD, CC, JL and RN participated in conducting experiments and acquisition of data. ZL curated data and performed formal analyses. VA and AM drafted original manuscript. All authors assisted in editing the manuscript, approve of the final version, and take responsibility for the accuracy and integrity of the work.Funding for these studies was provided in part by the NIH . This work was also supported in part by Indiana University School of Medicine, the Indiana Center for Musculoskeletal Health, the Stark Neuroscience Research Institute, and the Department of Orthopaedic Surgery. This material is also the result of work supported with resources and the use of facilities at the Richard L. Roudebush VA Medical Center, Indianapolis, IN: VA Merit #BX003751. The presented contents are solely the responsibility of the authors and do not necessarily represent the official views of any of the aforementioned agencies.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.


The study aimed to evaluate the contribution of building problems to mold proliferation in dwellings. We investigated 503 dwellings of patients suffering from respiratory diseases, whose attending physicians had requested a home inspection if the area of mold was equal to at least one square meter. After careful visual evaluation and basic environmental measurements performed by a trained technician, environmental issues were classified into building defects, accidental water damage, and condensation. Data analysis demonstrated that building defects were the pre-eminent cause of mold proliferation. Among the building defects, water infiltration through leaks in roofs or walls was the leading cause. These results highlight the need for health professionals managing patients with respiratory diseases to be able to request a home inspection, and for city health authorities to commission professionals who can focus on building problems and find ways to address them. Mold proliferation has been identified in around 20% of dwellings in Europe  and the Several governmental and nongovernmental organizations around the world have issued guidelines on the health impacts of indoor mold ,4,5. HomIn practical terms, when a home inspection is performed, the technician should be able to identify the leading cause of mold proliferation.This paper aimed to identify the leading causes of mold proliferation, following home visits to buildings with mold problems. This issue is crucial because it affects the way in which the building will be managed: either through remediation undertaken by professionals in the case of structural defects, or simple adjustments or counseling in the case of inappropriate occupant behavior.Five hundred and three dwellings with mold were studied. A dwelling was considered to have mold if the sum of the surfaces with mold in the living areas  was equal to at least 1 square meter . The inThe technician first performed a careful visual inspection of the dwelling. In each room, they located possible sources of air pollution by checking the condition of the joinery and heaters, maintenance of combustion appliances, looking for spots of mold, and checking the ventilation. They also had a discussion with the tenant concerning the living habits of the family and, in particular, the use of cleaning products.The technician then completed a questionnaire regarding the above items, and also the number of occupants.-A Qtrack to measure ambient temperature and relative humidity ;-A mural hygrometer );-A light paper sheet and a smoke canister to check the efficiency of mechanical ventilation .Then, various types of equipment were used in the living room and the patient’s bedroom, as follows:-Building defects: rising dampness, water infiltration through leaks in the roof and/or external walls, thermal bridge;-Accidental water damage resulting from a sudden disruption of the water pipes;-Vapor condensation on walls and/or ceiling. Measurement of wall humidity allowed the cause of mold development to be identified as either vapor condensation  or a water leak .At the end of the visit, environmental issues were classified into three different groups:The study group included 67 dwellings without structural defects, and 436 with at least one defect. The mean number of occupants per dwelling was equal to 6.25, although the mean figure for this area is 2.5 [This study has strengths and limitations. Its strengths are the large sample size and the standardization of the visits performed by a single trained technician. Nevalinen et al., have demonstrated that occupants missed signs of current or previous mold problems in 15% of houses surveyed . In addiExisting literature has highlighted many different risk factors for mold development: age of the building , number From the abovementioned results, it is clear that building problems are the number one cause of mold proliferation. Indeed, in addition to the “building problems” category, such problems also feature in the “condensation” category when condensation is caused by thermal bridges, and a lack of proper ventilation systems.To the best of our knowledge, this is the first demonstration of the pre-eminence of building problems as the main causes of mold proliferation. Inappropriate building usage is shown to be merely a contributory factor. Therefore, city health authorities and home inspections should first focus on possible building defects. Similarly, health professionals who manage patients suffering from respiratory diseases that may be related to mold proliferation should advise these patients to ask for a home inspection, because it is unlikely that the common advice they offer, concerning ventilation and cleaning moldy surfaces, will resolve the issue.In this study, we aimed to identify the causes of mold proliferation in dwellings. In inner-city high-rise buildings, building problems are the main cause of mold proliferation. This result is likely to apply to this type of building in other locations, but not necessarily to other types of housing in other climates. These findings point to the need for home inspections by professionals so that the defects identified can be correctly addressed.


Graphene field-effect transistors (GFETs) exhibit unique switch and sensing features. In this article, GFETs are investigated within the tight-binding formalism, including quantum capacitance correction, where the graphene ribbons with reconstructed armchair edges are mapped into a set of independent dual channels through a unitary transformation. A new transfer matrix method is further developed to analyze the electron transport in each dual channel under a back gate voltage, while the electronic density of states of graphene ribbons with transversal dislocations are calculated using the retarded Green’s function and a novel real-space renormalization method. The Landauer electrical conductance obtained from these transfer matrices was confirmed by the Kubo–Greenwood formula, and the numerical results for the limiting cases were verified on the basis of analytical results. Finally, the size- and gate-voltage-dependent source-drain currents in GFETs are calculated, whose results are compared with the experimental data. Graphene is a one-atom-thick planar sheet of carbon atoms in a honeycomb structure, whose free-standing monolayer was isolated and characterized for the first time in 2004 by Andre Geim and Konstantin Novoselov . This maWhen the semiconductor layer of a traditional metal–oxide–semiconductor field-effect transistor (MOSFET) is replaced by a graphene sheet, it is called as graphene field-effect transistor (GFET), whose source-drain current is extremely sensitive to the adsorbed molecules, and it is employable for use in gas and bio sensing ,7,8. Morsed CMOS . Among psed CMOS .Additionally, GFET has a singular capacitor behavior, since the graphene ribbon, being one of the parallel plates, possesses a very low charge density around the Dirac point, which may induce large variations in its chemical potential when a small amount of electric charge is introduced into the capacitor. This fact could significantly increase the potential difference between the back gate and the graphene, because it is equal to the difference between the chemical potentials at each plate. Such small quantities of charge generating a large potential difference lead to a lower capacitance compared to geometric or classic ones  , while tIn this article, the source-drain current  of an ideal parallel-plate capacitor with a dielectric material of thickness d and a relative permittivity e is the magnitude of the electron charge.In general, the capacitance per unit of area  in each transversal zig-zag line, as illustrated in W and L are integer numbers, H) of this graphene ribbon with null on-site energies can be written ast is the hopping integral of pristine graphene sheet and l and Let us consider an armchair-edged graphene ribbon with an even number of atoms n2D=∫f energy . CombiniT is given by , while R in turn increases with the ribbon length. Therefore, the asymmetry of Note in  in ref. , which i in ref. . In factThe correlation between source-drain current and gate voltage in graphene field-effect transistors (GFETs) is investigated by means of an atomic-scale tight-binding model, where mesoscopic graphene ribbons with edge reconstruction defects and transversal dislocations are addressed by an independent dual-channel transformation, which converts armchair-edged graphene ribbons into a set of independent dual channels.The electronic transport was studied within the Landauer formalism, and a new transfer matrix method has been further developed for each dual channel with nearest- and next-nearest-neighbor interactions, including aperiodically placed dislocations following a dilute Rudin–Shapiro (RS) sequence, which is one of the closest aperiodic arrangements to the random distribution. The Landauer conductance was confirmed by that obtained from the Kubo–Greenwood formula see , while tQuantized electrical conductance spectra are observed even in presence of edge reconstruction defects along mesoscopic graphene ribbons without dislocations, whose maximum value corresponds to the number of dual channels, while the multi-step behavior is softened at finite temperatures. In addition, the back gate voltage ( lengths .The approach of independent dual-channel plus renormalization method presented in this article can be used to study biosensor devices based on GFETs by considering adsorbed molecules as Fano impurities . This st


Due to its superhydrophobicity and sensitivity, this wearable sensor demonstrates efficient and stable monitoring of various underwater activities, including pressure, blowing, and tapping. Our approach provides an alternative idea for highly sensitive wearable sensors while broadening the practical application scope.Wearable pressure sensors have received widespread attention owing to their potential applications in areas such as medical diagnosis and human–computer interaction. However, current sensors cannot adapt to extreme environments  or show moderate sensitivity. Herein, a highly sensitive and superhydrophobic fabric sensor is reported based on graphene/PDMS coating. This wearable sensor exhibits great superhydrophobicity (water contact angle of 153.9°) due to the hydrophobic alkyl long chains and rough structure introduced by the Ar plasma. Owing to the network structure created by the electric-induced alignment of graphene sheets, an enhanced sensitivity (ΔI/I Wearable pressure sensors ,4,5,6,7 Researchers have introduced superhydrophobic material on the sensor surface to improve its resistance to harsh conditions. For example, Ni et al.  establisIn this experiment, a simple strategy was used to introduce an electric field into the preparation process of the conductive fabric to obtain a graphene interlayer-amplified structure. When an external force was applied, the additional structural deformation of the graphene sheet increased the sensor’s output. For underwater sensing, the polyester fabric obtained after plasma treatment had excellent superhydrophobicity and could be placed underwater to sense underwater motion (water contact angle (WCA) = 153.9°). New ideas were proposed for the design and development of future underwater intelligent equipment.2) were purchased from Miandu Textile Co., Ltd. . All fabrics were washed with Detergent 209 aqueous solution . The poly(dimethylsiloxane) precursor (MW770) was purchased from Alfa Aesar Chemical Company. Multilayer graphene (thickness less than 100 nm) was purchased from Kaisa  Materials Co., Ltd. Anhydrous ethanol (AR ≥ 99.7%) and argon (99.99%) were purchased from Changzhou Hongsheng Fine Detail Co., Ltd.  and Shanghai Haoqi Gas Co., Ltd. .Polyester fabrics  treatment. First, graphene and ethanol solutions were ultrasonically dispersed and exfoliated for 2 h, and the poly(dimethylsiloxane) precursor was then added to ultrasonic and mixed for 1 h . The preThe surface morphology of the polyester fabrics was tested using a field emission electron microscope . Water contact and rolling angles were measured using a professional A-200 instrument . The test system consists of an electrochemical workstation (CHI660E), a sensor, and a lab-built applied pressure device . An abra3 as -Si-CH2- under high-energy plasma and subsequent polymerization into -Si-CH2-CH2-Si- [-CH2-Si- ,21,22,23An electric field is applied to graphite nanosheets on PDMS substrates induce directional alignment of nanosheets. To clarify the effect of the electric field on the sensitivity of the fabric sensor, the response and recovery time of the sensor are measured when pressure is applied and removed from the sensor. As shown in μ). Here the dipole moment can be divided into two directions—the component parallel to the graphene sheet ||μ and the component perpendicular to the graphene sheet ⊥μ—and the force acting on the graphene sheet is the superposition of the two effects [However, the graphene nanosheets are not twisted completely parallel to the electric field, which may be due to the joint influence of the viscosity of macromolecules and the force between graphene nanosheets, and the movement of graphene nanosheets is greatly hindered by space. Therefore, the graphene nanosheets tend to move in one direction as a whole.  effects .T = μ × E, which is the driving force for the electric field to align the graphene, and the torque will try to twist the graphene, so that the graphene is aligned parallel to the electric field. However, due to the large aspect ratio of graphene, the generated dipole moment usually differs from the direction of the applied electric field force. The overall force acting on a graphene sheet is the superposition of moments caused by fields parallel and perpendicular to its axis; note that ||E = E × cosθ; ⊥E= E × sinθ, and θ is the angle between the direction of the DC electric field and sheet axis.The interaction between the electric field force and dipole moment produces an electric field-induced torque θ satisfies certain conditions, the total force can deflect the graphene sheet against the resistance in the system. If the directional arrangement of graphene is achieved under an electric field, the main consideration is the relationship between the torque and resistance of the system, and graphene starts to deflect when the torque generated under the electric field exceeds the sum of the resistance of the graphene in the system. Therefore, the choice of electric field strength is an important starting point for achieving the directional deflection of graphene.When 0, ΔI = I − I0, where I is the instantaneous current, I0 is the initial current at 0 kPa) is defined as the signal response of the pressure sensor, which can indirectly represent the sensitivity of the pressure sensor. The ΔI/I0 values initially increased and reaches an equal value then decreased as the dispersion concentration increased  and fast response time (~100 ms) due to the additional structural deformation obtained by the three-dimensional alignment of graphene under electric fields. The sensor can be used for pressure, water level, and underwater motion detection because it exhibits excellent superhydrophobicity (WCA = 153.9°) and good repeatability due to the formation of alkyl long chains and the rough surface under plasma. Moreover, stable superhydrophobic surfaces maintained good electrical conductivity when immersed in acid–alkali solutions with ultrasonic treatment. The sensor can be integrated into electronic circuits to provide warnings for preset pressures, or the fabric sensor could be used in underwater wearable devices to detect the pressure environment in which the human body is subjected to being underwater in real-time. We believe that this highly sensitive superhydrophobic fabric sensor provides an efficient strategy for wearable sensors, especially in wet or underwater environments.In summary, we have demonstrated a fabric pressure sensor with high sensitivity, good stability and good superhydrophobicity using the electric field synergistic plasma approach. The fabric sensor exhibits higher sensitivity (ΔI/I


Dysregulated brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signalling is implicated in several neurodegenerative diseases, including Alzheimer’s disease. A failure of neurotrophic support may participate in neurodegenerative mechanisms, such as ferroptosis, which has likewise been implicated in this disease class. The current study investigated whether modulators of TrkB signalling affect ferroptosis. Cell viability, C11 BODIPY, and cell-free oxidation assays were used to observe the impact of TrkB modulators, and an immunoblot assay was used to detect TrkB expression. TrkB modulators such as agonist BDNF, antagonist ANA-12, and inhibitor K252a did not affect RSL3-induced ferroptosis sensitivity in primary cortical neurons expressing detectable TrkB receptors. Several other modulators of the TrkB receptor, including agonist 7,8-DHF, activator phenelzine sulphate, and inhibitor GNF-5837, conferred protection against a range of ferroptosis inducers in several immortalised neuronal and non-neuronal cell lines, such as N27 and HT-1080 cells. We found these immortalised cell lines lack detectable TrkB receptor expression, so the anti-ferroptotic activity of these TrkB modulators was most likely due to their inherent radical-trapping antioxidant properties, which should be considered when interpreting their experimental findings. These modulators or their variants could be potential anti-ferroptotic therapeutics for various diseases. NTRK2; tropomyosin receptor kinase family) is a receptor for brain-derived neurotrophic factor (BDNF) and neurotrophin 4. The binding of ligands to the full-length TrkB receptor leads to ligand–receptor dimerisation and autophosphorylation of tyrosine residues in the intracellular kinase domain of the receptor [Tropomyosin receptor kinase B  ,5,6. BDNII(ATSM)), and by iron chelators such as deferiprone (DFP) [Ferroptosis, a unique form of iron-mediated regulated cell death, is executed by the build-up of lethal lipid hydroperoxides ,11,12. Ine (DFP) . Ferroptne (DFP) . TherefoGrowing evidence suggests that the ligand-activated TrkB signalling pathway mediates neuroprotection via amelioration of aberrant cellular responses such as inflammation ,17, oxidWe first screened different immortalised cell lines, such as N27, HT-22, HT-1080, and U-251, as well as primary cortical neurons for their TrkB expression and found that only mature primary cortical neurons express detectable levels of TrkB receptors a. Mature50 against RSL3: 19.2 μM) than in rescuing N27 cells (EC50 against RSL3: 72.1 μM).Independent studies have shown that the TrkB activators 7,8-DHF ,30 and p50 of 7,8-DHF: 24.3 μM; phenelzine: 40.9 μM), which was commensurate to the reference anti-ferroptotic RTA, Lip-1 (IC50 =22.7 μM)  c.50: 36~116 nM) and RSL3 (EC50: 29~73 nM), commensurate to Lip-1 in the following immortalised cell lines: N27, HT-22, and HT-1080  in the same cell lines: N27, HT-22, and HT-1080 a. We alsGrowing evidence of altered BDNF-TrkB signalling in neurodegeneration has spurred interest in understanding the possible underlying mechanisms. Ferroptosis is also implicated in neurodegeneration, and TrkB trophic signalling conceivably interferes with this pathway. Here, we reveal that several modulators of TrkB inhibit ferroptosis via a receptor-independent RTA mechanism.While BDNF is a potent agonist of the TrkB receptor, systemic therapy with BDNF has several pharmacokinetic limitations, such as short half-life, large molecular size, and poor blood–brain barrier (BBB) penetration. As an orally bioactive flavonoid, 7,8-DHF penetrates the BBB to act as an agonist of the TrkB receptor, and numerous preclinical studies ,30 demonTypical and fast-acting antidepressants bind to the TrkB receptor to exert biological and behavioural actions ,34. AntiANA-12 is a selective and potent antagonist of the TrkB receptor that penetrates the BBB , and K25GNF-5837 is a potent and orally bioavailable oxindole pan-Trk inhibitor with a mechanism similar to K252a . In our RPMI 1640 culture media (Cat# 72400120), penicillin and streptomycin (Cat#15140122), B27 (Cat#17504044), and MTT (Cat#M6494) were purchased from Thermo Fisher Scientific ; Foetal Bovine Serum ; Erastin (Cat#S7242), RSL3 (Cat#S8155), and 7,8-DHF (Cat#S8319) were purchased from Selleckchem ; FINO2 ; tBH solution (Cat#416665), BSO (Cat#B2515), deferiprone (Cat#379409), Lip-1 (Cat#SML1414), K252a (Cat#05288), recombinant human BDNF protein (Cat#B3795), phenelzine sulphate salt (Cat#P6777), GNF-5837 (Cat#SML0844), and ANA-12 (Cat#SML0209) were purchased from Sigma-Aldrich .2.N27 rat dopaminergic neural cell line harvested from E12 rat mesencephalic tissue and HT-22 mouse hippocampal cell line subcloned from the HT-4 cell line , U-251 human glioblastoma—a kind gift from Associate Professors Andrew Morokoff and Kate Drummond—, and HT-1080 human fibrosarcoma cells  were cultured in RPMI 1640 media (supplemented with 10% FBS and 1% penicillin and streptomycin). As previously described, primary cortical neurons were isolated from E14 C57BL/6J mice (RRID: IMSR_JAX:000664) . Upon is4 cells/well, while primary cortical neurons were 1 × 105 cells/well. Cells were then co-treated with tested compounds and ferroptosis inducers for 24 h (but tBH was for 4 h), and 20  μL of MTT (5  mg/mL) was added to each well and incubated for 2–4 h. The overlying media were aspirated from each well, and DMSO was added to the wells to dissolve formed formazan crystals in viable cells. The absorbance at 570 nm of the dissolved formazan demonstrated viable cells and was measured by a PowerWave XS microplate spectrophotometer , and cell viability was expressed as a percentage of control cells.The colourimetric MTT assay is routinely used to measure cell viability in the context of ferroptosis ,11,13. TABTS radical scavenging assay was used to evaluate the possible RTA properties of tested compounds in cell-free conditions. The assay was done according to the described protocol by Lee et al. (2015)  with theC11 BODIPY (581/591), a fluorophore that responds to lipid peroxidation with an increase in fluorescence emitted in the green range and a parallel decrease in fluorescence emitted in the red range, was employed to quantify lipid peroxides. After seeding cells per the viability assay, cells were co-treated with test compounds  for 24 h. Cells were washed once with PBS and then supplemented with PBS before measuring fluorescence at 565/600 nm (excitation/emission) for red fluorescence and 477/525 nm for green fluorescence using a CLARIOstar microplate reader . Lipid peroxidation was presented as the ratio of green to red C11 BODIPY fluorescence [We evaluated the possible RTA property of GNF-5837 in cell-free conditions by a fluorescence-based lipid peroxidation assay using the MDA Assay Kit  as previously reported [w/v) skimmed milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against TrkB  and beta-actin , and then following incubation with the normal isotype-matched IgG-HRP conjugated antibodies (Dako) with a dilution ratio of 1:2000 for 1 h. Finally, immunoblots were visualised by a chemiluminescence reagent (Amersham ECL Start Western Blotting Detection Reagent) and imaged using a Fujifilm Luminescent Image Analyser LAS-3000 and then quantified by Image J software.Total protein was extracted from the cells by a RIPA lysis buffer containing 50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100 and cOmplete™, and an EDTA-free protease inhibitor cocktail . Protein was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, which was then transferred to polyvinylidene difluoride membranes using the iBlot Gel Transfer Device (Invitrogen). The membranes were blocked in tris-buffered saline containing 0.1% Tween-20 (TBST) and additionally containing 5% (www.graphpad.com) were used to perform statistical analysis. Some test compounds were coloured; the operator and data assessment were not blinded, and samples were not randomised. At least 2–3 technical replicates were used to ensure the dependability of single values within each independent experiment.Microsoft Excel v16 and GraphPad Prism 9.1 software for windows . The corresponding fitted regression curve is shown on the data where an EC50 or IC50 is reported. One- or two-way ANOVA was used for all multiple comparisons to calculate statistical significance, and p < 0.05 was deemed statistically significant.Non-linear regression analysis with a variable slope model (four parameters) was employed to fit a logistic curve to dose–response data to determine ECWe found that several potent modulators of TrkB signalling, such as BDNF, K252a, and ANA-12, did not affect ferroptotic lethality in TrkB-expressing mature primary cortical neurons. BDNF mimetic 7,8-DHF, TrkB activator phenelzine, and inhibitor GNF-5837 showed TrkB-independent anti-ferroptotic activity, possibly acting as RTAs. Caution should be applied when interpreting the results of these compounds in prior and future studies since the potent RTA effect may confound the result. As we detected TrkB receptor expression only in mature cortical neurons, this study was limited to characterising the effect of TrkB signalling on RSL3-induced ferroptotic lethality. We do not exclude the possibility that TrkB modulators affect ferroptotic stress caused by cystine/glutamate transporter inhibitors such as erastin in TrkB-expressing cell lines.


E − EF = 0.2 and 0.65 eV from the pd-hybridization and controllable Dirac cone of graphene, respectively. In the parallel configuration, the transmission probability calculation showed a profile similar to that of the pristine graphene. High and colossal magnetoresistance ratios of 284% and 3100% were observed at E − EF = 0.65 eV and 0.2 eV, respectively. Furthermore, a magnetoresistance beyond 3100% was expected at E − EF = 0.65 eV when the width of the Ni(111) nanostructures on the nanometer scale was considered.In this study, we present a theoretical study on the in-plane conductance of graphene partially sandwiched between Ni(111) nanostructures with a width of ∼12.08 Å. In the sandwiched part, the gapped Dirac cone of the graphene was controlled using a pseudospin by changing the magnetic alignment of the Ni(111) nanostructures. Upon considering the antiparallel configuration of Ni(111) nanostructures, the transmission probability calculation of the in-plane conductance of graphene shows a gap-like transmission at  In this study, we present a theoretical study on the in-plane conductance of graphene partially sandwiched between Ni(111) nanostructures with a width of ∼12.08 Å. The equipotential of the C atoms in sublattices A and B of the graphene layer causes a Dirac cone energy band at zero energy. This Dirac cone energy band causes the electrons in graphene to behave peculiarly, having the same velocity and no inertia.2 Growing graphene on top of a transition metal substrate enables the sensitive tuning of the Dirac cone energy band. When graphene is placed on a metal substrate, hybridization occurs, breaking the chiral symmetry of graphene and creating a gapped Dirac cone.5 Another unique property of graphene is its magnetic response when grown on a ferromagnetic substrate.6–8 This induces a magnetic moment in graphene due to charge transfer and has an orientation opposite to that of the ferromagnetic substrate.8 Additionally, the weak spin–orbit coupling and long spin scattering length of graphene9 make it a prospective material for spintronic devices. Ni(111) surfaces are the most commonly used contacts for studying graphene-based spintronic devices due to their similar structures  and strong hybridization with graphene.6,7,10 Studies show that graphene has served as a bridge between two Ni electrodes11–13 because of its long spin relaxation lifetime9 and as a tunnel barrier in Ni/graphene/Ni magnetic tunnel junctions (MTJs).14–23 Although further results are expected, a new strategy for achieving low resistance and overcoming the absence of a bandgap is necessary to realize the application of graphene in spintronic devices.The extraordinary in-plane charge mobility of graphene24 When the magnetic moments of the upper and lower Ni(111) slabs have an antiparallel configuration (APC), the bandgap at the Dirac cone is open. However, the bandgap is closed in parallel configuration (PC). This unique characteristic is because the most stable arrangement of the Ni/graphene/Ni heterostructure occurs when Ni atoms of the upper and lower Ni(111) slabs at the interfaces hybridize with different graphene sublattices. In other words, Ni atoms from the lower Ni(111) slab hybridized with C atoms in sublattice A , and Ni atoms from the upper Ni(111) slab hybridized with C atoms in sublattice B . Although the C atoms of graphene bond with the Ni atoms, this special hybridization preserves the equipotential between sublattices A and B. Meanwhile, a magnetic moment is induced on the graphene layer by charge transfer from the Ni to C atoms. The induced magnetic moments of the CA and CB atoms depended on the magnetic alignment of the Ni(111) slabs. Moreover, the induced magnetic moments in APC and PC between the CA and CB atoms exhibit antiferromagnetic and ferromagnetic orders, respectively. This implies that the pseudospin between sublattices A and B can be controlled to preserve or break the chiral symmetry, thereby making the gap of the Dirac cone is also controllable. A controllable-gapped Dirac cone help realize a high in-plane magnetoresistance (MR) ratio for graphene.Previous studies showed that for a Ni/graphene/Ni heterostructure, the gapped Dirac cone of graphene can be controlled using the magnetic alignment of the Ni slabs.This study presents a theoretical study on the in-plane conductance and magnetoresistance of graphene partially sandwiched between Ni(111) nanostructures. Here, we investigate the effectiveness of a controllable Dirac cone on graphene conductance to realize a high MR ratio. For that purpose, we proposed a system consisting of Ni(111) nanostructures with a finite size and atomic-scale width of ∼12.08 Å sandwiched the middle part of graphene. Both the APC and PC states of the upper and lower Ni(111) structures are considered. First-principles quantum transport calculations, which coupled density functional theory (DFT) with the nonequilibrium Green's function, were performed. Our calculation results observed a high and colossal in-plane MR ratio of graphene about 284% and 3100%. A higher MR ratio beyond 3100% can be obtained when the width of the Ni(111) structures on a nanometer scale are considered.24 The finite size width of ∼12.08 Å for Ni(111) nanostructures was considered for understanding the effectiveness of the in-plane magnetoresistance of graphene created from the Ni/graphene/Ni magnetic junctions. Wider Ni(111) nanostructure, e.g., width in nanometer scale, is expected to exhibit high performance. Here, graphene was used as the buffer layer and electrodes in the calculation. We considered the same stacking arrangement used in the previous study, where Ni atoms at the interfaces of the lower and upper Ni(111) slabs hybridized with C atoms in sublattices A and B, respectively.24 Both APC and PC were considered for Ni(111) nanostructures.In this theoretical study, we considered Ni(111) nanostructures with three Ni atoms layer thickness and an atomic-scale width of ∼12.08 Å, as shown in 25 to obtain the structural equilibrium and spin-charge density properties of the proposed system. Furthermore, we described the electron–ion interaction using a revised Perdew–Burke–Ernzerhof functional for a densely packed solid surface 26 and ultrasoft pseudopotentials27 within the generalized gradient approximation (GGA). From our previous studies, the PBESol pseudopotential is essential to successfully achieve the interlayer distance between Ni slabs and graphene , which is consistent regardless the thickness of Ni slabs and agrees with experimental study.24,28,29 This interlayer distance at the interface is important because it determine the electronic state at the interface. The atomic positions were relaxed, with a total force tolerance of 0.001 eV Å−1. A 45 × 45 × 1 Monkhorst–Pack k-mesh was used for calculations. First-principles quantum transport calculations, which coupled DFT with the nonequilibrium Green's function, were performed using the Siesta and Transiesta packages30–34 to calculate the transport properties at a zero-bias voltage. The spin-dependent current through our proposed system was calculated using the Landauer–Buttiker given by:fL) is the right (left) moving electron injected from the left (right) lead in the form of the Fermi–Dirac function. μL(μR) denotes the chemical potentials of the left (right) electrodes. Since zero-bias voltage was considered, thus μL = μR = EF. In addition, the ballistic transmission T as a function of energy E is described with respect to the Green's function form asΓL(ΓR) is the coupling matrix of the left (right) electrode, GR(GA) is the retarded (advanced) Green's functions of the central region. Finally, the in-plane MR ratio for graphene was calculated as follows:A spin-polarized plane-wave-based DFT calculations were performed using the Quantum ESPRESSO package31 were used within GGA. A double-zeta plus polarization basis set35–37 was employed, and the temperature was set to 300 K. A perpendicular k-point of 1 × 901 with respect to the transmission direction was considered to obtain a good accuracy for the transmission probability.For the transmission probability calculation, the PBESol functional and Troullier–Martins pseudopotentialWe investigated the characteristics of the induced magnetic moment on graphene, which is sandwiched between the Ni(111) nanostructures. Particularly we focused on the boundaries between the bare and sandwiched graphene. Mapping of the spin-charge density, as shown in nAσ = nBσ with σ = ↑ or ↓. Meanwhile, the sandwiched graphene has an induced magnetic moment depending on the magnetic alignment of the Ni(111) nanostructures. For the Ni(111) nanostructures in APC state, A and CB atoms of the sandwiched graphene have an antiferromagnetic order. Thus,A and CB atoms of the sandwiched graphene have the same magnetic orientation and almost the same value , which is ∼0.04 μB. This means that the decrease in the magnetic moment amplitude along the boundary direction corresponds to the characteristics found in the finite size of the Ni nanostructures. From the structure of the system, this decrease is attributed to the coordination of Ni(111) nanostructures, which compresses their shape toward the center but maintains their bond with C atoms at the interfaces. An edged shape was formed on the Ni(111) nanostructures at the boundaries, which increases the lattice mismatch between the Ni layer at the interfaces and the graphene layer, and in turn, decreases the charge transfer from Ni atoms to C atoms. Thus, the reduced charge transfer from the Ni atoms to the C atoms at the interface decreases the induced magnetic moment on the C atoms. Since the lattice mismatch increases along the direction of the boundary, the induced magnetic moment also decreases along the same direction. However, the profile of the induced magnetic moment on sandwiched graphene is unique in PC. The induced magnetic moment initially decreases from the center of the sandwich to the boundaries. However, it then increases even higher than that of the center part before finally decreasing again near the boundaries. The magnetic moment of the C atoms in the center of the sandwiched graphene was approximately equal to the induced magnetic moment of the C atoms of the Ni/graphene/Ni MTJ, which was ∼0.022 μB. Furthermore, the decrease in the induced magnetic moment on the neighboring C atoms, which shifted from the center of the sandwich graphene to the boundary, corresponds to the increasing lattice mismatch, similar to the case of APC. Additionally, the amplitude of the magnetic moment on the C atoms increased even more than that in the center part at the boundaries due to the reduction in the antiferromagnetic configuration between sublattices A and B near the boundary. In the periodic system of Ni/graphene/Ni MTJ, we obtained higher amplitude of the induced magnetic moment of C atoms in APC is higher than that in PC24 since the carbon atoms of sublattices A and B in graphene had to have an antiferromagnetic (AFM) configuration because of the half-filled pz-orbital and Pauli's exclusion principle. This rule is often found in organic molecules in sp2-hybridization or magnetic alternant hydrocarbon systems.38 The contribution of the antiferromagnetic alignment between the hybridized C and Ni atoms near the boundaries is more dominant than that of the AFM alignment between the C atom sublattices A and B, leading to the increased induced magnetic moment. Finally, at the boundaries, the induced magnetic moment of the C atoms in the sandwiched part decreases again, indicating increased lattice mismatch.The magnetic moment characteristics of the CThese changes in the induced magnetic moment on sandwiched graphene affect the Dirac cone and mass-gapped Dirac-cone characteristics. For the Ni(111) nanostructures in the APC, the decreasing induced magnetic moment of the sandwiched graphene toward the boundary reduces the size of the mass-gapped Dirac cone. This implies that the Dirac cone mass-gapped is smaller at the boundaries of the sandwiched graphene than at the center. For Ni(111) nanostructures in the PC state, the changes in the induced magnetic moment affect the size of the Stoner gap between the spin majority and spin minority channels. These characteristics are discussed in detail in the next section.E − EF = 0.18–0.22 eV and 0.58–0.78 eV, with its lowest transmission probability at E − EF = 0.2 and 0.65 eV, respectively, to understand the origin of the gap-like transmission, local density of states (LDOS) and projected density of states (PDOS) calculations were performed on the Ni/graphene/Ni MTJ, which represents the sandwiched part of the proposed system. The LDOS of graphene, shown in E − EF = 0.2 eV corresponds to the gap-like transmission probability found at E − EF = 0.2 eV. This relation was observed in the spin majority and minority channel. This Dirac-cone-like DOS is originated from the hybridization between d2z-orbital of Ni atoms at the interface and pz-orbital of C atoms  nanostructures were in the APC state, as shown in  C atoms . The Dirdization .E − EF = 0.65 eV results from the opening mass-gapped Dirac cone of graphene  nanostructure does not optimize the induced magnetic moment on graphene and a constant value is not achieved, thereby resulting considerably high conductance at E − EF = 0.65 eV. However, this also implies that, when a width of Ni(111) nanostructures is considered in the nanometer scale, the transmission probability at E − EF = 0.65 eV will be as low as at E − EF = 0.2 eV.The gap-like transmission probability at graphene . HoweverE − EF = 0.2 and 0.65 eV, respectively nanostructures in APC and PC states, a high and colossal in-plane MR ratio of up to 3100% and 284% was observed at pectively. ApplyinMR ratio . InteresA and CB of the sandwiched graphene.In this study, we investigated the in-plane conductance of graphene, where the Ni(111) nanostructures with a width of ∼12.08 Å is sandwiched in the center of the graphene. Both the APC and PC states of the Ni(111) nanostructures were considered. The induced magnetic moment was observed on the sandwiched graphene and could be controlled using the magnetic alignment of the Ni(111) nanostructures. When APC (PC) state is considered, we observed that the carbon atoms of sublattices A and B of the sandwiched graphene had an antiferromagnetic (ferromagnetic) order. Both spin configurations lead to a controllable mass-gapped Dirac cone in the sandwiched graphene. The mass-gapped Dirac cone is open (closed) for APC (PC) state due to the modulated (equi-) potential between the CWhen the Ni(111) nanostructures were in the PC state, the transmission probability of the proposed system produces a profile similar to that of the pristine graphene. Furthermore, a slight increase in the total transmission probability was observed from spin-up electrons that transmit through graphene and the Ni(111) nanostructures. Thus, spin-up electrons have a higher transmission probability than the spin-down electrons.E − EF = 0.18–0.22 eV and 0.58–0.78 eV with the lowest transmission probability at E − EF = 0.2 and 0.65 eV, respectively. The gap-like transmission probability at E − EF = 0.2 eV comes from the Dirac-cone-like DOS shown in the local density of sandwiched graphene due to the hybridization property between the d2z-orbital of Ni atoms at the interface and pz-orbital of C atoms. On the other hand, the gap-like transmission probability at E − EF = 0.65 eV comes from the opening mass-gapped Dirac cone of graphene. However, the gap-like transmission probability was smaller than that found in the LDOS of graphene in Ni/graphene/Ni MTJ. This is because the induced magnetic moment decayed from the center part of the sandwiched graphene toward the boundary, leading to a parabolic shape for the transmission probability gap.However, for the Ni (111) nanostructures in the APC state, a unique gap-like transmission probability was observed from E − EF = 0.2 and 0.65 eV. By applying a gate voltage, the Fermi energy can be controlled resulting in a colossal in-plane MR ratio. Furthermore, by increasing the width of the Ni(111) nanostructure to the nanometer scale, a colossal MR ratio > 3100% can be expected because the gap-like transmission is nearly zero at E − EF = 0.65 eV in the APC state but having higher the transmission probability than that at E − EF = 0.2 eV in the PC state.Finally, high and colossal in-plane MR ratios of up to 3100% and 284% were observed at Conceptualization was performed by Y. W., K. K., H. H., and G. K. S. performed computer simulations of the atomic layered systems and M. A. M. conducted the theory of atomic layered materials. Y. W. determined the equilibrium structure of the proposed system and performed spin-charge density mapping and transmission probability analysis on the proposed system. YW wrote the original draft of the manuscript. All the authors have reviewed the manuscript. The manuscript was written through the contributions of all authors. All the authors approved the final version of the manuscript.There are no conflicts of interest to declare.


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by progressive loss of motor neurons and there is currently no effective therapy. Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein 43 kDa (TDP-43) within the CNS is a pathological hallmark in sporadic ALS and prion-like propagation of pathogenic TDP-43 is thought to be implicated in disease progression. However, cell-to-cell transmission of pathogenic TDP-43 in the human CNS has not been confirmed experimentally. Here we used induced pluripotent stem cells (iPSCs)-derived cerebral organoids as recipient CNS tissue model that are anatomically relevant human brain. We injected postmortem spinal cord protein extracts individually from three non-ALS or five sporadic ALS patients containing pathogenic TDP-43 into the cerebral organoids to validate the templated propagation and spreading of TDP-43 pathology in human CNS tissue. We first demonstrated that the administration of spinal cord extracts from an ALS patient induced the formation of TDP-43 pathology that progressively spread in a time-dependent manner in cerebral organoids, suggesting that pathogenic TDP-43 from ALS functioned as seeds and propagated cell-to-cell to form de novo TDP-43 pathology. We also reported that the administration of ALS patient-derived protein extracts caused astrocyte proliferation to form astrogliosis in cerebral organoids, reproducing the pathological feature seen in ALS. Moreover, we showed pathogenic TDP-43 induced cellular apoptosis and that TDP-43 pathology correlated with genomic damage due to DNA double-strand breaks. Thus, our results provide evidence that patient-derived pathogenic TDP-43 can mimic the prion-like propagation of TDP-43 pathology in human CNS tissue. Our findings indicate that our assays with human cerebral organoids that replicate ALS pathophysiology have a promising strategy for creating readouts that could be used in future drug discovery efforts against ALS. Amyotrophic lateral sclerosis (ALS) is a disease characterized by the degeneration of selected groups of neuronal cells. Mislocalization of the TAR DNA-binding protein 43 kDa (TDP-43) from the nucleus of neuronal cells toward the cytoplasm is a pathological hallmark of most cases of ALS. Owing to its structure TDP-43 has a high aggregation propensity when outside the nucleus. Here we used a a form of self-organizing three-dimensional cell culture model  to test if TDP-43 could contribute the progression of the disease that occurs across the central nervous system of patients. TDP-43 is hypothesized to contribute to the progression of the disease by inducing the propagation of its aggregated form across cells. Protein extracts prepared from postmortem spinal cord of ALS patients showing aggregated TDP-43 were separately injected into cerebral organoids derived from an ALS case and from a control individual. This approach demonstrated the spreading of aggregated TDP-43 across the cells of the injected organoids derived from an ALS case; by comparison, the same spreading was very limited within organoids from the control individual. Critically, protein extracts prepared from non-diseased individuals did not result in the propagation of aggregated TDP-43. Our finding could be used to evaluate treatments aiming to prevent the propagation of aggregated TDP-43 established to be pathogenic. Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by progressive muscle weakness due to the selective loss of motor neurons in the nervous system, ultimately leading to death from respiratory failure. Approximately 10% of ALS cases have a positive family history , and the remaining 90% of cases occur sporadically (sporadic ALS) ,3. In adOne of the key features of neurodegenerative diseases is the prion-like spreading of disease-related misfolded proteins such as tau in Alzheimer’s disease and α-synuclein in Parkinson’s disease . AccumulHuman induced pluripotent stem cells (iPSCs) are used to generate different disease models that can allow the study of disease pathogenesis . MoreoveHere we report that pathogenic TDP-43 extracted from postmortem ALS spinal cord tissue induces the formation of phosphorylated TDP-43 cytoplasmic aggregates in a time-dependent manner in human iPSC-derived cerebral organoids. We show that administration of pathogenic TDP-43 into cerebral organoids causes not only TDP-43 pathology but also astrogliosis, morphologically similar to what is seen in ALS postmortem tissue. We also show that pathogenic TDP-43 from ALS tissue facilitates cellular apoptosis and genomic damage. Our findings indicate that patient-derived pathogenic TDP-43 protein propagates in a prion-like manner and induces TDP-43 pathology in a time-dependent manner in a model that mimics human CNS environment.Human peripheral blood mononuclear cells (PBMCs) were collected to generate induced pluripotent stem cells (iPSCs) after obtaining written informed consent forms in accordance with the institutional human ethics committee guidelines. All the experimental procedures using postmortem human tissue samples were approved by McGill University Health Centre Research Ethics Board (IRB00010120).2. iPSCs colonies were passaged at approximately 70–80% confluency. Human iPSCs information is listed in Human iPSCs were generated from peripheral blood mononuclear cells (PBMCs) of consenting individuals. The PBMCs were reprogrammed into human iPSCs using the CytoTune iPS Sendai reprogramming kit (Thermo Fisher Scientific) according to the manufacture’s protocol. iPSCs were cultured on a Matrigel (Corning) coated plate with mTeSR1 medium (STEMCELL Technologies) in 100% humidity incubator at 37°C under 5% COCerebral organoids were differentiated from human iPSCs and cultured with STEMdiff Cerebral Organoid Kit (STEMCELL Technologies) according to the manufacture’s protocol. Once iPSCs colonies reached 70–80% confluency, iPSCs were placed in a 96-well round-bottom ultra-low attachment microplate  with EB formation medium  containing ROCK inhibitor to generate embryoid bodies (EBs) from iPSCs, and EB formation medium was added every other day. At 5 days in vitro (DIV), EBs were moved to a 24-well ultra-low attachment plate  and cultured with induction medium for 48 hours. At 7 DIV, EBs were embedded into 15 μL of Matrigel and placed into a 6-well ultra-low attachment plate  with expansion medium for 72 hours to develop expanded neuroepithelia. At 10 DIV, expansion medium was replaced to maturation medium, and the 6-well cell culture plate was placed onto an orbital shaker with 65 rpm in a cell culture incubator to differentiate cells into cerebral organoids. Maturation medium was replaced every 3–4 days until they are used for experimental process.Postmortem sporadic ALS and non-ALS spinal cord frozen specimens were obtained from the Montreal Neurological Institute-Hospital , the Douglas-Bell Canada Brain Bank  and the London Neurodegenerative Diseases Brain Bank . Tissue information is listed in The sarkosyl insoluble protein fraction from human postmortem frozen spinal cord was obtained by sequential protein extraction with increasing detergent strength buffers . AnterioStandardized automated immunohistochemistry is performed using the Benchmark Ultra IHC platform with OptiView DAB IHC detection  per manufacturer specification. Primary antibody (anti-phospho-TDP-43 (pS409); Cat#: TIP-PTD-P03, Cosmo Bio)  is incubDNA was extracted from a blood sample using standard salting out procedures. Whole genome sequencing (WGS) was performed at the Génome Québec Centre D’Expertise et de Services on a Novaseq 6000 sequencer , using 150bp paired-end reads. All bioinformatics analyses were performed on the Béluga computing cluster of Compute Canada and Calcul Québec. An average genomic coverage of 39.31X was generated on the hg38 human reference genome. Alignment and variant calling were performed using the Illumina DRAGEN Bio-IT v3.8 platform . Variant2.Cerebral organoids at 60 DIV were taken from maturation media and washed briefly with PBS at room temperature. 0.3 μL of sarkosyl-insoluble fraction of spinal cord extract was slowly injected into the cerebral organoids using a 32-gauge Hamilton Neurosyringe (Hamilton) at 1 mm depth. The injected cerebral organoids were placed back into maturation media and maintained in 100% humidity incubator at 37°C under 5% COAfter collected from maturation media and washed with cold PBS, cerebral organoids were briefly sonicated and lysed into 9x v/w cold RIPA buffer  containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and proteinase and phosphatase inhibitor cocktails (Roche). The protein extract was centrifuged at 25,000g for 30 minutes at 4°C, and the supernatant was used as RIPA-soluble fraction. The protein concentration of RIPA-soluble fraction was measured with Bradford assay (Biorad). The remaining pellet was washed with 5x v/w cold RIPA buffer and centrifuged at 25,000g for 30 minutes at 4°C. Subsequently, the pellet was resuspended and briefly sonicated in 4x v/w cold PBS, being taken as RIPA-insoluble fraction.Protein extract samples were eluted into 2x Laemmli sample buffer (Biorad) containing 2-mercaptethanol and denatured for 5 minutes at 95°C. The samples were loaded on a 12% TGX Stain-Free Fast Cast acrylamide gel (Biorad) and run for 30 minutes at 200 V. After electrophoresis, the protein samples were transferred onto a PVDF membrane (Millipore). The transferred PVDF membrane was blocked with 3% bovine serum albumin (BSA) in 0.1 M PBS/0.1% Tween 20 (0.1% PBS-T) at room temperature for 1 hour. The membrane was incubated with primary antibody diluted in 3% BSA/0.1% PBS-T at 4°C overnight. After washing with 0.1% PBS-T, the membrane was incubated with peroxidase-conjugated secondary antibody for 1 hour at room temperature. Targeted protein was detected by exposing the membrane to a mixture of the Clarity western ECL substrate (Biorad) and imaged using ChemiDoc MP Imager (Biorad).Cerebral organoids were immersed and fixed in 4% PFA at 4°C overnight and incubated in 30% sucrose/PBS at 4°C overnight. Subsequently the samples were embedded in 7.5% porcine skin gelatin solution with 10% sucrose/PBS after being incubated at 37°C for 1 hour, and frozen in liquid nitrogen. Frozen sections were cut into 14 μm thickness and mounted on glass slides. The frozen sections were stored at -80°C until experimental procedures.After washing slides with PBS at 37°C for 10 minutes to remove gelatin from slides, cerebral organoid tissue was blocked with 5% BSA/0.1% PBS-T at room temperature for 1 hour. Tissue was reacted with primary antibody diluted in 5% BSA/0.1% PBS-T containing 0.05% sodium azide at 4°C overnight in a humidified chamber. After washing with 0.1% PBS-T, tissue was incubated with Alexa Fluor-conjugated secondary antibody at room temperature for 1 hour in the dark. After washing with 0.1% PBS-T, a coverslip was mounted with antifade mounting medium containing DAPI (Vector Laboratories). Images were obtained with Leica TCS SP8 confocal microscope (Leica) using the Leica LAS-X software.TUNEL stain assay was evaluated with BrdU-Red TUNEL assay kit (Abcam) according to the manufacture’s protocol. After removing gelatin from a frozen cerebral organoid section slide with PBS at 37°C for 10 minutes, cerebral organoid tissue was added with 20 μg/mL proteinase K solution (100 mM Tris-HCl pH 8.0 and 50 mM EDTA) at room temperature for 5 minutes, and subsequently mounted with 4% PFA at room temperature for 5 minutes. After washing the slide, the tissue was reacted with DNA labeling solution containing TdT enzyme and Br-dUTP at 37°C for 1 hour in a dark and humidified incubator. The tissue was next incubated with anti-BrdU-Red antibody at room temperature for 30 minutes in the dark. After washing the slide, a coverslip was mounted with antifade mounting medium containing DAPI (Vector Laboratories). Images were obtained with Leica TCS SP8 confocal microscope using the Leica LAS-X software.t-test was performed when two groups were compared, and two-way ANOVA with Tukey’s multiple comparisons test was used when multiple groups were compared in different conditions. A P value of < 0.05 was considered statistically significant.Statistical analysis definitions were described in each figure legends and calculated using GraphPad Prism 7 software. Unpaired Antibodies used in this study are listed in the et al. ,3,22C9orTo determine if patient-derived pathogenic TDP-43 propagates in our human CNS tissue model, sarkosyl-insoluble protein extracts containing pTDP-43 were administrated as pathogenic seeds into mature cerebral organoids at 60 DIV through a fine needle injection. At 8 weeks post injection (p.i.) with ALS patient-derived protein extract, OrgCtrl cerebral organoids had few pTDP-43 aggregates . In contTDP-43 is an RNA/DNA binding protein that localizes predominantly in the nuclei. However, in most sporadic ALS patients pathological pTDP-43 aggregates can be found in the cytosol of neurons and glia cells with loss of nuclear TDP-43 –3. We neIn TDP-43-associated FTLD and sporadic ALS, propagation of pathogenic TDP-43 has been demonstrated in adherent human cultured cells –14, and Immunofluorescence analysis of OrgCtrl cerebral organoids at 2, 4, and 8 weeks p.i. with ALS patient-derived protein extract had no formation of pTDP-43 aggregates at either 2 or 4 weeks p.i., and minor amounts of pTDP-43 aggregates at 8 weeks p.i. . Analysi4C2 repeat hexanucleotide expansion in the C9orf72 gene is reported as the most common genetic cause of ALS and FTLD [4C2 repeat is thought to play a crucial role in inducing TDP-43 pathology through the disruption of nucleocytoplasmic transport and stress granule dynamics [C9orf72 gene-expanded ALS (C9orf72-ALS) contained GR repeat protein  repair defects in ALS ,42. DSBsOverall, our data demonstrates that pathogenic TDP-43 from sporadic ALS patient-derived protein extracts has the potential to seed and propagate in ALS-derived cerebral organoids used as human CNS tissue model, inducing astrogliosis and cellular apoptosis concomitant with DSBs.in vitro and in vivo [We demonstrate that ALS patient-derived protein extracts containing pathogenic TDP-43 have the potential to spread cell-to-cell and to form phosphorylated TDP-43 cytoplasmic aggregates replicating ALS pathology in a time-dependent manner in recipient human cerebral organoids. We also showed that the TDP-43 pathology causes astrogliosis, cellular apoptosis and genomic damage. Our findings support the recent reports describing that pathogenic TDP-43 show prion-like propagation in murine brain and spinal cord of TDP-43 transgenic animal administrated with ALS patient-derived tissue ,16 and t in vivo ,42. ThesWe use mature iPSCs-derived cerebral organoids as a model of the human brain. These organoids have a complex three-dimensional structure with a wide variety of neuronal and glial cell types, that resembles human brain tissue.et al. [Interestingly, the cerebral organoids derived from sporadic ALS-FTLD patient , but not cerebral organoids from a control individual, showed increased cytoplasmic pTDP-43 aggregates and the TDP-43 pathology as early as two weeks after the administration of ALS patient-derived protein extracts. Cerebral organoids differentiated from an unaffected healthy control  formed small amounts of cytoplasmic pTDP-43 accumulation only eight weeks after the injection. Our findings raise the possibility that cells from someone who developed ALS carry some factors making them more susceptible to TDP-43 induced pathology. All known genetic factors have been ruled out as a possible cause for this susceptibility. Perhaps some unknown genetic factors, or some residual epigenetic marks, are responsible for this susceptibility to prion-like spread of pathogenic TDP-43. Based on the experiments for TDP-43 propagation in transgenic mice, Porta et al.  describeAlbeit we did not discriminate directly between exogenous and endogenous TDP-43 in recipient cerebral organoids, our results showing the reduction of nuclear TDP-43 and the time-dependent increase of pTDP-43 aggregates in the cerebral organoids administrated with ALS patient-derived protein extracts suggest that exogenous pathogenic TDP-43 acts as seeds and propagates cell-to-cell to form de novo TDP-43 pathology by altering normal endogenous TDP-43 to pathological conformations. The addition of a protein marker such as a Flag-tag to TDP-43 protein extracts may provide the evidence more clearly that exogenous pathological TDP-43 can template endogenous TDP-43. Although the specific mechanisms for prion-like propagation of pathogenic TDP-43 remains enigmatic, accumulating evidence indicates that exosomes play a role in transferring several pathological proteins associated with neurodegenerative disorders such as tau, α-synuclein, and SOD1 –47. As fAstrogliosis characterized by hypertrophy and proliferation of astrocytes as well as upregulation of GFAP expression is one of the key features observed in ALS pathology . Our modWe show here that the administration of ALS patient-derived protein extracts also induces cellular apoptosis and DSBs in recipient cerebral organoids, suggesting a link between apoptotic cellular death and genomic damage in TDP-43 pathology. Genome damage-mediated neuronal apoptosis in ALS has been reported in several studies. TDP-43 is a critical component of the DSBs repair pathway and physiologically acts as scaffold for the recruitment of DSBs repair proteins at DSB sites. Loss of nuclear TDP-43 as well as ALS-linked TDP-43 mutations are associated with DSB repair defects that trigger neuronal apoptosis in ALS –42. Morein vitro do not contain as many mature neurons as an adult human. The ideal cerebral organoids would need to have achieved the same level of maturity as adult CNS tissue, an objective that has yet to be achieved, would be preferable to verify pathogenic TDP-43 propagation. We used cerebral organoids as a CNS tissue model resembling the human brain, while another critical site for TDP-43 pathology in ALS is the spinal cord. In addition, microglia derived from mesoderm, which is an important factor involved in ALS pathophysiology, is deficient in cerebral organoids composed only of cells differentiated from ectoderm. However, robust protocols for creating well-structured spinal cord organoids harboring microglia have not yet been established. It is necessary to wait for such protocols that create organoids that reproduce the anatomical structures and physiological functions of human spinal cord tissue to verify our results.There are some limitations to this study. We used only two cell lines of human iPSCs to create recipient cerebral organoids. Additional iPSCs lines, especially some derived from ALS cases, are required to provide a better understanding of pathogenic TDP-43 propagation in cerebral organoids and possible host factors. Moreover, given that ALS is a late-onset disease, our cerebral organoids at around 10–20 weeks growth In conclusion, our findings provide evidence that pathogenic TDP-43 in ALS spinal cord tissue has the potential to spread cell-to-cell to induce TDP-43 pathology as well as astrogliosis and to cause cell cytotoxicity due to DNA damage in human CNS tissue. Although additional investigation is required to elucidate the key factors that induce pathogenic TDP-43 transmission, our model could be used to test treatment strategies aimed at blocking of pathogenic TDP-43 propagation.S1 TableHuman iPSCs used in this study were generated from peripheral blood mononuclear cells (PBMCs) of a healthy control  or sporadic ALS-FTLD patient . N.A. indicates “not assessed”.(PDF)Click here for additional data file.S2 TablePostmortem frozen spinal cords tissue specimens were collected from the Montreal Neurological Institute-Hospital (MNI), the Douglas-Bell Canada Brain Bank (DBCBB) and the London Neurodegenerative Diseases Brain Bank (LNDBB). N.A. indicates “not assessed”.(PDF)Click here for additional data file.S3 TableIF, immunofluorescence; IHC, immunohistochemistry; WB, Western blot; DB, dot blot; N.A. indicates "not assessed".(PDF)Click here for additional data file.S1 FigA and B) Immunohistochemistry of anterior horn spinal cords stained by TDP-43 antibody. The blue arrow shows punctate intraneuronal inclusion (A) and the orange arrow shows skein-like intraneuronal inclusion (B). Scale bar = 50 μm.((TIF)Click here for additional data file.S2 FigA) Representative bright-field (left) and immunofluorescence images (right) of cerebral organoids differentiated from OrgCtrl or OrgALS iPSCs line at 60 DIV. Scale bars = 1 mm. (B) Double-label immunofluorescence images of CTIP2 and PAX6 staining OrgCtrl or OrgALS cerebral organoids at 30 DIV and day 60 DIV. Scale bars = 100 μm. (C) Double-label immunofluorescence images of CTIP2 and TUJ1 (upper panels) or SOX2 (lower panels) staining OrgCtrl or OrgALS cerebral organoids at 60 DIV. Sections were counterstained with DAPI to label the nuclei. Scale bars = 50 μm.((TIF)Click here for additional data file.S3 Figupper panel) and 10 μm (lower panel).Immunofluorescence images of OrgALS cerebral organoid double-labelled with pTDP-43 and GFAP at 8 weeks post injection of protein extracts from ALS (patient 5). The lower panels are higher magnifications of the white-line boxes in the upper panels. Scale bars = 50 μm ((TIF)Click here for additional data file.S4 FigA) Dot blot analysis of sarkosyl-insoluble protein extracts from non-ALS control (control 1) and C9orf72-ALS spinal cords (patient 3), immunoblotted with GR repeat antibody. (B) Immunofluorescence images of OrgALS cerebral organoids double-labelled with pTDP-43 and GR repeat proteins at 8 weeks post injection of protein extracts from C9orf72-ALS (patient 3) (upper panels) or non-C9orf72-ALS (patient 4) (lower panels). Scale bars = 10 μm.((TIF)Click here for additional data file.