Mucilago crustacea was investigated and found to contain seven group I introns, including a direct repeat-containing intron at insertion site S1389 in the small subunit ribosomal RNA gene. We collected, analyzed, and compared 72 S1389 group IC1 introns representing diverse myxomycete taxa. The consensus secondary structure revealed a conserved ribozyme core, but with surprising sequence variations in the guanosine binding site in segment P7. Some S1389 introns harbored large extension sequences in the peripheral region of segment P9 containing direct repeat arrays. These repeats contained up to 52 copies of a putative internal guide sequence motif. Other S1389 introns harbored homing endonuclease genes in segment P1 encoding His-Cys proteins. Homing endonuclease genes were further interrupted by small spliceosomal introns that have to be removed in order to generate the open reading frames. Phylogenetic analyses of S1389 intron and host gene indicated both vertical and horizontal intron transfer during evolution, and revealed sporadic appearances of direct repeats, homing endonuclease genes, and guanosine binding site variants among the myxomycete taxa.Nuclear group I introns are restricted to the ribosomal DNA locus where they interrupt genes for small subunit and large subunit ribosomal RNAs at conserved sites in some eukaryotic microorganisms. Here, the myxomycete protists are a frequent source of nuclear group I introns due to their unique life strategy and a billion years of separate evolution. The ribosomal DNA of the myxomycete  Tetrahymena thermophila group IC1 ribozyme and the Didymium iridis group IE ribozyme, respectively, have been published . Here, exoG attacks the 5′ SS and becomes covalently ligated to the 5′ end of intron RNA. In the second reaction the terminal intron nucleotide (ωG) replaces exoG at the G-site and becomes attacked by the free 3′ hydroxyl group of the upstream exon. The outcome of splicing is exon ligation and intron excision. In addition, some nuclear group I introns generate circular intron RNA molecules, either as truncated circles  or full-Physarum polycephalum and D. iridis [Homing at the DNA-level is an efficient intron mobility process linked to sexual mating. Group I intron homing is initiated by a double-strand break at the intron-lacking allele generated by an intron-encoded homing endonuclease and completed by gene conversion using the intron-containing allele as a template [reviewed in 3]. There are several distinct families of homing endonucleases, and mobile nuclear group I introns encode homing endonucleases of the His-Cys family ,13. Homi. iridis ,15. Reve. iridis . Reverse. iridis ,18, but Mucilago is a genus in the order Physarales that contains only one species, M. crustacea [M. crustacea collected in sub-Arctic Norway. DNA sequencing revealed seven group I introns; two in the SSU rRNA gene (insertion sites S788 and S1389) and five in the LSU rRNA gene . The intron at insertion site S1389 contained a complex structural organization and was studied in more detail. In an earlier report we showed that the myxomycete S1389 introns constituted a monophyletic clade (the S1389 family) among nuclear group I introns [Myxomycetes  are unicellular eukaryotes classified as a distinct and ancient protist phylum . The myxrustacea . We have introns . Here weM. crustacea rDNA revealed seven group I introns at conserved insertion sites in the SSU and LSU rRNA genes  contained direct repeat sequence motifs in peripheral regions of paired segments P1, P2.1, and P9, respectively.Sequencing analysis of the sub-Arctic isolate No-K94 of NA genes . Two sitrding to  based onructures , where fIntron Mcr.S1389 has a complex structural organization at the RNA level a. The 12Tetrahymena intron) , 15 genera, and 37 species were identified. The majority of the SSU rRNA gene sequences were retrieved from the NCBI database, but six Didymiceae sequences  were genM. crustacea intron , Lamproderma arcyriodes and Lamproderma cacographicum  contain 52 copies and 17 copies, respectively, of ψIGS  isolates and one in Brefeldia maxima   .T. varia, and 188 amino acids in B. maxima  [Physarum (I-PpoI) [The S1389 HEGs were organized in a sense orientation in P1 compared to that of the intron ribozyme and host SSU rRNA and encode His-Cys homing endonuclease proteins of 244 amino acids and 247 amino acids in . maxima a. Interend 53 bp . An amin(I-NjaI)  and Phys(I-PpoI)   appeared embedded within the Diderma cluster. Phylogenetic analysis of the corresponding S1389 intron from the same 72 taxa was challenging due to a large dataset and only 166 nt aligned positions. A neighbor-joining tree of the S1389 introns is presented in S1389 introns were identified in distantly related taxa representing four myxomycete orders . To gainD. iridis (AJ938151), Lamproderma columbinum (HQ687200), and L. arcyrioides JQ031973) appear horizontally inherited. (3) L. tigrinum (DQ903678) appears misannotated in GenBank and should most likely be reclassified as a Diderma sp. (4) The ψIGS-containing direct repeats in introns from M. crustacea, L. arcyriodes, and L. cacographicum have most likely been independently gained during the evolution. (5) HEGs noted in B. maxima and T. varia were also independently gained, but we were not able to conclude about evolutionary history of the two T. varia HEG variants. (6) The G-site variants  in P7 appear sporadically in closely related S1389 introns. An example is the U1 variant in D. squamulosum that appeared generated from the A1 variant by one point mutation  revealed that each insertion site represents a distinct intron family and that introns were removed in a non-restricted order [F. septica introns without in vitro self-splicing activities. The L1949 and L2449 introns are compulsory in Physarales myxomycetes [M. crustacea. The truncated catalytic core lacking segment P8 in the M. crustacea L1949 intron  and the closing base pair (Tetrahymena C262:G312) [Tetrahymena intron and found active in self-splicing [S1389 introns in myxomycetes constitute a phylogenetically distinct family of nuclear group I introns with a common origin , a notioA intron . This inA intron , and theA intron . The mos62:G312) ,42, form62:G312) . Among tsplicing .M. crustacea S1389 intron harbors a complex direct repeat pattern in peripheral segment P9, and direct repeats are also present in two additional Mucilago introns (S788 and L1949). Direct repeat insertions have been reported in several nuclear group I introns in myxomycetes, especially in introns lacking in vitro self-splicing activity. The F. septica, P. flavicomum, and D. niveum L1949 introns contained repeated copies of 45-bp, 101-bp, and 20-bp motifs, respectively, in segment P1 [Didymium dubium that harbored two copies of two different motifs (71 bp and 303 bp) in segment P1 [Physarum didermoides with direct repeats in segments P2 (5 copies of 60 bp) and P9 (5 copies of 100 bp) [M. crustacea S1389 intron and in two additional myxomycete S1389 introns (L. arcyriodes and L. cacographicum) may suggest a functional role. On the other hand, large extension sequences in segments P1, P2, or P9 do not interfere with group I ribozyme core structures and may be accepted within introns as long as the introns are efficiently removed, probably aided by host-encoded factors. Since direct repeat arrays have sporadic occurrences and probably are evolutionary neutral, they could be considered as non-functional junk RNA similar to what is proposed for the majority of vertebrate long non-coding RNAs [The gment P1 ,35,36. F 100 bp) . Functioing RNAs .B. maxima and T. varia, harbor S1389 introns with HEGs in segment P1. These HEGs encode His-Cys-type homing endonucleases typical for nuclear group I introns [T. varia HEGs were interrupted by small spliceosomal introns of 44 bp and 53 bp. Related spliceosomal introns have been reported to splice out and restore functional HEGs in two D. iridis group I introns [Two myxomycete species,  introns  and indi introns ,45,46. A introns ,48.M. crustacea (isolate No-K94) was collected as spores in sub-Arctic Norway . Germinated amoebae from M. crustacea and some other Physarales myxomycetes , accessed 1 May 2021). SSU rDNA sequences (with introns removed) were assembled and aligned in the software program Geneious prime® 2019.2.3 . Multiple sequence alignments were generated using MAFFT version 7.450 [® software program. The intron datasets 1 and 2 corresponded to the SSU datasets 1 and 2, except that T. thermophila L1925 and M. crustacea S788 introns were included as outgroups.Six of the SSU rDNA sequences, including group I introns, were generated in this work , and 67 on 7.450  with defon 7.450 ,52 and eon 7.450 ,49 using® 2019.2.3. BI was run with default settings with the substitution model, GTR + G (4 categories), including the chain length , subsampling frequency (200), heated chains (4), heated chain temperature (0.2), burn-in length , and random seed . The evolutionary history of SSU dataset 2 was reconstructed with NJ with Kimura 2 (K2) evolutionary model [The tree-building methods of neighbor joining (NJ), maximum likelihood (ML), and maximum parsimony (MP) in MEGA X  with defry model , ML withThe evolutionary history of intron dataset 1 was reconstructed with NJ and the Jukes-Cantor (JC) model using MEGA X  with defM. crustacea was found to contain seven rDNA group I introns, two in the SSU rRNA gene and five in the LSU rRNA gene. The M. crustacea S1389 intron has a complex structural organization and we collected, analyzed, and compared 72 S1389 intron from a wide spectrum of myxomycetes. The S1389 intron sequences and catalytic core structures were clearly related and supported the notion that introns occupying a specific insertion site represent a distinct group I intron family. However, sequence variation at the highly conserved G-site suggested that group I introns may accept more structural changes at the catalytic site than previously anticipated for natural group I ribozymes. Peripheral segments, on the other hand, may harbor large structural insertions containing homing endonuclease genes or direct repeat arrays, RNA sequences that apparently do not interfere with the splicing reaction and the ribozyme core. An interesting and unusual feature was that a second class of introns, the spliceosomal introns, were present within some of the S1389 group I introns. These small introns may have a biological role, probably linked to homing endonuclease gene expression.Myxomycete protists are a frequent source of nuclear group I introns and


To sum up, a feasible design of a biaxial graphene resonant accelerometer is proposed in this work, which provides a theoretical reference for the fabrication of a graphene accelerometer with high precision and stability.In this paper, a novel two-axis differential resonant accelerometer based on graphene with transmission beams is presented. This accelerometer can not only reduce the cross sensitivity, but also overcome the influence of gravity, realizing fast and accurate measurement of the direction and magnitude of acceleration on the horizontal plane. The simulation results show that the critical buckling acceleration is 460 g, the linear range is 0–89 g, while the differential sensitivity is 50,919 Hz/g, which is generally higher than that of the resonant accelerometer reported previously. Thus, the accelerometer belongs to the ultra-high sensitivity accelerometer. In addition, increasing the length and tension of graphene can obviously increase the critical linear acceleration and critical buckling acceleration with the decreasing sensitivity of the accelerometer. Additionally, the size change of the force transfer structure can significantly affect the detection performance. As the etching accuracy reaches the order of 100 nm, the critical buckling acceleration can reach up to 5 × 10 An accelerometer is a sensor that measures acceleration by calculating the inertia force or displacement of proof mass under external acceleration. It is widely used in inertial navigation, attitude measurement, and other scientific and industrial fields . AccordiReducing the size of the oscillator is of great significance to meet the requirements of a faster response speed, higher accuracy, and wider range of acceleration measurement . The emeIn 2007, J. S. Bunch et al.  fabricatmeasured  fabricatmeasured  designedmeasured .On these grounds, a novel biaxial graphene resonant accelerometer and its measurement performance were studied in this work. More importantly, in order to avoid non-axial applied force of graphene due to gravity and acceleration in other directions, transmission beams were designed to indirectly connect graphene with the proof mass. Thus, the cross sensitivity between two axes is reduced, realizing the independent measurement on the orthogonal biaxial. At the same time, two graphene resonant beams working in a differential state were designed on each axis, which improves the sensitivity of acceleration measurement. In addition, the measurement performance of the accelerometer was evaluated through finite element simulation. By optimizing the corresponding parameters of graphene and the size of the proof mass and transmission beams, the accelerometer was designed to meet the requirements of a proper measuring range and high sensitivity.As shown in When there is external acceleration, the proof mass is subjected to the inertia force In order to improve the sensitivity of acceleration measurement, a pair of graphene resonant beams working in a differential state are placed on each axis. The inertia force produced by axial acceleration stretches one graphene beam while it shrinks the other at the same time, resulting in reverse resonant frequency shifts of these two graphene beams. Graphene has a fracture strength up to 42 N/m , so it cdge mode . HoweverIn this paper, the accelerometer was modeled and simulated by using the finite element simulation software COMSOL Multiphysics . The modThe initial size of the accelerometer is shown in Here, we list the sensitivity, cross sensitivity, and other parameters of the resonant accelerometers in this work and other published work, as shown in The biaxial accelerometer can also measure the direction of the acceleration in the plane . Since tBy adjusting the structure of the accelerometer, the measurement range and sensitivity of the acceleration can change to a great extent but inversely. The structure of the accelerometer can be optimized for different measurement requirements and practice engineering conditions. Two methods are usually used to approach this purpose: adjusting the corresponding parameters of the graphene beam and the size of the force transfer structure, which contains a proof mass and transmission beams.Firstly, the influence of the corresponding parameters of graphene on the measurement performance was studied. It is reported that the prepared graphene is generally under the built-in tension. The graphene strain is reported in the order of 0−5~10−4 , and the0−5~10−4 : T=Et/ of the graphene beam is shown in Secondly, the size of the proof mass and transmission beams affects the measurement performance of acceleration by changing the corresponding stretching and shrinking length of the graphene under acceleration. Since the relative size of each structure was designed considering the independence of measurement and avoiding off-axis crosstalk, we only studied the synchronous magnification or reduction of these structures. We studied the measurement performance of the accelerometer in the range of 10 times reduction to 3 times magnification based on the original size, and the results are shown in d impact . At thisd impact . When thIn this work, a novel two-axis differential resonant accelerometer based on graphene was proposed, in which the design of the transmission beams enables the accelerometer to not only reduce the cross sensitivity and realize independent measurement on the orthogonal axis, but also overcome the influence of gravity on acceleration measurement. Therefore, in practical application, the accelerometer can quickly and accurately detect the magnitude and direction of acceleration. In addition, two graphene resonant beams work in the differential state on each axis, which improves the measurement sensitivity of acceleration. The accelerometer was modeled using finite element simulation software COMSOL Multiphysics, and the modeling process can be divided into the displacement calculation part and the resonance frequency calculation part, which completely simulates the working process of the accelerometer. The calculation results showed that the critical buckling acceleration is 460 g, and the linear range is 0–89 g. When the accelerometer works in the differential mode, its measurement sensitivity is 50,919 Hz/g, belonging to the ultra-high sensitivity accelerometer. In addition, the range and sensitivity of the accelerometer can be adjusted by optimizing the corresponding parameters of graphene and the size of the force transfer structure. For example, increasing the length and tension of graphene can significantly increase the critical linear acceleration and critical buckling acceleration, but the sensitivity will also decrease. Furthermore, when the size of the force transfer structure is reduced by 10 times synchronously, which requires the etching accuracy to reach 100 nm, the critical buckling acceleration range of the accelerometer will be close to


In this study, the interactions of magnesium (Mg) atom and Mg(001) surface with different metal-atom-doped graphene were investigated using a density functional theory (DFT) method. For the interactions of magnesium with Al-, Mn-, Zn-, and Zr-doped and intrinsic graphene, it was found that the magnesium atoms were physisorbed into the hollow sites of the intrinsic graphene with only the smallest interaction energy (approximately −1.900 eV). However, the magnesium atoms tended to be chemisorbed on the doped graphene, which exhibited larger interaction energies and charge transfers. Additionally, the Zn-doped graphene displayed the largest interaction energy with the Mg atom (approximately −3.833 eV). For the interactions of Mg(001) with Al-, Mn-, Zn-, and Zr-doped and intrinsic graphene (intrinsic and doped graphene/Mg interface), doped atoms interacted with a Mg layer to make graphene wrinkle, resulting in a higher specific surface area and better stability. Mg–C chemical bonds were formed at the Al-, Zn-, and Zr-doped interface, and Mg–Mn chemical bonds were formed at the Mn-doped interface. This study provided the fundamental research for future research into doped atoms on graphene reinforced magnesium matrix composites. Graphene is currently one of the most prominent nanoscale materials due to its excellent mechanical, structural, and electrical properties . Doping +, and Mg2+) on graphene. The results showed that the binding nature of the Mg ions (Mg+ and Mg2+) was caused by charge transfer interactions. Additionally, the Mg atoms were observed to interact with the graphene surfaces via a van der Waals (vdW) force. The distances between the Mg atoms and the graphene surfaces were calculated to be 1.80 Å (Mg2+), 2.16 Å (Mg+), and 4.17 Å (Mg). In a related study, Tachikawa et al. [Graphene-reinforced magnesium matrix composites have wide applications in both aerospace and aeronautical fields . Therefoa et al.  used dirIn this study, a DFT method was used to calculate the interaction energy, total electron density, and electron density differences between the magnesium atoms and the graphene or doped graphene . Based on the interactions of magnesium atoms with intrinsic and doped graphene, the interface properties of intrinsic graphene/Mg(001) and doped graphene/Mg(001) are revealed by calculating the interface adhesion work, electron density, Mulliken populations, and partial density of states (PDOS) using a DFT method. This study could reveal the interaction mechanism between magnesium and doped graphene through DFT calculation, and provide the fundamental research for future research into doped atoms on graphene-reinforced magnesium matrix composites.−6 Ha/atom, energy tolerance of 1.0 × 10−5 Ha/atom, maximum force tolerance of 0.002 Ha/Å, and maximum displacement tolerance of 0.005 Å [In this research study, all of the calculations were performed using an efficient ab initio computer code: DMol3. This code was used to investigate the interactions between the magnesium atoms and the doped or intrinsic graphene. A double-numerical plus polarization (DNP) basis set was employed to produce highly accurate results, while keeping the costs of the computational processes fairly low. A DFT semicore pseudopotential (DSPP) was used to describe the core electrons . A gener 0.005 Å . For theIn a DMol3 module, the parameters to be tested for convergence include lattice constant, k-point, and orbital cutoff. As can be seen from The system was modeled as a 4 × 4 graphene supercell, which contained 32 atoms with a periodic boundary condition. A vacuum space of 60 Å was set in the normal direction to the sheets in order to avoid interactions between the periodic images. The optimized structural model of the intrinsic graphene is shown in The energy variations in the doped graphene were systematically studied in order to investigate the effects of the doped atoms  on the microstructures and electronic structures of the graphene. The energies of a single doped atom and C atom were calculated. It is clear that the energy of the doped atom  is greater than that of the substituted C atom (−1028.68 eV). As shown in In this research study, a Mg atom was added above the intrinsic and doped graphene in order to investigate the influences of the different doped graphene on the Mg atoms. Then, in order to obtain the most stable interaction configuration, the Mg atom was initially placed at different positions above the graphene. Following a relaxation period, the optimized configurations obtained from the different initial states were compared for the purpose of identifying the most favorable state.The configurations of the modeling systems following the DFT calculation are shown in The bond length of the C–Mg between the Mg and the intrinsic graphene was determined to be 3.029 Å. This was too long to form any chemical bonds. Additionally, the structures experienced small charge transfer values, which confirmed that there was only a weak interaction between the intrinsic graphene and the Mg atoms . As prevgraphene+Mg, EMg, and Egraphene represent the system total energy, the Mg atoms energy, and the graphene sheet energy, respectively. A negative Eads value corresponded to the stable interactions. The higher the negative Eads value was, the more stable the adsorbed structure was.The Eads indicated the interaction intensity between the Mg atoms and the intrinsic or doped graphene, and was derived according to the following equation:ads between the Mg atoms and the intrinsic graphene was approximately −1.900 eV , as shown in The E1.900 eV . The dop3) of the total electron density indicated that the Zn-doped graphene and Mg atoms had a high strength joint, as well as the largest isosurface area. This was followed by the Al-, Zr-, and Mn-doped graphene sheets. These results were found to be in accordance with the Eads values that had been previously calculated (3.1.2: Interaction energy of magnesium with doped and intrinsic graphene). Although an overlapping of the electrons was observed between the Mg atoms and the doped graphene sheets, detailed electron transfers were not observed. Therefore, the electron density differences between intrinsic and atom-doped graphene were used to illustrate the gain and loss of electrons during the interactions. As shown in 3; blue = +0.02 e/Å3), the electron densities of both the Mg atoms and the Zn atoms demonstrated electron losses. These lost electrons formed intense interactions between the doped atom, Mg atom, and C atoms in the Zn-doped graphene. The electron accumulation areas were observed to be small in the Al-, Zr-, and Mn-doped graphene, as detailed in The electron densities, as well as the electron density differences, were examined in this study for the purpose of illustrating the electron transfers during the interactions of the Mg atoms and the intrinsic or metal-atom-doped graphene. As shown in The graphene/Mg interface microstructure has a significant impact on the properties of the composites. Therefore, the interface properties of the intrinsic graphene/Mg and four-doped graphene/Mg were calculated based on the interactions of magnesium atom with intrinsic and doped graphene. In the study of magnesium, the Mg(001) surface can be used as the preferred surface of interface structure ,31. As sgraphene, EMg(001), and Einterface represent the energy of graphene, four-layer Mg(001) slab model, and interface model, respectively, and A represents the interface area. The larger the interface adhesion work is, the smaller the interface spacing is, which means the stronger the interface stability is. As shown in Interface adhesion work is an important index to measure the strength of interface stability, which is calculated by the following formula :Wad =  of the graphene layer in the intrinsic graphene/Mg interface model are zero, indicating that the existence of a magnesium matrix does not make the graphene layer change into metallicity, while the electronic states at the Fermi level of the graphene layer in the doped interface are not zero , indicating that the doped atoms can change the graphene layer into metallicity. In the Al-doped interface  was higher than that of Mn-doped graphene (−2.590 eV). The result indicated that the Zn-doped graphene had the largest capability to capture the Mg atom, whereas the Mn-doped graphene displayed the lowest capability to capture the Mg atom. Additionally, the electron transfers between the doped graphene and the Mg atom were examined. The results indicated that the interaction between Zn-doped graphene and magnesium atoms was strongest.For the interactions of a Mg(001) surface with Al-, Mn-, Zn-, and Zr-doped and intrinsic graphene (intrinsic and doped graphene/Mg interface), the doped atoms are far away from or near the Mg atoms because of the influence of several Mg atoms on the Mg(001) surface, and the constraint of the doped atom–C chemical bond results in a great change in the local structure around the doped atoms, resulting in the wrinkles of the graphene layer, which also increases the specific surface area of the graphene layer. The results indicated that the doped atoms could improve the interface adhesion work and thus make the interface more stable. There was no chemical bond at the intrinsic graphene/Mg interface due to the maximum interface spacing (3.8801 Å). Mg–C chemical bonds were formed at the Al-, Zn-, and Zr-doped interface, and a Mg–Mn chemical bond was formed at the Mn-doped interface.Compared with the interactions of a magnesium atom with intrinsic and doped graphene, the movement directions of doped atoms are consistent in the interface model. For example, an Al atom, Zn atom, and Zr atom are far away from Mg atoms due to the gain of charge, and Mn is close to Mg atoms due to the loss of charge.The interaction abilities of an isolated Mg atom and Mg(001) surface with the intrinsic and doped graphene were theoretically investigated using DFT methods. The interactions between the Mg atom and the Mg(001) surface on intrinsic and doped graphene are revealed by calculating the energy, electron density, electron density differences, and partial density of state of the model system. The specific results are as follows:


In such a strategy, dual-functional 1D Ni-MOF nanorods not only act as the gelation agent but also afford the doping elements (N and S) originated from the organic species and the precursor for metal sulfide. Due to the synergistic effects of good impedance matching and multiple losses, the optimal reflection loss (RL) of as-prepared Ni3S2@N, S-codoped graphene aerogel reaches −46.9 dB at 17.1 GHz with only 2.0 mm and ultralow filling content (1.75 wt%). The maximum effective absorption bandwidth (EAB) reaches 6.3 GHz (11.7–18.0 GHz) at 2.38 mm, covering the whole Ku band. Moreover, the value of EAB with the RL less than −30 dB can be tuned to 12.2 GHz (5.8–18 GHz) at the absorber thickness ranging from 1.9 to 5.0 mm. This work provides insight for rational design and fabrication of multicomponent-containing graphene aerogels, showing the potential application in lightweight and high-performance microwave absorption.To develop high-performance microwave absorption materials with the features of lightweight, thin thickness, broad bandwidth, and strong absorption, an ultralight Ni Graphene aerogels (GAs) offer a distinctive combination of high porosity, low density, and tunable conductive properties, which make it grab considerable attention in various applications ,2,3,4,5,2 domains in a hexagonal lattice of graphene, improving the conduction loss along with additional polarization relaxation. References [max of 9.29 GHz, which is attributed to the magnetic-dielectric synergy. Currently, graphene aerogels, either doped with heteroatoms or incorporated with second dielectric loss or magnetization loss materials, have been fabricated to achieve high-performance EM absorbers ,11. As aferences ,13,14 Liferences . On the ferences ,17,18,193O4@C nanoparticles. This aerogel shows an outstanding minimum RL value of −71.5 dB at 9.5 GHz with a thickness of 2.95 mm, which attributes to multiple loss mechanisms [To further improve the microwave attenuation, the graphene-based aerogels integrated with multiple effects, including conductive loss, dipole polarization, interfacial polarization, magnetic loss, and multiple scattering and reflection have been developed ,21,22,23chanisms . HoweverMetal-organic framework (MOF) derivatives possess tunable electrical conductivity and abundant terminal groups of carbon matrix, facilitating strong conductive loss and dipole polarization. In addition, MOF derivatives exhibit a prominent feature of high dispersion of nanomaterials into carbon matrix, creating heterogeneous phase interfaces to improve the interfacial polarization loss and optimize the impedance matching ,26. 3S2@N, S-codoped graphene aerogels by further pyrolysis processes. In such a strategy, dual-functional 1D Ni-MOF nanorods not only act as the gelation agent, promoting the gelation of graphene oxide, but also afford the doping elements (N and S) originated from the organic species and the precursor for metal sulfide. The as-prepared Ni3S2@N, S-codoped graphene aerogels possess satisfactory features, including lightweight, thin thickness, broad bandwidth, and strong absorption. Notably, the reflection loss reaches −46.9 dB at 17.1 GHz with a matching thickness of only 2.0 mm and ultralow filling content (1.75 wt%). The maximum effective absorption bandwidth (EABmax) reaches 6.3 GHz (11.7–18.0 GHz) at 2.38 mm, covering the whole Ku band. Therefore, the presented MOF-induced gelation strategy provides an effective pathway to fabricate multicomponent absorbers with a 3D porous structure for achieving excellent EM absorption performance.In this work, a facile and low-cost approach based on 1D Ni-MOF nanorods directly induced gelation strategy has been developed for the preparation of 3D graphene/Ni-MOF aerogels, which are converted to Ni3)2·6H2O, and KOH were obtained from Aladdin Bio-Technology Co., Ltd. . Graphene oxide (GO) nanosheets were purchased from XFNANO . 2-Mercaptopyridine, Ni(NO3)2·6H2O  were mixed with 16 mL distilled water in a 50 mL Teflon-lined autoclave under stirring until all solid powder dissolved. Subsequently, 236 mg KOH was added 16 mL distilled water to acquire a lucent solution, and then the solution was added dropwise to the above mixture. After stirring for 10 min, the autoclave was transferred to an oven and heated at 75 °C to obtain Ni-MOF nanoparticles. The products were filtered and washed in distilled water and ethanol three times, individually. Finally, the samples were dried in an oven at 80 °C overnight. In a typical synthesis, 2-Mercaptopyridine  and Ni was dispersed into 2 mL of GO suspension (5 mg/mL) by vigorously shaking for 2 min. The mixed suspension was then transferred into glass bottles, which were heated at 95 °C for 2 h in an oven to obtain Ni-MOF/graphene hydrogels. The hydrogels were frozen for 24 h and freeze-dried for 24 h to obtain Ni-MOF/graphene aerogels. The as-prepared Ni-MOF/graphene aerogels were put into a glass tube and thermally treated at 600, 650, and 700 °C for 1 h in Ar with a heating rate of 3 °C/min to achieve Niin: 3.04 mm, Φout: 7.00 mm).The microstructure and composition of as-prepared samples were examined by X-ray diffraction , Raman spectroscopy (LabRam HR Evolution) with a 532 nm laser, X-ray photoelectron spectroscopy , scanning electron microscopy  with an energy dispersive X-ray spectroscopy (EDS) system, and transmission electron microscopy . Electromagnetic parameters were tested by an Agilent PNA-N5244A vector network analyzer . The aerogel samples were infiltrated in molten paraffin wax and cut into a standard annulus specimen (Φ3)2·6H2O in a molar ratio of 2:1 in water at 75 °C. Ni-MOFs were prepared by the hydrothermal reaction of 2-Mercaptopyridine and Ni of GO, the GO nanosheets can prevent insoluble 1D Ni-MOF nanorods from precipitation, forming stable suspensions. The mixed suspensions gradually transform into colloids and form stable gels in 1 h under 95 °C. By prolonging the gelation time, the volume of the gels gradually decreases  and cubic Ni3S2 (JCPDS No. 74-1336) [−1 are corresponding to the D band and G band, respectively. The intensity ratio of the D band and G band (ID/IG) gradually increases from 1.06, 1.11 to 1.17 with the increased pyrolysis temperature, indicating that there are much more defects or lattice distortion generated in graphene sheets due to elements doping. This can lead to more defect polarization, which is beneficial to improving the attenuation property of the aerogels.The crystallographic structures and composition of the prepared aerogels with the gelation time of 2 h were characterized by XRD. 74-1336) . Figure 3S2@NSGA-600. No obvious changes of these elements are found in Ni3S2@NSGAs prepared at different temperatures. 3S2@NSGAs including C–C (284.6 eV), C–S (285.1 eV), C–N (285.7 eV), and C–O (288.9 eV) [3/2 and S 2p1/2 located at 161.1 and 163.1 eV, respectively, originate from the Ni−S bond. The peak at 164.5 eV is allocated to C−S bonding  and inverse fast fourier transition (IFFT) images  increases from 86.49, 88.31 to 90.43 at % with the increased pyrolysis temperature. While the intensity of the O 1s peaks located at ca. 532.4 eV continually decreases, the oxygen content declines from 9.23, 7.62 to 5.53 at %, confirming the further reduction of graphene oxide sheets in a high temperature. In addition to C and O elements, N, S, and Ni elements also can be detected in as-prepared aerogels c. The su bonding f. Regard bonding g, two pe to Ni3+ ,28. The mappings b–f revea surface g–l. The  surface m. The cr) images n,o [14,23S2@NSGAs pyrolyzed at different temperatures are calculated based on the transmission line theory by the following equations:inZ is the input characteristic impedance of the absorber, Z0 stands for the impedance of air, c is the velocity of light, f is the frequency, and d is the absorber thickness.Reflection loss (RL) curves, 3D, and 2D representations of the RL for Nimax) values of Ni3S2@NSGA-600, Ni3S2@NSGA-650, and Ni3S2@NSGA-700 can reach 5.3 GHz (12.7–18 GHz), 6.3 GHz (11.7–18 GHz), and 7.0 GHz (11–18 GHz), respectively. Especially, the RLmin of Ni3S2@NSGA-650 reaches −46.9 dB at 17.1 GHz with a thickness of 2.0 mm, and the effective bandwidth is 6.3 GHz at 2.38 mm, covering the whole Ku band (12–18 GHz). Additionally, the effective absorption bandwidth with the RL less than −30 dB can be tuned to 12.2 GHz (5.8–18 GHz) at the absorber thickness of 1.9–5.0 mm, indicating the tunability of the frequency band at different thickness values. The EM performance for Ni3S2@NSGA-650 is compared with corresponding values in representative recent reports . The eddy current loss can be evaluated by the following equations:μ0 represents vacuum permeability, C0 is positively correlated with 2d (thickness squared) and σ (conductivity). The values of C0 keep constant in the range of 14–18 GHz in C0–f curves, which indicates that the magnetic loss comes from the eddy current loss. In contrast, the significant vibration area (blue dotted frame) of C0 at a low frequency (2–7 GHz) shown in The complex permeability and magnetic loss tangent of Niin/Z0|) for Ni3S2@NSGA samples are presented in 3S2@NSGA-600 and Ni3S2@NSGA-700 is apparent due to that the impedance matching characteristic values of both samples are far away from 1. The Ni3S2@NSGA-650 possesses the optimal Z values in the whole frequency range. Although it exhibits a moderate attenuation ability, impedance matching allows EM waves to enter into the absorbing materials as much as possible, which is a prerequisite for the Ni3S2@NSGA-650 aerogel to provide a high EM absorption performance. In the case of Ni3S2@NSGA-700, though its attenuation ability increases, the missed impedance matching leads to the weaker microwave absorption performance.Besides the intrinsic microwave attenuation capacity, impedance matching is another key factor for evaluating high-performance absorbing materials ,35. The 3S2@NSGA-650 is attributed to the synergistic effects of good impedance matching and multiple losses, which can be revealed from the perspective of the multiscale. (1) Microscopic scale, multiple random reflections, and the scattering of EM waves repeatedly occur at the internal interfaces of the cell walls, leading to the transfer of EM energy to be dissipated as heat, thereby realizing the efficient absorption of EM waves. In addition, the 3D interconnected porous reduced graphene oxide network can establish more conductive paths for electronic transport, endowing the aerogel with high conduction loss. (2) Nanoscale: Ni3S2 nanoparticle is a typical magnetic loss material, including natural resonance, exchange resonance, and eddy current loss. Despite causing a weak magnetic loss of the material, the low Ni3S2 content can substantially reduce the effective permittivity of the material, which is favorable for the highly conductive carbon-based absorbers to achieve good impedance matching. The multiple interfaces between Ni3S2 nanoparticles and reduced graphene oxide nanosheets also enhance the interface polarization. (3) Molecular scale: the introduction of N and S elements into graphene lattice improves the dipolar polarization and the intrinsic defect polarization. The dipolar polarization is ascribed to the different electronegativity between C, N, and S atoms or the residual oxygen functional groups in unsaturated coordination, and the intrinsic defect polarization is resulted from the charge unbalance around the carbon vacancies in the graphene lattice under an alternating electromagnetic field.Based on the aforementioned discussion, the high-performance microwave absorption property of as-prepared Ni3S2@N, S-codoped graphene aerogel-based microwave absorber has been fabricated by a facile approach based on 1D Ni-MOF nanorods directly induced gelation strategy. Dual-functional 1D Ni-MOF nanorods not only act as the gelation agent but also afford the doping elements for the generation of N, S-codoped reduced graphene oxide and the precursor for the formation of metal sulfide. The as-prepared Ni3S2@N, S-codoped graphene aerogel shows a comprehensive absorption performance with an RLmin value of −46.9 dB at a thin thickness of 2.0 mm and a broad EAB reaching 6.3 GHz (11.7–18.0 GHz) at 2.38 mm at a low filler loading of 1.75 wt%. It is postulated that the excellent microwave absorption performance is facilitated by the 3D multi-component porous structure, which provides enhanced conductive and polarization loss, magnetic loss, and multiple scattering and reflection. The results found in this work motivate further investigations into multicomponent-containing graphene such as EM absorbers for stealth camouflage techniques.In summary, a high-performance Ni


Gpr161 locus in mice to generate a variant (Gpr161mut1), which was ciliary localization defective but cAMP signaling competent. Tissue phenotypes from hedgehog signaling depend on downstream bifunctional Gli transcriptional factors functioning as activators or repressors. Compared to knockout (ko), mut1/koGpr161 had delayed embryonic lethality, moderately increased hedgehog targets, and partially down-regulated Gli3 repressor. Unlike ko, the mut1/Gpr161ko neural tube did not show Gli2 activator-dependent expansion of ventral-most progenitors. Instead, the intermediate neural tube showed progenitor expansion that depends on loss of Gli3 repressor. Increased extraciliary receptor levels in mut1/mut1Gpr161 prevented ventralization. Morphogenesis in limb buds and midface requires Gli repressor; these tissues in mut1/mut1Gpr161 manifested hedgehog hyperactivation phenotypes—polydactyly and midfacial widening. Thus, ciliary and extraciliary Gpr161 pools likely establish tissue-specific Gli repressor thresholds in determining morpho-phenotypic outcomes.The role of compartmentalized signaling in primary cilia during tissue morphogenesis is not well understood. The cilia localized G protein-coupled receptor, Gpr161, represses hedgehog pathway via cAMP signaling. We engineered a knock-in at the  The primary cilium is a paradigmatic organelle for studying compartmentalized cellular signaling in morphogenesis and disease . The priThe mechanisms by which cilia-specific signals are maintained and propagated to direct downstream pathways during morphogenesis is not well understood. Particularly, non-ciliary signaling by cilia localized proteins can be a confounding factor in understanding how signaling at and by cilia contributes to tissue phenotypes. Studying signaling at cilia requires mechanistic understanding of trafficking to cilia, isolating ciliary from extraciliary functions of signaling molecules, and studying functional consequences directly in tissues without disrupting cilia . DecodinThe primary cilium mediates cellular signaling responses to Hh morphogens in vertebrates . An intrGpr161 are embryonic lethal by embryonic day 10.5 (E10.5) and exhibit increased Hh signaling and expansion of ventral progenitors throughout the rostrocaudal extent of the neural tube, without disrupting cilia. Gpr161 determines Gli3R formation via cAMP-PKA signaling , Gpr161 functions as a negative regulator of Hh signaling during early neural tube development in mice . Mice knignaling . Howeverignaling , the intignaling . We receignaling , embryonignaling , and forignaling . Tissuesignaling , where G of GliR . Thus, GGpr161 deletion were rescued from concomitant loss of cilia  that is ciliary localization defective but signaling competent. We demonstrated the tubby family protein Tulp3 . Wildtype Gpr161 predominantly localized to cilia. In contrast, Gpr161mut1 was not localized to cilia but was observed in vesicles, some of which surrounded the base of cilia (We previously described the (V/I)KARK motif in the third intracellular loop of Gpr161 to be necessary and sufficient for targeting to cilia by the tubby family protein Tulp3 . We usedGpr161  to stablof cilia .mut1  to promote Gpr161 accumulation in cilia or prevent Gpr161 trafficking out of cilia and detect Gpr161mut1 by immunostaining for GFP. These experiments conclusively demonstrate that Gpr161mut1 does not at all transit through cilia.We previously showed that Tulp3 regulates trafficking of Gpr161 into cilia but does not affect trafficking out of cilia . We usedmut1 transits through cilia but is too low to detect at steady state, we would expect to see it accumulating upon Inpp5e loss. By stably expressing the mutant LAP-tagged wild type (wtGpr161) and mut1Gpr161 in RPE hTERT cells, we confirmed the mut1Gpr161 fusion to be not localizing to cilia unlike wtGpr161  subunits and Gpr161 in the ciliary membrane  likely fGpr161wt . As expein cilia . Howeverin cilia , despitein cilia .Arrb1/Arrb2 dko) MEFs as β−arrestins regulate Gpr161 removal from cilia  for estimating cAMP levels. These assays eliminate the background noise due to FRET-based estimation of signals, and the use of long-lived fluorophores (in this case lathanides), combined with time-resolved FRET detection (a delay between excitation and emission detection) minimizes prompt intrinsic fluorescence interferences  in the endogenous locus and confirmed the endogenous integration by southern blotting (mut1Gpr161 exon four sequence (Gpr161 knockout (ko) allele. Quantitative RT-PCR of Gpr161 transcripts in E9.5 embryos suggested that the mut1Gpr16 transcript was expressed at similar levels compared to wild type and was reduced to 50% compared to wildtype in ko/mut1Gpr161 embryos . In whole embryo extracts at E9.5, we detected increased levels of both transcripts in the ko/koGpr161 embryos, intermediate levels in mut1/koGpr161 embryos, and slightly elevated levels but statistically insignificant from wild type in mut1/mut1Gpr161 embryos , but not that of mut1,Gpr161 rescued the basal Gli2 ciliary accumulation in Gpr161 ko cells. Thus, basal Gli2 accumulation in ciliary tips occurs from the absence of ciliary Gpr161 pools.Upon Hh pathway activation, Gli2 proteins accumulate at the tips of cilia . While ~reatment . Stable ko/koGpr161 at E9.5  in posterior limb buds from E9.75  results increased expression of Ptch1/Gli1 in both anterior and posterior forelimb fields and ectopic expression of Shh anteriorly. In contrast to ko/koGpr161, forelimb buds were formed in mut1/mut1Gpr161 embryos, and as in f/fPrx-Cre; Gpr161 there was increased expression of Ptch1/Gli1 in both anterior and posterior forelimb and hindlimb fields, as detected by RNA in situ hybridization (ISH)  . Severalon (ISH) , a likelsenchyme . Second,at E10.5 , a likelat E10.5 . Third, at E12.5 .f/fPrx1-Cre; Gpr161 had increased number of digit fields resulting in polysyndactylous phenotypes  expresses a fusion protein containing the first 699 aa of Gli3 and extra residues from thymidine kinase used for generating the allele    in the bSubcellular expression of Gpr161 variants is, however, likely to lead to increased specificity of localized cAMP action compared to optogenetic and chemogenetic approaches increasing diffusible cAMP. The textbook model of cAMP causing dissociation of PKA catalytic subunits from regulatory subunits  is unlikmut1/mut1Gpr161 embryos increasing processing compared to mut1/koGpr161 embryos at E9.5  (vlGpr161 truncation mutant (pr161 ko . Recentl bifida) . While m bifida) , cross-r bifida)  and BMP  bifida)  could den mutant  is also n mutant  and couln mutant . Role ofHoxd13 from lack of Gli3R, which likely contributes to the polydactyly. We do not see ectopic Shh expression as in f/fPrx1-Cre; Gpr161  in the medial nasal processes . Midfaciwidening . The facary loss , suggestenotypes .In conclusion, the results presented here suggest a role for Gpr161-mediated signaling propagated by ciliary and extraciliary compartments in establishing tissue specific GliR thresholds regulating morpho-phenotypic outcomes from Hh pathway hyperactivation. By establishing an allelic series for Gpr161-mediated Hh pathway repression, we are also poised to unravel neomorphic phenotypic outcomes from varying signaling strength of Hh signaling.Gpr161 genomic locus was engineered by recombineering using an FRT-PGKneo-FRT selection cassette. The required mutation (with an engineered NotI site) was separated from the selection cassette by 536 bp (mut1Gpr161 targeting construct were generated using this engineered BAC (#RP23-235-E18) by further cloning into a modified pGKneoloxP2.DTA.2 vector as follows. The pGKneoloxP2.DTA.2 vector was digested with SacII and NheI and religated. The left arm consisting of 4100 bp upstream and the right arm consisting of 4800 bp downstream of the FRT-PGKneo-FRT cassette in the BAC were PCR cloned into the HindIII site of this modified vector .A BAC overlapping the mouse y 536 bp . Recombiy 536 bp . The exod vector . The engd vector . From ammut1-neo/+Gpr161 ES cells (5F9 clone) were injected into host embryos of the C57BL/6 albino strain by the transgenic core . Mice with germline transmission were crossed with Flp-O  for deleting the FRT-PGKneo-FRT cassette to generate the mut1Gpr161 allele. The Gpr161 knockout and conditional allele targeting the third exon crossed has been described before (tm1AljGli2 (ko) allele  allele . Prx1-Crase line , in whicGpr161 ko NIH 3T3 Flp-In cell line was a gift from Rajat Rohatgi (Gpr161 inserts followed by antibiotic selection. RPE hTERT cells were grown in DMEM F12 media with 10% FBS (Sigma-Aldrich), 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine. RPE hTert cells were retrovirally infected with C-term LAP-tagged Gpr161 wild type or mutant constructs and flow sorted for GFP after puromycin selection. Stable doxycycline-inducible NIH 3T3 Tet-on 3 G cells expressing untagged Gpr161 and mut1Gpr161 receptor variants were generated by retroviral infection. These cell lines were grown in Tet-free serum (Clontech) until induction by Doxycycline  and intron 4–5 (5’ ACCCTGACACTGCCCTTAGC). The PCR product of wild type and mut1 allele bands was 927 bp, but only the PCR product from the mut1 allele was digested into 400 and 527 bp products with NotI. Genotyping of Gpr161 knockout or floxed alleles were performed using primers in the deleted 4th exon (5’ CAAGATGGATTCGCAGTAGCTTGG), flanking the 3’ end of the deleted exon (5’ ATGGGGTACACCATTGGATACAGG), and in the Neo cassette (5’ CAACGGGTTCTTCTGTTAGTCC). Wild type, floxed and knockout bands were 816, 965, and 485 bp, respectively  and Cre-R (5’-GAA AAT GCT TCT GTC CGT TTG C-3’) primers (100 bp amplicon). To genotype Gli2 mice, Gli2 sense (5’-AAA CAA AGC TCC TGT ACA CG-3’), Gli2 antisense (5’-CAC CCC AAA GCA TGT GTT TT-3’) and pPNT (5’-ATG CCT GCT CTT TAC TGA AG-3’) primers were used. Wild type and knockout bands were 300 bp and 600 bp, respectively.Genotyping of ectively . Cre allGGAAUUAAAAGACGGAUUU-3′ and has been validated before  for immunofluorescence.RPE hTERT cells were passaged on glass coverslips and transfected with siRNA using Lipofectamine RNAiMax (Invitrogen). OTP non-targeting pool  was used as control siRNAs in all experiments. The siRNA sequences for INPP5E (J-020852–05) is 5′-d before . The siRd before . For INPGpr161 or mut1Gpr161 were performed according to manufacturer’s instructions (Cisbio)  and values for unknowns were interpolated using Graphpad Prism.We used a TR-FRET cAMP assay (Cisbio) for estimating cAMP as these assays eliminate the background noise due to FRET-based estimation of signals. Also, the use of long-lived fluorophores (in this case lathanides), combined with time-resolved detection (a delay between excitation and emission detection) minimizes prompt intrinsic fluorescence interferences . TR-FRET(Cisbio) . Cells wMouse embryos fixed in 4% PFA overnight at 4°C and processed for cryosectioning. For cryosectioning, the embryos were incubated in 30% sucrose at 4°C until they were submerged in the solution. Embryos were mounted with OCT compound. Embryos in OCT were cut into 15 µm frozen sections. The sections were incubated in PBS for 15 min to dissolve away the OCT. Sections were then blocked using blocking buffer  for 1 hr at room temperature. Sections were incubated with primary antibodies against the following antigens; overnight at 4°C: FoxA2 , Nkx6.1 , Pax6 , Pax7 . After three PBS washes, the sections were incubated in secondary antibodies  for 1 hr at room temperature. Cell nuclei were stained with Hoechst 33342 (Life Technologies). Slides were mounted with Fluoromount-G  and images were acquired with a Zeiss AxioImager.Z1 microscope. For immunofluorescence experiments in cell lines, cells were cultured on coverslips until confluent and starved for 48 hr. To quantify ciliary Gli2 and Gpr161 levels, cells were treated with 500 nM SAG or DMSO for 24 hr after 24 hr of serum starvation. In some experiments, Transferrin conjugated with Alexa Fluor 568  was added (10 μg/ml) in starvation media at 37°C for 30 min before fixation. Cells were fixed with 4% PFA for 10 min at room temperature and postfixed for 5 min with methanol at −20°C for γ-tubulin immunostaining. After blocking with 5% normal donktey serum, the cells were incubated with primary antibody solutions for 1 hr at room temperature followed by treatment with secondary antibodies for 30 min along with Hoechst. Primary antibodies used were against Gpr161  , acetylaGli1 .RNA was extracted using the GenElute mammalian total RNA purification kit . Genomic DNA was eliminated by DNase I . qRT-PCR was performed with SYBR Green Quantitative RT-qPCR Kit  or Kicqstart One-Step Probe RT-qPCR ReadyMix . Gli1  and Ptch1 , Sox9, Col2a1 , Hoxd13 . Whole mount in situ hybridization using digoxigenin-labeled probes was performed on embryos using standard protocols. Images were acquired using a Leica stereomicroscope (M165 C) with digital camera (DFC500) or Zeiss stereomicroscope (Discovery.V12) and AxioCam MRc.Antisense riboprobes were made using the following templates: t-test for comparing two groups using GraphPad Prism.Cilia positive for acetylated α-tubulin and GPR161/TULP3 were counted and expressed as % of GPR161/TULP3-positive cilia. To quantify ciliary pools of GPR161, TULP3, GFP, fluorescence levels were measured using the ‘Measure’ tool of Fiji software. Fluorescence levels of neighboring background areas were subtracted from that of the selected ciliary areas and expressed as corrected fluorescence. Quantification of dorsoventral extent of Nkx6.1 expression to that of the neural tube was done by averaging from two or more sections at thoracic region from two to four embryos. Statistical analyses were performed using Student’s  public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:This paper will be of interest to scientists interested in GPR161 biology and its ciliary regulation of Hedgehog (Hh) signaling. The phenotypes observed in a new GPR161 mutant mouse carrying a hypomorphic allele provide additional information about which developing tissues are more sensitive to Gpr161 function.Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Suzanne Pfeffer as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Maxence V Nachury (Reviewer #2).Thank you for submitting your article "Deficient ciliary Gpr161 pools cause high hedgehog signaling phenotypes by reducing Gli repression but not by activation" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.As you will see from the detailed reviewer comments, the referees did not think that the data at this stage support the model that the ciliary role of Gpr161 is related to its effect on Gli3R, while its extra-ciliary role is related to regulation of GliA. Measurement of ciliary, peri-ciliary and cytoplasmic cAMP levels would help but this is not a standard experiment. They wrote, "One way to make the paper work is to moderate the claims and focus on the conclusion that "Gpr161 suppresses Hh signaling through both ciliary and non-ciliary mechanisms". The authors should focus on the mouse phenotypes without linking these two mechanisms to specific biochemical (ie. Gli3R and GliA) or cell biological (ie. ciliary and extra-ciliary cAMP) processes ." In addition, one wrote, "The authors should try a few simple experiments to strengthen the basic premise of the paper, that GPR161mut1 is truly never in cilia. Depleting β arrestin and/or the BBSome in their MEFs will give a rapid answer to whether this mutant exits cilia more rapidly than the WT."Reviewer #1 (Recommendations for the authors):All recommendations are in public review.Reviewer #2 (Recommendations for the authors):Quantitation of WB signals . Are signals within the linear range of measurement? While manufacturers often claim instrument linearity within 4 or 5 orders of magnitudes, empiric validation often finds these claims to be exaggerated. A simple immunoblot with varying amounts of samples loaded would alleviate the concern of non-linear response.Figure 4C: The mut1/mut1 data on the Gli3FL/R ratio do not support the statement 'the Gpr161 mutants exhibit gradually decreasing severity of Gli3 processing defects compared to ko, commensurate with monoallelic or biallelic expression of Gpr161mut1.' The Gli3FL/R ratio is lower in mut1/mut1 than in any other allele combination, including WT. Why does the mut1/mut1 allele combination cause an increased production of Gli3R relative to the amount of Gli3FL? It is interesting that the signaling defects of the mut1/mut1 animals are extremely modest. One interpretation is that GPR161 ciliary localization plays no detectable role at E9.5. Another interpretation is that GPR161mut1 does localize to cilia in cells  of E9.5 embryos. The authors need to discuss the reduction of the Gli3FL/R ratio in mut1/mut1 animals  and may wish to stain E9.5 embryos to reject the hypothesis that GPR161mut1 localizes to cilia at this stage of development.On the other hand, the production of Gli3R is clearly compromised in the mut1/mut1 mouse limbs at E12.5. It is comforting that the morphological and molecular phenotypes are congruent.Figure 8 is helpful in orienting the reader through a complex set of data. The authors could add the 'no GPR161' situation to echo the discussion. In the same vein, adding diagrams showing the author's thinking of how ciliary vs. non ciliary pools of GPR161 are affecting Gli3R and Gli2A production would be helpful. The point that I have the hardest time reconciling with the presented data is that cilia are required to generate both activator and repressor forms of Gli. Yet, extra ciliary pools of GPR161 play a role in generating GLI2A.Some text constructions are ambiguous or grammatically incorrect. A few examples are given below but the list is not exhaustive. In general, the text would benefit from careful editing.'High hedgehog signaling phenotypes in Gpr161 knockout occur from Gli-repressor lack or Gli-activator generation.''Here we demonstrate distinctive roles for ciliary Gpr161 pools in preventing hedgehog hyperactivation phenotypes, contingent upon dependence of the morpho-phenotypic spectrum on downstream Gli repression'Gli2-dependent''Ventral expansion in Ankmy2 knockout is severe than Gpr161 knockout, but even then, is fully 'Gpr161 deletion showed GliR lack''However, lack of Gpr161 in cilia was incapable of sufficient Gli-activator generation required in causing ventral-most progenitor expansion in the neural tube.''Gpr161 ciliary pools regulate Hh repression regulated morpho-phenotypic spectrum'Typo'Gpr16mut/mut11 embryos exhibit high Hh signaling 1 and polydactyly in limb buds'Some inaccurate statements of facts:'Gpr161 C-tail has been proposed to be an A-kinase anchoring protein (AKAP) for PKA activation in cilia by binding to PKA regulatory subunit RIa and RIb .''Direct Gpr161 C-tail binding to PKA RIa/RIb regulatory subunits  facilitates PKA activation in close proximity to Gpr161-mediated cAMP production in cilia for GliR processing.' The Bachmann reference shows that the AKAP motif of GPR161 is specific for PKA RIalpha'Agonist-independent constitutive signaling.' The author may want to leave open the possibility that the tonic activity of GPR161 is caused by an agonist endogenous to the experimental system.Gli2/3 into Gli2/3 repressor (GliR) forms.' PKA does not mediate proteolysis'protein kinase A (PKA)-mediated limited proteolysis of full length 'Increased extracellular Gpr161 pools in Gpr161mut1/mut1 also prevent Nkx6.1 ventralization from likely restoration of Gli3R processing.' Extra-ciliary?eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Suzanne Pfeffer as the Senior Editor.Thank you for resubmitting your work entitled "Ciliary and extraciliary Gpr161 pools repress hedgehog signaling in a tissue-specific manner" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below.The reviewers felt that dissecting the ciliary vs. non-ciliary role of this GPCR is an important advance. However, as discussed below, it will be necessary to revise both the abstract and conclusion to explicitly acknowledge that you have not shown that their mutant Gpr161 changes ciliary cAMP levels and that you have not demonstrated conclusively that the phenotypes seen in these mutants are due to loss of Gli3 repressor. One reviewer wrote, "The text of the paper still leaves one with the (erroneous) impression that they have altered "cAMP signaling". I think all they can say is that they have identified a mutant of Gpr161 that has reduced localization in cilia and the phenotypes caused by this mutant are different from the null (depending on the tissue examined). I think they should add a separate paragraph to the conclusion highlighting these limitations."Please prepare a carefully revised version that addresses these concerns:Reviewer #1:I appreciate the effort the authors have made to improve data presentation of phenotypes, clarify localization of Gpr161 and discuss the recent paper in zebrafish showing the importance of ciliary cAMP pools for Hedgehog signaling.However, the authors have not addressed two major issues raised in my previous review:1. Does the mutation in Gpr161 (or its loss) cause a change in the ciliary levels of cAMP ? While there is an extensive discussion of the ciliary cAMP pools throughout the paper (and even a mention of this in the last sentence of the abstract), a demonstration that Gpr161 mutations changes the cAMP pool in cilia is lacking. Without additional data, the text must be carefully toned down as not to mislead the reader.2. Again, while there is extensive discussion in this area, the authors do not show that Gpr161 mutation causes phenotypes in some tissues that are Gli3 repressor dependent. A simple experiment to address this is to show that the phenotypes in question can be rescued by re-expression of Gli3R. This would establish cause-effect rather than correlation. Without this, the limitations of the study must be stated clearly and conclusions softened.Reviewer #2:A major point raised in the first round of review was the limited evidence that GPR161mut1 fails to enter cilia. The added data considerably strengthen this point. GPR161mut1 is still absent from cilia in the exit mutants Arrb1/Arrb2 DKO and in cells depleted of BBS4 by siRNA.In addition, the authors now show that GPR161mut1 is present in pericentriolar recycling endosomes, a notable point that enriches the discussion. This observation introduces the possibility that percentriolar cAMP generated by GPR161mut1 finds its way into cilia to raise ciliary cAMP levels to a level that suppresses formation of GliA.The text has been modified to temper some of the conclusions and more accurately describe the results. However, there are many instances where the writing is too convoluted and lacks clarity. In particular, the extensive re-writing of the abstract has made for some rather complex statements that only a few specialists will be able to comprehend. The level of textual editing required falls outside of the scope of peer-review. I had listed a few instances in my initial review but indicated that the list was not exhaustive. Yet, it appears that the authors only addressed the instances listed. It is a pity that clarity is still lacking as the lasting impact of this paper would be diminished if the paper were published in its current state. The authors may seek to engage the service of a professional editor.One instance is listed below but there are many more.'We conclusively ruled out that LAP-tagged Gpr161mut1  does not at all transit through cilia'. What do the authors rule out? The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.As you will see from the detailed reviewer comments, the referees did not think that the data at this stage support the model that the ciliary role of Gpr161 is related to its effect on Gli3R, while its extra-ciliary role is related to regulation of GliA. Measurement of ciliary, peri-ciliary and cytoplasmic cAMP levels would help but this is not a standard experiment. They wrote, "One way to make the paper work is to moderate the claims and focus on the conclusion that "Gpr161 suppresses Hh signaling through both ciliary and non-ciliary mechanisms". The authors should focus on the mouse phenotypes without linking these two mechanisms to specific biochemical (ie. Gli3R and GliA) or cell biological (ie. ciliary and extra-ciliary cAMP) processes ."mut1 mouse model and other optogenetic/chemogenetic methods for generating cAMP in cilia. These limitations arise from the cilioplasm not being strictly restricted from the cytoplasm. Therefore, the second messengers cAMP and ca2+ are freely diffusible between ciliary and extraciliary compartments . A paper published in Cell during revision of this study used optogenetic tools to show that ciliary, but not cytoplasmic, production of cAMP functions through PKA localized in cilia  to repress sonic hedgehog-mediated somite patterning in zebrafish . We have also compared and discussed these results with our study. Our study highlights that the effects of ciliary loss of Gpr161 pools are tissue specific and dependent on the requirements of the tissues on GliR vs GliA in the morpho-phenotypic spectrum. Overall, our results using Gpr161mut1 allele are strikingly complementary to the optogenetic study by showing that lack of ciliary Gpr161 pools result in Hh hyperactivation phenotypes arising mainly from lack of GliR, in the limb buds, mid-face and intermediate neural tube.We have revised the title of the paper and the focus of our discussion based on these suggestions. We have also discussed the limitations of our Gpr161In addition, one wrote, "The authors should try a few simple experiments to strengthen the basic premise of the paper, that GPR161mut1 is truly never in cilia. Depleting β arrestin and/or the BBSome in their MEFs will give a rapid answer to whether this mutant exits cilia more rapidly than the WT."mut1 is not present in cilia in β-arrestin1/2 (Arrb1/2) double ko MEFs or upon RNAi of BBS4. We already showed that knockdown of the 5’phosphpatase INPP5E that causes accumulation of Gpr161 in cilia does not show any accumulation of Gpr161mut1 in cilia. Based on all these experiments, we surmise that Gpr161mut1 does not transit through cilia.We have now shown that Gpr161Reviewer #2 (Recommendations for the authors):Quantitation of WB signals . Are signals within the linear range of measurement? While manufacturers often claim instrument linearity within 4 or 5 orders of magnitudes, empiric validation often finds these claims to be exaggerated. A simple immunoblot with varying amounts of samples loaded would alleviate the concern of non-linear response.Levels of Gli proteins in Figure 4 were normalized to α-tubulin. 30 µg of mouse embryo lysates were used in immunoblotting, well within the linear range of detection of α-tubulin levels using the DM1A monoclonal antibody and LI-COR infrared laser-based detection  of E9.5 embryos. The authors need to discuss the reduction of the Gli3FL/R ratio in mut1/mut1 animals  and may wish to stain E9.5 embryos to reject the hypothesis that GPR161mut1 localizes to cilia at this stage of development.On the other hand, the production of Gli3R is clearly compromised in the mut1/mut1 mouse limbs at E12.5. It is comforting that the morphological and molecular phenotypes are congruent.mut1 generating Gli3-repressor. Morphogenesis in limb buds and midface require Gli-repressor. These tissues in Gpr161mut1 manifested hedgehog hyperactivation phenotypes—polydactyly and midfacial widening. We think that compartmentalized ciliary cAMP signaling establishes tissue-specific repression thresholds in dictating these distinct morpho-phenotypic outcomes. We have discussed the differences in GliR processing between tissues and also in the morpho-phenotypic outcomes in the updated Discussion.As the reviewer suggests, intermediate level neural tube ventralization manifested from loss of ciliary Gpr161 but was suppressed by increased extraciliary Gpr161Figure 8 is helpful in orienting the reader through a complex set of data. The authors could add the 'no GPR161' situation to echo the discussion. In the same vein, adding diagrams showing the author's thinking of how ciliary vs. non ciliary pools of GPR161 are affecting Gli3R and Gli2A production would be helpful. The point that I have the hardest time reconciling with the presented data is that cilia are required to generate both activator and repressor forms of Gli. Yet, extra ciliary pools of GPR161 play a role in generating GLI2A.Thanks for the suggestions. First, we have added a “no Gpr161” vs “no Gpr161 in cilia” comparison of phenotypes in revised Figure 8A-C. Second, we have also added a new diagram  showing how we think the ciliary and extraciliary pools of Gpr161 are affecting Gli3R and Gli2A production.Some text constructions are ambiguous or grammatically incorrect. A few examples are given below but the list is not exhaustive. In general, the text would benefit from careful editing.Thank you for all the corrections. We have revised the text accordingly.'High hedgehog signaling phenotypes in Gpr161 knockout occur from Gli-repressor lack or Gli-activator generation.''Here we demonstrate distinctive roles for ciliary Gpr161 pools in preventing hedgehog hyperactivation phenotypes, contingent upon dependence of the morpho-phenotypic spectrum on downstream Gli repression''Ventral expansion in Ankmy2 knockout is severe than Gpr161 knockout, but even then, is fully Gli2-dependent''Gpr161 deletion showed GliR lack''However, lack of Gpr161 in cilia was incapable of sufficient Gli-activator generation required in causing ventral-most progenitor expansion in the neural tube.''Gpr161 ciliary pools regulate Hh repression regulated morpho-phenotypic spectrum'Typo'Gpr16mut/mut11 embryos exhibit high Hh signaling 1 and polydactyly in limb buds'Some inaccurate statements of facts:'Gpr161 C-tail has been proposed to be an A-kinase anchoring protein (AKAP) for PKA activation in cilia by binding to PKA regulatory subunit RIa and RIb .'The Bachmann paper, although focused on RIα, does show that Gpr161 C-tail binds to PKA regulatory subunit RIβ . They also find corresponding decrease in binding in a neurodegenerative patient mutation in RIβ (L50D) ). We do realize that much of the paper discussed RIα interactions. We have thus simplified the text throughout to reflect Gpr161 C-tail interactions with type I PKA regulatory subunits.'Direct Gpr161 C-tail binding to PKA RIa/RIb regulatory subunits  facilitates PKA activation in close proximity to Gpr161-mediated cAMP production in cilia for GliR processing.' The Bachmann reference shows that the AKAP motif of GPR161 is specific for PKA RIalphaPlease see previous comment.'Agonist-independent constitutive signaling.' The author may want to leave open the possibility that the tonic activity of GPR161 is caused by an agonist endogenous to the experimental system.Thanks, we now discuss this specific limitation in Discussion.'protein kinase A (PKA)-mediated limited proteolysis of full length Gli2/3 into Gli2/3 repressor (GliR) forms.' PKA does not mediate proteolysisThanks, corrected.'Increased extracellular Gpr161 pools in Gpr161mut1/mut1 also prevent Nkx6.1 ventralization from likely restoration of Gli3R processing.' Extra-ciliary?Thanks, corrected.References, et al. (2016). Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling. Proc Natl Acad Sci U S A 113, 7786-7791.Bachmann, V.A., Mayrhofer, J.E., Ilouz, R., Tschaikner, P., Raffeiner, P., Rock, R., Courcelles, M., Apelt, F., Lu, T.W., Baillie, G.S.182, 1519-1530 e1517.Bock, A., Annibale, P., Konrad, C., Hannawacker, A., Anton, S.E., Maiellaro, I., Zabel, U., Sivaramakrishnan, S., Falcke, M., and Lohse, M.J. (2020). Optical Mapping of cAMP Signaling at the Nanometer Scale. Cell 531, 656-660.Delling, M., Indzhykulian, A.A., Liu, X., Li, Y., Xie, T., Corey, D.P., and Clapham, D.E. (2016). Primary cilia are not calcium-responsive mechanosensors. Nature 63, 279-282.Gerk, P.M. (2011). Quantitative immunofluorescent blotting of the multidrug resistance-associated protein 2 (MRP2). J Pharmacol Toxicol Methods , et al. (2019). Primary Cilia Signaling Promotes Axonal Tract Development and Is Disrupted in Joubert Syndrome-Related Disorders Models. Dev Cell 51, 759-774 e755.Guo, J., Otis, J.M., Suciu, S.K., Catalano, C., Xing, L., Constable, S., Wachten, D., Gupton, S., Lee, J., Lee, A., et al. (2020). Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium. eLife 9.Hansen, J.N., Kaiser, F., Klausen, C., Stuven, B., Chong, R., Bonigk, W., Mick, D.U., Moglich, A., Jurisch-Yaksi, N., Schmidt, F.I.152, 210-223.Mukhopadhyay, S., Wen, X., Ratti, N., Loktev, A., Rangell, L., Scales, S.J., and Jackson, P.K. (2013). The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic hedgehog pathway via cAMP signaling. Cell Truong, M.E., Bilekova, S., Choksi, S.P., Li, W., Bugaj, L.J., Xu, K., and Reiter, J.F. (2021). Vertebrate cells differentially interpret ciliary and extraciliary cAMP. Cell.104, 12365-12370.Wang, Y.V., Wade, M., Wong, E., Li, Y.C., Rodewald, L.W., and Wahl, G.M. (2007). Quantitative analyses reveal the importance of regulated Hdmx degradation for p53 activation. Proc Natl Acad Sci U S A 13, 1169-1181.Wolff, C., Roy, S., and Ingham, P.W. (2003). Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo. Curr Biol , et al. (2020). Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling. Cell 182, 1531-1544 e1515.Zhang, J.Z., Lu, T.W., Stolerman, L.M., Tenner, B., Yang, J.R., Zhang, J.F., Falcke, M., Rangamani, P., Taylor, S.S., Mehta, S.The reviewers felt that dissecting the ciliary vs. non-ciliary role of this GPCR is an important advance. However, as discussed below, it will be necessary to revise both the abstract and conclusion to explicitly acknowledge that you have not shown that their mutant Gpr161 changes ciliary cAMP levels and that you have not demonstrated conclusively that the phenotypes seen in these mutants are due to loss of Gli3 repressor. One reviewer wrote, "The text of the paper still leaves one with the (erroneous) impression that they have altered "cAMP signaling". I think all they can say is that they have identified a mutant of Gpr161 that has reduced localization in cilia and the phenotypes caused by this mutant are different from the null (depending on the tissue examined). I think they should add a separate paragraph to the conclusion highlighting these limitations."mut1 functioning through cAMP signaling in cilia in the abstract. We have also added a separate section in Discussion highlighting the limitations of the current Gpr161 allele termed as “Limitations of the mut1Gpr161 allele.”We have extensively revised the abstract and the discussion explicitly addressing the above concerns. We have removed any reference to Gpr161mut1Gpr161 allele with regards to our lack of understanding of its role in ligand-induced cAMP signaling, ciliary cAMP signaling, and through loss of Gli3 repressor formation.This section explicitly addresses the limitations of mut1Gpr161allele arises from the fact that a physiological ligand, if any, for Gpr161 is unknown. Thus, we are unable to test role of Gpr161mut1 in ligand-induced cAMP signaling and formally rule out if this mutant is functioning as a hypomorph, in addition to being extraciliary.”For e.g., here we mention that “Currently, a limitation of the mut1 on ciliary cAMP levels.”We further mention that “In addition, there have been conflicting reports of ciliary cAMP concentration in kidney epithelial cells ranging from levels comparable to that in the cytoplasm  to supraphysiological levels ~4.5 µM . In the absence of a known ligand for Gpr161, and lack of consensus regarding efficacy of the currently available ciliary targeted cAMP probes, we are unable to show impact of Gpr161We also mention that:mut1Gpr161 are from loss of Gli3 repressor. The commonly used Gli3R allele (Gli3∆699) expresses a fusion protein containing the first 699 aa of Gli3 and extra residues from thymidine kinase used for generating the allele . The Gli3∆699 allele is of limited potency as a Gli3R, because it does not resemble Shh ko during limb development  as would be expected from Gli3R antagonizing Shh activity . Introducing improved Gli3R alleles with potency equal to that of the natural repressor (Gli3∆701)  in the background of Gpr161 and/or Gli2 mutants will be required in the future to conclusively show role of GliR in ko/Gpr161mut1Gpr161 phenotypes.”“Currently, we have not demonstrated conclusively that the resultant phenotypes in mut1 is cAMP signaling competent using expression in non-ciliated cells and TR-FRET assays measuring total cellular cAMP, measuring cAMP levels in cilia is thus technically limited by lack of any known ligand for Gpr161 and available probes. However, there is growing consensus in the field that Gpr161 functions through Gαs-cAMP signaling, PKA activation and GliR formation, at least in the context of developmental pathways  and across the vertebrate lineage .While we show that Gpr161Please prepare a carefully revised version that addresses these concerns:Reviewer #1:I appreciate the effort the authors have made to improve data presentation of phenotypes, clarify localization of Gpr161 and discuss the recent paper in zebrafish showing the importance of ciliary cAMP pools for Hedgehog signaling.Thank you.However, the authors have not addressed two major issues raised in my previous review:1. Does the mutation in Gpr161 (or its loss) cause a change in the ciliary levels of cAMP ? While there is an extensive discussion of the ciliary cAMP pools throughout the paper (and even a mention of this in the last sentence of the abstract), a demonstration that Gpr161 mutations changes the cAMP pool in cilia is lacking. Without additional data, the text must be carefully toned down as not to mislead the reader.mut1Gpr161 allele with regards to our lack of understanding of its role in ligand-induced cAMP signaling. We have also removed any reference to Gpr161mut1 functioning through cAMP signaling in cilia in the abstract. Please note that there have been conflicting reports of ciliary cAMP concentration ranging from levels comparable to that in the cytoplasm  to supraphysiological levels ~4.5 µM . Thus, clearly, the cAMP probes need further optimization. While we show that Gpr161mut1 is cAMP signaling competent using expression in non-ciliated cells, measuring cAMP levels in cilia is technically limited by lack of any known ligand for Gpr161.We have now added a separate section in Discussion addressing the limitations of 2. Again, while there is extensive discussion in this area, the authors do not show that Gpr161 mutation causes phenotypes in some tissues that are Gli3 repressor dependent. A simple experiment to address this is to show that the phenotypes in question can be rescued by re-expression of Gli3R. This would establish cause-effect rather than correlation. Without this, the limitations of the study must be stated clearly and conclusions softened.mut1Gpr161 are from loss of Gli3 repressor. Further demonstration would require introducing improved Gli3R alleles with potency equal to the natural repressor  in the background of mut1Gpr161/Gpr161 and/or Gli2 loss. Please note that the commonly used Gli3R allele (Gli3∆699) expresses a fusion protein containing the first 699 aa of Gli3 and extra 21 residues from the thymidine kinase gene sequence used for generating the mutant allele . This allele does not resemble Shh ko during limb development , as expected from Gli3R antagonizing Shh activity , suggesting limited potency as a Gli3R. These experiments using Gli3R alleles with potency equal to the natural repressor (Gli3∆701)  in the background of ko/Gpr161mut1Gpr161 and/or Gli2 loss are currently beyond the scope of the current manuscript. In addition, Gpr161 and Gli2 are on the same chromosome, so generating a double mutant for mut1Gpr161 and Gli2 knockout will require linking through genetic recombination by breeding mut1Gpr161with ko/+Gli2animals, as described for Gpr161 floxed and Gli2 knockout alleles in the Methods. We have discussed these limitations in the current manuscript in the separate section in Discussion on limitations of the mut1Gpr161 allele.We agree with the reviewer that we have not demonstrated conclusively that the resultant phenotypes in Reviewer #2:A major point raised in the first round of review was the limited evidence that GPR161mut1 fails to enter cilia. The added data considerably strengthen this point. GPR161mut1 is still absent from cilia in the exit mutants Arrb1/Arrb2 DKO and in cells depleted of BBS4 by siRNA.Thank you.In addition, the authors now show that GPR161mut1 is present in pericentriolar recycling endosomes, a notable point that enriches the discussion. This observation introduces the possibility that percentriolar cAMP generated by GPR161mut1 finds its way into cilia to raise ciliary cAMP levels to a level that suppresses formation of GliA.Thank you.The text has been modified to temper some of the conclusions and more accurately describe the results.Thank you.However, there are many instances where the writing is too convoluted and lacks clarity. In particular, the extensive re-writing of the abstract has made for some rather complex statements that only a few specialists will be able to comprehend. The level of textual editing required falls outside of the scope of peer-review. I had listed a few instances in my initial review but indicated that the list was not exhaustive. Yet, it appears that the authors only addressed the instances listed. It is a pity that clarity is still lacking as the lasting impact of this paper would be diminished if the paper were published in its current state. The authors may seek to engage the service of a professional editor.Our apologies if the text seemed complex. We have now carefully revised the text throughout the Abstract and Discussion.One instance is listed below but there are many more.'We conclusively ruled out that LAP-tagged Gpr161mut1  does not at all transit through cilia'. What do the authors rule out?Thank you for pointing to this gaffe and we are embarrassed by the mistake. We have corrected the statement as follows:mut1 does not at all transit through cilia.”“These experiments conclusively demonstrate that Gpr161References, et al. (2016). Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling. Proc Natl Acad Sci U S A 113, 7786-7791.Bachmann, V.A., Mayrhofer, J.E., Ilouz, R., Tschaikner, P., Raffeiner, P., Rock, R., Courcelles, M., Apelt, F., Lu, T.W., Baillie, G.S.11, 1129-1135.Bose, J., Grotewold, L., and Ruther, U. (2002). Pallister-Hall syndrome phenotype in mice mutant for Gli3. Hum Mol Genet 379, 221-228.Cao, T., Wang, C., Yang, M., Wu, C., and Wang, B. (2013). Mouse limbs expressing only the Gli3 repressor resemble those of Sonic hedgehog mutants. Dev Biol 145.Hwang, S.H., White, K.A., Somatilaka, B.N., Shelton, J.M., Richardson, J.A., and Mukhopadhyay, S. (2018). The G protein-coupled receptor Gpr161 regulates forelimb formation, limb patterning and skeletal morphogenesis in a primary cilium-dependent manner. Development 116, 12066-12071.Jiang, J.Y., Falcone, J.L., Curci, S., and Hofer, A.M. (2019). Direct visualization of cAMP signaling in primary cilia reveals up-regulation of ciliary GPCR activity following Hedgehog activation. Proc Natl Acad Sci U S A 418, 979-983.Litingtung, Y., Dahn, R.D., Li, Y., Fallon, J.F., and Chiang, C. (2002). Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity. Nature 220.May, E.A., Kalocsay, M., D'Auriac, I.G., Schuster, P.S., Gygi, S.P., Nachury, M.V., and Mick, D.U. (2021). Time-resolved proteomics profiling of the ciliary Hedgehog response. J Cell Biol 113, 13069-13074.Moore, B.S., Stepanchick, A.N., Tewson, P.H., Hartle, C.M., Zhang, J., Quinn, A.M., Hughes, T.E., and Mirshahi, T. (2016). Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics. Proc Natl Acad Sci U S A 152, 210-223.Mukhopadhyay, S., Wen, X., Ratti, N., Loktev, A., Rangell, L., Scales, S.J., and Jackson, P.K. (2013). The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic hedgehog pathway via cAMP signaling. Cell 16, 2865-2878.Persson, M., Stamataki, D., te Welscher, P., Andersson, E., Bose, J., Ruther, U., Ericson, J., and Briscoe, J. (2002). Dorsal-ventral patterning of the spinal cord requires Gli3 transcriptional repressor activity. Genes Dev , et al. (2018). Basal Suppression of the Sonic Hedgehog Pathway by the G-Protein-Coupled Receptor Gpr161 Restricts Medulloblastoma Pathogenesis. Cell reports 22, 1169-1184.Shimada, I.S., Hwang, S.H., Somatilaka, B.N., Wang, X., Skowron, P., Kim, J., Kim, M., Shelton, J.M., Rajaram, V., Xuan, Z.450, 47-62.Shimada, I.S., Somatilaka, B.N., Hwang, S.H., Anderson, A.G., Shelton, J.M., Rajaram, V., Konopka, G., and Mukhopadhyay, S. (2019). Derepression of sonic hedgehog signaling upon Gpr161 deletion unravels forebrain and ventricular abnormalities. Dev Biol 148.Tschaikner, P.M., Regele, D., Rock, R., Salvenmoser, W., Meyer, D., Bouvier, M., Geley, S., Stefan, E., and Aanstad, P. (2021). Feedback control of the Gpr161-Galphas-PKA axis contributes to basal Hedgehog repression in zebrafish. Development


Single-crystal monolayer graphene with a lateral size of 800 μm can be grown on the back of copper foils after 60 min of growth. Raman spectroscopy revealed the single-crystal graphene to be in uniform monolayers with a low D-band intensity.The nucleation of graphene at different locations in the quartz boat was studied, and the lowest nucleation density of graphene in the quartz boat was found. The nucleation density of graphene is the lowest at the bottom of the quartz boat near the gas inlet side. Based on the above results, a simple and reproducible way is proposed to significantly suppress the nucleation density of graphene on the copper foil during the chemical vapor deposition process. Placing the copper foil with an area of 1.3 cm × 1 cm in the middle of the bottom of the quartz boat or further back, and placing two copper pockets in front of the copper foil, an ultra-low nucleation density of ~42 nucleus/cm Graphene is composed of two-dimensional carbon atoms assembled in a hexagonal structure, akin to a honeycomb, and has exceptional chemical stability, superior mechanical stability, and very high thermal conductivities and electronic conductivities ,2,3. BecIt is still a challenge to grow large-size single-crystal graphene. There are two ideas to improve the size of single-crystal graphene, which are the single-seed method and the multi-seed method . The key2 was achieved. After increasing the growth time to 60 min, single-crystal monolayer graphene with a lateral size of 800 μm can be grown on the back of copper foils under the optimized growth condition. In addition, the Raman study indicates that the single-crystal graphene synthetized by our method has a very low D-band intensity on the silicon dioxide grown on Si wafers, which is of high quality.In this paper, the nucleation of graphene at different locations in the quartz boat isexplored through three groups of comparative experiments. Based on the results of these experiments, we report a simple and reproducible way to significantly suppress the nucleation density by placing the copper foil with an area of 1.3 cm × 1.0 cm in the middle of the bottom of the quartz boat, or further back, and placing two copper pockets in front of the copper foil. Nucleation density of graphene on the back of copper foils can be significantly reduced and an ultra-low nucleation density of ~42 nucleus/cm2:CH4 = 50:50:0.5 (in the unit of sccm), and growth time of 45 min. After studying the nucleation of graphene at different locations in the quartz boat, a new growth mode was used. A piece of copper foil with an area of 1.3 cm × 1.0 cm was prepared and then placed at the middle position of the bottom of the quartz boat. The distance between the copper foil and the bottom of the quartz boat was 0.5 cm, and the center of the copper foil was about 4 cm away from the tail of quartz boat . At the same time, two copper pockets were placed in front of the copper foil in order to further reduce the methane flow at the bottom of the quartz boat, as shown in Figure 6a. Finally, the quartz boat with copper foils was placed into the quartz tube and the growth test was carried out in the center of the heating furnace. The growth parameters were as follows: furnace temperature 1030 °C, Ar:H2:CH4 = 50:50:0.5 (in the unit of sccm), and growth time of 60 min. Each group of experiments was repeated multiple times, until the results were stable.Graphene was synthesized by the low-pressure CVD in a tube furnace. The copper foils were folded into copper pockets, and a Cu pocket was made by first bending a copper foil  and then crimping or pressing the three open edges carefully by using metal tweezers . The cop4)2S2O8) aqueous solution . After etching, the PMMA/graphene was rinsed in DI water several times. The PMMA/graphene was dried in a desiccator and then placed on the SiO2/Si substrate. The PMMA layer was dissolved using hot acetone .A thin film of polymethyl methacrylate (PMMA)  was spin-coated onto the graphene/Cu (600 r/min for 6 s then 3000 r/min for 40 s). The etching of Cu usually takes 3 h. Then, the PMMA/graphene/Cu thin film was placed into 30 mg/mL ammonium persulfate  and the nucleation density of graphene was very low. There were only about 102 nucleation sites of graphene in the area of 3.5 cm × 2 cm, and the nucleation density of graphene is 27 nucleus/cmThe above four groups of experiments were carried out multiple times in each group. The distribution of the nucleation density of graphene at four locations in the quartz boat was obtained after stable results appeared. We found that the nucleation density of graphene at the bottom of the quartz boat (location 2 or location 4) was lower than that at the top (location 1 or location 3), and the closer to the gas inlet side (location 1 or location 2), the lower the nucleation density of the graphene in the copper pockets method. In the copper pockets method, the growth of single-crystal graphene depends on the smooth inner surface of the copper pockets during the growth process. The smoothness of the inner surface is created by the dynamic balance of the evaporation and deposition of copper inside copper pockets . In addi2  were placed at the bottom of quartz boat, and two copper pockets were in front of this, single-crystal graphene could be stably obtained on the surfaces of copper foils. 2 under this condition. Based on the above results, we proposed a simple and reproducible way to synthesize single-crystal graphene. A copper foil with an area of 1.3 cm × 1.0 cm was placed on the middle of the bottom of the quartz boat, or further back, and two copper pockets were placed in front of the copper foil, as shown in 2D/IG intensity ratio was ~1.2, and there was no obvious D peak. High-quality graphene was obtained by our growth and transfer method.By placing copper foils on the bottom of the quartz boat . The single-crystal graphene with a diameter of 800 μm can be obtained directly on copper foils by our method after growing for 60 min. Raman spectroscopy revealed the single-crystal graphene to be in uniform monolayers with a low D-band intensity. The single-crystal graphene prepared in our work can be used in many kinds of 2D electronics, such as flexible electrodes and flexible field effect transistors, because of its ultra-high carrier mobility and excellent flexibility.