Based on theeutectic composition of the NaFSA–KFSA (56:44 mol/mol) binarysystem, the superconcentrated solutions of 35 mol kg–1 Na0.55K0.45FSA/H2O and 33 mol kg–1 Na0.45K0.55FSA/H2O are found to form at 25 °C. As both electrolytes demonstratea wider potential window of ∼3.5 V compared to that of eithersaturated 20 mol kg–1 NaFSA or 31 mol kg–1 KFSA solution, we applied the 33 mol kg–1 Na0.45K0.55FSA/H2O to two different batteryconfigurations, carbon-coated Na2Ti2(PO4)3∥K2Mn[Fe(CN)6] andcarbon-coated Na3V2(PO4)3∥K2Mn[Fe(CN)6]. The former cell showshighly reversible charge/discharge curves with a mean discharge voltageof 1.4 V. Although the latter cell exhibits capacity degradation,it demonstrates 2 V-class operations. Analysis data of the two cellsconfirmed that Na+ ions were mainly inserted into the negativeelectrodes passivated by a Na-rich solid electrolyte interphase, andboth Na+ and K+ ions were inserted into thepositive electrode. Based upon the observation, we propose new sodium-/potassium-ionbatteries using the superconcentrated NaFSA–KFSA aqueous electrolytes.Superconcentratedaqueous electrolytes containing NaN(SO In 2015,Suo et al. reported that the superconcentrated aqueous electrolyteof 21 mol kg–1 lithium bis(trifuluoromethanesulfonyl)amide (N(SO2CF3)2–, TFSA–)/H2O demonstrated a wide potentialwindow of 3.0 V, which realized a Mo6S8∥LiMn2O4 full cell with an operation voltage close to2 V.2 Soon after that, Yamada et al. reporteda binary anion system using TFSA– and bis(perfluoroethysulfonyl)amide(N(SO2C2F5)2–, BETA–) anions to form a 27.8 mol kg–1 Li(TFSA)0.7(BETA)0.3 aqueous solution (Li(TFSA)0.7(BETA)0.3·2H2O), which demonstrateda Li4Ti5O12∥LiNi0.5Mn1.5O4 full cell with an operation voltageof ∼3.0 V.3 The wide potential windowof superconcentrated aqueous solutions is achieved by both higheroxidation and reduction stability compared with the conventional concentrationof electrolytes. The improved oxidation stability is attributed tothe HOMO energy decrease caused by the coordination of water moleculesto cations, whereas the reduction stability improvement would be dueto the formation of anion-derived solid electrolyte interphase (SEI)induced by the higher LUMO energy of anions that have formed ionicpairs with cations.3AqueousLi-ion batteries using nonflammable aqueous electrolytesare promising for energy storage technology due to their intrinsicsafety.7 In general, aqueous Na and K solutions show an ionic conductivityhigher than that of Li counterparts because of their weak Lewis acidity,i.e., weak interaction between the cations and solvents/anions andconsequent small Stokes radii of Na+ and K+ ions.Moreover, these cations with weak Lewis acidity allow the use of N(SO2F)2– (FSA–)as a counteranion. The FSA– anion has weak Lewisbasicity, leading to high solubility and ionic conductivity, and formsa stable SEI derived from the decomposition products in nonaqueousLi, Na, and K batteries.11 However, the severe hydrolysis of FSA– anions in the LiFSA aqueous solution to generate HF limits the utilizationof LiFSA in aqueous electrolytes.12 Inthe concentrated NaFSA and KFSA electrolytes, the FSA– anion exhibits higher stability against hydrolysis due to the weakinteraction between the cation and the sulfonyl oxygen of FSA–.12 Although the long-term stabilityagainst hydrolysis still needs to be demonstrated, the FSA– anion could be utilized for Na and K superconcentrated aqueous solutions.However, the demonstration of the 2 V-class aqueous Na or K batteriesis still challenging owing to the narrower electrochemical stabilitywindows of the electrolytes compared to those of the Li ones. Thenarrower electrochemical stability windows would be due to the followingtwo reasons. First, the weaker interaction between the Na+/K+ ion and water molecules results in less HOMO energyand LUMO energy shift of the water molecules and anions, respectively.13 Second, there is a higher solubility of thecorresponding fluorides, which are common SEI components, in Na andK aqueous solutions compared to that in Li solutions, with solubilitiesof LiF, NaF, and KF of 0.13, 4.13, and 102 g, respectively, in 100mL of water.14 Recently, Zheng et al. reportedNa and K superconcentrated aqueous solutions based on eutectic compositionsof ternary anions, namely, TFSA–, (pentafluoroethanesulfonyl)(trifluoromethanesulfonyl)amide(N(SO2CF3)(SO2F5)−, PTFSA–), and trifluoromethanesulfonate.6 The superconcentrated solutions of18.5 mol kg–1 Na(PTFSA)0.65(TFSA)0.14(OTf)0.21 and 27.7 mol kg–1 K(PTFSA)0.12(TFSA)0.08(OTf)0.8 exhibitedionic conductivity of 14.0 and 34.6 mS cm–1, respectively.6 The conductivities are higher than that of 27.8mol kg–1 Li(TFSA)0.7(BETA)0.3/H2O (3 mS cm–1).6 Onthe other hand, the potential window of the superconcentrated Na andK solutions was 2.7 and 2.5 V, which is still narrower than >3.0Vfor the Li superconcentrated solutions.3Similar to the Li electrolytes,superconcentrated aqueous Na andK electrolytes have been recently studied for battery application.17 because the larger entropyincrease from the solid mixtures to the liquid mixtures at the eutecticmixture makes the liquid phase more stable.18 In dual-cation electrolytes such as Na–K systems, both cationscould be inserted into active materials, realizing a wide varietyof positive and negative electrode configurations. Moreover, the cationmixing should affect SEI stability due to the different solubilityof the alkali metal compounds. Thus, it is important to clarify theeffect of cation mixing on the solubility of the salts, potentialwindow, reaction mechanism of active materials, and the SEI compositionand stability. In this study, we prepared NaFSA–KFSA/H2O electrolytes based on the eutectic composition of the NaFSA–KFSA(56:44 mol/mol) binary system. A series of superconcentrated NaxKx1–FSAaqueous solutions were evaluated in terms of solubility, ionic transportproperties, FSA– stability against hydrolysis, electrochemicalstability, and SEI formation to prove the impact of mixed Na+ and K+ ions. Through the use of superconcentrated NaxKx1–FSAsolution, 2 V-class dual-cation batteries of carbon-coated NaTi2(PO4)3 (NTP-C)∥K2MnFe(CN)6] (KMnHCF) and carbon-coated Na3V2(PO4)3 (NVP-C)∥KMnHCF were demonstrated on thebasis of understanding the electrode reaction including mobile ionicspecies and the surface layer of the electrode.Not onlyanionic mixing but also cationic mixing decrease the meltingpoint and increase the solubilityx1–KxFSA aqueous solutionswere prepared by dissolving NaFSA (Solvionic) and KFSA (Solvionic)into deionized water at various concentrations at 25 ± 1 °C.A series of Na2Mn[Fe(CN)6] positive electrode material was synthesized via a chelate-assistedprecipitation method.19 Briefly, 4 mmol K4Fe(CN)6·3H2O and 4 mmol MnCl2·4H2O were separately dissolved in 100 mL of 0.2M potassium citrate solutions. Then these two solutions were mixedby dropping the MnCl2 solution at 0.5 mL min–1 into the K4Fe(CN)6 solution with magneticstirring under a N2 atmosphere at 25 ± 3 °C.After being stirred for 15 h, the precipitate was centrifuged, filtered,and washed thoroughly with 400 mL of deionized water. Finally, thefinal product was obtained after vacuum drying at 100 °C for24 h.The K2(PO4)3 (NTP) and Na3V2(PO4)3 (NVP) were used for the negative electrode. The NTPwas synthesized via a sol–gel method.21 Specifically, 0.01 M titanium butoxide (Aldrich) was dissolved in40 mL of 30% H2O2 and 15 mL of 28% NH3 under constant agitation. Citric acid was added to the solution at a molar ratio of citric acid to titaniumions of 2:1. After sufficient stirring, stoichiometric amounts ofNH4H2PO4 in 10 mL of distilled water and Na2CO3  in 10 mL of distilled water were added. Then ethylene glycol was added to the mixture to give a molar ratioof 1:1 with the citric acid. The resulting mixture was kept at 80°C for 2 h under continuous stirring. The obtained transparentgel was heated at 140 °C for 2 h. The gel precursor was subsequentlydecomposed at 350 °C for 3 h in air, which led to the eliminationof the organic contents. The residual powders were ground and calcinedat 800 °C for 12 h in air to obtain NaTi2(PO4)3 powders. To improve the electronic conduction of NTPby carbon coating, the synthesized NTP powders were dispersed in a50% sucrose aqueous solution, and the solution was heated in a polytetrafluoroethylene(PTFE)-lined autoclave at 180 °C for 6 h. The precipitates werefiltered, washed, and recalcined at 800 °C for 1 h in an Ar atmosphereto obtain carbon-coated NTP (NTP-C). The amount of carbon in the NTP-Cwas estimated to be 3% by thermogravimetric analysis using a thermogravimetricanalyzer .NASICON-type materials of NaTi22 Cationic surfactantof cetyltrimethylammonium bromide was dissolved in a mixture of deionized water and ethanol. To thissolution was added a stoichiometric mixture of CH3COONa, vanadium(III) acetylacetonate (VO(C5H7O2)2, Strem Chemicals), and NH4H2PO4 with continuous and vigorous stirring.The resulting precipitate was then continuously stirred for severalhours and dried at 70 °C. The precipitate was calcined in anAr atmosphere at 750 °C for 6 h. The carbon content in NVP-Cwas estimated to be 1.9% by thermogravimetric analysis.The NVP-C composite was directlysynthesized via the soft templatemethod as reported before.The KMnHCF electrodes consistingof a mixture of 70 wt % KMnHCF, 20 wt % Ketjen black , and 10 wt % poly(vinylidene fluoride) were prepared with a coating mixture slurry on Ti foil and dryingthe coat at 150 °C under vacuum. The electrodes consisting ofa mixture of 70 wt % NTP-C and NVP-C as active material, 25 wt % acetyleneblack , and 5 wt % PVDF were prepared by coatinga mixture slurry on Al foil and drying the coat at 150 °C undervacuum. Activated carbon electrodes consisting of 80 wt % activatedcarbon , 10 wt % KB, and 10 wt % PTFE (Daikin) wereformed on Al-expanded metal and dried at 200 °C under vacuum.Voltammetry was conducted in the three-electrode cell in which the activated carbon counter electrode and Ag/AgCl referenceelectrode were used. Charge–discharge tests were conductedin three-electrode cells  with a Ag/AgCl referenceelectrode and glass fiber separator .2-filled glovebag to avoid air exposure.Then the electrodes were dried at room temperature under vacuum andtransferred using a transfer vessel to avoid air exposure. The detailedsetup and condition of the HAXPES measurement are described in ourprevious paper.24 The binding energy of the obtainedspectrum was calibrated with the binding energy of sp2 carbonof graphite being 284.3 eV.HardX-ray photoelectron spectroscopy (HAXPES) spectra of the tested NTP-Celectrodes were acquired by high excitation energy of hard X-ray,7939 eV, and a photoelectron energy analyzer of R-4000 (Scienta Omicron)at BL46XU in SPring-8, Japan. The photoelectron detection angle andpass energy of the analyzer were set to 80° and 200 eV, respectively.Electrochemically tested NTP-C electrodes were taken out from cycledthree-electrode cells and rinsed with propylene carbonate and dimethylcarbonate in a N2-filled glovebagand dried under vacuum.The structural change of KMnHCFelectrodes after the electrochemical test was investigated using anX-ray diffractometer  equipped with a high-speedone-dimensional X-ray detector D/teX Ultra. The chemical compositionof the electrodes was characterized by energy-dispersive X-ray (EDX)spectroscopy using a scanning electron microscope . In these ex situ experiments, the KMnHCF electrodes were immediatelytaken out from the three-electrode cells within 3 min after reachingthe target potentials without potential holding to avoid the relaxationand ionic exchange. Then the electrodes were rinsed with dimethylsulfoxide and dimethyl carbonate in a Nx1–KxFSA mixture in water was examinedat different Na/K ratios. x1–KxFSAsolutions at 25 °C. The concentrations of the saturated NaFSAand KFSA solutions as end-members are 20 and 31 mol kg–1, respectively. By mixing Na+ and K+ cations,higher concentration solutions of 35 mol kg–1 Na0.55K0.45FSA/H2O and 33 mol kg–1 Na0.45K0.55FSA/H2O were demonstrated.The NaFSA/KFSA ratio for the highest concentration solution in theNaFSA–KFSA–H2O system is quite close to theeutectic composition of the binary NaFSA/KFSA = 56/44 in molar ratio,25 which should be mainly due to the largest entropyincrease from the solid mixtures to the liquid mixtures, i.e., themolten salt or aqueous solution, at this NaFSA/KFSA ratio.18 On the other hand, Na0.25K0.75FSA shows solubility lower than that of the KFSA in H2O, which is different from the melting point behavior of the binaryNaFSA–KFSA system. Similar behavior to the ternary system hasbeen reported for the LiNO3–NaNO3–H2O26 and Mg(NO3)2–NaNO3–H2O27 systems, and the lower solubility of Na0.25K0.75FSA would be due to existence of a stableNaFSA hydrate phase.28x1–KxFSAsolutions. To compare the effect of cations on ionic conductivity,we also prepared 20 mol kg–1 solutions of NaFSA,Na0.55K0.45FSA, and KFSA. The 20 mol kg–1 KFSA solution showed the highest ionic conductivityof 75.3 mS cm–1, whereas the 20 mol kg–1 NaFSA solution showed the lowest ionic conductivity (41.3 mS cm–1). The high ionic conductivity of the K solution isdue to the weak interaction of K+ ions with either watermolecules or anions. The 20 mol kg–1 Na0.55K0.45FSA solution delivers ionic conductivity of 49.8mS cm–1, which was lower than the ionic conductivitylinearly predicted from the Na/K ratio (∼57 mS cm–1). The significant decrease in ionic conductivity from the KFSA solutionto Na0.55K0.45FSA solution corresponds to anincrease in viscosity, which may be due to ion-pair formation betweenNa+ and FSA–. Indeed, the viscositiesof the 31 mol kg–1 KFSA solution and 20 mol kg–1 NaFSA were almost identical despite a much higherconcentration of the KFSA solution. The 31 mol kg–1 KFSA solution delivered a relatively high ionic conductivity of43.4 mS cm–1. The 35 mol kg–1 Na0.55K0.45FSA and 33 mol kg–1 Na0.45K0.55FSA solutions exhibited ionic conductivitiesof 22.0 and 25.2 mS cm–1, respectively. It is worthmentioning that the ionic conductivities are much higher than 3 mScm–1 for 27.8 mol kg–1 Li(TFSA)0.7(BETA)0.3.3 The 33mol kg–1 Na0.45K0.55FSA solutionshowed a viscosity of about 34 mPa·s, which was lower than the47 mPa·s of the 35 mol kg–1 Na0.55K0.45FSA solution. The reason behind the low viscosityof the Na0.45K0.55FSA solution is the higherK ratio as well as the slightly lower concentration. Notably, theseNax1–KxFSA superconcentrated solutions showed viscosity far lower than thatof the Li superconcentrated solution (203 mPa·s), despite thehigher concentration for Nax1–KxFSA. Moreover, preliminary semiquantitativeanalysis of F– ion in the electrolytes using theZr-EDTA complex29 showed that the 33 molkg–1 Na0.45K0.55FSA solutioncontains negligible F– ion (∼3 ppm) after2 weeks storage at 25 ± 3 °C . The F– ion concentrationis much lower than that of 31 mol kg–1 LiFSA solution. Therefore, it was proved that the utilization of Na+ and K+ ions, which have Lewis acidity weaker thanthatof Li+ ions, demonstrates superconcentrated electrolytesshowing higher ionic conductivity, lower viscosity, and higher FSA– stability.First, thesolubility of the Na–1 usingthe working electrodes of Ti and Al foils, respectively. First, 1,20, and 35 mol kg–1 Na0.55K0.45 solutions were evaluated to check the concentration effect, as shownin –1 solution exhibited acurrent increase from 1.5 V vs Ag/AgCl, and the current reached 5μA cm–2 at 1.60 V vs Ag/AgCl. In this study,5 μA cm–2 was used to simply define the oxidationand reduction stability limit potentials of the electrolytes. In contrastto the 1 mol kg–1 solution, the higher concentrationsolutions of 20 and 35 mol kg–1 exhibited oxidationstabilities of ∼1.8 V vs Ag/AgCl that were higher than thoseof the 1 mol kg–1 one . In contrast to the anodic stability, significant differences duringthe cathodic sweep were found in reduction stability between 20 and35 mol kg–1 electrolytes. The 1 and 20 mol kg–1 solutions showed lower stability against electroreduction,showing an increase in cathodic current below −1.0 V vs Ag/AgCl measurements at a scan rate of 0.5 mV skg–1 one 2a, and nlectrode S2. In co–1 NaFSA solution showed current flow from∼1.5 V vs Ag/AgCl. The higher concentration solutions of 33mol kg–1 Na0.45K0.55FSA, 35mol kg–1 Na0.55K0.45FSA, and31 mol kg–1 KFSA exhibited anodic stability higherthan that of the NaFSA solution  and a contact ionpair (CIP).34 In the 20 mol kg–1 NaFSA, 31 mol kg–1 KFSA, and 33 mol kg–1 Na0.45K0.55FSA solutions, the peak exhibitedan apparent blue shift −1 −1 5a. Thef−1 and 137 mAh (g-KMnHCF)−1. Most importantly, the full cell exhibited two voltageplateaus at 2.2 and 1.8 V in the discharge process. Thus, a 2 V-classaqueous Na/K dual-ion battery was demonstrated using 33 mol kg–1 Na0.45K0.55FSA. However, thereversible capacity slowly decreased to 125 mAh g–1 during 10 cycles, which would be due to partial electrolyte decompositionas the Coulombic efficiency remained around 94% in the subsequentcycles , suggesting preferential Na+-ion insertion. However, K+-ion insertion into NTP andNVP electrodes could occur in aqueous solutions as K+-ioninsertion into NTP electrodes has been reported in a highly concentratedK solution.7 Thus, future studies shouldreveal the thermodynamic and kinetic effects on the insertion speciesin the dual-cation electrolytes. Highly reversible Na+ andK+ ion insertion into KMnHCF has been proven in nonaqueousNa44 and K41 cells, respectively. Thus, the charge–discharge mechanismof KMnHCF in the Na0.45K0.55FSA solution wasexamined by conducting ex situ XRD and EDX measurements.As the electrolyte contains superconcentrated Na+ ion to the Na+ ion. The KMnHCF electrode had a cubic structure at the endof the lower-voltage plateau (sample B), followed by phase transitioninto a tetragonal structure at the end of the charge process . This trend of structural evolution is identicalto that of nonaqueous K cells.41 In thedischarge process, KMnHCF reversibly transformed back into a cubicstructure at the potential step (sample D). However, diffraction linesof 200C and 220C (in which C denotes the cubicphase) are located at a lower angle than the peaks of sample B. Thedifferent pattern suggests an asymmetric insertion/extraction mechanism.At the end of discharge (sample E), the electrode had a monoclinicstructure identical to that in the initial state, showing reversiblestructural evolution to K∼2Mn[Fe(CN)6]. These results imply that the KMnHCF electrode undergoes asymmetricstructural changes during the insertion/extraction process, thoughthe initial monoclinic structure fully recovers after discharge.+ ionswere extracted from KMnHCF. The ex situ XRD and EDX measurements showthe evolution of the crystal structure from monoclinic to tetragonalthrough the cubic structure during K+-ion extraction, asshown in the schematic illustration , and thatof K was the same as sample A. Therefore, K+ ions weremainly inserted for charge compensation during the redox reactionat the lower potential plateau, and the inserted Na+ ionswere spontaneously exchanged to K+ ions (2Mn[Fe(CN)6] is thermodynamically more favorable than Na2Mn[Fe(CN)6].45 Compared with the A2Mn[Fe(CN)6] (A = Na or K) case, the thermodynamic stabilitydifference between Na1Mn[Fe(CN)6] and K1Mn[Fe(CN)6] would be minor because the interactionbetween the alkali metal and framework is smaller in the lower alkalimetal contents. Indeed, the NaxMn[Fe(CN)6] electrode showed a redox potential accompanying Na+ insertion between Na2Mn[Fe(CN)6] and Na1Mn[Fe(CN)6] much lower than that of the K counterparts,whereas the NaxMn[Fe(CN)6]electrode delivered a redox potential between Na1Mn[Fe(CN)6] and Na0Mn[Fe(CN)6] competitive withor even higher than that of the K counterparts . In the unique electrolyte of Na+ andK+ coexistence, therefore, both Na+ and K+ ions were inserted simultaneously at the higher voltage plateau,and K+ ions were mainly inserted at the lower dischargeplateau, accompanied by Na+/K+-ion exchange.However, it should be noted that the insertion species may dependon kinetics such as charge/discharge rate. Future work should clarifythe thermodynamic and kinetic effects on the insertion species intothe positive electrode as well as the negative electrodes.To further understand the charge–dischargemechanism, EDXanalysis was carried out for each point to probe the change in sodiumand potassium contents 6b. All istration 7. The Na K+ ions 7. The co–1 Na0.45K0.55FSA electrolyte, and tested for 40 cycles in theNTP-C∥KMnHCF cell. Intensities of all spectra were correctedby relative sensitivity factors46 and normalizedby the integrated intensity of sp2 C peak at 284.3 eV toenable a semiquantitative analysis of the chemical species.47 In the C 1s/K 2p spectra of the pristine andsoaked electrodes, peaks attributed to sp2 C, −CH2–CF2–, and CF2 (290.5eV), which are derived from NTP-C/AB and PVDF, were observed  and NaF/KF(684.0 eV) were observed for the cycled electrode, whereas only thepeak attributed to PVDF-derived CF2 was observed in thepristine and soaked electrodes . The new peaks,such as −SO2–, −SOxF, and NaF/KF, for the cycled electrode are attributed to thedecomposition products of FSA– anions.50 Moreover, clear peaks in Na 1s and K 1s spectra supported the formationof Na and K compounds like NaF/KF on the tested negative electrodesurface  was much higher than that of K/sp2 C (0.08), indicating that SEI contains more Na componentssuch as NaF than K ones. The formation of a Na-rich SEI would be dueto the lower solubility of NaF compared to that of KF as well as preferableNa insertion into NTP electrodes. Therefore, the addition of Na+ ions to K-ion electrolytes would be an effective strategyfor stable SEI formation, which is supported by the cathodic stabilityof Nax1–KxFSA dual-ion electrolytes being higher than that of KFSA electrolyte,as shown in Finally, HAXPES measurements were conducted to investigatesurfacechemistry on the NTP-C electrode surface. observed 8a.48 The3–298eV 8a, suggeectrodes 8b. Moreolectrode S5a,b. Thlectrode S5a,b. Thlectrode S5a,b. Th–1 Na0.55K0.45FSA/H2O and 33 mol kg–1 Na0.45K0.55FSA/H2O. Both Nax1–KxFSAelectrolytes show ionic conductivity of 20–25 mS cm–1, which is much higher than the reported superconcentrated Li solution.The 35 and 33 mol kg–1 Nax1–KxFSA solutions demonstrate thewider potential window of ∼3.5 V compared to that of the end-members.Based on the superior performance of the 33 mol kg–1 K0.55Na0.45FSA/H2O electrolyte,we demonstrated two different aqueous full cells of NTP-C∥KMnHCFand NVP-C∥KMnHCF configurations. The NTP∥KMnHCF cellshows reversible charge/discharge curves with mainly two dischargevoltage plateaus located at 1.7 and 1.3 V and delivers high capacityretention. Although the NVP-C∥KMnHCF cell exhibits capacitydegradation, it demonstrates 2 V-class operations. In these cells,Na+ ions were mainly inserted/extracted into/from the negativeelectrode, and both Na+ and K+ ions were insertedinto the positive electrodes. The HAXPES measurements proved thatthe Na-rich SEI layer was formed on the negative electrode. The SEIwould improve the cathodic stability of the electrolyte.Utilization of the eutectic composition of NaFSA–KFSA realizedhigh concentration solutions of 35 mol kg


The GEI showed a high impact (p < 0.01) on pod yield and an effect (p < 0.05) on seed yield. The environment presented the most significant influence on pod and seed yield variations, followed by genetics and GEI. The total variation in seed yield was 64.22%, composed of PC1 and PC2 values at 45.71% and 18.51%, respectively. The GGE biplot analysis of the yield potentials at each location indicated that KUP12BS029-1-1-3 was the ideal genotype, with a high yield potential and most stability at multilocations, followed by KUP12BS030-3-4-1 and KUP12BS030-1-4-3. These promising lines will be released as new peanut varieties in central Thailand and are recommended as parental lines in breeding programs for large-seeded and yield potential in Thailand and Southeast Asia.The demand by industries for large-seeded peanuts is increasing in Thailand and Southeast Asia. New large-seeded peanut lines were recently developed in Thailand to respond to the demand. In this study, a multilocation yield trial was performed to identify the best genotype(s) in Thailand’s central region and investigate the genotype–environment interaction (GEI) on peanut production. Twelve promising large-seeded peanut lines and two check varieties (KU50 and KK6) were planted at 12 different planting locations during the dry and rainy seasons of 2018 and the dry season of 2019. This study found significant yield potential variability in the promising lines of peanuts evaluated at different planting locations. A combined analysis of variance presented that the environment and genotypes had a considerable impact ( Arachis hypogaea L.) is an important legume crop in Southeast Asia. The market demand for large-seeded peanuts has been increasing, and better quality large-seeded peanut varieties are needed for peanut products, particularly by farmers. Therefore, breeding for yield has been the primary strategy to improve peanut productivity in peanut-growing countries  variety  and Khon Kean 6 (KK6) variety .The two large-seeded check varieties were KK6 and Kasetsart 50 (KU50), recommended by the Thailand Department of Agriculture and often used by Thai farmers. KU50 is resistant to drought and foliar diseases and has a high yield and high seed dormancy.−1 of NPK (13-13-21). Gypsum (CaSO4·2H2O) was applied at early pegging at 312.50 kg·ha−1. The peanut fields were irrigated under drought conditions (Twelve promising large-seeded peanut lines and two check varieties (KU50 and KK6)  were planditions . The podIn the dry season of 2018, the average temperature ranged from 30.5°C to 30.9°C, and the total rainfall was approximately 166.3–380.6 mm. During the rainy season of 2018, the average temperature ranged from 27.7°C to 28.5°C, and the total rainfall was approximately 459.0–732.1 mm. During the dry season of 2019, the average temperature ranged from 30.7°C to 30.9°C, and the total rainfall was approximately 166.6–262.5 mm .The planting area trials were conducted at 12 locations with known environmental differences, namely, 1) plains, 2) river plains, 3) piedmont plains, and 4) upland . The soiThe pod and seed yields were determined based on sampling in the two center rows of each plot, excluding plants at the head and end of each row. The pod yield was based on sun-dried measurements to reduce the moisture content. Dry pod and seed weights were measured and recorded at 8% moisture content.https://www.R-project.org/] for pod and seed yield variance at multiple locations. The analysis of variance was used to compare the means in each environment. Significant differences among the means of yields were compared using Fisher’s protected least significant difference (LSD) at the 5% level of probability. Evaluation for the yield potential and stability was performed based on a GGE biplot graphical user interface package (GGE biplot GUI) using principal component 1 (PC1) and PC2 scores. Visualization of “which-won-where/what” patterns for the multilocation yield trials was used to study genotypes (G) and genotype–environment interactions (GEI).The collected data were analyzed using the R program version 3.6.1  to 4.95 t·ha−1 [Chon Phrai (CP)] (−1) was higher than that of KU50 (2.42 t·ha−1) at Lam Sonthi (LST). The pod yield of the KUP12BS030-4-2-1 (1.42 t·ha−1) peanut line was higher than that of KK6 (0.82 t·ha−1) and KU50 (0.53 t·ha−1) at WT.The number of pods, seeds, 100-seeds weight, pod yield, and seed yield of the 14 large-seeded peanut lines in the 12 planting locations differed significantly . The aveai (CP)] . The pod−1. The averaged seed yield of the KUP12BS029-1-1-3 line was higher than for that of KK6 (20.70%) and KU 50 (26.80%) (−1 (CP) (−1) was higher than that for KU50 (0.82 t·ha−1) and KK6 (0.53 t·ha−1) at SB. The seed yield of KUP12BS030-4-2-1 (0.48 t·ha−1) was higher than that for KU50 (0.18 t·ha−1) and KK6 (0.25 t·ha−1) at WT. However, the seed yield of KUP12BS029-1-1-3 was not different from that of KK6 and KU 50 at each location.KUP12BS029-1-1-3 showed a high performance for the number of pods, number of seeds, pod yield, and seed yield. The average seed yields from the 12 planting locations were significantly different, ranging from 1.45 to 2.17 t·ha(26.80%) . At each(−1 (CP) . The seeThe results of the GGE biplot showed that the total variation of the pod yields was 70.96%, composed of PC1 (47.04%) and PC2 (23.92%) . The PC1The total variation in the seed yield was 64.26%, composed of PC1 and PC2 values of 45.73% and 18.53%, respectively . The linThe analysis for the pod yield of the peanut lines studied at multilocations was facilitated using a “which-won-where” pattern, showing the interaction of genotype to the data sets of different environments at the multilocation yield trials . The polConsidering the seed yield of the lines at each location, the KUP12BS029-1-1-3 (G6) had the highest seed yield at Lam Sonthi (LST), Si Thep (ST), Wang Phloeng (WP), and Khok Charoen (KC) locations . KUP12BSThe average environment axis (AEA) of the biplot  is the lThe combined analysis of variance showed significant effects for all sources of variation for both pod and seed yields, indicating the differential behavior of genotypes that were not consistent with the different environments. These results were consistent with other published reports of peanut yield trials . In the via selection. Similar results were found for the peanut pod weight  and Si Thep (ST) . These rThis study shows the variability of yields in large-seeded peanut lines at multiple locations. Locational variations influenced yield more than variations of GEI and G. For the testing locations, Si Thep (ST) was both discriminating and a representative location that can be considered a suitable testing location for selecting genotypes. The KUP12BS029-1-1-3 line presented a high yield at multilocations. Therefore, the KUP12BS029-1-1-3 line is the large-seeded peanut genotype that is most suitable and should be introduced to farmers because its genotype shows high yield and high stability at multiple locations. These promising large-seeded peanut lines are released as new peanut varieties and used as parental lines in breeding programs for large-seeded yields in Thailand and Southeast Asia.


Loss of RNase H1 is embryonic lethal and to further study its role in mtDNA expression we characterized a conditional knockout of Rnaseh1 in mouse heart. We report that RNase H1 is essential for processing of RNA primers to allow site-specific initiation of mtDNA replication. Without RNase H1, the RNA:DNA hybrids at the replication origins are not processed and mtDNA replication is initiated at non-canonical sites and becomes impaired. Importantly, RNase H1 is also needed for replication completion and in its absence linear deleted mtDNA molecules extending between the two origins of mtDNA replication are formed accompanied by mtDNA depletion. The steady-state levels of mitochondrial transcripts follow the levels of mtDNA, and RNA processing is not altered in the absence of RNase H1. Finally, we report the first patient with a homozygous pathogenic mutation in the hybrid-binding domain of RNase H1 causing impaired mtDNA replication. In contrast to catalytically inactive variants of RNase H1, this mutant version has enhanced enzyme activity but shows impaired primer formation. This finding shows that the RNase H1 activity must be strictly controlled to allow proper regulation of mtDNA replication.The  Rnaseh1 knockout causes severe mtDNA depletion and developmental arrest at embryonic day (E) 8.5 (in vitro study of mtDNA replication initiation at the light-strand origin of replication (OL) with recombinant replication factors revealed an important role for RNase H1 in RNA primer removal  uses for replication initiation  and ptosis, and central nervous system involvement with cerebellar atrophy and ataxia (The ribonuclease H (RNase H) family of enzymes are endonucleases that cleave the RNA component of RNA:DNA hybrids without sequence specificity, and are found in prokaryotes, archaea and eukaryotes . Two mai (E) 8.5 . A study (E) 8.5 . In supp removal . However removal , consist removal . A subseitiation . In addiitiation . Furtheritiation . Constititiation . Althoug RNASEH1 . A ranged ataxia . In thesd ataxia .in vivo role of mouse RNase H1 and report that it is essential both during embryonic development and for the function of differentiated organs, such as the heart and skeletal muscle. Loss of RNase H1 in the heart leads to mtDNA replication stalling with accumulation of linear deleted mtDNA fragments and mtDNA depletion. Ablation of RNase H1 also leads to low levels of mtDNA-encoded transcripts and deficient oxidative phosphorylation (OXPHOS), which is likely related to the marked mtDNA depletion as no transcription elongation or RNA processing defects are observed. We provide evidence that RNase H1 is necessary to restrict RNA replication primer formation to specific sites at the heavy-strand origin of replication (OH) and OL in conditional knockout hearts of the mouse. Finally, we report a mitochondrial disease patient with altered mtDNA replication carrying a novel homozygous pathogenic mutation that makes RNase H1 more active in processing RNA:DNA hybrids than the wild-type enzyme. These findings demonstrate that the RNase H1 activity needs to be carefully balanced to promote accurate mtDNA replication.In this study, we have extensively characterized the Rnaseh1 knockout mice were generated by Taconic Artemis by targeting the gene in embryonic stem cells derived from C57BL/6N using BAC clones from C57BL/6J RPCI-23 BAC library. To generate the Rnaseh1 knockout allele, exon II of the Rnaseh1 locus was flanked by loxP sites. The puromycin resistance cassette was introduced as a selection marker and removed by mating +/loxP-purRnaseh1 mice with transgenic mice ubiquitously expressing Flp recombinase. To obtain germline knockout mice, +/loxPRnaseh1 mice were mated with mice ubiquitously expressing Cre recombinase under the β-actin promotor to generate heterozygous knockout +/−Rnaseh1 mice. The tissue-specific conditional knockout of RNase H1 in heart and skeletal muscle was obtained by crossing +/loxPRnaseh1 mice with transgenic mice expressing Cre recombinase under the control of the muscle creatinine kinase promoter (Ckmm-cre). Animals were housed in a 12-h light/dark cycle at 21°C and fed with a normal chow diet ad libitum. Animal studies were approved by the animal welfare ethics committee and performed in compliance with National and European law. Mice at the age of 8, 16 and 24 weeks were used for biochemical analyses.in vitro studies. Cells were cultured in DMEM GlutaMax (Invitrogen) medium supplemented with 10% fetal bovine serum (FBS) and 1× PenStrep and grown in an incubator at 37°C and 5% CO2. The patient fibroblast line carrying a homozygous c.424G > A (V142I) mutation in the H-domain of RNase H1 was obtained from a patient diagnosed at Karolinska University Hospital. This patient presented with PEO and muscular fatigue. A muscle biopsy specimen from the tibialis anterior muscle at 41 years of age showed a general reduction in the mitochondrial ATP production rate and a combined respiratory enzyme complex deficiency. Histochemical analysis revealed numerous ragged-red and COX-negative muscle fibers. This mutation has been previously reported . This patient was diagnosed at the IRCCS Istituto delle Scienze Neurologiche di Bologna in Italy and had early-onset PEO and ptosis, with mild mitochondrial myopathy and MRI of the brain showed evidence of cerebellar atrophy. The control fibroblast lines, C4 and C5, were obtained from healthy individuals at IRCCS Istituto delle Scienze Neurologiche di Bologna in Italy. For all individuals recruited in Italy, informed consent was obtained for fibroblast cell line establishment and the study was approved by the institutional ethical board .Human embryonic kidney cells (HEK293T) and patient-derived fibroblast lines were used for reported . Controlg for 10 min at 4°C to remove the cell debris and nuclei. The supernatant was collected and spun at 10 000 g for 15 min at 4°C to isolate mitochondria. Crude mitochondrial pellets were resuspended in mitochondria isolation buffer and protein concentration was determined with the Qubit 4.0 fluorometer (Invitrogen).Mitochondria were isolated from mouse heart using differential centrifugation as previously described . Followig for 10 min at 4°C to remove the cell debris. The supernatant was collected and centrifuged at 10 000 g for 10 min at 4°C. The mitochondrial pellet was collected and resuspended in mitochondria isolation buffer without BSA and centrifuged at 10 000 for 10 min at 4°C. Protein quantification was performed with the Qubit 4.0 fluorometer (Invitrogen).Mitochondria were isolated from cultured cells harvested after reaching 80–90% confluency. Cells were washed two times in cold PBS. The cell pellet was resuspended in 8 ml of mitochondria isolation buffer consisting of 20 mM HEPES pH 7.6, 220 mM mannitol, 70 mM sucrose, 1 mM EDTA and freshly added 1× Complete protease inhibitor cocktail (Roche) and 2 mg/ml of fatty acid-free bovine serum albumin (BSA). To facilitate swelling, the cell suspension was incubated on ice for 30 min. Subsequently, homogenization was performed with 40 strokes by using a manual homogenizer. The cell homogenate was centrifuged at 800 g for 7 min at 4°C and washed two times in cold PBS. Mitochondria-, cytosol- and nuclei-containing fractions were obtained by using the Abcam Cell Fractionation Kit according to the instructions. The fractions were further analyzed by SDS-PAGE and western blots. The antibodies used for this experiment are listed in HEK293T cells were cultured until reaching 80–90% confluency. Cells were then harvested, pelleted by centrifugation at 800 ) from HEK293T cells were pelleted, resuspended and incubated for 30 min in 400 μl mitochondria isolation buffer , hypotonic swelling buffer  or mitochondria isolation buffer supplemented with 1% Triton X-100. Each of the three aliquots of mitochondria in different buffers were further subdivided into two aliquots that contained proteinase K at a final concentration of 60 μg/ml or no proteinase K. After 20 min of incubation on ice, protease K activity was inhibited with 2 μl of 0.1 M phenylmethylsulfonyl fluoride (PMSF) added to each sample. Proteins were precipitated with 0.02% sodium deoxycholate and 12% trichloroacetic acid. Finally, samples were analyzed by SDS-PAGE and western blots. The antibodies used for this experiment are listed in For each treatment, 1 mg of isolated mitochondria  according to instructions. Total DNA from E8.5 embryos was isolated by incubation in 24 mM NaOH, 0.2 mM EDTA at 96°C for 40 min. The extracts were neutralized by adding 40 mM Tris (pH 7.8). DNA quantification was performed with the Qubit 4.0 fluorometer (Invitrogen). The mtDNA copy number was measured by qPCR in a QuantStudio 6 Flex Real-Time PCR System using the TaqMan™ Universal Master Mix II with UNG and TaqMan probes for escribed . The mtDd MT-ND1 . The 7S d MT-ND1 .32P]-dCTP-labelled dsDNA probes to detect total mtDNA , 7S DNA, or nuclear 18S rDNA as a loading control. Radioactive signals were visualized using PhosphorImager screens and a Typhoon 7000 FLA .Murine genomic DNA (or mtDNA where indicated), 2 μg (or more where indicated), was digested with one of the following enzymes: SacI, XhoI, BglII or EagI. Human genomic DNA was digested with the EcoRI and BamHI enzymes. Linearized DNA fragments were separated in 0.8% agarose gels and transferred to Hybond-N+ membranes . To release 7S DNA, samples were heated for 3 min at 93°C prior to loading. Next, membranes were hybridized with α- supplemented with fatty acid-free BSA (1 mg/ml) and 1 mM ADP. Mitochondria were then pelleted at 8000 g for 4 min and resuspended in 0.5 ml of prewarmed incubation buffer supplemented with 1 mg/ml of fatty acid-free BSA (1 mg/ml), 1 mM ADP, 50 μM each of dTTP, dCTP and dGTP and 20 μCi α-[32P]-dATP. Next, samples were incubated for 2 h at 37°C on a rotating wheel. After incubation mitochondria were pelleted at 8000 g for 4 min and washed three times with washing buffer . mtDNH-containing (nt 12 034–16 180) or the OL-containing (nt 3102–7084) region.Fresh heart mitochondria were isolated and mtDNA was extracted using the Gentra Puregene Tissue Kit without RNase A treatment. The mtDNA was digested with BclI, precipitated and 3 μg from each sample was loaded onto the first-dimension gels (0.4% agarose without ethidium bromide). Electrophoresis was carried out at 27 V for 18 h at room temperature. The lanes containing DNA were excised, turned 90° counterclockwise and cast with 1% agarose (500 ng/ml ethidium bromide). Second dimension gels were separated at constant 260 mA for 6 h at 4°C. Gels were transferred to nitrocellulose membranes by Southern blotting, and probed with PCR-generated DNA fragments detecting either the O32P]-ATP using PNK (NEB). The primer extension reaction was initiated for 5 min at 95°C and then continued for 25 cycles with 30 s at 95°C, 30 s at 58°C, 45 s at 72°C and 5 min at 72°C. The reactions were terminated and precipitated with ethanol.Total DNA was isolated from the mouse heart tissue with the Gentra Puregene Tissue Kit without RNase A treatment. An 3 μg aliquot of DNA was incubated for 1 h at 37°C with or without RNase H2 (NEB). Primer extension was performed with 2 U Taq DNA polymerase (NEB) in 1× ThermoPol buffer, 200 μM dNTPs and 1.5 pmol labelled primer. The primer, corresponding to nt 15 181–15 201, was 5′-end labelled with γ--dCTP-labelled dsDNA probe to detect 5.8S rRNA and a strand-specific oligonucleotide probe labeled with α-[32P]-dATP to detect 7S RNA. The membrane was used to expose a PhosphorImager screen.Total RNA from mouse heart was extracted using the TRIzol/chloroform extraction method. RNA was quantified with the Qubit 4.0 fluorometer (Invitrogen). RT-qPCR was used to determine in organello replication assay) supplemented with 1 mg/ml of fatty acid-free BSA and 1 mM ADP. Next, mitochondria were resuspended in 1 ml of prewarmed incubation buffer supplemented with 1 mg/ml of fatty acid-free BSA, 1 mM ADP and 60 μCi α-[32P]-UTP. Samples were further incubated 1.5 h at 37°C on a rotating wheel. Mitochondria were pelleted, resuspended again in prewarmed incubation buffer containing 1 mg/ml of fatty acid-free BSA, 1 mM ADP and non-radioactive UTP and incubated for an additional 10 min at 37°C. Finally, samples were washed three times in washing buffer (see the in organello replication assay). The mtRNAs were extracted using the TRIzol/chloroform extraction method. Samples were separated in 1% MOPS-formaldehyde agarose gel, transferred to Hybond-N+ membrane and the signal was visualized by autoradiography. To ensure equal input, aliquots of mitochondrial proteins, taken before mtRNA isolation, were separated by SDS-PAGE and transferred to a PVDF membrane which was hybridized with an antibody against the mitochondrial HSP60 protein.Freshly isolated mitochondria (1 mg) from mouse heart were washed twice in 0.5 ml of cold incubation buffer  with HISAT2  were resuspended in 4x Lämmli buffer , separated by SDS-PAGE  and transferred to PVDF membranes. Immunoblotting was done according to standard procedures. Primary antibodies used for immunoblotting are listed in the in-gel activity staining for complexes I, II, and IV. In-gel activity assays were performed as previously described (Around 75 μg of isolated mitochondria were lysed in 50 μl of solubilization buffer  and mixed with loading dye . Blue native–polyacrylamide gel electrophoresis samples were resolved on 3–13% gradient gels and further subjected to escribed .Skeletal muscle sections were incubated in freshly prepared COX medium . Following incubation for 45 min at 37°C, slides were washed three times in PBS. Next, slides were incubated in freshly prepared SDH medium  for 30 min at 37°C. Finally, slides were washed three times with PBS, dehydrated  and mounted for bright-field microscopy.4 pH 7.1, 10% glycerol, 1 mM DTT and 1× protease inhibitors), and the proteins were eluted in a 20-ml salt gradient (0.2–1.2 M NaCl in purification buffer). All other recombinant proteins were expressed and purified as described previously  was cloned into a pBacPAK9 vector (Clontech). The mutant RNase H1 versions, Y29C and V142I were generated by site-directed mutagenesis (Agilent Technologies). Recombinant wild-type and mutant human RNase H1 proteins were expressed in Sf9 insect cells as previously described . The proeviously .2 and 10 mM DTT), 20 fmol substrate, and increasing concentrations of wild-type or mutant RNase H1 proteins (10–300 fmol). The reactions were incubated at 37°C for 60 min and then stopped by the addition of 5 μl stop buffer  followed by incubation at 42°C for 45 min. The samples were recovered by ethanol precipitation in the presence of 0.5-volume ammonium acetate (7.5 M) and dissolved in 10 μl of TE buffer. The samples were then treated with 300 mM KCl or KOH for 2 h at 55°C and stopped with 2× stop buffer .Nuclease activity reactions were performed on an 80 nt DNA template annealed to a 3′ end labelled 52 nt chimeric oligonucleotide that contained 26 ribonucleotides followed by 26 deoxyribonucleotides (26RNA:26DNA). The reactions (20 μl) contained 1× RNase H buffer , 4 nM of indicated plasmid template, 20 nM POLRMT, 200 nM TFAM, 60 nM TFB2M and 40 nM TEFM when indicated. The reactions were incubated at 32°C for 5 min and stopped by the addition of 200 μl stop buffer  followed by incubation at 42°C for 45 min. The transcripts were recovered by ethanol precipitation and the pellets were dissolved in 20 μl gel loading buffer  and heated at 95°C for 3 min. The samples were analyzed on 4% denaturing polyacrylamide gels (1× TBE and 7 M urea) followed by exposure on photo film. Low Molecular Weight DNA Ladder (NEB) was used as a size marker.All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl pH 8.0, 10 mM MgCl2, 50 mM NaCl, 100 μg/ml BSA, 10 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 150 μM UTP, 100 μM dATP, 100 μM dGTP, 100 μM dCTP (or ddCTP if indicated), 10 μM dTTP, 0.027 μM α-[32P]-dTTP (3000 Ci/mmol) and 8 nM of indicated template. All reactions (unless otherwise stated) contained 200 nM of TFAM, 60 nM of TFB2M, 20 nM of POLRMT, 20 nM of D274A POLγA exo- and 40 nM POLγB. Wild-type or mutant RNase H1 proteins were added in increasing concentrations as indicated , and mtSSB was added at 40 nM unless otherwise stated. The reactions were incubated at 32°C for 30 min. Experiments with dCTP were stopped and evaluated as described for transcription reactions. Experiments with ddCTP were stopped by the addition of 5 μl stop buffer to a final concentration of 10 mM Tris-HCl pH 8.0, 0.2 M NaCl, 1 mM EDTA, 660 μg/ml glycogen (Roche) and 100 μg/ml proteinase K (Ambion) followed by incubation at 42°C for 45 min. The reactions were treated with KOH (300 mM) for 2 h at 55°C. The samples were recovered by ethanol precipitation in the presence of 0.5 volumes ammonium acetate (7.5 M), dissolved in 10 μl gel loading buffer , and heated at 95°C for 3 min. The products were analyzed on 6% denaturing polyacrylamide gels (1× TBE and 7 M urea) for samples with dCTP or 12% denaturing polyacrylamide sequencing gels (1× TBE and 7 M urea) for samples with ddCTP. The gels were exposed on photo film.All reaction volumes were 20 μl and contained 25 mM Tris–HCl pH 8.0, 10 mM MgClin vitro studies, oligonucleotides were PAGE purified (Eurofins MWG Operon) with the exception of the primer extension oligonucleotide. Oligonucleotides were radiolabelled on the 3′-end. A 51 nt long 26RNA:25DNA oligonucleotide was radiolabelled to generate a 52 nt long 26RNA:26DNA oligonucleotide using terminal transferase (NEB) and α-[32P]-dCTP, followed by 3′ overhang removal using T4 DNA polymerase (NEB).For all Key resources and reagents are listed in supplementary materials, RNase H1 has been reported to be localized to both the nucleus and mitochondria in mice and humans ,15. Howein vivo role of RNase H1, we generated a conditional mouse knockout allele where exon II of the Rnaseh1 gene was flanked with loxP sites  were obtained by crossing +/loxPRnaseh1 mice with β-actin-cre expressing mice. Intercrosses of +/–Rnaseh1 mice produced no viable homozygous knockouts . In line with a previous study  were small and poorly developed in comparison with wild-type (+/+Rnaseh1) embryos at E9.5 . Reverse transcription PCR (RT-PCR) analyses showed that exon 2 was lacking in Rnaseh1 transcripts in the knockout hearts , progressing to a profound reduction at 24 weeks of age (∼4% of wild-type levels) Figure . The mtDRnaseh1 knockout mice prompted us to further investigate the regulation of mtDNA replication. Analysis by in organello replication assays showed that linear deleted mtDNA fragments were generated as by-products during ongoing mtDNA replication in heart mitochondria lacking RNase H1   high-throughput computational pipeline for analysis , as previously described  Figure . We also1 Figure . To deveanalysis . We founs Figure , whereasescribed . Our resRnaseh1 knockout heart DNA to map the ends of the linear deleted mtDNA molecules. The linear deletions were shown to contain sequences corresponding to the major arc region of mtDNA  generated terminated products at different locations in the D-loop region in Rnaseh1 knockout hearts that were absent in control mice  enzyme activity in skeletal muscle showed profound COX deficiency in Rnaseh1 knockout mice  domain or compound heterozygous for mutations in the H-domain and the connection domain (CD)  of RNase H1  Figure . In a re1 Figure . No mutas Figure , multipls Figure  but norms Figure . Southers Figure  23). Ho. Hoin vis Figure , demonsts Figure . As a cos Figure  caused bs Figure  and skels Figure , similars Figure . Interesescribed . A normaescribed . The patescribed  consisteA Figure , D. The s Figure , showingin vitro. First, we characterized the RNase H1, RNase H1:Y29C and RNase H1:V142I variants on a defined RNA:DNA hybrid template. RNase H1:Y29C was more active than the wild-type enzyme  strand synthesis initiates. Replication initiation at OH depends on transcription from LSP that results in an R-loop that is processed by RNase H1 to form RNA primers  of mtDNA replication , replica primers . The 3′  primers . In the 1 Figure . In agreRNase H1  and anotRNase H1 . MoreoveRNase H1 .H by promoting the formation and stabilization of the R-loop transcribed from LSP (in vivo. Moreover, RNA-seq shows that RNase H1 is not important for removing R-loops, that potentially would block transcription elongation, or for ribosomal RNA processing, at variance with other reports (We have previously shown that mtSSB plays a major role in controlling the replication/transcription switch at Ofrom LSP . In the from LSP . These ffrom LSP . Our dat reports . Given t reports , we propIn addition to the well documented role for RNase H1 in replication initiation, it is also likely important for mtDNA replication completion. When mtDNA replication has progressed around the circle, POLγ will encounter and displace the 5′ end where replication started to create a ∼100 nucleotides long DNA flap ,58. OnceE. coli RNase H1 enzyme (in vivo. Skin-derived fibroblasts from mitochondrial disease patients carrying mutations in the HBD or H-domain have defects in de novo mtDNA replication. Interestingly, mutations in the HBD and H-domain have opposite effects on the RNase H1 cleavage activity in vitro, causing significantly increased or drastically decreased enzyme activity, respectively. These results show that the RNase H1 activity must be tightly regulated in vivo. The HBD of RNase H1 may therefore not only have a role in controlling enzyme processivity, as previously proposed (Eukaryotic RNase H1 has an HBD, a CD and an H-domain  Figure . The HBD1 enzyme ,66 that proposed ,66, but in vivo, i.e. replication initiation and replication completion. Lack of RNase H1 activity leads to mtDNA replication defects and the formation of linear deleted mtDNA fragments but does not directly affect transcription or posttranscriptional RNA processing in mammalian mitochondria.In conclusion, we demonstrate that a carefully balanced RNase H1 activity is essential for regulating two crucial steps of mtDNA replication The mtDNA sequencing data, HydEn sequencing data and RNA sequencing data have been deposited in Gene Expression Omnibus under accession number GSE199771, GSE199198 and GSE199147, respectively.gkac661_Supplemental_FileClick here for additional data file.


The Ni1−xO based RRAM devices exhibited both unipolar and bipolar resistive switching characteristics without an electroforming step. Auger electron spectroscopy showed nickel deficiency in the Ni1−xO films, and X-ray photoemission spectroscopy showed that the Ni3+ valence state in the Ni1−xO films increased with increasing oxygen partial pressure. Conductive atomic force microscopy showed that the conductivity of the Ni1−xO films increased with increasing oxygen partial pressure during deposition, possibly contributing to the reset-first switching of the Ni1−xO films.Reset-first resistive random access memory (RRAM) devices were demonstrated for off-stoichiometric Ni It wa2 [2O3 ,7, HfO2  [2O3 [2 ,9,10, NiO3 [2 [x ,12,13,14[2 [x [x ,16, TaOx[x [x [x ,18, Nb2O [x [2O5 ,20, and xCaxMnO3 ,22,23 shxCaxMnO3 ,26,27,28xCaxMnO3  and pulsxCaxMnO3  were usexCaxMnO3 ,32. NiO behavior ,34. It id states . Therefod states ,37. The d states . It was filament ,40,41. Tfilament . Moreovefilament ,44,45.1−xO films for unipolar and bipolar RSs . Particularly, it was demonstrated that nickel-deficient Ni1−xO films deposited under excessive oxygen partial pressure exhibit a reset-first RS without an electroforming step. An RRAM device with a reset-first RS could be an alternative to overcome the limitations of RRAM requiring an electro-forming step.This study investigated the RS characteristics of off-stoichiometric Ni2 on a Si substrate using DC magnetron sputtering. Pt or TiN films were then deposited as bottom electrodes (BE). BE with various areas of 0.18~4.0 µm2 were formed to investigate the area-dependence of the electrical characteristics. After BE formation, off-stoichiometric Ni1−xO films with a thickness of 10 nm were deposited via reactive RF magnetron sputtering using a Ni target under various O2 partial pressures. During sputtering, the base and working pressures were less than 3 × 10−3 and 3 mTorr, respectively. During deposition, the RF power and temperature of the substrate were main-tained at 100 W and 400 °C, respectively. The fraction of the O2 partial pressure in the mixture of Ar and O2 varied from 10% to 50% for deposition. Finally, Pt top electrodes (TEs) with a thickness of 100 nm were formed using DC magnetron sputtering and a lift-off process. The electrical characteristics of the device were characterized using a Keysight B1500A analyzer at 21~23 °C. RS under DC bias was measured with a com-pliance current of 10 mA to avoid hard breakdown of the Ni1-xO films. The spatial distribution of conductivity in the pristine state was investigated using conductive atomic force microscopy (C-AFM)  with a measurement bias of 3 V [1−xO films, respectively.MIM devices with Pt/NiO/Pt and Pt/NiO/TiN stacks were fabricated for electrical characterization. First, Ti/TiN adhesive layers with thicknesses of 10–50 nm were deposited onto SiOs of 3 V ,47. Graz1−xO films. The XRD patterns of Ni1−xO films deposited under various O2 fractions are illustrated in 2 partial pressure fraction of 50% showed lower intensity with a more comprehensive full-width half maximum (FWHM), implying poorer crystallinity of NiO films. The XRD peak of the (111) plane shifted to lower diffraction with increasing O2 partial pressure, indicating an increase in the lattice constant with increasing O2 partial pressure, as shown in 2 partial pressure [1−xO films with various O2 partial pressures during deposition. The volume of Ni is gradually reduced with increasing O2 partial pressure, resulting in a Ni-deficient Ni1−xO film. The compositions of nickel oxide at 10% and 50% O2 partial pressures were estimated to be Ni0.89O and Ni0.86O, respectively.XRD analysis was conducted to investigate the crystallinity of Nipressure ,49,50. F1−xO/Pt stacks. The pristine Ni1−xO films deposited under an O2 partial pressure fraction of 10% offered an initial high resistivity [form) and set voltage (Vset) was approximately 0.57 V. In contrast, pristine Ni1−xO films deposited under the 30% or 50% O2 ratio showed low resistance in the pristine state without the electroforming step and reset-first RS behavior, where the initial LRS state was changed to the HRS state, as shown in set is similar to that of Ni1−xO films for the O2 partial pressure fraction of 10%, the IHRS/ILRS ratio decreased because of the overall high current level in the HRS state. In particular, the IHRS between these oxygen partial pressure fractions showed that the 50% O2 ratio was 10 times higher than that of 30% O2. The I-V curves of TiN/Ni1−xO/Pt stacks are plotted in 1−xO film deposited under a 10% O2 partial pressure fraction show BRS [1−xO film showed high resistivity, and the resistance state changed to LRS after the electroforming step with a negative bias on TE. The difference between Vform (−4.0 V) and Vset (−0.7 V) was approximately 3.3 V. On the contrary, the Ni1−xO film deposited under the 30% or 50% O2 partial pressure fraction showed reset-first BRS behavior for a positive voltage on the TE, as shown in istivity  at an apshow BRS  characte1−xO/TiN stacks in the LRS and HRS states, where Ni1−xO films were deposited at various O2 partial pressures. The mean values of IHRS and ILRS (red line) increased with the O2 ratio, suggesting that the Ni1−xO film conductivity depends on the O2 partial pressure, as shown in 1−xO films with a 10% O2 fraction required electroforming for resistive switching, but the Ni1−xO films with a 30% O2 fraction or higher showed reset-first RS behavior without electroforming. HRS with O2 partial pressure, but the slope was lower than that of the IHRS state. The IHRS and ILRS showed the highest values for Ni1−xO films deposited under the 50% O2 partial pressure fraction.2 at a bias of ±0.48 V. 1−xO films deposited by 10% O2 partial pressure fraction. The resistance of the HRS remained almost constant with decreasing geometric device area, while that of the LRS is almost independent of the device area. These area-independent characteristics imply that resistance switching through the device occurs in local regions, such as filament paths, rather than homogeneously distributed switching paths [1−xO films deposited at 50% O2 partial pressure showed increased dependence on the device area, as shown in 1−xO films with 50% O2 partial pressure is close to that of Ni1−xO films with 10% O2 partial pressure, the nature of the RS is filamentary in the local area. The significant dependence of HRS on the Ni1−xO films with 50% O2 partial pressure is attributed to the reduced resistance of the Ni1−xO films, as shown in To understand the nature of resistance switching, HRS and LRS resistances were measured from devices with BE of 0.18, 0.38, 2.00, and 3.69 μmng paths ,55,56,571−xO device are shown in read) of ±0.25 V under a compliance current of 10 mA. The measured IHRS/ILRS ratio is higher than 101 even after 103 cycles. read of 0.3 V conditions. The device has a uniform IHRS/ILRS ratio even after 105 cycles, which results in a stable RS property.The DC, and AC endurance characteristics of the Ni1−xO film conductivity. 2/Pt stacks were simultaneously formed on a sample to compare the differences during the current image mapping. Cross-sectional TEM images of the Ni1−xO films for C-AFM measurements are shown in 1−xO thickness on the SiO2/Pt stacks was estimated to be within 15%. Therefore, we ignore the difference in conductivity due to thickness variation. 1−xO films deposited under various O2 partial pressures. The left region of each mapping image represents the reference of the insulating SiO2 between the BEs and Ni1−xO films. The regions on the right represent the Ni1−xO films on the Pt BEs in their pristine state. Similar to the I-V characteristics of MIM devices, C-AFM showed an increased current through the Ni1−xO films with increasing O2 partial pressure. The conductive regions in the Ni1−xO film regions increased with increasing O2 partial pressure fraction, as shown in 1−xO film with a 50% O2 fraction. In contrast, films deposited under 10% O2 partial pressure fraction showed improved resistivity, as shown in C-AFM measurements investigated the two-dimensional (2D) variation of the Ni2 partial pressure on the chemical bonding states in the Ni1−xO films is investigated through XPS analysis. 3/2 peaks of Ni1−xO films deposited with various O2 partial pressures. Ni0, Ni2+ and Ni3+ states with binding energies of 852.5, 853.7, and 855.5 eV, respectively, are used for deconvolution of Ni 2p3/2 peaks [The effect of the O/2 peaks ,59.3+ state was estimated from the ratio of the Ni3+ peak area to the Ni2+ peak area. The Ni3+ valence state increased while the fraction of Ni2+ ions decreased with increasing O2 partial pressure 2Nve paths ,60,61. F1−xO films were investigated. The RS behavior without the electroforming step was observed in the unipolar and bipolar off-stoichiometric Ni1−xO films. Ni3+ distribution contributes significantly to the conductivity of the pristine Ni1−xO films. The conductivity and Ni deficiency of pristine Ni1−xO films increased as the O2 partial pressure increased during a deposition as revealed by the C-AFM and AES results. Moreover, Ni2+ was further oxidized to Ni3+ as the O2 partial pressure increased, as revealed by the XPS results.In this study, the reset-first RS characteristics of off-stoichiometric Ni2O3 bonding by Ni3+ ions is related to the reset-first RS behavior without the electroforming step. This is advantageous in terms of device scale-down, making Ni1−xO films promising candidates for memory applications by overcoming the limitations of the electroforming step in RRAM.The Ni


SIX3 cause variable holoprosencephaly in humans and mice. We identified two children with neonatal hypopituitarism and thin pituitary stalk who were doubly heterozygous for rare, likely deleterious variants in the transcription factors SIX3 and POU1F1. We used genetically engineered mice to understand the disease pathophysiology. Pou1f1 loss-of-function heterozygotes are unaffected; Six3 heterozygotes have pituitary gland dysmorphology and incompletely ossified palate; and the Six3+/−; Pou1f1+/dw double heterozygote mice have a pronounced phenotype, including pituitary growth through the palate. The interaction of Pou1f1 and Six3 in mice supports the possibility of digenic pituitary disease in children. Disruption of Six3 expression in the oral ectoderm completely ablated anterior pituitary development, and deletion of Six3 in the neural ectoderm blocked the development of the pituitary stalk and both anterior and posterior pituitary lobes. Six3 is required in both oral and neural ectodermal tissues for the activation of signaling pathways and transcription factors necessary for pituitary cell fate. These studies clarify the mechanism of SIX3 action in pituitary development and provide support for a digenic basis for hypopituitarism.Congenital hypopituitarism is a genetically heterogeneous condition that is part of a spectrum disorder that can include holoprosencephaly. Heterozygous mutations in   The anterior and posterior lobes of the pituitary gland develop from oral and neural ectoderm, respectively, under the control of embryonic signaling pathways and lineage-specific transcription factors. Variants in more than 60 genes have been reported in patients with combined pituitary hormone deficiency (CPHD), and most of the genes have roles in pituitary and/or hypothalamic development . BecauseOTX2), aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), chromodomain helicase DNA binding protein 7 (CHD7), paired box 6 (PAX6), roundabout guidance receptor 1 (ROBO1) and LIM homeobox 4 (LHX4)  is a congenital midline defect defined as thinning or disappearance of the pituitary stalk, hypoplasia of the anterior pituitary and ectopic posterior pituitary . PSIS is4 (LHX4) . The extSHH, Sine oculis homeobox homolog 3 (SIX3), zinc finger protein of the cerebellum 2 (ZIC2), TGFB induced factor homeobox 1 (TGIF1) and patched 1 (PTCH1) (SHH and TGIF) also cause PSIS  is a developmental defect characterized by a failure of the forebrain to cleave into cerebral hemispheres and ventricles during the prenatal period . This de (PTCH1) . Mutatiouse PSIS .Six3 and Hesx1, a transcription factor associated with CPHD and septo-optic dysplasia, has been reported in mice  and Foxg1  in two children with PSIS and CPHD. Here we report that SIX3 p.P74R/+ and POU1F1 p.S50A/+ , and mutations in either the SHH enhancer or SIX3 can cause HPE (FOXG1), and this process is evolutionarily conserved . There is no significant difference in the weight of male or female +/−; Pou1f1Six3+/dw double heterozygotes compared with single heterozygotes and wild-type controls at 3 or 6 weeks (P = 0.11–0.87), (+/−Six3 (1%) or the other genotypes  (P = 0.0094). The animals with poor viability appeared sickly and frail and/or had hydrocephaly. We analyzed pituitaries of surviving mice at 6 weeks (N = 106). Modest abnormalities were noted in some +/−Six3 mice (12%). However, more obvious pituitary dysmorphology, including extension of pituitary tissue through the palate, was observed in 34% of +/−Six3; Pou1f1+/dw mutants , . However mutants , Table 2N = 48)  and +/−Six3; Pou1f1+/dw mice  compared with either wild type  or Pou1f1+/dw mice   . The pal66 ± 4%) . Thus, S+/−Six3 and +/−Six3; Pou1f1+/dw embryos had an abnormal number of mesenchymal cells between these tissue layers and a highly bifurcated marginal zone at E14.5, consistent with an epithelial to mesenchymal transition defect , a marker of cell cycle exit, are normally concentrated in a small area on the ventral side of the lumen, and both +/−Six3 and +/−Six3; Pou1f1+/dw mutants have abundant p57 positive cells on the dorsal side of the lumen  and adult mouse pituitaries. The neural and oral ectoderm are normally juxtaposed throughout pituitary development, but both n defect . At E14.n defect . Cells in defect . We anal embryos . Considehe lumen . We did hat time . There what time .Six3 haploinsufficiency can substantially affect pituitary morphology and growth, and haploinsufficiency for both Six3 and Pou1f1 exacerbates the phenotype.Each of the expected hormone-producing cell types was present in both newborn  and adulSix3 function in pituitary development, we used a well-characterized Prop1-cre bacterial artificial chromosome transgenic mouse line to disrupt Six3 in Rathke’s pouch (Prop1-cre and the Rosa tdTomato reporter gene (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J) exhibited tdTomato expression in Rathke’s pouch and not the adjacent ventral diencephalon. In addition, SIX3 immunostaining was not detected in Rathke’s pouch of fl/fl; Prop1-creSix3 mutants at E12.5, but it was present the ventral diencephalon (N = 7) and E11.5 (N = 14) (To investigate the mechanism of ’s pouch . We conf’s pouch . Embryos(N = 14) .fl/fl; Prop1-creSix3 mutants, and LHX3 expression was absent (N = 4/5) or markedly reduced (N = 1/5), indicating early disruption of pituitary fate specification (N = 7/16) to multiple ectopic invaginations extending rostrally along the oral ectoderm (N = 9/16)   . To deteN = 4/8) . The morSix3 can interact genetically with Hesx1, causing activation of WNT signaling and hypopituitarism associated with pituitary gland dysmorphology  at E12.5  or reduced (N = 2/6) in fl/fl; Prop1-creSix3 mutants. The canonical WNT signaling pathway stabilizes β-catenin and activates downstream genes such as Axin2. Axin2 is expressed in the dorsal hypothalamus, and there were no differences in expression among genotypes , fibroblast growth factor (FGF), SHH and WNT signaling are important for induction of Rathke’s pouch . We analcre mice . The pat and FGF . Shh traenotypes . Six6 is mutants . Thus, Sfl/fl; Prop1-creSix3 mutants continue to exhibit varying levels of rudimentary pouch development with multiple invaginations  to clarify the role of hypothalamic Six3 expression in pituitary development. The efficacy and penetrance of cre-mediated recombination with the Nkx2.1-cre strain is excellent  or expressed in only a few cells in the rostral and ventral side of invaginated oral ectoderm (N = 2/3)  . The arefl/fl; Nkx2.1-creSix3 embryos had no obvious formation of infundibulum and no Rathke’s pouch structure, or only a tiny rudiment marked by PITX1 and LHX3  or was weakly expressed dorsal to the expected area in the infundibulum (N = 1/4 samples) . TL. TLSix3fsamples) .fl/+; Nkx2.1-creSix3 embryos exhibit dysmorphology . Thus, fl/fl; Nkx2.1-creSix3 embryos are not viable, possibly due to a lack of pituitary hormone production  underlying the disappearance of the pituitary primordium. Apoptotic cells, defined as cleaved caspase-3-positive cells, were increased in the invaginated oral ectoderm, especially in the caudal side, in at E10.5 . At E11.’s pouch . There wtuicytes . The exptuicytes .fl/fl; Nkx2.1-creSix3 embryos did express Otx2 in the expected area of the infundibulum and diencephalon  than other genotypes  is a transcription factor that is involved in the early patterning of the mammalian hypothalamus . Rax knopon Six3 . We hypopon Six3 . RNAscop signals . MYCN is signals ,76. Exce signals ,78.Hes1, a gene downstream of Notch signaling, is required for normal placement of AVP neurons  regulates the specification of the infundibulum and organization of differentiated cells within the anterior and intermediate lobes of the pituitary  or weak expression (N = 2/5) of Lhx2 in the infundibulum, implying that low Lhx2 expression could contribute to the failure of posterior lobe formation  is expressed in the ventral diencephalon and is another essential signaling molecule and induces the formation of Rathke’s pouch from the oral ectoderm . Express embryos .Shh by SOX2 at the distal Shh enhancer, SBE2. Tbx3-deficient mice lack a posterior lobe, and Rathke’s pouch degenerates  or weak (N = 3/5) Tbx3 expression in the ventral diencephalon  or normal (N = 3/5) Shh expression in the diencephalon , and activation of differentiation through regulation of Tbx3 and Lhx2. Six3 is necessary for expression of Otx2, which is in turn necessary for stimulating BMP and FGF signaling. fl/flOtx2; Nkx2.1-cre mutants had no changes in Shh expression —Conception Hospital , and a declaration was made to the National Commission for Data Protection and Liberties (CNIL-France): 1991429 v 0. Patients or their parents signed a written informed consent to participate. The University of Michigan Institutional Review Board (UM) found the study exempt because patient DNA samples were anonymized before exome sequencing at UM. Exome sequencing was done as described .A non-consanguineous family had five children, and three of them had congenital hypoglycemia and were diagnosed with TSH and GH deficiency . II-2 had mild dysmorphic features and attended normal school. She entered puberty at 9 1/2 years and obtained a final height of 149 cm with GH and thyroid hormone replacement. An MRI in the neonatal period indicated the possibility of PSIS or thin stalk, and at 2 years of age, her MRI showed thin stalk, anterior pituitary hypoplasia and small olfactory bulb. II-3 also had mild dysmorphic features and attended normal school. His MRI detected no abnormalities other than a thin pituitary. He entered puberty at 10 years 3 months and was prescribed GnRH antagonist therapy. He is still growing with GH and thyroid hormone replacement. II-5 had neonatal GH and thyroid hormone deficiency, some dysmorphic features and delayed language acquisition. MRI was not performed. The two children without hormone deficiencies had some syndromic features. Individual II-1 had congenital nystagmus and severe visual impairment with normal development and learning, while II-4 had congenital nystagmus and autism with normal brain MRI. The father had a son from a previous marriage who had no known medical condition. The father’s height is 180 cm and the mother’s height is 165 cm.4 cells per well were seeded in a 24-well plate 24 h before transfection. Plasmids were transiently transfected into 3 T3 cells using ViaFect Transfection Reagent . Forty-eight hours after transfection, cells were washed with 1× phosphate-buffered saline  and treated with lysis buffer for luciferase assays. Luciferase activities were measured according to the manufacturer’s recommendation . Statistical analyses were performed using JMP Statistical Database Software version 12.2.0 . Data are presented as mean ± standard error of the mean calculated from triplicate wells. Significant differences between samples were assessed using the analysis of variance (ANOVA). P-values < 0.05 were considered statistically significant.3T3 cells were obtained from the American Type Culture Collection. Cells were maintained in Dulbecco’s modified eagle medium  containing 10% fetal bovine serum (Gibco). Human SIX3 expression vectors  were subcloned into pcDNA3.1+/C-DYK . SHH brain enhancer 2 (SBE2) sequence was obtained from Dr Douglas J. Epstein, U. Penn . This seSix3+/− mice and Six3fl/+ mice , and we received them on outbred genetic backgrounds, CD1 and NMRI, respectively. The DW/J- +/dwPou1f1, C57BL/6 J and B6-Tg(Nkx2.1-cre)2Sand/J, Nkx2.1-cre mice (stock number 008661) (Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (stock number 007909) were obtained from Jackson Laboratory, Bar Harbor, ME. The DW/J inbred genetic background has single nucleotide polymorphisms that match C57 substrains better than other inbred strains tested , referred to here as Prop1-cre, were generated at the University of Michigan by injecting fertilized eggs from a cross of C57BL/6 J and B6D2F1 mice and then maintained on a C57BL/6 J background  , and C57s tested . Six3+/−Six3 null allele with the forward oligo (5′- CTTGGGTGGAGAGGCTATTCG-3′) and reverse allele (5’-GATCCTGCAGGTACCACTCC-3′), using the following amplification parameters: 92°C 2 min, followed by 35 cycles of 92°C, 30 s; 57.5°C, 30 s; 72°C, 30 s and 1 cycle of 72°C, 10 min Six3 null allele produces a band of 933 bp. PCR was used for the genotyping for Six3 flox allele with the following primer pair (forward/reverse): 5′- CGGCCCATGTACAACGCGTATT -3′/5’-CCCCTAGCCTAACCCAAACATTCG -3′, using the following amplification parameters: 95°C 2 min, followed by 35 cycles of 95°C, 30 s; 59.2°C, 30 s; 72°C, 30 s and 1 cycle of 72°C, 4 min. Six3 wild type and flox alleles produced 405 and 487 bp, respectively. +/dwPou1f1 genotyping was previously described  with or without MOM kit . Antibody information, dilutions and detection methods are listed in In situ hybridization (ISH) was performed on sections of formaldehyde-fixed paraffin-embedded tissues as previously described . Probe iAxin2, cat#400331; 45 min, Ctgf, cat#314541, 45 min; Cyr61, cat#429008; Bmp4, cat#425011, 1 h.RNAscope was performed using the protocol and reagents, including the RNAscope 2.5 HD Red Detection kit, from Advanced Cell Diagnostics (Cat#322360). Sections were treated with the hydrogen peroxide reagent for 10 min at room temperature and then rinsed in water. Antigen retrieval was performed with the target retrieval reagent in the microwave for 15 min, immediately removed and then washed in 100% ethanol. Sections were dried at room temperature. Samples were treated with Protease Plus for 30 min at 40°C, washed in MilliQ water and then incubated with probe or a negative control for 2 h. Samples were washed in 1× wash buffer, and then steps AMP1–AMP4 were performed at 40°C as per the manufacturer’s protocol washing with 1× wash buffer in between steps. Sections were incubated in AMP5 at room temperature for 30 min to 1 h depending on the probe, see notes below. After washing, sections were incubated in AMP6 for 15 min, followed by a 10 min incubation in Fast Red A:Fast Red B (60:1). After washing in MilliQ water, samples were counterstained with 1:8 dilution of hematoxylin in water. The probes used and incubation times in AMP5 are as follows: Images of immunostaining and ISH obtained with a DFC7000 T . Images of the skeletal preps were obtained with DFC310 FX (Leica). Images were processed using LasX software (Leica Microsystems).P-value of <0.05 was considered statistically significant.Statistical analyses were performed using JMP Statistical Database Software version 12.2.0 (SAS Institute). ANOVA, Fisher’s exact test and Pearson’s chi-square test were used as appropriate. A Experiments in mice were conducted under the IACUC approval number PRO00008702. Studies on patient samples were conducted with written informed consent prior to the start of experiments. Patient studies are approved by the National Commission for Data Protection and Liberties (CNIL-FRANCE), number 1991429.Bando_SupplementalMaterial_revised_ddac192Click here for additional data file.