The functionalizationwith a hydrophilic and soft polymer modifies the hydrophobicity ofgraphene and might enhance its biocompatibility. We have characterizedthese hybrid materials by atomic force microscopy (AFM), X-Ray photoelectronspectroscopy (XPS) and Raman spectroscopy and studied their electricalresponse, confirming that the graphene/p-AP/PHEAarchitecture is anchored covalently by the sp3 hybridizationand controlled polymerization reaction on graphene, retaining itssuitable electronic properties. Among all the possibilities, we assessthe proof of concept of this graphene-based hybrid platform as a humiditysensor. An enhanced sensitivity is obtained in comparison with pristinegraphene and related materials. This functional nanoarchitecture andthe two-step strategy open up future potential applications in sensors,biomaterials, or biotechnology fields.The modificationof the surface properties of graphene with polymersprovides a method for expanding its scope into new applications asa hybrid material. Unfortunately, the chemical inertness of graphenehinders the covalent functionalization required to build them up.Developing new strategies to enhance the graphene chemical activityfor efficient and stable functionalization, while preserving its electronicproperties, is a major challenge. We here devise a covalent functionalizationmethod that is clean, reproducible, scalable, and technologicallyrelevant for the synthesis of a large-scale, substrate-supported graphene–polymerhybrid material. In a first step, hydrogen-assisted plasma activationof  Otherwise, PA dissociation hasbeen limited to mono-atomic functionalization,20 but these limitations can be overcome if PA low-vacuumtechniques are used to promote the dissociation of organic moleculesin gas phase that immediately link to the graphene lattice. Thus,PA is a promising strategy for the covalent functionalization of graphene,as we address in this work. In addition, the PA low-vacuum processis fast, clean, cost-effective, and scalable.Graphene, an sp28 It is also a promising polymer with soft and hydrophilic propertiesand high water adsorption capacity,30 one of thekey factors for biocompatible and bioengineering applications. Therefore,we expect this hybrid material to be a great candidate to study themodification of the graphene hydrophobicity and its improvement inthe biocompatible behavior as in other cases where a polymer is combinedwith other materials.28Acrylate polymersare a type of significant vinyl polymers usedin a wide range of industrial products, ranging from contact lensesor bone cement to cosmetics, coatings, adhesives orthopedics, textilesor drug administration. Acrylates in combination with other components form interesting newhybrid materials for bioengineering. PHEA is a polymer broadly usedas a hydrogel for lens, hemotherapy, in bioactive scaffolds, controlledrelease and applications that prove its biocompatible character.p-aminophenol(p-AP) and the carrier H2 gas dissociatein a low-vacuum plasma chamber and the graphene layer is activated,enabling the covalent functionalization of the basal plane with theorganic moiety. We functionalize the substrate-supported graphenewith organic molecules in a low-vacuum clean plasma environment, asan unexplored alternative to the wet chemistry methods5 and UHV8 approaches. This (p-AP) organic molecule incorporates an end-group (phenol)which is intended to be the linker to further anchor more complexmolecules and subsequently obtain a hybrid material. The key advantageof this strategy is the versatility of the method that enables touse specific linker molecules to functionalize graphene on demand.18 Recently, a similar platform has been demonstrated,especially suitable for high-performance bioelectronic devices.31Here, we devise a new versatiletwo-stage functionalization approachto build large-area graphene–polymer hybrid nanoarchitecturesas platforms with potential applications in biotechnology and sensing.In a first stage, the organic linker molecule 32 from the OH group provided bythe p-AP molecule, resulting in a robust and stablegraphene/p-AP/PHEA hybrid functional material. Ourmethod keeps the suitable properties of functionalized graphene, sinceno solvents or high temperatures that could lead to the material degradationare used enhancing, in contrast, the versatility of graphene.36 This approach is highly versatile and opens the door to the combinationof a wide range of linkers and polymers with potential applicationsin several fields.41 For instance, graphene is a material with a large potential in high-performancehumidity sensors, as reported in the literature.44 Therefore,the combination of the graphene sensitivity, the robustness of thecovalent functionalization, and the water absorption capacity of thepolymer make the hybrid platform proposed as a potential candidateto be an efficient humidity sensor. Accordingly, we show in this worka proof of concept of such application.Afterwards, we carry out an in situ surfacepolymerization of 2-hydroxyethylacrylate (HEA) monomer that grows in a controlled and reproduciblemannerp-AP/PHEA architecture is anchoredcovalently, in a stable and robust way, by sp3 hybridizationof graphene bonds and controlled polymerization. Finally, we assessthe sensitivity of our graphene–polymer hybrid platform developedby measuring the surface electrical properties by the four-point probeanalysis after different relative humidity exposures as a proof ofconcept of a humidity sensor. This study describes a new versatileand reproducible route to design functional robust nanoarchitecturesbased on graphene that have shown breakthrough applications in differentfields.The topographic changesin graphene are sequentially characterizedby atomic force microscopy (AFM), and the chemical functionalizationis analyzed by X-ray spectroscopy (XPS) and Raman spectroscopy inboth graphite surfaces and supported graphene samples. We ascertainthat the graphene/22.12.1.145 During functionalization,the chamber pressure is 5.4 × 10–2 mbar, andthe plasma power is 200 W. HOPG purchased from Tip Nano OÚand Si/SiOx wafer-supported CVD graphene purchased from GrapheneaInc. are used as substrates. The p-aminophenol (p-AP) powder precursor is purchased from Sigma-Aldrich (H2NC6H4OH ≥ 98%), and H2 is used as a gas carrier. The second stage of functionalizationand the final step to build up the hybrid material is carried outin an oven with a constant nitrogen flow. The monomer and initiator,2-hydroxyethyl acrylate (HEA)  and 2,2-azobis(isobutyronitrile)(AIBN), the reactants used to carry out in situ free-radical (FR)polymerization on the surface, are purchased and used as receivedfrom Sigma-Aldrich.Thefirst stage of covalent graphene functionalization is carried outby using an ASTEX AX 4500 electron cyclotron resonance (ECR) plasma-assistedequipment. A comprehensive description of the plasma system can befound elsewhere.2.1.2Room-temperatureAFM measurements for topographical analysis are performed with twocommercial equipment: (a) Nanoscope IIIa from Veeco (United States):Dynamic operation mode is selected, and silicon cantilevers (Bruker)with a nominal radius of curvature of 8 nm and nominal constant forceof 1–5 N/m are used. (b) Instrument and software from NanotecElectrónica S.L: Dynamic operation mode is employed, excitingthe tip at its resonance frequency (∼75 kHz) to acquire topographicinformation of the samples. The structure of our graphene films isaddressed by Raman spectroscopy using a confocal Raman microscope(S&I Monovista CRS+). Raman spectra have been obtained using a532 nm excitation laser, a 100× objective lens (NA = 0.9), andan incident laser power of 6 mW. The chemical nature of the hybridmaterial is determined by XPS. XPS measurements are carried out underUHV conditions using a PHOIBOS 100 1D delay line detector electron/ionanalyzer, monochromatic Al Kα anode (1486.6 eV), and pass energyof 30 eV.2.1.3–6 mbar). The 94 ± 3% RH data were acquired after2 h of continuous exposure to a humidity-saturated environment usinga wet paper in an airtight Petri dish . The room RH (27 ± 3%) correspondsto the laboratory ambient conditions. Measurements correspond to I–V curves in a four-point probesystem JANDEL RMS2 Universal Probe with continuous current (from 0.1to 10 μA). We apply the standard equation R□ = 4.53·ΔV/I for sheet resistance (R□) calculations (ΔV being the change in voltagemeasured between the inner probes and I the currentapplied between the outer probes).The water vapor absorption ability onto a sample surfacebecauseof the polymer presence allows using the hybrid material as a humiditysensor that is assessed at 0% relative humidity (RH), room RH (27± 3%), and top RH (94 ± 3%). The 0% RH data were acquiredafter the samples were placed 2 h in a vacuum environment (102.2p-aminophenol (p-AP) and the 2-hydroxyethyl acrylate (HEA) in situ polymerizationon the surface.The build-up strategy includestwo main stages: the linker attachment consisting of a covalent functionalizationwith the organic molecule 2.2.1p-AP precursor powder isevaporated into the chamber at 100 °C during 10 min, while thegraphene or HOPG samples are at room temperature. After the p-AP gas pressure is stabilized, we introduce the H2 gas carrier and switch on the plasma during 30 s . The plasma activationdissociates atomic H and the organic precursor in radicals, as depictedin 3 soft hybridization of the basal plane.47 Note that this ECR plasma produces around 5–10% atomic H.This process enables the attachment of the p-AP organicradicals previously activated by plasma. In this way, a high densityof functionalization can be achieved, controlling time and dose . One plausible reaction path is theplasma-assisted dehydrogenation48 or dissociation49 of the amino group in plasma, leaving behindaromatic groups with hydroxide moieties that anchor to the buckledgraphene lattice, as pointed out by our XPS results below. We alsotested the attachment of the molecule by carrying out an ethanol washand taking AFM images before and after the process. We observe thatthe molecule remains intact . As a result, the phenol end-groupbecomes the linking moiety to anchor more complex molecules.2.2.22 flow in order to perform a slow andcontrolled polymerization based on previous kinetic studies reportedin the bibliography.32 We are interested in a slow polymerizationrate to obtain a low surface coverage. In this work, two in situ polymerizationsare carried out where one sample aims to have the lowest amount ofpolymer on the surface (L-PHEA) and the other intends to achieve higherpolymer coverage on graphene (H-PHEA). For L-PHEA, the reaction timeis 30 min, while for H-PHEA, it is 1 h, and in addition, the polymerizationprocess was repeated once.p-AP linkermolecule anchored on the graphene surface. Similar processes havebeen studied by the radical polymerization of acrylate monomers, suchas poly(hydroxyethyl methacrylate), PHEMA, or poly(butyl methacrylate),PBMA, to obtain nanocomposites based on the reaction of the monomerwith nanosilica or by the addition of graphene oxide (GO).52 This reaction is selective and requires the presence of oxygen-containinggroups in order to accomplish the attachment to the acrylate and,therefore, can be a marker of the presence of free −OH groupsin the linked p-AP. The resulting graphene–polymersamples are washed using ethanol to remove the possible physisorbedmonomer and polymer . The final anchoring of the monomer to thelinker molecule covalently bonded on the graphene surface is depictedin x substrates.The process of anchoring the firstmonomer unit to our functionalized graphene surface proceeds as follows.Initially, as the temperature increases, the initiator (AIBN) fragmentsinto primary radicals by the cleavage of weak bonds. These radicalsfind a monomer molecule to react with their double bond and startthe reaction. During polymerization, several monomer molecules areadded from the cleaved double bond, thus forming macroradical chainsattached to this first monomer represented in 33.1p-AP/L-PHEA)and another with a higher polymer coverage (HOPG/p-AP/H-PHEA). Two types of samples were polymerized in situ with HEA, one witha low amount of polymer on the surface . For the HOPG/p-AP sample, this peak iswider and shifted to slightly higher values because of the surfaceroughening and the p-AP molecules. The rougheningas well as the different morphology allows us to confirm the incorporationof p-AP molecules onto the surface. p-AP/L-PHEA) and highest (HOPG/p-AP/H-PHEA) polymer coverage on graphene, respectively.The wide images in p-AP/L-PHEA, a surface coverage close to 20% and a surface densityof about 350 structures/μm2 are observed. A highdensity of features, with heights between 0.3 nm and 4.6 nm is found,a majority of them being of globular morphology with an average heightaround 1.2 nm. These changes confirm that polymerization has takenplace and that it is stable as the samples were measured after beingwashed with water. Finally, for the HOPG/p-AP/H-PHEAsample, the large image in p-AP molecule through the hydroxyl group, and then fromthere the polymer grows. Larger aggregates represent a larger polymerchain. In addition to the domains observed as aggregates in 3.2p-AP-graphite and PHEA-p-AP-graphitearchitecture. p-AP functionalization (HOPG/p-AP), and HOPG samples after the two polymerization processes(HOPG/p-AP/L-PHEA and HOPG/p-AP/H-PHEA).The C 1s core level . We also check that the main sp2 component remainsalmost unchanged, confirming that no damage was induced in the sampleby plasma-assisted processing. After the polymerization, new contributionsappear at 285.2 and 286.0 eV, which are ascribed to C–OH andC=O, respectively, from the hydroxyls and carboxylic groupsfrom PHEA. Finally, relative to the last C 1s spectrum of p-AP/H-PHEA sample reveals the increment of polymer conversionon the graphite surface with the enhancement of the contributionsat 285.2, 286.0, and 286.8 eV, assigned to the oxygen bonds, C–OH,C=O, and O–C=O respectively, from the hydroxylsand carboxylic groups from the PHEA structure.58 This signalenhancement is a direct consequence of the increase in the polymercoverage, in good agreement with the previous morphology seen in The chemical nature of the hybrid functional material has beencharacterized by XPS. Freshly cleaved HOPG is used as a referencesubstrate to study the chemical variations induced in the consecutivestages, the 52 The next spectrumcorresponds to the p-AP covalent functionalized sample,HOPG/p-AP, where, in addition to the adsorbed species,a new component at 532.8 eV appears. We ascribe this new componentto the OH groups of the p-AP molecule, confirmingits presence.60 We also see the increase of adsorbed species,which is easy to explain considering the plasma activation of theHOPG surface and exposure to ambient air before the XPS analysis.This activation enhances the adsorption of molecules as the samplesurface is not completely saturated during the covalent functionalizationexperiment. This intentioned saturation control allows us to maintainthe proper electronic properties of graphene, as we see below . Regardingthe functionalized surface after the polymerization reaction, as expectedfor a PHEA coverage, there are multiple components in the O 1s peak:531.6, 532.8, and 533.7 eV, assigned to the carbonyl (C=O),hydroxyl (OH), and ester (O–C=O) species, respectively.61 Two similar spectra are observedin both L-PHEA and H-PHEA polymerizations, with the only differencein the intensity of each characteristic contribution being largerafter H-PHEA polymerization, as expected. We also observe a smallremaining contribution due to the adsorbed species at 529.9 eV thatmay be a slight surface oxidation due to the existence of steps, defects,grain boundaries or exposed atomic sites after repeated surface washings.For the pristine sample, the spectrumpresents two small componentsthat can be assigned to the environmental adsorbed species as a consequenceof the exfoliation in air.3.3Figures S1 and S2 in the SupportingInformation). The analyses confirm, overall, that the functionalizationprocess takes place similarly on graphene as remarkable differencesin the final morphology of the samples or in the chemical mechanisminvolved are not observed. We conclude that the synthesis of a polymer/graphenehybrid functional material successfully works, which opens the doorto its application in devices.The study from HOPG has allowed us todevelop, characterize and validate the efficiency of a two-stage graphenefunctionalization process based on a linker and functional moleculeattachment on the surface to obtain a graphene–polymer hybridplatform. However, from the large-scale application point of view,the relevant next step consists of the transfer of this protocol toa graphene sample. In this scenario, the substrate morphology andthe structure of functionalized graphene samples have been sequentiallycharacterized by AFM and Raman spectroscopy in every step of the process(see R□) of thelayer by means of a four-point probe analysis at room temperature. R□ variation of the samples, extractedfrom the analysis of the slope of the corresponding I–V curves performed at several points ofthe samples under room conditions . Theinitial pristine graphene layer exhibits an average sheet resistancevalue of 0.55 kΩ after the cleaning procedure that we applyto our samples, 2 h at 340 °C, in a high vacuum ,being a standard result due to the high quality of the material. Thisresistance value increases to 1.05 kΩ on average after the functionalizationwith p-AP, as expected, considering the induced sp3 hybridization of the graphene lattice. As mentioned above,the functionalization density, and thus the hybridization degree,obviously depends on the plasma-assisted processing time, 30 s, asthis parameter determines the precursor dose. The processing timeused is selected after studying its influence on the electrical propertiesand the structure of graphene as a balance between the functionalizationdensity desired and the reduction of conductivity allowed for theintended application . After the polymerization step, the sheetresistance increases, up to 1.35 kΩ, as seen in the last pointof I–V curves measured on each sample. Finally, 64 A similar behavior is observed when p-AP is covalentlybonded to graphene, G/p-AP in red, which does showa small increase in resistivity as the humidity increases, in agreementwith previous reports on this effect as a function of graphene doping65 and band gap opening.66One of the main challenges ofcovalent graphene functionalization is to retain suitable electricalproperties of the graphene layer after processing. In order to studythe changes in the conductivity of graphene throughout the differentstages, we have measured systematically before and after each stagethe sheet resistance  shows that the 2D peak position is around 2682 cm–1 pointing out toward n-doping.68 The water absorption is known to induce p-doping in graphene, aswater molecules behave as electron acceptors69 when directly into contact with the graphenesurface. As the Raman 2D peak of the functionalized samples is inthe same position as that of pristine graphene in Figure S2d in our samples, the water-induced charge transferdecreases the graphene n-doping, increasing its resistivity.However, when the polymer is anchored to the surface,the sensitivityof this graphene-based hybrid platform, in blue, becomes outstanding.This is related to the hydrophilic character of the polymer and itsability to absorb water into its structure.p-AP exhibit a low interactionwith the dynamic humidity change in the environment (red and greenlines). However, a remarkable improvement in the detection performanceof the G/p-AP/L-PHEA material is observed due tothe natural adsorption and release of water molecules on the polymer(blue line). From a quantitative point of view, the resistance variationsor sensitivity, SR, exceed 29% (R□ (94%RH) = 1.57 kΩ, R□ (0%RH) = 1.21 kΩ, from SR in Table S1). Table S1 compiles a performance comparison ofthis work with a number of resistive humidity sensors reported inthe literature70 that confirms the high sensitivityof the hybrid material to the humidity conditions. It is thus demonstratedthat chemical modification by poly-2-hydroxyethyl acrylate on thesurface is a feasible way to improve the sensitivity of graphene tohumidity.Regarding the reproducibility and sensitivity of the materialsas a sensing platform, 4p-AP). This unexploredphysical protocol is clean, reproducible, easily scalable, and cost-effective.The second stage consists of in situ radical polymerization on thefunctionalized graphene surface, leading to a PHEA/graphene hybridmaterial. This strategy is highly versatile and opens the door forthe design of a wide range of graphene–polymer or biopolymerhybrid platforms for biosensing devices. AFM, XPS, Raman, and four-pointprobe sheet resistance analyses confirm that the graphene/p-AP/PHEA architecture is covalently anchored by sp3 hybridization of graphene, retaining suitable electronicproperties after the polymerization process. We have successfullytested the hybrid material as a resistive humidity sensor since thehydrophilic PHEA polymer modifies the hydrophobicity of graphene,finding an outstanding sensitivity to humidity variations, SR = 29%, competing with previous sensors basedon graphene, graphene oxide, reduced graphene oxide, or polymer-functionalizedgraphene. In addition, this proposed graphene–polymer hybridfunctional material leads the way for further biotechnological applicationsthat will be studied in future research projects in order to expandits field of potential applications.In summary,we present a novel route to synthesize a polymer/graphenehybrid functional material that consists of a two-stage process. Inthe first stage, a low-vacuum hydrogen-assisted plasma activationprocess produces a stable and covalent graphene-functionalized platformwith a small linker molecule (


The results show that the sensor provides direct electrical readout from temperature to resistance transduction by the nano piezoresistive effect in graphene. And the cavity structure can weaken the substrate impurity scattering and thermal resistance effect, which results in better sensitivity and wide-range temperature sensing. In addition, monolayer graphene is almost no temperature sensitivity. And the few-layer graphene temperature sensitivity, lower than that of the multilayer graphene cavity structure (3.50%/°C), is 1.07%/°C. This work demonstrates that piezoresistive in suspended graphene membranes can effectively enhance the sensitivity and widen the temperature sensor range in NEMS temperature sensors.The substrate impurities scattering will lead to unstable temperature-sensitive behavior and poor linearity in graphene temperature sensors. And this can be weakened by suspending the graphene structure. Herein, we report a graphene temperature sensing structure, with suspended graphene membranes fabricated on the cavity and non-cavity SiO For example, the preparation of suspended graphene on Si/SiOsitivity . Bolotinsitivity . Jian-Hasitivity  revealedsitivity . Howeversitivity . That isIn this work, a cavity graphene structure a on a si2 surface. And the graphene will seal the gas in the cavity, which is led by excellent air tightness and van der Waals forces between graphene and SiO2. The gas pressure in the cavity increases as the temperature rises, leading to the gas pressure difference as shown in As shown in 5 Pa is the initial air pressure at the two sides of the cavity. The gas pressure formula is given as follows [The pressure strain of multilayer graphene with PMMA was simulated using COMSOL Multiphysics 5.5. In the simulation of air pressure in the cavity, we assume that gases are in standard atmospheric pressure. And the  follows :(1)T1p1=5 Pa. The size of the cavity is 1.5 mm × 1.5 mm × 50 μm, and the volume of the gas in the cavity is 1.12 × 10−12 m3. The range of gas pressure values in the cavity is converted to Newtonian mechanical units of 0.2 N–9.6 N. The atmospheric pressure on the suspended graphene is 0.2 N, and the pressure difference on the suspended graphene from the previous analysis is 0 N−9.4 N. The experimental conditions were chosen to remain within the expected tearing limits of the graphene membrane. It can be seen from SEM images that the maximum stretch of the graphene film reaches about 20% [Under the temperature sensing range of 25 °C–120 °C, the pressure range of suspended graphene is 0–3.84 × 10bout 20% ,34,35.2 = 500 nm, Young’s modulus E = 1.6 GPa, Poisson’s ratio γ = 0.22. The simulation results are shown in The PMMA layer in the device designed in this work protects the graphene. PMMA covering the graphene should be unavoidable, which is contrary to the argument. Therefore, the PMMA layer was considered in the simulation. The detailed parameter settings in the simulation are as follows. The thickness of suspended graphene film t = 10 nm, Young’s modulus E = 1 TPa, Poisson’s ratio γ = 0.16, The thickness of PMMA tIf a pressure difference is present between the inside and the outside of the cavity compare c, the grIgnoring the substrate impurity scattering and the thermal resistance effect of graphene. The piezoresistive simulation shows that the device resistance will increase with rising temperature. Piezoresistive effects, substrate scattering effects, and thermal resistance effects all contribute to the output resistance of the sensor. In this work, piezoelectric effect simulations are performed to theoretically verify the role of strain and resistance of graphene. Therefore, the effect of other factors on the resistance is not considered in the simulation. Moreover, the thermal resistance effect of graphene is not the core of the sensor in this article. On the contrary, the thermal resistance effect of graphene is an effect that the paper hopes to eliminate. The substrate scattering effect of silicon substrates on graphene has a significant impact on the performance of the monolayer sensors in this work. However, there is no complete theoretical system for the research results on the scattering effect of silicon substrates on graphene . TherefoThe CVD graphene on copper foil used in the device preparation process is transferred to the target substrate by wetting transfer technology. The PMMA is a protective layer for graphene during the wet transfer process. In addition, PMMA, a sacrificial layer in the wet transfer process, is not removed and continues to protect graphene during the temperature sensing process. The detailed: (a) Removal of residual organic matter from the back side of graphene copper foil using a glue remover. (b) The PMMA solution was spin-coated on the front side of the copper-based graphene and cured on a hot plate at less than 60 °C. (c) The cured copper-based graphene is put into the configured copper sulfate solution to etch the copper base. (d) After all the copper corroded, the graphene film with PMMA is first fished into deionized water with silicon wafers and left to stand for a few minutes. The graphene film with PMMA is then transferred to the target substrate and treated with natural dry. In this work, we prepared monolayer, few-layer, and multilayer graphene for temperature sensing, respectively. Different layers of graphene are used for the preparation of devices.2 film on a silicon substrate. This CVD graphene on copper foil was gotten by wet etching. And the wet etching allows the transfer of graphene from the copper foil to the target substrate. This graphene on copper foil was purchased from Sixcarbon Tech Shenzhen. The thickness of the Si layer is 500 μm, the thickness of the SiO2 layer is 300 nm, and the Si/SiO2 substrate is from Shenzhen Rigorous Technology. The wafer is a heavily-doped monocrystalline silicon. The details of the fabrication shown in 2 substrate needs to be cleaned by acid cleaning and alkali cleaning to remove organic impurities on the surface of the substrate; Removal of SiO2 from the substrate backside using BOE. It is used for subsequent verification of the conductivity of the bottom electrode and the electrodes on two sides of the graphene. (b) A cavity is etched on the prepared sheet using the photolithography technique and RIE etching method. And the depth of the trench is 50 μm. (c) Au electrodes are sputtered on both sides of the cavity using Magnetron Sputtering equipment. (d) CVD graphene, with PMMA, is transferred onto the substrate by wetting transfer technology [In experiments, graphene membranes made from CVD graphene on copper foil are suspended over cavities etched into a Si/SiOchnology ,37. The Preliminary verification of the conductivity of the sample is required because the graphene may be damaged during the transfer process. The finished samples need to select resistance values in the range of 1 kΩ–10 kΩ for subsequent testing. The device yield by the fabrication process is about 90%. Also, the suspension of graphene on the device needs to be verified. The device designed in this work can also be used as a transistor. When the graphene is not suspended but in contact with the silicon substrate at the bottom of the cavity, then the source/drain and the gate will be conductive. Then verifying the conductivity between the source and drain can be a preliminary verification of whether graphene is suspended or not. Then, the qualified samples need to be wire-bonded. In addition, the sensing structure in our work is monolayer, few-layer, and multilayer graphene. The SEM image and Raman test data of the device are shown in In our work, the graphene temperature sensor provides a direct electrical readout of temperature to strain transduction. The hot plate simulated ambient temperature. And Keithley 2611B is for monitoring current change under different temperatures. The specific measurements are as follows: (1) The temperature sensor is fixed on the hot plate with high-temperature tape; (2) The temperature was set as 25 °C. Then, save the I-V curve data after the device value becomes stable. (3) Increase the temperature of the hot plate to 120 ℃ and save the I-V data after the temperature reaches stability. And the temperature is changed in steps at 5 °C. (4) Export the test data and analyze the sensing property between graphene resistance and temperature. Finally, the testing data changed from I-V to R-V data for the benefit of the analysis.The suspended graphene sensors data in The resistance does not show a regularity with temperature on non-cavity samples in From the data in Finally, the sensitivity and linearity of the sensor were analyzed and calculated. In resistance-based temperature sensors, the initial resistance  and the temperature coefficient of resistance (TCR) are essential parameters for temperature measurement . A higheA linear fit of the tested resistance data with temperature performed by Origin is in In this work, the sensor provides a direct electrical readout of temperature to resistance transduction by the nano piezoresistive effect in graphene. And the cavity structure can weaken the substrate impurity scattering and thermal resistance effect, which achieves better sensitivity and wide-range temperature sensing. The sensor’s sensitivity is 3.50%/°C, the linearity is 0.98, and the sensing temperature range is 25 °C–120 °C. Compared with conventional thermal resistance graphene temperature sensors, the sensors have orders of magnitude higher sensitivity. Further, the experiments suggest that the graphene layers affect the sensitivity, linearity, sensing range, and other sensor parameters. In the experiments, the monolayer, few-layer, and multilayer graphene sensors have different sensor parameters. This work can provide theoretical and technical support for NEMS temperature sensors.


K145Q resulted in stress-induced nuclear TDP-43 foci and loss of TDP-43 function in primary mouse and human-induced pluripotent stem cell (hiPSC)-derived cortical neurons. Mice harboring the TDP-43K145Q mutation recapitulated key hallmarks of FTLD, including progressive TDP-43 phosphorylation and insolubility, TDP-43 mis-localization, transcriptomic and splicing alterations, and cognitive dysfunction. Our study supports a model in which TDP-43 acetylation drives neuronal dysfunction and cognitive decline through aberrant splicing and transcription of critical genes that regulate synaptic plasticity and stress response signaling. The neurodegenerative cascade initiated by TDP-43 acetylation recapitulates many aspects of human FTLD and provides a new paradigm to further interrogate TDP-43 proteinopathies.TDP-43 proteinopathies including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders characterized by aggregation and mislocalization of the nucleic acid-binding protein TDP-43 and subsequent neuronal dysfunction. Here, we developed endogenous models of sporadic TDP-43 proteinopathy based on the principle that disease-associated TDP-43 acetylation at lysine 145 (K145) alters TDP-43 conformation, impairs RNA-binding capacity, and induces downstream mis-regulation of target genes. Expression of acetylation-mimic TDP-43 TDP-43 proteinopathies are characterized by the dysfunction and aggregation of transactivation response element DNA-binding protein of 43 kDa (TDP-43), with ~95% of all amyotrophic lateral sclerosis (ALS) and ~50–60% of all frontotemporal lobar dementia (FTLD-TDP) cases harboring TDP-43 pathology . There iTARDBP mutations that are causative for FTLD/ALS  and gene transcription . StructuFTLD/ALS . In sporFTLD/ALS . Both TDFTLD/ALS .TARDBP mutations that disrupt RNA binding, and thereby may act in a similar manner to TDP-43 acetylation, have been identified in FTLD-TDP patients  (K145Q) is sufficient to neutralize the positive charge, disrupt RNA binding, and induce several hallmarks of TDP-43 pathology in vitro , such as phosphorylation, acetylation, and ubiquitination may play a role. PTMs modulate TDP-43’s biochemical properties leading to conformational changes, modulation of nucleic acid-binding affinity, regulation of liquid–liquid phase separation, and propensity to form insoluble TDP-43 aggregates, all of which are disease-associated phenomena . Among td K181E) , supportin vitro , supportTardbp gene, thereby generating acetylation-mimic TDP-43K145Q knock-in mice, which enabled us to investigate the pathophysiological impacts of an aberrant TDP-43 PTM while leaving native upstream and downstream genomic elements intact. Using mouse cortical neurons, human hiPSC-derived cortical neurons, and aged cohorts of TDP-43K145Q homozygous mice, we found that acetylation-mimic TDP-43K145Q-induced nuclear TDP-43 foci and cytoplasmic TDP-43 accumulation, which coincided with several disease-associated and loss-of-function measures including widespread transcriptome and splicing alterations. Finally, we observed prominent FTLD-like cognitive and behavioral deficits in acetylation-mimic TDP-43 mice that correlated with biochemical and splicing alterations in affected brain regions. Our study supports lysine acetylation of TDP-43 as a driver of dysfunction in sporadic TDP-43 proteinopathies.Here, we used CRISPR/Cas9 genome editing to introduce a K145Q substitution into the endogenous mouse wt), acetylation-deficient (TDP-43K145R), and acetylation-mimic (TDP-43K145Q) variants to overexpress TDP-43 species in neurons, and then examined their subcellular localization by immunofluorescence microscopy. In the absence of acute cellular stress, most neurons overexpressing TDP-43K145Q showed distinct nuclear foci that were more prominent compared to TDP-43wt or TDP-43K145R constructs  mRNA splice variants, as a more sensitive endogenous indicator of loss of TDP-43-dependent splicing function. Functional nuclear TDP-43 results in the production of a mature spliced Sort1 mRNA transcript, however, in the setting of TDP-43 depletion or loss of function, TDP-43 is unable to repress the inclusion of exon 17b, generating a longer Sort1+ex17b transcript variant , acetylation-deficient TDP-43 (TDP-43K145R.2 and TDP-43K145R.12), or unmodified TDP-43   . We confquencing , verifiequencing , and thequencing . We thenlization . All hiPlization . Followiive foci . In compK145Q cortical neurons show more robust functional deficits compared to the mouse neuron data above. We again employed RT-qPCR to measure levels transcripts associated with TDP-43 loss of function, beginning with TARDBP as an indicator of impaired TDP-43 autoregulation. In contrast to mouse neurons, we observed an approximately twofold increase TARDBP mRNA in TDP-43K145Q.12 and TDP-43K145Q.18 neurons and a slight but significant reduction in TARDBP mRNA in TDP-43K145R.12 neurons  was reduced about 50% in both acetylation-mimic TDP-43K145Q lines but unchanged in TDP-43K145R.2 neurons  failed to detect the abnormal splice product in TDP-43wt and TDP-43K145R.2 lines, with only low levels observed in some TDP-43K145R.12 samples. In contrast, both TDP-43K145Q lines dramatically accumulated the UNC13A cryptic exon containing mRNA (UNC13A WT) were similar across all five lines  buffer-extracted) and insoluble (Urea-extracted) protein fractions. TDP-4310 locus  -TDP-43 -TDP-43  and mode)-TDP-43  in the s animals . We thuswt or TDP-43KQ/KQ mice, representing the regions and timepoints with the most striking biochemical and behavioral and abnormalities and identified nearly 400 differentially expressed genes (DEGs) in each brain region in TDP-43KQ/KQ mice compared to TDP-43wt, after correcting for underlying batch effects. As expected by acetylation-induced loss of TDP-43 function and subsequent autoregulation, the Tardbp transcript was increased , gliogenesis , and myelination . Sema5b was the most dramatically reduced transcript in the hippocampus, and the third most highly reduced in the cortex  were selectively downregulated in the neocortex, while trans-synaptic signaling and ion transport mechanisms  were decreased in the hippocampus.We then clustered the DEGs based on their up- or downregulation and the brain region affected , which re cortex  with a tSesn1, Nrros, Plat, Klf15) and many apoptotic regulators  in both the hippocampus and neocortex, along with an over-representation of GO terms related to metabolism, localization, and cell adhesion . Interestingly, while genes associated with trans-synaptic signaling were decreased in the hippocampus, another set of genes involved in this same pathway was upregulated in the cortex   .KQ/KQ mice, we sought to draw parallels between the TDP-43KQ/KQ mouse and the human FTLD-TDP transcriptome. Comparison of the DEGs in TDP-43KQ/KQ mice to the mouse orthologs of those found in FTLD-TDP frontal or temporal cortex tissue , with 81.7% of loci containing at least one cryptic splice junction, and 29.8% containing two cryptic splice junctions   . Of all identified DSGs, the most substantially altered transcript was Sort1, encoding the Sortilin-1 (Sort1) protein. We noticed that exclusion of a Sort1 3′ exon was reduced by 55.9% in TDP-43KQ/KQ cortex and by 57.0% in the hippocampus  , transcr, Lrrk2) , and alspocampus , such thSORT1 exon 17b inclusion, this leads to the production of a soluble and putatively toxic SORT1 variant that is increased in FTLD-TDP patients , and a primer pair that recognizes all Sort1 variants  on tissues isolated from TDP-43KQ/KQ or WT mice  and regulates endosomal/lysosomal function through a pathway that is genetically linked to FTLD-TDP . Becausepatients . To conf WT mice . We founeocortex  and hippeocortex  of TDP-4with age .Sort1+ex17b transcript in TDP-43KQ/KQ mice leads to the generation of a distinct Sort1 protein product, we immunoblotted hippocampus and cortex homogenates and identified a higher molecular weight Sort1 variant in TDP-43KQ/KQ compared to WT mice . Using a combination of molecular, cellular, and transcriptomic readouts combined with in vivo behavioral assays, we provide evidence that aberrant TDP-43K145Q recapitulates key hallmarks of TDP-43 proteinopathies, including altered TDP-43 solubility and localization, impaired autoregulation, and abnormal splicing function. Taken together, our findings suggest that TDP-43 acetylation-induced dysfunction likely contributes to disease progression.Here, we developed novel neuronal and mouse models of RNA-binding deficient acetylation-mimic TDP-43  and likely other factors. Based on our original description of these TDP-43 foci, and several more recent studies of acetyl-mimic TDP-43 variants  The levels of insoluble phosphorylated TDP-43 in the spinal cord have not crossed a critical threshold required for functional decline. (2) There is a time-dependent delay in the accumulation of TDP-43 and the onset of motor symptoms that could emerge beyond 18 months, which will require additional studies in aged cohorts of mice. (3) Spinal motor neurons in mice may possess some intrinsic resilience that suppresses functional decline. (4) It is possible that TDP-43KQ/KQ mice do indeed show subtle motor deficits that could be detected by more sensitive measures, such as electromyography-based techniques  and altered splicing of TDP-43 targets   . Similar/KQ mice . Hippoca/KQ mice . This al/KQ mice , the K14nockdown . This isutations , and TDPKQ/KQ mouse brains. Many up- and downregulated transcripts identified in acetylation-mimic mice were also differentially expressed in FTLD-TDP human tissue  was significantly altered in TDP-43KQ/KQ mice, and abnormalities in LRP8 can cause learning and memory defects, likely by disrupting Reelin-mediated synaptic plasticity and long-term potentiation (LTP)  protein that regulates LTP and synaptic plasticity via the production of nitric oxide  and other significantly altered splice variants  have been linked to AD and other neurodegenerative diseases  . Similaric oxide , showed diseases . Our traGiven the correlation between TDP-43 acetylation and FTLD-TDP presented in this study, it is perhaps surprising that acetylated TDP-43 (detected with an acetylation-specific ac-K145 antibody) is found within ALS spinal cord inclusions but has not yet been detected in FTLD-TDP cortex . HoweverK145Q mutation results in an age-dependent, dementia-like phenotype characterized by signatures of FTLD, including progressive cognitive deficits, TDP-43 mislocalization and phosphorylation, impaired autoregulation, and prominent RNA dysregulation. TDP-43 acetylation affected many known FTLD-associated pathways, including STMN2, SORT1, and stress response signaling, while also pinpointing new putative pathways as mediators of disease pathogenesis. By developing models of TDP-43 acetylation, we expand our understanding of sporadic TDP-43 proteinopathies and provide new avenues to identify therapeutics that target the pathogenic mechanisms underlying TDP-43 proteinopathies.In conclusion, disrupting RNA binding with an acetylation-mimic TDP-43Mice were housed in ventilated microbarrier cages on racks providing high efficiency particulate air (HEPA)-filtered air supply to each cage. Animals were kept on a 12-hr light–dark cycle with access to food and water ad libitum. All animal husbandry, experiments, and procedures were performed in strict compliance with animal protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill (Protocol #21.257).wt/KQ breeding pairs. Timed pregnant females at embryonic days 15–16 were lethally anesthetized with isoflurane. The abdominal cavity was opened, and the uterus incised to remove the placentas and embryonic sacs, which were washed briefly in ice-cold 70% ethanol and then placed in cold 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered Bank’s balanced salt solution (HBSS). The embryos were transferred into a 10-cm dish containing cold HBSS, the embryos removed from the amniotic sac, and the brains were extracted from the cranium. For TDP-43wt/KQ dissections, process was paused for approximately 2 hr to permit genotyping of the fetuses, during which the brains were stored at 4°C protected from light in a Hibernate-E (BrainBits NC9063748) solution supplemented with B27 (Gibco 17504044) and GlutaMAX (Thermo Fisher 35050061). After genotyping, if applicable, the cerebral cortices from each brain were isolated under a stereomicroscope, pooled by genotype, minced with forceps, and digested for 30 min at 37°C in a filter-sterilized HBSS solution containing 20 U/ml papain , 1 mM EDTA, 0.2 mg/ml l-cysteine, and 5 U/ml DNAse (Promega M6101). The enzyme solution was removed, and the digested tissue was washed twice with sterile HBSS. Warm plating media  containing 5 U/ml DNAse, was added and the tissue was dissociated mechanically using a P1000 pipette. The resulting cell suspension was spun down for 5 min at 1.5 rcf to pellet the cells, resuspended in plating media, and filtered through a 40-mm cell strainer. Cells were counted using a hemocytometer and plated onto poly-D-lysine (PDL)-coated 12-well tissue culture plates (Corning 356470) at 300 K cells/well (for RNA extraction) or 96-well glass bottom black wall plates (Cellvis P96-1.5H-N) at 30 K/well (for immunofluorescence and microscopy). 16–24 hr after plating, all plating media was removed and replaced with neuronal cell media . Cultures were incubated at 37°C, 5% CO2 and 95% humidity with half-media exchanges every 3 days for the duration of all experiments.Murine primary cortical neurons were performed using wild-type C57Bl/6 mice (Charles River) or TDP-43http://n2t.net/addgene:24129; RRID:Addgene_24129)  . To genee_24129)  and a pc plasmid  as the tg for 10 min. Lentiviral particles were purified using a double-sucrose gradient method. Briefly, the supernatants were loaded onto a 70–60−30–20% sucrose gradient and centrifuged at 70,000 × g for 2 hr at 17°C . The 30–60% fraction containing the viral particles was retrieved, resuspended in phosphate-buffered saline (PBS), filtered with a 0.45-μm filter flask before loaded onto a 20% sucrose cushion, and centrifuged a second time at 70,000 × g for 2 hr at 17°C. The supernatants were carefully discarded, and the viral particles present in the pellet were resuspended in PBS, aliquoted and stored at −80°C.Lentiviral production was performed by co-transfecting  37.5 μg lenti-plasmid with 25 μg psPAX2, 12.5 μg VSVG, and 6.25 μg REV for each 15 cm dish of lenti-X 293T cells (Takara 632180) and 3 dishes of cells were used for each lentiviral production. Three days after transfection, culture media were collected and centrifuged at 2000 × Lentivirus aliquots were evaluated for neuronal transduction ability prior to each experiment and diluted to approximately equal concentrations of effective virus. Lentiviral transductions were performed on DIV10 for a DIV14 harvest or DIV24 for a DIV28 harvest. In brief, lentiviruses were diluted to a 2× concentration in neuronal media, and then added to plates via half-media exchange. Twenty-four hours after viral transduction, all virus-containing media was removed and replaced with 50% fresh neuronal media and 50% conditioned media from plates containing WT non-transduced neurons. Neurons were then cultured to DIV14 or DIV28 as the experiment required.The HPC26 iPSCs were maintained on Matrigel-coated dishes (Corning 354480) in StemFlex Medium (Thermo Fisher Scientific A3349401) and passaged every 3–4 days with 0.5 mM EDTA dissociation solution as previously described .5 iPSCs were electroporated on the Neon electroporation system (Thermo Fisher Scientific) with recombinant protein complexes made of Cas9 v2 protein (Thermo Fisher Scientific A36498), 900 ng sgRNA-TARDBP and 2700 ng of single-stranded donor oligonucleotide-K145Q or donor oligonucleotide-K145R  supplemented with 10 µM Y27632 (PeproTech 1293823) and cultured in suspension on an orbital shaker at 37°C and 5% CO2. After 48 hr, cells were dissociated with Accutase and 3 × 105 cells differentiated into NPCs as neutrosphere in StemScale PSC medium supplemented with 10 µM Y27632 (PeproTech 1293823) and 1.5 µM CHIR99021 (PeproTech 2520691), 10 µM SB431542 (PeproTech 3014193) and 50 nM LDN-193189 (Sigma SML0755). CHIR99021 was removed after 24 hr and cells were cultured for 10 days with daily medium changes and neurospheres dissociated at 1:3 ratio twice a week. Then NPCs were expanded for 7 days in the presence of 20 ng/ml FGF (Peprotech 100-18B) in Neuronal Expansion Medium [1:1 Advanced Dulbecco's Modified Eagle Medium (DMEM)/F12 (12634028) and Neural Induction Supplement (A1647801)]. NPCs were plated for maturation on PDL/Laminin-coated plates or coverslips in cortical neuron maturation medium  supplemented with 10 ng/ml BDNF, 10 ng/ml glial cell-derived neurotrophic factor (GDNF), and 10 µM N-[N-- L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Seventy-two hours after plating, cells were treated with 1 µg/ml Mitomycin C for (Sigma M5353) for 1 hr. Cellular identity was assessed specific markers of neuronal progenitor , and mature neurons   . All rea, neurons were treated for 2 hr with 200 µM sodium arsenite (NaAsO2) or vehicle (molecular biology grade water). Following treatment, neurons were fixed with 4% paraformaldehyde (PFA) in 1× PBS and washed with 1× PBS. Neurons were permeabilized with 0.3% Triton X-100 in 1× PBS for 15 min at RT, blocked in an 8% normal goat serum (NGS) 0.2% Triton X-100 in 1xPBS solution for 1–2 hr at RT, and incubated with primary antibodies (On DIV14 (mouse primary neurons) or mature hiPSC-derived cortical neurons (~DIV50)tibodies  diluted tibodies  diluted Mouse primary cortical neurons in 96-well plates were visualized via automated fluorescence microscopy using EVOS M7000 Imaging System (Thermo Fisher AMF7000) equipped with Olympus ×20/0.75 NA UPlanSApo and Olympus ×40/0.95 NA UPlanSApo objectives, and the following filter cubes: DAPI , GFP , Texas Red , and Cy5.5 . Images were acquired in an automated fashion, using DAPI fluorescence as the autofocus substrate, taking 16–32 images per well of 2–3 wells per experimental condition. Automated quantitative image analysis was performed using CellProfiler 4.0  to measuTardbp K145 codon were identified using Benchling software . Three guide RNAs were selected for activity testing. Guide RNAs were cloned into a T7 promoter vector followed by in vitro transcription and spin column purification . Guide RNAs were tested for cleavage activity by in vitro cleavage assay. Each guide RNA was incubated with Cas9 protein (UNC Protein Expression and Purification Core Facility) and PCR-amplified guide RNA target site. The products were run on an agarose gel for analysis of target site cleavage. Based on this assay, the guide RNA selected for genome editing in embryos was Tardbp-g79T  containing (1) 20 ng/µl Cas9 mRNA, 10 ng/µl g79T guide RNA, and 50 ng/µl donor oligonucleotide (founder #9) or (2) 20 ng/µl Cas9 mRNA, 400 nM Cas9 protein, 10 ng/µl g79T guide RNA, and 50 ng/µl donor oligonucleotide . Injected embryos were implanted in recipient pseudopregnant females and resulting pups were screened by PCR and sequencing for the presence of the desired mutant allele. Founder lines were propagated as heterozygotes and regularly sequenced to confirm retention of the K145Q and silent mutations.Tardbp locus.We assessed the likelihood of off-target mutations using prediction algorithms to ensure low likelihood of off-target effects. The Benchling MIT off-target score for Tardbp-g79T is 72 , and theTardbp locus was performed with ApexRed master mix (Genesee Scientific 42-138B) and TDPKQ F′ and TDPKQ R′ primers  crossed by a grid of photobeams . Counts were taken of the number of photobeams broken during the trial in 5-min intervals, with separate measures for locomotor activity  and vertical rearing movements. Time spent in the center region was used as an index of anxiety-like behavior.Training. On day 1, mice were placed in the test chambers, contained in sound-attenuating boxes. The mice were allowed to explore the novel chambers for 2 min before presentation of a 30-s tone (80 dB), which co-terminated with a 2-s scrambled foot shock (0.4 mA). Mice received two additional shock-tone pairings, with 80 s between each pairing, and were removed from the test chambers 80 s following the third shock. Context- and cue-dependent learning. On day 2, mice were placed back into the original conditioning chambers for a test of contextual learning. Levels of freezing (immobility) were determined across a 5-min session. On day 3, mice were evaluated for associative learning to the auditory cue in another 5 min session. The conditioning chambers were modified using a Plexiglas insert to change the wall and floor surface, and a novel odor (dilute vanilla flavoring) was added to the sound-attenuating box. Mice were placed in the modified chamber and allowed to explore. After 2 min, the acoustic stimulus (an 80-dB tone) was presented for a 3-min period. Levels of freezing before and during the stimulus were obtained by the image tracking system.Mice were evaluated for conditioned fear using the near-infrared image tracking system . The procedure had the following phases: training on day 1, a test for context-dependent learning on day 2, and a test for cue-dependent learning on day 3. Grip strength was evaluated using precision force gauges. Measures were based on paw grasp of a metal grid by a mouse gently pulled by the tail. Digital force meters  were mounted on an acrylic platform (San Diego Instruments), with two different grids: a left-hand grid for the front paws, and a right-hand grid for all-four paws. Each grid connected to a force transducer, which provided measures of peak force (newtons). Each mouse was given three trials, with at least 1 min between each trial. Each trial had two components: front-paw measures from the left-hand grid, immediately followed by all-four-paw measures from the right-hand grid.Subjects were tested for motor coordination and motor learning on an accelerating rotarod . For the first test, mice were given three trials, with 45 s between each trial. Two additional trials were given 48 hr later. Rpm (revolutions per minute) was set at an initial value of 3, with a progressive increase to a maximum of 30 rpm across 5 min . Measures were taken for latency to fall from the top of the rotating barrel.The water maze was used to assess spatial and reversal learning, swimming ability, and vision. The water maze consisted of a large circular pool (diameter = 122 cm) partially filled with water , located in a room with numerous visual cues. The procedure involved three separate phases: a visible platform test, acquisition in the hidden platform task, and a test for reversal learning (an index of cognitive flexibility).Each mouse was given 4 trials per day, across 2 days, to swim to an escape platform cued by a patterned cylinder extending above the surface of the water. For each trial, the mouse was placed in the pool at one of four possible locations (randomly ordered), and then given 60 s to find the visible platform. If the mouse found the platform, the trial ended, and the animal was allowed to remain 10 s on the platform before the next trial began. If the platform was not found, the mouse was placed on the platform for 10 s, and then given the next trial. Measures were taken of latency to find the platform and swimming speed via an automated tracking system (Noldus Ethovision).Following the visible platform task, mice were tested for their ability to find a submerged, hidden escape platform (diameter = 12 cm). Each mouse was given 4 trials per day, with 1 min per trial, to swim to the hidden platform. The criterion for learning was an average group latency of 15 s or less to locate the platform. Mice were tested until the group reached criterion, with a maximum of 9 days of testing. When the group reached criterion (on day 5 in the present study), mice were given a 1-min probe trial in the pool with the platform removed. Selective quadrant search was evaluated by measuring the number of crosses over the location where the platform (the target) had been placed during training, versus the corresponding area in the opposite quadrant. Following the acquisition phase, mice were tested for reversal learning, using the same procedure as described above. In this phase, the hidden platform was re-located to the opposite quadrant in the pool. As before, measures were taken of latency to find the platform. On day 5 of testing, the platform was removed from the pool, and the group was given a probe trial to evaluate reversal learning.This procedure was used to assess auditory function, reactivity to environmental stimuli, and sensorimotor gating. The test was based on the reflexive whole-body flinch, or startle response, that follows exposure to a sudden noise. Measures were taken of startle magnitude and PPI, which occurs when a weak prestimulus leads to a reduced startle in response to a subsequent louder noise.Mice were placed into individual small Plexiglas cylinders within larger, sound-attenuating chambers. Each cylinder was seated upon a piezoelectric transducer, which allowed vibrations to be quantified and displayed on a computer (San Diego Instruments SR-Lab system). The chambers included a ceiling light, fan, and a loudspeaker for the acoustic stimuli. Background sound levels (70 dB) and calibration of the acoustic stimuli were confirmed with a digital sound level meter (San Diego Instruments). Each session began with a 5-min habituation period, followed by 42 trials of 7 different types: no-stimulus (NoS) trials, trials with the acoustic startle stimulus  alone, and trials in which a prepulse stimulus  occurred 100 ms before the onset of the startle stimulus. Measures were taken of the startle amplitude for each trial across a 65 ms sampling window, and an overall analysis was performed for each subject’s data for levels of PPI at each prepulse sound level  × 100]).At each end point, mice were anesthetized deeply with isoflurane and euthanized via rapid decapitation. The hippocampus and neocortex were dissected out of the brain on a cold surface using clean surgical tools, placed into cryo-safe nuclease-free microcentrifuge tubes, flash frozen in liquid nitrogen (LN2), and stored at −80°C until processing. Frozen tissue was then pulverized in LN2-cooled stainless steel Cryo-Cups using cold stainless-steel pestles and transferred into cold nuclease-free microcentrifuge tubes and immediately stored at −80°C until use.Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride (TPCK) (Sigma T7254), 1 µg/ml Trypsin inhibitor (Sigma T9003), 1 µg/mL N-p-Tosyl-l-phenylalanine chloromethyl ketone (TLCK) (Sigma T4376), 0.67 µg/ml trichostatin A, 10 mM nicotidamide, 1 mM phenyline thanosulfyl fluoride, 1 mM phenylmethylsulfonyl fluoride]. The solution was homogenized by sonication and centrifuged at 4°C for 45 min at 18,000 × rcf. The supernatant was removed and saved as the RIPA-soluble (soluble) protein fraction. The pellet was resuspended in RIPA buffer with inhibitor mixture, sonicated, and centrifuged as described above, and the supernatant discarded. The resulting pellet of RIPA-insoluble material was resuspended in approximately 1 µl/µg of Urea buffer  with the inhibitor mixture (as above), sonicated to homogenize, and centrifuged at RT for 45 min at 21,000 × rcf. The resulting supernatant of RIPA-insoluble, urea-soluble protein fraction was saved as the ‘insoluble’ protein fraction.All steps of protein fractionation were performed on ice unless otherwise indicated. For solubility fractionation, pulverized tissue was suspended in 5 µl/mg of ice-cold 1× RIPA buffer  containing a mix of protease, phosphatase, and deacetylase inhibitors [1 µg/ml Peptstatin A (Sigma P4265), 1 µg/ml Leupeptin (Sigma L2023), 1 µg/ml Nuclear and cytoplasmic soluble proteins were isolated using the Thermo Fisher NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific 78835) per the manufacturer’s instructions, with the addition of the protease, phosphatase, and deacetylase inhibitors (as above) to each buffer.All soluble protein fractions were analyzed by Bicinchoninic acid (BCA) assay (Thermo Scientific 23225) to determine protein concentration. Equal quantities of protein per sample were run onto 4–20% Tris-Glycine SDS–polyacrylamide gel electrophoresis gels (Bio-Rad 5671095) under reducing conditions and then transferred onto nitrocellulose membranes. Total transferred protein (TTP) was assessed using PonceauS  protein stain on nitrocellulose membranes per the manufacturer’s instructions. Membranes were washed three times in Tris-buffered saline with 0.1% Tween-20 (TBST), once in 1x Tris-buffered saline (TBS), and blocked in 2% nonfat milk in 1× TBS for 1–2 hr at RT. Membranes were incubated with primary antibodies  diluted Mouse brain tissue was isolated, flash frozen, and pulverized as described above. Approximately 20 mg of pulverized brain tissue per sample was used to isolate RNA. 1 ml of TRIzol (Invitrogen 15596018) was added to each nuclease-free Eppendorf tube containing pulverized tissue, and tissues were lysed via trituration with a P1000 pipette tip, followed by trituration with a 21-G and then a 25-G needle on a 1-ml syringe. Samples were centrifuged at 4°C for 5 min at 10,000 rcf to remove tissue debris. The supernatant was removed and added to a new tube containing 200 µL of chloroform, which were mixed by inversion and cooled on ice for 5 min. Samples were centrifuged at 4°C for 15 min at 10,000 rcf, and the upper aqueous phase containing RNA was transferred into a new tube, followed by the addition of 100 µl of isopropanol to precipitate RNA and overnight incubation at −20°C. The next day, samples were centrifuged at top speed  for 20 min at 4°C to pellet the RNA. The pellets were washed twice with 1 ml of ice-cold 70% molecular biology grade ethanol and then air dried for 15 min at RT. The RNA pellets were resuspended in 20 µl of nuclease-free water. On-column DNAse digestion and RNA clean-up were then performed using the QIAGEN RNeasy mini kit  per the manufacturer’s instructions, followed by elution in nuclease-free water.RNA concentration was assessed using Qubit RNA BR Assay Kit (Q10210) and a Qubit 3.0 Fluorometer. RNA integrity was assessed using an Agilent 4150 TapeStation system and associated RNA screen tape reagents (Agilent 5067-5576). Only samples with an estimated RNA integrity number (RIN) ≥7.0 were sent to the New York Genome Center (NYGC) for bulk total RNA sequencing. Upon receipt at NYGC, RNA samples were re-evaluated for quantification and integrity, using Ribogreen and Fragment Analyzer 5300, respectively. Total RNA libraries were prepped using Kapa Total library prep with Ribo-Erase, in accordance with manufacturer recommendations. Briefly, 500 ng of total RNA was used for ribosomal depletion and fragmentation of total RNA. Depleted RNA underwent first- and second-strand cDNA synthesis. cDNA was then adenylated, ligated to Illumina sequencing adapters, and amplified by PCR (using nine cycles). The cDNA libraries were quantified using Fragment Analyzer 5300  kit FA-NGS-HS (Agilent DNF-474-1000) and Spectramax M2 (Molecular Devices) kit Picogreen (Life Technologies P7589). Libraries were sequenced on an Illumina NovaSeq sequencer, using paired end sequencing (2 × 100 bp cycles) to a depth of >75 M read pairs per sample.Raw reads were then trimmed and filtered of adapter sequencing using cutadapt  and filtadj < 0.05) were then separated into 6 groups based on their intersections between the two brain regions using UpSetR v1.4.0  experiments involving mouse primary cortical neurons or human iPSC-derived cortical neurons, cells were washed with 1× PBS, harvested from the culture dish, and RNA was extracted using the QIAGEN RNeasy mini kit  with on-column DNAse digestion per the manufacturer’s instructions. RNA concentration was determined using a NanoDrop 2.0 spectrophotometer, 250 ng to 1 µg of RNA was used to generate cDNA using Applied Biosystems High-capacity RNA-to-cDNA kit (#4387406) per the manufacturer’s instructions. cDNA was generated from 250 to 500 ng of RNA using Applied Biosystems High-capacity RNA-to-cDNA kit (#4387406) per the manufacturer’s instructions. Quantitative PCR was performed on a QuantStudio 6 Real Time Polymerase Chain Reaction (PCR) system  using PowerUp SYBR Green master mix (Applied Biosystems A25776). The PCR phase consisted of 40 cycles of 15 s at 95°C and 1 min at 60°C. Forward and reverse primer sequences for imerBank  and syntMice were transcardially perfused with 1× PBS followed by 4% PFA, and brains were removed and post-fixed for 24 hr in 4% PFA. Brains were cryoprotected in 15% sucrose in 1× PBS for 24 hr followed then 30% sucrose in 1× PBS for 48 hr, and then embedded in Fisher Tissue-Plus OCT compound (4585). Forebrain (~Bregma +1.78 mm), midbrain (~Bregma +0.50 mm), and hippocampal (~Bregma –1.94 mm) 10 mm cryosections were collected onto Superfrost Plus charged slides using a Leica CM1950 cryostat.Slides were acclimated to RT and rinsed in distilled water. They were then incubated in 70% ethanol for ~60 hr followed by 95% ethanol for 30 min. Tissues were stained with 0.1% Luxol Fast Blue Solution overnight at 60°C, rinsed in distilled water, and differentiated as needed by 0.05% Lithium Carbonate followed by 70% ethanol. They were then counterstained with 0.1% Cresyl Echt Violet, rinsed in distilled water, and differentiated again by 95% ethanol. Finally, slides were dehydrated in 100% ethanol, cleared in Xylene, and coverslipped with DPX mountant (Millipore-Sigma 44581).Slides were acclimated to RT and rinsed in distilled water. Heat-induced epitope retrieval was performed at 120°C using pH 6.0 buffer . The tissues were then blocked in 10% NGS for 1 hr. Primary antibodies were applied overnight at 4°C: Mouse Anti-NeuN (clone A60) conjugated to Alexa Fluor 555 , and Rabbit Anti-TDP-43  or Rabbit anti-Iba1  and mouse anti-GFAP conjugated to Alexa Fluor 488  . After rBrain sections stained with Cresyl Violet (CV) and brain sections labeled with Iba1 and GFAP anatibodies were imaged on a Nikon Eclipse Ti2 widefield microscope equipped with a Nikon DS-Fi3 CMOS color camera (for CV imaging) and a pco.edge 4.2Q High QE sCMOS camera  using a ×20/0.5 NA Plan Fluor objective and NIS-Elements software . Immunofluorescently labeled brain sections were imaged on a Leica SP8X Falcon confocal microscope equipped with hybrid GaAsP detectors using a ×40/1.30 NA Plan Apochromatic (oil) objective and Leica Application Suite X Life Sciences software . Cell counting (density of CV- and NeuN-positive cells), as well as TDP-43 fluorescence intensity, TDP-43 localization measurements, GFAP fluorescence intensity and area, and Iba1 fluorescence intensity and area were performed using CellProfiler . Prior thttps://www.graphpad.com. For datasets with n > 10, outliers were identified and removed (if any) using the ROUT method , as well as >95% of amyotrophic lateral sclerosis (ALS). To determine if acetylation is a primary, disease-driving event, they generated a TDP-43 mutant harboring an acetylation-mimicking mutation (K145Q). Animals carrying the acetylation-mimic mutation (K145Q) displayed key pathological features of disease, including more cytoplasmic TDP43 and impaired TDP43 splicing activity, together with behavioral phenotypes reminiscent of FTLD.This is a well-written and well-illustrated manuscript, with clear and convincing findings. The observations are significant and emphasize the importance of post-translational modifications to TDP-43 function and disease phenotypes. In addition, the TDP43(K145Q) mice may prove to be a valuable model for studying TDP-43-related mechanisms of neurodegeneration and therapeutic strategies.Comments on the latest version:The authors have addressed most concerns. The additional analysis demonstrating a lack of neuron loss is quite different from the original study -- it is good that the authors pursued this question. In addition, new data focusing on native TDP-43 splice targets, rather than the splicing reporter, are excellent. This paper extends prior work demonstrating the importance of K145 acetylation of TDP-43 as a post-translational modification that impacts its RNA-binding capacity and may contribute to pathology in FTLD-ALS. The main strengths of this paper are the generation of a novel mouse model, using CRISPR gene editing, in which an acetylation-mimetic mutation (K to Q) is introduced at position 145. Behavioral, biochemical, and genetic analyses indicate that these mice display phenotypes relevant to TDP-43-associated disease and will be a valuable contribution to the field. Numerous experimental models are phenotyped in this manuscript including mouse neurons, iPSC-derived human neurons, knock-in mice, and knock-in iPSCs. Expression of acetylation-mimic or acetylation-null TDP-43 protein is achieved either with overexpression or CRISPR-Cas9-based knock-in. A complex phenotype is observed including loss of TDP-43 function  and a gain of TDP-43 (increased insoluble TDP-43 protein). These correlate with downstream neurobehavioral changes which are most consistent with a cortical/hippocampal phenotype without a motor phenotype. Post-translational modifications of disease-associated proteins are thought to contribute to neurodegenerative disease pathogenesis, and this study succeeds in demonstrating that TDP-43 acetylation results in downstream molecular and behavioral phenotypes.TDP-43 acetylation is a post-translational modification that is known to be associated with TDP-43 inclusions that are characteristic of human diseases. An important strength is the rigorous use of multiple different experimental models  with overall consistent results. Moreover, multiple orthogonal endpoints are presented including histology/cytology/immunostaining, biochemistry, molecular biology, and neurobehavioral assays. As TDP-43 acetylation is known to block RNA binding, these novel cellular and mouse models represent interesting albeit complex tools to study the functional consequences of a partial loss of function. As TDP-43 regulates its own expression (i.e. autoregulation), the complexity lies at least in part due to the loss of RNA binding leading to a functional loss of TDP-43 function which includes the increased expression of the TARDBP transcript and TDP-43 protein.Conceptually, there is a disconnect in that the mouse model exhibits primarily a cortical/hippocampal phenotype more akin to frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), while TDP-43 acetylation is only seen in ALS tissues and not in FTLD-TDP tissues because most of the pathologic protein in the latter is N-terminally truncated (i.e. the acetylation site is not present). That being said, there is no mouse model which completely and faithfully recapitulates the human disease, and this mouse model avoids overt overexpression  and avoids the use of synthetic/artificial mutation .This revision addresses most of my prior comments including documenting the lack of neurodegeneration in this model, the use of more appropriate statistical methods, and the use of more robust/quantitative aberrant splicing measures. The one thing which would still be helpful is sequencing the top predicted off target genomic loci for their various CRISPR'd models irrespective of whether these loci are exonic or noncoding. Having actual sequencing verification of the lack of mutations at these loci is preferable over relying only on computational likelihood estimates. The following is the authors’ response to the original reviews.Reviewer #11.1 Fig. 1: A good control for these studies would be a TDP-43 variant with an RRM1 mutation that impairs RNA binding, but not an acetylation mimic In our original paper , we already characterized TDP-43 acetylation and employed a complete RNA-deficient mutant (F147/149L), as the reviewer suggested. In that original study, this mutant showed maximal RNA binding-deficiency, and therefore we proposed that acetylation mimic mutations represent a comparable RNA-binding deficient variant.1.2 Fig. 1: time and expression level can influence nuclear TDP-43 puncta formation. It is important that the authors take these into account when measuring puncta number/frequency.All expression levels and transfection/transduction times were identical across samples. We chose the optimal times to express TDP-43 constructs yet minimize toxicity and found that neuronal transduction at DIV10 and arsenite exposure on DIV14 in mature neurons was optimal.1.3 Fig. 2: to accurately refer to the nuclear foci as anisosomes, the authors will need to conduct higher-resolution imaging.We agree with the reviewer and since anisosomes are not well characterized in terms of their relationship to TDP-43 nuclear foci (and may represent only a subset of foci), we have now omitted any mention of anisosomes but instead refer to them in the discussion, where we suggest that TDP-43 K145Q foci may partially represent anisosomes.1.4 Fig. 2D: it seems as though the splicing reporter should have a fluorescence-based readout . Is this the case, and is the ratio informative?We have now removed the splicing reporter data and replaced this with much more robust data showing RT-qPCR of downstream TDP-43 targets including Sortilin-1 .1.5 Line 145: "Overall, these results indicate that a single endogenously expressed acetylation-mimic TDP-43(K145Q) mutation is sufficient to alter TDP-43 localization, induce TDP-43 phase separation, and impair splicing in a murine primary neuron culture model." The authors did not assess phase separation in this study. Moreover, it would be more convincing to assess native splice targets of TDP-43 in K145Q primary neurons, rather than an exogenous splicing reporter.See comment 1.1 above. We have now avoided mentioning phase separation in the main text but mention this as a potential mechanism in the discussion. In addition, we have now evaluated native TDP-43 splice targeted in primary neurons.1.6 Fig. 4A: is the loss of neurons selective for a specific layer or region of the cortex?Since we did not observe any gliosis, we have gone back and completely re-evaluated neuronal loss since the concept of neurodegeneration is a critical question in the TDP-43KQ/KQ mice. We do not find any significant neuronal loss in the homozygous TDP-43KQ/KQ mice .1.7 Fig. 6: The authors suggest that the large majority of splicing changes are direct results of the TDP43(K145Q) mutation and impaired RNA binding by TDP-43. However, without a direct assessment of TDP43(K145Q) target RNAs in comparison to those of TDP-43(WT), this is only an assumption. Moreover, given the fact that RNA-seq was performed in aged animals, the potential for indirect gene expression changes is very high.In our original study , we showed that the K145Q is severely deficient in RNA binding. In this study, we now show strong evidence that many known targets of direct TDP-43 binding are dysregulated, supporting the expected loss of function if TDP-43 K145Q mutation abrogated RNA binding. Although we have not performed direct RNA binding studies to the Sort1 transcript, for example, other studies have clearly indicated that wild-type TDP-43 binds these targets. We infer that loss of function mutations  impact direct targets of TDP-43. Future studies employing RNA-immunoprecipitation followed by RNA sequencing (RIP-seq) could be useful in this regard and will be required to mechanistically address this point.1.8 Sup Fig. 8 is very interesting and suggests that any TDP-43 variant that is unable to bind RNA may lead to upregulation of TDP43 RNA and phenotypes similar to those observed n K145Q animals. This is alluded to in the discussion but never specifically tested.Yes, we agree with this reviewer’s comment. Loss of RNA binding, whether due to acetylation  or otherwise is expected to cause autoregulatory up-regulation of the TARDBP transcript and impact other targets, potentially yielding phenotypes similar to the TDP-43KQ/KQ mice. However, new in vivo models would be needed to prove this point. For example, in the future, we will consider this possibility by characterizing recently identified RNA-binding deficient familial TARDBP mutants .1.9 The authors should also provide some comment or potential explanation for why TDP43(K145Q) animals show no signs of motor neuron disease.We now show a moderate level of TDP-43 aggregation and hyper-phosphorylation in spinal cord of mutant mice in Figure 6 – Figure Supplement 3. We also speculate in the discussion why we observe aspects of TDP-43 dysfunction in spinal cord without overt motor phenotypes up until 18 months old.1.10 Line 79: "However, TARDBP mutations that disrupt RNA binding, and thereby may act in a similar manner to TDP-43 acetylation, have been identified in FTLD-TDP patients." Evidence suggests that the D169G mutation does not interfere with RNA binding. See Furukawa et al., 2016.We thank the reviewer for pointing this out. We have now removed the D169G mutation from the discussion.1.11 It is unclear why the authors focused solely on homozygous K145Q animals, rather than heterozygous mice.We focused initially on homozygous mutant mice to provide better statistical power to detect small effect sizes. However, we have now included a thorough analysis of heterozygous mice including molecular analysis of brain tissue and mouse behavior, as shown in Figure 4 – Figure Supplements 1-2 and Figure 6 – Figure Supplements 1-3.Reviewer #22.1 A strength of this paper is the generation of a new mouse model to study the effects of K145 acetylation in TDP-43 proteinopathy. While the authors note an absence of a behavioral phenotype on neuromuscular testing in aged animals, it would be appropriate to include some analysis of spinal cord and skeletal muscle in this initial description of their model. At a minimum, I wonder if there is pathology in the cord  or muscle (fiber atrophy) if insoluble p-TDP-43 is detectable in these tissues, and whether dysregulated splicing of TDP-43 target genes  occurs at these sites.See comment 1.9 above. We analyzed TDP-43 aggregation, localization, and splicing in the spinal cord of TDP-43KQ/KQ mice and found mild loss of TDP-43 function that was comparable, though not to same extent, as that seen in hippocampus and cortex. We discussed these findings in the discussion and provide several possibilities for why there are no overt motor phenotypes in these mice. We note that TDP-43 Q331K knock-in mice also have cognitive but no motor deficits, suggesting TDP-43 dysfunction may preferentially  impact cognitive function :552-563).2.2 Fig 2: Differences in the splicing reporter are hard to appreciate from the images shown in panel E. Is the quantification shown in panel F corroborated by an analysis of green vs yellow fluorescence or by another method? Quantification of results shown in panel 2G  should be included.We have now removed the splicing reporter data in lieu of the more robust RT-qPCR data shown in Figure 2E and 3B-I. We have also now included more biological replicates from our iPSC neuron imaging, as shown in Figure 3A. Due to time and resource constraints, we were not able to quantify the images shown in figure 3A, and reinforce in the text that our statements are qualitative. However, we were able to add quantitative analysis of TDP-43 dysfunction, by detecting genotype-dependent splicing changes in hiPSC neurons, as mentioned above, which strengthens our claim that TDP-43 dysfunction is prominent in this culture modee.2.3 Fig 4: Differences in NeuN quantification without changes in cresyl violet staining or gliosis are surprising and a bit difficult to understand. Is there confirmation of neuron loss through another metric? Is it possible that NeuN expression is lower in mutants without frank neuron loss? Also, although no significant differences were seen by IF for TDP-43 staining, did IF for phospho TDP-43 show differences? One might expect this to be the case given the biochemical findings in Fig 5.See comment 1.6 above. After a much more in-depth and rigorous assessment, we find little evidence for neurodegeneration. Given the transcriptome data showing that TDP-43 regulates a subset of synaptic genes, we suggest that synaptic deficits underlie the behavioral phenotype rather than neuronal loss.Regarding phospho-TDP-43 pathology by immunofluorescence (IF) staining, after much effort, we have not been able to detect phospho-TDP-43 pathology by IF in TDP-43KQ/KQ mice. Currently available phospho-TDP-43 antibodies (including those acquired from collaborators) do not work well to detect endogenous mouse TDP-43 by histology or IF staining, and therefore we are somewhat limited technically. Nonetheless, given the increase in phospho-TDP-43 in the insoluble fractions by western blotting combined with the increase in cytoplasmic TDP-43 via biochemical fractionation, our data suggest that phospho-TDP-43 is the relevant species accumulating in the cytoplasm of TDP-43KQ/KQ mice.2.4 Fig 5: Probing the NC fractions for phospho TDP-43 would be an interesting addition to support the conclusion that increased cytoplasmic localization of the KQ mutant occurs prior to its phosphorylation.We agree that this would be an excellent addition to our data. Unfortunately, after rigorous antibody validation experiments, we were not able to find a phospho-TDP-43 antibody that specifically detected phosphorylated TDP-43 and did not cross-react with unphosphorylated TDP-43 in the buffers used for N-C fractionations. We tested phospho-TDP antibodies in RIPA (soluble), Urea (detergent-insoluble), and the N-C fractionation buffers, using samples treated or untreated with lambda phosphatase (to de-phosphorylate TDP-43). Only one antibody reliably detected the phosphorylated TDP-43 and not the lambda phosphatase-treated TDP-43 samples, and only did so in the Urea buffer, which is shown by straight westerns in our manuscript. Because of these technical difficulties with the phospho-TDP-43 antibodies, this was a challenging point to address at the moment. As better phospho-TDP antibodies become available, we hope to be able to address this. We therefore cannot definitively conclude that cytoplasmic phospho-TDP-43 pathology is present in these mice, but nonetheless the total phospho-TDP-43 levels are significantly elevated in urea (insoluble) fractions.2.5 Fig 1: What quantitative criteria were used to distinguish between puncta and foci, as highlighted in panel A? What is the biological significance of this distinction? From the images in panel A, it is difficult to see the TDP-43 foci in wt and K145R expressing cells.Although the size of nuclear TDP-43 foci can be quite variable, and we are certainly interested in the biological significance of this parameter, we did not focus this study on size profiles of K145Q-induced foci, only their accelerated formation and abundance. Therefore, in the revised manuscript we chose not to explicitly state any differences in “foci” vs. “puncta” and now refer to all nuclear TDP-43 structures as “foci” (removed the word “puncta” throughout).2.6 Fig 3: In describing the results of context-dependent fear testing, it is more appropriate to state that significant deficits appeared at 18 months, deleting the word "more" on line 186.We have deleted the work “more”.Reviewer #33.1 Multiple figures  include data with multiple measurements per field of view and multiple fields of view per condition. It appears that each measurement was considered an "n" for ANOVA or t-tests, but the data structure violates the requirement that data points are independent. More rigorous statistical methods such as mixed effect models should be considered (see DOI: 10.1016/j.neuron.2021.10.030) which in many cases provide more statistical power. Mixed effects models are the more appropriate statistical method for much of their data. Should the authors want to reanalyze their data with this method, they can reach out to me for an introduction to this statistical model.We have now re-evaluated the figures mentioned using linear mixed effects models, similar to what the reviewer has mentioned. The new statistical measurements have been incorporated into the revised Figures 1, 2, and 5 . A description of the statistical methods used is now provided in the revised methods section.3.2 In the introduction, the authors write "we avoid both TDP-43 overexpression and disruption of autoregulatory genomic elements of the endogenous Tardbp transcript" but they show that autoregulation is altered. So shouldn't the acetylation sites be considered a genomic element that regulates autoregulation?We agree and have now stated that our knock-in approach avoids disrupting surrounding genomic elements  in order to retain the native Tardbp gene in its unaltered form.3.3 Suggest editing the language regarding potential neurodegeneration/neuron loss as the same results could be obtained with tissue volume and/or developmental effects independent of progressive neurodegeneration.See comments 1.6 and 2.3 above. The language has been edited to reflect no apparent neurodegeneration.3.4 Sequencing the top predicted off-target loci in CRISPR'd mice and iPSC cell lines would help show the absence of off-target mutations.We described in the methods how potential off-target effects were avoided. We assessed the likelihood of off-target mutations using prediction algorithms to ensure low likelihood. All of the predicted exonic off-target sites have 4 mismatches, making them extremely unlikely to be mutated.3.5 The authors describe a subtle shift in electrophoretic mobility of the SORT1 protein band in figure 7d/e, but it is unclear why the entire SORT1 band should be shifted up in mutant mice given that the RNA analysis suggests that WT species  is still the major RNA that is expressed. In addition, others have shown that the WT versus +ex17b bands can be resolved (see DOI: 10.1073/pnas.1211577110). Perhaps knockout/knockdown cells can facilitate by providing a positive control for sizing/separation of Sort1 by immunoblotting.Please refer to our RNA-seq data shown in Figure 8A. In WT mice, nearly 80% of Sort1 transcripts lack exon17b, while this number drops to 23% in the TDP-43KQ/KQ mice. Therefore, the abnormally spliced +ex17b becomes the dominant transcript in TDP-43KQ/KQ mice. Given the prominent +ex17b inclusion that we are observing at the transcript level, it is not surprising that we mostly observe the up-shifted ex17b-containing Sort1 protein band. We have been unable to resolve two distinct bands by immunoblotting in mouse tissues using multiple Sort1 antibodies, including those used in Prudencio et al Proc Natl Acad Sci U S A. 2012 Dec 26;109(52):21510-5. Nonetheless, the up-shifted Sort1 protein is clearly the abnormal variant, as it becomes destabilized in our mice. Another possibility is that partial loss of TDP-43 function, as we suspect occurs in the TDP-43KQ/KQ mice, may magnify (or enhance) the effects on Sort1 such that the dominant Sort1 variant observed is the +ex17b containing variant. We suspect this to be true since this phenomenon was also observed in the Prudencio et al study .3.6 The authors may try to corroborate their CFTR splicing results by examining fluorescence as it appears that the construct allows for analysis of splicing differences using GFP vs mCherry expression. This is a minor point as RNA-seq analysis demonstrates abundant splicing changes in acetylation-mimic expression models.We have now removed the CFTR splicing data entirely and replaced it with more robust readouts of endogenous TDP-43 splicing targets both in vitro  and in vivo .3.7 Should the bars in figure 3d for 1 and 2 min be colored in grey/pink? It is unclear why they are clear and only outlined in color.This point is clarified in the revised Figure 4D legend. In our cue-dependent conditioned fear testing, the filled bars beyond 2 min represents the presence of the auditory cue (tone) and the period of statistical analysis.3.8 The statistical test used (Fisher's exact test?) for determining overlap between transcriptome datasets should be stated.We clarified our comment in the results section to reflect the use of over-enrichment analysis. In the methods section, it reads “Previously published differentially expressed genes from Hasan et al95 and Polymenidou et al96 were retrieved from the respective publications; significant over-enrichments as well as human gene symbol mappings to mouse orthologs were performed using gprofiler2 (g:Orth).”


The coupling effects of surface plasmon resonance (SPR) from metamaterials induce variation in both the frequency and intensity of plasmonic modes. Here, we report an angular-dependent THz modulator with hybrid metal–graphene metastructures. The metastructures composed of the period gold split-rod arrays on top of a monolayer graphene, which show redshift modulation in the THz region with an increasing incident angle due to the strong out-of-plane magnetic flux introduced by the clockwise circular current at the oblique incidence. By utilizing graphene-based actively tunable conductor with ion-gel electrical gating, the THz transmission can be significantly modified. The modulation depth of the hybrid metal–graphene metastructure modulator can reach ~37.6% at 0.62 THz with a gate voltage of −3 V. The theoretical modeling of transmitted dependency on frequency and incident angle is demonstrated at different Fermi energies, which fits well with the experimental results. This hybrid device can offer a useful method for THz applications (such as angle sensors or angular-resolved spectroscopy), where angle-dependent modulation is needed. The terahertz (THz) spectral region, serving as a bridge between electronics and optics, has attracted tremendous interest over the past few decades for its potential applications in noninvasive imaging , high-sp2 hybridized carbon atoms, possesses exceptional gapless electronic band structure [fE) and well described by the Drude model [Graphene, a two-dimensional structure arranged in a hexagonal lattice with sptructure , high motructure  and linetructure  for electructure ,14. The tructure . In the tructure , which cde model . Such unMetamaterials composed of discrete sub-wavelength structures have attracted enormous attention for versatile applications, such as perfect lensing , invisibHere, we demonstrate an angular-dependent modulator with hybrid metal–graphene metastructures in the THz region. By applying ion-gel doping, the transmission characteristics of the gated hybrid metal–graphene metastructures can be tuned, which consequently induces modulation of the transmitted wave. We observe that the resonance increases with the incident angle due to the enhanced localized field in the split-rod arrays. The THz modulation enhancement of the hybrid device is verified by both experimental and theoretical results, which shows dependency on the incidence angles and gating voltages. It turns out that the maximum modulation depth can reach −37.6% with a gate voltage of −3 V at an incident angle of 30°. Our results show that the hybrid metal–graphene metastructures can be useful in various active devices for angle-dependent photonic applications.xP and yP) of 120 µm, length (L) of 100 µm, split width (w) of 10 µm and split gap (g) of 6 µm are designed to generate the optical resonance at range of 0–1 THz, which is to fit with the operation wavelength of our THz measurement setup. The configuration of the metamaterials is sketched in 2 and a beam resolution of 0.1 µm. After e-beam lithography, the sample has to be immersed in AZ351  for 10 min to remove Al firstly and developed in a solution made of Methyl Iso Butyl Ketone (MIBK):Isopropanol (IPA) = 1:3 for 1 min. The morphology of the gold metamaterials is depicted in −1) triblock copolymer and 20 mL dichloromethane. By using the mask with apertures, the electrode with proper scale can be obtained by engaging with the e-beam evaporation. Finally, wire-bonding technology is used to apply the electrical connection, and the scheme of the measured device is shown in Experimentally, the gold split-rod metamaterials are fabricated on a sapphire substrate with further covered with chemical-vapor-deposition (CVD) graphene sheet, and the fabrication diagram is displayed in An angular-solved THz-time domain spectroscopy (TDS) setup is used to measure the transmission properties at oblique incidence from the hybrid metal–graphene metastructure. The source laser is a Ti/Sapphire laser  with a pulse width of 200 fs and repetition rate of 75.9 MHz at 800 nm, which further impinge onto a photoconductive switch integrated on low-temperature grown GaAs (LT-GaAs) at an average power of 30 mW. The switch is biased at 40 V and produces the linear polarized THz pulses that propagate in a nitrogen-purged environment through the sample, with probe detection performed via an LT-GaAs-based photoconductive switch as well. The spot size of the THz pulse is approximately 3 mm with a frequency resolution of ~50 GHz. By rotating the sample, the incident angle of the light is typically set to 0–30° for the angular-resolved THz transmission measurement.The morphology of the metastructure is characterized by field emission scanning electron microscopy . Graphene properties are characterized by Raman spectroscopy . The electrical properties of the device are measured using a sourcemeter (Agilent 2400 and 2401).gσ) can be described by altering the Fermi energy (fE) of graphene using the Kubo formula, which is present with both intraband and interband transitions [bk and T is the Boltzmann constant and temperature, Г is the carrier relaxing time, e is the charge of an electron, and ω is angular frequency, respectively.In calculations, the optical conductivity of graphene σgГ defined by μ and the Fermi velocity μ of 10,000 cm2/(V•s) and 6 m/s throughout the entire calculations.In the lower THz region, the optical conductivity of graphene is mainly governed by the intraband transition due to the Fermi level of graphene increasing above half of the photo energy. Here we only consider graphene with the ke model :(4)σg(ω)X- and Y-directions. In simulations, the thickness of the metal and substrate are 50 nm and 2 µm, respectively. The permittivity of gold in the THz region is described by the Drude model with a plasma frequency of pω = 1.37 × 1016 s−1 and damping constant dω = 4.08 × 1013 s−1, respectively [By using the frequency domain solver of CST Microwave Studio, the numerical calculations at oblique incidence can be performed with the unit cell boundary condition only supplying in ectively . The dieectively . The traBy investigating the electromagnetic response of the split-rod arrays, it is obvious that the gold split-rod metamaterials can be viewed as an LC resonance in which the effective inductance arises from the loop formed by the split-rod and effective capacitance due to the gap region between split-rod arms. The degree of asymmetry is increased by increasing the incident angle and further leads to the redshift of the resonance frequency. As a result, the electric field can effectively couple to the metastructures and induces a circular current, which accounts for a magnetic dipole perpendicular to the plane of metastructures.gV), the electrical measurement is employed to study the chemical doping states of the device and helps to define the charge neutral point (CNP) of graphene, as given in dsV = 1.0 V. It is apparent that the leakage current (gsI) (black line) is stabilized around zero, while the source–drain current (dsI) (blue line) varies conspicuously with the applied gate voltage. This behavior reveals that the electric doping of graphene can be efficiently tunable, and the Fermi level of graphene can be adjusted approaching the Dirac point with a gate voltage of 0.5 V , which implies a natural p-type doping of our graphene samples. It is worth noting that the asymmetry response in the hole and electron transportation regions is observed, which is affected by current leakage through the electrical gating [gV, the theoretical relation between fE and gV is given by [a = 1.45 × 1013/m2V is the capacitance per charge of the ion-gel [Due to the fact that carrier doping in graphene can be controlled with the gate voltage /T0, gT and 0T represent the transmission of the device with applied at gV and CNPV) is defined as the relative transmittance normalized to the value near the CNP for a selection of spectra for CNPV = 0.5 V under normal incidence, as shown in gV = −3.0 V under normal incidence. The relative transmittance with incident angles of 10°, 20°and 30° are present in In order to evaluate the modulation of the gated metal-graphene metastructure device more comprehensively, the modulation depth  at around 0.62 THz with gV = −3.0 V.To verify the angular-dependent modulation more intuitively, we investigate the transmission dip as a function of incident angle, as shown in To gain a better understanding of the physical mechanisms of the angular-dependent modulation characteristics, the simulated transmission spectra of hybrid metal–graphene metastructure device are applied by numerical results versus the incident angle and the Fermi energy of graphene layer, as shown in z-component magnetic field and x-component electric field at frequencies centered within resonant modes under normal incidence, respectively. It is evident that the magnetic fields are mainly localized at the edge of individual split-rods with TE mode, which lead to a magnetic dipole perpendicular to the surface plane of metamaterials. Hence the circulating currents can be excited, which naturally induces opposite electrical fields in the x-direction. Similarly, the distribution of magnetic fields at oblique incidence shows further localization at the edge of individual split-rods as portrayed in gV = −3 V at an incident angle of 30°. To further reveal the physical mechanism of the modulation properties, a theoretical analysis for transmitted dependency on the incident angle and Fermi energy of graphene is proposed. It is observed that the LC resonant model is employed to describe the near-field interaction in this hybrid device, which agrees well with the experimental results. The distribution of magnetic and electric fields further illuminates that the modulated behaviors lie in the recombination effect of the conductive graphene. These results prove that the angular-dependent modulation of the gated graphene-metastructure device can potentially provide a platform for strong light–matter interactions. Further tunable modulation of graphene-based metastrctures can be achieved by applying vertical or broadside coupling, which can be effective routes through exploiting the interlayer near-field electromagnetic coupling. Our works indicate that the graphene-based optical modulator can be used under multi-angle incident conditions.In conclusion, in this work we investigate the angle-dependent modulation of electrostatic gating for a hybrid metal–graphene metastructure device. Through an experiment performed on an angle-resolved TDS setup, the maximum angular-dependent modulation depth is obtained as −37.6% with