We report on the development of hybrid organic–inorganicmaterial-based flexible memristor devices made by a fast and simpleelectrochemical fabrication method. The devices consist of a bilayerof poly(methyl methacrylate) (PMMA) and Te-rich GeSbTe chalcogenidenanoscale thin films sandwiched between Ag top and TiN bottom electrodeson both Si and flexible polyimide substrates. These hybrid memristorsrequire no electroforming process and exhibit reliable and reproduciblebipolar resistive switching at low switching voltages under both flatand bending conditions. Multistate switching behavior can also beachieved by controlling the compliance current (CC). We attributethe switching between the high resistance state (HRS) and low resistancestate (LRS) in the devices to the formation and rupture of conductiveAg filaments within the hybrid PMMA/GeSbTe matrix. This work providesa promising route to fabricate flexible memory devices through anelectrodeposition process for application in flexible electronics. This involvesa melt-quenching reset process that requires a high programming current,which has become the major obstacle for the integration of PCMs.26 Resistive switching, however, does not involvemajor structural phase changes, and the switching between the HRSand LRS and vice versa can be achieved at lower voltages upon changethe polarity of the applied electric field.27 Resistive switching effects have been observed in several chalcogenidessuch as AgGeSe, AgGeTe, AgInSbTe, and GeSbTe, with switching mechanismsattributed to a formation and rapture of conductive filaments eitherfrom the migration of metal cations 36 in the electrochemical metallization (ECM) process or from migrationof Sb and Te ions inside the chalcogenide materials.41Typically, a resistive memristor consists of a simple metal–insulator–metal(MIM) structure where the resistive switching material is sandwichedbetween two electrodes. The switching between the low resistance state(LRS) and high resistance state (HRS) is achieved by the creationand disruption of conductive filaments which are made of either metallicatoms from the active electrode or the defects within the switchinglayer.42 These “top-down”approaches often require vacuum equipment, which leads to high depositioncosts and slow deposition rates. Additionally, their high thermalbudgets can also be prohibitive for their application in flexibleapplication. Electrodeposition is a well-established deposition methodin the electronic industry that offers a low-cost and fast alternativesolution for chalcogenide deposition.44 Our grouphas previously reported the electrodeposition of amorphous ternaryGeSbTe thin films from a single, highly tunable, nonaqueous electrolyte,46 demonstrating both phase-change and resistive switching propertiesin highly scalable cross-bar architectures.47 More importantly, electrodeposition is a low-temperature depositiontechnique which makes it highly suitable for deposition onto flexiblesubstrates. However, the deposition of a nanoscale thin film chalcogenide-basedmemristor onto flexible substrate via electrodeposition has neverbeen reported.Conventionally, chalcogenide materials are deposited via techniquessuch as physical vapor deposition, chemical vapor deposition, andatomic layer deposition.53 In comparison to inorganic material-based memristors, hybrid memristorshave demonstrated improved switching properties such as high ON/OFFratio, ultralow operating voltage, reduced power consumption, as wellas having various advantages such as multilevel data storage, analogueswitching, and flexibility.59In addition, hybrid organic–inorganicmaterial-based memristorshave attracted attention since they combine the electronic propertiesof inorganic components and the solution processing advantages oforganic components.In this work, we report the development of high-quality hybridmemristor devices that are fabricated by electrodeposition. The hybridmemristor devices consist of a bilayer of electrodeposited GeSbTenanoscale thin films combined with different thicknesses of an insulatingPMMA layer (the thickness is controlled by changing the PMMA concentration)sandwiched between a TiN bottom electrode and an Ag top electrodeusing Si and polyimide substrates. The hybrid devices demonstratedesirable memristor properties such as a free forming process, lowoperation voltages, good cycling endurance, multistate switching behavior,and, importantly, mechanical flexibility. These properties open theroute to realize fully the potential of memristor devices for low-costand high-performance flexible electronics.2nBu4]Cl in dichloromethane (CH2Cl2) as a reference electrode. Electrodeposition of GeSbTethin film was carried out in a dichloromethane (DCM) solution usingprecursors for Ge, Sb, and Te synthesized in-house.60 Prepatterned TiN thin films on Si/SiO2 substrateswere used as the deposition electrode. The details of TiN electrodefabrication were reported in our previous work.46 Prior to the film deposition, cyclic voltammetry (CV) wasrecorded as shown in 4+ to Ge2+, Sb3+, Te4+, and Ge2+ to Ge(0), respectively.61 To ensure the incorporation of all three elements, the depositionpotential of −1.75 V vs Ag/AgCl was selected. The detailedprocess of the electrodeposition is provided in the Supporting Information Figure S1. The memristorfabrication process is completed by patterning Ag top electrodes onthe hybrid organic–inorganic structure using thermal evaporationtechnique.The composition of the electrodeposited GeSbTefilm was characterizedby energy dispersive X-ray analysis (EDX) and the spectrum is presentedin 63 The absence of the CC during the RESET process switches the single-layerGeSbTe-based device to a higher operating current, resulting in breakdownof the device.41 Subsequent DC-IV cycles(gray lines) are similar to the initial switching cycle (red line).This indicates that no electroforming process is required to initializethe devices for the resistive switching effect. Note that the electroformingprocess is needed to initiate the switching in oxides.64 However, we expect the lack of the forming processin our devices due to a large amount of Te atoms within the Te-richGeSbTe matrix, which can play a role in the formation of Te conductivefilament(s). Both SET and RESET processes are characterized by gradualcurrent change, and the device only operates at high current. Suchhigh operating current is likely due to the formation of strong filament(s)within the GeSbTe layer. This is not surprising, as the granule-liketexture of the as-deposited GeSbTe layer implies a certain amountof porosity in the film.41 It has been suggestedthat the voids or pores in the electrolyte layer could serve to facilitateion migration in the thin film for the electrochemical metallization(ECM) memories.65 The voids or pores withinthe GeSbTe electrolyte can likely act as diffusion pathways for Agcations, enabling the formation of conductive nano Ag filament(s)across the device.32 This high operatingcurrent leads to high power consumption which is highly undesirablefor memristors.Figure S2a–e. Retention testing for the hybrid TiN/PMMA/GeSbTe/Agmemristor (50% PMMA concentration) in both the HRS and LRS were examinedat a continuous read voltage of 0.1 V for over 4000 s and are presentedin Figure S2f.The hybrid PMMA/GeSbTe-based memristor exhibitssimilar forming-free,bipolar resistive switching behavior to the single layer device shownin 3c–f.HowPStandby = IHRS × Vread), was calculatedat a read voltage of (100 mV) and the OFF currents of the devices.This notable control of the currents results in a significant improvementin the separation between the OFF and ON states and therefore enhancesthe ON/OFF resistance ratio from 8 to 836, as shown in To better understandthe impact of the additional PMMA layer onthe memristor switching properties, we plot the operating currents(at 0.1 V), the power consumption, and the ON/OFF resistance ratioof each device as a function of the PMMA concentration. Figure S3.To explore the conduction mechanism in our controlAg/GeSbTe/TiNmemristor and hybrid Ag/PMMA/GeSbTe/TiN memristor, the current–voltagecurves were replotted in double-logarithmic scale as shown in A switching mechanism consistent with the electricaldata is proposedand displayed in +) at the top Ag/PMMA interface. Under high electricfield, the Ag+ ions can move across the PMMA and GeSbTeto the bottom electrode, where the neutral Ag atoms are accumulatedand start to form a filament. Once the grown filament connects tothe top Ag electrode, the device switches to the LRS (SET). In contrast,applying a negative potential moves the Ag+ ions formedat the bottom electrode to the top electrode and ruptures the preformedfilament, switching the device back to the HRS (RESET).In the case of the hybrid memristor, both PMMA/GeSbTematrix canact as electrolyte, where a resistive switching through oxidation/reductionreactions (ECM process) can occur upon altering the polarity of theelectric field 6b. The h66 that can result in undesirable device properties such asswitching the device to a higher current level and breakdown in theabsence of CC during RESET process.41 Protectingthe GeSbTe with PMMA not only improves the resistive switching propertiesbut also reduces the standby power consumption, which potentiallysuppresses the sneak current and devices disturbance issues.18Theresults obtained suggest that the PMMA layer plays a key rolein improving the resistive switching properties. As a barrier betweenthe GeSbTe and top electrode, the PMMA diminishes the penetrationof the Ag atoms into the voids of the GeSbTe thin film. The PMMA alsochanges the switching between the two resistance states from a smoothtransition for the GeSbTe device to a sharp transition for the hybridPMMA/GeSbTe devices. For the devices containing only GeSbTe, multiple,large Ag filaments can easily be formed across the high porosity GeSbTethin film upon application of high electric field. The large Ag filament(s)results in the high operating currents and is also responsible forthe CC requirement during the RESET process. For the devices containinghybrid PMMA/GeSbTe material, the formation of Ag filaments acrossthe hybrid material tends to be more restricted at high PMMA concentration,resulting in a sharp switching with 2 orders of magnitude differencebetween the LRS and HRS state. In addition, the PMMA makes the switchingfrom the HRS to the LRS slower, as the SET voltage increases from∼0.6 V for the devices containing only GeSbTe thin films to∼1.7 V for the devices containing hybrid PMMA (50%)/GeSbTematerial. The PMMA may also smooth the GeSbTe interface region andmask local surface defects and surface roughness70 Such CC level-dependent controlhas been widely demonstrated in resistive switching memories and canbe ascribed by the formation of filaments with different dimensionsacross the device.71 Although similar multistate switchingbehavior was previously observed in our crossbar GeSbTe-based memorydevices, the ON/OFF resistance ratio was smaller (by about 1 orderof magnitude), and the CC was needed for both the SET and RESET processesto protect the devices.71Multistate switching can be achieved inour hybrid PMMA/GeSbTedevices by controlling the CC level. Figure S4. The 33% PMMA concentration solutionwas selected to deposit the PMMA layer on the GeSbTe thin film toobtain the hybrid material structure.We will now demonstrate the development of hybridPMMA/GeSbTe material-basedmemristor devices on flexible substrate. The GeSbTe thin film waselectrodeposited on a polyimide substrate with a TiN layer as thebottom electrode. The cyclic voltammetry and deposition transientare shown in R) of 15 mm. The effect of mechanicalflexibility testing on the electronic properties of the flexible memristordevice was investigated by performing I–V sweeps before bending (black curve), during bending (bluecurve), and after bending (red curve), as shown in A photographic image of the developed flexiblehybrid memory deviceswith the structure of Ag/PMMA/GeSbTe/TiN under bending is shown in VSET and VRESET were also analyzed under flat and bendingconditions as shown in Supporting Information Figure S5. This suggestselectrodeposition is a viable approach for fabricating flexible memristordevices.To evaluate operational reliability of our hybridflexible memristor,the memory performance properties including the DC endurance and cumulativeprobability for the ON/OFF resistance ratio were analyzed. As shownin 3In conclusion, we havesuccessfully demonstrated the developmentof a flexible memristor that shows reliable bipolar resistive switchingproperties at low switching voltages. This is achieved by utilizinga hybrid organic–inorganic materials approach by incorporatingPMMA with resistive switching electrodeposited GeSbTe nanoscale thinfilms. The switching mechanism in these devices is explained by theformation and rapture of conductive Ag filament(s) upon applicationof different electric field polarity. The resistance states can becontrolled by CC, demonstrating the multistate switching behavior.Our approach for the fabrication of flexible memristor devices couldhave potential applications in future nonvolatile data storage electronics.4The memristordevices consist of a vertical thin film stack withthe following order of materials, Ag/PMMA/GeSbTe/TiN 3a. Prior2Cl2 , dried and degassed byrefluxing with CaH2, followed by distillation and thenstored in the glovebox. A 0.1 M [NnBu4]Cl  was used as the supporting electrolyte. Theelectrolyte contains 2.5 mM [NnBu4][GeCl5], 1 mM [NnBu4][SbCl4], and2 mM [NnBu4]2[TeCl6],which were synthesized as described previously.61Electrolytes were prepared in anhydrous CH72 A Pt/Ir (90%:10%) disk was employed as the counter electrode andan Ag/AgCl wire (0.1 M [NnBu4]Cl in anhydrousCH2Cl2) was used as the reference electrode.Silicon and polyamide substrates with predepositedTiN bottom electrodes serve as the electrode for electrodeposition.The detailed fabrication process can be found in our previous work.2Cl2 solution containing 2.5 mM [NnBu4]GeCl5, 1 mM [NnBu4]SbCl4, and 2 mM [NnBu4]2TeCl6 at a scan rate of 50 mV s–1, 47 Electrodeposition of GeSbTe was carried outat −1.75 V vs Ag/AgCl with a cutoff charge of 5 μC toallow a complete depositing GeSbTe thin film, as shown in 2Cl2 and stored in the glovebox.All cyclic voltammetry (CV) and electrodepositionexperiments were carried out in a recirculating glovebox (Belle) toexclude moisture and air. Oxygen levels were kept below 10 ppm. AmicroAutolab 3 potentiostat and a Nova 1 software package were usedfor all electrochemical measurements. Prior to GeSbTe electrodeposition,voltammetry was recorded on a TiN bottom contact in CH495 poly(methylmethacrylate) (PMMA) solution, purchased from Sigma-Aldrich, was dilutedin anisole in different ratios designed to give predefined PMMA concentrationsolutions, 20%, 25%, 33%, and 50%. The solutions were spin coatedonto the pre-electrodeposited GeSbTe thin films at 4000 rpm for 60s. The samples were then annealed at 120 °C for 3 min in airto remove any remaining solvent. Ag top electrodes (200 nm thick)were thermally deposited under vacuum conditions through a shadowmask containing 250-μm-diameter circles. Note that a controldevice consisting of GeSbTe (no PMMA) with a structure of Ag/GeSbTe/TiNwas also fabricated. The electrical characteristics were measuredat room temperature and ambient pressure using a probe connected toa Keysight (B1500) system. For all measurements, the voltage was appliedto the top electrode Ag contacts, while the bottom electrode TiN contactswere grounded. SEM, EDX and AFM were used to characterize the thinfilms.


The object of this study was to evaluate the use of −SCN as a counter anion for cationic manganese porphyrin (MnP) catalytic antioxidants, which could increase the parent compound’s antioxidant spectrum against hypohalous acids while supplementing innate immunity. The antioxidant activities of the parent compound were examined, as its chloride salt was compared with the −SCN-anion exchanged compound, (MnP(SCN) versus MnP(Cl)). We measured the superoxide dismutase activity spectrophotometrically and performed hydrogen peroxide scavenging using oxygen and hydrogen peroxide electrodes. Peroxidase activity was measured using an amplex red assay. The inhibition of lipid peroxidation was assessed using a thiobarbituric acid reactive species (TBARS) assay. The effects of the MnP compounds on macrophage phagocytosis were assessed by flow cytometry. The abilities of the MnP(Cl) formulations to protect human bronchiolar epithelial cells against hypochlorite (HOCl) and glycine chloramine versus their MnP(SCN) formulations were assessed using a cell viability assay. We found that anions exchanging out the chloride for −SCN improved the cellular bioavailability but did not adversely affect the cell viability or phagocytosis and that they switched hydrogen-peroxide scavenging from a dismutation reaction to a peroxidase reaction. In addition, the −SCN formulations improved the ability of MnPs to protect human bronchiolar epithelial cells against hypochlorous acid (HOCl) and glycine chloramine toxicity. These novel types of antioxidants may be more beneficial in treating lung disease that is associated with chronic infections or acute infectious exacerbations.The innate immune response to infection results in inflammation and oxidative damage, creating a paradox where most anti-inflammatory and antioxidant therapies can further suppress an already inadequate immune response. We have previously reported the beneficial effects of the exogenous supplementation of innate immunity with small pseudohalide thiocyanate ( Diseases associated with recurrent or chronic infections result in tissue damage due to the prolonged inflammation and oxidative stress initiated by the innate immune system. Cystic fibrosis (CF) is characterized by chronic recurrent lung infections and progressive lung destruction, due in part to chronic lung neutrophilia . Lung-re−SCN) is an endogenous pseudohalide anion that is abundant in bodily fluids and a natural product from plant-based diets [−SCN levels are associated with glucoside and glucosinolate-containing plants such as those found in the Brassicaceae family [−SCN is utilized by haloperoxidases, including neutrophil myeloperoxidase (MPO) and epithelial lactoperoxidase (LPO), to generate hypothiocyanous acid (HOSCN), a wide-spectrum antimicrobial agent [−SCN/HOSCN redox couple exhibits selective antimicrobial toxicity. HOSCN has been shown to be more toxic to some pathogens than the host because of its ability to be metabolized by the host thioredoxin reductase, but not by the pathogen thioredoxin reductase [−SCN also can readily react with all hypohalous acids and their secondary products, forming HOSCN that can be readily deactivated by the host [−SCN the ability to act as an antioxidant while retaining host defense properties and could be combined with more traditional antioxidants and anti-inflammatory agents to enhance treatments for chronic infectious disease.Thiocyanate  have been developed as catalytic antioxidants with the ability to perform one and two electron dismutation reactions with superoxide and hydrogen peroxide [meso-group N-alkyl pyridine or N, N-alkyl imidazole pendants (−SCN (MnP(SCN)) would affect their known antioxidant activities and improve their ability to protect lung epithelial cells against hypochlorous acid (HOCl) and glycine chloramine.Manganese (III) peroxide . The catpendants . These aMaterials and reagents: Frozen rabbit brains were purchased from Pel-Freeze. Cytochrome c from horse heart, xanthine oxidase from bovine milk, thiobarbituric acid, L-ascorbate, ferrous chloride, glycine, sodium thiocyanate, sodium hypochlorite (10–15%), 1,1,3,3-tetramethoxypropane, cytochalasin D, and dimethyl sulfoxide were purchased from Sigma–Aldrich. Hydrogen peroxide (30%) and 1-butanol were purchased from Fisher Chemicals. DMEM and phosphate buffered saline were purchased from Corning Cellgro. Fetal calf serum was purchased from Hyclone. Amplex red, 3--2,5-dipenyl tetrazolium bromide (MTT), imaging buffer, and Staphylococcus aureus pH rhodo red bioparticles were purchased from Invitrogen. All reagents used were the highest reagent grade available and at least >95% purity, unless otherwise stated.meso-porphyrins anion exchange:Manganese  MnPs, their chloride (MnP(Cl)) and −SCN (MnP(SCN)) salts, were provided as kind gifts by Aeolus Pharmaceuticals. The −SCN content in the MnPs was determined by using HPLC with electrochemical detection  as previously described [−SCN to MnP was determined using MnP soret band extinction coefficients.escribed . The molSuperoxide dismutase (SOD) assay: Superoxide was generated by xanthine oxidase and detected spectrophotometrically  by a reduction of cytochrome c that was assessed following the change in absorbance at 550 nm in PBS buffer at pH 7.4. A single unit of SOD activity is defined as the amount of compound needed to decrease the cytochrome c reduction rate by one half [one half .Hydrogen peroxide assay: Hydrogen peroxide (H2O2) consumption was measured with an electrode (WPI). The hydrogen peroxide (H2O2) electrode  was calibrated with a 5-point hydrogen peroxide standard curve. Increasing concentrations of H2O2 were incubated with 25 μM AEOL10150 as its chloride or -SCN salt formulation at room temperature in PBS buffer at pH 7.4. H2O2 consumption was measured over 1 min using a free radical detector (WPI) and digitally recorded (Lab Scribe3). Oxygen formation assay: Oxygen formation was measured with an oxygen electrode (WPI). The oxygen probe was calibrated to a room oxygen-saturated buffer solution and oxygen formation measured in a nitrogen-purged buffer containing 1 mM H2O2 and 50 μM of AEOL10150 chloride salt formulation in the presence or absence of 1 mM NaSCN. Oxygen formation was measured over 5 min using a free radical detector (WPI) and digitally recorded (Lab Scribe 3).Amplex red peroxidase assay: The assay was performed as described using the amplex red peroxidase kit microplate format with horseradish peroxidase (1 mU/mL HRP) as a positive control (Invitrogen). Reactions contained 50 μM amplex red, 1 mM H2O2, and increasing concentrations of AEOL10150(Cl). The total reaction volume was 100 μL. Reactions were incubated at room temperature in the dark for 10 min and read on a fluorescence plate reader  with an excitation of 530 nm and an emission of 590 nm. AEOL10150(Cl) alone at concentrations of up to 5 μM did not alter the background fluorescence in the assay.Lipid peroxidation assay: Rabbit brain homogenates (1 mg/mL protein) were treated with iron and ascorbate for 60 min to generate hydroxyl radial-mediated lipid peroxidation in the presence of increasing concentrations of MnPs, as previously described [50). escribed . Lipid pMouse macrophage cell line  was grown in DMEM with 10% fetal calf serum. J774A.1 cells were passed into 24 well plates and treated after reaching 90% confluence. Phagocytosis Assay: J774A.1 cells were incubated at 37 °C for 1 h in a cell image buffer (Invitrogen) with Staphylococcus aureus bioparticles labeled with pH-sensitive red fluoroprobe (1 mg/mL) in the presence or absence of 400 μM NaSCN, 100 μM AEOL10150(Cl), 100 μM AEOL10150(SCN), 100 μM AEOL 10113(Cl), 100 μM AEOL 10113(SCN), 100 μM AEOL 10158(Cl), 100 μM AEOL 10158(SCN), or 40 μg/mL cytochalasin D treatments. The cells were scraped and resuspended in fresh cell-imaging buffer for a flow cytometry analysis . The cells were gated on side scatter and PE (red) signals. Cytochalasin D-treated cells were used to set the negative gate. The total inhibition of Staphylococcus aureus bioparticle phagocytosis by cytochalasin D treatment was confirmed with fluorescent microscopy . The image-buffer-only group was used to normalize the data to 100% phagocytosis and the cytochalasin D group as 0% phagocytosis. Data were analyzed using FlowJo software version 10 (TreeStar).Human lung bronchial epithelial cell line (16HBE41o-) was grown in DMEM with 10% fetal-calf serum. HBE cells were passed in 24 well plates. The treatment groups contained 8 well replicates. The cells were treated with oxidant for 0.5 to 1 h in PBS and washed and replaced with fresh media. Cell viability was assessed 24 h later using a 3--2,5-dipenyl tetrazolium bromide (MTT) assay.Manganese porphyrin (MnP) HPLC assay: The cellular levels of MnPs were determined by HPLC in cellular lysates as previously described with modifications [g for 12 min. This extraction resulted in an extraction efficiency of MnPs greater than 90%. MnP concentrations were determined by HPLC with spectrophotometric detection (Shimadzu). A 25 μL sample or standard was injected and MnPs were measured at 446 nm with retention times of 2.3 to 4.8 min. MnP concentrations were determined from standard curves that were linear over the concentrations reported.ications . BrieflyHOCl and glycine chloramine cytotoxicity assay: 16HBE41o- cells were treated with 200 μM of glycine chloramine, made as previously described [50) determinations and their respective 95% confidence intervals.escribed , or HOClMTT cell viability assay: The cytotoxicity of lung epithelial cells was determined using MTT, as previously described [escribed . BrieflyStatistically analysis: Pair wise data were analyzed for statistical differences using a two-tailed t-test. Data sets with more than two treatment groups were analyzed for statistical differences using a one-way ANOVA with a Tukey’s range test for data with equal variances or a Kruskal–Wallis test with a Dunn’s range test for nonparametric data sets. Significance was established at p < 0.05 . −SCN for chloride. The anion-exchanged MnPs were assayed for −SCN incorporation using HPLC with electrochemical detection. The compounds were found to contain on average 3.5 moles of −SCN per mole of MnP. The MnP AEOL10150, in the form of its −SCN salt AEOL10150(SCN), was further investigated for any changes in antioxidant properties compared with its chloride salt analog AEOL10150(Cl).MnPs have been synthesized and screened for antioxidant activity over the last two decades. Several of the cationic MnPs were found to have high antioxidant activity  Figure . Three o2O2 was measured using a H2O2-calibrated electrode, and the first order rate constants were determined for each salt formulation. Both salt formulations of AEOL10150 had similar rate constants for scavenging H2O2  was formed in the reaction. The ability of AEOL10150 to function as a peroxidase was further assessed using an amplex red peroxidase assay. AEOL10150(Cl) exhibited dose-dependent peroxidase activity when compared to horseradish peroxidase (HRP)  A. NaSCN se (HRP) B. 50). Both salt formulations of AEOL10150 had similar IC50s for the inhibition of lipid peroxidation  assay and the concentration of AEOL10150 salt formulations was determined that inhibited TBA formation by 50%  were incubated with 100 μM of MnPs for 24 h. Cells were extensively washed, and lysates were made to determine the cellular levels of MnPs by HPLC . The MnPThe finding that MnPs may exhibit peroxidase activity with the generation of HOSCN suggests the MnP(SCN) formulations could impact cell viability . The MnP−SCN and chloride salts were investigated for any changes in the ability of murine macrophages (J774A.1 cells) to perform phagocytosis on Staphylococcus aureus (SA) bioparticles. The SA bioparticles used exhibited maximum fluorescence upon acidification within the phagolysosome [We next examined whether the MnPs in their different salt formulations interfered with innate immunity. A key process of the innate immune system is the ability of the leukocytes to phagocytose pathogens. The MnPs in the form of their lysosome . Flow cy50 of 17.7 μM (95% CI 14–23 μM), than against glycine chloramine-mediated injury, with an IC50 of 33 μM (95% CI 31–36 μM). Next, the MnP(Cl) and MnP(SCN) formulations were compared for efficacy in this HOCl and glycine chloramine cytotoxicity assay. The MnP(SCN) formulations were in general more potent in protecting against both HOCl and glycine chloramine-mediated cell injury than their respective MnP(Cl) analogs (50s that ranged from 6 to 23 μM for HOCl-mediated injury with a potency ranking of AEOL10158(Cl) > AEOL10150(Cl) > AEOL10113(Cl). MnPs as their −SCN salts had protective IC50s that ranged from 1 to 4 μM for HOCl-mediated injury with a potency ranking of AEOL10158(SCN) > AEOL10113(SCN) > AEOL10150(SCN). MnPs as their chloride salts had variable protective IC50s that ranged from 16 to 500 μM for glycine chloramine-mediated injury with a potency ranking of AEOL10158(Cl) >>> AEOL10113(Cl) > AEOL10150(Cl). MnPs as their −SCN salts were much more protective, with IC50s that ranged from 4 to 9 μM for glycine chloramine-mediated injury with a potency ranking of AEOL10150(SCN) > AEOL10113(SCN) > AEOL10158(SCN).The innate immune system generates antimicrobial oxidants using haloperoxidases that are activated in phagolysomes or released extracellularly. A common oxidant generated by myeloperoxidase is hypochlorous acid (HOCl) and subsequently, chloramines as reaction by-products from the HOCl reaction with amino groups. The MnP chloride and -SCN salt formulations were tested for their ability to protect human bronchial epithelial cells against HOCl and glycine chloramine-mediated cytotoxicity. NaSCN has been previously shown to protect against these oxidants. However, MnPs have not been examined in this system before. NaSCN was first tested in this cell cytotoxicity assay against HOCl and glycine chloramine-mediated injury. NaSCN protected human bronchial epithelial cells against both HOCl and glycine chloramine-mediated injury . NaSCN w analogs . MnPs in−SCN, which can be utilized by the innate immune system, to generate an antimicrobial and potentially cell-signaling oxidant with an antioxidant. We demonstrate that the incorporation of −SCN as a pharmacologic salt with cationic MnP antioxidants does not adversely affect the level of safety while switching the H2O2 scavenging mechanism from a dismutation reaction to a peroxidase reaction, thus mimicking the host’s haloperoxidases. In addition, the −SCN salts of MnPs increased the level of protective capability against oxidants generated by the innate immune system that can damage the host and promote tissue inflammation both in vitro and in vivo. Although −SCN can be protective against oxidants generated by the innate immune system, such as HOCl and chloramines, −SCN lacks antioxidant activity against superoxide and H2O2 and their downstream oxidants, such as lipid peroxides. MnPs in terms of their chloride and −SCN salts retain their ability to effectively scavenge superoxide and hydrogen peroxide, as well as to inhibit lipid peroxidation. It was interesting to note that the MnP mechanism for H2O2 scavenging switched in the presence of −SCN from a catalase dismutation reaction to a peroxidase reaction. The peroxidase reaction would be more prominent in vivo, given that the catalase reaction operates better at high concentrations of H2O2. Endogenous −SCN concentration in bodily fluids ranges from 5 to 3000 μM [2O2 concentration is generally in the 10–100 nM levels. The peroxidase activity of MnPs in the presence of exogenous or endogenous -SCN may support innate immune defense against pathogens by converting H2O2 to HOSCN. The reaction of MnPs with H2O2 has been shown to form relatively stable dioxomanganese (V) porphyrin complexes similar to haloperoxidases [−SCN and H2O2. Given these findings, it was also interesting that the MnP(SCN) formulations were as equally well tolerated by lung epithelial cells as the MnP(Cl) formulations. MnPs have been reported to possess some antimicrobial activities [−SCN present as the counter anion. The −SCN salt formulation improved MnP cellular bioavailability, which may also contribute to improving the efficacy of antioxidants. It is known that when NaSCN reacts with hypochlorous acid or chloramine, the product of this reaction generates hypothiocyanous acid [The goal of this study was to combine the pseudohalide  3000 μM , whereastivities , and thious acid  that is ous acid . This reous acid  that may2O2 and are efficacious in a wide variety of in vitro and in vivo models for oxidative stress [−SCN formulations. Some of this improved activity could be due to its cellular bioavailability, since it is better at accumulating in epithelial cells than the other two MnPs. Longer alkyl substitutions on the porphyrin pendants of AEOL10158 may be able to account for the improvement in cellular bioavailability. We observed that all the porphyrins tested showed that their −SCN salt formulations had lower water solubility than their chloride salt formulations. This may be due to the lower dissociation of −SCN anion from the cationic porphyrins than the chloride anion, thus making the −SCN formulations more hydrophobic with better bioavailability. The increased solubility of the −SCN formulations did not appear to account for all the enhanced potency of the MnPs, given that AEOL10150SCN was the most potent against glycine chloramine but the least affected by a change in cellular bioavailability. However, all three MnP(SCN)s were more effective at protecting lung epithelial cells against both hypochlorous acid and glycine chloramine than their respective chloride salts. This is likely due to −SCN’s unique ability to act as a cloaked oxidant in the presence of hypohalous acids (−SCN/HOSCN) [MnPs are effective catalytic antioxidants that efficiently scavenge superoxide and He stress . HoweverN/HOSCN) . −SCN/HOSCN system is the selective metabolism of HOSCN by the host thioredoxin reductase but not the pathogen thioredoxin reductase [2O2-mediated cell-signaling mechanism [One of the features of the eductase . Anothereductase . This maeductase . Thioredeductase . HOSCN ieductase ,35. Thisechanism ,37. In fechanism . −SCN as a pharmaceutical salt with MnPs may restore some of the beneficial effects that are removed during antioxidant treatments. This could only be possible if the HOSCN formed were less damaging than the other hypohalous acids. This has been demonstrated in vitro and in animal models, where inhaled or oral administration of NaSCN had positive outcomes in lung infections [−SCN in vitro hold up in more complex animal models and improve the outcomes of antioxidants in models of infectious disease and human diseases associated with chronic infections or infectious exacerbations, such as those seen in cystic fibrosis and chronic obstructive pulmonary disease.The use of antioxidants to treat infectious diseases associated with chronic inflammation has been disappointing, given the abundant evidence for the role of oxidative stress in these diseases . For insfections ,39, ischfections , and athfections . It will


In this work, weinvestigate whether stiffening in compressionis a feature of single cells and whether the intracellular polymernetworks that comprise the cytoskeleton  stiffen or soften when subjected to compressivestrains. We find that individual cells, such as fibroblasts, stiffenat physiologically relevant compressive strains, but genetic ablationof vimentin diminishes this effect. Further, we show that unlike networksof purified F-actin or microtubules, which soften in compression,vimentin intermediate filament networks stiffen in both compressionand extension, and we present a theoretical model to explain thisresponse based on the flexibility of vimentin filaments and theirsurface charge, which resists volume changes of the network undercompression. These results provide a new framework by which to understandthe mechanical responses of cells and point to a central role of intermediatefilaments in response to compression. Here, we compare the responses of purified cross-linkednetworks formed by F-actin, microtubules, and vimentin intermediatefilaments and show that actin and MT networks soften in compression,but vimentin networks stiffen. The unique effects of vimentin networksare consistent with a theoretical model, and their contribution towhole cell mechanics is evident in studies of a single mEF cell compressionusing AFM. Mechanical testing of nuclei isolated from vimentin expressingand vimentin null cells show that this effect is not the result ofnucleus compression but suggests that the vimentin networks that surroundthe nucleus enable compression-stiffening of cells and protect thenucleus from mechanical damage.Cells and tissues are subjectedto different types of external mechanical stresses, including compressiveloads, pressure gradients, or shear. Although the fibrous cytoskeletonprovides the mechanical strength of an individual cell,7 as well as those of synthetic semiflexible polymer networks,11 and is explained by the buckling of stiff filamentswithin compressed networks.6 In contrast,networks of vimentin intermediate filaments (VIFs) become stifferas they are compressed, as shown by the increase in shear modulusin response to axial strain. The abundance of VIFs within mesenchymalcells such as the fibroblast shown in the inset of 12 a highly charged semiflexiblepolysaccharide that is often interspersed in the ECM with collagenand is the dominant ECM element in tissues such as the brain and thevitreous body.13When cross-linked networksof actin filaments or microtubules aresubject to increasing compression, the shear modulus measured at lowstrain decreases as the axial strain magnitude becomes larger (morenegative) 1A. The s7 VIFs differfrom F-actin and microtubules in that they have a significantly largernegative surface charge,14 and they areat least an order of magnitude more flexible.15 Consequently, networks of VIFs might resist changes in volume becauseof the osmotic effect of their mobile counterions, and because theirpersistence length is no longer larger than the network mesh size.When the increased flexibility of the VIFs and the incompressibilityof the network are considered in the theoretical model  with the amount of the compressive force applied. 19 and the observed difference incompressibility between WT mEFs and vim null mEFs cannot be attributedto such alterations.To test how uniaxial compressive forceis transferred to the cagelikevimentin network surrounding the cell nucleus, we used NIH 3t3 fibroblastsexpressing GFP-vimentin. Time-lapse images of the fluorescently labeledvimentin network inside the cell was recorded simultaneously withAFM compression, allowing for correlation of the image of real-timevimentin cage deformation .To determinea possible biological significance of the unique compressionstiffening behavior of VIF networks, monolayers of cells were subjectedto varying amounts of compressive stress, and the morphology and nuclearintegrity of the cells were quantified  required for microtubule dependent rearrangements of vimentinnetwork during migration or kinesin-1, dynein, and plectin that participatein vimentin assembly.30Intermediate filaments acquired their name because they wereintermediatein size between the thin filaments (F-actin) and thick filaments (acto-myosin)of muscle cells.20 The importance of protectingthe nucleus against compressive stress might also be related to thefact that intermediate filaments are not expressed in plant or fungalcells, where compressive stresses are taken up by the rigid cell wallthat provide nucleus protection. Although our experiments were performedon single, isolated cells residing in an environment that is far fromphysiological, the effects explored using AFM-based whole cell compressionsshow the uniqueness of vimentin networks in preserving nucleus mechanicalintegrity and cell resilience upon large compressive strains.The unique contribution of vimentin networks,their stiffeningin compression, and their concentration around the nucleus were previouslyshown to protect the nucleus against damage as cells squeeze throughnarrow spaces.31 and biotin-modifiedactin was purchased from Molecular Probes . Monomericactin was prepared by dialysis against 5 mM Tris-HCl pH 8.0 and 0.2mM CaCl2. Actin was polymerized by addition of 2 mM Mg2+ and 100 mM KCl and cross-linked by addition of avidin  as previously described.32 Vimentin purified from Ehrlich ascites tumor cells wasprovided by Peter Traub33 polymerized at4 mg/mL as previously described25 and cross-linkedwith 2 mM Cu2+, which has been shown to be an efficientcross-linker of vimentin filaments and other similarly charged polyelectrolytefilaments.34 Microtubule protein (MTP)was purified from bovine brain through cycles of temperature-inducedpolymerization and depolymerization as previously described and pelletby centrifugation.36 Further details can be foundin Actin was purified from rabbitskeletal muscle,G′) at 2% shear strain and 1 Hz frequencywere made with an RFS3  ora Kinexus  rheometer equippedwith 20 mm diameter parallel plates. Further details can be foundin Measurements of the dynamic shear storagemodulus  the gel is entirely compressible and undergoes homogeneous uniaxialstrain, or (2) the gel is incompressible and bonded to the upper andlower plates, such that the compressed sample bulges outward at theedges with a parabolic profile.40 Sketches of the two deformationvarieties are shown in To explorewhether incompressibility can explain the stiffeningobserved in the experiments, we calculate the expected dependenceof the shear modulus on axial strain for a gel of semiflexible filaments2 with saturated humidity.Twocell lines were used, wild-type mouseembryonic fibroblasts (mEF WT) and vimentin-null fibroblast (mEF vimnull). Cells were cultured in DMEM (ATCC) with 10% fetal bovine serum(FBS), penicillin (50 μg/mL) and streptomycin (50 μg/mL).Cells were maintained at 37 °C in an atmosphere containing 5%CO–1 with a 25 μmdiameter sphere attached (Novascan) according to previously publishedprotocols.41 Both round and spread cells were measuredwith 20 cells per condition tested. Further details can be found in Experiments withwhole cell compression of wild-type mouse embryonic fibroblasts (mEFWT) and vimentin-null fibroblasts (mEF vim null) were performed usinga JPK Nanowizard 4 atomic force microscope equipped with cantileversof nominal stiffness 2.4 N m43 with the modifications detailed in Nuclei were isolated as previouslydescribedAFM measurements were made with a DAFM-LNBioscope  mounted on an Axiovert 100 microscope .Isolated nuclei were compressed using tipless silicon nitride cantilevers with a nominal spring constant of0.12 N/m at 1 Hz and a maximum applied force of 16 nN. Cytoplastswere indented using silicon nitride cantilevers with a 1 μmdiameter spherical bead attached and a spring constant of 0.06 N/m. Indentations were made at three pointsin the center of the cytoplast at a frequency of 1 Hz and a maximumapplied force of 6 nN. All measurements were made at room temperaturein PBS (plus Ca and Mg) with a minimum of 20 cells per condition measured.Supplementary Figure 1). Beforemeasurements the top slide with cells was drained by gently touchingthe edge of the slide to a dust-free wipe to pull off excess medium.Selected pressures were converted into a unit of force. Weights inthe shape of an upper slide were applied on top. For nuclei observation,a Zeiss Axio Observer D1 microscope was used. Further details of theloading tests performed are found in Supporting Information.The device used to assess alteration ofcell nuclei under pressure consisted of two slides and a microscopeholder . p < 0.05was considered to be statistically significant. Data are presentedas mean values ± SD.The significanceof differenceswas determined using the two-tailed Student’s


Similar correlations were demonstrated for FIP, SigFIP, and SevFIP acquired from the lower leg. Our MRI parameters obtained through semiquantitative analyses of muscles significantly correlated with clinical parameters in CMT1A patients, suggesting their potential applicability as imaging markers for clinical severity.We aimed to derive comprehensive MRI parameters that reflect intramuscular fat infiltration severity for designated lower extremity levels, based on semiquantitative analyses in Charcot-Marie-Tooth disease type 1A (CMT1A) patients. We reviewed lower extremity MRIs of 116 CMT1A patients. Intramuscular fat infiltration grading using the Mercuri scale was performed for the non-dominant lower extremity at three levels  for the thigh and at two levels  for the lower leg. Based on MRI results, the following parameters were calculated for each level and for entire muscles: fat infiltration proportion (FIP), significant fat infiltration proportion (SigFIP), and severe fat infiltration proportion (SevFIP). The relationships between the MRI parameters and clinical data were evaluated using Spearman’s correlation analysis. FIP, SigFIP, and SevFIP measured for entire muscles significantly correlated with Charcot-Marie-Tooth Neuropathy Score ( The most common type of CMT, CMT type 1A (CMT1A), results from a duplication of the peripheral myelin protein 22 (PMP22) gene on chromosome 171. There is a substantial variability in the clinical course of patients with CMT1A, and a significant age-dependent increase in neurologic deficits can occur3; hence, these patients should be examined carefully. Although there is currently no cure for the disease, several recent clinical and preclinical studies using therapeutic agents have shown encouraging results6. In this regard, it is necessary to develop and establish an objective and reproducible evaluation method for patients with CMT1A, including the evaluation of treatment response.Charcot-Marie-Tooth disease (CMT), the most common hereditary neuromuscular disorder, comprises a group of genetically and clinically heterogeneous disorders characterized by symmetric distal muscle wasting, muscle weakness, and sensory loss1. Owing to the demand for additional clinical information and objective evaluation tools, various diagnostic imaging studies have been increasingly employed in clinical practice8. Several studies have targeted a number of anatomical structures using different imaging techniques in patients with CMT1A and reported promising results relating to the use of potential imaging biomarkers for these patients13. An MRI evaluation of fat infiltration in extremity muscles has been the most widely used of these techniques not only for patients with CMT1A, but also for those with other neuromuscular disorders, as extremity muscle fat infiltration is the central pathophysiological mechanism directly linked to the clinical manifestations of CMT1A17. The evaluation of intramuscular fat infiltration using MRI can be accomplished via a semiquantitative or quantitative approach. The semiquantitative approach is performed by evaluating each muscle using a classification system such as the Mercuri or Goutallier scale, which grades a muscle based on the relative amount of fat tissue present within the muscle16. The quantitative approach is performed by obtaining an additional imaging sequence containing a fat fraction map that enables a direct quantitative measurement of the fat percentage of each muscle9. Both approaches focus on the evaluation of individual muscles, rather than providing comprehensive information on the overall severity of fat infiltration at the designated level. To our knowledge, few studies have evaluated imaging parameters that comprehensively reflect the degree of muscular fat infiltration based on semiquantitative MRI in patients with CMT.The clinical assessment of patients with CMT1A has traditionally relied on physical examination and electrophysiological studiesIn this study, we aimed to derive comprehensive MRI parameters that reflect the severity of intramuscular fat infiltration for designated levels of the lower extremity, based on semiquantitative analyses using the Mercuri scale in patients with CMT1A. We assessed the potential value of these MRI parameters as imaging biomarkers by correlating them with clinical parameters. In addition, we evaluated the degree and pattern of intramuscular fat infiltration at multiple levels of the lower extremity muscles.18, we chose the oldest member of each family for analysis, considering the potential similarity of muscular fat infiltration among family members (thereby excluding 65 subjects). We additionally excluded 42 patients who had missing clinical assessment data. Furthermore, we sought to exclude patients with previous lower extremity surgery or suboptimal MRI quality; however, no such patients were present.The institutional review board of our institution  approved this retrospective study and waived the requirement for informed consent due to the retrospective nature of the study. The study was conducted in accordance with the declaration of Helsinki. Initially, we enrolled 223 patients diagnosed with CMT1A, who were confirmed to have PMP22 duplication by genetic analysis at our neurology department, and who underwent lower extremity MRI between September 2013 and March 2020. Although significant intrafamilial variability in clinical manifestations has been reported in patients with CMT1ALower extremity MRI scans were obtained using a 1.5-T or 3.0-T MRI system  with a 16-channel anterior coil and a posterior built-in coil. Axial T1-weighted images encompassing the pelvic girdle, bilateral thighs, and lower legs were obtained at different levels from the anterior inferior iliac spine through the distal tibia. Imaging parameters were as follows: repetition time, 613.7 ms; echo time, 16.7 ms; flip angle, 90 19 as follows: stage 0, normal appearance; stage 1, scattered small areas of increased signal; stage 2a, numerous discrete areas of increased signal comprising less than 30% of the muscle; stage 2b, numerous discrete areas of increased signal comprising 30–60% of the muscle; stage 3, washed-out appearance due to confluent areas of increased intensity with muscle still present at the periphery; and stage 4, end-stage appearance, muscle entirely replaced by areas of increased signal.Two radiologists  performed a consensus-based analysis of the thigh and lower leg muscles using T1-weighted images. Intramuscular fat infiltration grading was performed using the Mercuri scaleEvaluations were performed for the non-dominant lower extremity at three levels  for the thigh muscles and at two levels  for the lower leg muscles. Levels were determined based on anatomical landmarks on axial T1-weighted images: gluteus maximus tendon insertion ; just inferior to the inferior margin of the gluteus maximus, where the muscle is no longer visualized ; just inferior to the inferior margin of the adductor longus, where the muscle is no longer visualized ; just inferior to the inferior margin of the popliteus, where the muscle is no longer visualized ; and the uppermost part of the gastrocnemius tendon, where the gastrocnemius muscle is no longer visualized  ; sartorius, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, adductor longus, adductor brevis, adductor magnus, gracilis, semitendinosus, semimembranosus, and biceps femoris ; sartorius, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gracilis, semitendinosus, semimembranosus, and biceps femoris ; tibialis anterior, extensor digitorum longus, peroneus longus, gastrocnemius medialis, gastrocnemius lateralis, soleus, and tibialis posterior ; tibialis anterior, extensor digitorum longus, extensor hallucis longus, peroneus longus, soleus, tibialis posterior, flexor digitorum longus, and flexor hallucis longus .Fat infiltration proportion (FIP) = number of muscles with stage 1 or higher/total number of evaluated muscles × 100 (%)Significant fat infiltration proportion (SigFIP) = number of muscles with stage 2a or higher/total number of evaluated muscles × 100 (%)Severe fat infiltration proportion (SevFIP) = number of muscles with stage 3 or higher/total number of evaluated muscles × 100 (%)Based on the MRI results, we defined the following quantitative parameters to measure the degree of muscular fat infiltration at each level Fig. .Fat infiEach parameter was also assessed for the entire muscles (46 muscles) of the lower extremity .20. A nine-point FDS was used to determine disease severity in terms of the ability to walk and run as follows: 0 = normal; 1 = normal, but with cramps and fatigability; 2 = inability to run; 3 = walking difficult, but still possible unaided; 4 = able to walk with a cane; 5 = able to walk with crutches; 6 = able to walk with a walker; 7 = wheelchair-bound; and 8 = bedridden18. For the 10-MWT, which was performed to evaluate locomotor ability, patients were asked to walk a 10-m distance, and the time taken was measured in seconds.We assessed clinical data for patients with CMT1A, including age at onset, disease duration, CMT Neuropathy Score version 2 (CMTNSv2), 10 m walk test (10-MWT) time, and functional disability scale (FDS) score. The age at onset was determined by asking the patient when symptoms such as foot deformity, distal muscle weakness, and/or sensory changes initially appeared. The CMTNSv2 is a composite score consisting of neurological symptoms, clinical signs, and electrophysiological parameters. It ranges from 0 (no deficit) to 36 (maximum deficit)p value < 0.05 was considered statistically significant. Spearman’s correlation coefficients were interpreted as follows: r < 0.2, negligible correlation; 0.2 ≤ r < 0.4, weak correlation; 0.4 ≤ r < 0.6, moderate correlation; 0.6 ≤ r < 0.8, strong correlation; and r ≥ 0.8, very strong correlation21.Relationships between MRI parameters  and clinical data  were evaluated using Spearman’s correlation analysis in patients with CMT1A. Descriptive statistics for MRI parameters, clinical variables, and demographic variables are presented as mean ± standard deviation, median [interquartile range], and range. Statistical analyses were performed using SAS version 9.4 . A Clinical and demographic data of patients with CMT1A are shown in Table The results of the imaging analyses, including measured FIP, SigFIP, and SevFIP at each level, are summarized in Supplementary Tables The results of the correlation analysis between the MRI and clinical parameters are summarized in Table r = 0.5292), followed by FDS score and SigFIP at level 5 (r = 0.5170). Degree of other significant correlations between MRI parameters and clinical parameters were weak or negligible correlation.All three MRI parameters acquired from the entire muscles and those acquired from the lower leg level (levels 4 and 5) showed moderate correlations with FDS score. In addition, SigFIP at level 5 and total SigFIP were moderately correlated with CMTNSv2 and disease duration, respectively. FDS score and SigFIP at level 4 had the strongest correlation  showed moderate positive correlations with clinical parameters; FDS score showed the strongest correlation with SigFIP at level 4.22. Our result does not seem to imply that only the proportion of muscles with substantial or severe fat infiltration possess more grave clinical significance. It may be necessary conduct a large-scale study to evaluate which MRI parameter is more strongly correlated with clinical parameters and to assess the potential efficacies of these MRI parameters as imaging biomarkers in patients with CMT1A.Our proposed MRI parameters were calculated as proportions of muscles that showed fat infiltration among all individual muscles at designated levels. We devised three parameters—FIP, SigFIP, and SevFIP—to represent proportions of muscles showing fat infiltration (Mercuri stage 1 or higher), significant fat infiltration (Mercuri stage 2a or higher), and severe fat infiltration (Mercuri stage 3 or higher), respectively. We devised separate imaging parameters to verify if the proportion of muscles with significant (SigFIP) or severe fat infiltration (SevFIP) showed a more significant correlation with clinical parameters compared to the correlation between proportions of any individual muscles that display fat infiltration (FIP). All three parameters showed meaningful correlations with the clinical parameters, without significant differences among each other. In clinical practice, it may be challenging to determine whether mild intramuscular fat infiltration identified on MRI is related to the normal aging process or results from denervation in patients with neuromuscular disorders2, and therefore our findings may provide scope for further studies to enhance the application of MRI analyses at multiple levels in the lower leg level than in the thigh level.Our results showed moderate positive correlations between MRI parameters and FDS score as well as weak positive correlations between MRI parameters and 10-MWT time. Both the FDS score and 10-MWT time are used to assess disease severity in terms of the ability to walk and run. Our results may suggest a potential applicability of MRI parameters in evaluating motor function in patients with CMT1A. In addition, our findings revealed that clinical parameters were more strongly correlated with MRI parameters obtained from the lower leg level than with those obtained from the thigh level. Distally predominant limb muscle wasting is a characteristic feature in patients with CMT1A20. The intramuscular fat fraction of the lower extremity, obtained using quantitative analyses of MRI fat fraction maps, reportedly correlates with CMTNSv2 in patients with CMT1A23. Quantitative analyses require the acquisition of an imaging sequence containing a fat fraction map that enables a direct quantitative measurement of the fat percentage of each muscle, which is not possible with conventional imaging sequences. Our proposed parameters obtained by a simple calculation of semiquantitative analyses result of muscles on conventional T1-weighted imaging sequence were also significantly correlated with CMTNSv2, which may imply a potential applicability of these parameters as imaging biomarkers. Studies with a larger number of participants are warranted to confirm our results.MRI parameters were correlated with CMTNSv2, which is a composite scoring system comprising several factors including clinical signs and electrophysiological parameters3. Earlier age at onset is reportedly related to a more severe disease course18, although we did not demonstrate any significant negative correlation between age at onset and MRI parameters.We found positive correlations between MRI parameters measured for entire muscles and muscles at multiple levels and disease duration; nevertheless, these parameters were not significantly correlated with age at onset. These results may reflect the progressive nature of intramuscular fat infiltration in patients with CMT1A24. Generally, patients with CMT1A present a peroneal-type pattern of fat infiltration, although superficial posterior compartment muscles are also commonly affected26. Our findings corroborate with previous study findings. Among the thigh muscles, the highest median Mercuri stage was found in the semitendinosus muscle at level 2 (stage 2a). Little has been reported on the pattern of thigh muscle denervation in patients with CMT1A. Hence, it would be interesting to investigate the tendency of muscle involvement based on disease severity in the future.The median Mercuri stage of the evaluated muscles was highest in the extensor hallucis longus and peroneus longus at level 5 (stage 2b). Other anterior and superficial posterior compartment muscles at level 5, as well as the peroneus longus and the gastrocnemius medialis at level 4, demonstrated a median stage of 2a. Price et al. divided the denervated muscles in the lower legs into ‘‘peroneal-type” and ‘‘tibial-type” muscles, based on the predominantly affected musclesIn addition to intrinsic limitations of retrospective studies, we acknowledge several limitations in our study. First, interobserver agreement was not evaluated due to the consensus-based analyses performed in this study. Second, imaging analyses were performed on non-dominant lower extremities. It may be more desirable to obtain imaging parameters that reflect comprehensive information on both lower extremity muscles. Third, the median FDS score of our cohort was 2 [1–2.5], indicating that the majority of patients could walk unaided. Thus, future studies should investigate whether our proposed MRI parameters are significantly correlated with clinical parameters in patients with more advanced disease stages. Fourth, it is not clear whether performing MRI analyses for individual muscles has clinical benefit over performing MRI analyses for individual muscle groups. Fifth, although our study results showed significant correlations between MRI parameters and clinical parameters, only mild to moderate correlations were demonstrated. It would be interesting to assess the relationships of MRI parameters with other clinical parameters such as those acquired using CMT pediatric scale, CMT functional outcome measure, 6-min walk test, or dynamometry. Sixth, the age of onset which was determined by asking the patients may not have been accurate due to inaccurate recall of initial symptom. In addition, we intended to reflect the severity of fat infiltration involving entire muscles at a designated level, and the degree of contribution of individual muscle compartments to the clinical severity could not be determined. This could be an interesting subject for investigation in future studies. Moreover, it would be beneficial to examine these patients in the long term, to assess whether these MRI parameters have potential applications in prognosis estimation.In conclusion, we proposed preliminary MRI parameters that may comprehensively reflect the severity of intramuscular fat infiltration at designated levels of the lower extremity; these parameters were obtained through semiquantitative analyses of muscles based on the Mercuri scale in patients with CMT1A. These parameters were positively correlated with clinical parameters, thereby indicating their potential applicability as imaging biomarkers that clinically reflect disease severity in these patients. Regarding the degree and pattern of intramuscular fat infiltration, median Mercuri stage assessment showed a preferential involvement of the anterior and lateral compartment muscles in the distal lower leg.Supplementary Information.


Genetic evidence indicates disrupted epigenetic regulation as a major risk factor for psychiatric disorders, but the molecular mechanisms that drive this association remain to be determined. EHMT1 is an epigenetic repressor that is causal for Kleefstra Syndrome (KS), a genetic disorder linked with neurodevelopmental disorders and associated with schizophrenia. Here, we show that reduced EHMT1 activity decreases NRSF/REST protein leading to abnormal neuronal gene expression and progression of neurodevelopment in human iPSC. We further show that EHMT1 regulates NRSF/REST indirectly via repression of miRNA and leads to aberrant neuronal gene regulation and neurodevelopment timing. Expression of a NRSF/REST mRNA that lacks the miRNA-binding sites restores neuronal gene regulation to EHMT1 deficient cells. Significantly, the EHMT1-regulated miRNA gene set not only controls NRSF/REST but is enriched for association for Intellectual Disability (ID) and schizophrenia. This reveals a broad molecular interaction between H3K9 demethylation, NSRF/REST regulation and risk for ID and Schizophrenia. Genetic evidence points to an association of chromatin remodellers, mediators of epigenetic regulation, as a substantial risk factor for many common psychiatric disorders , 2. Genode novo postzygotic EHMT1 mutation and an ASD and neurocognitive dysfunctions in adults  Study). Procedures included clinical and cognitive testing, where possible, and blood sampling for generation of iPSCs and were approved by the South-East Wales Research Ethics Committee. Where participants did not have capacity to consent, as in this case, a representative (next of kin) provided written informed consent on their behalf. Peripheral blood mononuclear cells (PBMCs) from each donor were reprogrammed using a CytoTune-iPS 2.0 Sendai reprogramming kit  . KaryotyEhmt1+/− cell lines mutant mouse ES cells (mESCs), obtained from the European Mouse Mutant Cell Repository Centre (EuMMCR); each had a single copy of a ‘knockout first’ conditional allele Ehmt1tm1a(EUcOMM)Hmgu allele ..GluN1) gHere, we report a regulatory pathway that connects the molecular lesion in EHMT1 activity to altered neuronal cell development and neuronal function in human iPSC-derived neurons. The key mechanistic components of the pathway are the transcriptional regulator NRSF/REST and its control via miRNA, with reduced EHMT1-mediated H3K9me2 resulting in elevated miRNA transcription. As a consequence, gene expression of both NRSF/REST-regulated and general neuronal specific markers is elevated with lower levels of EHMT1 activity but can be reduced to control levels by expression of a NRSF/REST mRNA that lacks the miRNA regulation sites. Although KS presents a strong genetically penetrant case for this pathway, our genetic association analysis suggests a broader association for ID and schizophrenia, with implications for therapeutic intervention.NeuroD1, Tuj1, and DCX [−/+ cells is associated with elevated expression of the human pro-neural transcription factors MASH1 and NGN2.NRSF/REST plays a key role in repression of neuronal gene expression to maintain stem cells in the undifferentiated state , 48, mak and DCX . In the +/− hiPSC at these later stages of neurodifferentiation is unclear, but it is noteworthy that NRSF/REST-suppressed genes include cell death-inducing genes that may directly induce apoptosis [ehmt1 [rest genes increase cell proliferation and adult neurogenesis, but the prolonged loss of NRSF/REST leads to a functional depletion of the adult neuronal stem cells and decreased granule neuron production [The linkage between NRSF/REST and mental health is not well explored, but there are some mechanistic observations reported. In human neuronal culture, decreased nuclear NRSF/REST has been observed in neuronal cultures derived from sporadic Alzheimer’s Disease (AD) patient cells and again leads to accelerated neural differentiation and increased excitability, which can be reversed by exogenous NRSF/REST expression . In the poptosis . We alsos [ehmt1  or rest oduction . Finallyoduction .Nonetheless, GWAS has not strongly associated NRSF/REST with psychiatric disorders. This may be because of a combination of network robustness that protects against minor fluctuations of its upstream regulatory pathway, and the severity caused by major changes in NRSF/REST expression, as reported with its association with dementias , ischemic shock and some NDD. Our cross-disorder analysis of the miRNA under EHMT1-regulation and their association with GWAS-significant miRNA genes suggests hitherto unexplored linkage with NSRF/REST not only for KS but across a broader range of ID and schizophrenia cases and may offer significant insights for alternative therapeutic strategy.In summary, this study identifies a mechanism that couples EHMT1 activity to the neuronal regulator NRSF/REST through miRNA-dependent pathway, which leads to altered neurodevelopment. It suggests NRSF/REST as a key node within a miRNA-mediated gene regulatory network and offers a mechanism for the specific case of KS. Importantly it also reveals the presence of a more extensive pathway centred around NRSF/REST regulation of the neurodevelopmental gene regulation programme, which has broader significance for neurodevelopmental and psychiatric disorders, such as ID and schizophrenia.Supplemental Material


N-Methyl-D-aspartate (NMDA) subtype of glutamate receptors is expressed in the human lungs and central nervous system.  NMDA receptor potentiation could increase calcium ion influx and promote downstream signaling mechanisms associated with cellular contractions that are disrupted in severe acute respiratory syndrome. Pharmacological effects generated by triggering glutamate receptor function in the brain, coupled with concurrent stimulation of the respiratory tract, may produce a synergetic effect, improving the airway smooth muscle function. A novel multipronged intervention to simultaneously potentiate NMDA receptors expressed both in the central nervous system and airway muscles would be helpful for the treatment of severe acute respiratory syndrome that deteriorates peripheral and central nervous system function before causing death in humans. Functional NMDA receptors are hetero-tetramers composed of two identical glycine binding GluN1 subunits and two identical or different glutamate binding GluN2 subunits, of which there are four subtypes, GluN2A-D.,Glutamate is the major neurotransmitter of the central nervous system and it has diverse roles in the periphery. The N-methyl-D-aspartate (NMDA) receptor is a major subtype of glutamate receptors, which are predominantly expressed throughout the nervous system and in all vital organs in the human body. This finding corroborates a large number of previous reports on the extraneuronal expression of NMDA receptors in various animals.,– NMDA, when applied to perfused tracheal segments of guinea pigs, increased resting muscle tone and enhanced the contractile response to acetylcholine., In whole guinea pig lungs, when administered through the trachea, NMDA increased airway perfusion pressure and this increase was abolished by NMDA receptor channel blocker MK-801(4). Following systemic MK-801 administration, adult cats developed apneusis., In addition, recent studies reveal the critical role of endogenous glutamate in NMDA receptor function during acute lung injury and airway inflammation.,,, An NMDA receptor blocker could impair fetal rat lung development., NMDA receptor activation mediates lung fibroblast proliferation and differentiation in hyperoxia-induced chronic lung disease in newborn rats. Acute lung injury, acute respiratory distress syndrome and severe acute respiratory syndrome (SARS) all imply the occurrence of lung injury resulting from direct or indirect respiratory insult.The Human Proteome Project identified abundant expression of NMDA receptor subunits in various organs outside the CNS, including lungs, esophagus, and T-helper cells. The GluN1/2D subunit was predominantly expressed in the peripheral, gas-exchange zone of the lungs and in alveolar macrophages; this expression was upregulated in lungs treated with NMDA. GluN1 and all four GluN2 subunits were also expressed in the human pulmonary artery smooth muscle cells. Overall, these findings indicate that NMDA receptors could control the respiratory tract function in vertebrate animals.The expression of GluN1/2A and 1/2B subtypes were not confirmed in the lung cells, whereas the GluN1/2C subtype was found to be expressed in peripheral and middle-lobe lung samples.2+ ions. NMDA receptor mediated transport of calcium and sodium ions into the cytoplasm is essential for excitatory cellular events that result in human airway smooth muscle contraction. Each non-GluN1 subunit confers distinct spatiotemporal expression and biophysical properties that result in varying agonist affinity, magnesium sensitivity, ion conductance, activation kinetics, open probability, mean open time, cellular localization, and downstream signaling mechanisms. In general, diheteromeric NMDA receptors (GluN1/2) exhibit deactivation time constants that span about a 50-fold range, with the following order (from fastest to slowest): NR2A < 2C < 2B << 2D. The GluN1/2A subunit-containing NMDA receptor deactivation time constant is about ~50 ms, GluN1/2B ~400 ms, GluN1/2C ~290 ms and GluN1/2D is >1second. Since GluN1/2C&D subunits of NMDA receptors are predominantly expressed in the lung epithelial cells and macrophages, and are the slowest channel (among other glutamate receptors) to deactivate, these receptors can conduct a large amount of calcium and sodium ions into the cells and trigger cellular contractions.–Glutamate, with concurrent binding of the co-agonist D-serine or glycine, activates NMDA receptors that non-selectively conduct ions across the cells at depolarizing membrane potential which unbinds the otherwise blocking Mg This drug is also an FDA-approved drug of choice  for the treatment of dyskinesia in patients with Parkinson’s disease. An analog of amantadine, memantine , is one of two FDA-approved clinically used drugs for the treatment of moderate to severe symptoms of Alzheimer’s disease. Since both amantadine and memantine are chemically similar adamantane derivatives, memantine also exerts antiviral effects as previously reported. Presumably, these effects could be a collective outcome of activities on host cell glutamate receptors and viral proteins like M2-viroporin.One of the clinically used antiviral agents, amantadine, is a potent NMDA receptor antagonist.– These compounds have been largely studied for their activities in neuronal NMDA receptor populations, with the aim of developing treatments for neurological and psychiatric disorders; however, these compounds and their analogs might have therapeutic potential for non-CNS disorders, but this has not yet been explored.In recent years, a variety of NMDA receptor modulators have been identified, and they exhibit a broad spectrum of subunit selectivity and mechanisms of action.3794169), coded as CNS4, and studied its activity on NMDA receptors. CNS4 selectively potentiates GluN1/2D receptor currents up to 8-fold, when activated by 100 μM glycine and 0.3μM glutamate, and produces minimal effects on GluN1/2A or 1/2B receptors. CNS4 has a variety of other biological activities as reported by the National Center for Advancing Translational Sciences (NCATS); for example: an inconclusive anti-viral activity against influenza-A virus non-structural protein-1 (PubChem AID# 2326); anti-malarial, as an inhibitor of apical membrane antigen-1 ofPlasmodium falciparum (AID# 720542); antiprotozoal, as an inhibitor of fructose 1,6- bisphosphate aldolase fromGiardia Lamblia (AID#lamblia (2451); and inhibition of nuclear receptor ROR-gamma in the immune cells (AID# 2551 & 2546). The chemical structure of CNS4 and more details on its activities are available atPubChem.Through our ongoing NMDA receptor drug discovery project, we have identified a compound from PubChem  function with anti-viral properties could be beneficial for treating disorders that involve NMDAR dysfunctions. The author first summarized known facts for distinct NMDAR subunits expressed in lung cells . The author then introduced amantadine (a potent NMDAR antagonist) and its analog (memantine) that have been clinically used for treatment of dyskinesia patients. The author also detailed a novel NMDAR modulator  that has a possibly therapeutic potential for non-CNS disorders and SARS. I believe that the author has done a wonderful job in concisely providing a logical opinion: a newly developed NMDAR modulator could be clinically promising for treating various non-neurological disorders that are linked to NMDAR dysfunctions.Is the topic of the opinion article discussed accurately in the context of the current literature?YesAre arguments sufficiently supported by evidence from the published literature?YesAre all factual statements correct and adequately supported by citations?YesAre the conclusions drawn balanced and justified on the basis of the presented arguments?YesReviewer Expertise:Synaptic neuroscience.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In severe acute respiratory distress syndrome, cellular contractions may be disrupted.  By triggering glutamate receptor function in the brain and with stimulation of the respiratory tract, a synergetic effect may improve airways smooth muscle function and neurologic function. Stimulation of NMDA receptors in the brain and in the airway muscles could be beneficial in the treatment of severe respiratory distress syndrome and associated neurologic disease. The authors excellently outline how an NMDA receptor modulator with antiviral properties could be a novel treatment strategy for SARS. This avenue of treatment has the potential to prevent or treat the neurological dysfunction often associated with SARS. At the same time, NMDA receptor modulation may improve pulmonary status by improving smooth muscle airways function, often disrupted in this syndrome. The multifaceted potential benefits of stimulating glutamate receptors could involve both the lungs and the nervous system. The authors have clearly and concisely outlined the rationale and hypothesis for the use of NMDA modulators in the treatments of SARS and associated neurologic dysfunction.Is the topic of the opinion article discussed accurately in the context of the current literature?YesAre arguments sufficiently supported by evidence from the published literature?YesAre all factual statements correct and adequately supported by citations?YesAre the conclusions drawn balanced and justified on the basis of the presented arguments?YesReviewer Expertise:PulmonologistI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


GAD1 gene using cerebral organoids generated from induced pluripotent stem cells (iPSCs) from adults with a diagnosis of ASD and from age- and gender-matched healthy individuals. Both groups showed high levels of methylation across the majority of CpG sites within the profiled GAD1 region of interest. The ASD group exhibited a higher number of unique DNA methylation patterns compared to controls and an increased CpG-wise variance. We detected six differentially methylated CpG sites in ASD, three of which reside within a methylation-dependent transcription factor binding site. In ASD, GAD1 is subject to differential methylation patterns that may not only influence its expression, but may also indicate variable epigenetic regulation among cells.DNA methylation profiling has become a promising approach towards identifying biomarkers of neuropsychiatric disorders including autism spectrum disorder (ASD). Epigenetic markers capture genetic risk factors and diverse exogenous and endogenous factors, including environmental risk factors and complex disease pathologies. We analysed the differential methylation profile of a regulatory region of the  The methylation of DNA at distinct nucleotides, most commonly cytosines followed by a guanine residue (CpG site), is an essential epigenetic modification that provides a reversible mechanism for regulating gene expression without altering the DNA sequence . During Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterised by challenges in social interaction and communication, as well as repetitive or restrictive behaviours or interests . VariousGAD1, which encodes a glutamic acid decarboxylase enzyme isoform that catalyses GABA synthesis and is an excellent biomarker in the assessment of E/I balance at the molecular level.The balance between excitation (E) and inhibition (I) is a key principle for neuronal network organisation and information processing ,14,15. CGAD1 DNA methylation patterns are disturbed during development [GAD1 methylation pattern during embryonic development may differ in ASD, we generated cerebral organoids from iPSCs derived from individuals with and without ASD, and performed targeted DNA methylation profiling of a known regulatory target region of the GAD1 gene [It is clear that there is evidence for epigenetic dysregulation being implicated in the imbalance of E/I in ASD ,21, and elopment ,23. To iAD1 gene .GAD1 gene approximately 3400 bp downstream of the transcription start site (TSS)   by bisulgulation . The cergulation .We observed distinct methylation profiles within the control samples, where the majority (94.3%) of the randomly picked clones had high methylation levels throughout the amplicon. Clones with <50% methylation across the amplicon were only detected in one out of the five control organoids A. In theStrikingly, the diversity in DNA methylation profiles across the amplicon was increased in the ASD group. To compare this methylation pattern diversity, we analysed the occurrence of distinct patterns in each of the dominant >50% methylation populations. While the control group exhibited only five distinct patterns (2 distinct patterns in organoids from control subjects #1–#4), sixteen patterns were observed from the ASD group . In linep = 0.052, one-sided Wilcoxon rank sum test).For the much smaller group of rarer clones with <50% methylation, sample numbers are too small for a detailed analysis as above. However, the percentage of CpG sites within the amplicon that are methylated is lower in the control group as compared to the ASD group , and an increased overall CpG-wise variability in ASD (p < 0.001 Wilcoxon rank sum). Five out of the six differentially methylated CpG sites are hypomethylated in ASD. Notably, three of these differentially methylated CpG sites lie within a CTCF binding site and the remaining sites are in close proximity (within 100 base-pairs up- and downstream), suggesting that this regulatory region is prone to differential methylation in ASD.To further compare the diversity of DNA methylation signatures, we analysed the methylation variability of each CpG site (for clones with >50% methylation across the amplicon). We detected a total of six CpG sites that are significantly differentially methylated in ASD relative to the control group  enzymes, is significantly increased in the brain [GAD1 in a region upstream of our target region [Methylation signatures vary not only between individuals, but also in a tissue- and cell-type dependent manner . We, thehe brain . Moreovet region . DNMT ist region ,47. TherCGCGCGGGGGCGCTTT) contains four CpG sites and is known to be sensitive to DNA methylation [GAD1 transcription and mRNA expression [GAD1 transcription is decreased in ASD organoids. This would, therefore, corroborate the decreased expression of GAD1 and GAD-67 that is seen in ASD patients [Within our region of interest resides a binding motif for CTCF, a transcriptional factor protein able to regulate transcription through its interactions with enhancers and promoters. The binding sequence present in our region of interest  according to the protocol previously described by Ilieva et al. and Kamand et al. ,30,31, aThe cerebral organoids were generated according to the protocol described by Lancaster and Knoblich  and modiGAD1 amplicon was 560 bp. The following PCR conditions were used: 95 °C for 5 min; 40 cycles of 94 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min; 72 °C for 30 min.The genomic DNA was bisulfite converted using EpiTect Bisulfite Kit (Qiagen), then amplified using HotStar Taq Plus Master Mix (Qiagen). The target region and primer sequences are shown in PCR products were subcloned using a TOPO™ TA Cloning™ Kit with One Shot™ TOP10 chemically competent E. coli cells (Invitrogen). Up to 20 colonies per organoid were picked after blue-white selection. The DNA was purified using QIAprep Spin Miniprep Kit (Qiagen) and used for the Sanger sequencing (Genewiz UK).http://quma.cdb.riken.jp/ (accessed 30 April 2022) [http://gattaca.imppc.org/methylation_plotter/ (accessed 30 April 2022) [The trace files were analysed using the QUMA online tool, available at il 2022) . The lolil 2022) . The difsignrank. For the statistical test on the amplicon-wide methylation differences (for <50% methylated clones), we calculated the percentage of methylated CpG sites across the amplicon and performed a one-sided Wilcoxon rank sum test between the control and ASD conditions using the MATLAB command ranksum.The binary clone sequence data from different organoids of the same condition were pooled for the statistical analysis. The statistical analysis was performed using MATLAB with Statistics and Machine Learning Toolbox. For the CpG-wise differential methylation, Wilcoxon signed rank test was performed for each CpG site between the control vs. ASD conditions using the MATLAB command