Thus, epidermal sensing ofH2O2 should be useful to monitorthe progression of skin pathologies. We have evaluated epidermal sensing ofH2O2 in vitro, by visualisingH2O2 permeation through the skin.Skin membranes were mounted in Franz cells, and a suspension of Prussian whitemicroparticles was deposited on the stratum corneum face of the skin. UponH2O2 permeation, Prussian whitewas oxidised to Prussian blue, resulting in a pattern of blue dots. Comparison ofskin surface images with the dot patterns revealed that about 74% of the blue dotswere associated with hair shafts. The degree of the Prussian white to Prussian blueconversion strongly correlated with the reciprocal resistance of the skin membranes.Together, the results demonstrate that hair follicles are the major pathways ofH2O2 transdermal penetration. Thestudy recommends that the development ofH2O2 monitoring on skin should aimfor pathway-specific epidermal sensing, allowing micrometre resolution to detect andquantify this ROS biomarker at hair follicles.Elevated amounts of reactive oxygen species (ROS) including hydrogenperoxide (HGraphical abstractThe online version contains supplementary material available at 10.1007/s00604-020-04633-9. Robust, non-invasive, or minimally invasive sensing on skin, oftenreferred to as epidermal sensing, is one of the tools required to realisepersonalised healthcare, at point-of-care units or home self-monitoring. Epidermalsensing is carried out using epidermal electronics –3, microEpidermal non-invasive sensing that does not relay on sweating or othertypes of skin interrogation is less developed . In suchCurrent technologies employed to make epidermal electronics and sensorsallow manufacturing of nano- and micrometre-sized sensors, which could targetspecific transdermal biomarker permeability pathways, e.g. the hair follicularpathway , 18. One2O2, across the SC has aspecific dominant pathway. Since H2O2is a biomarker of many inflammatory disorders, this discovery might be important fordevelopment of epidermal sensing of several common skin disorders. Specifically,increased epidermal H2O2 levels arereported for vitiligo ), 30% HCl,FeCl3, soluble Prussian blue nanoparticles, ascorbicacid, and chloroform were obtained from Sigma-Aldrich . KCl,NaCl, Na2HPO4, andKH2PO4 were purchased fromMerck . H2O235% w/w was obtained from Alfa Aesar , and ferricyanide(K3Fe(CN)6) from AppliChem,PanReac . Phosphate buffer saline (PBS) was prepared usinghigh-purity, 18.2 MΩ cm resistivity, Milli-Q water. PBS (pH 7.4) comprised130.9 mM NaCl, 5.1 mM Na2HPO4, and1.5 mM KH2PO4.Sodium azide, potassium ferrocyanide, and circular membranes (1.6 cm diameter) werepunched out from the stripes. The video of skin membrane preparation issubmitted as electronic supporting information. For preparing full-thicknessskin membranes, a scalpel was used to remove all tissue attached to the earcartilage (resulting in 3–4-mm-thick skin membranes).2O2 permeation mechanism,lipid-extracted skin membranes were prepared and used forH2O2 permeation studies,similarly to studies of intact skin. Lipid extraction from skin membranes wasperformed following a protocol published elsewhere solution in water was degassed by mixing and applying vacuum for 20 min. Thesolution was then transferred to a Teflon-lined stainless steel autoclave and was additionally purged withnitrogen to remove dissolved oxygen. The autoclave was sealed and maintained at160 °C for 48 h; when room temperature was reached, the supernatant wasdiscarded and the white particulate precipitate was collected. The particleswere washed several times with 0.1 M KCl in distilled water and acetone, andfinally dried under vacuum for 12 h. Commercially available PB nanoparticleswere reduced to PW by washing them with saturated ascorbic acid solution in0.1 M KCl.Air-stable , equipped with a LaB6filament. Briefly, different dilutions of the particles were drop-casted on Leitcarbon tapes (Agar Scientific) and left to dry. Prior to SEM measurements, thesamples were covered with gold, layer thickness < 10 nm, using an Auto AgarSputter Coater : parameters I = 30 mA, t = 40 s,and SEM images of PW particles Fig. a were ann = 4, measurementsusing different dilutions and intensity weighting). To be able to comparecommercially available PB particles with the synthesised PW particles, the PBsize distribution determined by DLS was number-weighted and expressed in termsof number percent. The size distribution of PB particles was fitted to theLogNormal distribution model.The size of commercially available PB particles was measured bydynamic light scattering . The samples were prepared in deionised water. The average particle sizeequalled 164.7 ± 4.5 nm (3 (20 mM for full-thickness skin). Thetop chamber was filled with an identical solution (0.6 mL). The dermal skin sidefaced the lower chamber. Skins were left to equilibrate for 30 min in the Franzcell, and the electrical impedance was measured as described above. Afterequilibration, the PBS solution in the top chamber (stratum corneum plane of theskin) was removed and the skin surface structure was imaged using a digital USBmicroscope . Upon emptying of the top chamber, the Franz cellposition remained fixed throughout the experiment. Two milligrammes of PWsuspended in 200 μL of 0.1 M KCl was spread on the skin surface and a definedamount of H2O2 was pipetted intothe lower chamber of the Franz cell. The resulting solution (4 mMH2O2) in the lower chamber wascontinuously agitated using a magnetic stirrer. Directly after theH2O2 introduction, the skinsurface with deposited PW particles was imaged and the photo was assigned as theskin/PW image at time 0 (0 min). The images were then recorded at regular timepoints. To overcome the disturbance from external light, an illumination ring was mounted on the microscope and theentire Franz cell–microscope set-up was shielded from external light by acardboard box.PW particles were deposited on the surface of the membraneScheme . Dermato2O2 concentration (0.5 mM)were supplemented by an external peristaltic pump  connected tothe lower chamber of the Franz cell. This allowed refreshing the PBSbuffer/0.5 mM H2O2 each hour, toavoid possible degradation ofH2O2. All measurements wereconducted at room temperature, 22 °C.Penetration experiments with a lowerHThe ImageJ software  incombination with an adapted script  wasused for image processing. Briefly, skin surface images were cropped andconverted to cyan, magenta, yellow, and black (CMYK) images. Then, cyan imageswere compared with an original photographic image; the B&W threshold wasadjusted for each image (the threshold for different images variedslightly).2O2 on theskin surface using the Prussian white–Prussian blue redox couple 3)to PB (FeIII4[FeII(CN)6]3),and consequently the PW crystal colour changes from white to blue [2O2 penetrationthrough skin membranes. This in vitro approach can be considered as a good modelfor assessing epidermal sensing ofH2O2; we consider the approachas being a necessary step before testing the sensing in vivo using livinganimals or humans.Htion Eq. , is an o to blue . Hence, −2).H2O2 permeability through skinresulted in PW turning blue. The colour change was visualised by a USBmicroscope. The results revealed that PW microparticles could be adopted as asensing material for monitoringH2O2 penetration through skin.The observed H2O2 permeability wasrationalised by considering transdermal penetration pathway theory [The experimental set-up comprised a skin membrane placed in a Franzcell Scheme . The skiy theory .2O2 detoxifying enzyme,converts H2O2 into water andoxygen [2O2 concentrations in theepidermis. Working with skin in vitro and aiming to monitorH2O2 presence on the skinsurface, or to monitor H2O2transdermal penetration, the skin catalase is an obvious problem since itconverts H2O2 toH2O and O2. Therefore, toassure H2O2 penetration, skincatalase in skin membranes has been inhibited. The inhibition process wasconfirmed by using skin membranes firmly attached to oxygen electrodes. Theset-up is known as skin-covered oxygen electrode (SCOE), which measuresO2 liberated by skin catalase when the electrode isexposed to a solution of H2O2[2O2 in theabsence and in the presence of a catalase inhibitor, specifically sodium azide,NaN3. Amperometric measurements with the SCOE didshow that 14 mM of NaN3 is sufficient to inhibit morethan 90% of skin catalase. Sufficient NaN3 amounts werethus always present in solutions of the Franz cell experiments.In healthy skin, catalase, the mainHdoxygen . In in vdoxygen , xeroderdoxygen  etc.), c of H2O2, 28, 37.2O2monitoring in clinical situations might be equally time demanding. Hence, it isan absolute requirement that PW particles are stable against oxidation byambient O2 and O2 dissolved insolution. PW oxidation by O2 is rarely reported, butdepending on the particle structure, O2 can oxidise PW toPB in a few minutes or months. Various synthesis trials showed (data not shown)that micrometre-sized PW particles are more O2-stable.Specifically, we found that PW particles with an average size of 4.4 μm ± 1.2 μm(mean ± SD)   Fig.  areO2-sSD) Fig. , were un2 can be supported by the results of Hu andco-workers (2011), who performed Mössbauer spectroscopy measurements on the PWparticles synthesised by the same synthesis protocol that was used in our study.In their work, Mössbauer spectra of PW particles were unchanged after exposureto air for 2 months [II, indicating that PW ofthis size is stable against oxidation by O2 inair.These visual observations of PW particle stability towardsO2 months . ThespeThe size stability of our PW particles over time was assessed usinglight microscopy. A few PW particles were re-suspended in KCl solution and wereimaged every day for 5 days. With no apparent change in shape and size of theparticles, it was concluded that the PW particles are stable for at least 5 daysin KCl solution Fig. . In addi2O2 penetration was done byperforming Franz cell permeation assays; vide supra, Scheme −2 PW coverage. The skin membrane wasperiodically photographed, which allowed monitoring of PW oxidation to PB due tothe transdermal permeation of H2O2from the dermal side of skin membrane onto its SC surface . Control experiments with no addition ofH2O2 into the Franz cellsshowed no PW oxidation to PB over 24 h.The analysis of the images Fig.  was carrAs can be judged from longer measurements  values; an example of impedance data is shownin Fig. 2, was 12.6 kΩ ± 7.3 kΩ  on average, while lipid-extractedskins had an average resistance value of 0.20 kΩ ± 0.02 kΩ . As can be seen from Fig. mem = 6.4 kΩ,3.4% for Rmem = 8.1 kΩ, 2.4% forRmem = 9.5 kΩ, 1.2% forRmem = 14.9 kΩ, and 0.04% forRmem = 24.3 kΩ, respectively. The observed correlationbetween H2O2 permeation (i.e. bluecolour development) across the skin membrane and reciprocal of skin resistanceindicates that H2O2 penetrates theskin through the same pathway as hydrated ions. Additionally, the blue colourdevelopment kinetics on non-treated skin membranes, e.g. median resistancemembranes , was attenuated compared to lipidextracted membranes . On lipid-extracted skin, more than90% of the image did turn blue in less than 30 min  of the experiments  images  of the blue dots are associated with hair shafts  of all hairshafts were associated with blue colour development. The notable variation andrelatively low fraction of hair follicles associated with facileH2O2 penetration (55%) mightbe due to structure and growth cycle differences of a particular hair. It isknown that approximately 26% of the hair follicles present in human forearm skinare inactive; i.e. they do not excrete sebum and are filled with a corneocyteplug which restricts penetration of both hydrophilic and hydrophobic molecules[After membrane lipid extraction, the dot features of the bluecolour were not observed Fig. ;rather,ns Table , only55olecules. The stu2O2 penetration pathways,additional experiments were carried out. First, epidermal monitoring of thepermeation from a lower, more physiologically relevant concentration (0.5 mM) ofH2O2 was evaluated indicate slower PW particle oxidation,compared to measurements on skin with similar resistance but with eight timeshigher H2O2 concentration. When the skin membrane was exposed to0.5 mM H2O2, after 8 h only 4.6%of the area was coloured blue  tocross the barrier maintained by the SC. Despite the fact that many researcharticles [2 [2; the PWmicroparticles stay white (i.e. white-grey) for more than 2 months when kept ina closed Eppendorf tube. Based on experience with different synthetic routes, itwas concluded that smaller particles (< 0.2 μm) are highly prone to oxidationby O2 in air or solution, and are thus not suitable foroptical epidermal sensing of H2O2.Thus, micrometre-sized PW particles were used to develop epidermalH2O2 sensing and to visualisepathways of H2O2 penetrationthrough skin.Another observation made during our studies was that the PW/PBredox pair has a considerable sensitivity to Oarticles , 45 and articles , 45 focuicles [2 ,46. Theicles [2  yielded 2O2 penetration carried outin this work relied on (i) skin membranes mounted in Franz cells; (ii)deposition of a H2O2-sensitive,but O2-insensitive PW microparticle layer on the SC faceof skin membranes; (iii) recording the blue colour development of the dispersedPW microparticles, due to reaction with permeatedH2O2; and (iv) complementingthe colour-based assay with skin integrity measurements using electricalimpedance spectroscopy. Repeated epidermal monitoring ofH2O2 with different skinsamples revealed that it takes 3 to 10 h until about 4% of the skin area iscovered with blue colour vs. an area with low density of hair follicles (6hairs/cm2). They found that the follicle-denseskin allowed almost nine times higher flux of glucose if compared with the fluxacross the follicle-poor area of the porcine skin [In a simplified view of permeation across a skin barrier, at leasttwo separate routes of penetration can be distinguished. First, skin appendagescomprised sweat ducts and hair follicles, and, second, intercellular andtranscellular penetration pathways –49. The ine skin .2O2 penetration through skingenerates a blue colour dot-like pattern in theH2O2-sensitive PW particlelayer deposited on the surface of skin membranes  appeared at skin sections withoutvisible hair, which might be due to invisible smaller hairs or other appendages(e.g. sweat ducts). Even given this uncertainly, our results show thatH2O2 penetration through skinis dominated by the hair follicle pathway. This strongly favours the argumentthat permeation of hydrophilic molecules via the hair follicle pathway issubstantial and should be targeted by epidermal sensing [Our results show thatHes Figs. , and5. yer Fig., with th sensing , 52–54.2O2 preferentially penetratesskin through hair follicles, the permeability experiments were performed usingfull-thickness skin (4 mm thick). The same blue dot pattern in theH2O2-sensitive PWmicroparticle layer developed  skinmembranes.To reinforce the statement thatHped Fig. .This co2O2 penetration assays. Insuch a case, H2O2 penetrating theskin initially will meet PW particles lodged inside the hair follicle,explaining the observed blue dot pattern. As can be seen in Fig. 2O2 seems tocross the skin evenly/homogenously. Additional experiments have also been doneby first depositing a filter paper on the skin membrane and then pipetting asuspension of PW particles on the top of the filter. Similar dot-like patterns(data not shown) of PB appeared even for thisH2O2-sensing layerconfiguration. Altogether, the results strongly suggest thatH2O2 has a strong preferenceto penetrate through the appendages of skin, particularly through hairfollicles. This knowledge is important in the development of clinically robustepidermal sensing of H2O2 withmicrometre resolution. Our results show that micrometre resolution would allowfaster biomarker detection: probably in 1–2 h as judged from the appearance ofthe first blue dots in Figs. Additionally, micrometre-sized particles (< 10 μm) can enter thefollicular orifices ; hence,2O2 penetration through skinmembranes in order to model and rationalise strategies for the development ofclinically relevant, epidermal sensing of ROS biomarkers, specificallyH2O2. We conducted epidermalmonitoring of H2O2 penetration throughskin by using H2O2-sensitive Prussianwhite microparticles. The PW particle layer was deposited on the surface of skinmembranes and after some time the PW particles turned blue, signallingH2O2 permeation through the skin.We found that PW particles with an average size of 4.4 μm ± 1.2 μm (mean ± SD) weresufficiently O2-insensitive to allow prolonged (at least upto 24 h) epidermal monitoring of H2O2.The monitoring of H2O2 penetration wasonly possible after inhibition of skin catalase. Unfortunately, in most clinicalcases, skin catalase inhibition will not be allowed; however, diseased skin isusually characterised by downregulation of catalase. Thus, epidermal sensing ofH2O2 in clinically relevantsituations might still be possible with a PW microparticle layer on the skin.Additionally, we found that the penetration ofH2O2 through skin generates a bluedot pattern in the PW layer. About 74% ± 7% of the dots were associated with visiblehair shafts. This observation, for the first time, firmly establishes that hairfollicles are the dominant H2O2penetration pathways in skin. In general, the results suggest that targeting hairfollicles with micrometre resolution should provide more sensitive and rapidmonitoring of low molecular weight, hydrophilic biomarkers of skin disorders. Thisis especially important to realise for development of epidermal sensing ofH2O2.H2O2 is one of the common ROS,elevated in many inflammatory and autoimmune skin disorders, and its epidermalmonitoring would be extremely valuable in managing these pathologicalconditions.This study aimed to visualiseHESM 1(PDF 1238 kb)ESM 2


Resting membrane potential determines the excitability of the cell and is essential for the cellular electrical activities. The NALCN channel mediates sodium leak currents, which positively adjust resting membrane potential towards depolarization. The NALCN channel is involved in several neurological processes and has been implicated in a spectrum of neurodevelopmental diseases. Here, we report the cryo-EM structure of rat NALCN and mouse FAM155A complex to 2.7 Å resolution. The structure reveals detailed interactions between NALCN and the extracellular cysteine-rich domain of FAM155A. We find that the non-canonical architecture of NALCN selectivity filter dictates its sodium selectivity and calcium block, and that the asymmetric arrangement of two functional voltage sensors confers the modulation by membrane potential. Moreover, mutations associated with human diseases map to the domain-domain interfaces or the pore domain of NALCN, intuitively suggesting their pathological mechanisms. The NALCN channel mediates sodium leak currents, which in turn adjusts resting membrane potential and neuronal excitability. Here the authors describe a cryo-EM structure of mammalian NALCN-FAM155A channel complex, showing how selectivity filter contributes to sodium permeation and calcium block and how the voltage sensors contribute to current modulation. It mediates sodium leak currents that are essential for setting the membrane excitability2. NALCN channel is involved in several crucial physiological processes including maintaining normal circadian rhythms3 and respiratory rhythm4. NALCN channel functions as a multi-protein complex, which consists of NALCN, FAM155, UNC79, UNC80, and other proteins5. Mutations in the genes of human NALCN or UNC80 lead to a spectrum of neurological diseases6, including infantile hypotonia, psychomotor retardation, and characteristic facies (IHPRF)8 and congenital contractures of the limbs and face, hypotonia, and developmental delay (CLIFAHDD)9. In addition, a gain-of-function mutation of NALCN in the mouse causes the dreamless phenotype, suggesting the function of NALCN in rapid eye movement sleep10.NALCN channel is a voltage-modulated sodium-selective ion channelV or calcium channels CaV. Four consecutive voltage sensor-pore modules are fused into a single polypeptide chain. However, unique to the NALCN channel, the positive charges on voltage sensors of NALCN are much fewer than that of NaV or CaV. Moreover, residues in the selectivity filter of NALCN are “EEKE” instead of “DEKA” of NaV or “EEEE” of CaV. These features render NALCN to be a special clade in the voltage-gated channel superfamily11. In addition to the NALCN subunit, FAM155, UNC79, and UNC80 subunits are indispensable for the function of NALCN channel. The co-expression of NALCN, FAM155, UNC79, and UNC80 proteins in a heterologous expression system, such as Xenopus laevis oocytes or HEK293 cells, is necessary and sufficient to generate robust voltage-modulated sodium-selective currents1. FAM155 is a transmembrane protein family with a cysteine-rich domain (CRD). There are two FAM155 family members in human, namely, FAM155A and FAM155B, both of which could support the currents of the NALCN complex1. FAM155 is the homolog of NLF-1 in C. elegans, which is required for the membrane localization of NALCN12. It is proposed that the interaction between NALCN and FAM155 is similar to the interaction between yeast calcium channel Cch1 and its regulator Mid114. The other two proteins in the complex, UNC79 and UNC80, are large conserved proteins without any known domains but they are essential for the function of NALCN in vivo17. During the preparation of this manuscript, the structure of human NALCN–FAM155A at 2.8 Å in nanodiscs was published18 and another group reported a similar structure at 3.1 Å in detergent on preprint sever19. Here, we describe the structure of rat NALCN and mouse FAM155A in detergent to the resolution of 2.7 Å, which provides enhanced details and additional structural insights into the mechanism of this important channel complex.Sequence analysis shows NALCN protein shares a similar domain arrangement to the pore subunit of eukaryotic voltage-gated sodium channels Na1 and FAM155 are required for correct trafficking of NALCN12, we reasoned the structure of NALCN–FAM155A complex would provide clues of how NALCN channel works. Therefore, we started with the cryo-EM studies of the NALCN–FAM155 core complex. We prepared the NALCN–FAM155A complex in a buffer with sodium chloride and GDN detergent, mimicking the symmetric sodium solution used in electrophysiology recording 18  which is folded by three consecutive α helices and the connecting loops in between. The CRD of FAM155A is highly cross-linked by disulfide bonds owing to the high content of cysteines in this domain. Most of the Cys residue pairs are conserved from NLF-1 of CRD Fig. .Fig. 2StI, L5III, L5IV, and L6IV) in NALCN form a platform for FAM155A binding , E554 (domain II), and K1115 (domain III) point to the center of the pore  but different from CaV   Fig. , providiPCs Fig. , suggest30, but its function in NALCN remains unclear. The constriction close to the cytoplasmic side is at the level of bundle crossings which represents the putative intracellular gate of NALCN. The gate is tightly sealed by several hydrophobic residues on S6 helices  and followed by a positively charged residue, forming the E1-X2-F3-E4-R5 structural motif. Together with a negatively charged residue on S3 helix, F3 and E4 on S2 form the so-called charge-transfer center (CTC) at the bottom of VSV1.4 in depolarizing state (PDB ID: 6AGF) is at a similar level to R3 (R225), suggesting S4 of NALCN DI-VS is even “upper” than NaV1.4  has a consensus S2 helix. On the S4 helix, positively charged residues of R2, R3, and R5 are conserved, while the middle R4 is replaced by Ile (I149) Fig. . We obse1.DII-VS has an intact CTC and three consecutive gating charges on S4 (R3-R5) and the Cα of F3 (F429) is at a similar level to Cα of R3 (R481), which is similar to DII-VS of Nav1.4 (PDB ID: 6AGF) Fig. . These o1. We observed S4 residues above R3 (R989) adopt α helical structure while residues below R3 form a 310 helix. R3 interacts with E906 on S1–S2 linker and E1 (E922). R4 interacts with E1 (E922) and F925. R5 is stabilized by interactions with E4 and D958 on S3. The Cα of F3 (M929) is at the same level as Cα of R5 (R995), which is similar to Nav1.4 (PDB ID: 6AGF) , it has a defective CTC on S2 and the essential aromatic residue at F3 is replaced by M929 Fig. . This isGF) Fig. , suggest1. Notably, it is reported the W1287L mutation on S3 of DIV-VS can leads to the loss of currents and IHRRF34  and R6 (K1317) on S4. Moreover, the majority of S4 adopts an α helical structure on which the spatial arrangement of gating charges is not optimal for movements upon membrane potential changes, indicating DIV-VS is defective. In agreement with this, mutation of R1310Q has no effect on the voltage sensitivity of NALCNF34 Fig. , probablIII helix is connected to the well-ordered III–IV linker via two sharp turns at G1164 and L1168. These structures reroute the intracellular exit for sodium towards the direction of domain III 35 and NaVPaS (PDB ID: 5X0M)36 channels but distinct from NaV1.2 (PDB ID: 6J8E)37, NaV1.4 (PDB ID: 6AGF)38, NaV1.5 (PDB ID: 6UZ3)39, and NaV1.7 (PDB ID: 6J8G)40 35 and NaVPaS (PDB ID: 5X0M)36, except the C-terminal C5 helix orients differently 18, probably due to the poor local map quality.The III–IV helix, together with the intracellular surface of NALCN forms a platform for the binding of CTD. The resolved portion of NALCN CTD consists of five helixes (C1-C5) and forms a calmodulin-like domain that packs below the III–IV linker Fig. . The NALThe high-resolution structure of the NALCN–FAM155A complex presented here shows the architecture of the NALCN channel core components, reveals the structure of FAM155 CRD, and depicts the detailed inter-molecular interface between NALCN and FAM155A subunits. More importantly, the non-canonical configuration of selectivity filter suggests the mechanism of sodium permeation and calcium block. Furthermore, the asymmetric spatial arrangement of both functional and degenerate voltage sensors provides mechanistic clues for the voltage sensitivity of NALCN. In addition, our structure also provides a template for drug discovery targeting the NALCN channel in related human diseases.2 and 70% humidity. HEK293T (ATCC) cells were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% FBS and 1% penicillin-streptomycin (ThermoFisher Scientific) at 37 °C with 5% CO2. The cell lines were routinely checked to be negative for mycoplasma contamination.Sf9 insect cells (ThermoFisher Scientific) were cultured in SIM SF  at 27 °C. HEK293F suspension cells (ThermoFisher Scientific) were cultured in Freestyle 293 medium (ThermoFisher Scientific) or SMM 293T-I medium  supplemented with 1% FBS at 37 °C with 6% CO41) at a ratio of 2:1:1:1 using Lipofectamine 3000 (ThermoFisher Scientific) and incubated for 24 h before recording. Patch electrodes were pulled with a horizontal microelectrode puller  to a resistance of 2–3 MΩ. Whole-cell patch clamps were performed using an Axon-patch 200B amplifier , and data were collected with pClamp 10 software  and an Axon Digidata 1550B digitizer . Pipette solution containing (mM): 10 HEPES , 136 NaCl, 10 NaF, 5 EGTA, 2 Na2ATP and bath solution containing (mM): 10 HEPES , 150 NaCl, 30 glucose. In some experiments, 10 µM GdCl3 (Sigma) was added to the bath solution to block the inward current, as indicated. For ion selectivity experiments, the bath solution was exchanged to 10 mM HEPES , 150 mM XCl , or NMDG and 30 mM glucose by local perfusion equipment . The membrane potential was increased from holding potential (0 mV) to +80 mV for 0.5 s to activate the channel and then stepped to various testing potentials (−80 to +80 mV). The peak of tail currents was used to generate I–V curves for the determination of reversal potentials (Erev). When Na+ in the bath solution, the measured Erev is close to the theoretical Erev (0 mV). To improve the accuracy of our measurements, patches with loose seals (<−100 pA at −80 mV when exposing to NMDG+ bath solution) or small currents (<1.5 nA at +80 mV when exposing to Na+ bath solution) were discarded. The liquid junction potentials were calculated using pClamp 10  and corrected to calculate the reversal potentials. Signals were acquired at 5 kHz and low-pass filtered at 1 kHz. The ion permeability ratios were calculated with the following equation:Erev, F, R, and T are the reversal potential, Faraday constant, gas constant, and absolute temperature, respectively. The data were processed with Microsoft Excel and GraphPad Prism 6.Constructs containing desired point mutations of rNALCN were generated by Quick Change. HEK293T cells were co-transfected with rNALCN-CGFP, mFAM155A-FLAG, mScarlet-mUNC80, and HA-mUNC79 plasmids  for 10 min, and washed with 50 mM Tris (pH 7.5), 150 mM NaCl, 2 μg/ml aprotinin, 2 μg/ml pepstatin, 2 μg/ml leupeptin, flash-frozen, and storage at −80 °C.Mouse FAM155A was cloned from the cDNA that was reverse transcribed from the total mRNA of the mouse brain. The cDNAs of rat NALCN and mouse FAM155A were cloned into a modified BacMam expression vector with C-terminal GFP-strep and FLAG-tag, respectivelyg  for 10 min. The supernatant was ultra-centrifuged at 126,100×g  for 40 min. The solubilized proteins were loaded onto 3 ml Streptactin Beads 4FF (Smart-Lifesciences) column and washed with 15 ml W buffer, which containing 20 mM Tris (pH 7.5), 150 mM NaCl, 2 μg/ml aprotinin, 2 μg/ml pepstatin, 2 μg/ml leupeptin, 10% glycerol and 0.02% GDN. The column was washed with 50 ml W buffer plus 10 mM MgCl2 and 2 mM adenosine triphosphate (ATP) to remove contamination of heat shock proteins. Then the column was washed with 20 ml W buffer again to remove residual MgCl2 and ATP. The target protein was eluted with 12 ml W buffer plus 40 mM Tris (pH 8.0) and 5 mM d-desthiobiotin (IBA). Eluted protein was concentrated using 100-kDa cutoff concentrator (Millipore) and further purified by Superose 6 increase  running in a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 0.02% GDN. Fractions corresponding to monomeric NALCN–FAM155A complex were pooled and concentrated to A280 = 5.For each batch of protein purification, cell pellet corresponding to 0.75 l culture was thawed and extracted with 54 ml lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 2 μg/ml aprotinin, 2 μg/ml pepstatin, 2 μg/ml leupeptin, 10% (v/v) glycerol, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 1% (w/v) glyco-diosgenin  at 4 °C for 80 min. 1 mg/ml iodoacetamide was added during the detergent extraction procedure to reduce non-specific cysteine crosslinking. Then the cell debris was removed by centrifugation at 30,966×n-dodecyl β-d-maltoside , and detected by a fluorescence detector  at room temperature.10 µl of purified proteins were injected onto a Superose 6 increase 5/150 column , running in a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM 2 and 75% Ar. Aliquots of 3 μl concentrated protein sample were applied on glow-discharged grids and the grids were blotted for 2 s before plunged into liquid ethane using Vitrobot Mark IV (ThermoFisher Scientific).Holey carbon grids  were glow-discharged by Solarus advanced plasma system (Gatan) for 120 s using 25% O−/Å2/s, resulting in a total dose of about 50.9 e−/Å2.Cryo-grids were firstly screened on a Talos Arctica electron microscope (ThermoFisher Scientific) operating at 200 kV with a K2 Summit direct electron camera (ThermoFisher Scientific). The screened grids were subsequently transferred to a Titan Krios electron microscope (ThermoFisher Scientific) operating at 300 kV with a K2 Summit direct electron camera and a GIF Quantum energy filter set to a slit width of 20 eV. Images were automatically collected using SerialEM in super-resolution mode at a nominal magnification of ×130,000, corresponding to a calibrated super-resolution pixel size of 0.5225 Å with a preset defocus range from −1.5 to −1.8 μm. Each image was acquired as a 7.12 s movie stack of 32 frames with a dose rate of 7.15 e43. Contrast transfer function (CTF) parameters were estimated with Gctf44. Micrographs with ice or ethane contamination and empty carbon were removed manually. A total of 2,226,376 particles were auto-picked using Gautomatch from 5,428 micrographs. All subsequent classification and reconstruction were performed in Relion 3.145 unless otherwise stated. Two rounds of reference-free 2D classification were performed to remove contaminants, yielding 567,397 particles. The particles were subjected to 57 iterations K = 1 global search 3D classification with an angular sampling step of 7.5° to determine the initial alignment parameters using the initial model generated from cryoSPARC46. For each of the last seven iterations of the global search, a K = 6 multi-reference local angular search 3D classification was performed with an angular sampling step of 3.75° and a search range of 30°. The multi-reference models were generated using reconstruction at the last iteration from global search 3D classification low-pass filtered to 8 , 15, 25, 35, 45, and 55 Å, respectively. The classes that showed obvious secondary structure features were selected and combined. Duplicated particles were removed, yielding 292,877 particles in total. These particles were subjected to additional three rounds of multi-reference local angular search 3D classification using the same parameters. Each round had 30 iterations, and the particles corresponding to good classes from the last 15 iterations of each round were combined, and duplicated particles were removed, yielding 201,654 particles. These particles were subsequently subjected to local search 3D auto-refinement, which resulted in a map with a resolution of 3.0 Å. In order to further clean up the data set, three rounds of random-phase 3D classification47 were performed with K = 2, an angular sampling step of 1.875°, and a local search range of 10°, using two reference models, which are the model obtained from previous refinement and the phase-randomized model. Phase-randomized models were generated from the model obtained from the previous refinement using randomize software (from the lab of Nikolaus Grigorieff) for phase-randomization beyond 40, 30, and 20 Å for the first, second, and third round of 3D classification. For random-phase 3D classification, each round had 30 iterations, and the particles corresponding to good class from the last 15 iterations of each round were combined, and duplicated particles were removed, yielding 135,043 particles, resulting in a 2.9 Å map. CTF refinement was then performed with Relion 3.1, the resolution was improved to 2.8 Å. The particles were then re-extracted, re-centered, and re-boxed from 240 pixels to 320 pixels. After 3D auto-refinement, the resolution reached 2.7 Å.The image processing workflow is illustrated in Supplementary Fig. 45. The local resolution was estimated with Relion 3.145.All of the resolution estimations were based on a Fourier shell correlation (FSC) of 0.143 cutoff after correction of the masking effect. B-factor used for map sharpening was automatically determined by the post-processing procedure in Relion 3.1v1.1-α1 subunit (PDB 5GJV)35. The model was then fitted into the cryo-EM map using Chimera48 and rebuilt manually using Coot49. Model of FAM155A was built manually using Coot49. Model refinement was performed using phenix.real_space_refine in PHENIX29. Images were produced using Pymol and Chimera48.The initial model of NALCN alone was generated by SWISS-MODEL based on the structure of rabbit Cag for 10 min, the supernatants were mixed with Glutathione Sepharose 4B  and incubated at 4 °C for 1 h. Then the beads were washed with 20 mM Tris (pH 7.5) and 150 mM NaCl six times. Bound proteins were eluted with 50 mM Tris (pH 8.0), 150 mM NaCl, and 10 mM reduced glutathione.HEK293F cells cultured in SMM 293T-I medium were transfected with indicated plasmids  membranes. Membranes were blocked using 5% nonfat milk in TBST  for 1 h at room temperature and incubated with primary antibodies  for 2 h at room temperature. Then membranes were incubated with horseradish-peroxidase (HRP) labeled secondary antibody  for 1 h at room temperature and developed using High-Sig ECL Western Blotting Substrate (Tanon).E. coli  with 0.2 mM isopropyl-β-d-thiogalactoside (IPTG) when the cell density reached OD600 = 0.6 for 18 h at 16 °C, and overexpression of GFP and GST was induced with 1 mM IPTG for 4 h at 37 °C. The cells were collected and sonicated in lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 2 μg/ml aprotinin, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 1 mM PMSF. Then the cell debris was removed by centrifugation at 30,966×g  for 30 min. For purification of GST and GST-CTD, the supernatants were loaded onto Glutathione Sepharose 4B and washed with 20 mM Tris (pH 7.5), 150 mM NaCl and eluted with 50 mM Tris (pH 8.0), 50 mM NaCl and 10 mM reduced glutathione. For purification of GFP and CIH-GFP, the supernatants were loaded onto TALON (Clontech) and washed with W1 buffer (20 mM Tris (pH 7.5), 300 mM NaCl) and W2 buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 10 mM imidazole) and eluted with 250 mM imidazole (pH 8.0) and 50 mM NaCl. All proteins were further purified with an anion-exchange column  and the peak fractions were pooled for GST pull-down assay. The purified proteins were mixed (10 µM for each) as indicated (Supplementary Fig. For GST pull-down assay using purified proteins from Supplementary InformationPeer Review File


Electroacupuncture (EA) can improve myocardial ischemia (MI) injury; nevertheless, the mechanism is not entirely clear. And there were disagreements about whether the effect of EA at acupoint in disease-affected meridian is better than EA at acupoint in non-affected meridian and sham acupoint. Here, we showed that the effect of EA at Neiguan (PC6) is better than EA at Hegu (LI4) and sham acupoint in affecting RPP and ECG, increasing ATP and ADO production, decreasing AMP production, and upregulating the mRNA expression levels of A1AR, A2aAR, and A2bAR; knockdown of A1AR or A2bAR reversed the effect of EA at PC6 in alleviating MI injury; knockdown of A2aAR had no influence on the cardiac protection of EA at PC6; thus, the cardioprotective effect of EA at PC6 needs A1AR and A2bAR, instead of A2aAR; considering that the cardio protection of adenosine receptor needs activation of other adenosine receptors, one of the reasons may be that after silence of A1AR or A2bAR, EA at PC6 could not impact the expression levels of the other two adenosine receptors, and after silence of A2aAR, EA at PC6 could impact the expression levels of A1AR and A2bAR. These results suggested that EA at PC6 may be a potential and effective treatment for MI by activation of A1AR and A2bAR. Myocardial ischemia (MI) is one of heart conditions caused by lack of coronary blood flow , which iMale and female Sprague-Dawley (SD) rats , weighing 200–220 g, received ad libitum food and water, and were housed in a temperature-controlled room (25 ± 3 °C) under a 12-h light/dark cycle. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals  and was approved by ethical committee of Chengdu University of Traditional Chinese Medicine (no. 2014–03).Recombinant virus plasmid and the carriers of auxiliary packaging original plasmids were purchased from Shanghai Genechem Co., Ltd. . Adora1, adora2a, and adora2b mRNA in situ hybrization kits were purchased from Boster Biological Technology Co., Ltd. . 293 T cell was purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences .After 1-week acclimatization, 140 rats were randomly divided equally into 14 groups, namely normal control (NC) group, sham operation (SO) group, model (M) group, Neiguan (N) group, Hegu (H) group, sham acupoint (SA) group, negative lentivirus infected (NLI) group, A1AR lentivirus-infected (A1LI) group, A2aAR lentivirus-infected (A2aLI) group, A2bAR lentivirus-infected (A2bLI) group, negative lentivirus-infected Neiguan (NLIN) group, A1AR lentivirus-infected Neiguan (A1LIN) group, A2aAR lentivirus-infected Neiguan (A2aLIN) group, and A2bAR lentivirus-infected Neiguan (A2bLIN) group. In NC group, rats received no operation; in SO group, rats were only threaded, but not ligated left anterior descending coronary artery (LADCA); in M group, rats received ligation of LADCA; in N group, rats received ligation of LADCA and electroacupuncture (EA) treatment at bilateral PC6; in H group, rats received ligation of LADCA and EA treatment at bilateral LI4; in SA group, rats received ligation of LADCA and EA treatment at bilateral sham acupoint; in NLI, A1LI, A2aLI, and A2bLI groups, rats received corresponding lentivirus injection and ligation of LADCA; in NLIN, A1LIN, A2aLIN, and A2bLIN groups, rats received corresponding lentivirus injection, ligation of LADCA, and EA treatment at bilateral PC6.The rats were anesthetized with 10% chloral hydrate (0.4 ml/100 g IP) and were positioned on an operating table for small animals  was injected into left ventricle anterior wall at four different points. Finally, the chest was closed.The rats were anesthetized by intraperitoneal injection of chloral hydrate and received above thoracotomy. After exposure of heart, 50 μl corresponding lentivirus solution  was inserted to a depth of 1–2 mm. The acupuncture needle was connected to the HANS Acupuncture Point Nerve Stimulator . The frequency was set at 2/100 Hz, and the intensity of stimulation was 0.1–1 mA. The procedures of all EA groups  were the same. The location of acupoints, which refers to previous literatures , 24, is Left ventricular developed pressure (LVDP) and heart rate (HR) obtained by Data Acquisition System  were used to calculate rate pressure product (RPP), according to the previously described method : RPP = L4 (20 μl/mg). After centrifugation at 4 °C and 20,000×g for 10 min, aliquots of the supernatant were collected. To collect supernatant to regulate PH value to 6.5–7.0, 1 mol/l K2CO3 was added. After blending, the mixture was stood on ice for 5 min. After centrifugation at 4 °C and 20,000×g for 10 min, the supernatant was collected for HPLC determination of ATP, AMP, ADP, and ADO concentrations. The chromatographic conditions were as follows: (1) chromatographic column: hypersil C18, 5 μm, 4.6 × 200 mm; (2) mobile phase: 0.2 mol/l phosphate potassium salt buffer (pH 6.0); and (3) flow rate 0.9 ml/min. Ultraviolet-visible detector  was adopted to carry out chromatographic analysis.HPLC was used to determine the production of ATP, AMP, ADP, and ADO in myocardial tissue. The samples were determined by 2 mol/l HClOCardiac tissue was fixed in 4% paraformaldehyde overnight and was soaked in phosphate buffer solution (PBS) 4–5 times for 5 min. Cardiac tissue was put into 30% sucrose/0.1 M PBS. Two days later, cardiac slice was soaked in 0.1 M PBS (once for 5 min), 0.1 M glycine/0.1 M PBS (once for 5 min), 0.3% Triton X-100/0.1 M PBS (once for 10 min), and 0.1 M PBS (three times for 5 min). Fifty microliter proteinase was added to incubate at 37 °C for 30 min. Cardiac slice was soaked in 4% paraformaldehyde for 5 min and was washed by 0.1 M PBS twice for 5 min. Cardiac slice was put into 0.25% acetic anhydride/0.1 M triethanolamine for 10 min. Cardiac slice was pre-hybridized by pre-hybridization solution at 42 °C for 30 min and was hybridized by hybridization solution at 42 °C overnight. Four times SSC, 2× SSC, 1× SSC, 0.5× SSC, 0.2× SSC, 0.2× SSC/0.1 M PBS, 0.05 M PBS were, respectively, used to wash cardiac slice. Then, cardiac slice was coated by 3% BSA/0.05 M PBS at 37 °C for 30 min. Anti-digoxin/antiserum alkaline phosphatase was added to cardiac slice, and cardiac slice was, respectively, washed by 0.05 M PBS, TSM1, and TSM2. ImagePro Plus 6.0 software  was adopted to analyze results.Short hairpin RNA (shRNA) interference sequences were designed for A1AR, A2aAR, and A2bAR  to construct recombinant shuttle plasmids and packaging plasmids (pFU-GW-RNAi). The target sequences for A1AR, A2aAR, A2bAR, and negative control are as follows: 5′-CTTCTTTGCGTTCGTGTTA-3′, 5′-GATTTGGAATGACCACTTC-3′, 5′-GTGTCTCTTTGAGAACGTA-3′, 5′-TTCTCCGAACGTGTCACGT-3′. 293 T cells were transfected by sequenced plasmids and transfection reagent RNA-mate. The supernatants with lentivirus particles were concentrated and purified.P values were less than 0.05.Statistical analysis was made using SPSS 26.0 software . All data were expressed as mean ± SD and were analyzed using one-way ANOVA with post hoc Tukey tests or Games-Howell tests. Differences were considered statistically significant when P < 0.01); thus, MI injury model is successful. Compared with the M group, the value of RPP was significantly increased and ST segment was depressed in the N group  and the value of RPP and ST segment had no significant change in the H group and the SA group . Compared with the N group, the value of RPP was reduced and ST segment were elevated in the H group and the SA group .As shown in Fig. P < 0.01); compared with the A1LI group, the value of RPP and ST segment in the NLIN group had no significant change ; compared with the A2aLI group, the A2aLIN group had significantly increase the value of RPP and depress ST segment ; compared with the A2bLI group, the A2bLIN group had no significant change in the value of RPP and ST segment .In addition, compared with the NLI group, the value of RPP was increased and ST segment was depressed in the NLIN group  and no change in the concentration of ADP  compared with the NC group and the SO group. EA treatment at PC6 significantly increased the concentrations of ATP and ADO, and decreased the concentration of AMP ; however, EA treatment at PC6 did not significantly impacted the concentration of ADP . EA treatment at LI6 and sham acupoint had no significant impact on the concentrations of ATP, AMP, ADP, and ADO . Compared with the H group and the SA group, the concentrations of ATP and ADO were increased, and the concentration of AMP was reduced by EA treatment at PC6 , and the concentration of ADP was not impacted .The results showed that MI injury caused decrease in the concentrations of ATP and ADO and increase in the concentration of AMP , and the concentration of ADP is not different from that of the NLI group (P > 0.05); the A1LI group and the A1LIN group have no statistical difference in the concentrations of ATP, AMP, ADP, and ADO ; the concentrations of ATP and ADO are significantly increased, and the concentration of AMP is decreased , and the concentration of ADP is not changed (P > 0.05) in the A2aLIN group compared with the A2aLI group; the A2bLI group and the A2bLIN group also have no significant difference in the concentrations of ATP, AMP, ADP, and ADO .Figure P < 0.01). EA treatment at PC6 could significantly upregulate the mRNA expression levels of A1AR, A2aAR, and A2bAR ; however, EA treatment at LI6 and sham acupoint could not significantly upregulate the mRNA expression levels of A1AR, A2aAR, and A2bAR . The mRNA expression levels of A1AR, A2aAR, and A2bAR were higher in the N group than those of the H group and the SA group .Compared with the NC group and the SO group, the mRNA expression levels of A1AR, A2aAR, and A2bAR were significantly upregulated in the M group ; no significant differences were observed in the mRNA expression levels of A2aAR and A2bAR between the A1LI group and the A1LIN group ; compared with the A2aLI group, the mRNA expression levels of A1AR and A2bAR were upregulated in the A2aLIN group ; there was no statistical difference in the mRNA expression levels of A1AR and A2aAR between the A2bLI group and the A2bLIN group .The mRNA expression levels of A1AR, A2aAR, and A2bAR in the NLIN group were higher than those of the NLI group  EA at PC6 has better effect than EA at LI6 and sham acupoint in affecting RPP and ECG, increasing ATP and ADO production, decreasing AMP production, and upregulating the expression levels of A1AR, A2aAR, and A2bAR; (b) A1AR and A2bAR are involved in EA-induced cardio protection in MI injury; and (c) silence of A1AR or A2bAR reverses the influence of EA at PC6 in upregulating the other two adenosine receptors and silence of A2aAR has no influence on the effect of EA at PC6 in upregulating A1AR and A2bAR.MI induced depletion of ATP, and the decrease of ATP is due to inadequate rate of production of high-energy phosphate relative to the demand of the heart for energy . ATP canKnockdown of A1AR or A2bAR abolished EA at PC6-induced functional protection via affecting RPP and ECG. Moreover, knockdown of A1AR and A2bAR reversed influences of ATP, AMP, and ADO production caused by EA at PC6. The cardio protection of adenosine receptor needs activating other adenosine receptors –35. In oActivating adenosine receptors can improve MI , 37. ForThe protective effect of A1AR, A2aAR, and A2bAR needs other adenosine receptors, namely only one adenosine receptor cannot play its role. A2aAR and A2bAR must be simultaneously activated for cardiac protection to occur . The antIn conclusion, this study indicated that EA at PC6 is effective in alleviating MI injury, EA at PC6 is more effective than EA at LI6 and sham acupoint; after silence of A1AR or A2bAR, EA at PC6 could not improve MI injury; thus, the effect of EA at PC6 may depend on A1AR and A2bAR. In addition, one of the reasons may be that after the silence of A1AR or A2bAR, EA at PC6 could not impact the expression levels of the other two adenosine receptors, and after the silence of A2aAR, EA at PC6 could impact the expression levels of A1AR and A2bAR.


This work introduces a multidisciplinary strategy to characterize the structure, folding status, and function of the RNA G-quadruplex in APP 3′UTR. in vitro and in cells. We apply this strategy to a biologically important rG4 in amyloid precursor protein (APP) transcript and reveal distinct structural features of APP rG4. Notably, we visualize the formation of APP rG4 in cells using an APP-specific G-quadruplex-triggered fluorogenic hybridization (GTFH) probe and report that the regulatory role of APP rG4 in translation is dependent on rG4 thermostability, providing evidence to the existence and significance of the stable rG4 structure in gene regulation.RNA G-quadruplexes (rG4s) are emerging structural motifs that are of pivotal importance in chemistry and biology; however, the current structural information of rG4s is limited, with their folding status and functions in cells remaining elusive. Here, we develop and employ a multi-disciplinary approach to characterize the structure, formation and function of an individual rG4 of interest  In this work, we employ cross-disciplinary methods to characterize the rG4 in APP 3′UTR, reporting its structural features, as well as visualizing its formation in cells for the first time  is ubiquitously expressed in humans and its proteolytic product amyloid beta (Aβ) can form amyloid plaque, whose accumulation is one of the characteristic pathological features of Alzheimer's Disease (AD).rst time . Importanslation .APP 3′UTR, we have initially performed multiple spectroscopic assays. First, circular dichroism (CD) data on the APP 3′UTR rG4wt motif (Table S1+ (G4-stabilizing) with Li+ (G4-non-stabilizing)  of the APP 3′UTR rG4wt motif was determined to be 78 °C under 1 mM K+ and >95 °C under 150 mM K+ conditions, much higher than the physiological temperature of 37 °C  Fig. S1. This CDm Fig. S2, which iC Fig. S2. In addin Fig. S3, suggest+ Fig. S4, which iAPP 3′UTR rG4 in the presence of flanking sequences, as there are so far only limited rG4 studies that consider sequences beyond the rG4 motif itself.APP 3′UTR sequence to our constructs and designed both rG4wt and rG4mut versions  assay, originally developed by us,+ and K+ conditions  and >95 °C (150 mM K+), as evidenced by our UV melting data on the APP 3′UTR rG4wt motif ,t region , but nott region . A closet region , zoom int region , red, ant region , green. ′UTR rG4 , providie Fig. S7, and fou compare  and S7†,APP 3′UTR sequences in vitro and in cell lysate. Notably, our findings on the APP 3′UTR rG4 structure are new and the distinct rG4 spectroscopic and structural profiles revealed can likely be used as general structural signatures to identify and characterize rG4s in different contexts.Collectively, our biophysical characterization and structural analysis have offered substantial evidence for the formation of rG4 in a construct that contains native APP 3′UTR rG4 in cells, we next attempted to visualize its formation in HEK293T cells  in HEK293T cells. Our results showed that compared to the rG4 mutant construct, the rG4 wildtype construct has a 2.7-fold lower normalized luciferase activity  that changed APP 3′UTR rG4 from three G-quartet planes in the wildtype into rG4 with only two G-quartet planes . Vectors used in the dual luciferase reporter gene assay were synthesized by iGENE (China).IVT RNAs were prepared using DNA hemi-duplex  and HiScribe T7 High Yield RNA Synthesis Kit . RNA was purified using precast 15% denaturing polyacrylamide gel and the gel band with the desired RNA product was cut under UV. The gel was then crushed and soaked in 1× 10 mM Tris–HCl pH 7.5, 1 mM EDTA, and 800 mM LiCl (1× TEL800) in a thermoshaker at 4 °C and 1300 rpm overnight, and the RNA was gel extracted, purified and concentrated using RNA Clean & Concentrator-5 . RNA was suspended in 20 μL water and kept at –20 °C.–1. They were then normalized to molar residue ellipticity and smoothed over 5 nm.CD spectroscopy was conducted using a Jasco J-1500 CD spectrophotometer, and a 1 cm path length quartz cuvette  was employed in a volume of 2 mL. Samples with 5 μM RNA  were prepared in 10 mM LiCac (pH 7.0) and 150 mM KCl/LiCl. Each of the RNA samples was then thoroughly mixed and denatured by heating at 95 °C for 5 minutes and cooled to room temperature for 15 minutes for renaturation. RNA samples were excited and scanned from 220 to 310 nm at 25 °C and spectra were acquired every 1 nm. All spectra reported were average of 2 scans with a response time of 2 s nm2 gas and cooled down to 5 °C for 5 minutes. After the sample solutions were loaded to the cuvettes, they were sealed with 3 layers of Teflon tape to prevent vaporization at high temperature. The samples were scanned from 5 to 95 °C with a temperature increment rate of 0.5 °C min–1. The temperature was held at 95 °C for 5 minutes before a reversed scan was performed, scanning from 95 to 5 °C with a rate of 0.5 °C min–1. The unfolding and folding transitions in both scans were monitored at 295 nm. From the raw data obtained we subtracted data of the blank solutions, which contained the same concentrations of LiCac buffer (pH 7.0) and corresponding salt solution only. It was then smoothed over 10 data points and its first derivative was plotted in Microsoft Excel.Samples were prepared to a concentration of 10 mM LiCac buffer, 150 mM KCl (unless otherwise stated) and 5 μM RNA, with a total volume of 2 mL. For the concentration dependent experiments, the samples were prepared with RNA concentrations ranging from 1 to 10 μM. Each of the samples was mixed thoroughly and heated at 95 °C for 5 minutes to denature the RNA. It was then cooled for 15 minutes at room temperature for renaturation. All UV melting experiments were performed on an Agilent Cary 100 UV-vis Spectrophotometer, using a 1 cm path length quartz cuvette. Before the experiment started, the sample block was first flushed with dry NFluorescence spectroscopy was performed using a HORIBA FluoroMax-4, and a 1 cm path length quartz cuvette  was used with a volume of 2 mL. Samples with 5 μM DNA/RNA were prepared in 10 mM LiCac (pH 7.0) and 150 mM KCl/LiCl. The samples were then denatured at 95 °C for 5 minutes and cooled to room temperature for 15 minutes for renaturation. For the measurement of the intrinsic fluorescence of G-quadruplexes, the samples were excited at 260 nm and the emission spectra were acquired from 300 to 500 nm. Spectra were acquired every 2 nm at 25 °C. The bandwidth of the entrance and exit slits was 5 nm. All data were smoothed over 5 nm. Results here are from three independent experiments and analyzed using Microsoft Excel.N-Methyl-mesoporphyrin IX (NMM) and Thioflavin T (ThT) were purchased from Frontier Scientific and Solarbio Life Sciences, respectively. ISCH-oa1 was produced based on a scheme published before.Sample solutions containing 0.5 μM RNA were prepared in 150 mM LiCl/KCl, 10 mM LiCac buffer (pH 7.0) and 0.5 μM ligand (NMM/ThT/ISCH-oa1). Fluorescence spectroscopy was performed using a HORIBA FluoroMax-4, and a 1 cm path length quartz cuvette  was used with a sample volume of 100 μL. Before the measurement, the samples (ligand not added) were denatured at 95 °C for 3 minutes and allowed to cool down at room temperature for 15 minutes. The samples were excited at 395 nm for NMM, 425 nm for ThT and 570 nm for ISCH-oa1. The emission spectra were acquired from 550 to 750 nm, 440 to 700 nm and 600 to 750 nm for NMM, ThT and ISCH-oa1, respectively. Data were collected every 2 nm at 25 °C with 5 nm entrance and exit slit widths. Raw ligand enhanced fluorescence spectra were first blanked with the corresponding sample spectra that resembled all chemical conditions except in the absence of the ligand. The blanked spectra were then smoothed over 10 nm (5 data points).2, 20 mM Tris pH 7.5, 1 mM DTT, and 0.5 mM dNTPs. The mixture was first heated to 75 °C for 3 min and then 35 °C for 5 min and 0.5 μL of Superscript III (200 U μL–1)  was added to make up the 10 μL reaction mixture. The reverse transcription was maintained at 50 °C for 15 min, and then 0.5 μL of 2 M NaOH was added at the end of the step. The temperature was immediately ramped up to 95 °C for 10 min. Finally, 10 μL of home-made 2× denaturing formamide dye was added to the reaction mixture.5 pmol of IVT RNA was added up to 4.5 μL with nuclease-free water, and 1 μL of 5 μM Cy5-labelled DNA primer was subsequently added. 3 μL reverse transcription buffer was added to give a final concentration of 150 mM LiCl/KCl, 4 mM MgCl2, and 40 mM Tris–HCl (pH 7.5). The final reaction volume was 19 μL. The reaction mixture was incubated at 37 °C, 10 min for system equilibration. NAI was then added to the reaction mixture at a final concentration of 0.05 M, and allowed to react with the RNA for 5 min at 37 °C. Dithiothreitol (DTT) was added to a final concentration of 0.2 M to quench the reaction, followed by RNA Clean & Concentrator-5  to clean up the RNA.IVT RNA (5 pmol) was renatured at 95 °C for 1.5 min, then cooled to 4 °C for 1.5 min, and incubated at 37 °C in 150 mM KCl/LiCl, 0.5 mM MgCl2, 0.5% Nonidet P-40) and 1 μL Riboblock RNase inhibitor . IVT RNA (200 pmol) was first denatured at 95 °C for 1.5 min, then cooled to 4 °C for 1.5 min for renaturation, and incubated at 37 °C in 200 mM KCl, and 10 mM Tris–HCl (pH 7.5). IVT RNA was added into the cell lysate and shaken at 37 °C for 5 min, and NAI  was added and reacted for 5 min. The reaction was quenched by adding 4 μL of 2 M DTT and RNA was cleaned using a RNeasy Plus Kit  following the manufacturer's protocol. Minus NAI treatment was performed by adding anhydrous DMSO.One million cells were resuspended with 40 μL native cell lysis buffer   was added to make up the 10 μL reaction mixture. The reverse transcription was performed at 50 °C for 15 min, and then 0.5 μL of 2 M NaOH was added. The temperature was immediately raised to 95 °C for 10 min. Finally, 10 μL of home-made 2× denaturing formamide dye was added to the reaction mixture.NAI-modified or untreated RNA was added up to 5.5 μL with nuclease-free water, and 1 μL of 5 μM Cy5-labelled DNA primer was then added. 3 μL of reverse transcription buffer was added to give a final concentration of 150 mM LiCl, 4 mM MgCl–1. All chemicals were purchased from commercial sources unless otherwise specified. All the solvents were of analytical reagent grade and were used without further purification.The GFTH probe was synthesized according to the scheme in Chen and co-workers' paper.2 atmosphere. Cells were seeded in a glass bottom 96-well plate  and grew overnight. Oligonucleotide transfections were performed using 50 nM RNA oligonucleotides and Lipofectamine 3000 Transfection Reagent  for 4 h. Cells were fixed with 4% paraformaldehyde in DEPC–PBS at room temperature for 15 min. After rinsing with DEPC–PBS, cells were permeabilized in 0.5% TritonX-100/DEPC/PBS at 37 °C for 30 min. After rinsing with 2× SSC, probes were diluted at 0.3 μM in hybridization buffer  and applied to the cells. Hybridization was done at 37 °C overnight. After hybridization, cells were washed in 2× SSC for 15 min twice and subsequently stained with (0.5 μg mL–1) DAPI for 15 min at 37 °C. For the RNase A treatment, cells were incubated with 200 units per mL–1 RNase A before hybridization at 37 °C for 1 h. Digital images were recorded using an LSM 710 laser scanning confocal microscope with a 63× objective lens, and analyzed with Imaris software . The Cellomics ArrayScan Vti  high-content imaging platform was used for the quantification of the fluorescence signal in cells. The high-content analysis automatically focused on the fluorescence channel of DAPI and captured the channel of ISCH-app.The authenticated HeLa and HEK293T cells with no mycoplasma contamination were grown in DMEM media  containing 10% heat inactivated fetal bovine serum  at 37 °C, with 5% COCells were seeded in a glass bottom 96-well plate  and grew overnight. 10 μM RNA oligonucleotides were hybridized with a 10 μM ISCH-app rG4 imaging probe by heating at 95 °C for 5 min and cooling down to room temperature for 2 h. The mixture was then transfected into cells using Lipofectamine 3000 Transfection Reagent  for over 4 h. Digital images were recorded using an Olympus FV3000 laser scanning confocal microscope with a 63× objective lens, and analyzed with Imaris software .The wildtype and mutant APP 3′UTR DNA sequences were ligated to the firefly/Renilla luciferase reporter vector, psiCHECK-2 , between NotI and XhoI restriction enzyme sites at the 3′ end of the Renilla luciferase gene by IGENE (China). Authenticated HEK293T/HeLa cells with no mycoplasma contamination were seeded in 96-well black-wall optical plates  at the density of 50 000 cells per well. In each well, the cells were transfected with 10 ng luciferase reporter plasmid using Lipofectamine® 2000 according to the manufacturer's protocol . After 5 and 24 hours, the medium was replaced with fresh medium. The cells were harvested 48 hours after transfection using the Dual Luciferase Reporter Assay System  according to the manufacturer's manual. Firefly and Renilla luciferase activities were determined using the Synergy H1 microplate reader . Renilla luciferase activity was normalized to firefly luciferase activity  and presented as the average of three transfection repeats.–1)  by reverse transcription. qRT-PCR analysis of mRNAs was performed using an SsofastTM Evagreen (SYBR Green) qPCR master mix (Bio-Rad). All qPCR reactions were performed by using a CFX96 Touch™ Real-Time PCR Detection System .Total RNA was extracted, cleaned and concentrated from cells using RNase Plus Mini Kit  according to the manufacturer's manuals. RNA samples were quantified using NanoDrop analysis . 100 ng total RNA was converted to cDNA with the Superscript III first-strand synthesis system . For quantification, each experiment was usually repeated at least three times with cells from three cell passages.The authors declare no competing financial interests.Supplementary informationClick here for additional data file.


The transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2) regulates the expression of genes involved in antioxidant defenses to modulate fundamental cellular processes such as mitochondrial function and GSH metabolism. Previous reports proposed that mitochondrial reactive oxygen species production and disruption of the GSH pool activate the Nrf2 pathway, suggesting that Nrf2 senses mitochondrial redox signals and/or oxidative damage and signals to the nucleus to respond appropriately. However, until now, it has not been possible to disentangle the overlapping effects of mitochondrial superoxide/hydrogen peroxide production as a redox signal from changes to mitochondrial thiol homeostasis on Nrf2. Recently, we developed mitochondria-targeted reagents that can independently induce mitochondrial superoxide and hydrogen peroxide production mitoParaquat (MitoPQ) or selectively disrupt mitochondrial thiol homeostasis MitoChlorodinitrobenzoic acid (MitoCDNB). Using these reagents, here we have determined how enhanced generation of mitochondrial superoxide and hydrogen peroxide or disruption of mitochondrial thiol homeostasis affects activation of the Nrf2 system in cells, which was assessed by the Nrf2 protein level, nuclear translocation, and expression of its target genes. We found that selective disruption of the mitochondrial GSH pool and inhibition of its thioredoxin system by MitoCDNB led to Nrf2 activation, whereas using MitoPQ to enhance the production of mitochondrial superoxide and hydrogen peroxide alone did not. We further showed that Nrf2 activation by MitoCDNB requires cysteine sensors of Kelch-like ECH-associated protein 1 (Keap1). These findings provide important information on how disruption to mitochondrial redox homeostasis is sensed in the cytoplasm and signaled to the nucleus. Oxidative stress and damage are involved in the development and progression of many diseases , 2. CellIn addition to its role in overall cellular redox homeostasis, Nrf2 is also critical for the maintenance of mitochondrial antioxidant defenses and redox homeostasis . This isThe Nrf2 activity enhances the expression of antioxidant systems , 29, incvia peroxidases (A) (B) (N-acetyl-L-cysteine (NAC) and in cells expressing mutant Keap1 that lacked particular sensor cysteine residues. These results indicate that elevated mitochondrial superoxide generation alone does not activate Nrf2 but provide confirmation of direct signaling to the cytosol as a stress response to disrupted mitochondrial thiol redox homeostasis.In exploring how mitochondrial oxidative stress and damage activate Nrf2 , it has oxidases , 12, 13.oxidases , suggestoxidases A 42). I. Ivia peases (A) B  (B) . Our hyp (A) (B) . We founA). Treatment with H2O2 as a positive control  increased the protein levels of Nrf2 within the cell ∼2-fold  and caused its redistribution to the nucleus, as assessed by immunocytochemistry , as did the positive control SFN  and led to its translocation to the nucleus, as assessed by immunocytochemistry  and by subcellular fractionation , with nearly 90% of cells having a clear nuclear distribution of Nrf2 upon MitoCDNB treatment . To determine whether this effect of MitoCDNB was due to its reaction with mitochondrial thiols, or by a nonspecific disruption of mitochondria by accumulation of the triphenylphosphonium-targeting group, we synthesized a MitoCDNB control compound (A–C). This control compound had no effect on Nrf2 protein levels or nuclear translocation (A–D). Thus, the activation of Nrf2 by MitoCDNB is dependent on its thiol reactivity and not a nonspecific interaction of the mitochondria-targeting moiety.We first assessed if selective disruption of mitochondrial thiol redox homeostasis activated Nrf2. To do this, we used the mitochondria-targeted thiol reagent MitoCDNB, which we had previously shown selectively depletes GSH and inhibits TrxR2 within mitochondria, without directly affecting their cytosolic counterparts . Under cFig. 2trol SFN . Similarcompound . The concompound , A–C. Thvia the antioxidant response element. Immunoblotting showed that MitoCDNB, but not its corresponding control compound, led to a time-dependent increase in the levels of the Nrf2 downstream targets glutamate–cysteine ligase catalytic subunit (GCLC), GSH synthetase (GSS), and heme oxygenase-1 (HO-1). HO-1 was increased at 8 and 12 h after exposure, while GCLC and GSS levels increased later, at 12 h (A–B). To further confirm Nrf2 activation, we used the quantitative NAD(P)H:quinone oxidoreductase-1 (NQO1) inducer assay (C), whereas the control compound had no effect. We conclude that the selective disruption of mitochondrial thiol homeostasis by MitoCDNB activates Nrf2.To determine whether the nuclear accumulation of Nrf2 by MitoCDNB activates transcription of Nrf2-dependent genes, we assessed the levels of proteins known to be under Nrf2 control  at 12 h , A–B. Toer assay , 47. Theer assay . The poter assay C, whereaA). MitoPQ did not cause an increase in Nrf2 protein levels (A) nor was there any localization of Nrf2 to the nucleus assessed by cell subfractionation followed by immunoblotting (B), in contrast to the positive control SFN (C–D), compared with the positive control H2O2. Under these conditions, MitoPQ did not elicit any changes in NQO1 activity (E) or in the expression of the Nrf2 targets, GCLC, GSS and HO-1 . Increasing MitoPQ concentrations 5- to 10-fold did not enhance cell levels of Nrf2 , indicating that the lack of effect on Nrf2 was not due to insufficient MitoPQ. We conclude that under these conditions, the selective production of superoxide and hydrogen peroxide within the mitochondrial matrix does not activate Nrf2.It has been previously reported that increasing mitochondrial oxidative damage and/or stress activates the Nrf2 pathway . It was Fig. S3n levels A nor wasblotting B, in control SFN , or by itrol SFN , C–D, coactivity E or in tA) and its translocation to the nucleus  in C2C12 cells. Hepa1c1c7 cells exposed to CDNB and MitoCDNB both increased NQO1 activity . As MitoCDNB acts in part by depleting mitochondrial GSH levels, we next assessed the effect of NAC, a GSH precursor that also increases cell thiol levels and can thereby directly ameliorate cellular oxidative stress that impacts thiols . As expected, NAC did not have an influence on SFN-induced Nrf2 nuclear localization (A). The MitoCDNB-mediated induction of NQO1 was also diminished when cells were either pretreated (24 h) or cotreated with NAC . Treatment with NAC at concentrations up to 10 mM had no effect on the activity of NQO1 (B). As NAC and MitoCDNB do not interact directly (A), this suggests that NAC prevents the MitoCDNB-mediated Nrf2 activation by boosting thiol defenses within the cell. This was confirmed by showing that NAC did prevent depletion of whole-cell GSH by CDNB and MitoCDNB (B) and increased mitochondrial GSH even in the presence of MitoCDNB (C). To investigate further how MitoCDNB affected the cytosol, we used CellROX, which is a fluorescent probe that responds to a wide range of oxidative processes, enabling us to measure changes in whole-cell oxidative stress, but did not observe an increase with MitoCDNB .From the previous results, we observed that MitoCDNB, but not MitoPQ, led to Nrf2 stabilization, nuclear localization, and enhanced expression of its downstream targets. Previously  it has bFig. 5s thiols , on Nrf2lization A. The Mi of NQO1 B. As NACdirectly A, this sMitoCDNB B and incMitoCDNB C. To invvia the reaction of the activators with thiols on Keap1, with specific thiols likely having particular reactivity with different species. These reactions disrupt the substrate adaptor function of Keap1 and enable Nrf2 to escape ubiquitination, migrate to the nucleus, and activate gene expression. To interrogate the involvement of Keap1 in the mechanism of Nrf2 activation by MitoCDNB, we used mouse embryonic fibroblast (MEF) cells expressing two different Keap1 cysteine mutants or their WT counterpart (C151S and Keap1C226S/C613S (A). Cys151 on Keap1 is the main sensor for SFN, 4-hydroxynonenal, and nitric oxide, whereas Cys226 and Cys613 respond to H2O2 (A) (B). In WT cells, MitoCDNB induced NQO1 in a concentration-dependent manner, but this induction was diminished in MEF cells expressing either of the Keap1 mutants (B). The NQO1 inducer potency of the classical Nrf2 activator SFN was also greatly reduced in the Keap1C151S mutant cells in comparison with their WT or Keap1C226S/C613S counterparts, in agreement with previous reports that Cys151 is the main sensor for SFN (C). The use of Keap1 mutants confirms that MitoCDNB activates Nrf2 through multiple cysteine sensors in Keap1 and further suggests the involvement of both electrophiles and oxidants as potential mediators.The above analysis indicates that MitoCDNB, but not MitoPQ, generates signals that activate Nrf2. Under homeostatic conditions, Nrf2 is bound to Keap1 and targeted for ubiquitination and proteolysis by the proteasome. The canonical pathway for the activation of Nrf2 by oxidants or electrophiles is nterpart . Specifi6S/C613S A. Cys151 to H2O2 A (51). TH2O2 (A) B. In WT  mutants B. The NQ for SFN ,52  with high glucose (4.5 g/l D-glucose), supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Hepa1c1c7 cells  were grown at 37 °C in a humidified atmosphere of 95% air and 5% CO2 in α-minimum essential medium supplemented with 10% (v/v) heat- and charcoal-inactivated fetal bovine serum with no antibiotics.C2C12 . After appropriate incubations, cells were detached using trypsin, pelleted , and resuspended in a 1-ml fractionation buffer (FB) . Cells were homogenized by being passed through a 27-gauge needle 10 times and a sample was taken as a whole-cell fraction. The homogenate was centrifuged , with the whole-cell supernatant stored, and the nuclear pellet was washed and passed through a 25-gauge needle 10 times. The subsequent nuclear homogenate was centrifuged again , with the supernatant discarded, and the pellet containing nuclei was kept as the nuclear fraction. The aforementioned whole-cell supernatant containing the mitochondria, membranes, and cytoplasm was recentrifuged , and the pellet was discarded, with this step repeated 3 times at which point no pellet was observed. The resulting supernatant was subsequently centrifuged , collecting the supernatant (S) and resuspending the mitochondrial pellet (P) in 1 ml of the FB. The centrifugation step was repeated on the supernatant. The resuspended pellet (P) was centrifuged , the supernatant was discarded, and the pellet was resuspended in 50-μl FB . The supernatant (S) underwent an ultracentrifugation step , and the resulting supernatant was collected as the cytosolic fraction. The protein concentration of each fraction was measured with a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific).Cells were grown up to 20 × 10C2C12 cells were plated at 15,000 cells/well in 24-well plates containing glass coverslips. After 16 h, the compounds of interest were added. At the end of the incubation, the media was removed, cells were washed twice in cold PBS (Gibco), treated with 4% (w/v) paraformaldehyde for 15 min at 37 °C followed by three 5-min washes with PBS. Cells were then permeabilized with 0.1% (v/v) Triton X-100 in PBS for 10 min at RT, washed with PBS (3 × 10 min), blocked with 10% (w/v) bovine serum albumin (BSA) in PBS for 20 min at RT, and then incubated with primary antibody in 5% (w/v) BSA in PBS for 2 h at RT. The primary antibody solution was removed, and cells washed with PBS (3 × 10 min) and incubated with secondary antibodies in 5% (w/v) BSA in PBS for 1 h at RT. This solution was then removed, and the cells were washed with PBS (3 × 10 min). Coverslips were then mounted onto slides using Dako Mounting Medium  and analyzed using Zeiss LSM880 confocal system equipped with a Zeiss Plan-Achromat 63x/1.4 NA oil-immersion objective. Z-stacks were acquired at 0.5-μm steps.Representative images for each condition were taken from >30 cells. This process was repeated on three separate cell passages to ensure biological replication. Primary antibody was rabbit anti-Nrf2 . The secondary antibody was AlexaFluor488 goat anti-rabbit .6 cells per ml and treated as described previously. Thirty minutes before the end of the stimulation, CellROX (1 μM) was added directly into the cell culture medium. Cells were shielded from light with tinfoil and incubated at 37 °C for 30 min. Fifteen minutes before the end of CellROX incubation, SYTOX Red (1 μM) was added into the cell culture medium. Cells were washed with PBS, scraped in PBS (0.5 ml), and transferred to polypropylene fluorescence activated cell sorting tubes. At least 10,000 total events were acquired using a BD LSRFortessa cell analyzer (BD Biosciences). CellROX Green and SYTOX Red were excited by laser at 488 nm and 640 nm, and the data were collected at forward scatter, side scatter, 530/30 nm (CellROX), and 660/20 (SYTOX) detector. The data were analyzed using FlowJo software (version 10.4.2), with the cell debris, represented by forward and side scatters, gated out for analysis. The median fluorescence intensity of the 530/30 nm channel was measured as a measure of cellular oxidative stress.C2C12 cells were seeded at 0.5 × 10g for 10 min at 4 °C, and the supernatants were collected for protein quantification using the BCA assay (Thermo Fisher Scientific) with BSA as a standard. Samples were mixed with 4× loading buffer , 8% (w/v) SDS, 0.4% (w/v) bromophenol blue, 40% (v/v) glycerol), 400-mM DTT as a reductant. Typically, 10 to 40 μg of protein was loaded onto 12% or 4 to 20% Tris-Glycine SDS-PAGE gels (Bio-Rad) and run at 120 V for 1 h. Protein was transferred to polyvinylidene difluoride membranes (Immobilon-FL or Bio-Rad Trans-Blot Turbo Mini) by wet transfer  methanol, pH (8.4)) or semidry transfer (Bio-Rad Trans-Blot Turbo) before blocking for 1 h at RT with Odyssey blocking buffer (LI-COR). Primary antibody incubation was completed in 4% (v/v) Odyssey buffer in PBS + 0.1% (v/v) Tween-20 (PBST) overnight at 4 °C. Membranes were then washed 3 × 15 min in PBST, followed by secondary antibody incubation in 4% Odyssey buffer in PBS for 1 h at RT, washed 2 × 15 min in PBST and 1 × 15 min in PBS. Membranes were then visualized using a LI-COR Odyssey CLx system or enhanced chemiluminescence (Amersham ECL Prime Western blotting reagent) and analyzed using Image Studio Lite. Primary antibodies: rabbit anti-GAPDH , rabbit anti-Nrf2 , mouse anti–histone-4 , mouse anti-Tubulin , rabbit anti-GCLC , rabbit anti-GSS , and mouse anti–HO-1 . Secondary antibodies were goat anti-rabbit IRDye800  and goat anti-mouse IRDye680 , or goat anti-rabbit IgG HRP conjugate  and goat anti-mouse IgG HRP conjugate .C2C12 cells were plated at 300,000 cells/well in 6 well plates (Thermo Fisher Scientific) and treated with test reagents after a day of growth. At the end of the incubation, the media was removed, and cells were washed twice in cold PBS (Gibco), before the addition of 100 μl of RIPA buffer (Merck) supplemented with protease inhibitors  and 15-min incubation on ice. The samples were centrifuged at 17,0006–9 cells) or mitochondrial fractions (isolated with the hypotonic method as described (g for 10 min), and the supernatant analyzed by the GSH recycling assay as described  whole cells (1 × 10escribed ) were trescribed , 60. Dat4 per well of a 96-well plate) were grown for 24 h and exposed (n = 8) to serial dilutions of compounds for the time periods indicated in the figure legends. NQO1 enzyme activity was quantified in cell lysates using menadione as a substrate. Protein concentrations were determined in aliquots from the same cell lysates by the BCA assay (Thermo Scientific). The CD value was used as a measure of inducer potency. The same assay was used to determine the NQO1 enzyme activity in MEF cells, but the starting number of cells was 2 × 104 per well.Inducer potency was quantified by use of the NQO1 bioassay in Hepa1c1c7 mouse hepatoma cells , 47. Celg for 3 min. The supernatant was collected and centrifuged at 10,000g for 10 min. Mitochondrial pellets were resuspended in 10 ml and recentrifuged at 10,000g for 10 min before final resuspension in 5 ml per liver. In all cases, the protein concentration was measured using the BCA assay with BSA as a standard.For isolated mitochondria studies, mitochondria were prepared from rat livers. In all cases, mitochondria were isolated by homogenization using a Dounce homogenizer followed by differential centrifugation at 4 °C. Liver mitochondria were prepared in STE buffer . Homogenates were pelleted by centrifugation at 3000g for 10 min) and pellets and supernatants analyzed as necessary. Supernatants were immediately snap-frozen, whereas pellets were extracted in 250-μl acetonitrile (ACN) + 0.1% TFA before centrifugation , and the resulting supernatant snap-frozen.Mitochondrial incubations were in KCl buffer  at 37 °C, with succinate (10 mM), rotenone (4 μg/ml), and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (0.5 μM) added as appropriate, unless stated otherwise. After incubations, mitochondria were pelleted by centrifugation (7500g for 10 min) and filtration of the supernatant through a syringe-driven 0.22-μm polyvinylidene difluoride filter unit . Samples were then loaded into a 2-ml sample loop and separated by reversed-phase HPLC using a C18 column  attached to a Wide Pore C18 guard column (Phenomenex), all driven by a Gilson 321 pump. A flow rate of 1 ml/min was used with a gradient of 0.1% (v/v) TFA in water (buffer A) and 0.1% (v/v) TFA in ACN (buffer B) at (%B), 0 to 2 min; 5%, 2 to 17 min; 5 to 100%, 17 to 19 min; 100%, 19 to 22 min; 100 to 5%. Absorbance was measured at 220 (triphenylphosphonium) and 340 (GSH adduct) nm using a UV-Visible detector (Gilson 151).Samples for reversed-phase HPLC were prepared by diluting to 25% (v/v) ACN containing 0.1% TFA, followed by centrifugation . The organic layer was washed with 1 M hydrochloric acid (2 × 100 ml), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography using a 12 g Agela cartridge eluting with CH2Cl2:MeOH (100:0 increasing to 90:10 over 10 column volumes) to give the chloride salt of MitoCDNB control as a yellow solid foam  (H (400 MHz: CDCl3): 1.74 to 1.84 , 2.08 to 2.15 , 2.71 , 3.80 , 4.05 to 4.12 , 7.25 , 7.64 to 7.69 , 7.74 to 7.79 , 7.86 to 7.91 , 8.29 , 8.97 . δC (101 MHz: CDCl3): 19.83 , 22.27 , 22.27 (CH3), 29.07 , 42.44 (CH2), 117.83 (CH), 118.34 , 125.79 (CH), 128.59 (C), 130.60 , 133.84 , 135.15 , 137.06 (C), 143.81 (C), 147.25 (C). δP (162 MHz: CDCl3): 24.66 (s). m/z (ESI): Found: 514.1881. C29H29O4N3P requires M+, 514.1890.Diisopropylamine  was added to a stirred solution of 5-chloro-2,4-dinitrotoluene  and (4-aminobutyl)triphenylphosphonium bromide  in dry ACN (10 ml) at RT under argon. The solution was stirred overnight and poured into 1 M hydrochloric acid and extracted into CHmg, 69%) . δH .Statistical comparison between two groups was carried out using GraphPad Prism 7 software using two-tailed, unpaired Student’s t-tests or one-way ANOVA with the appropriate correction for multiple comparisons. The number of biological replicates (n) and statistical values are stated in each figure legend. n = number of replicates. Individual significant All data from this study are contained within the manuscript, including Supplemental Information.The authors declare that they have no conflicts of interest with the contents of this article.


Herein, the nanostructured CoMnO2 were directly deposited onto carbon fibers by a chemical oxidation route without high temperature treatment in presence of ammonium persulfate (APS) as an oxidizing agent. FE-SEM analysis confirmed that the CoMnO2-coated carbon fiber electrode exhibited the porous hierarchical interconnected nanosheet structures, depending on the added amount of APS, and Fe2O3-coated carbon fiber electrode showed a uniform distribution of porous Fe2O3 nanorods over the surface of carbon fibers. The electrochemical properties of the CoMnO2-coated carbon fiber with the concentration of 6 mmol APS presented the enhanced electrochemical activity, probably due to its porous morphologies and good conductivity. Further, to reduce the interfacial contact resistance as well as improve the adhesion between transition metal nanostructures and carbon fibers, the carbon fibers were pre-coated with the Ni layer as a seed layer using an electrochemical deposition method. The fabricated ASC device delivered a specific capacitance of 221 F g−1 at 0.7 A g−1 and good rate capability of 34.8% at 4.9 A g−1. Moreover, the wire-type device displayed the superior energy density of 60.2 Wh kg−1 at a power density of 490 W kg−1 and excellent capacitance retention of 95% up to 3000 charge/discharge cycles.In this work, we report the carbon fiber-based wire-type asymmetric supercapacitors (ASCs). The highly conductive carbon fibers were prepared by the carbonized and graphitized process using the polyimide (PI) as a carbon fiber precursor. To assemble the ASC device, the CoMnO Accordingly, the electrode design of the pseudocapacitors with hierarchically nanostructured and battery-type bimetal oxides has been most important issues to improve the specific capacitance value and energy density. The extending of the potential window can be attained by the fabrication of asymmetric supercapacitors (ASCs), which effects an expanded potential window up to ~2V in aqueous electrolytes   . The oth43-1003) . The fit2/N20@PICF-6, XPS studies were carried out ((0), Co2+, and Co3+ at the 778.5–795.1 eV, 780.5 eV, and 779.5–797.3 eV, respectively. The deconvoluted Mn 2p spectrum displayed two peaks at 653.9 and 642.2 eV, which were ascribed to the presence of mixed Mn 2p1/2 and Mn 2p3/2 with a spin-energy separation of 11.7eV. The O 1s spectrum at 529.5 eV, 531.58 eV, and 533.28 eV can be attributed to metal oxygen bond , the hydrated trivalent oxide bond (M-O-H), and the residual water bond (H-O-H), respectively. In the C 1s spectrum of CoMnO2@PICFs, the sharp peak at around 286 eV can be spilt to three peaks at 284.4, 286.6, and 288.9 eV, which represent the sp2 graphitic carbon, C–O and O–C=O bonds. The Co and Mn contents of CoMnO2/N20@PICFs reached 15.87 and 14.57 atomic%, suggesting the successful formation of CoMnO2 on the fiber substrate.To confirm the chemical states of CoMnOried out b–f. The 2@PICF-6, CV and EIS tests of bare PICF, CoMnO2@PICF-6, and CoMnO2/N20@PICF-6 were carried out in three-electrode configuration. As seen in 2/N20@PICF-6 electrode showed clearly large integrated area than those of other samples, suggesting an enhanced electrochemical storage ability. As expected, the CoMnO2/N20@PICF-6 displayed the longer discharge time  were further investigated by EIS. Compared to the CoMnO2@PICF-6 (14.1 Ω), the CoMnO2/N20@PICF-6 showed the lower RCT value (3.4 Ω), suggesting that Ni seed layer provided a good interfacial contact between CoMnO2 and PICF, giving the lower interfacial resistance and a fast reversible redox reaction.In order to investigate the effect of Ni seed layer on the electrochemical properties of CoMnOrge time b, as com density c. The Co2O3-decorated N20@PICF with Ni seed layer (Fe2O3/N20@PICF) was prepared by hydrothermal method and then used as anode material. FE-SEM image showed a uniform distribution of porous Fe2O3 nanorods over the surface of PICF, as can be seen in inset of 2O3/N20@PICF sample which contained Fe, O, Ni, and C elements. The Fe 2p spectrum  of the cathode and anode materials was calculated by using Equation (2), and it turned out to be 1:0.7. The CV profiles of the CoMnO2/N20@PICF6 and Fe2O3/N20@PICF electrodes were shown in −1 at the current density of 0.7 A g−1. Moreover, the capacitance value started to decrease as the current density further increased. The ASC device showed stable electrochemical performances at different voltage windows, as seen in −1 to 177 F g−1. The Nyquist plot  of the CoMnO2@PICF-6//Fe2O3@PICF device without Ni seed layer, ascribed to the reduced interfacial contact resistance by an introduced Ni layer  [−1 at 790 W kg−1) [−1 at 8000 W kg−1) [−1 at 500 W kg−1) [−1 at 2625 W kg−1) [−1 at 850 W kg−1) [−1 at 1080 W kg−1) [To test the feasibility of the CoMnOist plot f showed Ni layer g. FurtheNi layer g. The asone plot h. Compar W kg−1) , NiCoMn- W kg−1) , CoMn-HWot −1 at 7 W kg−1  W kg−1) , NCM//AC W kg−1) , Ni-Mn L W kg−1) , and Co/ W kg−1) , as summ3COO)2·4H2O), manganese(II) acetate tetrahydrate (Mn(CH3COO)2·4H2O), iron(III) chloride hexahydrate(FeCl3·6H2O), nickel(II) chloride hexahydrate (NiCl2·6H2O), ammonium persulfate 2S2O8)), and ammonium chloride (NH4Cl) were purchased from Sigma-Aldrich . All the reagents were an analytical grade and used without further purification. All aqueous solutions used in these experiments were prepared with deionized (DI) water .Poly(amic acid) /4,4′-diaminodiphenyl ether , 230,000 cPs of viscosity, PI Advanced Materials Co., Ltd., Anyang, Korea)-based carbon fibers (PICFs)  were kin3COO)2·4H2O and 4.5 mmol Mn(CH3COO)2·4H2O were dissolved in 30 mL of DI water and stirred for 1 h to make a clear bright red solution. Then the PICF was immersed in the above solution and sonicated slightly to remove the microbubbles of the solution. Afterwards, APS solution with various concentrations of 1, 3, 6, and 9 mmol was further added and reacted at 60 °C for 12 h  and labelled as CoMnO2@PICF-1, CoMnO2@PICF-3, CoMnO2@PICF-6, and CoMnO2@PICF-9, respectively. The obtained CoMnO2-decorated PICFs (CoMnO2@PICFs) as cathode material was washed with DI water gently, and dried at 60 °C. The mass loading of CoMnO2 metal oxide deposited onto PICF was about 2 mg. For the preparation of anode material, 6 mmol FeCl3·6H2O was dissolved in 60 mL of DI water and stirred for 1 h at room temperature to make a homogeneous solution. Then, PICF and the above solution were transferred into 80 mL Teflon-lined stainless steel autoclave, and hydrothermal treatment was carried out at 140 °C for 12 h in an oven. The resultant product was carefully rinsed with DI water and dried at 60 °C for 12 h. Finally, the obtained sample was further calcinated at 350 °C for 2 h at a heating rate of 2 °C min−1 to achieve the Fe2O3-decorated PICF (Fe2O3@PICF). The mass loading of Fe2O3 metal oxides deposited onto PICF was about 1 mg.At first, 4.5 mmol Co at a deposition time of 20 s (denoted as Ni20@PICF) using a DC power supply. After deposition, the samples were washed with DI water and dried at 60 °C for 12 h. The schematic illustration for the preparation of CoMnO2@PICFs was represented in For the deposition of Ni seed layer, 2M NHα radiation operating at 40 keV/40 mA at a scanning rate of 15° per min in the 2θ range from 10° to 90°. The chemical state of the elements linked with the surface chemical composition of the sample was analyzed by X-ray photoelectron spectroscopy . The specific surface area (SSA) and pore size of the samples were computed using the Brunauer–Emmett–Teller (BET) equation.The surface morphologies were examined by field emission scanning electron microscopy  along with energy dispersive X-ray (EDX) system. The X-ray diffraction studies were performed using Rigaku diffractometer, CuK−1). The GCD tests were carried out within the potential range of 0 to 0.5 V in 1M KOH. The specific capacitance of the fabricated electrodes was calculated from the discharge curves using the following Equation (1).−1), I is the discharge current (mA), m is the mass (mg) of the electroactive material, ∆t and ∆V are the discharge time (s) and potential window (V). The EIS measurement was carried out at open circuit potential in the frequency range of 0.1 Hz to 100 kHz. The ZView software was employed to fit the EIS data. The impedance data are presented in the form of Nyquist plot, representing the resistive and capacitive behavior of electrodes. The values of the charge transfer resistance (RCT) and internal resistance (RS) were determined using Zsimpwin software simulations.The electrochemical performance was investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and galvanostatic charge–discharge (GCD). The CV and EIS measurements were carried out in a three-electrode system at room temperature using an electrochemical workstation . Here, Pt wire was used as the counter electrode, Ag/AgCl as the reference electrode and the prepared samples as the working electrode, respectively. The CV curves were recorded within the potential window from 0 to 0.6 V (vs. Ag/AgCl) in 1M KOH electrolyte at various scan rates  device was constructed using CoMnO2/N20@PICF and Fe2O3/N20@PICF as cathode and anode materials, respectively. The PVA/KOH gel electrolyte was used for both electrolyte and separator in ASC device. To prepare the PVA/KOH gel electrolyte, 2 g PVA and 2g KOH were added to the 20 mL DI water and it was kept under stirring and then heated slowly to 90 °C until it became clear and transparent. After it was cooled down to room temperature, the CoMnO2/N20@PICF and Fe2O3/N20@PICF electrodes were coated with PVA/KOH gel electrolyte and carefully assembled together and then sealed in the plastic tube to fabricate the wire-type ASC device. To obtain optimum energy and power densities, the optimal mass ratio of both electrodes was determined based on the charge balance relationship  provided by the following Equation (2).+, m−, C+, C−, V+, and V− signifies the mass (g), specific capacitance (F g−1) and working potential window (V) for the cathode and anode materials, respectively. The energy  and power densities  of as-assembled ASC device were computed by the following Equations (3) and (4).−1), ΔV is the working potential window (V) and Δt is the discharging time (s).For the practical applications, the asymmetric supercapacitor  with the concentration of 6 mmol APS presented the enhanced electrochemical activity, due to its porous morphologies and good conductivity. Moreover, additional Ni seed layer provided a good interfacial contact between CoMnO2 and PICF, giving the lower interfacial resistance and a fast reversible redox reaction. The fabricated ASC device delivered a specific capacitance of 221 F g−1 at 0.7 A g−1 and good rate capability of 34.8% at 4.9 A g−1. The wire-type device displayed the superior energy density of 60.2 Wh kg−1 at a power density of 490 W kg−1 and excellent capacitance retention of 95% up to 3000 charge/discharge cycles.We have prepared the carbon fiber-based wire-type asymmetric supercapacitors (ASCs). The nanostructured and porous CoMnO